Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Pharmacologic resistance in colorectal cancer: a review:

Pharmacologic resistance in colorectal cancer: a review: 614530 TAM0010.1177/1758834015614530Therapeutic Advances in Medical OncologyW. A. Hammond et al. review-article2015 Therapeutic Advances in Medical Oncology Review Ther Adv Med Oncol Pharmacologic resistance in colorectal 2016, Vol. 8(1) 57 –84 DOI: 10.1177/ cancer: a review © The Author(s), 2015. Reprints and permissions: http://www.sagepub.co.uk/ William A. Hammond, Abhisek Swaika and Kabir Mody journalsPermissions.nav Abstract: Colorectal cancer (CRC) persists as one of the most prevalent and deadly tumor types in both men and women worldwide. This is in spite of widespread, effective measures of preventive screening, and also major advances in treatment options. Despite advances in cytotoxic and targeted therapy, resistance to chemotherapy remains one of the greatest challenges in long-term management of incurable metastatic disease and eventually contributes to death as tumors accumulate means of evading treatment. We performed a comprehensive literature search on the data available through PubMed, Medline, Scopus, and the ASCO Annual Symposium abstracts through June 2015 for the purpose of this review. We discuss the current state of knowledge of clinically relevant mechanisms of resistance to cytotoxic and targeted therapies now in use for the treatment of CRC. Keywords: colon adenocarcinoma, colon cancer, resistance Correspondence to: Introduction in determining initial and subsequent lines of Kabir Mody, MD Colorectal cancer (CRC) remains a significant treatment. Innate resistance is typically noted Division of Hematology/ Oncology, Mayo Clinic cause of morbidity and mortality worldwide with during early drug development or in early phase Cancer Center, Mayo high disease incidence and, in spite of large-scale clinical trials of biologic efficacy, however some- Clinic, 4500 San Pablo Rd S, Jacksonville, screening efforts recommended for all US adults, times innate resistance is not understood until FL 32224, USA significant numbers of patients presenting with retrospective analyses of in vivo studies. For mody.kabir@mayo.edu advanced, metastatic disease [Siegel et al. 2014b]. instance, resistance to EGFR antagonists was not William A. Hammond, MD Abhisek Swaika, MD Metastatic disease is considered incurable, with well understood and from initial studies only 10– Division of Hematology/ the exception of patients presenting with oligo- 20% of patients exhibited a response to the EGFR Oncology, Mayo Clinic, Jacksonville, FL, USA metastatic lesions confined to the liver or lung targeted therapies cetuximab or panitumumab amenable to resection, or metastasectomy [Cunningham et  al. 2004; Bardelli and Siena, [Abdalla et  al. 2004; Adam et  al. 2004]. When 2010]. The subsequent elucidation of RAS muta- treatment with curative intent is not possible, tions in CRC clarified a marker of innate resist- patients are typically given a combination of cyto- ance to these therapies, and changed their clinical toxic chemotherapy often in conjunction with a use. targeted therapy. In spite of advances in systemic therapy, the 5-year survival rate is still a mere Different mechanisms of acquired resistance can 12.5% [Siegel et al. 2014a], and the primary rea- exist for each cytotoxic therapy and each targeted son for treatment failure is believed to be acquired pathway, but often acquired resistance to one resistance to therapy which occurs in 90% of drug confers resistance to other drugs which may patients with metastatic cancer [Longley and work by different mechanisms of action, a con- Johnston, 2005]. Resistance to targeted therapy cept referred to as multidrug resistance (MDR). noted by disease progression is often noted within In general, resistance to traditional cytotoxic ther- 3–12 months on epidermal growth factor receptor apy is accomplished by decreasing the delivery of (EGFR) antagonists [Cunningham et  al. 2004; drug to the cancer cell, either by increased efflux Van Cutsem et  al. 2007], necessitating a change out of the cell mediated by ATP-dependent trans- in treatment. porters, by decreased uptake into the cell, or by a change in enzymes involved in metabolism. Malignant tumors can have intrinsic resistance Alternately, resistance can be conferred by and/or acquired resistance and each is important changes within the cell itself by genetic or http://tam.sagepub.com 57 Therapeutic Advances in Medical Oncology 8(1) epigenetic modifications that can alter drug sensi- or replication error (RER+). Microsatellites are tivity [Gottesman et al. 2002]. repetitive genetic sequences, typically 1–5 base pairs repeated 15–30 times, and instability in these Resistance to targeted therapies occurs by different regions due to either insertion or deletion of mechanisms including upregulation, mutation, or repeated units causes alteration in the DNA repli- activation of downstream signaling molecules cation process. Sporadic mutations in mismatch within specific pathways; pathway bypass mecha- repair (MMR) genes leading to MSI occur in 10– nisms; or increased cross-talk between analogous 20% of CRCs [Carethers et  al. 2004]. Mutated pathways [Longley and Johnston, 2005; Tejpar MMR genes, such as MLH1, MSH2, MSH6, et  al. 2012]. Understanding the mechanisms of PMS1, or PMS6 and loss of their respective pro- acquired drug resistance to targeted therapies is teins, can be caused by germline mutations as seen critical for the development of novel, rational, and in nearly all cases of hereditary nonpolyposis colon more effective treatment combinations and will cancer (Lynch syndrome), or by sporadic muta- help guide future therapies. tions [Wheeler et al. 2000]. The loss of MMR pro- teins causes diffuse errors in repetitive DNA Since the development of markedly improved sequences, resulting from loss of scanning and rec- genomic sequencing and molecular biology tech- ognizing errors during DNA replication and failure niques, a plethora of new putative mechanisms of to edit these errors to maintain an intact genetic resistance and potential therapies have been real- code [Fink et al. 1998]. The loss of these proteins ized. We will review some of these mechanisms is typically a result of epigenetic hypermethylation and their clinical relevance. of the promoter regions of both alleles of the MLH1 genes, which halts gene expression [Wheeler et al. 2000]. Deficiency in the subsequent Cytotoxic/cytostatic chemotherapy protein products of the hMSH2, hMLH1 and Cytotoxic chemotherapy has long been the back- hMSH6 MMR genes and loss of detection of mis- bone of treatment for CRC in patients with lymph matched and unpaired bases is thought to be a pri- node positive and metastatic disease. Efficacy was mary mechanism of inherent resistance to FPs first recognized in the US Intergroup INT-0035 because cells become tolerant to DNA damage trial in 1990 which showed the beneficial effect of and do not undergo apoptosis [Carethers et  al. 5-fluorouracil (5-FU) over surgery alone [Moertel 2004]. In one study, MMR deficient cell lines that et al. 1990]. Since the adoption of widespread use of had MMR function restored by inserting a cor- chemotherapy for advanced CRC in the adjuvant rected clone of hMLH1 gene led to increased sen- and metastatic setting, other cytotoxic drugs have sitivity to FPs [Meyers et al. 2001]. been studied and validated including capecitabine, tegafur, irinotecan, and oxaliplatin, leading to their incorporation into practice guidelines and wide- 5-FU spread use in clinical practice. The details of this 5-FU (Adrucil; Pfizer, Teva Pharmaceuticals) is pathway described below are depicted in Figure 1. the prototypical FP, a synthetic fluorinated pyrimidine analog, administered intravenously. It Fluoropyrimidines (FPs) such as 5-FU and works by multiple mechanisms to create fluori- capecitabine exert antitumor activity by inducing nated nucleotides that are incorporated into DNA a state of thymidylate deficiency and creating in place of thymidine (dTMP, or deoxythymidine imbalances in the nucleotide pool, which leads to monophosphate) thus inhibiting DNA replication impaired DNA replication, transcription, and and causing cell death. It is a prodrug that requires repair and subsequent cell death [Wilson et  al. intracellular conversion to its active metabolites. 2014]. 5-FU was one of the first chemotherapeu- The primary active metabolite is FdUMP (fluoro- tic drugs reported to have anticancer activity deoxyuridine monophosphate) which inhibits the [Heidelberger et al. 1957]. Following the earliest enzyme thymidylate synthase (TS), a key enzyme publications noting its efficacy in colon cancer, in the creation of the DNA nucleotide dTMP. 5-FU became the mainstay of therapy and is still Inhibition of TS prevents conversion of dUMP to given as a standard part of most treatments for dTMP, which in turn impairs DNA synthesis in advanced or metastatic CRC (mCRC). the S phase of the cellular replication cycle. In addition, other 5-FU metabolites 5-fluorouridine One inherent mechanism of resistance to FPs in a triphosphate (5-FUTP) and 5-fluorodeoxy subset of tumors is microsatellite instability (MSI), triphosphate (5-FdUTP) are created by alternate 58 http://tam.sagepub.com WA Hammond, A Swaika et al. Figure 1. Schematic representation of the fluoropyrimidine pathway. 5,10-CH2-FH4, 5,10-methylenetetrahydrofolate; 5-CH3-FH4, 5-methyltetrahydrofolate; 5’dFCR, 5’-deoxy-5- fluorocytidine; 5’dFUR, 5’-deoxy-5-fluorouridine; 5-FdUDP, 5-fluorodeoxyuridine diphosphate; 5-FdUMP, 5- fluorodeoxyuridine monophosphate; 5-FdUTP, 5-fluorodeoxyuridine triphosphate; 5-FU, 5-fluorouracil; 5- FUDP, 5-fluorodeoxyuridine; 5-FUDR, 5-fluorodeoxyuridine, 5-FUMP,5-fluorouridine monophosphate; 5- FUR, 5-fluorouridine; 5-FUTP, 5-fluorouridine triphosphate; CDHP, 5-chloro-2,4-dihydroxypyridine; DHFU, dihydrofluorouracil; DPD, dihydropyrimidine dehydrogenase; dTTP, deoxythymidine triphosphate; FBAL, fluoro-β-alanine; FH4, tetrahydrofolate; FH2, dihydrofolate; FUPA, α-fluoro-β-ureidopropionic acid; NDK, nucleoside diphosphate kinase; ORPT, orotate phosphoribosyltransferase; TFT, trifluorothymidine; TFT-MP, trifluorothymidine monophosphate; TFT-TP, trifluorothymidine triphosphate; TK, thymidine kinase; UMP-CMPK, uridine monophosphate-cytidine monophosphate kinase. enzymatic pathways and create false nucleotides and improved response rates to 5-FU [Salonga which are incorporated into DNA and interfere et al. 2000; Johnston et al. 1995; Leichman et al. with normal protein production leading to cell 1995]. TS is encoded by the TYMS gene, and death [Nicum et al. 2000]. genetic alterations occur by copy number varia- tions or by variations in the TYMS promoter One of the most well established mechanisms of region [Muhale et al. 2011]. In one study, patients resistance to 5-FU, and other antifolates, is with CRC with low TYMS gene expression had increased expression of TS, the primary target of improved median survival compared with those the metabolite FdUMP. TS expression is a key with higher TYMS expression [Leichman et  al. predictor of 5-FU activity [Longley et  al. 2003], 1997]. This concept was further validated by at and its expression has long been recognized as a least two published meta-analyses showing an primary determinant of resistance [Berger et  al. inverse relationship of TYMS gene expression 1985]. This is supported by studies showing an with survival and response to FP therapy [Popat inverse association between low tumor TS levels et al. 2004; Qiu et al. 2008]. http://tam.sagepub.com 59 Therapeutic Advances in Medical Oncology 8(1) Increased expression of TS is also an acquired TP is an enzyme encoded by the TYMP gene and mechanism of resistance after exposure to 5-FU is responsible for converting 5-FU to 5-fluoro- therapy [Chu et  al. 1993]. Normally, unbound 2-deoxyuridine (5-FUDR), an intermediate in the TS (not bound by the metabolite dUMP) binds conversion of 5-FU to the active metabolite to its own mRNA in a negative feedback loop to 5-FdUMP. It has been observed that expression inhibit its own translation, in turn reducing levels of TP correlates with response to 5-FU therapy of the enzyme. It is hypothesized that TS bound [Panczyk, 2014]. Cells with higher levels of TP by FdUMP cannot enact this negative feedback, theoretically should have greater sensitivity to resulting in increased protein expression and 5-FU due to increase in the concentration of decreased sensitivity to 5-FU [Longley et  al. FdUMP. To date, however, studies have shown 2002]. mixed results regarding response to 5-FU-based chemotherapy and level of TP expression. Low The inhibition of TS by the metabolite FdUMP TP expression, measured in one study by reverse requires formation of a complex between FdUMP transcriptase polymerase chain reaction (RT-PCR) and 5, 10-methylenetetrahydrofolate (CH THF). and in another by immunohistochemistry (IHC) Normally, multiple enzymes regulate the intracel- and tissue microarrays, correlated with improved lular level of folate in order to maintain an appro- treatment outcomes, in terms of overall survival priate pool of tetrahydrofolate, which is formed (OS), in patients with mCRC treated with adju- by the reduction of CH THF to 5-methyltetrahy- vant 5-FU [Soong et al. 2008; Salonga et al. 2000]. drofolate (CH THF) by the enzyme methylene- A more recent study analyzed tumor tissue from tetrahydrofolate reductase (MTHFR). MTHFR mCRC by RT-PCR and found longer time to pro- activates a unidirectional reaction to convert gression with high TP expression [Lindskog et al. CH THF to CH THF, thereby decreasing the 2014]. Further well-designed trials are needed to 2 3 amount of CH THF and in turn decreasing the establish a definitive link between TP expression activity of TS. Decreased enzymatic activity of and resistance to FP therapy. MTHFR increases the concentration of the reduced cofactor CH THF and increases the OPRT is the protein product of the uridine inhibition of TS by increased concentration of the monophosphate synthase (UPMS) gene. This FdUMP-CH THF complex. Several single enzyme catalyzes the conversion of 5-FU to nucleotide polymorphisms (SNPs) have been 5-fluoro-uridine monophosphate (5-FUMP), an shown to affect the activity of MTHFR. Two in intermediate but necessary step in the production particular are the 677C>T and 1298A>C poly- of the active metabolites 5-FUTP and 5-FdUTP. morphisms. These decrease the activity of the Increased expression of UPMS gene and increased enzyme and were observed in vivo to show mixed levels of OPRT in tumor tissue has been shown in results, with one study demonstrating a lack of multiple trials to increase the chemo-sensitivity of independent correlation to response [Marcuello cells to 5-FU [Koopman et  al. 2009; Tokunaga et  al. 2006] but numerous others showing a et  al. 2007; Isshi et  al. 2002]. Only one study, greater response to 5-FU based therapy [Sohn however, has demonstrated that decreased et  al. 2004; Etienne-Grimaldi et  al. 2010]. expression, by means of UPMS knockdown cell Although it seems intuitive that increased activity lines, leads to resistance to 5-FU [Muhale et  al. of this enzyme would lead to resistance, studies 2011]. More research is needed to determine if have not shown that upregulation or amplifica- this is a clinically significant biomarker of sensi- tion of the MTHFR gene or protein product cor- tivity or mechanism of resistance in vivo. relate with chemoresistance. Dihydropyrimidine dehydrogenase (DPD) is the Fluorouracil must go through several enzymatic enzyme primarily responsible for catabolism of steps before it is converted to its other active 5-FU to 5-fluorodihydrouracil (5-FUH ), also metabolites 5-FdUTP, and 5-FUTP. It has been referred to as dihydrofluorouracil (DHFU), the shown that activity of three of the necessary first step in 5-FU elimination. 5-FUH is then enzymes for these conversions, thymidine phos- converted into the soluble molecule 5-fluoro-β- phorylase (TP), uridine phosphorylase (UP), and alanine and eliminated in urine. Intrinsic overex- orotate phosphoribosyl transferase (OPRT), cor- pression of DPD by malignant cells has been relate with the sensitivity of CRC cells to the cyto- shown in vitro to extend resistance to 5-FU toxic effects of 5-FU [Schwartz et  al. 1985; [Longley and Johnston, 2005; Takebe et  al. Houghton and Houghton, 1983]. 2001]. High levels of DPD mRNA expression in 60 http://tam.sagepub.com WA Hammond, A Swaika et al. CRC cells have also been associated with 5-FU randomized trials completed in the mid-1990s resistance [Salonga et  al. 2000]. This has been comparing capecitabine to weekly bolus 5-FU/LV demonstrated as an intrinsic mechanism of resist- [Van Cutsem et al. 2001; Hoff et al. 2001]. These ance, but available data is not yet conclusive on results have been subsequently confirmed in a DPD as a means of acquired resistance. In addi- meta-analysis of six randomized trials that showed tion, DPYD gene expression has been investi- equivalent OS in patients treated with single gated as a biomarker of treatment resistance, but agent and matched capecitabine-containing regi- this has also not been shown to be clinically rele- mens to single agent and matched 5-FU-containing vant [Yanagisawa et  al. 2007; Vallbohmer et  al. regimens [Cassidy et al. 2011]. 2006]. Since capecitabine is eventually converted into Numerous variable number of tandem repeats 5-FU within the tumor, many of the mechanisms (VNTRs) and single nucleotide polymorphisms of resistance are identical to those implicated for (SNPs) of the TYMS, MTHFR, DPYD, and UPMS 5-FU. Several of these mechanisms have been genes have been identified as contributors to 5-FU evaluated specifically in capecitabine treated cells. resistance, and could serve as potential targets for As shown in relation to 5-FU, DPD expression future directed therapy to combat drug resistance, has been implicated in resistance to capecitabine. or potentially for gene therapy [Panczyk, 2014]. In one study, higher gene expression, as meas- ured by mRNA analysis from tumor tissues fol- lowing capecitabine treatment, correlated with Oral FPs resistance to therapy as evidenced by shorter pro- Capecitabine, S-1, and tegafur-uracil are three gression-free survival (PFS) and lower response oral FPs demonstrating similar efficacy as intra- rate in patients [Vallbohmer et al. 2007]. In a ret- venous 5-FU, with the potential for more con- rospective review of 556 tumors from the CAIRO venience by reducing time spent in an infusion (CApecitabine, IRinotecan, Oxaliplatin) study, suite or on an infusion pump. Capecitabine is the DPD expression inversely correlated with PFS only one of these currently approved for use in the and OS [Koopman et  al. 2009]. This study also United States. All of these therapies are prodrugs investigated the predictive value of multiple other which, by a series of enzymatic reactions, are markers including OPRT, TP, TS, and ERCC1, eventually converted to 5-FU in the tumor micro- and found that high OPRT in stromal cells was environment. Because of this, they have many of favorable for response to therapy, but elevated the same mechanisms of resistance as 5-FU, how- expression in tumor cells correlated with worse ever with additional layers needed for activation PFS and OS. The reason for this is unclear and come new potential sources of resistance. has not been fully elucidated. Capecitabine. Capecitabine (Xeloda; Genentech, TP converts 5-FU prodrugs into active metabolites Roche, Switzerland) is an oral FP carbamate that exert antitumor effect. This enzyme is which was developed to mimic continuous 5-FU expressed in higher concentration in tumors, but infusion, but with activation occurring primarily was shown to be expressed in the tumor microenvi- at the tumor site. After absorption it is converted ronment rather than the tumor cells. TP has other to fluorouracil by three enzymes, two of which, TP actions related to carcinogenesis, including promo- and UP, are present in higher concentrations tion of metastasis, tumor infiltration, and angiogen- within malignant cells compared to normal cells esis which correlate with poor prognosis [Ye and [Wilson et al. 2014]. This orally administered drug Zhang, 2013]. High expression of TP, however, was designed to function similar to infusional correlates with better response to capecitabine, and 5-FU, giving a consistent level of drug exposure to loss of function confers resistance [Petrioli et  al. tumor cells over time. Since the final steps in the 2010], a fact that has also been shown to have clini- pathway occur preferentially in the tumor it has a cal significance with improved response to CAPIRI theoretical advantage of increased efficacy and chemotherapy by extending time to progression decreased systemic toxicity, however this has not [Meropol et al. 2006]. This is seen in contrast to the been the case in clinical trials [ Cassidy et al. 2011]. inverse effect of TP expression on response to 5-FU. One putative mechanism of loss of function The efficacy of capecitabine in advanced CRC, of TP is abnormal pre-mRNA splicing by increased including stage III and metastatic disease, was levels heterogeneous nuclear RNP (hnRNP) H/F originally demonstrated by two phase III splicing factors which leads to loss of function of http://tam.sagepub.com 61 Therapeutic Advances in Medical Oncology 8(1) the TYMP gene [Stark et al. 2011]. Further research TAS-102. TAS-102 (Taiho Pharmaceutical, Tokyo, is ongoing in regards to exact mechanisms of devel- Japan) is an oral nucleoside antitumor agent with opment of resistance specifically to capecitabine multiple components. Trifluorothymidine (TFT) that may be distinct from other FPs. is the active ingredient and is a FP that is active in inhibiting DNA replication. TFT is phosphory- S-1 and tegafur-uracil. S-1 (TS-1; Taiho Pharma- lated by thymidine kinase (TK) and in turn inhib- ceutical, Tokyo, Japan) is a fourth generation oral its thymidine synthase (TS) in a similar fashion to FP available worldwide outside of the United other FPs. It can also form the triphosphate form, States. It consists of tegafur (UFT), gimeracil TFT-TP, which is incorporated into DNA thus (5-chloro-2, 4-dihydroxypyridine) and oteracil interfering directly with replication [Wilson et  al. (potassium oxonate). Tegafur is a prodrug that is 2014]. In a phase II trial comparing monotherapy converted to fluorouracil within tumor cells. with placebo, TAS-102 showed improvement in Gimeracil is an inhibitor of DPD, the primary both PFS and OS in a small subset of patients enzyme responsible for fluorouracil metabolism. with mCRC refractory to traditional therapy Oteracil inhibits the phosphorylation of fluoro- [Yoshino et al. 2012]. The results of the phase III uracil in the gastrointestinal tract and serves to RECOURSE trial comparing TAS-102 to placebo reduce toxic side effects of 5-FU [Sakuramoto in pretreated patients showed improvement in OS et  al. 2007]. Tegafur–uracil is another oral FP and PFS in European patients with mCRC refrac- agent and is approved in 50 countries worldwide. tory to at least two prior lines of therapy [Mayer It consists of tegafur attached to uracil, which et al. 2015]. As yet there is no direct experimental blocks DPD degradation of fluorouracil’s pyrimi- evidence supporting a process for development of dine base. Tegafur is not well tolerated by patients resistance, however one would presume it may fol- as they experience consistently high toxicity low a pattern very similar to other FPs. negating any benefit, which is the primary reason for lack of approval in the United States. Numerous gene polymorphisms of OPRT, MTHFR, UGT1A1, and DPD have been investi- The approval of S-1 is based on two trials in East gated in relation to toxicity [Tsunoda et al. 2011; Asian patients. It is considered an acceptable Choi et al. 2012], but to date none of these have alternative to 5-FU when combined with oxalipl- demonstrated clinical significance in relation to atin as part of FOLFOX or XELOX [Hong et al. chemoresistance. These oral FPs may prove use- 2012], or an alternative to FOLFIRI when com- ful for patients in the United States if they gain bined with irinotecan [Muro et al. 2010] in East FDA approval. Asian patients. A subsequent meta-analysis of eight trials from Japan and China in Asian patients with either mCRC or advanced gastric cancer Irinotecan demonstrated essentially equivalent efficacy to Irinotecan (Camptosar, formerly CPT-11; Pfizer, the 5-FU-containing regimens [Cao et al. 2014]. Pharmacia) is a semisynthetic analog of the natural alkaloid camptothecin, a DNA topoisomerase I Tegafur is metabolized by the cytochrome P-450 inhibitor. This enzyme relaxes super-coiled double- isoenzyme encoded by the CYP2A6 gene. The stranded DNA by inducing single strand breaks. By expression of the variants CYP2A6*4 and inhibiting its action, irinotecan interferes with DNA CYP2A6*1B seem to be the primary determi- replication and transcription. Topoisomerase I nants for the degree of conversion into 5-FU, and inhibitors stabilize intermediate cleavage complexes are required for the anticancer activity of tegafur formed between the inhibitor, the enzyme, and the [Wang et al. 2011]. This has been shown to cor- DNA single strand and subsequently prevent DNA relate with toxicity, a putative reason why white re-ligation, leading to cell death. Irinotecan must patients experience intolerable toxicity compared be converted to its active metabolite SN-38 to exert with East Asian patients [Shirao et  al. 2004]. It its anticancer effect [Xu and Villalona-Calero, has been shown that patients with wild-type 2002], and SN-38 reversibly binds to topoisomer- CYP2A6 have improved efficacy outcomes com- ase-1 and stabilizes the complex. It was approved in pared with mutants in gastric cancer, and also an accelerated fashion by the FDA in 1996 and showed increased response to TIROX compared received full approval for use in CRC in 1998. with several polymorphisms [Kim et  al. 2013], presumably related to decreased conversion to The addition of irinotecan to adjuvant therapy for 5-FU and exposure of tumor cells. advanced CRC and to mCRC was supported by 62 http://tam.sagepub.com WA Hammond, A Swaika et al. multiple trials starting in the early 1990s irinotecan in in vitro studies [Kojima et  al. 1998; [Douillard et  al. 2000; Saltz et  al. 2000b; Boyer et al. 2004], however, outside of associations Giacchetti et  al. 2000]. Single-agent irinotecan with specific SNPs with cytotoxicity, no direct cor- has only a modest response rate of 11–27% in relation has been shown between carboxylesterase clinical trials in 5-FU refractory patients with activity or expression and chemoresistance. SN-38 mCRC [Cunningham et  al. 1998], but a larger is metabolized by glucuronidation in the liver by effect when given in combination with FPs, 5-FU the enzyme uridine diphosphate glucuronosyl- and capecitabine, and/or oxaliplatin [Shimada transferase (UGT) to form SN-38 glucuronide et al. 1996]. In the first-line setting response rates (SN-38G). Increased glucuronidation-mediated range from 39% to 49% [Saltz et  al. 2000a; clearance in CRC cells may contribute to tumor Douillard et  al. 2000]. It has been approved as resistance to irinotecan [Cummings et  al. 2002]. first-line therapy in advanced or metastatic dis- Genetic variants of both liver and plasma UGT1A, ease, but not for adjuvant treatment as this was by VNTRs and other polymorphisms, have been shown to provide no additional benefit to FP shown to have effects on CPT-11 toxicity includ- monotherapy [Van Cutsem et  al. 2009]. The ing neutropenia and diarrhea, however these have addition of irinotecan to FP-based regimens also not been shown to correlate with response to ther- shows a significant benefit in PFS from 4.3 to apy [Marcuello et al. 2004]. Any association with 7 months, and OS from 12 to 18–21 months, and poor response to treatment is believed to be a result its efficacy as monotherapy in patients with FP of having to reduce the dose of the drug due to side refractory disease indicates activity in spite of effects, not from the polymorphism [Carlini et al. acquired resistance to FP therapy [Cunningham 2005; Rouits et al. 2004]. et al. 1998; Rougier et al. 1998]. Irinotecan itself is metabolized by oxidation, pri- Irinotecan resistance in CRC appears to develop marily by two hepatic cytochrome P-450 enzymes, by a few mechanisms including low intratumor CYP3A4 and CYP3A5. These enzymes metabolize level of the active metabolite SN-38, a decrease in irinotecan to APC ((7-ethyl-10-(4-N-aminopenta- expression of topoisomerase I, change in the noic acid)-1-piperidino) carbonyloxycamptoth- activity of the SN-38-Topo I- DNA complex, and ecin) and NPC (7-ethyl-10-(4-amino-1-piperidino) changes in downstream events such as suppres- carbonyloxycamptothecin), respectively. NPC is an sion of apoptosis, cell cycle alterations, or inactive metabolite but can be hydrolyzed by enhancement of DNA repair. hepatic carboxylesterase back to SN-38 [Panczyk, 2014]. Variations in CYP3A4 activity have been The level of intratumoral SN-38 can be altered by investigated as a source for resistance to treatment increased efflux or increased metabolism of either with irinotecan, however current research, includ- irinotecan or the metabolite SN-38. Active trans- ing studies measuring in vivo activity of these port out of cells by the multidrug resistance pro- enzymes, is inconclusive about the role of numer- tein (MRP), an ATP-binding cassette (ABC) ous described polymorphisms [Xie et  al. 2004; transporter protein, has been shown in cancer Fujiwara and Minami, 2010]. cells to result in resistance to irinotecan and SN-38 [Longley and Johnston, 2005; Thomas As described previously, SN-38 binds to topoi- and Coley, 2003]. Numerous in vitro studies of somerase I as it binds to DNA to relieve strand polymorphisms of transporter proteins, such as tension during replication. The action of irinote- ABCC1/MRP1, ABCC2/MRP2, and ABCG2/ can is dependent on normal functioning topoi- BCRP have shown results that explain variation somerase I, encoded by the TOP1 gene. Level of in drug toxicity in patients, and the development TOP1 gene expression and copy number was of drug resistance against irinotecan and SN-38 demonstrated as a potential cause of intrinsic [Zhao et  al. 2014]. However, these results have resistance by an in vitro study of colon cancer cell been inconsistent in the literature and there are lines [McLeod and Keith, 1996]. A more recent no in vivo studies to support development of these Scandinavian retrospective analysis of tumor tis- proteins as targets for therapy. sue from irinotecan pre-treated patients identified an increased objective response in tumors with The active metabolite SN-38 is created by hydrol- increased copy number of TOP1 gene, although ysis of CPT-11 by carboxylesterases CES1 and this did not reach statistical significance [Nygard CES2. Carboxylesterase activity in cancer cells has et  al. 2014]. Increased copy number has been been shown to correlate with sensitivity to noted in as many as two-thirds of tumors from a http://tam.sagepub.com 63 Therapeutic Advances in Medical Oncology 8(1) cohort of patients with stage III CRC [Smith et al. with response rates as high 50% [de Gramont 2013], so low levels of TOP1 gene copy number is et  al. 2000]. The GERCOR study published in implicated as a means of intrinsic resistance. 