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A phase II study of temsirolimus and liposomal doxorubicin for patients with recurrent and refractory bone and soft tissue sarcomas

A phase II study of temsirolimus and liposomal doxorubicin for patients with recurrent and... Background: Relapsed and refractory sarcomas continue to have poor survival rates. The cancer stem cell (CSC) theory provides a tractable explanation for the observation that recurrences occur despite dramatic responses to upfront chemotherapy. Preclinical studies demonstrated that inhibition of the mechanistic target of rapamycin (mTOR) sensitizes the CSC population to chemotherapy. Methods: Here we present the results of the Phase II portion of a Phase I/II clinical trial that aimed to overcome the chemoresistance of sarcoma CSC by combining the mTOR inhibitor temsirolimus (20 mg/m weekly) with the chemo‑ therapeutic agent liposomal doxorubicin (30 mg/m monthly). Results: Fifteen patients with relapsed/refractory sarcoma were evaluable at this recommended Phase 2 dose level. The median progression free survival was 315 days (range 27–799). Response rate, defined as stable disease or better for 60 days, was 53%. Nine of the patients had been previously treated with doxorubicin. Therapy was well tolerated. In a small number of patients, pre‑ and post‑ treatment tumor biopsies were available for assessment of ALDH expres‑ sion as a marker of CSCs and showed a correlation between response and decreased ALDH expression. We also found a correlation between biopsy‑ proven inhibition of mTOR and response. Conclusions: Our study adds to the literature supporting the addition of mTOR inhibition to chemotherapy agents for the treatment of sarcomas, and proposes that a mechanism by which mTOR inhibition enhances the efficacy of chemotherapy may be through sensitizing the chemoresistant CSC population. Further study, ideally with pre‑ and post‑ therapy assessment of ALDH expression in tumor cells, is warranted. Trial registration The trial was registered on clinicaltrials.gov (NCT00949325) on 30 July 2009. http://www.edito rialm anage r.com/csrj/defau lt.aspx Keywords: mTOR, Cancer stem cell, Chemoresistance, Sarcoma, Aldehyde dehydrogenase *Correspondence: dloeb@montefiore.org Department of Pediatrics, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, 3411 Wayne Ave., Room 910, Bronx, NY 10467, USA Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 2 of 9 published the results of the dose finding portion of this Background study [6] and are reporting here the results of the Phase Therapeutic advances in the treatment of localized high- II portion of the clinical trial, including pharmacody- grade sarcomas have dramatically improved survival for namic data regarding mTOR inhibition and targeting of these patients over the past two decades, but patients high ALDH sarcoma cells. with recurrent or refractory disease continue to have a dismal prognosis. Although many high grade sarcomas Methods respond well to initial therapy, recurrence is common and Patient eligibility requirements is usually fatal. The cancer stem cell (CSC) hypothesis Eligible patients were at least 1  year of age, had a histo- provides an explanation for the discrepancy between ini- logically confirmed diagnosis of sarcoma that was either tial treatment response and overall survival. This model recurrent or refractory to conventional therapy, and had proposes that a small subpopulation of chemotherapy- measurable disease amenable to percutaneous image- resistant tumor cells with a “stem cell-like” phenotype is guided biopsy. Additionally, patients were required responsible for both regrowth of the tumor in its original to have an adequate performance status (ECOG ≤ 2; site and/or for metastatic dissemination [1]. Therapies Karnofsky or Lansky ≥ 60% for children), a life expec- that target cancer stem cells would therefore be expected tancy greater than 3  months, and adequate organ func- to decrease the risk of local and metastatic recurrence, tion (absolute neutrophil count ≥ 1500/µl, platelet dramatically improving the survival of patients with count ≥ 100,000/µl, total bilirubin ≤ 1.5× institutional high-risk disease. upper limit of normal, AST and ALT ≤ 2.5× institu- Cancer cells expressing high levels of aldehyde dehy- tional upper limit of normal, and creatinine ≤ 1.5× insti- drogenase (ALDH) have been shown to possess a phe- tutional upper limit of normal for age or a creatinine notype reminiscent of stem cells in numerous cancers clearance ≥ 60  ml/min/1.73  m ). Since temsirolimus can [2]. In particular, our laboratory has demonstrated that affect both lipid and glucose metabolism, patients were Ewing sarcoma cells expressing high levels of ALDH, as high required to have a fasting cholesterol ≤ 350  mg/dl, fast- determined by cell sorting using Aldefluor (ALDH ing serum triglycerides ≤ 400  mg/dl, amylase and lipase cells), exhibit a variety of stem cell properties, includ- within normal limits (unless elevations were related ing clonogenic growth in soft agar, the ability to grow to tumor involving the pancreas), and a hemoglobin as spheres under non-adherent conditions, and expres- A1c ≤ 10%. Patients were excluded if they had a history of sion of so-called “stem cell genes” such as OCT4 and high pulmonary hypertension or