2004 [Tournigand et  al. 2004] showed equiva- lence of the two primary regimens FOLFOX and SN-38 forms non-covalent, but stable bonds to FOLFIRI, which was subsequently confirmed by the Topo-1-DNA complex to exert its cytostatic Colucci and colleagues the following year effect that leads to cell death. Changes in the [Colucci et  al. 2005]. Since the FOLFIRI regi- binding site of topoisomerase I prevent SN-38 men was shown to be ineffective in the adjuvant from creating a stable bond. One study of irinote- setting, FOLFOX has become the mainstay of can-treated tumor samples identified that point therapy in postoperative patients. mutations in the Top1 gene, as detected by RT-PCR of mRNA from tumor samples, altered The mechanisms of resistance of oxaliplatin the binding of SN-38 to the enzyme, implicating appear to be somewhat different from those seen acquired resistance to therapy [Tsurutani et  al. with cisplatin and carboplatin. In fact, it has been 2002]. Further in vitro analyses of SN-38 resist- shown to be active in cancer cell lines resistant to ant tumor cell clones showed that resistance may earlier generation platinum compounds [Raymond develop by decreased affinity of TOP1/SN-38 et al. 2002]. It has also been well established that binding or by mutations in the linker domain enhanced replicative bypass and loss of MMR are which lead to decreased flexibility of the complex mechanisms of resistance to cisplatin but not to [Gongora et al. 2011]. oxaliplatin by both in vitro and in vivo studies. One study comparing similar platinum resistant cell Again, numerous SNPs have been discovered to lines exposed to cisplatin and oxaliplatin showed help explain the genetics behind these mecha- that oxaliplatin exerted cytotoxic effects at a much nisms of resistance, as well as explain potential lower concentration than did cisplatin, and also variations amongst individuals with apparently created fewer DNA-Pt adducts [Hector et  al. similar tumor types [Panczyk, 2014]. The clinical 2001]. These adducts are likely different than relevance of these numerous polymorphisms is those created by cisplatin, supported by the fact not yet certain based on the current body of that MMR deficient cells demonstrate resistance literature. to cisplatin but not oxaliplatin [Fink et al. 1997], implying that even MMR deficient cells are able to recognize oxaliplatin DNA-Pt adducts and Oxaliplatin undergo apoptosis. Oxaliplatin (Eloxatin; Sanofi-Aventis Pharma- ceuticals) is a third generation platinum com- Like other chemotherapy, resistance can occur by pound that works by inducing DNA cross-linkages decreased entrance into or increased efflux out of the leading to apoptotic cell death. It is a square pla- tumor cells. The transporters regulating platinum nar platinum, distinct from other platinums in efflux are ABC-type MDR proteins as well as cop- that it contains a bidentate ligand 1,2-diaminocy- per-transporting p-type ATPases [Panczyk, 2014]. clohexane in lieu of two monodentate ammine A few transporters have been recognized to regulate ligands. It causes inter- and intra-strand DNA influx of platinum compounds, including copper cross-links that halt replication and transcription transporter (CTR) proteins, organic cation trans- [Hector et  al. 2001]. It was approved for use in porters (OCTs), and an undefined cis-specific plati- Europe in 1996 and it was granted accelerated num influx transporter that has not been implicated approval in 2002 by the US FDA, with full in oxaliplatin resistance. Some transporters have approval granted in 2004 for use in combination been investigated, including members of the SCL22 with 5-FU for advanced CRC or mCRC. family (such as OCT2), as well as CTRs [Zhang et  al. 2006] (including CTR1 and CTR2 [Holzer Oxaliplatin has been shown to have minimal sin- et al. 2006]), and these have been shown in preclini- gle agent activity, with response rates of around cal studies to be potential targets of modulation of 20–24%, in several phase II trials [Becouarn et al. influx and efflux. No in vivo studies have shown 1998; Diaz-Rubio et  al. 1998] in the front-line actual clinical significance of these postulated resist- setting. In combination with 5-FU and leucov- ance mechanisms. In vitro studies have shown that orin (FOLFOX), however, it has been shown in oxaliplatin requires lower concentrations to exert multiple trials to contribute to increased PFS and anticancer effects, but these transports have not been OS compared with 5-FU and leucovorin alone shown to be major mechanism of resistance. 64 http://tam.sagepub.com WA Hammond, A Swaika et al. A mechanism of platinum compound inactivation Epigenetic changes have also been implicated in is the formation of conjugates, or covalent link- development of resistance, primarily hypermeth- ages, between the drug and the thiol glutathione ylation. Although oxaliplatin works by inducing (GSH, a tripeptide of glutamic acid, cysteine, and single-strand breaks by intra-strand adduct for- glycine) [Meijer et  al. 1992]. Glutathione is a mation, it also forms inter-strand adducts and can potent antioxidant that functions to prevent oxi- cause double-stranded breaks. These abnormali- dative damage to DNA and RNA. This also serves ties are repaired by the NER process, as well as by as a mechanism of increased clearance, however, BRCA1, which works by homologous recombina- because GSH–platinum conjugates become a sub- tion to repair double-strand breaks [Fedier et  al. strate for the ABC transporter proteins which pro- 2003]. Inactivation of the BRCA1 interactor motes drug efflux out of the cell [Ishikawa and SRBC by hypermethylation of the SRBC1 gene is Ali-Osman, 1993]. It has been shown that some associated with oxaliplatin resistance [Moutinho tumors may be resistant to platinums due to their et  al. 2014]. Little has been definitively discov- having higher levels of GSH [Kelland, 1993]. ered about the impact of epigenetic alterations on There are also studies demonstrating the potential CRC resistance, however, as in other tumor influence of several polymorphisms of the glu- types, this is likely a mechanism. tathione s-transferases, however this is not con- sistently shown and has not been demonstrated in Resistance to cytotoxic chemotherapy occurs by in vivo studies [Panczyk, 2014]. A meta-analysis several variations on similar themes, such as of five clinical studies showed no correlation of a decreased intracellular drug concentration, common polymorphism, 313A>G, with response altered metabolism, or alterations to targets of the to oxaliplatin-based therapy [Ye et al. 2013]. therapy. These mechanisms of resistance are summarized in Table 1. Some of these concepts Platinum compounds exert their antitumor effect translate into mechanisms of resistance to novel by creating adducts in the DNA, typically intra- targeted therapies, however often resistance is strand cross-links [Hector et al. 2001], that lead to more complex, at times not involving mutation apoptosis once recognized by MMR mechanisms. but molecular pathway alteration, something not Nucleotide excision repair (NER) is a mechanism seen with evasion of traditional chemotherapy. by which cells repair DNA damage and one of the ways in which cancer cells overcome chemother- apy effects. This may be particularly true for plati- Targeted therapy nums by removing DNA-Pt adducts from the As knowledge about cancer biology and genetics strand. Excision repair cross-complementation expands, new treatment targets have been discov- group 1 and 2 (ERCC1 and 2) proteins are two of ered and drugs developed to affect tumors in the main effectors of the NER mechanism. They more elegant and rational fashion than impacting recognize DNA-Pt adducts and coordinate the all cells actively in the cell cycle. This has led to base excision process. High expression of ERCC1 treatments with good response and often less- mRNA has been associated with poor response to toxic side-effect profiles than cytotoxic/cytostatic FOLFOX in 5-FU resistant tumors [Shirota et al. therapy. Two broad categories of targeted thera- 2001], implicating that enhanced DNA repair pies include monoclonal antibodies and small decreases the benefit of platinum therapy. molecule inhibitors, different in their target of Oxaliplatin-resistant tumors have upregulation of action and mode of administration. ERCC1 as shown by higher mRNA expression in retrospective analysis of tumor samples [Baba Although targeted agents provide hope for more et  al. 2012] implicating that this may also be a effective therapy, studies have shown modest ben- form of acquired resistance. Numerous polymor- efit and, like more traditional chemotherapy, are phisms in the ERCC1 gene have been identified subject to both primary and secondary resistance and investigated as predictive, however most have which ultimately leads to treatment failure. Protein shown inconclusive results [Panczyk, 2014]. The tyrosine kinases, such as EGFR and vascular ERCC1 (354C>A) SNP, in conjunction with the endothelial growth factor receptor (VEGFR) are XRCC1 (1196A>G) SNP have an independent some of the most well-understood pathways of predictive effect on disease control rate and OS potential therapy used to treat CRC. Resistance is [Liang et  al. 2010]. This, however, must still be often seen through constitutive pathway activation, taken in the context of a retrospective analysis and perhaps by receptor overexpression or mutation. we await further prospective analyses. By identifying specific ligands or receptors involved http://tam.sagepub.com 65 Therapeutic Advances in Medical Oncology 8(1) Table 1. Reported mechanisms of resistance to chemotherapy agents. Chemotherapy Enzyme/pathway Mechanism of resistance (MoR) Reference References contrary to proposed MoR 5-Fluorouracil Thymidylate synthase Increased expression leading to Popat et al. [2004], (5-FU) (TS) increased target of 5-FU inhibition Qiu et al. [2008] Decreased negative feedback by Longley et al. TS-FdUMP on its own expression [2002] Methylene Increased activity of MTHFR, Sohn et al. [2004], Marcuello et al. [2006] tetrahydrofolate decreasing CH THF availability Etienne-Grimaldi reductase (MTHFR) required for inhibition of TS et al. [2010] (postulated) Thymidine phosphorylase Increased expression (postulated), Soong et al. [2008], Lindskog et al. [2014] (TP) may lead to increased salvage Salonga et al. pathway of nucleotides. Low [2000] expression correlates with increased response to 5-FU therapy. Orotate phosphoribosyl Decreased expression Muhale et al. transferase (OPRT) (postulated), as high expression [2011] correlates with sensitivity Dihydropyridine Increased expression leading to Salonga et al. Yanagisawa et al. dehydrogenase (DPD) increased degradation [2000] [2007], Meropol et al. [2006], Vallbohmer et al. [2006] Capecitabine Thymidine phosphorylase Decreased expression leading Meropol et al. (TP) to decreased formation of active [2006], Petrioli metabolite et al. [2010] Dihydropyridine Increased expression leading to Vallbohmer et al. dehydrogenase (DPD) increased metabolism [2007], Koopman et al. [2009] Irinotecan Multidrug resistance Increased expression leading to Zhao et al. [2014] protein (MRP) increased efflux Uridine diphosphate Increased expression leading to Cummings et al. glucuronosyltransferase increased metabolism of SN-38 [2002] (UGT) Carboxylase Decreased expression Kojima et al. (postulated) as enzyme is required [1998], Boyer et al. to create active metabolite [2004] Topoisomerase-I Decreased copy number of TOP1 Nygard et al. gene (postulated), as increased [2014] copy number Alterations in binding site Tsurutani et al. [2002], Gongora et al. [2011] Oxaliplatin Multidrug resistance Increased expression leading to Zhang et al. [2006] protein (MRP) increased efflux Glutathione (GSH) Increased levels of GSH Kelland [1993] Ye et al. [2013] inactivating the platinum and increasing export from cell ERCC1 Increased expression leading to Shirota et al. increased nucleotide excision [2001], Baba et al. repair [2012] 66 http://tam.sagepub.com WA Hammond, A Swaika et al. Table 2. Reported mechanisms of resistance to targeted therapies. Targeted Enzyme/ Mechanism of resistance Per cent present Reference therapy gene/pathway in de novo tumors [Bardelli and Siena, 2010] EGFR EGFR Decreased expression (postulated) Sartore-Bianchi et al. [2007], antagonists as increased expression is shown to Cappuzzo et al. [2008] correlate with improved response KRAS Activating mutation 35–45%, at least Lievre et al. [2006], De Roock 50% of secondary et al. [2010], Douillard et al. mutations* [2013], *Misale et al. [2012] Amplification 0.7% Misale et al. [2014] NRAS Activating mutation 3–5% Douillard et al. [2013], Meriggi et al. [2014] PTEN Loss of function (mutation or loss of 27–30% Frattini et al. [2007], Sartore- expression), leads to constitutively Bianchi et al. [2009] activated AKT PI3K Activating mutation 14–17% De Roock et al. [2010], Sood et al. [2012] HER2 Amplification, serves as a bypass, or 3% Perrone et al. [2009], Sood escape mechanism et al. [2012], Rajput et al. [2007] MET Amplification, serves as a bypass, or 2% Bardelli et al. [2013] escape mechanism BRAF Activating mutation, constitutive 5–10% Di Nicolantonio [2008], activation of MAPK pathway Hirschi et al. [2014] Paracrine Increased serum levels and binding Hobor et al. [2014] activation of alternate ligands, TGF-α and amphiregulin Increased binding of ligands (e.g. Yonesaka et al. [2011] heregulin, HGF) to parallel activating pathways, (e.g. HER2, MET) Cetuximab EGFR Confers resistance to cetuximab after Esposito et al. [2013] S492R point exposure, but not to panitumumab mutation VEGF VEGF Autocrine signaling, with increased Mesange et al. [2014] antagonists expression caused by positive feedback (increased HIF induced by hypoxia) Alternate Increased expression of PlGF, IL-8, Kopetz et al. [2010], Lieu ligands VEGF-D, et al. [2013], Mizukami et al. [2005] Vascular Recruitment of pro-angiogenic Mitchell [2013], Pollard protection factors, e.g. BMDCs, tumor-associated [2004], De Palma et al. macrophages, TIE2, and VEGFR1 positive [2005], Hattori et al. [2002] hemagiocytes Increased pericyte coverage Kamba and McDonald [2007] Increased Increased local invasion and metastasis Du et al. [2008] invasiveness by co-opting local vasculature Aflibercept VEGF-C Increased binding Li et al. [2014] BMDC, bone-marrow-derived cell; EGFR, epidermal growth factor receptor; HGF, hepatocyte growth factor; IL, interleukin; KRAS, Kristen rat sarcoma; NRAS, neuronal rat sarcoma; PTEN, phosphatase and tensin homolog; TGF-α, transforming growth factor alpha; VEGF, vascular endothelial growth factor. http://tam.sagepub.com 67 Therapeutic Advances in Medical Oncology 8(1) Figure 2. Schematic representation of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) and receptor (VEGF-R) pathways. BAD, Bcl-2 associated death promotor; EGF, epidermal growth factor; EIF-4, eukaryotic initiation factor-4; Grb2,growth factor receptor bound protein 2; MAPK, mitogen activated protein kinase; MEK, mitogen/extracellular signal related kinase; mTOR, mammalian target of rapamycin; MDM2, mouse double minute 2; NFκB, nuclear factor κ-light chain enhancer of activated B cells; TGF-α, transforming growth factor alpha; TF, transcription factor; P, phosphate; PI3K, phosphoinositide 3 kinase; PIP2, phosphatidylinositol (4, 5)-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-triphosphate; RAS, rat sarcoma; RAF, rapidly accelerated fibrosarcoma; SOS, son of sevenless. in cell growth or survival, both the activating ligand kinase domain, a transmembrane hydrophobic seg- and receptor can potentially be targeted. Common ment, and an extracellular receptor to which ligands mechanisms of resistance are related to alterations bind for activation. It is a member of the ErbB of the target itself, bypass mechanisms, upregula- tyrosine kinase receptor (TKR) family, and is also tion and activation of downstream effectors, or referred to as ErbB1, or HER1. This family also cross-talk between associated pathways which acti- includes human epidermal growth factor receptor 2 vate complementary cell survival and growth path- (HER2) which is implicated in other cancer types, ways. The details of these pathways described also known as ErbB2. Other members of the ErbB below are depicted in Figure 2. family include Erb3 and 4. All four ErbB family members can interact with each other to induce signaling depending on which ligand is bound. Epidermal growth factor EGFR is activated primarily by the binding of epi- EGFR is a transmembrane signaling protein on dermal growth factor (EGF) or transforming human epidermal cells involved in cellular signaling growth factor-alpha (TGF-α) [Roskoski, 2014]. for proliferation and survival, and its over-expres- sion is linked to cancer development and prolifera- Inactive EGFR exists as a monomer, however tion. It is composed of an intracellular tyrosine once a ligand binds to the extracellular domain, 68 http://tam.sagepub.com WA Hammond, A Swaika et al. this induces either homodimerization with another Phosphatase and tensin homolog (PTEN) is a EGFR monomer or heterodimerization with a dif- negative regulator of the PIP3KCA/Akt pathway, ferent ErbB family receptor. This then induces and is one of the most frequently mutated tumor transphosphorylation of several intracellular tyros- suppressor genes [Yamada and Araki, 2001]. Its ine kinase (TK) domains which propagate down- normal function is to regulate the activity of PIP3 stream signaling by several well-described by dephosphorylation, and thus inhibit Akt activ- pathways, most notably the Ras/Raf/MEK/ ity. Mutation of PTEN and loss of its activity thus ERK1/2/MAPK and PI3K/Akt/mTOR pathways leads to constitutive activation of Akt and promo- [Roskoski, 2014; Spano et  al. 2005]. The PI3K/ tion of cell survival. Akt pathway has been shown to participate pri- marily in mediating cell survival and motility inva- Upregulation or activation of EGFR is present in sion, where the Ras/Raf/MEK/ERK1/2 pathway is 60–80% of CRCs [Messa et  al. 1998; Porebska implicated in cellular proliferation. Both of these et al. 2000; Salomon et al. 1995] and overexpres- pathways are also involved in angiogenesis, cellu- sion of EGFR is associated with poor prognosis in lar adhesion, cell motility, development, and mCRC [Mayer et al. 1993]. As such, this receptor organogenesis [Roskoski, 2012]. has been a focus of drug development and targeted therapies against this receptor are approved for use Initiation of the Ras/Raf/MEK/ERK pathway by in CRC, including cetuximab and panitumumab. EGFR begins with activation of Grb2 (growth factor receptor bound protein 2) adaptor protein Cetuximab. Cetuximab (Erbitux; Bristol-Meyer which binds to the phosphotyrosine residue of the Squibb, Eli Lilly and Company) is a chimeric ErbB receptor as well as SOS (son of sevenless), a mouse/human IgG1 monoclonal antibody that Ras-guanine nucleotide exchange factor binds to the extracellular domain of the EGFR. [Lowenstein et  al. 1992]. SOS activates Ras to Cetuximab was first approved by the FDA for use Ras-GTP which propagates downstream signal- in mCRC in February 2004 as monotherapy or in ing, starting with Raf and resulting in cell growth combination with irinotecan. Cetuximab was and differentiation [Roskoski, 2012]. shown to have a response rate of about 10% as monotherapy in a study of irinotecan pretreated Activation of the PI3K tyrosine kinase by EGFR patients by Cunningham and colleagues with induces phosphorylation of membrane-bound improved time to progression from 1.5 to phosphatidylinositol 4,5-bisphosphonate (PIP ) 4.1  months [Cunningham et  al. 2004]. In addi- to form phosphatidylinositol 3,4,5-triphosphate tion, Jonker and colleagues showed an 8% partial (PIP ). PIP attracts Akt (also known as protein response rate and improved PFS, as well as 3 3 kinase b, or PKB) to the plasma membrane improved overall quality of life compared with [Longley and Johnston, 2005]. Akt is a serine/ best supportive care in heavily pretreated patients threonine kinase that binds tightly to PIP and with mCRC [Jonker et al. 2007]. Interestingly, the interacts with various other kinases, including addition of cetuximab to irinotecan in irinotecan- phosphoinositide-dependent protein kinase 1 resistant/refractory disease showed prolongation (PDK1) and mammalian target of rapamycin of both PFS and OS compared with cetuximab complex 2 (mTORC2). These kinases both phos- monotherapy, suggesting a means of overcoming phorylate Akt and stimulate it to phosphorylate resistance to irinotecan [Cunningham et al. 2004]. and activate mTOR, another serine/threonine kinase which has multiple targets involved in cell The current understanding of the mechanism of survival. Activated Akt also phosphorylates, and action of cetuximab is that the drug binds to the thus inactivates, Bcl2 associate death promotor external domain of the EGFR and prevents ligand (BAD), which, in its unphosphorylated state, is binding, preventing cell growth and survival. pro-apoptotic by binding and sequestering the After binding, the receptor is internalized and antiapoptotic Bcl2. Akt also has the ability to degraded without activation or phosphorylation affect apoptosis by phosphorylating MDM-2 [Tabernero, 2007]. There is also evidence that which, once translocated to the nucleus, down- cetuximab-receptor binding also induces anti- regulates p53 expression [Feng et al. 2004]. Thus, body-mediated cytotoxicity, leading to tumor cell by activation of the PI3K/Akt pathway, EGFR death [Ciardiello and Tortora, 2008]. In addi- promotes cell survival via both mTOR and Bcl2 tion, cetuximab was shown to down-regulate activation and by p53 downregulation [Roskoski, VEGF expression, thus reducing tumor angio- 2014]. genesis [Ciardiello et al. 2000]. http://tam.sagepub.com 69 Therapeutic Advances in Medical Oncology 8(1) Early in the investigation into mechanisms of inherent resistance to both cetuximab and panitu- resistance, it was postulated that somatic EGFR mumab, leading to minimal clinical effect [De mutations may play a significant role in lack of Stefano and Carlomagno, 2014]. Lievre and col- response, as was demonstrated to other targeted leagues first noted that tumor response was dic- therapies, for example in lung and breast cancer tated by KRAS mutation status when they [Paez et al. 2004; Hudis, 2007]. It has since been examined 30 tumors and found that 13/30 tumors suggested by large cohort studies that EGFR harbored a KRAS mutation and two thirds (68%) mutations are not only uncommon in mCRC, but of the nonresponders had a mutation whereas this that they do not impact response when they are was found in none of the responders [Lievre et al. present [Barber et al. 2004; Moroni et al. 2005]. 2006]. The first large retrospective review of However, one specific point mutation of EGFR, a treated tumors found that 43.2% of tumors had at change of serine to arginine at codon 492 (S492R) least one mutation in exon 2 which correlated with leading to a change in the external domain of the lack of response to cetuximab [Karapetis et  al. EGFR was recently shown to confer resistance to 2008]. The predictive mutations were noted first cetuximab binding but not the other approved in exon 2, codons 12 and 13, however subsequent EGFR targeted therapy panitumumab [Van studies have shown that as many as 5–11% of Emburgh et al. 2014]. It has been shown that this additional tumors have mutations in exons 3 mutation is likely present only in patients follow- (codons 59 and 61) and 4 (codons 117 and 146) ing exposure to EGFR antagonism, and not in which are similarly predictive of lack of response treatment-naïve patients [Esposito et  al. 2013]. to EGFR antagonist therapy [Therkildsen et  al. Recently, the incidence of this mutation in cetuxi- 2014; Misale et al. 2014]. mab and panitumumab treated tumors was evalu- ated in a review of tumor samples from the phase The discovery of activating KRAS mutations led III ASPECCT trial of second-line treatment of to better patient selection, however, in spite of wild-type KRAS exon 2 mCRC [Price et  al. ‘wild type’ EGFR, still only about 40% of patients 2014]. Of the 999 patients, roughly half in each responded to EGFR targeted therapy, indicating group had post-treatment EGFR S492R status an alternate means of innate resistance. KRAS available, and the mutation was found in 1% of amplification has been identified as a cause of panitumumab treated tumors and 16% of cetuxi- resistance to anti-EGFR therapy as well, but is mab treated tumors. The mutation was not iden- likely present in only in ~1–2% of cases and it is tified in any of the pretreatment samples, mutually exclusive from KRAS mutations [Misale confirming that this is an acquired mutation con- et  al. 2014]. Resistance to EGFR antagonists is ferring resistance. In addition, these EGFR also influenced by neuronal RAS (NRAS) muta- mutant tumors demonstrated longer duration of tions [Douillard et al. 2013; Meriggi et al. 2014]. treatment before progression, but had lower median OS by almost 2 months [Price et  al. NRAS is a gene found on the short arm of chro- 2015]. mosome 1 which encodes for a GTPase mem- brane enzyme that shuttles between the Golgi and Although the relationship between EGFR muta- the cell membrane [De Stefano and Carlomagno, tions and response remains unclear, there is evi- 2014]. Mutations have been identified at exon 2 dence that increased copy number of the EGFR (codons 12 and 13), exon 3 (codons 59 and 61) receptor, as measured by fluorescent in situ and exon 4 (codons 117 and 146) and occur at a hybridization (FISH), is a positive predictor of frequency of approximately 2–5% among patients response to treatment with both cetuximab and with mCRC. These mutations have been shown panitumumab [Sartore-Bianchi et  al. 2007; to negatively impact response to anti-EGFR ther- Cappuzzo et al. 2008]. Likewise, low-level expres- apy, with either cetuximab or panitumumab sion is purported as a means of intrinsic resistance [Meriggi et  al. 2014; De Roock et  al. 2010; to therapy, yet this has not been shown clinically. Therkildsen et al. 2014]. Mutations in NRAS are mutually exclusive of mutations in KRAS and In early clinical trials, only about 10–20% of unse- BRAF. In spite of increasing knowledge, there are lected patients with mCRC responded to single still tumors that do not respond to treatment, agent EGFR antagonists. It is now known that indicating some yet unidentified innate or intrin- tumors with a mutation of the Kristen rat sarcoma sic resistance. For now, it is standard of care to (KRAS) gene found on chromosome 12, encod- test not only for KRAS mutations in exons 2 ing a small G-protein downstream of EGFR, have [Allegra et  al. 2009], 3 and 4, but also NRAS 70 http://tam.sagepub.com WA Hammond, A Swaika et al. mutations prior to exposing a patient to anti- contribute to EGFR therapy resistance, however EGFR therapy. this has not been definitively demonstrated in clinical trials. Activating KRAS mutations have been shown to be a mechanism of both intrinsic and acquired Many of the aforementioned mutations and alter- resistance. In a large analysis by Misale and col- ations play a central role in innate, or primary leagues, EGFR sensitive cell lines of primary resistance. It has been shown that approximately colon tumors and metastases showed that numer- 50% of tumors harbor a KRAS mutation at time ous molecular alterations, mostly point mutations of progression on treatment with anti-EGFR in KRAS gene, led to both cetuximab and panitu- therapies [Hobor et al. 2014; Misale et al. 2012], mumab resistance. This study also showed that implying that other means of resistance subvert KRAS mutations could be detected in blood sam- the effect of EGFR blockade. Heterogeneity ples as early as 10 months before radiographic within a given tumor is often observed where evidence of disease progression, implicating that mutated cells (e.g. KRAS, NRAS, BRAF this type of resistance may occur quite early in mutated) exist along with other sensitive, non- disease treatment [Misale et al. 2012]. mutated cells, implicating a potential protective mechanism. In addition, it is known that EGFR Changes in pathways related to the target of ther- has multiple ligands other than EGF, including apy, or parallel to it, may confer resistance. In TGF-α and amphiregulin, which bind to the regard to EGFR targeted therapies, PIK3CA receptor and activate downstream signaling. A activating mutations are seen in 14.5% of paracrine method of resistance has been postu- untreated tumors, most in exons 9 and 20 [De lated in which tumor cells can increase the secre- Roock et  al. 2010]. These mutations have been tion of these alternate ligands which serve to shown to predict lack of response to EGFR ther- protect the cells from EGFR-blockade. This was apy in pre-clinical models and early in vivo stud- shown in an in vitro study of cetuximab-resistant ies [Jhawer et  al. 2008; Sartore-Bianchi et  al. cells cultured with cetuximab-sensitive cells 2009; Sood et  al. 2012]. The largest analysis to where the sensitive cells grew in the presence of date, however, with more than 750 untreated cetuximab and increased secretion of ligands and CRC tumor samples from European centers increased EGFR signaling were observed [Hobor showed an association between exon 20 and et al. 2014]. worse response to cetuximab therapy, but not exon 9, perhaps implicating limitations of tumor Lastly, another mechanism of acquired resistance sample volume in prior studies that showed asso- may occur by alteration of complementary path- ciation with exon 9 [De Roock et  al. 2010]. ways which serve to bypass the EGFR antago- Additionally effecting the EGFR/PI3K/Akt path- nism, so-called escape mechanisms. In particular, way is epigenetic inactivation of PTEN phos- upregulation of ErbB2 (HER2) and MET can phatase which has been suggested to be predictive occur and may contribute to resistance. ErbB2 is of lack of response to EGFR targeted therapy a receptor with no known biologic ligand that [Perrone et al. 2009]. Loss of function in PTEN undergoes a conformational change that activates has been observed to occur by means of muta- the PI3K/Akt pathway [Rajput et  al. 2007]. tion, loss of gene expression, or hypermethylation Amplification of HER2 has been noted in only of the promotor region [Sood et al. 2012]. Studies 2–3% of untreated tumors, however has been have shown that patients with tumors having shown that cetuximab-resistant cell lines demon- preservation of PTEN and wild-type PIK3CA strate both HER2 amplification as well as an had improved OS and a trend toward improved increase in secretion of heregulin, a ligand which PFS [Sood et al. 2012], and that time to progres- binds to the HER2 receptor [Yonesaka et  al. sion was significantly shorter in tumors with 2011; Ciardiello and Normanno, 2011; Bertotti PIK3CA mutations and loss of PTEN [Saridaki et al. 2011]. MET gene amplification may act as et al. 2011]. In a retrospective analysis of cetuxi- another escape mechanism, as well as increased mab treated patients, none of the patients with binding by its ligand hepatocyte growth factor loss of function PTEN mutation showed response (HGF) [Bardelli et  al. 2013]. The vascular to cetuximab therapy [Frattini et  al. 2007]. In endothelial growth factor (VEGF) pathway, addition, BRAF mutations, present in about in described in detail later in this review, is also 9.6% of CRC [Hirschi et  al. 2014], have been closely related to the EGFR pathway. In fact, shown to confer a worse prognosis, and may overexpression of VEGF is one way in which http://tam.sagepub.com 71 Therapeutic Advances in Medical Oncology 8(1) tumor cells overcome resistance to EGFR inhibi- mechanisms, including mutations of KRAS, tion. It has been shown that EGFR over-expres- NRAS, PIK3CA, PTEN [Douillard et  al. 2013; sion leads to upregulation and increased signaling Sood et al. 2012]. One difference is that of a specific by VEGF, however the resistance seems to occur point mutation of the EGFR gene, S492R, which independent of EGFR signaling [Tabernero, was shown to confer resistance to cetuximab post- 2007]. This was shown in a study of gene expres- treatment, but not panitumumab. The incidence of sion of biomarkers from tumors pretreated with the S492R mutation in panitumumab-treated irinotecan or oxaliplatin, followed by single agent tumors has been reported from retrospective analy- cetuximab. Those tumors with elevated VEGF sis of tumors from patients treated in the ASPECCT expression were more resistant to cetuximab trial and found in only 1% of post-treatment evalu- [Vallbohmer et al. 2005]. able tumor samples, implying that this may not be a prominent mechanism of acquired resistance. No Panitumumab. The other approved EGFR mono- other specific polymorphisms are published that clonal antibody is panitumumab (Vectibix; show resistance unique to panitumumab [Van Amgen), a fully human IgG2 monoclonal anti- Emburgh et al. 2014]. body that binds with high affinity to the extracel- lular domain of the EGFR. It is thought that panitumumab prevents downstream activation VEGF signaling by competitive inhibition [Hohla et  al. The role of angiogenesis and lymphangiogenesis 2014]. It was approved by the FDA for use in in tumor growth is well established. Hypoxia in mCRC expressing EGFR in September 2006 in the tumor microenvironment has been shown to combination with other approved chemotherapy upregulate hypoxia inducible factor (HIF), which regimens for patients who had progressed on or then signals production of VEGF [Maxwell et al. after initial therapy. It has also now been approved, 1997]. Over-expression of VEGF gene and high as of May 2014, for use in the first-line setting levels of circulating VEGF protein are both associ- with FOLFOX therapy. ated with worse prognosis in CRC [Jurgensmeier et  al. 2013]. Several agents have been developed The efficacy of panitumumab was first demon- to inhibit angiogenesis and thus slow tumor strated by Van Cutsem and colleagues as mono- growth. VEGF has been identified as the promi- therapy [Van Cutsem et  al. 2007]. In the nent effector of angiogenesis, and has been the ASPECCT trial, a phase III randomized noninfe- target of drug developments since it was recog- riority trial, panitumumab was shown to have nized that its inhibition suppresses tumor growth similar OS and PFS as cetuximab, showing simi- [Kim et al. 1993]. The VEGF family is composed lar efficacy in patients with exon 2 KRAS wild- of at least nine ligands, VEGF-A through E, and type mCRC [Price et  al. 2014]. Perhaps the placental growth factor (PlGF) 1 through 4. These largest and most impactful study of this agent has ligands act upon at least one of three known recep- been the PRIME trial which compared FOLFOX4 tors, VEGFR-1 through 3. Blocking members of with and without panitumumab. In patients with this family, either the ligand or receptor, prevents KRAS wild-type tumors, there was a significantly tumor mitigation of local hypoxia and starvation. improved PFS of 9.6 months with the addition of The first VEGF/VEGFR targeted drug brought to panitumumab, compared with 8 months in the market was bevacizumab, and much of our under- control arm. Objective response rate was also standing about resistance to VEGF inhibition improved at 55%, compared with 48% in the comes from experience with this drug. FOLFOX4 alone group. Again, this effect was lost in those with mutated KRAS. In fact PFS was Bevacizumab. The monoclonal antibody bevaci- significantly worse in this group compared with zumab (Avastin; Genentech/Roche) is a recombi- FOLFOX4 alone [Douillard et al. 2010]. nant humanized IgG1 antibody against all isoforms of VEGF-A, a ligand for the VEGF Retrospective analyses of tumor samples from trials receptors 1 and 2 [Tejpar et al. 2012]. It was first of tumors treated with panitumumab have shown approved by the FDA in February 2004 for use in that KRAS mutations in exon 2, codons 12 and 13, combination with chemotherapy in the first-line were mutated at the same frequency, approximately treatment of mCRC. 43%, as in analogous reviews of trials with cetuxi- mab [Amado, 2008]. Resistance to panitumumab Bevacizumab has been shown to have clinical is akin to cetuximab occurring by many of the same activity in CRC in numerous studies, the first of 72 http://tam.sagepub.com WA Hammond, A Swaika et al. which was a trial by Hurwitz et al. [2004] which elevated pro-angiogenic biomarkers in tumors fol- showed an improved response rate, PFS and OS lowing treatment with bevacizumab, alternate pro- with the addition of bevacizumab to a first-line angiogenic pathways have been discovered. In one irinotecan-based regimen (IFL) in mCRC. It has study, PIGF, a ligand of VEGFR-1, was found to also been shown to have increased PFS in combi- be significantly elevated in tumors following treat- nation with oxaliplatin-based chemotherapy, ment with FOLFIRI plus bevacizumab [Kopetz combined with both 5-FU and capecitabine, as et al. 2010]. This ligand, as well as VEGF-D, was first-line therapy in mCRC [Saltz et  al. 2008]. shown in another study to be upregulated for about Both of these studies showed an improvement of 6 weeks following treatment with bevacizumab no more than 2 months in PFS. Interestingly, it plus chemotherapy, indicating this as a transient has also been shown that continuation of bevaci- alteration [Lieu et  al. 2013]. In addition, IL-8, a zumab after progression, while changing the cyto- chemokine with many functions including angio- toxic chemotherapy regimen, resulted in improved genesis, was shown to provide HIF-independent PFS and OS compared with post-progression angiogenic stimulus in vitro in CRC cell lines chemotherapy alone [Bennouna et al. 2013]. The [Mizukami et  al. 2005]. Elevated pretreatment reasons behind this are likely related to the differ- levels of IL-8 in blood were associated with shorter ent mechanisms of resistance to angiogenesis PFS in a phase II trial investigating biomarkers in inhibitors, including both evasive/acquired resist- relation to response to FOLFIRI plus bevaci- ance and intrinsic indifference [Bergers and zumab [Kopetz et  al. 2010]. Third, fibroblast Hanahan, 2008]. growth factor (FGF) upregulation has been noted in pancreatic neuroendocrine [Casanovas et  al. VEGF inhibitors such as bevacizumab work by 2005] and glioblastoma [Batchelor et  al. 2007] reducing the formation of new vasculature needed tumor cell lines, implicating this as a potential eva- by developing tumors for continued growth. They sive mechanism of resistance to bevacizumab, induce a state of local hypoxia, however hypoxia although this has never been shown explicitly in induces increased VEGF expression via HIF, so CRC. Additional biomarkers of putative signifi- its very inhibition induces its production. In addi- cance are platelet-derived growth factor-C tion, the binding of VEGF to its target leads to (PDGF-C) [Crawford et  al. 2009], neuropilin-1 downstream upregulation of VEGF, creating a (NRP-1) [Pan et al. 2007], and delta-like ligand-4 positive feedback loop of continued vascular (Dll4) [Ridgway et al. 2006]. growth promotion. CRC cells express VEGF receptor and demonstrate this autocrine signal- Another method of evasion of bevacizumab ther- ing, which promotes cell survival [Mesange et al. apy is by increased protection of vasculature by 2014]. This occurs particularly under external recruitment of alternative pro-angiogenic factors stress, including stress from treatment with 5-FU and by increasing protective barriers over existing [Samuel et al. 2011]. In vitro, bevacizumab resist- vasculature to increase its survival. Bone-marrow- ant cell lines show strong autocrine HIF-VEGF- derived cells (BMDCs), including vascular pro- VEGFR signaling as a response to exposure to genitors and vascular modulatory cells, are anti-VEGF therapy [Mesange et al. 2014]. recruited to the tumor environment in the setting of hypoxia to promote new vessel growth Acquired resistance to anti-angiogenic agents is [Mitchell, 2013]. Not only are progenitors of epi- often referred to as evasive resistance, because thelial cells and pericytes recruited to the area, unlike resistance to cytotoxic therapy or EGFR but so too are other vascular modulators such as antagonists, this typically occurs by adapting to tumor-associated macrophages [Pollard, 2004], the presence of anti-angiogenic agents in lieu of TIE2 [De Palma et  al. 2005], VEGFR-1+ more definitive mechanisms such as mutation of hemangiocytes [Hattori et al. 2002], and CD11b+ constituents within the pathway. This evasive myeloid cells [Yang et  al. 2004]. These function resistance occurs by at least three different mech- to promote vascular growth by producing anisms including revascularization by alternate cytokines, growth factors and proteases that pro- angiogenic pathways, development of protective mote vascular growth and development [Bergers mechanisms for vasculature, and enhanced ability and Hanahan, 2008]. It has also been noted that of tumors to invade or metastasize. tumors treated with anti-angiogenic agents have a change in the structure and concentration of peri- Continued or revascularization can occur by upreg- cyte coverage over existing vasculature [Kamba ulation of alternate VEGFR ligands. By recognizing and McDonald, 2007], presumably lending to http://tam.sagepub.com 73 Therapeutic Advances in Medical Oncology 8(1) the observed rapid reconstitution of vasculature Given the specificity of bevacizumab to the following removal of angiogenic blockade VEGF-A ligand, and the range of evasive mecha- [Mancuso et al. 2006]. Two such factors contrib- nisms within tumors, it is not surprising that uting to recruitment of BMDCs and monocytes/ resistance develops somewhat rapidly. Resistance macrophages to the tumor microenvironment are is not considered permanent, and as mentioned HIF1α and PlGF [Ribatti, 2008]. above, ongoing treatment with bevacizumab has shown prolonged PFS and OS compared with Some tumors treated with continuous anti-angio- chemotherapy alone. Perhaps the use of a differ- genic therapy may also develop augmented inva- ent agent to attempt broader angiogenic inhibi- siveness leading to increased local invasion and tion, including some of the evasive mechanisms of metastasis. The mechanism by which they are resistance, would be more efficacious. able to invade is by co-option of normal vascula- ture allowing this to provide nutrients necessary Aflibercept. Aflibercept (Zaltrap; Regeneron for continued growth. This was first seen in glio- Pharmaceuticals, Sanofi-Aventis), referred to in blastoma cell lines [Rubenstein et  al. 2000], and the United States as ziv-aflibercept, is a recombi- has been since recognized in pancreatic neuroen- nant decoy VEGFR1 and VEGFR2 fusion pro- docrine tumors as well [Casanovas et  al. 2005]. tein, each linked via the Fc segment of IgG1, that Tumor cells are able to migrate along the blood has anti-angiogenic and vascular permeability vessels, a process called perivascular invasion, activity by targeting multiple members of the allowing for subsequent direct invasion into sur- VEGF family, including VEGF-A, VEGF-B and rounding tissue [Du et al. 2008]. placental growth factor 2 (PlGF-2). Binding these growth factors prevents their activity at the In some trials, a minority of patients had rapid VEGFR-1 and VEGFR-2 receptors, which are progression in spite of angiogenic blockade, sug- found on the surface of endothelial cells and leu- gesting either rapid development of resistance or kocytes. Its activity results in regression of tumor the presence of intrinsic resistance. It has been vasculature, inhibition of new vascular growth postulated that some of the same mechanisms of and remodeling of surviving vasculature [Mitch- acquired resistance are present innately within ell, 2013]. It was approved for use in the US by some tumors, such as BMDCs such as CD11+ the FDA in August 2012. monocytes [Shojaei et  al. 2007]. Some tumors may also intrinsically possess the invasive pheno- The efficacy of aflibercept was demonstrated in type allowing them to co-opt surrounding normal the VELOUR trial, a phase III randomized, pla- vasculature to promote growth [Du et al. 2008]. cebo controlled trial of over 1200 patients with mCRC who had progressed on oxaliplatin-based Lastly, KRAS mutation status has been observed chemotherapy. Patients were randomized to to affect response to bevacizumab-containing reg- receive FOLFIRI plus aflibercept or FOLFIRI imens when examined in subgroup analyses. One plus placebo. The addition of aflibercept resulted example is the TLM trial of bevacizumab con- in improved response rate (20% versus 11%, tinuation after progression on a bevacizumab respectively), PFS (6.9 versus 4.7 months, respec- containing first-line treatment which was designed tively), and modestly improved OS by 1.5 months with an exploratory endpoint of evaluation of OS, [Van Cutsem et al. 2012]. This is to date the only PFS, and subsequent anticancer treatment published phase III trial demonstrating efficacy of according to KRAS mutation status [Bennouna aflibercept in any setting. Interestingly, subgroup et  al. 2013]. Patients treated with bevacizumab analysis has shown that the benefit of adding plus chemotherapy had improved PFS regardless aflibercept was seen regardless of whether the of KRAS mutation status, however patients with patient had received bevacizumab as part of first- KRAS wild-type cancer had improved OS with line treatment, potentially implicating efficacy addition of bevacizumab to second-line chemo- beyond development of resistance to VEGF inhi- therapy that was not seen in the patients with bition [Tabernero et al. 2014]. KRAS mutant cancer. These data seem to imply a mechanism of resistance, however the treatment Resistance to aflibercept is presumed to occur by by KRAS status interaction test for this subgroup many of the same mechanisms implicated in resist- was negative for PFS and OS, indicating no iden- ance to bevacizumab. It has been specifically tifiable KRAS mutation-independent treatment shown that VEGF-C is upregulated in tumors effect. treated with the VEGF-trap aflibercept, suggesting 74 http://tam.sagepub.com WA Hammond, A Swaika et al. that alternate angiogenesis pathways are triggered derived growth factor receptor) and β, and FGFR to promote resistance [Li et  al. 2014]. As afliber- (fibroblast growth factor receptor) 1 and 2, and cept is studied in earlier treatment lines for mCRC, p38 MAP kinase [Grothey et al. 2013; T ejpar et al. more data on acquired resistance may become 2012]. available upon review of treated tumors. It was approved by the FDA in September 2012 Ramucirumab. Ramucirumab (Cyramza; Eli Lilly for use in patients with previously treated mCRC and Company, US) is a recombinant, fully who had received all previously approved treat- humanized IgG1 monoclonal antibody directed ments including VEGF- and EGFR-active agents. against the extracellular domain of the VEFGR2. Its approval was based primarily on the results of It binds to this receptor with high affinity, and the CORRECT trial, a large international rand- thereby blocks ligand binding, primarily VEGF-A omized, double-blind, placebo controlled phase but others as well. It was approved in April 2015 III trial involving 760 patients who had previously for use in combination with FOLFIRI for the been treated with chemotherapy plus bevaci- treatment of patients with mCRC who have pro- zumab, as well as an anti-EGFR drug if the gressed on or after prior therapy with bevaci- patient’s tumor was KRAS wild type. The study zumab, oxaliplatin, and a FP. achieved its primary end point of improved median OS by showing an improvement of Approval was based on the results of the RAISE 1.4 months with regorafenib treatment over best trial, a phase III randomized, double-blind, supportive care. The study also demonstrated an multicenter international trial of 1072 patients improvement in PFS, though there was no differ- who had disease progression during or within ence in overall response rate [Grothey et al. 2013]. 6  months of the last dose of first-line therapy [Tabernero et  al. 2015]. The patients received Given the nature of this multi-targeted kinase, FOLFIRI plus either ramucirumab or placebo, potential mechanisms of resistance are difficult to randomized in a 1:1 ratio. The primary endpoint, elucidate. Presumably, mechanisms of resistance median OS, was improved by 1.6 months (13.3 previously shown for other specifically targeted versus 11.7 months) and the survival benefit was agents may be applicable to this agent as well, seen across all subgroups including KRAS however this agent simultaneously blocks multi- mutants and those with short time to progression ple members of a single pathway and members of (<6 months) after previous first-line treatment. parallel pathways between which cross-talk may PFS was also prolonged by 1.2 months (5.7 ver- occur, though likely not all isoforms of each tar- sus 4.5 months), but there was no appreciable get. The modest clinical benefit over best sup- difference in response rate. The results of this portive care (1.4 month improvement) implies trial indicate that VEGF receptor blockade after that there are very likely some mechanisms of progression through alternate angiogenesis resistance. This benefit was seen in heavily pre- blockade provides additional benefit, similar to treated patients, so it is likely the tumors had sub- what was seen in the TLM trial with bevaci- stantial acquired resistance to typical pathways of zumab [Bennouna et al. 2013] and the VELOUR targeted treatment. Further research on this topic trial with aflibercept [Tabernero et  al. 2014]. is certainly expected after its recent approval and Since this anti-angiogenic therapy is still new in with more patients being treated with this drug. the armamentarium of colon cancer treatments, there is no published data to specifically impli- cate unique mechanisms of resistance. Conclusions This review demonstrates that much is known about resistance to cytotoxic and targeted thera- Multi-kinase targeted agents pies in the treatment of CRC, though clearly Regorafenib. Regorafenib (Stivarga; Bayer more needs to be discovered. The goal of eluci- HealthCare Pharmaceuticals) is an oral multi- dating mechanisms of resistance is to develop kinase tyrosine kinase inhibitor that targets mul- methods to overcome the resistance to advance tiple angiogenesis targets including VEGFR1, our fight against this disease and achieve better, VEGFR2, VEGFR3, and TIE2, as well as multi- more durable treatment responses and longer ple oncogenic targets including KIT, RET, RAF1, patient survival. With so many potential targets BRAF, and BRAF-V600E, and tumor microenvi- for therapy, personalized treatments with existing ronment targets including PDGFRα (platelet drugs would be expected to show improved http://tam.sagepub.com 75 Therapeutic Advances in Medical Oncology 8(1) Amado, R., Wolf, M., Peeters, M., Van Cutsem, E., results. Early trials of personalized therapy based Siena, S., Freeman, D. et. al. (2008) Wild-type KRAS on mutation status of KRAS, BRAF, PI3KCA, is required for panitumumab efficacy in patients and expression of Topo-I, ERCC1, TS, and TP with metastatic colorectal cancer. J Clin Oncol 26: did not show any improvement in PFS [Cubillo 1626–34. et  al. 2014]. However, some authors have pro- posed using KRAS, BRAF, PI3KCA, and PTEN Baba, H., Watanabe, M., Okabe, H., Miyamoto, Y., Sakamoto, Y., Baba, Y. et al. (2012) Upregulation mutation status as a signature to guide therapy of ERCC1 and DPD expressions after oxaliplatin- [Bardelli and Siena, 2010; Sartore-Bianchi et  al. based first-line chemotherapy for metastatic colorectal 2009]. Other clinical trials involving personalized cancer. Br J Cancer 107: 1950–1955. therapies for CRC based on more comprehensive genomic and molecular profiling are underway. Barber, T., Vogelstein, B., Kinzler, K. and Velculescu, Certainly, in this era of more targeted therapies V. (2004) Somatic mutations of EGFR in colorectal cancers and glioblastomas. N Engl J Med 351: 2883. and much improved capabilities in genomic and molecular profiling, much is left to discover about Bardelli, A., Corso, S., Bertotti, A., Hobor, S., Valtorta, the effects that these targeted therapies have on E., Siravegna, G. et al. (2013) Amplification of the the intricate web of signaling and activities both MET receptor drives resistance to anti-EGFR therapies intracellularly and in the tumor microenviron- in colorectal cancer. Cancer Discov 3: 658–673. ment. Ultimately, new discoveries will continue Bardelli, A. and Siena, S. (2010) Molecular mechanisms to translate into improved treatment options and of resistance to cetuximab and panitumumab in important clinical outcomes for patients. colorectal cancer. J Clin Oncol 28: 1254–1261. Batchelor, T., Sorensen, A., Di Tomaso, E., Zhang, Acknowledgements W., Duda, D., Cohen, K. et al. (2007) AZD2171, We would like to acknowledge Margaret a pan-VEGF receptor tyrosine kinase inhibitor, McKinney for material support. normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer Cell 11: 83–95. Funding Becouarn, Y., Ychou, M., Ducreux, M., Borel, C., This research received no specific grant from any Bertheault-Cvitkovic, F., Seitz, J. et al. (1998) Phase funding agency in the public, commercial, or not- II trial of oxaliplatin as first-line chemotherapy in for-profit sectors. metastatic colorectal cancer patients. Digestive Group of French Federation of Cancer Centers. J Clin Oncol Conflict of interest statement 16: 2739–2744. The author(s) declare(s) that there is no conflict Bennouna, J., Sastre, J., Arnold, D., Osterlund, P., of interest. Greil, R., Van Cutsem, E. et al. (2013) Continuation of bevacizumab after first progression in metastatic colorectal cancer (ML18147): a randomised phase 3 trial. Lancet Oncol 14: 29–37. References Abdalla, E., Vauthey, J., Ellis, L., Ellis, V., Pollock, Berger, S., Jenh, C., Johnson, L. and Berger, F. R., Broglio, K. et al. (2004) Recurrence and outcomes (1985) Thymidylate synthase overproduction and following hepatic resection, radiofrequency ablation, gene amplification in fluorodeoxyuridine-resistant and combined resection/ablation for colorectal liver human cells. Mol Pharmacol 28: 461–467. metastases. Ann Surg 239: 818–825; discussion 825–827. Bergers, G. and Hanahan, D. (2008) Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer Adam, R., Delvart, V., Pascal, G., Valeanu, A., 8: 592–603. Castaing, D., Azoulay, D. et al. (2004) Rescue surgery for unresectable colorectal liver metastases Bertotti, A., Migliardi, G., Galimi, F., Sassi, F., downstaged by chemotherapy: a model to predict Torti, D., Isella, C. et al. (2011) A molecularly long-term survival. Ann Surg 240: 644–657; annotated platform of patient-derived xenografts discussion 657–658. (“xenopatients”) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal Allegra, C., Jessup, J., Somerfield, M., Hamilton, cancer. Cancer Discov 1: 508–523. S., Hammond, E., Hayes, D. et al. (2009) American society of clinical oncology provisional clinical opinion: Boyer, J., Mclean, E., Aroori, S., Wilson, P., Mcculla, testing for KRAS gene mutations in patients with A., Carey, P. et al. (2004) Characterization of p53 metastatic colorectal carcinoma to predict response wild-type and null isogenic colorectal cancer cell lines to anti-epidermal growth factor receptor monoclonal resistant to 5-fluorouracil, oxaliplatin, and irinotecan. antibody therapy. J Clin Oncol 27: 2091–2096. Clin Cancer Res 10: 2158–2167. 76 http://tam.sagepub.com WA Hammond, A Swaika et al. Cao, C., Zhang, X., Kuang, M., Gu, D., He, M., Ciardiello, F. and Tortora, G. (2008) EGFR Chen, J. et al. (2014) Survival benefit from S-1 as antagonists in cancer treatment. N Engl J Med 358: compared to Fluorouracil in Asian patients with 1160–1174. advanced gastrointestinal cancer: a meta-analysis. Colucci, G., Gebbia, V., Paoletti, G., Giuliani, Cancer Sci 105: 1008–1014. F., Caruso, M., Gebbia, N. et al. (2005) Phase III Cappuzzo, F., Finocchiaro, G., Rossi, E., Janne, randomized trial of FOLFIRI versus FOLFOX4 in the P., Carnaghi, C., Calandri, C. et al. (2008) EGFR treatment of advanced colorectal cancer: a multicenter FISH assay predicts for response to cetuximab in study of the Gruppo Oncologico Dell’Italia chemotherapy refractory colorectal cancer patients. Meridionale. J Clin Oncol 23: 4866–4875. Ann Oncol 19: 717–723. Crawford, Y., Kasman, I., Yu, L., Zhong, C., Wu, X., Modrusan, Z. et al. (2009) PDGF-C mediates the Carethers, J., Smith, E., Behling, C., Nguyen, angiogenic and tumorigenic properties of fibroblasts L., Tajima, A., Doctolero, R. et al. (2004) associated with tumors refractory to anti-VEGF Use of 5-fluorouracil and survival in patients treatment. Cancer Cell 15: 21–34. with microsatellite-unstable colorectal cancer. Gastroenterology 126: 394–401. Cubillo, A., Rodriguez-Pascual, J., Lopez-Rios, F., Plaza, C., Garcia, E., Alvarez, R. et al. (2014) Phase Carlini, L., Meropol, N., Bever, J., Andria, M., Hill, II trial of target-guided personalized chemotherapy in T., Gold, P. et al. (2005) UGT1A7 and UGT1A9 first-line metastatic colorectal cancer. Am J Clin Oncol. polymorphisms predict response and toxicity in colorectal cancer patients treated with capecitabine/ Cummings, J., Boyd, G., Ethell, B., Macpherson, irinotecan. Clin Cancer Res 11: 1226–1236. J., Burchell, B., Smyth, J. et al. (2002) Enhanced clearance of topoisomerase I inhibitors from human Casanovas, O., Hicklin, D., Bergers, G. and colon cancer cells by glucuronidation. Biochem Hanahan, D. (2005) Drug resistance by evasion of Pharmacol 63: 607–613. antiangiogenic targeting of VEGF signaling in late- stage pancreatic islet tumors. Cancer Cell 8: 299–309. Cunningham, D., Humblet, Y., Siena, S., Khayat, D., Bleiberg, H., Santoro, A. et al. (2004) Cetuximab Cassidy, J., Saltz, L., Twelves, C., Van Cutsem, monotherapy and cetuximab plus irinotecan in E., Hoff, P., Kang, Y. et al. (2011) Efficacy of irinotecan-refractory metastatic colorectal cancer. capecitabine versus 5-fluorouracil in colorectal and N Engl J Med 351: 337–345. gastric cancers: a meta-analysis of individual data from 6171 patients. Ann Oncol 22: 2604–2609. Cunningham, D., Pyrhonen, S., James, R., Punt, C., Hickish, T., Heikkila, R. et al. (1998) Randomised Choi, Y., Kim, T., Kim, K., Lee, S., Hong, Y., Ryu, trial of irinotecan plus supportive care versus M. et al. (2012) A Phase II study of clinical outcomes supportive care alone after fluorouracil failure for of 3-week cycles of irinotecan and S-1 in patients patients with metastatic colorectal cancer. Lancet 352: with previously untreated metastatic colorectal 1413–1418. cancer: influence of the UGT1A1 and CYP2A6 polymorphisms on clinical activity. Oncology 82: De Gramont, A., Figer, A., Seymour, M., Homerin, 290–297. M., Hmissi, A., Cassidy, J. et al. (2000) Leucovorin and fluorouracil with or without oxaliplatin as first- Chu, E., Koeller, D., Johnston, P., Zinn, S. and line treatment in advanced colorectal cancer. J Clin Allegra, C. (1993) Regulation of thymidylate Oncol 18: 2938–2947. synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. Mol Pharmacol De Palma, M., Venneri, M., Galli, R., Sergi, 43: 527–533. L., Politi, L., Sampaolesi, M. et al. (2005) Tie2 identifies a hematopoietic lineage of proangiogenic Ciardiello, F., Bianco, R., Damiano, V., Fontanini, monocytes required for tumor vessel formation and G., Caputo, R., Pomatico, G. et al. (2000) a mesenchymal population of pericyte progenitors. Antiangiogenic and antitumor activity of anti- Cancer Cell 8: 211–226. epidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial De Roock, W., Claes, B., Bernasconi, D., De growth factor antisense oligonucleotide in human Schutter, J., Biesmans, B., Fountzilas, G. et al. GEO colon cancer cells. Clin Cancer Res 6: (2010) Effects of KRAS, BRAF, NRAS, and 3739–3747. PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic Ciardiello, F. and Normanno, N. (2011) HER2 colorectal cancer: a retrospective consortium analysis. signaling and resistance to the anti-EGFR monoclonal Lancet Oncol 11: 753–762. antibody cetuximab: a further step toward personalized medicine for patients with colorectal De Stefano, A. and Carlomagno, C. (2014) Beyond cancer. Cancer Discov 1: 472–474. KRAS: Predictive factors of the efficacy of http://tam.sagepub.com 77 Therapeutic Advances in Medical Oncology 8(1) anti-EGFR monoclonal antibodies in the treatment of decreased ubiquitination is mediated by protein kinase metastatic colorectal cancer. World J Gastroenterol 20: B/Akt-dependent phosphorylation. J Biol Chem 279: 9732–9743. 35510–35517. Di Nicolantonio, F., Martini, M., Molinari, F., Fink, D., Aebi, S. and Howell, S. (1998) The role of Sartore-Bianchi, A., Arena, S., Saletti, P., De Dosso, DNA mismatch repair in drug resistance. Clin Cancer S. et. al. (2008) Wild-type BRAF is required for Res 4: 1–6. response to panitumumab or cetuximab in metastatic Fink, D., Zheng, H., Nebel, S., Norris, P., Aebi, S., colorectal cancer. J Clin Oncol 26: 5705–5712. Lin, T. et al. (1997) In vitro and in vivo resistance to Diaz-Rubio, E., Sastre, J., Zaniboni, A., Labianca, cisplatin in cells that have lost DNA mismatch repair. R., Cortes-Funes, H., De Braud, F. et al. (1998) Cancer research 57: 1841–1845. Oxaliplatin as single agent in previously untreated Frattini, M., Saletti, P., Romagnani, E., Martin, V., colorectal carcinoma patients: a phase II multicentric Molinari, F., Ghisletta, M. et al. (2007) PTEN loss of study. Ann Oncol 9: 105–108. expression predicts cetuximab efficacy in metastatic Douillard, J., Cunningham, D., Roth, A., Navarro, colorectal cancer patients. Br J Cancer 97: 1139–1145. M., James, R., Karasek, P. et al. (2000) Irinotecan Fujiwara, Y. and Minami, H. (2010) An overview of combined with fluorouracil compared with the recent progress in irinotecan pharmacogenetics. fluorouracil alone as first-line treatment for metastatic Pharmacogenomics 11: 391–406. colorectal cancer: a multicentre randomised trial. Lancet 355: 1041–1047. Giacchetti, S., Perpoint, B., Zidani, R., Le Bail, N., Faggiuolo, R., Focan, C. et al. (2000) Phase III Douillard, J., Oliner, K., Siena, S., Tabernero, J., multicenter randomized trial of oxaliplatin added to Burkes, R., Barugel, M. et al. (2013) Panitumumab- chronomodulated fluorouracil-leucovorin as first-line FOLFOX4 treatment and RAS mutations in treatment of metastatic colorectal cancer. J Clin Oncol colorectal cancer. N Engl J Med 369: 1023–1034. 18: 136–147. Douillard, J., Siena, S., Cassidy, J., Tabernero, J., Burkes, R., Barugel, M. et al. (2010) Randomized, Gongora, C., Vezzio-Vie, N., Tuduri, S., Denis, phase III trial of panitumumab with infusional V., Causse, A., Auzanneau, C. et al. (2011) New fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) Topoisomerase I mutations are associated with versus FOLFOX4 alone as first-line treatment resistance to camptothecin. Molecular Cancer 10: 64. in patients with previously untreated metastatic Gottesman, M., Fojo, T. and Bates, S. (2002) colorectal cancer: the PRIME study. J Clin Oncol 28: Multidrug resistance in cancer: role of ATP- 4697–4705. dependent transporters. Nat Rev Cancer 2: 48–58. Du, R., Lu, K., Petritsch, C., Liu, P., Ganss, R., Grothey, A., Van Cutsem, E., Sobrero, A., Siena, Passegue, E. et al. (2008) HIF1alpha induces the S., Falcone, A., Ychou, M. et al. (2013) Regorafenib recruitment of bone marrow-derived vascular monotherapy for previously treated metastatic modulatory cells to regulate tumor angiogenesis and colorectal cancer (CORRECT): an international, invasion. Cancer Cell 13: 206–220. multicentre, randomised, placebo-controlled, phase 3 Esposito, C., Rachiglio, A., La Porta, M., Sacco, A., trial. Lancet 381: 303–312. Roma, C., Iannaccone, A. et al. (2013) The S492R Hattori, K., Heissig, B., Wu, Y., Dias, S., Tejada, EGFR ectodomain mutation is never detected in R., Ferris, B. et al. (2002) Placental growth factor KRAS wild-type colorectal carcinoma before exposure reconstitutes hematopoiesis by recruiting VEGFR1(+) to EGFR monoclonal antibodies. Cancer Biol Ther 14: stem cells from bone-marrow microenvironment. Nat 1143–1146. Med 8: 841–849. Etienne-Grimaldi, M., Milano, G., Maindrault- Hector, S., Bolanowska-Higdon, W., Zdanowicz, Goebel, F., Chibaudel, B., Formento, J., Francoual, J., Hitt, S. and Pendyala, L. (2001) In vitro studies M. et al. (2010) Methylenetetrahydrofolate reductase on the mechanisms of oxaliplatin resistance. Cancer (MTHFR) gene polymorphisms and FOLFOX Chemother Pharmacol 48: 398–406. response in colorectal cancer patients. Br J Clin Pharmacol 69: 58–66. Heidelberger, C., Chaudhuri, N., Danneberg, P., Mooren, D., Griesbach, L., Duschinsky, R. et al. Fedier, A., Steiner, R., Schwarz, V., Lenherr, L., (1957) Fluorinated pyrimidines, a new class of Haller, U. and Fink, D. (2003) The effect of loss of tumour-inhibitory compounds. Nature 179: 663–666. Brca1 on the sensitivity to anticancer agents in p53- deficient cells. Int J Oncol 22: 1169–1173. Hirschi, B., Gallmeier, E., Ziesch, A., Marschall, Feng, J., Tamaskovic, R., Yang, Z., Brazil, D., Merlo, M. and Kolligs, F. (2014) Genetic targeting of A., Hess, D. et al. (2004) Stabilization of Mdm2 via B-RafV600E affects survival and proliferation and 78 http://tam.sagepub.com WA Hammond, A Swaika et al. identifies selective agents against BRAF-mutant cells to the epidermal growth factor receptor inhibitor colorectal cancer cells. Mol Cancer 13: 122. cetuximab. Cancer Res 68: 1953–1961. Johnston, P., Lenz, H., Leichman, C., Danenberg, K., Hobor, S., Van Emburgh, B., Crowley, E., Misale, S., Allegra, C., Danenberg, P. et al. (1995) Thymidylate Di Nicolantonio, F. and Bardelli, A. (2014) TGF- synthase gene and protein expression correlate and are alpha and amphiregulin paracrine network promotes associated with response to 5-fluorouracil in human resistance to EGFR blockade in colorectal cancer colorectal and gastric tumors. Cancer Res 55: 1407–1412. cells. Clin Cancer Res 20: 6429–6438. Jonker, D., O’Callaghan, C., Karapetis, C., Zalcberg, Hoff, P., Ansari, R., Batist, G., Cox, J., Kocha, W., J., Tu, D., Au, H. et al. (2007) Cetuximab for the Kuperminc, M. et al. (2001) Comparison of oral treatment of colorectal cancer. N Engl J Med 357: capecitabine versus intravenous fluorouracil plus 2040–2048. leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized Jurgensmeier, J., Schmoll, H., Robertson, J., Brooks, phase III study. J Clin Oncol 19: 2282–2292. L., Taboada, M., Morgan, S. et al. (2013) Prognostic and predictive value of VEGF, SVEGFR-2 and CEA Hohla, F., Winder, T., Greil, R., Rick, F., Block, N. in mCRC studies comparing cediranib, bevacizumab and Schally, A. (2014) Targeted therapy in advanced and chemotherapy. Br J Cancer 108: 1316–1323. metastatic colorectal cancer: current concepts and perspectives. World J Gastroenterol 20: 6102–6112. Kamba, T. and McDonald, D. (2007) Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Holzer, A., Manorek, G. and Howell, S. (2006) Cancer 96: 1788–1795. Contribution of the major copper influx transporter CTR1 to the cellular accumulation of cisplatin, Karapetis, C., Khambata-Ford, S., Jonker, D., carboplatin, and oxaliplatin. Mol Pharmacol 70: O’Callaghan, C., Tu, D., Tebbutt, N. et al. (2008) 1390–1394. K-Ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 359: Hong, Y., Park, Y., Lim, H., Lee, J., Kim, T., Kim, 1757–1765. K. et al. (2012) S-1 plus oxaliplatin versus capecitabine plus oxaliplatin for first-line treatment of patients with Kelland, L. (1993) New platinum antitumor metastatic colorectal cancer: a randomised, non- complexes. Crit Rev Oncol Hematol 15: 191–219. inferiority phase 3 trial. Lancet Oncol 13: 1125–1132. Kim, K., Li, B., Winer, J., Armanini, M., Gillett, Houghton, J. and Houghton, P. (1983) Elucidation of N., Phillips, H. et al. (1993) Inhibition of vascular pathways of 5-fluorouracil metabolism in xenografts of endothelial growth factor-induced angiogenesis human colorectal adenocarcinoma. Eur J Cancer Clin suppresses tumour growth in vivo. Nature 362: Oncol 19: 807–815. 841–844. Hudis, C. (2007) Trastuzumab - mechanism of action Kim, S., Hong, S., Shim, K., Kong, S., Shin, and use in clinical practice. N Engl J Med 357: 39–51. A., Baek, J. et al. (2013) S-1 plus irinotecan and oxaliplatin for the first-line treatment of patients with Hurwitz, H., Fehrenbacher, L., Novotny, W., metastatic colorectal cancer: a prospective phase II Cartwright, T., Hainsworth, J., Heim, W. et al. study and pharmacogenetic analysis. Br J Cancer 109: (2004) Bevacizumab plus irinotecan, fluorouracil, and 1420–1427. leucovorin for metastatic colorectal cancer. N Engl J Med 350: 2335–2342. Kojima, A., Hackett, N. and Crystal, R. (1998) Reversal of CPT-11 resistance of lung cancer cells Ishikawa, T. and Ali-Osman, F. (1993) Glutathione- by adenovirus-mediated gene transfer of the human associated cis-diamminedichloroplatinum(II) carboxylesterase cDNA. Cancer Res 58: 4368–4374. metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione- Koopman, M., Venderbosch, S., Van Tinteren, H., platinum complex and its biological significance. Ligtenberg, M., Nagtegaal, I., Van Krieken, J. et al. J Biol Chem 268: 20116–20125. (2009) Predictive and prognostic markers for the outcome of chemotherapy in advanced colorectal Isshi, K., Sakuyama, T., Gen, T., Nakamura, Y., cancer, a retrospective analysis of the phase III Kuroda, T., Katuyama, T. et al. (2002) Predicting randomised CAIRO study. Eur J Cancer 45: 1999– 5-FU sensitivity using human colorectal cancer specimens: comparison of tumor dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase Kopetz, S., Hoff, P., Morris, J., Wolff, R., Eng, C., activities with in vitro chemosensitivity to 5-FU. Int J Glover, K. et al. (2010) Phase II trial of infusional Clin Oncol 7: 335–342. fluorouracil, irinotecan, and bevacizumab for Jhawer, M., Goel, S., Wilson, A., Montagna, C., Ling, metastatic colorectal cancer: efficacy and circulating Y., Byun, D. et al. (2008) PIK3CA mutation/PTEN angiogenic biomarkers associated with therapeutic expression status predicts response of colon cancer resistance. J Clin Oncol 28: 453–459. http://tam.sagepub.com 79 Therapeutic Advances in Medical Oncology 8(1) Leichman, C., Fleming, T., Muggia, F., Tangen, Marcuello, E., Altes, A., Menoyo, A., Del Rio, E., C., Ardalan, B., Doroshow, J. et al. (1995) Phase II Gomez-Pardo, M. and Baiget, M. 2004. UGT1A1 gene study of fluorouracil and its modulation in advanced variations and irinotecan treatment in patients with colorectal cancer: a Southwest Oncology Group study. metastatic colorectal cancer. Br J Cancer 91: 678–682. J Clin Oncol 13: 1303–1311. Marcuello, E., Altes, A., Menoyo, A., Rio, E. Leichman, C., Lenz, H., Leichman, L., Danenberg, and Baiget, M. (2006) Methylenetetrahydrofolate K., Baranda, J., Groshen, S. et al. (1997) Quantitation reductase gene polymorphisms: genomic predictors of intratumoral thymidylate synthase expression of clinical response to fluoropyrimidine-based predicts for disseminated colorectal cancer response chemotherapy? Cancer Chemother Pharmacol 57: and resistance to protracted-infusion fluorouracil and 835–840. weekly leucovorin. J Clin Oncol 15: 3223–3229. Maxwell, P., Dachs, G., Gleadle, J., Nicholls, L., Li, D., Xie, K., Ding, G., Li, J., Chen, K., Li, H. Harris, A., Stratford, I. et al. (1997) Hypoxia-inducible et al. (2014) Tumor resistance to anti-VEGF therapy factor-1 modulates gene expression in solid tumors through up-regulation of VEGF-C expression. Cancer and influences both angiogenesis and tumor growth. Lett 346: 45–52. Proc Natl Acad Sci U S A 94: 8104–8109. Liang, J., Jiang, T., Yao, R., Liu, Z., Lv, H. and Qi, Mayer, A., Takimoto, M., Fritz, E., Schellander, G., W. (2010) The combination of ERCC1 and XRCC1 Kofler, K. and Ludwig, H. (1993) The prognostic gene polymorphisms better predicts clinical outcome significance of proliferating cell nuclear antigen, to oxaliplatin-based chemotherapy in metastatic epidermal growth factor receptor, and MDR gene colorectal cancer. Cancer Chemother Pharmacol 66: expression in colorectal cancer. Cancer 71: 2454–2460. 493–500. Mayer, R., Van Cutsem, E., Falcone, A., Yoshino, T., Lieu, C., Tran, H., Jiang, Z., Mao, M., Overman, Garcia-Carbonero, R., Mizunuma, N. et al. (2015) M., Lin, E. et al. (2013) The association of alternate Randomized trial of TAS-102 for refractory metastatic VEGF ligands with resistance to anti-VEGF therapy colorectal cancer. N Engl J Med 372: 1909–1919. in metastatic colorectal cancer. PloS One 8: e77117. McLeod, H. and Keith, W. (1996) Variation in Lievre, A., Bachet, J., Le Corre, D., Boige, V., Landi, topoisomerase I gene copy number as a mechanism B., Emile, J. et al. (2006) KRAS mutation status for intrinsic drug sensitivity. Br J Cancer 74: 508–512. is predictive of response to cetuximab therapy in Meijer, C., Mulder, N., Timmer-Bosscha, H., colorectal cancer. Cancer Res 66: 3992–3995. Sluiter, W., Meersma, G. and De Vries, E. (1992) Lindskog, E., Derwinger, K., Gustavsson, B., Falk, P. Relationship of cellular glutathione to the cytotoxicity and Wettergren, Y. (2014) Thymidine phosphorylase and resistance of seven platinum compounds. Cancer expression is associated with time to progression in Res 52: 6885–6889. patients with metastatic colorectal cancer. BMC Clin Meriggi, F., Vermi, W., Bertocchi, P. and Zaniboni, Pathol 14: 25. A. (2014) The emerging role of NRAS mutations Longley, D., Boyer, J., Allen, W., Latif, T., Ferguson, in colorectal cancer patients selected for anti-EGFR P., Maxwell, P. et al. (2002) The role of thymidylate therapies. Rev Recent Clin Trials 9: 8–12. synthase induction in modulating p53-regulated Meropol, N., Gold, P., Diasio, R., Andria, M., gene expression in response to 5-fluorouracil and Dhami, M., Godfrey, T. et al. (2006) Thymidine antifolates. Cancer Res 62: 2644–2649. phosphorylase expression is associated with response to Longley, D., Harkin, D. and Johnston, P. (2003) capecitabine plus irinotecan in patients with metastatic 5-fluorouracil: mechanisms of action and clinical colorectal cancer. J Clin Oncol 24: 4069–4077. strategies. Nat Rev Cancer 3: 330–338. Mesange, P., Poindessous, V., Sabbah, M., Longley, D. and Johnston, P. (2005) Molecular Escargueil, A., De Gramont, A. and Larsen, A. (2014) mechanisms of drug resistance. J Pathol 205: Intrinsic bevacizumab resistance is associated with 275–292. prolonged activation of autocrine VEGF signaling and hypoxia tolerance in colorectal cancer cells and can be Lowenstein, E., Daly, R., Batzer, A., Li, W., overcome by nintedanib, a small molecule angiokinase Margolis, B., Lammers, R. et al. (1992) The SH2 inhibitor. Oncotarget 5: 4709–4721. and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to Ras signaling. Cell 70: Messa, C., Russo, F., Caruso, M. and Di Leo, A. 431–442. (1998) EGF, TGF-alpha, and EGF-R in human colorectal adenocarcinoma. Acta Oncol 37: 285–289. Mancuso, M., Davis, R., Norberg, S., O’Brien, S., Sennino, B., Nakahara, T. et al. (2006) Rapid vascular Meyers, M., Wagner, M., Hwang, H., Kinsella, T. regrowth in tumors after reversal of VEGF inhibition. and Boothman, D. (2001) Role of the hMLH1 DNA J Clin Invest 116: 2610–2621. mismatch repair protein in fluoropyrimidine-mediated 80 http://tam.sagepub.com WA Hammond, A Swaika et al. cell death and cell cycle responses. Cancer Res 61: Paez, J., Janne, P., Lee, J., Tracy, S., Greulich, H., 5193–5201. Gabriel, S. et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib Misale, S., Di Nicolantonio, F., Sartore-Bianchi, therapy. Science 304: 1497–1500. A., Siena, S. and Bardelli, A. (2014) Resistance to anti-EGFR therapy in colorectal cancer: from Pan, Q., Chanthery, Y., Liang, W., Stawicki, S., Mak, heterogeneity to convergent evolution. Cancer Discov J., Rathore, N. et al. (2007) Blocking neuropilin-1 4: 1269–1280. function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer Cell 11: 53–67. Misale, S., Yaeger, R., Hobor, S., Scala, E., Janakiraman, M., Liska, D. et al. (2012) Emergence of Panczyk, M. (2014) Pharmacogenetics research on KRAS mutations and acquired resistance to anti-EGFR chemotherapy resistance in colorectal cancer over the therapy in colorectal cancer. Nature 486: 532–536. last 20 years. World J Gastroenterol 20: 9775–9827. Perrone, F., Lampis, A., Orsenigo, M., Di Mitchell, E. (2013) Targeted therapy for metastatic Bartolomeo, M., Gevorgyan, A., Losa, M. et al. colorectal cancer: role of aflibercept. Clin Colorectal (2009) PI3KCA/PTEN deregulation contributes Cancer 12: 73–85. to impaired responses to cetuximab in metastatic Mizukami, Y., Jo, W., Duerr, E., Gala, M., Li, J., colorectal cancer patients. Ann Oncol 20: 84–90. Zhang, X. et al. (2005) Induction of interleukin-8 Petrioli, R., Bargagli, G., Lazzi, S., Pascucci, A., preserves the angiogenic response in HIF-1alpha- Francini, E., Bellan, C. et al. (2010) Thymidine deficient colon cancer cells. Nature Med 11: 992–997. phosphorylase expression in metastatic sites is Moertel, C., Fleming, T., Macdonald, J., Haller, D., predictive for response in patients with colorectal Laurie, J., Goodman, P. et al. (1990) Levamisole and cancer treated with continuous oral capecitabine and fluorouracil for adjuvant therapy of resected colon biweekly oxaliplatin. Anticancer Drugs 21: 313–319. carcinoma. N Engl J Med 322: 352–358. Pollard, J. (2004) Tumour-educated macrophages Moroni, M., Veronese, S., Benvenuti, S., Marrapese, promote tumour progression and metastasis. Nat Rev G., Sartore-Bianchi, A., Di Nicolantonio, F. et al. Cancer 4: 71–78. (2005) Gene copy number for epidermal growth Popat, S., Matakidou, A. and Houlston, R. (2004) factor receptor (EGFR) and clinical response to Thymidylate synthase expression and prognosis in antiEGFR treatment in colorectal cancer: a cohort colorectal cancer: a systematic review and meta- study. Lancet Oncol 6: 279–286. analysis. J Clin Oncol 22: 529–536. Moutinho, C., Martinez-Cardus, A., Santos, C., Porebska, I., Harlozinska, A. and Bojarowski, T. Navarro-Perez, V., Martinez-Balibrea, E., Musulen, (2000) Expression of the tyrosine kinase activity E. et al. (2014) Epigenetic inactivation of the BRCA1 growth factor receptors (EGFR, ERB B2, ERB B3) in interactor SRBC and resistance to oxaliplatin in colorectal adenocarcinomas and adenomas. Tumour colorectal cancer. J Natl Cancer Inst 106: djt322. Biol 21: 105–115. Muhale, F., Wetmore, B., Thomas, R. and McLeod, Price, T., Newhall, K., Peeters, M., Kim, T., Li, J., H. (2011) Systems pharmacology assessment of Cascinu, S. et al. (2015) Prevalence and outcomes the 5-fluorouracil pathway. Pharmacogenomics 12: of patients (pts) with EGFR S492R ectodomain 341–350. mutations in ASPECCT: Panitumumab (PMAB) Muro, K., Boku, N., Shimada, Y., Tsuji, A., vs. cetuximab (CMAB) in pts with chemorefractory Sameshima, S., Baba, H. et al. (2010) Irinotecan plus wild-type KRAS exon 2 metastatic colorectal cancer S-1 (IRIS) versus fluorouracil and folinic acid plus (mCRC). J Clin Oncol (Suppl. 3): abstr 740. irinotecan (FOLFIRI) as second-line chemotherapy Price, T., Peeters, M., Kim, T., Li, J., Cascinu, S., for metastatic colorectal cancer: a randomised phase Ruff, P. et al. (2014) Panitumumab versus cetuximab 2/3 non-inferiority study (FIRIS study). Lancet Oncol in patients with chemotherapy-refractory wild- 11: 853–860. type KRAS exon 2 metastatic colorectal cancer (ASPECCT): a randomised, multicentre, open- Nicum, S., Midgley, R. and Kerr, D. (2000) label, non-inferiority phase 3 study. Lancet Oncol 15: Chemotherapy for colorectal cancer. J R Soc Med 93: 569–579. 416–419. Qiu, L., Tang, Q., Bai, J., Qian, X., Li, R., Liu, B. Nygard, S., Christensen, I., Nielsen, S., Nielsen, H., et al. (2008) Predictive value of thymidylate synthase Brunner, N. and Spindler, K. (2014) Assessment of expression in advanced colorectal cancer patients the topoisomerase I gene copy number as a predictive receiving fluoropyrimidine-based chemotherapy: biomarker of objective response to irinotecan in evidence from 24 studies. Int J Cancer 123: 2384– metastatic colorectal cancer. Scand J Gastroenterol 49: 84–91. http://tam.sagepub.com 81 Therapeutic Advances in Medical Oncology 8(1) Rajput, A., Koterba, A., Kreisberg, J., Foster, in combination with oxaliplatin-based chemotherapy J., Willson, J. and Brattain, M. (2007) A novel as first-line therapy in metastatic colorectal cancer: a mechanism of resistance to epidermal growth factor randomized phase III study. J Clin Oncol 26: 2013–2019. receptor antagonism in vivo. Cancer Res 67: 665–673. Saltz, L., Cox, J., Blanke, C., Rosen, L., Raymond, E., Faivre, S., Chaney, S., Woynarowski, Fehrenbacher, L., Moore, M. et al. (2000a) Irinotecan J. and Cvitkovic, E. (2002) Cellular and molecular plus fluorouracil and leucovorin for metastatic pharmacology of oxaliplatin. Mol Cancer Ther 1: colorectal cancer. Irinotecan Study Group. N Engl J 227–235. Med 343: 905–914. Ribatti, D. (2008) The discovery of the placental Saltz, L., Cox, J., Blanke, C., Rosen, L., growth factor and its role in angiogenesis: a historical Fehrenbacher, L., Moore, M. et al. (2000b) Irinotecan review. Angiogenesis 11: 215–221. plus fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med 343: 905–914. Ridgway, J., Zhang, G., Wu, Y., Stawicki, S., Liang, W., Chanthery, Y. et al. (2006) Inhibition of Dll4 Samuel, S., Fan, F., Dang, L., Xia, L., Gaur, P. and signalling inhibits tumour growth by deregulating Ellis, L. (2011) Intracrine vascular endothelial growth angiogenesis. Nature 444: 1083–1087. factor signaling in survival and chemoresistance of human colorectal cancer cells. Oncogene 30: 1205–1212. Roskoski, R., Jr. (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res 66: Saridaki, Z., Tzardi, M., Papadaki, C., Sfakianaki, 105–143. M., Pega, F., Kalikaki, A. et al. (2011) Impact of KRAS, BRAF, PIK3CA mutations, PTEN, AREG, Roskoski, R., Jr. (2014) The ErbB/HER family of EREG expression and skin rash in >/= 2 line protein-tyrosine kinases and cancer. Pharmacol Res 79: cetuximab-based therapy of colorectal cancer patients. 34–74. PloS One 6: e15980. Rougier, P., Van Cutsem, E., Bajetta, E., Niederle, Sartore-Bianchi, A., Di Nicolantonio, F., Nichelatti, N., Possinger, K., Labianca, R. et al. (1998) M., Molinari, F., De Dosso, S., Saletti, P. et al. Randomised trial of irinotecan versus fluorouracil (2009) Multi-determinants analysis of molecular by continuous infusion after fluorouracil failure in alterations for predicting clinical benefit to EGFR- patients with metastatic colorectal cancer. Lancet 352: targeted monoclonal antibodies in colorectal cancer. 1407–1412. PloS One 4: e7287. Rouits, E., Boisdron-Celle, M., Dumont, A., Guerin, Sartore-Bianchi, A., Moroni, M., Veronese, S., O., Morel, A. and Gamelin, E. (2004) Relevance Carnaghi, C., Bajetta, E., Luppi, G. et al. (2007) of different UGT1A1 polymorphisms in irinotecan- Epidermal growth factor receptor gene copy number induced toxicity: a molecular and clinical study of 75 and clinical outcome of metastatic colorectal cancer patients. Clin Cancer Res 10: 5151–5159. treated with panitumumab. J Clin Oncol 25: 3238–3245. Rubenstein, J., Kim, J., Ozawa, T., Zhang, M., Schwartz, P., Moir, R., Hyde, C., Turek, P. and Westphal, M., Deen, D. et al. (2000) Anti-VEGF Handschumacher, R. (1985) Role of uridine antibody treatment of glioblastoma prolongs survival phosphorylase in the anabolism of 5-fluorouracil. but results in increased vascular cooption. Neoplasia 2: Biochem Pharmacol 34: 3585–3589. 306–314. Shimada, Y., Rougier, P. and Pitot, H. (1996) Sakuramoto, S., Sasako, M., Yamaguchi, T., Efficacy of CPT-11 (irinotecan) as a single agent in Kinoshita, T., Fujii, M., Nashimoto, A. et al. (2007) metastatic colorectal cancer. Eur J Cancer 32A(Suppl. Adjuvant chemotherapy for gastric cancer with S-1, an 3): S13–S17. oral fluoropyrimidine. N Engl J Med 357: 1810–1820. Shirao, K., Hoff, P., Ohtsu, A., Loehrer, P., Hyodo, Salomon, D., Brandt, R., Ciardiello, F. and I., Wadler, S. et al. (2004) Comparison of the efficacy, Normanno, N. (1995) Epidermal growth factor- toxicity, and pharmacokinetics of a uracil/tegafur related peptides and their receptors in human (UFT) plus oral leucovorin (LV) regimen between malignancies. Crit Rev Oncol Hematol 19: 183–232. Japanese and American patients with advanced colorectal cancer: joint United States and Japan study Salonga, D., Danenberg, K., Johnson, M., Metzger, of UFT/LV. J Clin Oncol 22: 3466–3474. R., Groshen, S., Tsao-Wei, D. et al. (2000) Colorectal tumors responding to 5-fluorouracil have low gene Shirota, Y., Stoehlmacher, J., Brabender, J., expression levels of dihydropyrimidine dehydrogenase, Xiong, Y., Uetake, H., Danenberg, K. et al. (2001) thymidylate synthase, and thymidine phosphorylase. ERCC1 and thymidylate synthase mRNA levels Clin Cancer Res 6: 1322–1327. predict survival for colorectal cancer patients Saltz, L., Clarke, S., Diaz-Rubio, E., Scheithauer, receiving combination oxaliplatin and fluorouracil W., Figer, A., Wong, R. et al. (2008) Bevacizumab chemotherapy. J Clin Oncol 19: 4298–4304. 82 http://tam.sagepub.com WA Hammond, A Swaika et al. Shojaei, F., Wu, X., Malik, A., Zhong, C., Baldwin, Ramucirumab versus placebo in combination with M., Schanz, S. et al. (2007) Tumor refractoriness to second-line FOLFIRI in patients with metastatic anti-VEGF treatment is mediated by CD11b+Gr1+ colorectal carcinoma that progressed during or after myeloid cells. Nat Biotechnol 25: 911–920. first-line therapy with bevacizumab, oxaliplatin, and a fluoropyrimidine (RAISE): a randomised, double- Siegel, R., Desantis, C. and Jemal, A. (2014a) blind, multicentre, phase 3 study. Lancet Oncol 16: Colorectal cancer statistics, 2014. CA Cancer J Clin 499–508. 64: 104–117. Takebe, N., Zhao, S., Ural, A., Johnson, M., Siegel, R., Ma, J., Zou, Z. and Jemal, A. (2014b) Banerjee, D., Diasio, R. et al. (2001) Retroviral Cancer statistics, 2014. CA Cancer J Clin 64: 9–29. transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to Smith, D., Christensen, I., Jensen, N., Markussen, B., 5-fluorouracil in murine hematopoietic progenitor Romer, M., Nygard, S. et al. (2013) Mechanisms of cells and human CD34+-enriched peripheral blood topoisomerase I (TOP1) gene copy number increase progenitor cells. Cancer Gene Ther 8: 966–973. in a stage III colorectal cancer patient cohort. PloS One 8: e60613. Tejpar, S., Prenen, H. and Mazzone, M. (2012) Overcoming resistance to antiangiogenic therapies. Sohn, K., Croxford, R., Yates, Z., Lucock, M. and Oncologist 17: 1039–1050. Kim, Y. (2004) Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity Therkildsen, C., Bergmann, T., Henrichsen-Schnack, of colon and breast cancer cells to 5-fluorouracil and T., Ladelund, S. and Nilbert, M. (2014) The methotrexate. J Natl Cancer Inst 96: 134–144. predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic Sood, A., Mcclain, D., Maitra, R., Basu-Mallick, A., colorectal cancer: A systematic review and meta- Seetharam, R., Kaubisch, A. et al. (2012) PTEN gene analysis. Acta Oncol 53: 852–864. expression and mutations in the PIK3CA gene as predictors of clinical benefit to anti-epidermal growth Thomas, H. and Coley, H. (2003) Overcoming factor receptor antibody therapy in patients with multidrug resistance in cancer: an update on the KRAS wild-type metastatic colorectal cancer. Clin clinical strategy of inhibiting p-glycoprotein. Cancer Colorectal Cancer 11: 143–150. Control 10: 159–165. Soong, R., Shah, N., Salto-Tellez, M., Tai, B., Soo, Tokunaga, Y., Sasaki, H. and Saito, T. (2007) R., Han, H. et al. (2008) Prognostic significance Clinical role of orotate phosphoribosyl transferase and of thymidylate synthase, dihydropyrimidine dihydropyrimidine dehydrogenase in colorectal cancer dehydrogenase and thymidine phosphorylase protein treated with postoperative fluoropyrimidine. Surgery expression in colorectal cancer patients treated with 141: 346–353. or without 5-fluorouracil-based chemotherapy. Ann Tournigand, C., Andre, T., Achille, E., Lledo, G., Oncol 19: 915–919. Flesh, M., Mery-Mignard, D. et al. (2004) FOLFIRI Spano, J., Fagard, R., Soria, J., Rixe, O., Khayat, followed by FOLFOX6 or the reverse sequence in D. and Milano, G. (2005) Epidermal growth factor advanced colorectal cancer: a randomized GERCOR receptor signaling in colorectal cancer: preclinical data study. J Clin Oncol 22: 229–237. and therapeutic perspectives. Ann Oncol 16: 189–194. Tsunoda, A., Nakao, K., Watanabe, M., Matsui, N., Stark, M., Bram, E., Akerman, M., Mandel- Ooyama, A. and Kusano, M. (2011) Associations of Gutfreund, Y. and Assaraf, Y. (2011) Heterogeneous various gene polymorphisms with toxicity in colorectal nuclear ribonucleoprotein H1/H2-dependent unsplicing cancer patients receiving oral uracil and tegafur of thymidine phosphorylase results in anticancer drug plus leucovorin: a prospective study. Ann Oncol 22: resistance. J Biol Chem 286: 3741–3754. 355–361. Tabernero, J. (2007) The role of VEGF and EGFR Tsurutani, J., Nitta, T., Hirashima, T., Komiya, T., inhibition: implications for combining anti-VEGF and Uejima, H., Tada, H. et al. (2002) Point mutations in anti-EGFR agents. Mol Cancer Res 5: 203–220. the topoisomerase I gene in patients with non-small cell lung cancer treated with irinotecan. Lung Cancer Tabernero, J., Van Cutsem, E., Lakomy, R., 35: 299–304. Prausova, J., Ruff, P., Van Hazel, G. et al. (2014) Aflibercept versus placebo in combination with Vallbohmer, D., Kuramochi, H., Shimizu, D., fluorouracil, leucovorin and irinotecan in the Danenberg, K., Lindebjerg, J., Nielsen, J. et al. (2006) treatment of previously treated metastatic colorectal Molecular factors of 5-fluorouracil metabolism in cancer: prespecified subgroup analyses from the colorectal cancer: analysis of primary tumor and VELOUR trial. Eur J Cancer 50: 320–331. lymph node metastasis. Int J Oncol 28: 527–533. Tabernero, J., Yoshino, T., Cohn, A., Obermannova, Vallbohmer, D., Yang, D., Kuramochi, H., Shimizu, R., Bodoky, G., Garcia-Carbonero, R. et al. (2015) D., Danenberg, K., Lindebjerg, J. et al. (2007) DPD http://tam.sagepub.com 83 Therapeutic Advances in Medical Oncology 8(1) is a molecular determinant of capecitabine efficacy in Xie, H., Wood, A., Kim, R., Stein, C. and Wilkinson, colorectal cancer. Int J Oncol 31: 413–418. G. (2004) Genetic variability in CYP3A5 and its possible consequences. Pharmacogenomics 5: 243–272. Vallbohmer, D., Zhang, W., Gordon, M., Yang, D., Yun, J., Press, O. et al. (2005) Molecular Xu, Y. and Villalona-Calero, M. (2002) Irinotecan: determinants of cetuximab efficacy. J Clin Oncol 23: mechanisms of tumor resistance and novel strategies 3536–3544. for modulating its activity. Ann Oncol 13: 1841–1851. Van Cutsem, E., Labianca, R., Bodoky, G., Yamada, K. and Araki, M. (2001) Tumor suppressor Barone, C., Aranda, E., Nordlinger, B. et al. PTEN: modulator of cell signaling, growth, migration (2009) Randomized phase III trial comparing and apoptosis. J Cell Sci 114: 2375–2382. biweekly infusional fluorouracil/leucovorin alone Yanagisawa, Y., Maruta, F., Iinuma, N., Ishizone, or with irinotecan in the adjuvant treatment of stage S., Koide, N., Nakayama, J. et al. (2007) Modified III colon cancer: PETACC-3. J Clin Oncol Irinotecan/5FU/Leucovorin therapy in advanced 27: 3117–3125. colorectal cancer and predicting therapeutic efficacy Van Cutsem, E., Peeters, M., Siena, S., Humblet, Y., by expression of tumor-related enzymes. Scand J Hendlisz, A., Neyns, B. et al. (2007) Open-label phase Gastroenterol 42: 477–484. III trial of panitumumab plus best supportive care Yang, L., Debusk, L., Fukuda, K., Fingleton, B., compared with best supportive care alone in patients Green-Jarvis, B., Shyr, Y. et al. (2004) Expansion with chemotherapy-refractory metastatic colorectal of myeloid immune suppressor Gr+CD11b+ cells cancer. J Clin Oncol 25: 1658–1664. in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 6: 409–421. Van Cutsem, E., Tabernero, J., Lakomy, R., Prenen, H., Prausova, J., Macarulla, T. et al. (2012) Addition Ye, D. and Zhang, J. (2013) Research development of aflibercept to fluorouracil, leucovorin, and of the relationship between thymidine phosphorylase irinotecan improves survival in a phase III randomized expression and colorectal carcinoma. Cancer Biol Med trial in patients with metastatic colorectal cancer 10: 10–15. previously treated with an oxaliplatin-based regimen. Ye, F., Liu, Z., Tan, A., Liao, M., Mo, Z. and Yang, J Clin Oncol 30: 3499–3506. X. (2013) XRCC1 and GSTP1 polymorphisms Van Cutsem, E., Twelves, C., Cassidy, J., Allman, and prognosis of oxaliplatin-based chemotherapy in D., Bajetta, E., Boyer, M. et al. (2001) Oral colorectal cancer: a meta-analysis. Cancer Chemother capecitabine compared with intravenous fluorouracil Pharmacol 71: 733–740. plus leucovorin in patients with metastatic colorectal Yonesaka, K., Zejnullahu, K., Okamoto, I., Satoh, T., cancer: results of a large phase III study. J Clin Oncol Cappuzzo, F., Souglakos, J. et al. (2011) Activation 19: 4097–4106. of ERBB2 signaling causes resistance to the EGFR- Van Emburgh, B., Sartore-Bianchi, A., Di directed therapeutic antibody cetuximab. Sci Transl Nicolantonio, F., Siena, S. and Bardelli, A. (2014) Med 3: 99ra86. Acquired resistance to EGFR-targeted therapies in Yoshino, T., Mizunuma, N., Yamazaki, K., Nishina, colorectal cancer. Mol Oncol 8: 1084–1094. T., Komatsu, Y., Baba, H. et al. (2012) TAS-102 Wang, H., Bian, T., Liu, D., Jin, T., Chen, Y., Lin, monotherapy for pretreated metastatic colorectal A. et al. (2011) Association analysis of CYP2A6 cancer: a double-blind, randomised, placebo- genotypes and haplotypes with 5-fluorouracil controlled phase 2 trial. Lancet Oncol 13: 993–1001. formation from tegafur in human liver microsomes. Zhang, S., Lovejoy, K., Shima, J., Lagpacan, L., Shu, Pharmacogenomics 12: 481–492. Y., Lapuk, A. et al. (2006) Organic cation transporters Wheeler, J., Bodmer, W. and Mortensen, N. (2000) are determinants of oxaliplatin cytotoxicity. Cancer Res DNA mismatch repair genes and colorectal cancer. 66: 8847–8857. Gut 47: 148–153. Zhao, J., Li, W., Zhu, D., Yu, Q., Zhang, Z., Sun, M. et al. (2014) Association of single nucleotide Wilson, P., Danenberg, P., Johnston, P., Lenz, H. and Visit SAGE journals online polymorphisms in MTHFR and ABCG2 with Ladner, R. (2014) Standing the test of time: targeting http://tam.sagepub.com the different efficacy of first-line chemotherapy in thymidylate biosynthesis in cancer therapy. Nat Rev SAGE journals metastatic colorectal cancer. Med Oncol 31: 802. Clin Oncol 11: 282–298. 84 http://tam.sagepub.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Therapeutic Advances in Medical Oncology SAGE

Pharmacologic resistance in colorectal cancer: a review:

Loading next page...
 
/lp/sage/pharmacologic-resistance-in-colorectal-cancer-a-review-3073deU04z
Publisher
SAGE
Copyright
Copyright © 2022 by SAGE Publications Ltd unless otherwise noted. Manuscript content on this site is licensed under Creative Commons Licenses
ISSN
1758-8340
eISSN
1758-8359
DOI
10.1177/1758834015614530
Publisher site
See Article on Publisher Site

Abstract

614530 TAM0010.1177/1758834015614530Therapeutic Advances in Medical OncologyW. A. Hammond et al. review-article2015 Therapeutic Advances in Medical Oncology Review Ther Adv Med Oncol Pharmacologic resistance in colorectal 2016, Vol. 8(1) 57 –84 DOI: 10.1177/ cancer: a review © The Author(s), 2015. Reprints and permissions: http://www.sagepub.co.uk/ William A. Hammond, Abhisek Swaika and Kabir Mody journalsPermissions.nav Abstract: Colorectal cancer (CRC) persists as one of the most prevalent and deadly tumor types in both men and women worldwide. This is in spite of widespread, effective measures of preventive screening, and also major advances in treatment options. Despite advances in cytotoxic and targeted therapy, resistance to chemotherapy remains one of the greatest challenges in long-term management of incurable metastatic disease and eventually contributes to death as tumors accumulate means of evading treatment. We performed a comprehensive literature search on the data available through PubMed, Medline, Scopus, and the ASCO Annual Symposium abstracts through June 2015 for the purpose of this review. We discuss the current state of knowledge of clinically relevant mechanisms of resistance to cytotoxic and targeted therapies now in use for the treatment of CRC. Keywords: colon adenocarcinoma, colon cancer, resistance Correspondence to: Introduction in determining initial and subsequent lines of Kabir Mody, MD Colorectal cancer (CRC) remains a significant treatment. Innate resistance is typically noted Division of Hematology/ Oncology, Mayo Clinic cause of morbidity and mortality worldwide with during early drug development or in early phase Cancer Center, Mayo high disease incidence and, in spite of large-scale clinical trials of biologic efficacy, however some- Clinic, 4500 San Pablo Rd S, Jacksonville, screening efforts recommended for all US adults, times innate resistance is not understood until FL 32224, USA significant numbers of patients presenting with retrospective analyses of in vivo studies. For mody.kabir@mayo.edu advanced, metastatic disease [Siegel et al. 2014b]. instance, resistance to EGFR antagonists was not William A. Hammond, MD Abhisek Swaika, MD Metastatic disease is considered incurable, with well understood and from initial studies only 10– Division of Hematology/ the exception of patients presenting with oligo- 20% of patients exhibited a response to the EGFR Oncology, Mayo Clinic, Jacksonville, FL, USA metastatic lesions confined to the liver or lung targeted therapies cetuximab or panitumumab amenable to resection, or metastasectomy [Cunningham et  al. 2004; Bardelli and Siena, [Abdalla et  al. 2004; Adam et  al. 2004]. When 2010]. The subsequent elucidation of RAS muta- treatment with curative intent is not possible, tions in CRC clarified a marker of innate resist- patients are typically given a combination of cyto- ance to these therapies, and changed their clinical toxic chemotherapy often in conjunction with a use. targeted therapy. In spite of advances in systemic therapy, the 5-year survival rate is still a mere Different mechanisms of acquired resistance can 12.5% [Siegel et al. 2014a], and the primary rea- exist for each cytotoxic therapy and each targeted son for treatment failure is believed to be acquired pathway, but often acquired resistance to one resistance to therapy which occurs in 90% of drug confers resistance to other drugs which may patients with metastatic cancer [Longley and work by different mechanisms of action, a con- Johnston, 2005]. Resistance to targeted therapy cept referred to as multidrug resistance (MDR). noted by disease progression is often noted within In general, resistance to traditional cytotoxic ther- 3–12 months on epidermal growth factor receptor apy is accomplished by decreasing the delivery of (EGFR) antagonists [Cunningham et  al. 2004; drug to the cancer cell, either by increased efflux Van Cutsem et  al. 2007], necessitating a change out of the cell mediated by ATP-dependent trans- in treatment. porters, by decreased uptake into the cell, or by a change in enzymes involved in metabolism. Malignant tumors can have intrinsic resistance Alternately, resistance can be conferred by and/or acquired resistance and each is important changes within the cell itself by genetic or http://tam.sagepub.com 57 Therapeutic Advances in Medical Oncology 8(1) epigenetic modifications that can alter drug sensi- or replication error (RER+). Microsatellites are tivity [Gottesman et al. 2002]. repetitive genetic sequences, typically 1–5 base pairs repeated 15–30 times, and instability in these Resistance to targeted therapies occurs by different regions due to either insertion or deletion of mechanisms including upregulation, mutation, or repeated units causes alteration in the DNA repli- activation of downstream signaling molecules cation process. Sporadic mutations in mismatch within specific pathways; pathway bypass mecha- repair (MMR) genes leading to MSI occur in 10– nisms; or increased cross-talk between analogous 20% of CRCs [Carethers et  al. 2004]. Mutated pathways [Longley and Johnston, 2005; Tejpar MMR genes, such as MLH1, MSH2, MSH6, et  al. 2012]. Understanding the mechanisms of PMS1, or PMS6 and loss of their respective pro- acquired drug resistance to targeted therapies is teins, can be caused by germline mutations as seen critical for the development of novel, rational, and in nearly all cases of hereditary nonpolyposis colon more effective treatment combinations and will cancer (Lynch syndrome), or by sporadic muta- help guide future therapies. tions [Wheeler et al. 2000]. The loss of MMR pro- teins causes diffuse errors in repetitive DNA Since the development of markedly improved sequences, resulting from loss of scanning and rec- genomic sequencing and molecular biology tech- ognizing errors during DNA replication and failure niques, a plethora of new putative mechanisms of to edit these errors to maintain an intact genetic resistance and potential therapies have been real- code [Fink et al. 1998]. The loss of these proteins ized. We will review some of these mechanisms is typically a result of epigenetic hypermethylation and their clinical relevance. of the promoter regions of both alleles of the MLH1 genes, which halts gene expression [Wheeler et al. 2000]. Deficiency in the subsequent Cytotoxic/cytostatic chemotherapy protein products of the hMSH2, hMLH1 and Cytotoxic chemotherapy has long been the back- hMSH6 MMR genes and loss of detection of mis- bone of treatment for CRC in patients with lymph matched and unpaired bases is thought to be a pri- node positive and metastatic disease. Efficacy was mary mechanism of inherent resistance to FPs first recognized in the US Intergroup INT-0035 because cells become tolerant to DNA damage trial in 1990 which showed the beneficial effect of and do not undergo apoptosis [Carethers et  al. 5-fluorouracil (5-FU) over surgery alone [Moertel 2004]. In one study, MMR deficient cell lines that et al. 1990]. Since the adoption of widespread use of had MMR function restored by inserting a cor- chemotherapy for advanced CRC in the adjuvant rected clone of hMLH1 gene led to increased sen- and metastatic setting, other cytotoxic drugs have sitivity to FPs [Meyers et al. 2001]. been studied and validated including capecitabine, tegafur, irinotecan, and oxaliplatin, leading to their incorporation into practice guidelines and wide- 5-FU spread use in clinical practice. The details of this 5-FU (Adrucil; Pfizer, Teva Pharmaceuticals) is pathway described below are depicted in Figure 1. the prototypical FP, a synthetic fluorinated pyrimidine analog, administered intravenously. It Fluoropyrimidines (FPs) such as 5-FU and works by multiple mechanisms to create fluori- capecitabine exert antitumor activity by inducing nated nucleotides that are incorporated into DNA a state of thymidylate deficiency and creating in place of thymidine (dTMP, or deoxythymidine imbalances in the nucleotide pool, which leads to monophosphate) thus inhibiting DNA replication impaired DNA replication, transcription, and and causing cell death. It is a prodrug that requires repair and subsequent cell death [Wilson et  al. intracellular conversion to its active metabolites. 2014]. 5-FU was one of the first chemotherapeu- The primary active metabolite is FdUMP (fluoro- tic drugs reported to have anticancer activity deoxyuridine monophosphate) which inhibits the [Heidelberger et al. 1957]. Following the earliest enzyme thymidylate synthase (TS), a key enzyme publications noting its efficacy in colon cancer, in the creation of the DNA nucleotide dTMP. 5-FU became the mainstay of therapy and is still Inhibition of TS prevents conversion of dUMP to given as a standard part of most treatments for dTMP, which in turn impairs DNA synthesis in advanced or metastatic CRC (mCRC). the S phase of the cellular replication cycle. In addition, other 5-FU metabolites 5-fluorouridine One inherent mechanism of resistance to FPs in a triphosphate (5-FUTP) and 5-fluorodeoxy subset of tumors is microsatellite instability (MSI), triphosphate (5-FdUTP) are created by alternate 58 http://tam.sagepub.com WA Hammond, A Swaika et al. Figure 1. Schematic representation of the fluoropyrimidine pathway. 5,10-CH2-FH4, 5,10-methylenetetrahydrofolate; 5-CH3-FH4, 5-methyltetrahydrofolate; 5’dFCR, 5’-deoxy-5- fluorocytidine; 5’dFUR, 5’-deoxy-5-fluorouridine; 5-FdUDP, 5-fluorodeoxyuridine diphosphate; 5-FdUMP, 5- fluorodeoxyuridine monophosphate; 5-FdUTP, 5-fluorodeoxyuridine triphosphate; 5-FU, 5-fluorouracil; 5- FUDP, 5-fluorodeoxyuridine; 5-FUDR, 5-fluorodeoxyuridine, 5-FUMP,5-fluorouridine monophosphate; 5- FUR, 5-fluorouridine; 5-FUTP, 5-fluorouridine triphosphate; CDHP, 5-chloro-2,4-dihydroxypyridine; DHFU, dihydrofluorouracil; DPD, dihydropyrimidine dehydrogenase; dTTP, deoxythymidine triphosphate; FBAL, fluoro-β-alanine; FH4, tetrahydrofolate; FH2, dihydrofolate; FUPA, α-fluoro-β-ureidopropionic acid; NDK, nucleoside diphosphate kinase; ORPT, orotate phosphoribosyltransferase; TFT, trifluorothymidine; TFT-MP, trifluorothymidine monophosphate; TFT-TP, trifluorothymidine triphosphate; TK, thymidine kinase; UMP-CMPK, uridine monophosphate-cytidine monophosphate kinase. enzymatic pathways and create false nucleotides and improved response rates to 5-FU [Salonga which are incorporated into DNA and interfere et al. 2000; Johnston et al. 1995; Leichman et al. with normal protein production leading to cell 1995]. TS is encoded by the TYMS gene, and death [Nicum et al. 2000]. genetic alterations occur by copy number varia- tions or by variations in the TYMS promoter One of the most well established mechanisms of region [Muhale et al. 2011]. In one study, patients resistance to 5-FU, and other antifolates, is with CRC with low TYMS gene expression had increased expression of TS, the primary target of improved median survival compared with those the metabolite FdUMP. TS expression is a key with higher TYMS expression [Leichman et  al. predictor of 5-FU activity [Longley et  al. 2003], 1997]. This concept was further validated by at and its expression has long been recognized as a least two published meta-analyses showing an primary determinant of resistance [Berger et  al. inverse relationship of TYMS gene expression 1985]. This is supported by studies showing an with survival and response to FP therapy [Popat inverse association between low tumor TS levels et al. 2004; Qiu et al. 2008]. http://tam.sagepub.com 59 Therapeutic Advances in Medical Oncology 8(1) Increased expression of TS is also an acquired TP is an enzyme encoded by the TYMP gene and mechanism of resistance after exposure to 5-FU is responsible for converting 5-FU to 5-fluoro- therapy [Chu et  al. 1993]. Normally, unbound 2-deoxyuridine (5-FUDR), an intermediate in the TS (not bound by the metabolite dUMP) binds conversion of 5-FU to the active metabolite to its own mRNA in a negative feedback loop to 5-FdUMP. It has been observed that expression inhibit its own translation, in turn reducing levels of TP correlates with response to 5-FU therapy of the enzyme. It is hypothesized that TS bound [Panczyk, 2014]. Cells with higher levels of TP by FdUMP cannot enact this negative feedback, theoretically should have greater sensitivity to resulting in increased protein expression and 5-FU due to increase in the concentration of decreased sensitivity to 5-FU [Longley et  al. FdUMP. To date, however, studies have shown 2002]. mixed results regarding response to 5-FU-based chemotherapy and level of TP expression. Low The inhibition of TS by the metabolite FdUMP TP expression, measured in one study by reverse requires formation of a complex between FdUMP transcriptase polymerase chain reaction (RT-PCR) and 5, 10-methylenetetrahydrofolate (CH THF). and in another by immunohistochemistry (IHC) Normally, multiple enzymes regulate the intracel- and tissue microarrays, correlated with improved lular level of folate in order to maintain an appro- treatment outcomes, in terms of overall survival priate pool of tetrahydrofolate, which is formed (OS), in patients with mCRC treated with adju- by the reduction of CH THF to 5-methyltetrahy- vant 5-FU [Soong et al. 2008; Salonga et al. 2000]. drofolate (CH THF) by the enzyme methylene- A more recent study analyzed tumor tissue from tetrahydrofolate reductase (MTHFR). MTHFR mCRC by RT-PCR and found longer time to pro- activates a unidirectional reaction to convert gression with high TP expression [Lindskog et al. CH THF to CH THF, thereby decreasing the 2014]. Further well-designed trials are needed to 2 3 amount of CH THF and in turn decreasing the establish a definitive link between TP expression activity of TS. Decreased enzymatic activity of and resistance to FP therapy. MTHFR increases the concentration of the reduced cofactor CH THF and increases the OPRT is the protein product of the uridine inhibition of TS by increased concentration of the monophosphate synthase (UPMS) gene. This FdUMP-CH THF complex. Several single enzyme catalyzes the conversion of 5-FU to nucleotide polymorphisms (SNPs) have been 5-fluoro-uridine monophosphate (5-FUMP), an shown to affect the activity of MTHFR. Two in intermediate but necessary step in the production particular are the 677C>T and 1298A>C poly- of the active metabolites 5-FUTP and 5-FdUTP. morphisms. These decrease the activity of the Increased expression of UPMS gene and increased enzyme and were observed in vivo to show mixed levels of OPRT in tumor tissue has been shown in results, with one study demonstrating a lack of multiple trials to increase the chemo-sensitivity of independent correlation to response [Marcuello cells to 5-FU [Koopman et  al. 2009; Tokunaga et  al. 2006] but numerous others showing a et  al. 2007; Isshi et  al. 2002]. Only one study, greater response to 5-FU based therapy [Sohn however, has demonstrated that decreased et  al. 2004; Etienne-Grimaldi et  al. 2010]. expression, by means of UPMS knockdown cell Although it seems intuitive that increased activity lines, leads to resistance to 5-FU [Muhale et  al. of this enzyme would lead to resistance, studies 2011]. More research is needed to determine if have not shown that upregulation or amplifica- this is a clinically significant biomarker of sensi- tion of the MTHFR gene or protein product cor- tivity or mechanism of resistance in vivo. relate with chemoresistance. Dihydropyrimidine dehydrogenase (DPD) is the Fluorouracil must go through several enzymatic enzyme primarily responsible for catabolism of steps before it is converted to its other active 5-FU to 5-fluorodihydrouracil (5-FUH ), also metabolites 5-FdUTP, and 5-FUTP. It has been referred to as dihydrofluorouracil (DHFU), the shown that activity of three of the necessary first step in 5-FU elimination. 5-FUH is then enzymes for these conversions, thymidine phos- converted into the soluble molecule 5-fluoro-β- phorylase (TP), uridine phosphorylase (UP), and alanine and eliminated in urine. Intrinsic overex- orotate phosphoribosyl transferase (OPRT), cor- pression of DPD by malignant cells has been relate with the sensitivity of CRC cells to the cyto- shown in vitro to extend resistance to 5-FU toxic effects of 5-FU [Schwartz et  al. 1985; [Longley and Johnston, 2005; Takebe et  al. Houghton and Houghton, 1983]. 2001]. High levels of DPD mRNA expression in 60 http://tam.sagepub.com WA Hammond, A Swaika et al. CRC cells have also been associated with 5-FU randomized trials completed in the mid-1990s resistance [Salonga et  al. 2000]. This has been comparing capecitabine to weekly bolus 5-FU/LV demonstrated as an intrinsic mechanism of resist- [Van Cutsem et al. 2001; Hoff et al. 2001]. These ance, but available data is not yet conclusive on results have been subsequently confirmed in a DPD as a means of acquired resistance. In addi- meta-analysis of six randomized trials that showed tion, DPYD gene expression has been investi- equivalent OS in patients treated with single gated as a biomarker of treatment resistance, but agent and matched capecitabine-containing regi- this has also not been shown to be clinically rele- mens to single agent and matched 5-FU-containing vant [Yanagisawa et  al. 2007; Vallbohmer et  al. regimens [Cassidy et al. 2011]. 2006]. Since capecitabine is eventually converted into Numerous variable number of tandem repeats 5-FU within the tumor, many of the mechanisms (VNTRs) and single nucleotide polymorphisms of resistance are identical to those implicated for (SNPs) of the TYMS, MTHFR, DPYD, and UPMS 5-FU. Several of these mechanisms have been genes have been identified as contributors to 5-FU evaluated specifically in capecitabine treated cells. resistance, and could serve as potential targets for As shown in relation to 5-FU, DPD expression future directed therapy to combat drug resistance, has been implicated in resistance to capecitabine. or potentially for gene therapy [Panczyk, 2014]. In one study, higher gene expression, as meas- ured by mRNA analysis from tumor tissues fol- lowing capecitabine treatment, correlated with Oral FPs resistance to therapy as evidenced by shorter pro- Capecitabine, S-1, and tegafur-uracil are three gression-free survival (PFS) and lower response oral FPs demonstrating similar efficacy as intra- rate in patients [Vallbohmer et al. 2007]. In a ret- venous 5-FU, with the potential for more con- rospective review of 556 tumors from the CAIRO venience by reducing time spent in an infusion (CApecitabine, IRinotecan, Oxaliplatin) study, suite or on an infusion pump. Capecitabine is the DPD expression inversely correlated with PFS only one of these currently approved for use in the and OS [Koopman et  al. 2009]. This study also United States. All of these therapies are prodrugs investigated the predictive value of multiple other which, by a series of enzymatic reactions, are markers including OPRT, TP, TS, and ERCC1, eventually converted to 5-FU in the tumor micro- and found that high OPRT in stromal cells was environment. Because of this, they have many of favorable for response to therapy, but elevated the same mechanisms of resistance as 5-FU, how- expression in tumor cells correlated with worse ever with additional layers needed for activation PFS and OS. The reason for this is unclear and come new potential sources of resistance. has not been fully elucidated. Capecitabine. Capecitabine (Xeloda; Genentech, TP converts 5-FU prodrugs into active metabolites Roche, Switzerland) is an oral FP carbamate that exert antitumor effect. This enzyme is which was developed to mimic continuous 5-FU expressed in higher concentration in tumors, but infusion, but with activation occurring primarily was shown to be expressed in the tumor microenvi- at the tumor site. After absorption it is converted ronment rather than the tumor cells. TP has other to fluorouracil by three enzymes, two of which, TP actions related to carcinogenesis, including promo- and UP, are present in higher concentrations tion of metastasis, tumor infiltration, and angiogen- within malignant cells compared to normal cells esis which correlate with poor prognosis [Ye and [Wilson et al. 2014]. This orally administered drug Zhang, 2013]. High expression of TP, however, was designed to function similar to infusional correlates with better response to capecitabine, and 5-FU, giving a consistent level of drug exposure to loss of function confers resistance [Petrioli et  al. tumor cells over time. Since the final steps in the 2010], a fact that has also been shown to have clini- pathway occur preferentially in the tumor it has a cal significance with improved response to CAPIRI theoretical advantage of increased efficacy and chemotherapy by extending time to progression decreased systemic toxicity, however this has not [Meropol et al. 2006]. This is seen in contrast to the been the case in clinical trials [ Cassidy et al. 2011]. inverse effect of TP expression on response to 5-FU. One putative mechanism of loss of function The efficacy of capecitabine in advanced CRC, of TP is abnormal pre-mRNA splicing by increased including stage III and metastatic disease, was levels heterogeneous nuclear RNP (hnRNP) H/F originally demonstrated by two phase III splicing factors which leads to loss of function of http://tam.sagepub.com 61 Therapeutic Advances in Medical Oncology 8(1) the TYMP gene [Stark et al. 2011]. Further research TAS-102. TAS-102 (Taiho Pharmaceutical, Tokyo, is ongoing in regards to exact mechanisms of devel- Japan) is an oral nucleoside antitumor agent with opment of resistance specifically to capecitabine multiple components. Trifluorothymidine (TFT) that may be distinct from other FPs. is the active ingredient and is a FP that is active in inhibiting DNA replication. TFT is phosphory- S-1 and tegafur-uracil. S-1 (TS-1; Taiho Pharma- lated by thymidine kinase (TK) and in turn inhib- ceutical, Tokyo, Japan) is a fourth generation oral its thymidine synthase (TS) in a similar fashion to FP available worldwide outside of the United other FPs. It can also form the triphosphate form, States. It consists of tegafur (UFT), gimeracil TFT-TP, which is incorporated into DNA thus (5-chloro-2, 4-dihydroxypyridine) and oteracil interfering directly with replication [Wilson et  al. (potassium oxonate). Tegafur is a prodrug that is 2014]. In a phase II trial comparing monotherapy converted to fluorouracil within tumor cells. with placebo, TAS-102 showed improvement in Gimeracil is an inhibitor of DPD, the primary both PFS and OS in a small subset of patients enzyme responsible for fluorouracil metabolism. with mCRC refractory to traditional therapy Oteracil inhibits the phosphorylation of fluoro- [Yoshino et al. 2012]. The results of the phase III uracil in the gastrointestinal tract and serves to RECOURSE trial comparing TAS-102 to placebo reduce toxic side effects of 5-FU [Sakuramoto in pretreated patients showed improvement in OS et  al. 2007]. Tegafur–uracil is another oral FP and PFS in European patients with mCRC refrac- agent and is approved in 50 countries worldwide. tory to at least two prior lines of therapy [Mayer It consists of tegafur attached to uracil, which et al. 2015]. As yet there is no direct experimental blocks DPD degradation of fluorouracil’s pyrimi- evidence supporting a process for development of dine base. Tegafur is not well tolerated by patients resistance, however one would presume it may fol- as they experience consistently high toxicity low a pattern very similar to other FPs. negating any benefit, which is the primary reason for lack of approval in the United States. Numerous gene polymorphisms of OPRT, MTHFR, UGT1A1, and DPD have been investi- The approval of S-1 is based on two trials in East gated in relation to toxicity [Tsunoda et al. 2011; Asian patients. It is considered an acceptable Choi et al. 2012], but to date none of these have alternative to 5-FU when combined with oxalipl- demonstrated clinical significance in relation to atin as part of FOLFOX or XELOX [Hong et al. chemoresistance. These oral FPs may prove use- 2012], or an alternative to FOLFIRI when com- ful for patients in the United States if they gain bined with irinotecan [Muro et al. 2010] in East FDA approval. Asian patients. A subsequent meta-analysis of eight trials from Japan and China in Asian patients with either mCRC or advanced gastric cancer Irinotecan demonstrated essentially equivalent efficacy to Irinotecan (Camptosar, formerly CPT-11; Pfizer, the 5-FU-containing regimens [Cao et al. 2014]. Pharmacia) is a semisynthetic analog of the natural alkaloid camptothecin, a DNA topoisomerase I Tegafur is metabolized by the cytochrome P-450 inhibitor. This enzyme relaxes super-coiled double- isoenzyme encoded by the CYP2A6 gene. The stranded DNA by inducing single strand breaks. By expression of the variants CYP2A6*4 and inhibiting its action, irinotecan interferes with DNA CYP2A6*1B seem to be the primary determi- replication and transcription. Topoisomerase I nants for the degree of conversion into 5-FU, and inhibitors stabilize intermediate cleavage complexes are required for the anticancer activity of tegafur formed between the inhibitor, the enzyme, and the [Wang et al. 2011]. This has been shown to cor- DNA single strand and subsequently prevent DNA relate with toxicity, a putative reason why white re-ligation, leading to cell death. Irinotecan must patients experience intolerable toxicity compared be converted to its active metabolite SN-38 to exert with East Asian patients [Shirao et  al. 2004]. It its anticancer effect [Xu and Villalona-Calero, has been shown that patients with wild-type 2002], and SN-38 reversibly binds to topoisomer- CYP2A6 have improved efficacy outcomes com- ase-1 and stabilizes the complex. It was approved in pared with mutants in gastric cancer, and also an accelerated fashion by the FDA in 1996 and showed increased response to TIROX compared received full approval for use in CRC in 1998. with several polymorphisms [Kim et  al. 2013], presumably related to decreased conversion to The addition of irinotecan to adjuvant therapy for 5-FU and exposure of tumor cells. advanced CRC and to mCRC was supported by 62 http://tam.sagepub.com WA Hammond, A Swaika et al. multiple trials starting in the early 1990s irinotecan in in vitro studies [Kojima et  al. 1998; [Douillard et  al. 2000; Saltz et  al. 2000b; Boyer et al. 2004], however, outside of associations Giacchetti et  al. 2000]. Single-agent irinotecan with specific SNPs with cytotoxicity, no direct cor- has only a modest response rate of 11–27% in relation has been shown between carboxylesterase clinical trials in 5-FU refractory patients with activity or expression and chemoresistance. SN-38 mCRC [Cunningham et  al. 1998], but a larger is metabolized by glucuronidation in the liver by effect when given in combination with FPs, 5-FU the enzyme uridine diphosphate glucuronosyl- and capecitabine, and/or oxaliplatin [Shimada transferase (UGT) to form SN-38 glucuronide et al. 1996]. In the first-line setting response rates (SN-38G). Increased glucuronidation-mediated range from 39% to 49% [Saltz et  al. 2000a; clearance in CRC cells may contribute to tumor Douillard et  al. 2000]. It has been approved as resistance to irinotecan [Cummings et  al. 2002]. first-line therapy in advanced or metastatic dis- Genetic variants of both liver and plasma UGT1A, ease, but not for adjuvant treatment as this was by VNTRs and other polymorphisms, have been shown to provide no additional benefit to FP shown to have effects on CPT-11 toxicity includ- monotherapy [Van Cutsem et  al. 2009]. The ing neutropenia and diarrhea, however these have addition of irinotecan to FP-based regimens also not been shown to correlate with response to ther- shows a significant benefit in PFS from 4.3 to apy [Marcuello et al. 2004]. Any association with 7 months, and OS from 12 to 18–21 months, and poor response to treatment is believed to be a result its efficacy as monotherapy in patients with FP of having to reduce the dose of the drug due to side refractory disease indicates activity in spite of effects, not from the polymorphism [Carlini et al. acquired resistance to FP therapy [Cunningham 2005; Rouits et al. 2004]. et al. 1998; Rougier et al. 1998]. Irinotecan itself is metabolized by oxidation, pri- Irinotecan resistance in CRC appears to develop marily by two hepatic cytochrome P-450 enzymes, by a few mechanisms including low intratumor CYP3A4 and CYP3A5. These enzymes metabolize level of the active metabolite SN-38, a decrease in irinotecan to APC ((7-ethyl-10-(4-N-aminopenta- expression of topoisomerase I, change in the noic acid)-1-piperidino) carbonyloxycamptoth- activity of the SN-38-Topo I- DNA complex, and ecin) and NPC (7-ethyl-10-(4-amino-1-piperidino) changes in downstream events such as suppres- carbonyloxycamptothecin), respectively. NPC is an sion of apoptosis, cell cycle alterations, or inactive metabolite but can be hydrolyzed by enhancement of DNA repair. hepatic carboxylesterase back to SN-38 [Panczyk, 2014]. Variations in CYP3A4 activity have been The level of intratumoral SN-38 can be altered by investigated as a source for resistance to treatment increased efflux or increased metabolism of either with irinotecan, however current research, includ- irinotecan or the metabolite SN-38. Active trans- ing studies measuring in vivo activity of these port out of cells by the multidrug resistance pro- enzymes, is inconclusive about the role of numer- tein (MRP), an ATP-binding cassette (ABC) ous described polymorphisms [Xie et  al. 2004; transporter protein, has been shown in cancer Fujiwara and Minami, 2010]. cells to result in resistance to irinotecan and SN-38 [Longley and Johnston, 2005; Thomas As described previously, SN-38 binds to topoi- and Coley, 2003]. Numerous in vitro studies of somerase I as it binds to DNA to relieve strand polymorphisms of transporter proteins, such as tension during replication. The action of irinote- ABCC1/MRP1, ABCC2/MRP2, and ABCG2/ can is dependent on normal functioning topoi- BCRP have shown results that explain variation somerase I, encoded by the TOP1 gene. Level of in drug toxicity in patients, and the development TOP1 gene expression and copy number was of drug resistance against irinotecan and SN-38 demonstrated as a potential cause of intrinsic [Zhao et  al. 2014]. However, these results have resistance by an in vitro study of colon cancer cell been inconsistent in the literature and there are lines [McLeod and Keith, 1996]. A more recent no in vivo studies to support development of these Scandinavian retrospective analysis of tumor tis- proteins as targets for therapy. sue from irinotecan pre-treated patients identified an increased objective response in tumors with The active metabolite SN-38 is created by hydrol- increased copy number of TOP1 gene, although ysis of CPT-11 by carboxylesterases CES1 and this did not reach statistical significance [Nygard CES2. Carboxylesterase activity in cancer cells has et  al. 2014]. Increased copy number has been been shown to correlate with sensitivity to noted in as many as two-thirds of tumors from a http://tam.sagepub.com 63 Therapeutic Advances in Medical Oncology 8(1) cohort of patients with stage III CRC [Smith et al. with response rates as high 50% [de Gramont 2013], so low levels of TOP1 gene copy number is et  al. 2000]. The GERCOR study published in implicated as a means of intrinsic resistance. 2004 [Tournigand et  al. 2004] showed equiva- lence of the two primary regimens FOLFOX and SN-38 forms non-covalent, but stable bonds to FOLFIRI, which was subsequently confirmed by the Topo-1-DNA complex to exert its cytostatic Colucci and colleagues the following year effect that leads to cell death. Changes in the [Colucci et  al. 2005]. Since the FOLFIRI regi- binding site of topoisomerase I prevent SN-38 men was shown to be ineffective in the adjuvant from creating a stable bond. One study of irinote- setting, FOLFOX has become the mainstay of can-treated tumor samples identified that point therapy in postoperative patients. mutations in the Top1 gene, as detected by RT-PCR of mRNA from tumor samples, altered The mechanisms of resistance of oxaliplatin the binding of SN-38 to the enzyme, implicating appear to be somewhat different from those seen acquired resistance to therapy [Tsurutani et  al. with cisplatin and carboplatin. In fact, it has been 2002]. Further in vitro analyses of SN-38 resist- shown to be active in cancer cell lines resistant to ant tumor cell clones showed that resistance may earlier generation platinum compounds [Raymond develop by decreased affinity of TOP1/SN-38 et al. 2002]. It has also been well established that binding or by mutations in the linker domain enhanced replicative bypass and loss of MMR are which lead to decreased flexibility of the complex mechanisms of resistance to cisplatin but not to [Gongora et al. 2011]. oxaliplatin by both in vitro and in vivo studies. One study comparing similar platinum resistant cell Again, numerous SNPs have been discovered to lines exposed to cisplatin and oxaliplatin showed help explain the genetics behind these mecha- that oxaliplatin exerted cytotoxic effects at a much nisms of resistance, as well as explain potential lower concentration than did cisplatin, and also variations amongst individuals with apparently created fewer DNA-Pt adducts [Hector et  al. similar tumor types [Panczyk, 2014]. The clinical 2001]. These adducts are likely different than relevance of these numerous polymorphisms is those created by cisplatin, supported by the fact not yet certain based on the current body of that MMR deficient cells demonstrate resistance literature. to cisplatin but not oxaliplatin [Fink et al. 1997], implying that even MMR deficient cells are able to recognize oxaliplatin DNA-Pt adducts and Oxaliplatin undergo apoptosis. Oxaliplatin (Eloxatin; Sanofi-Aventis Pharma- ceuticals) is a third generation platinum com- Like other chemotherapy, resistance can occur by pound that works by inducing DNA cross-linkages decreased entrance into or increased efflux out of the leading to apoptotic cell death. It is a square pla- tumor cells. The transporters regulating platinum nar platinum, distinct from other platinums in efflux are ABC-type MDR proteins as well as cop- that it contains a bidentate ligand 1,2-diaminocy- per-transporting p-type ATPases [Panczyk, 2014]. clohexane in lieu of two monodentate ammine A few transporters have been recognized to regulate ligands. It causes inter- and intra-strand DNA influx of platinum compounds, including copper cross-links that halt replication and transcription transporter (CTR) proteins, organic cation trans- [Hector et  al. 2001]. It was approved for use in porters (OCTs), and an undefined cis-specific plati- Europe in 1996 and it was granted accelerated num influx transporter that has not been implicated approval in 2002 by the US FDA, with full in oxaliplatin resistance. Some transporters have approval granted in 2004 for use in combination been investigated, including members of the SCL22 with 5-FU for advanced CRC or mCRC. family (such as OCT2), as well as CTRs [Zhang et  al. 2006] (including CTR1 and CTR2 [Holzer Oxaliplatin has been shown to have minimal sin- et al. 2006]), and these have been shown in preclini- gle agent activity, with response rates of around cal studies to be potential targets of modulation of 20–24%, in several phase II trials [Becouarn et al. influx and efflux. No in vivo studies have shown 1998; Diaz-Rubio et  al. 1998] in the front-line actual clinical significance of these postulated resist- setting. In combination with 5-FU and leucov- ance mechanisms. In vitro studies have shown that orin (FOLFOX), however, it has been shown in oxaliplatin requires lower concentrations to exert multiple trials to contribute to increased PFS and anticancer effects, but these transports have not been OS compared with 5-FU and leucovorin alone shown to be major mechanism of resistance. 64 http://tam.sagepub.com WA Hammond, A Swaika et al. A mechanism of platinum compound inactivation Epigenetic changes have also been implicated in is the formation of conjugates, or covalent link- development of resistance, primarily hypermeth- ages, between the drug and the thiol glutathione ylation. Although oxaliplatin works by inducing (GSH, a tripeptide of glutamic acid, cysteine, and single-strand breaks by intra-strand adduct for- glycine) [Meijer et  al. 1992]. Glutathione is a mation, it also forms inter-strand adducts and can potent antioxidant that functions to prevent oxi- cause double-stranded breaks. These abnormali- dative damage to DNA and RNA. This also serves ties are repaired by the NER process, as well as by as a mechanism of increased clearance, however, BRCA1, which works by homologous recombina- because GSH–platinum conjugates become a sub- tion to repair double-strand breaks [Fedier et  al. strate for the ABC transporter proteins which pro- 2003]. Inactivation of the BRCA1 interactor motes drug efflux out of the cell [Ishikawa and SRBC by hypermethylation of the SRBC1 gene is Ali-Osman, 1993]. It has been shown that some associated with oxaliplatin resistance [Moutinho tumors may be resistant to platinums due to their et  al. 2014]. Little has been definitively discov- having higher levels of GSH [Kelland, 1993]. ered about the impact of epigenetic alterations on There are also studies demonstrating the potential CRC resistance, however, as in other tumor influence of several polymorphisms of the glu- types, this is likely a mechanism. tathione s-transferases, however this is not con- sistently shown and has not been demonstrated in Resistance to cytotoxic chemotherapy occurs by in vivo studies [Panczyk, 2014]. A meta-analysis several variations on similar themes, such as of five clinical studies showed no correlation of a decreased intracellular drug concentration, common polymorphism, 313A>G, with response altered metabolism, or alterations to targets of the to oxaliplatin-based therapy [Ye et al. 2013]. therapy. These mechanisms of resistance are summarized in Table 1. Some of these concepts Platinum compounds exert their antitumor effect translate into mechanisms of resistance to novel by creating adducts in the DNA, typically intra- targeted therapies, however often resistance is strand cross-links [Hector et al. 2001], that lead to more complex, at times not involving mutation apoptosis once recognized by MMR mechanisms. but molecular pathway alteration, something not Nucleotide excision repair (NER) is a mechanism seen with evasion of traditional chemotherapy. by which cells repair DNA damage and one of the ways in which cancer cells overcome chemother- apy effects. This may be particularly true for plati- Targeted therapy nums by removing DNA-Pt adducts from the As knowledge about cancer biology and genetics strand. Excision repair cross-complementation expands, new treatment targets have been discov- group 1 and 2 (ERCC1 and 2) proteins are two of ered and drugs developed to affect tumors in the main effectors of the NER mechanism. They more elegant and rational fashion than impacting recognize DNA-Pt adducts and coordinate the all cells actively in the cell cycle. This has led to base excision process. High expression of ERCC1 treatments with good response and often less- mRNA has been associated with poor response to toxic side-effect profiles than cytotoxic/cytostatic FOLFOX in 5-FU resistant tumors [Shirota et al. therapy. Two broad categories of targeted thera- 2001], implicating that enhanced DNA repair pies include monoclonal antibodies and small decreases the benefit of platinum therapy. molecule inhibitors, different in their target of Oxaliplatin-resistant tumors have upregulation of action and mode of administration. ERCC1 as shown by higher mRNA expression in retrospective analysis of tumor samples [Baba Although targeted agents provide hope for more et  al. 2012] implicating that this may also be a effective therapy, studies have shown modest ben- form of acquired resistance. Numerous polymor- efit and, like more traditional chemotherapy, are phisms in the ERCC1 gene have been identified subject to both primary and secondary resistance and investigated as predictive, however most have which ultimately leads to treatment failure. Protein shown inconclusive results [Panczyk, 2014]. The tyrosine kinases, such as EGFR and vascular ERCC1 (354C>A) SNP, in conjunction with the endothelial growth factor receptor (VEGFR) are XRCC1 (1196A>G) SNP have an independent some of the most well-understood pathways of predictive effect on disease control rate and OS potential therapy used to treat CRC. Resistance is [Liang et  al. 2010]. This, however, must still be often seen through constitutive pathway activation, taken in the context of a retrospective analysis and perhaps by receptor overexpression or mutation. we await further prospective analyses. By identifying specific ligands or receptors involved http://tam.sagepub.com 65 Therapeutic Advances in Medical Oncology 8(1) Table 1. Reported mechanisms of resistance to chemotherapy agents. Chemotherapy Enzyme/pathway Mechanism of resistance (MoR) Reference References contrary to proposed MoR 5-Fluorouracil Thymidylate synthase Increased expression leading to Popat et al. [2004], (5-FU) (TS) increased target of 5-FU inhibition Qiu et al. [2008] Decreased negative feedback by Longley et al. TS-FdUMP on its own expression [2002] Methylene Increased activity of MTHFR, Sohn et al. [2004], Marcuello et al. [2006] tetrahydrofolate decreasing CH THF availability Etienne-Grimaldi reductase (MTHFR) required for inhibition of TS et al. [2010] (postulated) Thymidine phosphorylase Increased expression (postulated), Soong et al. [2008], Lindskog et al. [2014] (TP) may lead to increased salvage Salonga et al. pathway of nucleotides. Low [2000] expression correlates with increased response to 5-FU therapy. Orotate phosphoribosyl Decreased expression Muhale et al. transferase (OPRT) (postulated), as high expression [2011] correlates with sensitivity Dihydropyridine Increased expression leading to Salonga et al. Yanagisawa et al. dehydrogenase (DPD) increased degradation [2000] [2007], Meropol et al. [2006], Vallbohmer et al. [2006] Capecitabine Thymidine phosphorylase Decreased expression leading Meropol et al. (TP) to decreased formation of active [2006], Petrioli metabolite et al. [2010] Dihydropyridine Increased expression leading to Vallbohmer et al. dehydrogenase (DPD) increased metabolism [2007], Koopman et al. [2009] Irinotecan Multidrug resistance Increased expression leading to Zhao et al. [2014] protein (MRP) increased efflux Uridine diphosphate Increased expression leading to Cummings et al. glucuronosyltransferase increased metabolism of SN-38 [2002] (UGT) Carboxylase Decreased expression Kojima et al. (postulated) as enzyme is required [1998], Boyer et al. to create active metabolite [2004] Topoisomerase-I Decreased copy number of TOP1 Nygard et al. gene (postulated), as increased [2014] copy number Alterations in binding site Tsurutani et al. [2002], Gongora et al. [2011] Oxaliplatin Multidrug resistance Increased expression leading to Zhang et al. [2006] protein (MRP) increased efflux Glutathione (GSH) Increased levels of GSH Kelland [1993] Ye et al. [2013] inactivating the platinum and increasing export from cell ERCC1 Increased expression leading to Shirota et al. increased nucleotide excision [2001], Baba et al. repair [2012] 66 http://tam.sagepub.com WA Hammond, A Swaika et al. Table 2. Reported mechanisms of resistance to targeted therapies. Targeted Enzyme/ Mechanism of resistance Per cent present Reference therapy gene/pathway in de novo tumors [Bardelli and Siena, 2010] EGFR EGFR Decreased expression (postulated) Sartore-Bianchi et al. [2007], antagonists as increased expression is shown to Cappuzzo et al. [2008] correlate with improved response KRAS Activating mutation 35–45%, at least Lievre et al. [2006], De Roock 50% of secondary et al. [2010], Douillard et al. mutations* [2013], *Misale et al. [2012] Amplification 0.7% Misale et al. [2014] NRAS Activating mutation 3–5% Douillard et al. [2013], Meriggi et al. [2014] PTEN Loss of function (mutation or loss of 27–30% Frattini et al. [2007], Sartore- expression), leads to constitutively Bianchi et al. [2009] activated AKT PI3K Activating mutation 14–17% De Roock et al. [2010], Sood et al. [2012] HER2 Amplification, serves as a bypass, or 3% Perrone et al. [2009], Sood escape mechanism et al. [2012], Rajput et al. [2007] MET Amplification, serves as a bypass, or 2% Bardelli et al. [2013] escape mechanism BRAF Activating mutation, constitutive 5–10% Di Nicolantonio [2008], activation of MAPK pathway Hirschi et al. [2014] Paracrine Increased serum levels and binding Hobor et al. [2014] activation of alternate ligands, TGF-α and amphiregulin Increased binding of ligands (e.g. Yonesaka et al. [2011] heregulin, HGF) to parallel activating pathways, (e.g. HER2, MET) Cetuximab EGFR Confers resistance to cetuximab after Esposito et al. [2013] S492R point exposure, but not to panitumumab mutation VEGF VEGF Autocrine signaling, with increased Mesange et al. [2014] antagonists expression caused by positive feedback (increased HIF induced by hypoxia) Alternate Increased expression of PlGF, IL-8, Kopetz et al. [2010], Lieu ligands VEGF-D, et al. [2013], Mizukami et al. [2005] Vascular Recruitment of pro-angiogenic Mitchell [2013], Pollard protection factors, e.g. BMDCs, tumor-associated [2004], De Palma et al. macrophages, TIE2, and VEGFR1 positive [2005], Hattori et al. [2002] hemagiocytes Increased pericyte coverage Kamba and McDonald [2007] Increased Increased local invasion and metastasis Du et al. [2008] invasiveness by co-opting local vasculature Aflibercept VEGF-C Increased binding Li et al. [2014] BMDC, bone-marrow-derived cell; EGFR, epidermal growth factor receptor; HGF, hepatocyte growth factor; IL, interleukin; KRAS, Kristen rat sarcoma; NRAS, neuronal rat sarcoma; PTEN, phosphatase and tensin homolog; TGF-α, transforming growth factor alpha; VEGF, vascular endothelial growth factor. http://tam.sagepub.com 67 Therapeutic Advances in Medical Oncology 8(1) Figure 2. Schematic representation of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) and receptor (VEGF-R) pathways. BAD, Bcl-2 associated death promotor; EGF, epidermal growth factor; EIF-4, eukaryotic initiation factor-4; Grb2,growth factor receptor bound protein 2; MAPK, mitogen activated protein kinase; MEK, mitogen/extracellular signal related kinase; mTOR, mammalian target of rapamycin; MDM2, mouse double minute 2; NFκB, nuclear factor κ-light chain enhancer of activated B cells; TGF-α, transforming growth factor alpha; TF, transcription factor; P, phosphate; PI3K, phosphoinositide 3 kinase; PIP2, phosphatidylinositol (4, 5)-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-triphosphate; RAS, rat sarcoma; RAF, rapidly accelerated fibrosarcoma; SOS, son of sevenless. in cell growth or survival, both the activating ligand kinase domain, a transmembrane hydrophobic seg- and receptor can potentially be targeted. Common ment, and an extracellular receptor to which ligands mechanisms of resistance are related to alterations bind for activation. It is a member of the ErbB of the target itself, bypass mechanisms, upregula- tyrosine kinase receptor (TKR) family, and is also tion and activation of downstream effectors, or referred to as ErbB1, or HER1. This family also cross-talk between associated pathways which acti- includes human epidermal growth factor receptor 2 vate complementary cell survival and growth path- (HER2) which is implicated in other cancer types, ways. The details of these pathways described also known as ErbB2. Other members of the ErbB below are depicted in Figure 2. family include Erb3 and 4. All four ErbB family members can interact with each other to induce signaling depending on which ligand is bound. Epidermal growth factor EGFR is activated primarily by the binding of epi- EGFR is a transmembrane signaling protein on dermal growth factor (EGF) or transforming human epidermal cells involved in cellular signaling growth factor-alpha (TGF-α) [Roskoski, 2014]. for proliferation and survival, and its over-expres- sion is linked to cancer development and prolifera- Inactive EGFR exists as a monomer, however tion. It is composed of an intracellular tyrosine once a ligand binds to the extracellular domain, 68 http://tam.sagepub.com WA Hammond, A Swaika et al. this induces either homodimerization with another Phosphatase and tensin homolog (PTEN) is a EGFR monomer or heterodimerization with a dif- negative regulator of the PIP3KCA/Akt pathway, ferent ErbB family receptor. This then induces and is one of the most frequently mutated tumor transphosphorylation of several intracellular tyros- suppressor genes [Yamada and Araki, 2001]. Its ine kinase (TK) domains which propagate down- normal function is to regulate the activity of PIP3 stream signaling by several well-described by dephosphorylation, and thus inhibit Akt activ- pathways, most notably the Ras/Raf/MEK/ ity. Mutation of PTEN and loss of its activity thus ERK1/2/MAPK and PI3K/Akt/mTOR pathways leads to constitutive activation of Akt and promo- [Roskoski, 2014; Spano et  al. 2005]. The PI3K/ tion of cell survival. Akt pathway has been shown to participate pri- marily in mediating cell survival and motility inva- Upregulation or activation of EGFR is present in sion, where the Ras/Raf/MEK/ERK1/2 pathway is 60–80% of CRCs [Messa et  al. 1998; Porebska implicated in cellular proliferation. Both of these et al. 2000; Salomon et al. 1995] and overexpres- pathways are also involved in angiogenesis, cellu- sion of EGFR is associated with poor prognosis in lar adhesion, cell motility, development, and mCRC [Mayer et al. 1993]. As such, this receptor organogenesis [Roskoski, 2012]. has been a focus of drug development and targeted therapies against this receptor are approved for use Initiation of the Ras/Raf/MEK/ERK pathway by in CRC, including cetuximab and panitumumab. EGFR begins with activation of Grb2 (growth factor receptor bound protein 2) adaptor protein Cetuximab. Cetuximab (Erbitux; Bristol-Meyer which binds to the phosphotyrosine residue of the Squibb, Eli Lilly and Company) is a chimeric ErbB receptor as well as SOS (son of sevenless), a mouse/human IgG1 monoclonal antibody that Ras-guanine nucleotide exchange factor binds to the extracellular domain of the EGFR. [Lowenstein et  al. 1992]. SOS activates Ras to Cetuximab was first approved by the FDA for use Ras-GTP which propagates downstream signal- in mCRC in February 2004 as monotherapy or in ing, starting with Raf and resulting in cell growth combination with irinotecan. Cetuximab was and differentiation [Roskoski, 2012]. shown to have a response rate of about 10% as monotherapy in a study of irinotecan pretreated Activation of the PI3K tyrosine kinase by EGFR patients by Cunningham and colleagues with induces phosphorylation of membrane-bound improved time to progression from 1.5 to phosphatidylinositol 4,5-bisphosphonate (PIP ) 4.1  months [Cunningham et  al. 2004]. In addi- to form phosphatidylinositol 3,4,5-triphosphate tion, Jonker and colleagues showed an 8% partial (PIP ). PIP attracts Akt (also known as protein response rate and improved PFS, as well as 3 3 kinase b, or PKB) to the plasma membrane improved overall quality of life compared with [Longley and Johnston, 2005]. Akt is a serine/ best supportive care in heavily pretreated patients threonine kinase that binds tightly to PIP and with mCRC [Jonker et al. 2007]. Interestingly, the interacts with various other kinases, including addition of cetuximab to irinotecan in irinotecan- phosphoinositide-dependent protein kinase 1 resistant/refractory disease showed prolongation (PDK1) and mammalian target of rapamycin of both PFS and OS compared with cetuximab complex 2 (mTORC2). These kinases both phos- monotherapy, suggesting a means of overcoming phorylate Akt and stimulate it to phosphorylate resistance to irinotecan [Cunningham et al. 2004]. and activate mTOR, another serine/threonine kinase which has multiple targets involved in cell The current understanding of the mechanism of survival. Activated Akt also phosphorylates, and action of cetuximab is that the drug binds to the thus inactivates, Bcl2 associate death promotor external domain of the EGFR and prevents ligand (BAD), which, in its unphosphorylated state, is binding, preventing cell growth and survival. pro-apoptotic by binding and sequestering the After binding, the receptor is internalized and antiapoptotic Bcl2. Akt also has the ability to degraded without activation or phosphorylation affect apoptosis by phosphorylating MDM-2 [Tabernero, 2007]. There is also evidence that which, once translocated to the nucleus, down- cetuximab-receptor binding also induces anti- regulates p53 expression [Feng et al. 2004]. Thus, body-mediated cytotoxicity, leading to tumor cell by activation of the PI3K/Akt pathway, EGFR death [Ciardiello and Tortora, 2008]. In addi- promotes cell survival via both mTOR and Bcl2 tion, cetuximab was shown to down-regulate activation and by p53 downregulation [Roskoski, VEGF expression, thus reducing tumor angio- 2014]. genesis [Ciardiello et al. 2000]. http://tam.sagepub.com 69 Therapeutic Advances in Medical Oncology 8(1) Early in the investigation into mechanisms of inherent resistance to both cetuximab and panitu- resistance, it was postulated that somatic EGFR mumab, leading to minimal clinical effect [De mutations may play a significant role in lack of Stefano and Carlomagno, 2014]. Lievre and col- response, as was demonstrated to other targeted leagues first noted that tumor response was dic- therapies, for example in lung and breast cancer tated by KRAS mutation status when they [Paez et al. 2004; Hudis, 2007]. It has since been examined 30 tumors and found that 13/30 tumors suggested by large cohort studies that EGFR harbored a KRAS mutation and two thirds (68%) mutations are not only uncommon in mCRC, but of the nonresponders had a mutation whereas this that they do not impact response when they are was found in none of the responders [Lievre et al. present [Barber et al. 2004; Moroni et al. 2005]. 2006]. The first large retrospective review of However, one specific point mutation of EGFR, a treated tumors found that 43.2% of tumors had at change of serine to arginine at codon 492 (S492R) least one mutation in exon 2 which correlated with leading to a change in the external domain of the lack of response to cetuximab [Karapetis et  al. EGFR was recently shown to confer resistance to 2008]. The predictive mutations were noted first cetuximab binding but not the other approved in exon 2, codons 12 and 13, however subsequent EGFR targeted therapy panitumumab [Van studies have shown that as many as 5–11% of Emburgh et al. 2014]. It has been shown that this additional tumors have mutations in exons 3 mutation is likely present only in patients follow- (codons 59 and 61) and 4 (codons 117 and 146) ing exposure to EGFR antagonism, and not in which are similarly predictive of lack of response treatment-naïve patients [Esposito et  al. 2013]. to EGFR antagonist therapy [Therkildsen et  al. Recently, the incidence of this mutation in cetuxi- 2014; Misale et al. 2014]. mab and panitumumab treated tumors was evalu- ated in a review of tumor samples from the phase The discovery of activating KRAS mutations led III ASPECCT trial of second-line treatment of to better patient selection, however, in spite of wild-type KRAS exon 2 mCRC [Price et  al. ‘wild type’ EGFR, still only about 40% of patients 2014]. Of the 999 patients, roughly half in each responded to EGFR targeted therapy, indicating group had post-treatment EGFR S492R status an alternate means of innate resistance. KRAS available, and the mutation was found in 1% of amplification has been identified as a cause of panitumumab treated tumors and 16% of cetuxi- resistance to anti-EGFR therapy as well, but is mab treated tumors. The mutation was not iden- likely present in only in ~1–2% of cases and it is tified in any of the pretreatment samples, mutually exclusive from KRAS mutations [Misale confirming that this is an acquired mutation con- et  al. 2014]. Resistance to EGFR antagonists is ferring resistance. In addition, these EGFR also influenced by neuronal RAS (NRAS) muta- mutant tumors demonstrated longer duration of tions [Douillard et al. 2013; Meriggi et al. 2014]. treatment before progression, but had lower median OS by almost 2 months [Price et  al. NRAS is a gene found on the short arm of chro- 2015]. mosome 1 which encodes for a GTPase mem- brane enzyme that shuttles between the Golgi and Although the relationship between EGFR muta- the cell membrane [De Stefano and Carlomagno, tions and response remains unclear, there is evi- 2014]. Mutations have been identified at exon 2 dence that increased copy number of the EGFR (codons 12 and 13), exon 3 (codons 59 and 61) receptor, as measured by fluorescent in situ and exon 4 (codons 117 and 146) and occur at a hybridization (FISH), is a positive predictor of frequency of approximately 2–5% among patients response to treatment with both cetuximab and with mCRC. These mutations have been shown panitumumab [Sartore-Bianchi et  al. 2007; to negatively impact response to anti-EGFR ther- Cappuzzo et al. 2008]. Likewise, low-level expres- apy, with either cetuximab or panitumumab sion is purported as a means of intrinsic resistance [Meriggi et  al. 2014; De Roock et  al. 2010; to therapy, yet this has not been shown clinically. Therkildsen et al. 2014]. Mutations in NRAS are mutually exclusive of mutations in KRAS and In early clinical trials, only about 10–20% of unse- BRAF. In spite of increasing knowledge, there are lected patients with mCRC responded to single still tumors that do not respond to treatment, agent EGFR antagonists. It is now known that indicating some yet unidentified innate or intrin- tumors with a mutation of the Kristen rat sarcoma sic resistance. For now, it is standard of care to (KRAS) gene found on chromosome 12, encod- test not only for KRAS mutations in exons 2 ing a small G-protein downstream of EGFR, have [Allegra et  al. 2009], 3 and 4, but also NRAS 70 http://tam.sagepub.com WA Hammond, A Swaika et al. mutations prior to exposing a patient to anti- contribute to EGFR therapy resistance, however EGFR therapy. this has not been definitively demonstrated in clinical trials. Activating KRAS mutations have been shown to be a mechanism of both intrinsic and acquired Many of the aforementioned mutations and alter- resistance. In a large analysis by Misale and col- ations play a central role in innate, or primary leagues, EGFR sensitive cell lines of primary resistance. It has been shown that approximately colon tumors and metastases showed that numer- 50% of tumors harbor a KRAS mutation at time ous molecular alterations, mostly point mutations of progression on treatment with anti-EGFR in KRAS gene, led to both cetuximab and panitu- therapies [Hobor et al. 2014; Misale et al. 2012], mumab resistance. This study also showed that implying that other means of resistance subvert KRAS mutations could be detected in blood sam- the effect of EGFR blockade. Heterogeneity ples as early as 10 months before radiographic within a given tumor is often observed where evidence of disease progression, implicating that mutated cells (e.g. KRAS, NRAS, BRAF this type of resistance may occur quite early in mutated) exist along with other sensitive, non- disease treatment [Misale et al. 2012]. mutated cells, implicating a potential protective mechanism. In addition, it is known that EGFR Changes in pathways related to the target of ther- has multiple ligands other than EGF, including apy, or parallel to it, may confer resistance. In TGF-α and amphiregulin, which bind to the regard to EGFR targeted therapies, PIK3CA receptor and activate downstream signaling. A activating mutations are seen in 14.5% of paracrine method of resistance has been postu- untreated tumors, most in exons 9 and 20 [De lated in which tumor cells can increase the secre- Roock et  al. 2010]. These mutations have been tion of these alternate ligands which serve to shown to predict lack of response to EGFR ther- protect the cells from EGFR-blockade. This was apy in pre-clinical models and early in vivo stud- shown in an in vitro study of cetuximab-resistant ies [Jhawer et  al. 2008; Sartore-Bianchi et  al. cells cultured with cetuximab-sensitive cells 2009; Sood et  al. 2012]. The largest analysis to where the sensitive cells grew in the presence of date, however, with more than 750 untreated cetuximab and increased secretion of ligands and CRC tumor samples from European centers increased EGFR signaling were observed [Hobor showed an association between exon 20 and et al. 2014]. worse response to cetuximab therapy, but not exon 9, perhaps implicating limitations of tumor Lastly, another mechanism of acquired resistance sample volume in prior studies that showed asso- may occur by alteration of complementary path- ciation with exon 9 [De Roock et  al. 2010]. ways which serve to bypass the EGFR antago- Additionally effecting the EGFR/PI3K/Akt path- nism, so-called escape mechanisms. In particular, way is epigenetic inactivation of PTEN phos- upregulation of ErbB2 (HER2) and MET can phatase which has been suggested to be predictive occur and may contribute to resistance. ErbB2 is of lack of response to EGFR targeted therapy a receptor with no known biologic ligand that [Perrone et al. 2009]. Loss of function in PTEN undergoes a conformational change that activates has been observed to occur by means of muta- the PI3K/Akt pathway [Rajput et  al. 2007]. tion, loss of gene expression, or hypermethylation Amplification of HER2 has been noted in only of the promotor region [Sood et al. 2012]. Studies 2–3% of untreated tumors, however has been have shown that patients with tumors having shown that cetuximab-resistant cell lines demon- preservation of PTEN and wild-type PIK3CA strate both HER2 amplification as well as an had improved OS and a trend toward improved increase in secretion of heregulin, a ligand which PFS [Sood et al. 2012], and that time to progres- binds to the HER2 receptor [Yonesaka et  al. sion was significantly shorter in tumors with 2011; Ciardiello and Normanno, 2011; Bertotti PIK3CA mutations and loss of PTEN [Saridaki et al. 2011]. MET gene amplification may act as et al. 2011]. In a retrospective analysis of cetuxi- another escape mechanism, as well as increased mab treated patients, none of the patients with binding by its ligand hepatocyte growth factor loss of function PTEN mutation showed response (HGF) [Bardelli et  al. 2013]. The vascular to cetuximab therapy [Frattini et  al. 2007]. In endothelial growth factor (VEGF) pathway, addition, BRAF mutations, present in about in described in detail later in this review, is also 9.6% of CRC [Hirschi et  al. 2014], have been closely related to the EGFR pathway. In fact, shown to confer a worse prognosis, and may overexpression of VEGF is one way in which http://tam.sagepub.com 71 Therapeutic Advances in Medical Oncology 8(1) tumor cells overcome resistance to EGFR inhibi- mechanisms, including mutations of KRAS, tion. It has been shown that EGFR over-expres- NRAS, PIK3CA, PTEN [Douillard et  al. 2013; sion leads to upregulation and increased signaling Sood et al. 2012]. One difference is that of a specific by VEGF, however the resistance seems to occur point mutation of the EGFR gene, S492R, which independent of EGFR signaling [Tabernero, was shown to confer resistance to cetuximab post- 2007]. This was shown in a study of gene expres- treatment, but not panitumumab. The incidence of sion of biomarkers from tumors pretreated with the S492R mutation in panitumumab-treated irinotecan or oxaliplatin, followed by single agent tumors has been reported from retrospective analy- cetuximab. Those tumors with elevated VEGF sis of tumors from patients treated in the ASPECCT expression were more resistant to cetuximab trial and found in only 1% of post-treatment evalu- [Vallbohmer et al. 2005]. able tumor samples, implying that this may not be a prominent mechanism of acquired resistance. No Panitumumab. The other approved EGFR mono- other specific polymorphisms are published that clonal antibody is panitumumab (Vectibix; show resistance unique to panitumumab [Van Amgen), a fully human IgG2 monoclonal anti- Emburgh et al. 2014]. body that binds with high affinity to the extracel- lular domain of the EGFR. It is thought that panitumumab prevents downstream activation VEGF signaling by competitive inhibition [Hohla et  al. The role of angiogenesis and lymphangiogenesis 2014]. It was approved by the FDA for use in in tumor growth is well established. Hypoxia in mCRC expressing EGFR in September 2006 in the tumor microenvironment has been shown to combination with other approved chemotherapy upregulate hypoxia inducible factor (HIF), which regimens for patients who had progressed on or then signals production of VEGF [Maxwell et al. after initial therapy. It has also now been approved, 1997]. Over-expression of VEGF gene and high as of May 2014, for use in the first-line setting levels of circulating VEGF protein are both associ- with FOLFOX therapy. ated with worse prognosis in CRC [Jurgensmeier et  al. 2013]. Several agents have been developed The efficacy of panitumumab was first demon- to inhibit angiogenesis and thus slow tumor strated by Van Cutsem and colleagues as mono- growth. VEGF has been identified as the promi- therapy [Van Cutsem et  al. 2007]. In the nent effector of angiogenesis, and has been the ASPECCT trial, a phase III randomized noninfe- target of drug developments since it was recog- riority trial, panitumumab was shown to have nized that its inhibition suppresses tumor growth similar OS and PFS as cetuximab, showing simi- [Kim et al. 1993]. The VEGF family is composed lar efficacy in patients with exon 2 KRAS wild- of at least nine ligands, VEGF-A through E, and type mCRC [Price et  al. 2014]. Perhaps the placental growth factor (PlGF) 1 through 4. These largest and most impactful study of this agent has ligands act upon at least one of three known recep- been the PRIME trial which compared FOLFOX4 tors, VEGFR-1 through 3. Blocking members of with and without panitumumab. In patients with this family, either the ligand or receptor, prevents KRAS wild-type tumors, there was a significantly tumor mitigation of local hypoxia and starvation. improved PFS of 9.6 months with the addition of The first VEGF/VEGFR targeted drug brought to panitumumab, compared with 8 months in the market was bevacizumab, and much of our under- control arm. Objective response rate was also standing about resistance to VEGF inhibition improved at 55%, compared with 48% in the comes from experience with this drug. FOLFOX4 alone group. Again, this effect was lost in those with mutated KRAS. In fact PFS was Bevacizumab. The monoclonal antibody bevaci- significantly worse in this group compared with zumab (Avastin; Genentech/Roche) is a recombi- FOLFOX4 alone [Douillard et al. 2010]. nant humanized IgG1 antibody against all isoforms of VEGF-A, a ligand for the VEGF Retrospective analyses of tumor samples from trials receptors 1 and 2 [Tejpar et al. 2012]. It was first of tumors treated with panitumumab have shown approved by the FDA in February 2004 for use in that KRAS mutations in exon 2, codons 12 and 13, combination with chemotherapy in the first-line were mutated at the same frequency, approximately treatment of mCRC. 43%, as in analogous reviews of trials with cetuxi- mab [Amado, 2008]. Resistance to panitumumab Bevacizumab has been shown to have clinical is akin to cetuximab occurring by many of the same activity in CRC in numerous studies, the first of 72 http://tam.sagepub.com WA Hammond, A Swaika et al. which was a trial by Hurwitz et al. [2004] which elevated pro-angiogenic biomarkers in tumors fol- showed an improved response rate, PFS and OS lowing treatment with bevacizumab, alternate pro- with the addition of bevacizumab to a first-line angiogenic pathways have been discovered. In one irinotecan-based regimen (IFL) in mCRC. It has study, PIGF, a ligand of VEGFR-1, was found to also been shown to have increased PFS in combi- be significantly elevated in tumors following treat- nation with oxaliplatin-based chemotherapy, ment with FOLFIRI plus bevacizumab [Kopetz combined with both 5-FU and capecitabine, as et al. 2010]. This ligand, as well as VEGF-D, was first-line therapy in mCRC [Saltz et  al. 2008]. shown in another study to be upregulated for about Both of these studies showed an improvement of 6 weeks following treatment with bevacizumab no more than 2 months in PFS. Interestingly, it plus chemotherapy, indicating this as a transient has also been shown that continuation of bevaci- alteration [Lieu et  al. 2013]. In addition, IL-8, a zumab after progression, while changing the cyto- chemokine with many functions including angio- toxic chemotherapy regimen, resulted in improved genesis, was shown to provide HIF-independent PFS and OS compared with post-progression angiogenic stimulus in vitro in CRC cell lines chemotherapy alone [Bennouna et al. 2013]. The [Mizukami et  al. 2005]. Elevated pretreatment reasons behind this are likely related to the differ- levels of IL-8 in blood were associated with shorter ent mechanisms of resistance to angiogenesis PFS in a phase II trial investigating biomarkers in inhibitors, including both evasive/acquired resist- relation to response to FOLFIRI plus bevaci- ance and intrinsic indifference [Bergers and zumab [Kopetz et  al. 2010]. Third, fibroblast Hanahan, 2008]. growth factor (FGF) upregulation has been noted in pancreatic neuroendocrine [Casanovas et  al. VEGF inhibitors such as bevacizumab work by 2005] and glioblastoma [Batchelor et  al. 2007] reducing the formation of new vasculature needed tumor cell lines, implicating this as a potential eva- by developing tumors for continued growth. They sive mechanism of resistance to bevacizumab, induce a state of local hypoxia, however hypoxia although this has never been shown explicitly in induces increased VEGF expression via HIF, so CRC. Additional biomarkers of putative signifi- its very inhibition induces its production. In addi- cance are platelet-derived growth factor-C tion, the binding of VEGF to its target leads to (PDGF-C) [Crawford et  al. 2009], neuropilin-1 downstream upregulation of VEGF, creating a (NRP-1) [Pan et al. 2007], and delta-like ligand-4 positive feedback loop of continued vascular (Dll4) [Ridgway et al. 2006]. growth promotion. CRC cells express VEGF receptor and demonstrate this autocrine signal- Another method of evasion of bevacizumab ther- ing, which promotes cell survival [Mesange et al. apy is by increased protection of vasculature by 2014]. This occurs particularly under external recruitment of alternative pro-angiogenic factors stress, including stress from treatment with 5-FU and by increasing protective barriers over existing [Samuel et al. 2011]. In vitro, bevacizumab resist- vasculature to increase its survival. Bone-marrow- ant cell lines show strong autocrine HIF-VEGF- derived cells (BMDCs), including vascular pro- VEGFR signaling as a response to exposure to genitors and vascular modulatory cells, are anti-VEGF therapy [Mesange et al. 2014]. recruited to the tumor environment in the setting of hypoxia to promote new vessel growth Acquired resistance to anti-angiogenic agents is [Mitchell, 2013]. Not only are progenitors of epi- often referred to as evasive resistance, because thelial cells and pericytes recruited to the area, unlike resistance to cytotoxic therapy or EGFR but so too are other vascular modulators such as antagonists, this typically occurs by adapting to tumor-associated macrophages [Pollard, 2004], the presence of anti-angiogenic agents in lieu of TIE2 [De Palma et  al. 2005], VEGFR-1+ more definitive mechanisms such as mutation of hemangiocytes [Hattori et al. 2002], and CD11b+ constituents within the pathway. This evasive myeloid cells [Yang et  al. 2004]. These function resistance occurs by at least three different mech- to promote vascular growth by producing anisms including revascularization by alternate cytokines, growth factors and proteases that pro- angiogenic pathways, development of protective mote vascular growth and development [Bergers mechanisms for vasculature, and enhanced ability and Hanahan, 2008]. It has also been noted that of tumors to invade or metastasize. tumors treated with anti-angiogenic agents have a change in the structure and concentration of peri- Continued or revascularization can occur by upreg- cyte coverage over existing vasculature [Kamba ulation of alternate VEGFR ligands. By recognizing and McDonald, 2007], presumably lending to http://tam.sagepub.com 73 Therapeutic Advances in Medical Oncology 8(1) the observed rapid reconstitution of vasculature Given the specificity of bevacizumab to the following removal of angiogenic blockade VEGF-A ligand, and the range of evasive mecha- [Mancuso et al. 2006]. Two such factors contrib- nisms within tumors, it is not surprising that uting to recruitment of BMDCs and monocytes/ resistance develops somewhat rapidly. Resistance macrophages to the tumor microenvironment are is not considered permanent, and as mentioned HIF1α and PlGF [Ribatti, 2008]. above, ongoing treatment with bevacizumab has shown prolonged PFS and OS compared with Some tumors treated with continuous anti-angio- chemotherapy alone. Perhaps the use of a differ- genic therapy may also develop augmented inva- ent agent to attempt broader angiogenic inhibi- siveness leading to increased local invasion and tion, including some of the evasive mechanisms of metastasis. The mechanism by which they are resistance, would be more efficacious. able to invade is by co-option of normal vascula- ture allowing this to provide nutrients necessary Aflibercept. Aflibercept (Zaltrap; Regeneron for continued growth. This was first seen in glio- Pharmaceuticals, Sanofi-Aventis), referred to in blastoma cell lines [Rubenstein et  al. 2000], and the United States as ziv-aflibercept, is a recombi- has been since recognized in pancreatic neuroen- nant decoy VEGFR1 and VEGFR2 fusion pro- docrine tumors as well [Casanovas et  al. 2005]. tein, each linked via the Fc segment of IgG1, that Tumor cells are able to migrate along the blood has anti-angiogenic and vascular permeability vessels, a process called perivascular invasion, activity by targeting multiple members of the allowing for subsequent direct invasion into sur- VEGF family, including VEGF-A, VEGF-B and rounding tissue [Du et al. 2008]. placental growth factor 2 (PlGF-2). Binding these growth factors prevents their activity at the In some trials, a minority of patients had rapid VEGFR-1 and VEGFR-2 receptors, which are progression in spite of angiogenic blockade, sug- found on the surface of endothelial cells and leu- gesting either rapid development of resistance or kocytes. Its activity results in regression of tumor the presence of intrinsic resistance. It has been vasculature, inhibition of new vascular growth postulated that some of the same mechanisms of and remodeling of surviving vasculature [Mitch- acquired resistance are present innately within ell, 2013]. It was approved for use in the US by some tumors, such as BMDCs such as CD11+ the FDA in August 2012. monocytes [Shojaei et  al. 2007]. Some tumors may also intrinsically possess the invasive pheno- The efficacy of aflibercept was demonstrated in type allowing them to co-opt surrounding normal the VELOUR trial, a phase III randomized, pla- vasculature to promote growth [Du et al. 2008]. cebo controlled trial of over 1200 patients with mCRC who had progressed on oxaliplatin-based Lastly, KRAS mutation status has been observed chemotherapy. Patients were randomized to to affect response to bevacizumab-containing reg- receive FOLFIRI plus aflibercept or FOLFIRI imens when examined in subgroup analyses. One plus placebo. The addition of aflibercept resulted example is the TLM trial of bevacizumab con- in improved response rate (20% versus 11%, tinuation after progression on a bevacizumab respectively), PFS (6.9 versus 4.7 months, respec- containing first-line treatment which was designed tively), and modestly improved OS by 1.5 months with an exploratory endpoint of evaluation of OS, [Van Cutsem et al. 2012]. This is to date the only PFS, and subsequent anticancer treatment published phase III trial demonstrating efficacy of according to KRAS mutation status [Bennouna aflibercept in any setting. Interestingly, subgroup et  al. 2013]. Patients treated with bevacizumab analysis has shown that the benefit of adding plus chemotherapy had improved PFS regardless aflibercept was seen regardless of whether the of KRAS mutation status, however patients with patient had received bevacizumab as part of first- KRAS wild-type cancer had improved OS with line treatment, potentially implicating efficacy addition of bevacizumab to second-line chemo- beyond development of resistance to VEGF inhi- therapy that was not seen in the patients with bition [Tabernero et al. 2014]. KRAS mutant cancer. These data seem to imply a mechanism of resistance, however the treatment Resistance to aflibercept is presumed to occur by by KRAS status interaction test for this subgroup many of the same mechanisms implicated in resist- was negative for PFS and OS, indicating no iden- ance to bevacizumab. It has been specifically tifiable KRAS mutation-independent treatment shown that VEGF-C is upregulated in tumors effect. treated with the VEGF-trap aflibercept, suggesting 74 http://tam.sagepub.com WA Hammond, A Swaika et al. that alternate angiogenesis pathways are triggered derived growth factor receptor) and β, and FGFR to promote resistance [Li et  al. 2014]. As afliber- (fibroblast growth factor receptor) 1 and 2, and cept is studied in earlier treatment lines for mCRC, p38 MAP kinase [Grothey et al. 2013; T ejpar et al. more data on acquired resistance may become 2012]. available upon review of treated tumors. It was approved by the FDA in September 2012 Ramucirumab. Ramucirumab (Cyramza; Eli Lilly for use in patients with previously treated mCRC and Company, US) is a recombinant, fully who had received all previously approved treat- humanized IgG1 monoclonal antibody directed ments including VEGF- and EGFR-active agents. against the extracellular domain of the VEFGR2. Its approval was based primarily on the results of It binds to this receptor with high affinity, and the CORRECT trial, a large international rand- thereby blocks ligand binding, primarily VEGF-A omized, double-blind, placebo controlled phase but others as well. It was approved in April 2015 III trial involving 760 patients who had previously for use in combination with FOLFIRI for the been treated with chemotherapy plus bevaci- treatment of patients with mCRC who have pro- zumab, as well as an anti-EGFR drug if the gressed on or after prior therapy with bevaci- patient’s tumor was KRAS wild type. The study zumab, oxaliplatin, and a FP. achieved its primary end point of improved median OS by showing an improvement of Approval was based on the results of the RAISE 1.4 months with regorafenib treatment over best trial, a phase III randomized, double-blind, supportive care. The study also demonstrated an multicenter international trial of 1072 patients improvement in PFS, though there was no differ- who had disease progression during or within ence in overall response rate [Grothey et al. 2013]. 6  months of the last dose of first-line therapy [Tabernero et  al. 2015]. The patients received Given the nature of this multi-targeted kinase, FOLFIRI plus either ramucirumab or placebo, potential mechanisms of resistance are difficult to randomized in a 1:1 ratio. The primary endpoint, elucidate. Presumably, mechanisms of resistance median OS, was improved by 1.6 months (13.3 previously shown for other specifically targeted versus 11.7 months) and the survival benefit was agents may be applicable to this agent as well, seen across all subgroups including KRAS however this agent simultaneously blocks multi- mutants and those with short time to progression ple members of a single pathway and members of (<6 months) after previous first-line treatment. parallel pathways between which cross-talk may PFS was also prolonged by 1.2 months (5.7 ver- occur, though likely not all isoforms of each tar- sus 4.5 months), but there was no appreciable get. The modest clinical benefit over best sup- difference in response rate. The results of this portive care (1.4 month improvement) implies trial indicate that VEGF receptor blockade after that there are very likely some mechanisms of progression through alternate angiogenesis resistance. This benefit was seen in heavily pre- blockade provides additional benefit, similar to treated patients, so it is likely the tumors had sub- what was seen in the TLM trial with bevaci- stantial acquired resistance to typical pathways of zumab [Bennouna et al. 2013] and the VELOUR targeted treatment. Further research on this topic trial with aflibercept [Tabernero et  al. 2014]. is certainly expected after its recent approval and Since this anti-angiogenic therapy is still new in with more patients being treated with this drug. the armamentarium of colon cancer treatments, there is no published data to specifically impli- cate unique mechanisms of resistance. Conclusions This review demonstrates that much is known about resistance to cytotoxic and targeted thera- Multi-kinase targeted agents pies in the treatment of CRC, though clearly Regorafenib. Regorafenib (Stivarga; Bayer more needs to be discovered. The goal of eluci- HealthCare Pharmaceuticals) is an oral multi- dating mechanisms of resistance is to develop kinase tyrosine kinase inhibitor that targets mul- methods to overcome the resistance to advance tiple angiogenesis targets including VEGFR1, our fight against this disease and achieve better, VEGFR2, VEGFR3, and TIE2, as well as multi- more durable treatment responses and longer ple oncogenic targets including KIT, RET, RAF1, patient survival. With so many potential targets BRAF, and BRAF-V600E, and tumor microenvi- for therapy, personalized treatments with existing ronment targets including PDGFRα (platelet drugs would be expected to show improved http://tam.sagepub.com 75 Therapeutic Advances in Medical Oncology 8(1) Amado, R., Wolf, M., Peeters, M., Van Cutsem, E., results. Early trials of personalized therapy based Siena, S., Freeman, D. et. al. (2008) Wild-type KRAS on mutation status of KRAS, BRAF, PI3KCA, is required for panitumumab efficacy in patients and expression of Topo-I, ERCC1, TS, and TP with metastatic colorectal cancer. J Clin Oncol 26: did not show any improvement in PFS [Cubillo 1626–34. et  al. 2014]. However, some authors have pro- posed using KRAS, BRAF, PI3KCA, and PTEN Baba, H., Watanabe, M., Okabe, H., Miyamoto, Y., Sakamoto, Y., Baba, Y. et al. (2012) Upregulation mutation status as a signature to guide therapy of ERCC1 and DPD expressions after oxaliplatin- [Bardelli and Siena, 2010; Sartore-Bianchi et  al. based first-line chemotherapy for metastatic colorectal 2009]. Other clinical trials involving personalized cancer. Br J Cancer 107: 1950–1955. therapies for CRC based on more comprehensive genomic and molecular profiling are underway. Barber, T., Vogelstein, B., Kinzler, K. and Velculescu, Certainly, in this era of more targeted therapies V. (2004) Somatic mutations of EGFR in colorectal cancers and glioblastomas. N Engl J Med 351: 2883. and much improved capabilities in genomic and molecular profiling, much is left to discover about Bardelli, A., Corso, S., Bertotti, A., Hobor, S., Valtorta, the effects that these targeted therapies have on E., Siravegna, G. et al. (2013) Amplification of the the intricate web of signaling and activities both MET receptor drives resistance to anti-EGFR therapies intracellularly and in the tumor microenviron- in colorectal cancer. Cancer Discov 3: 658–673. ment. Ultimately, new discoveries will continue Bardelli, A. and Siena, S. (2010) Molecular mechanisms to translate into improved treatment options and of resistance to cetuximab and panitumumab in important clinical outcomes for patients. colorectal cancer. J Clin Oncol 28: 1254–1261. Batchelor, T., Sorensen, A., Di Tomaso, E., Zhang, Acknowledgements W., Duda, D., Cohen, K. et al. (2007) AZD2171, We would like to acknowledge Margaret a pan-VEGF receptor tyrosine kinase inhibitor, McKinney for material support. normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer Cell 11: 83–95. Funding Becouarn, Y., Ychou, M., Ducreux, M., Borel, C., This research received no specific grant from any Bertheault-Cvitkovic, F., Seitz, J. et al. (1998) Phase funding agency in the public, commercial, or not- II trial of oxaliplatin as first-line chemotherapy in for-profit sectors. metastatic colorectal cancer patients. Digestive Group of French Federation of Cancer Centers. J Clin Oncol Conflict of interest statement 16: 2739–2744. The author(s) declare(s) that there is no conflict Bennouna, J., Sastre, J., Arnold, D., Osterlund, P., of interest. Greil, R., Van Cutsem, E. et al. (2013) Continuation of bevacizumab after first progression in metastatic colorectal cancer (ML18147): a randomised phase 3 trial. Lancet Oncol 14: 29–37. References Abdalla, E., Vauthey, J., Ellis, L., Ellis, V., Pollock, Berger, S., Jenh, C., Johnson, L. and Berger, F. R., Broglio, K. et al. (2004) Recurrence and outcomes (1985) Thymidylate synthase overproduction and following hepatic resection, radiofrequency ablation, gene amplification in fluorodeoxyuridine-resistant and combined resection/ablation for colorectal liver human cells. Mol Pharmacol 28: 461–467. metastases. Ann Surg 239: 818–825; discussion 825–827. Bergers, G. and Hanahan, D. (2008) Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer Adam, R., Delvart, V., Pascal, G., Valeanu, A., 8: 592–603. Castaing, D., Azoulay, D. et al. (2004) Rescue surgery for unresectable colorectal liver metastases Bertotti, A., Migliardi, G., Galimi, F., Sassi, F., downstaged by chemotherapy: a model to predict Torti, D., Isella, C. et al. (2011) A molecularly long-term survival. Ann Surg 240: 644–657; annotated platform of patient-derived xenografts discussion 657–658. (“xenopatients”) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal Allegra, C., Jessup, J., Somerfield, M., Hamilton, cancer. Cancer Discov 1: 508–523. S., Hammond, E., Hayes, D. et al. (2009) American society of clinical oncology provisional clinical opinion: Boyer, J., Mclean, E., Aroori, S., Wilson, P., Mcculla, testing for KRAS gene mutations in patients with A., Carey, P. et al. (2004) Characterization of p53 metastatic colorectal carcinoma to predict response wild-type and null isogenic colorectal cancer cell lines to anti-epidermal growth factor receptor monoclonal resistant to 5-fluorouracil, oxaliplatin, and irinotecan. antibody therapy. J Clin Oncol 27: 2091–2096. Clin Cancer Res 10: 2158–2167. 76 http://tam.sagepub.com WA Hammond, A Swaika et al. Cao, C., Zhang, X., Kuang, M., Gu, D., He, M., Ciardiello, F. and Tortora, G. (2008) EGFR Chen, J. et al. (2014) Survival benefit from S-1 as antagonists in cancer treatment. N Engl J Med 358: compared to Fluorouracil in Asian patients with 1160–1174. advanced gastrointestinal cancer: a meta-analysis. Colucci, G., Gebbia, V., Paoletti, G., Giuliani, Cancer Sci 105: 1008–1014. F., Caruso, M., Gebbia, N. et al. (2005) Phase III Cappuzzo, F., Finocchiaro, G., Rossi, E., Janne, randomized trial of FOLFIRI versus FOLFOX4 in the P., Carnaghi, C., Calandri, C. et al. (2008) EGFR treatment of advanced colorectal cancer: a multicenter FISH assay predicts for response to cetuximab in study of the Gruppo Oncologico Dell’Italia chemotherapy refractory colorectal cancer patients. Meridionale. J Clin Oncol 23: 4866–4875. Ann Oncol 19: 717–723. Crawford, Y., Kasman, I., Yu, L., Zhong, C., Wu, X., Modrusan, Z. et al. (2009) PDGF-C mediates the Carethers, J., Smith, E., Behling, C., Nguyen, angiogenic and tumorigenic properties of fibroblasts L., Tajima, A., Doctolero, R. et al. (2004) associated with tumors refractory to anti-VEGF Use of 5-fluorouracil and survival in patients treatment. Cancer Cell 15: 21–34. with microsatellite-unstable colorectal cancer. Gastroenterology 126: 394–401. Cubillo, A., Rodriguez-Pascual, J., Lopez-Rios, F., Plaza, C., Garcia, E., Alvarez, R. et al. (2014) Phase Carlini, L., Meropol, N., Bever, J., Andria, M., Hill, II trial of target-guided personalized chemotherapy in T., Gold, P. et al. (2005) UGT1A7 and UGT1A9 first-line metastatic colorectal cancer. Am J Clin Oncol. polymorphisms predict response and toxicity in colorectal cancer patients treated with capecitabine/ Cummings, J., Boyd, G., Ethell, B., Macpherson, irinotecan. Clin Cancer Res 11: 1226–1236. J., Burchell, B., Smyth, J. et al. (2002) Enhanced clearance of topoisomerase I inhibitors from human Casanovas, O., Hicklin, D., Bergers, G. and colon cancer cells by glucuronidation. Biochem Hanahan, D. (2005) Drug resistance by evasion of Pharmacol 63: 607–613. antiangiogenic targeting of VEGF signaling in late- stage pancreatic islet tumors. Cancer Cell 8: 299–309. Cunningham, D., Humblet, Y., Siena, S., Khayat, D., Bleiberg, H., Santoro, A. et al. (2004) Cetuximab Cassidy, J., Saltz, L., Twelves, C., Van Cutsem, monotherapy and cetuximab plus irinotecan in E., Hoff, P., Kang, Y. et al. (2011) Efficacy of irinotecan-refractory metastatic colorectal cancer. capecitabine versus 5-fluorouracil in colorectal and N Engl J Med 351: 337–345. gastric cancers: a meta-analysis of individual data from 6171 patients. Ann Oncol 22: 2604–2609. Cunningham, D., Pyrhonen, S., James, R., Punt, C., Hickish, T., Heikkila, R. et al. (1998) Randomised Choi, Y., Kim, T., Kim, K., Lee, S., Hong, Y., Ryu, trial of irinotecan plus supportive care versus M. et al. (2012) A Phase II study of clinical outcomes supportive care alone after fluorouracil failure for of 3-week cycles of irinotecan and S-1 in patients patients with metastatic colorectal cancer. Lancet 352: with previously untreated metastatic colorectal 1413–1418. cancer: influence of the UGT1A1 and CYP2A6 polymorphisms on clinical activity. Oncology 82: De Gramont, A., Figer, A., Seymour, M., Homerin, 290–297. M., Hmissi, A., Cassidy, J. et al. (2000) Leucovorin and fluorouracil with or without oxaliplatin as first- Chu, E., Koeller, D., Johnston, P., Zinn, S. and line treatment in advanced colorectal cancer. J Clin Allegra, C. (1993) Regulation of thymidylate Oncol 18: 2938–2947. synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. Mol Pharmacol De Palma, M., Venneri, M., Galli, R., Sergi, 43: 527–533. L., Politi, L., Sampaolesi, M. et al. (2005) Tie2 identifies a hematopoietic lineage of proangiogenic Ciardiello, F., Bianco, R., Damiano, V., Fontanini, monocytes required for tumor vessel formation and G., Caputo, R., Pomatico, G. et al. (2000) a mesenchymal population of pericyte progenitors. Antiangiogenic and antitumor activity of anti- Cancer Cell 8: 211–226. epidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial De Roock, W., Claes, B., Bernasconi, D., De growth factor antisense oligonucleotide in human Schutter, J., Biesmans, B., Fountzilas, G. et al. GEO colon cancer cells. Clin Cancer Res 6: (2010) Effects of KRAS, BRAF, NRAS, and 3739–3747. PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic Ciardiello, F. and Normanno, N. (2011) HER2 colorectal cancer: a retrospective consortium analysis. signaling and resistance to the anti-EGFR monoclonal Lancet Oncol 11: 753–762. antibody cetuximab: a further step toward personalized medicine for patients with colorectal De Stefano, A. and Carlomagno, C. (2014) Beyond cancer. Cancer Discov 1: 472–474. KRAS: Predictive factors of the efficacy of http://tam.sagepub.com 77 Therapeutic Advances in Medical Oncology 8(1) anti-EGFR monoclonal antibodies in the treatment of decreased ubiquitination is mediated by protein kinase metastatic colorectal cancer. World J Gastroenterol 20: B/Akt-dependent phosphorylation. J Biol Chem 279: 9732–9743. 35510–35517. Di Nicolantonio, F., Martini, M., Molinari, F., Fink, D., Aebi, S. and Howell, S. (1998) The role of Sartore-Bianchi, A., Arena, S., Saletti, P., De Dosso, DNA mismatch repair in drug resistance. Clin Cancer S. et. al. (2008) Wild-type BRAF is required for Res 4: 1–6. response to panitumumab or cetuximab in metastatic Fink, D., Zheng, H., Nebel, S., Norris, P., Aebi, S., colorectal cancer. J Clin Oncol 26: 5705–5712. Lin, T. et al. (1997) In vitro and in vivo resistance to Diaz-Rubio, E., Sastre, J., Zaniboni, A., Labianca, cisplatin in cells that have lost DNA mismatch repair. R., Cortes-Funes, H., De Braud, F. et al. (1998) Cancer research 57: 1841–1845. Oxaliplatin as single agent in previously untreated Frattini, M., Saletti, P., Romagnani, E., Martin, V., colorectal carcinoma patients: a phase II multicentric Molinari, F., Ghisletta, M. et al. (2007) PTEN loss of study. Ann Oncol 9: 105–108. expression predicts cetuximab efficacy in metastatic Douillard, J., Cunningham, D., Roth, A., Navarro, colorectal cancer patients. Br J Cancer 97: 1139–1145. M., James, R., Karasek, P. et al. (2000) Irinotecan Fujiwara, Y. and Minami, H. (2010) An overview of combined with fluorouracil compared with the recent progress in irinotecan pharmacogenetics. fluorouracil alone as first-line treatment for metastatic Pharmacogenomics 11: 391–406. colorectal cancer: a multicentre randomised trial. Lancet 355: 1041–1047. Giacchetti, S., Perpoint, B., Zidani, R., Le Bail, N., Faggiuolo, R., Focan, C. et al. (2000) Phase III Douillard, J., Oliner, K., Siena, S., Tabernero, J., multicenter randomized trial of oxaliplatin added to Burkes, R., Barugel, M. et al. (2013) Panitumumab- chronomodulated fluorouracil-leucovorin as first-line FOLFOX4 treatment and RAS mutations in treatment of metastatic colorectal cancer. J Clin Oncol colorectal cancer. N Engl J Med 369: 1023–1034. 18: 136–147. Douillard, J., Siena, S., Cassidy, J., Tabernero, J., Burkes, R., Barugel, M. et al. (2010) Randomized, Gongora, C., Vezzio-Vie, N., Tuduri, S., Denis, phase III trial of panitumumab with infusional V., Causse, A., Auzanneau, C. et al. (2011) New fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) Topoisomerase I mutations are associated with versus FOLFOX4 alone as first-line treatment resistance to camptothecin. Molecular Cancer 10: 64. in patients with previously untreated metastatic Gottesman, M., Fojo, T. and Bates, S. (2002) colorectal cancer: the PRIME study. J Clin Oncol 28: Multidrug resistance in cancer: role of ATP- 4697–4705. dependent transporters. Nat Rev Cancer 2: 48–58. Du, R., Lu, K., Petritsch, C., Liu, P., Ganss, R., Grothey, A., Van Cutsem, E., Sobrero, A., Siena, Passegue, E. et al. (2008) HIF1alpha induces the S., Falcone, A., Ychou, M. et al. (2013) Regorafenib recruitment of bone marrow-derived vascular monotherapy for previously treated metastatic modulatory cells to regulate tumor angiogenesis and colorectal cancer (CORRECT): an international, invasion. Cancer Cell 13: 206–220. multicentre, randomised, placebo-controlled, phase 3 Esposito, C., Rachiglio, A., La Porta, M., Sacco, A., trial. Lancet 381: 303–312. Roma, C., Iannaccone, A. et al. (2013) The S492R Hattori, K., Heissig, B., Wu, Y., Dias, S., Tejada, EGFR ectodomain mutation is never detected in R., Ferris, B. et al. (2002) Placental growth factor KRAS wild-type colorectal carcinoma before exposure reconstitutes hematopoiesis by recruiting VEGFR1(+) to EGFR monoclonal antibodies. Cancer Biol Ther 14: stem cells from bone-marrow microenvironment. Nat 1143–1146. Med 8: 841–849. Etienne-Grimaldi, M., Milano, G., Maindrault- Hector, S., Bolanowska-Higdon, W., Zdanowicz, Goebel, F., Chibaudel, B., Formento, J., Francoual, J., Hitt, S. and Pendyala, L. (2001) In vitro studies M. et al. (2010) Methylenetetrahydrofolate reductase on the mechanisms of oxaliplatin resistance. Cancer (MTHFR) gene polymorphisms and FOLFOX Chemother Pharmacol 48: 398–406. response in colorectal cancer patients. Br J Clin Pharmacol 69: 58–66. Heidelberger, C., Chaudhuri, N., Danneberg, P., Mooren, D., Griesbach, L., Duschinsky, R. et al. Fedier, A., Steiner, R., Schwarz, V., Lenherr, L., (1957) Fluorinated pyrimidines, a new class of Haller, U. and Fink, D. (2003) The effect of loss of tumour-inhibitory compounds. Nature 179: 663–666. Brca1 on the sensitivity to anticancer agents in p53- deficient cells. Int J Oncol 22: 1169–1173. Hirschi, B., Gallmeier, E., Ziesch, A., Marschall, Feng, J., Tamaskovic, R., Yang, Z., Brazil, D., Merlo, M. and Kolligs, F. (2014) Genetic targeting of A., Hess, D. et al. (2004) Stabilization of Mdm2 via B-RafV600E affects survival and proliferation and 78 http://tam.sagepub.com WA Hammond, A Swaika et al. identifies selective agents against BRAF-mutant cells to the epidermal growth factor receptor inhibitor colorectal cancer cells. Mol Cancer 13: 122. cetuximab. Cancer Res 68: 1953–1961. Johnston, P., Lenz, H., Leichman, C., Danenberg, K., Hobor, S., Van Emburgh, B., Crowley, E., Misale, S., Allegra, C., Danenberg, P. et al. (1995) Thymidylate Di Nicolantonio, F. and Bardelli, A. (2014) TGF- synthase gene and protein expression correlate and are alpha and amphiregulin paracrine network promotes associated with response to 5-fluorouracil in human resistance to EGFR blockade in colorectal cancer colorectal and gastric tumors. Cancer Res 55: 1407–1412. cells. Clin Cancer Res 20: 6429–6438. Jonker, D., O’Callaghan, C., Karapetis, C., Zalcberg, Hoff, P., Ansari, R., Batist, G., Cox, J., Kocha, W., J., Tu, D., Au, H. et al. (2007) Cetuximab for the Kuperminc, M. et al. (2001) Comparison of oral treatment of colorectal cancer. N Engl J Med 357: capecitabine versus intravenous fluorouracil plus 2040–2048. leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized Jurgensmeier, J., Schmoll, H., Robertson, J., Brooks, phase III study. J Clin Oncol 19: 2282–2292. L., Taboada, M., Morgan, S. et al. (2013) Prognostic and predictive value of VEGF, SVEGFR-2 and CEA Hohla, F., Winder, T., Greil, R., Rick, F., Block, N. in mCRC studies comparing cediranib, bevacizumab and Schally, A. (2014) Targeted therapy in advanced and chemotherapy. Br J Cancer 108: 1316–1323. metastatic colorectal cancer: current concepts and perspectives. World J Gastroenterol 20: 6102–6112. Kamba, T. and McDonald, D. (2007) Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Holzer, A., Manorek, G. and Howell, S. (2006) Cancer 96: 1788–1795. Contribution of the major copper influx transporter CTR1 to the cellular accumulation of cisplatin, Karapetis, C., Khambata-Ford, S., Jonker, D., carboplatin, and oxaliplatin. Mol Pharmacol 70: O’Callaghan, C., Tu, D., Tebbutt, N. et al. (2008) 1390–1394. K-Ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 359: Hong, Y., Park, Y., Lim, H., Lee, J., Kim, T., Kim, 1757–1765. K. et al. (2012) S-1 plus oxaliplatin versus capecitabine plus oxaliplatin for first-line treatment of patients with Kelland, L. (1993) New platinum antitumor metastatic colorectal cancer: a randomised, non- complexes. Crit Rev Oncol Hematol 15: 191–219. inferiority phase 3 trial. Lancet Oncol 13: 1125–1132. Kim, K., Li, B., Winer, J., Armanini, M., Gillett, Houghton, J. and Houghton, P. (1983) Elucidation of N., Phillips, H. et al. (1993) Inhibition of vascular pathways of 5-fluorouracil metabolism in xenografts of endothelial growth factor-induced angiogenesis human colorectal adenocarcinoma. Eur J Cancer Clin suppresses tumour growth in vivo. Nature 362: Oncol 19: 807–815. 841–844. Hudis, C. (2007) Trastuzumab - mechanism of action Kim, S., Hong, S., Shim, K., Kong, S., Shin, and use in clinical practice. N Engl J Med 357: 39–51. A., Baek, J. et al. (2013) S-1 plus irinotecan and oxaliplatin for the first-line treatment of patients with Hurwitz, H., Fehrenbacher, L., Novotny, W., metastatic colorectal cancer: a prospective phase II Cartwright, T., Hainsworth, J., Heim, W. et al. study and pharmacogenetic analysis. Br J Cancer 109: (2004) Bevacizumab plus irinotecan, fluorouracil, and 1420–1427. leucovorin for metastatic colorectal cancer. N Engl J Med 350: 2335–2342. Kojima, A., Hackett, N. and Crystal, R. (1998) Reversal of CPT-11 resistance of lung cancer cells Ishikawa, T. and Ali-Osman, F. (1993) Glutathione- by adenovirus-mediated gene transfer of the human associated cis-diamminedichloroplatinum(II) carboxylesterase cDNA. Cancer Res 58: 4368–4374. metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione- Koopman, M., Venderbosch, S., Van Tinteren, H., platinum complex and its biological significance. Ligtenberg, M., Nagtegaal, I., Van Krieken, J. et al. J Biol Chem 268: 20116–20125. (2009) Predictive and prognostic markers for the outcome of chemotherapy in advanced colorectal Isshi, K., Sakuyama, T., Gen, T., Nakamura, Y., cancer, a retrospective analysis of the phase III Kuroda, T., Katuyama, T. et al. (2002) Predicting randomised CAIRO study. Eur J Cancer 45: 1999– 5-FU sensitivity using human colorectal cancer specimens: comparison of tumor dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase Kopetz, S., Hoff, P., Morris, J., Wolff, R., Eng, C., activities with in vitro chemosensitivity to 5-FU. Int J Glover, K. et al. (2010) Phase II trial of infusional Clin Oncol 7: 335–342. fluorouracil, irinotecan, and bevacizumab for Jhawer, M., Goel, S., Wilson, A., Montagna, C., Ling, metastatic colorectal cancer: efficacy and circulating Y., Byun, D. et al. (2008) PIK3CA mutation/PTEN angiogenic biomarkers associated with therapeutic expression status predicts response of colon cancer resistance. J Clin Oncol 28: 453–459. http://tam.sagepub.com 79 Therapeutic Advances in Medical Oncology 8(1) Leichman, C., Fleming, T., Muggia, F., Tangen, Marcuello, E., Altes, A., Menoyo, A., Del Rio, E., C., Ardalan, B., Doroshow, J. et al. (1995) Phase II Gomez-Pardo, M. and Baiget, M. 2004. UGT1A1 gene study of fluorouracil and its modulation in advanced variations and irinotecan treatment in patients with colorectal cancer: a Southwest Oncology Group study. metastatic colorectal cancer. Br J Cancer 91: 678–682. J Clin Oncol 13: 1303–1311. Marcuello, E., Altes, A., Menoyo, A., Rio, E. Leichman, C., Lenz, H., Leichman, L., Danenberg, and Baiget, M. (2006) Methylenetetrahydrofolate K., Baranda, J., Groshen, S. et al. (1997) Quantitation reductase gene polymorphisms: genomic predictors of intratumoral thymidylate synthase expression of clinical response to fluoropyrimidine-based predicts for disseminated colorectal cancer response chemotherapy? Cancer Chemother Pharmacol 57: and resistance to protracted-infusion fluorouracil and 835–840. weekly leucovorin. J Clin Oncol 15: 3223–3229. Maxwell, P., Dachs, G., Gleadle, J., Nicholls, L., Li, D., Xie, K., Ding, G., Li, J., Chen, K., Li, H. Harris, A., Stratford, I. et al. (1997) Hypoxia-inducible et al. (2014) Tumor resistance to anti-VEGF therapy factor-1 modulates gene expression in solid tumors through up-regulation of VEGF-C expression. Cancer and influences both angiogenesis and tumor growth. Lett 346: 45–52. Proc Natl Acad Sci U S A 94: 8104–8109. Liang, J., Jiang, T., Yao, R., Liu, Z., Lv, H. and Qi, Mayer, A., Takimoto, M., Fritz, E., Schellander, G., W. (2010) The combination of ERCC1 and XRCC1 Kofler, K. and Ludwig, H. (1993) The prognostic gene polymorphisms better predicts clinical outcome significance of proliferating cell nuclear antigen, to oxaliplatin-based chemotherapy in metastatic epidermal growth factor receptor, and MDR gene colorectal cancer. Cancer Chemother Pharmacol 66: expression in colorectal cancer. Cancer 71: 2454–2460. 493–500. Mayer, R., Van Cutsem, E., Falcone, A., Yoshino, T., Lieu, C., Tran, H., Jiang, Z., Mao, M., Overman, Garcia-Carbonero, R., Mizunuma, N. et al. (2015) M., Lin, E. et al. (2013) The association of alternate Randomized trial of TAS-102 for refractory metastatic VEGF ligands with resistance to anti-VEGF therapy colorectal cancer. N Engl J Med 372: 1909–1919. in metastatic colorectal cancer. PloS One 8: e77117. McLeod, H. and Keith, W. (1996) Variation in Lievre, A., Bachet, J., Le Corre, D., Boige, V., Landi, topoisomerase I gene copy number as a mechanism B., Emile, J. et al. (2006) KRAS mutation status for intrinsic drug sensitivity. Br J Cancer 74: 508–512. is predictive of response to cetuximab therapy in Meijer, C., Mulder, N., Timmer-Bosscha, H., colorectal cancer. Cancer Res 66: 3992–3995. Sluiter, W., Meersma, G. and De Vries, E. (1992) Lindskog, E., Derwinger, K., Gustavsson, B., Falk, P. Relationship of cellular glutathione to the cytotoxicity and Wettergren, Y. (2014) Thymidine phosphorylase and resistance of seven platinum compounds. Cancer expression is associated with time to progression in Res 52: 6885–6889. patients with metastatic colorectal cancer. BMC Clin Meriggi, F., Vermi, W., Bertocchi, P. and Zaniboni, Pathol 14: 25. A. (2014) The emerging role of NRAS mutations Longley, D., Boyer, J., Allen, W., Latif, T., Ferguson, in colorectal cancer patients selected for anti-EGFR P., Maxwell, P. et al. (2002) The role of thymidylate therapies. Rev Recent Clin Trials 9: 8–12. synthase induction in modulating p53-regulated Meropol, N., Gold, P., Diasio, R., Andria, M., gene expression in response to 5-fluorouracil and Dhami, M., Godfrey, T. et al. (2006) Thymidine antifolates. Cancer Res 62: 2644–2649. phosphorylase expression is associated with response to Longley, D., Harkin, D. and Johnston, P. (2003) capecitabine plus irinotecan in patients with metastatic 5-fluorouracil: mechanisms of action and clinical colorectal cancer. J Clin Oncol 24: 4069–4077. strategies. Nat Rev Cancer 3: 330–338. Mesange, P., Poindessous, V., Sabbah, M., Longley, D. and Johnston, P. (2005) Molecular Escargueil, A., De Gramont, A. and Larsen, A. (2014) mechanisms of drug resistance. J Pathol 205: Intrinsic bevacizumab resistance is associated with 275–292. prolonged activation of autocrine VEGF signaling and hypoxia tolerance in colorectal cancer cells and can be Lowenstein, E., Daly, R., Batzer, A., Li, W., overcome by nintedanib, a small molecule angiokinase Margolis, B., Lammers, R. et al. (1992) The SH2 inhibitor. Oncotarget 5: 4709–4721. and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to Ras signaling. Cell 70: Messa, C., Russo, F., Caruso, M. and Di Leo, A. 431–442. (1998) EGF, TGF-alpha, and EGF-R in human colorectal adenocarcinoma. Acta Oncol 37: 285–289. Mancuso, M., Davis, R., Norberg, S., O’Brien, S., Sennino, B., Nakahara, T. et al. (2006) Rapid vascular Meyers, M., Wagner, M., Hwang, H., Kinsella, T. regrowth in tumors after reversal of VEGF inhibition. and Boothman, D. (2001) Role of the hMLH1 DNA J Clin Invest 116: 2610–2621. mismatch repair protein in fluoropyrimidine-mediated 80 http://tam.sagepub.com WA Hammond, A Swaika et al. cell death and cell cycle responses. Cancer Res 61: Paez, J., Janne, P., Lee, J., Tracy, S., Greulich, H., 5193–5201. Gabriel, S. et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib Misale, S., Di Nicolantonio, F., Sartore-Bianchi, therapy. Science 304: 1497–1500. A., Siena, S. and Bardelli, A. (2014) Resistance to anti-EGFR therapy in colorectal cancer: from Pan, Q., Chanthery, Y., Liang, W., Stawicki, S., Mak, heterogeneity to convergent evolution. Cancer Discov J., Rathore, N. et al. (2007) Blocking neuropilin-1 4: 1269–1280. function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer Cell 11: 53–67. Misale, S., Yaeger, R., Hobor, S., Scala, E., Janakiraman, M., Liska, D. et al. (2012) Emergence of Panczyk, M. (2014) Pharmacogenetics research on KRAS mutations and acquired resistance to anti-EGFR chemotherapy resistance in colorectal cancer over the therapy in colorectal cancer. Nature 486: 532–536. last 20 years. World J Gastroenterol 20: 9775–9827. Perrone, F., Lampis, A., Orsenigo, M., Di Mitchell, E. (2013) Targeted therapy for metastatic Bartolomeo, M., Gevorgyan, A., Losa, M. et al. colorectal cancer: role of aflibercept. Clin Colorectal (2009) PI3KCA/PTEN deregulation contributes Cancer 12: 73–85. to impaired responses to cetuximab in metastatic Mizukami, Y., Jo, W., Duerr, E., Gala, M., Li, J., colorectal cancer patients. Ann Oncol 20: 84–90. Zhang, X. et al. (2005) Induction of interleukin-8 Petrioli, R., Bargagli, G., Lazzi, S., Pascucci, A., preserves the angiogenic response in HIF-1alpha- Francini, E., Bellan, C. et al. (2010) Thymidine deficient colon cancer cells. Nature Med 11: 992–997. phosphorylase expression in metastatic sites is Moertel, C., Fleming, T., Macdonald, J., Haller, D., predictive for response in patients with colorectal Laurie, J., Goodman, P. et al. (1990) Levamisole and cancer treated with continuous oral capecitabine and fluorouracil for adjuvant therapy of resected colon biweekly oxaliplatin. Anticancer Drugs 21: 313–319. carcinoma. N Engl J Med 322: 352–358. Pollard, J. (2004) Tumour-educated macrophages Moroni, M., Veronese, S., Benvenuti, S., Marrapese, promote tumour progression and metastasis. Nat Rev G., Sartore-Bianchi, A., Di Nicolantonio, F. et al. Cancer 4: 71–78. (2005) Gene copy number for epidermal growth Popat, S., Matakidou, A. and Houlston, R. (2004) factor receptor (EGFR) and clinical response to Thymidylate synthase expression and prognosis in antiEGFR treatment in colorectal cancer: a cohort colorectal cancer: a systematic review and meta- study. Lancet Oncol 6: 279–286. analysis. J Clin Oncol 22: 529–536. Moutinho, C., Martinez-Cardus, A., Santos, C., Porebska, I., Harlozinska, A. and Bojarowski, T. Navarro-Perez, V., Martinez-Balibrea, E., Musulen, (2000) Expression of the tyrosine kinase activity E. et al. (2014) Epigenetic inactivation of the BRCA1 growth factor receptors (EGFR, ERB B2, ERB B3) in interactor SRBC and resistance to oxaliplatin in colorectal adenocarcinomas and adenomas. Tumour colorectal cancer. J Natl Cancer Inst 106: djt322. Biol 21: 105–115. Muhale, F., Wetmore, B., Thomas, R. and McLeod, Price, T., Newhall, K., Peeters, M., Kim, T., Li, J., H. (2011) Systems pharmacology assessment of Cascinu, S. et al. (2015) Prevalence and outcomes the 5-fluorouracil pathway. Pharmacogenomics 12: of patients (pts) with EGFR S492R ectodomain 341–350. mutations in ASPECCT: Panitumumab (PMAB) Muro, K., Boku, N., Shimada, Y., Tsuji, A., vs. cetuximab (CMAB) in pts with chemorefractory Sameshima, S., Baba, H. et al. (2010) Irinotecan plus wild-type KRAS exon 2 metastatic colorectal cancer S-1 (IRIS) versus fluorouracil and folinic acid plus (mCRC). J Clin Oncol (Suppl. 3): abstr 740. irinotecan (FOLFIRI) as second-line chemotherapy Price, T., Peeters, M., Kim, T., Li, J., Cascinu, S., for metastatic colorectal cancer: a randomised phase Ruff, P. et al. (2014) Panitumumab versus cetuximab 2/3 non-inferiority study (FIRIS study). Lancet Oncol in patients with chemotherapy-refractory wild- 11: 853–860. type KRAS exon 2 metastatic colorectal cancer (ASPECCT): a randomised, multicentre, open- Nicum, S., Midgley, R. and Kerr, D. (2000) label, non-inferiority phase 3 study. Lancet Oncol 15: Chemotherapy for colorectal cancer. J R Soc Med 93: 569–579. 416–419. Qiu, L., Tang, Q., Bai, J., Qian, X., Li, R., Liu, B. Nygard, S., Christensen, I., Nielsen, S., Nielsen, H., et al. (2008) Predictive value of thymidylate synthase Brunner, N. and Spindler, K. (2014) Assessment of expression in advanced colorectal cancer patients the topoisomerase I gene copy number as a predictive receiving fluoropyrimidine-based chemotherapy: biomarker of objective response to irinotecan in evidence from 24 studies. Int J Cancer 123: 2384– metastatic colorectal cancer. Scand J Gastroenterol 49: 84–91. http://tam.sagepub.com 81 Therapeutic Advances in Medical Oncology 8(1) Rajput, A., Koterba, A., Kreisberg, J., Foster, in combination with oxaliplatin-based chemotherapy J., Willson, J. and Brattain, M. (2007) A novel as first-line therapy in metastatic colorectal cancer: a mechanism of resistance to epidermal growth factor randomized phase III study. J Clin Oncol 26: 2013–2019. receptor antagonism in vivo. Cancer Res 67: 665–673. Saltz, L., Cox, J., Blanke, C., Rosen, L., Raymond, E., Faivre, S., Chaney, S., Woynarowski, Fehrenbacher, L., Moore, M. et al. (2000a) Irinotecan J. and Cvitkovic, E. (2002) Cellular and molecular plus fluorouracil and leucovorin for metastatic pharmacology of oxaliplatin. Mol Cancer Ther 1: colorectal cancer. Irinotecan Study Group. N Engl J 227–235. Med 343: 905–914. Ribatti, D. (2008) The discovery of the placental Saltz, L., Cox, J., Blanke, C., Rosen, L., growth factor and its role in angiogenesis: a historical Fehrenbacher, L., Moore, M. et al. (2000b) Irinotecan review. Angiogenesis 11: 215–221. plus fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med 343: 905–914. Ridgway, J., Zhang, G., Wu, Y., Stawicki, S., Liang, W., Chanthery, Y. et al. (2006) Inhibition of Dll4 Samuel, S., Fan, F., Dang, L., Xia, L., Gaur, P. and signalling inhibits tumour growth by deregulating Ellis, L. (2011) Intracrine vascular endothelial growth angiogenesis. Nature 444: 1083–1087. factor signaling in survival and chemoresistance of human colorectal cancer cells. Oncogene 30: 1205–1212. Roskoski, R., Jr. (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res 66: Saridaki, Z., Tzardi, M., Papadaki, C., Sfakianaki, 105–143. M., Pega, F., Kalikaki, A. et al. (2011) Impact of KRAS, BRAF, PIK3CA mutations, PTEN, AREG, Roskoski, R., Jr. (2014) The ErbB/HER family of EREG expression and skin rash in >/= 2 line protein-tyrosine kinases and cancer. Pharmacol Res 79: cetuximab-based therapy of colorectal cancer patients. 34–74. PloS One 6: e15980. Rougier, P., Van Cutsem, E., Bajetta, E., Niederle, Sartore-Bianchi, A., Di Nicolantonio, F., Nichelatti, N., Possinger, K., Labianca, R. et al. (1998) M., Molinari, F., De Dosso, S., Saletti, P. et al. Randomised trial of irinotecan versus fluorouracil (2009) Multi-determinants analysis of molecular by continuous infusion after fluorouracil failure in alterations for predicting clinical benefit to EGFR- patients with metastatic colorectal cancer. Lancet 352: targeted monoclonal antibodies in colorectal cancer. 1407–1412. PloS One 4: e7287. Rouits, E., Boisdron-Celle, M., Dumont, A., Guerin, Sartore-Bianchi, A., Moroni, M., Veronese, S., O., Morel, A. and Gamelin, E. (2004) Relevance Carnaghi, C., Bajetta, E., Luppi, G. et al. (2007) of different UGT1A1 polymorphisms in irinotecan- Epidermal growth factor receptor gene copy number induced toxicity: a molecular and clinical study of 75 and clinical outcome of metastatic colorectal cancer patients. Clin Cancer Res 10: 5151–5159. treated with panitumumab. J Clin Oncol 25: 3238–3245. Rubenstein, J., Kim, J., Ozawa, T., Zhang, M., Schwartz, P., Moir, R., Hyde, C., Turek, P. and Westphal, M., Deen, D. et al. (2000) Anti-VEGF Handschumacher, R. (1985) Role of uridine antibody treatment of glioblastoma prolongs survival phosphorylase in the anabolism of 5-fluorouracil. but results in increased vascular cooption. Neoplasia 2: Biochem Pharmacol 34: 3585–3589. 306–314. Shimada, Y., Rougier, P. and Pitot, H. (1996) Sakuramoto, S., Sasako, M., Yamaguchi, T., Efficacy of CPT-11 (irinotecan) as a single agent in Kinoshita, T., Fujii, M., Nashimoto, A. et al. (2007) metastatic colorectal cancer. Eur J Cancer 32A(Suppl. Adjuvant chemotherapy for gastric cancer with S-1, an 3): S13–S17. oral fluoropyrimidine. N Engl J Med 357: 1810–1820. Shirao, K., Hoff, P., Ohtsu, A., Loehrer, P., Hyodo, Salomon, D., Brandt, R., Ciardiello, F. and I., Wadler, S. et al. (2004) Comparison of the efficacy, Normanno, N. (1995) Epidermal growth factor- toxicity, and pharmacokinetics of a uracil/tegafur related peptides and their receptors in human (UFT) plus oral leucovorin (LV) regimen between malignancies. Crit Rev Oncol Hematol 19: 183–232. Japanese and American patients with advanced colorectal cancer: joint United States and Japan study Salonga, D., Danenberg, K., Johnson, M., Metzger, of UFT/LV. J Clin Oncol 22: 3466–3474. R., Groshen, S., Tsao-Wei, D. et al. (2000) Colorectal tumors responding to 5-fluorouracil have low gene Shirota, Y., Stoehlmacher, J., Brabender, J., expression levels of dihydropyrimidine dehydrogenase, Xiong, Y., Uetake, H., Danenberg, K. et al. (2001) thymidylate synthase, and thymidine phosphorylase. ERCC1 and thymidylate synthase mRNA levels Clin Cancer Res 6: 1322–1327. predict survival for colorectal cancer patients Saltz, L., Clarke, S., Diaz-Rubio, E., Scheithauer, receiving combination oxaliplatin and fluorouracil W., Figer, A., Wong, R. et al. (2008) Bevacizumab chemotherapy. J Clin Oncol 19: 4298–4304. 82 http://tam.sagepub.com WA Hammond, A Swaika et al. Shojaei, F., Wu, X., Malik, A., Zhong, C., Baldwin, Ramucirumab versus placebo in combination with M., Schanz, S. et al. (2007) Tumor refractoriness to second-line FOLFIRI in patients with metastatic anti-VEGF treatment is mediated by CD11b+Gr1+ colorectal carcinoma that progressed during or after myeloid cells. Nat Biotechnol 25: 911–920. first-line therapy with bevacizumab, oxaliplatin, and a fluoropyrimidine (RAISE): a randomised, double- Siegel, R., Desantis, C. and Jemal, A. (2014a) blind, multicentre, phase 3 study. Lancet Oncol 16: Colorectal cancer statistics, 2014. CA Cancer J Clin 499–508. 64: 104–117. Takebe, N., Zhao, S., Ural, A., Johnson, M., Siegel, R., Ma, J., Zou, Z. and Jemal, A. (2014b) Banerjee, D., Diasio, R. et al. (2001) Retroviral Cancer statistics, 2014. CA Cancer J Clin 64: 9–29. transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to Smith, D., Christensen, I., Jensen, N., Markussen, B., 5-fluorouracil in murine hematopoietic progenitor Romer, M., Nygard, S. et al. (2013) Mechanisms of cells and human CD34+-enriched peripheral blood topoisomerase I (TOP1) gene copy number increase progenitor cells. Cancer Gene Ther 8: 966–973. in a stage III colorectal cancer patient cohort. PloS One 8: e60613. Tejpar, S., Prenen, H. and Mazzone, M. (2012) Overcoming resistance to antiangiogenic therapies. Sohn, K., Croxford, R., Yates, Z., Lucock, M. and Oncologist 17: 1039–1050. Kim, Y. (2004) Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity Therkildsen, C., Bergmann, T., Henrichsen-Schnack, of colon and breast cancer cells to 5-fluorouracil and T., Ladelund, S. and Nilbert, M. (2014) The methotrexate. J Natl Cancer Inst 96: 134–144. predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic Sood, A., Mcclain, D., Maitra, R., Basu-Mallick, A., colorectal cancer: A systematic review and meta- Seetharam, R., Kaubisch, A. et al. (2012) PTEN gene analysis. Acta Oncol 53: 852–864. expression and mutations in the PIK3CA gene as predictors of clinical benefit to anti-epidermal growth Thomas, H. and Coley, H. (2003) Overcoming factor receptor antibody therapy in patients with multidrug resistance in cancer: an update on the KRAS wild-type metastatic colorectal cancer. Clin clinical strategy of inhibiting p-glycoprotein. Cancer Colorectal Cancer 11: 143–150. Control 10: 159–165. Soong, R., Shah, N., Salto-Tellez, M., Tai, B., Soo, Tokunaga, Y., Sasaki, H. and Saito, T. (2007) R., Han, H. et al. (2008) Prognostic significance Clinical role of orotate phosphoribosyl transferase and of thymidylate synthase, dihydropyrimidine dihydropyrimidine dehydrogenase in colorectal cancer dehydrogenase and thymidine phosphorylase protein treated with postoperative fluoropyrimidine. Surgery expression in colorectal cancer patients treated with 141: 346–353. or without 5-fluorouracil-based chemotherapy. Ann Tournigand, C., Andre, T., Achille, E., Lledo, G., Oncol 19: 915–919. Flesh, M., Mery-Mignard, D. et al. (2004) FOLFIRI Spano, J., Fagard, R., Soria, J., Rixe, O., Khayat, followed by FOLFOX6 or the reverse sequence in D. and Milano, G. (2005) Epidermal growth factor advanced colorectal cancer: a randomized GERCOR receptor signaling in colorectal cancer: preclinical data study. J Clin Oncol 22: 229–237. and therapeutic perspectives. Ann Oncol 16: 189–194. Tsunoda, A., Nakao, K., Watanabe, M., Matsui, N., Stark, M., Bram, E., Akerman, M., Mandel- Ooyama, A. and Kusano, M. (2011) Associations of Gutfreund, Y. and Assaraf, Y. (2011) Heterogeneous various gene polymorphisms with toxicity in colorectal nuclear ribonucleoprotein H1/H2-dependent unsplicing cancer patients receiving oral uracil and tegafur of thymidine phosphorylase results in anticancer drug plus leucovorin: a prospective study. Ann Oncol 22: resistance. J Biol Chem 286: 3741–3754. 355–361. Tabernero, J. (2007) The role of VEGF and EGFR Tsurutani, J., Nitta, T., Hirashima, T., Komiya, T., inhibition: implications for combining anti-VEGF and Uejima, H., Tada, H. et al. (2002) Point mutations in anti-EGFR agents. Mol Cancer Res 5: 203–220. the topoisomerase I gene in patients with non-small cell lung cancer treated with irinotecan. Lung Cancer Tabernero, J., Van Cutsem, E., Lakomy, R., 35: 299–304. Prausova, J., Ruff, P., Van Hazel, G. et al. (2014) Aflibercept versus placebo in combination with Vallbohmer, D., Kuramochi, H., Shimizu, D., fluorouracil, leucovorin and irinotecan in the Danenberg, K., Lindebjerg, J., Nielsen, J. et al. (2006) treatment of previously treated metastatic colorectal Molecular factors of 5-fluorouracil metabolism in cancer: prespecified subgroup analyses from the colorectal cancer: analysis of primary tumor and VELOUR trial. Eur J Cancer 50: 320–331. lymph node metastasis. Int J Oncol 28: 527–533. Tabernero, J., Yoshino, T., Cohn, A., Obermannova, Vallbohmer, D., Yang, D., Kuramochi, H., Shimizu, R., Bodoky, G., Garcia-Carbonero, R. et al. (2015) D., Danenberg, K., Lindebjerg, J. et al. (2007) DPD http://tam.sagepub.com 83 Therapeutic Advances in Medical Oncology 8(1) is a molecular determinant of capecitabine efficacy in Xie, H., Wood, A., Kim, R., Stein, C. and Wilkinson, colorectal cancer. Int J Oncol 31: 413–418. G. (2004) Genetic variability in CYP3A5 and its possible consequences. Pharmacogenomics 5: 243–272. Vallbohmer, D., Zhang, W., Gordon, M., Yang, D., Yun, J., Press, O. et al. (2005) Molecular Xu, Y. and Villalona-Calero, M. (2002) Irinotecan: determinants of cetuximab efficacy. J Clin Oncol 23: mechanisms of tumor resistance and novel strategies 3536–3544. for modulating its activity. Ann Oncol 13: 1841–1851. Van Cutsem, E., Labianca, R., Bodoky, G., Yamada, K. and Araki, M. (2001) Tumor suppressor Barone, C., Aranda, E., Nordlinger, B. et al. PTEN: modulator of cell signaling, growth, migration (2009) Randomized phase III trial comparing and apoptosis. J Cell Sci 114: 2375–2382. biweekly infusional fluorouracil/leucovorin alone Yanagisawa, Y., Maruta, F., Iinuma, N., Ishizone, or with irinotecan in the adjuvant treatment of stage S., Koide, N., Nakayama, J. et al. (2007) Modified III colon cancer: PETACC-3. J Clin Oncol Irinotecan/5FU/Leucovorin therapy in advanced 27: 3117–3125. colorectal cancer and predicting therapeutic efficacy Van Cutsem, E., Peeters, M., Siena, S., Humblet, Y., by expression of tumor-related enzymes. Scand J Hendlisz, A., Neyns, B. et al. (2007) Open-label phase Gastroenterol 42: 477–484. III trial of panitumumab plus best supportive care Yang, L., Debusk, L., Fukuda, K., Fingleton, B., compared with best supportive care alone in patients Green-Jarvis, B., Shyr, Y. et al. (2004) Expansion with chemotherapy-refractory metastatic colorectal of myeloid immune suppressor Gr+CD11b+ cells cancer. J Clin Oncol 25: 1658–1664. in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 6: 409–421. Van Cutsem, E., Tabernero, J., Lakomy, R., Prenen, H., Prausova, J., Macarulla, T. et al. (2012) Addition Ye, D. and Zhang, J. (2013) Research development of aflibercept to fluorouracil, leucovorin, and of the relationship between thymidine phosphorylase irinotecan improves survival in a phase III randomized expression and colorectal carcinoma. Cancer Biol Med trial in patients with metastatic colorectal cancer 10: 10–15. previously treated with an oxaliplatin-based regimen. Ye, F., Liu, Z., Tan, A., Liao, M., Mo, Z. and Yang, J Clin Oncol 30: 3499–3506. X. (2013) XRCC1 and GSTP1 polymorphisms Van Cutsem, E., Twelves, C., Cassidy, J., Allman, and prognosis of oxaliplatin-based chemotherapy in D., Bajetta, E., Boyer, M. et al. (2001) Oral colorectal cancer: a meta-analysis. Cancer Chemother capecitabine compared with intravenous fluorouracil Pharmacol 71: 733–740. plus leucovorin in patients with metastatic colorectal Yonesaka, K., Zejnullahu, K., Okamoto, I., Satoh, T., cancer: results of a large phase III study. J Clin Oncol Cappuzzo, F., Souglakos, J. et al. (2011) Activation 19: 4097–4106. of ERBB2 signaling causes resistance to the EGFR- Van Emburgh, B., Sartore-Bianchi, A., Di directed therapeutic antibody cetuximab. Sci Transl Nicolantonio, F., Siena, S. and Bardelli, A. (2014) Med 3: 99ra86. Acquired resistance to EGFR-targeted therapies in Yoshino, T., Mizunuma, N., Yamazaki, K., Nishina, colorectal cancer. Mol Oncol 8: 1084–1094. T., Komatsu, Y., Baba, H. et al. (2012) TAS-102 Wang, H., Bian, T., Liu, D., Jin, T., Chen, Y., Lin, monotherapy for pretreated metastatic colorectal A. et al. (2011) Association analysis of CYP2A6 cancer: a double-blind, randomised, placebo- genotypes and haplotypes with 5-fluorouracil controlled phase 2 trial. Lancet Oncol 13: 993–1001. formation from tegafur in human liver microsomes. Zhang, S., Lovejoy, K., Shima, J., Lagpacan, L., Shu, Pharmacogenomics 12: 481–492. Y., Lapuk, A. et al. (2006) Organic cation transporters Wheeler, J., Bodmer, W. and Mortensen, N. (2000) are determinants of oxaliplatin cytotoxicity. Cancer Res DNA mismatch repair genes and colorectal cancer. 66: 8847–8857. Gut 47: 148–153. Zhao, J., Li, W., Zhu, D., Yu, Q., Zhang, Z., Sun, M. et al. (2014) Association of single nucleotide Wilson, P., Danenberg, P., Johnston, P., Lenz, H. and Visit SAGE journals online polymorphisms in MTHFR and ABCG2 with Ladner, R. (2014) Standing the test of time: targeting http://tam.sagepub.com the different efficacy of first-line chemotherapy in thymidylate biosynthesis in cancer therapy. Nat Rev SAGE journals metastatic colorectal cancer. Med Oncol 31: 802. Clin Oncol 11: 282–298. 84 http://tam.sagepub.com

Journal

Therapeutic Advances in Medical OncologySAGE

Published: Dec 18, 2015

Keywords: colon adenocarcinoma; colon cancer; resistance

References