pneumonitis, prior therapy NANOG [3]. Most importantly, as few as 160 ALDH with an mTOR inhibitor, uncontrolled brain metasta- cells can generate a tumor in immune deficient mice, ses, a history of hypersensitivity to macrolide antibiot- as compared with 800,000 unsorted cells. Similar find - ics (because of the risk of crossreactions), or grade 3 or ings have been demonstrated in osteosarcoma and soft 4 proteinuria. The study was approved by the Johns Hop - tissue sarcomas [4, 5]. As predicted by the CSC model, high kins University Institutional Review Board and patients ALDH cells are resistant to doxorubicin and etoposide signed written informed consent according to institu- in  vitro when compared with unsorted cells. We subse- tional standards. The trial was registered with Clinical - quently demonstrated that inhibition of the mechanistic Trials.gov (Registration ID: NCT00949325). target of rapamycin (mTOR) by sirolimus increases the high sensitivity of ALDH cells to doxorubicin and causes Treatment plan synergistic cytotoxicity when unsorted cells are treated Temsirolimus and liposomal doxorubicin were admin- in  vitro [6]. Based on these findings, we conducted a istered intravenously in the outpatient clinic, and dos- Phase I/II study of the combination of liposomal doxoru- ing was based on body surface area to allow concurrent bicin (Doxil) with temsirolimus (Torisel), an intravenous enrollment of both children and adults. Temsirolimus mTOR inhibitor that is rapidly converted to sirolimus was given weekly, and liposomal doxorubicin was admin- in  vivo, for patients with recurrent and refractory bone istered every 28  days. Patients were pretreated with and soft tissue sarcomas, hypothesizing that the addition high diphenhydramine to avoid infusion-related hypersen- of temsirolimus will sensitize the A LDH population sitivity reactions. We are reporting here the results of to the liposomal doxorubicin. Encapsulating doxorubicin 18 subjects treated at the recommended phase II dose in pegylated liposomes allows improved localization of combination of liposomal doxorubicin 30  mg/m /dose drug to tumors, resulting in activity in chemotherapy- monthly with temsirolimus 20 mg/m /dose weekly. Three refractory disease. Pegylated liposomal doxorubicin is patients were treated at a higher dose of temsirolimus generally better tolerated than standard doxorubicin, (27 mg/m weekly), and they are included in some of the allowing treatment of patients who have already received analyses as well. substantial doses of anthracyclines. We have previously Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 3 of 9 Treatment efficacy using microscopy (Nikon E600), and photographed Subjects were considered evaluable for response if with digital camera (Nikon DXM1200F; ACT-1 they received therapy at least until the first scheduled software). radiologic evaluation. Event-free survival (EFS) and For immunocytochemical detection of ALDH, cells progression-free survival (PFS) were determined from were pelleted on slides using a cytospin. Cells were fixed the date of the first dose of study drug. EFS was defined with 4% paraformaldehyde for 20 min at room tempera- as the time to either documentation of disease progres- ture, washed with PBS, and blocked in PBS with normal sion or withdrawal from the study due to unacceptable goat serum for 1  h at room temperature. After block- toxicity. Subjects who withdrew from the study for rea- ing, cells were stained overnight with primary antibody sons other than toxicity or progression were censored against ALDH1 (BD Bioscience, clone 44)) at a 1∶100 for EFS on the date of withdrawal. PFS was defined as dilution in PBS with 5% normal goat serum and 0.02% the time to documentation of disease progression as Triton X-100 at 4  °C. The next day, cells were washed defined by RECIST criteria. Subjects who withdrew with PBS and stained with Alexa Fluor 555 goat anti- from the study were censored for PFS on the date of mouse antibody (Invitrogen) at 1:400 dilution. After sec- withdrawal. ondary labeling, cells were washed in PBS and mounted in Prolong Gold with DAPI. Flow cytometry Analysis of ALDH immunohistochemical staining Patients underwent core needle biopsies at study entry, Quantification of ALDH expression was performed using after 4  weeks of therapy, and at the time of progres- FRIDA [FRamework for Image Dataset Analysis; [3], a sion. Samples were digested using collagenase/dispase custom open source image analysis software package as previously described, and single cells isolated using (available at http://sourc eforg e.net/proje cts/frida jhu/)] a Ficoll gradient. These cells were then treated with for the analysis of RGB color images generated from Aldefluor (Stem Cell Technologies, Vancouver, BC) scanning of tissue microarray slides. Hue Saturation and according to the manufacturer’s instructions [6], and Brightness (HSB) segmentation ranges for DAB brown then separated by flow cytometry using FACSAria and staining and hematoxylin alone (cells not staining brown) FACSDiva software (BD Biosciences, Franklin Lakes, were defined by creating different color masks. Num - NJ) into populations with high aldehyde dehydroge- high low bers of cells were counted by using particle count filter nase expression (ALDH ), low expression (ALDH ), set with size limitation. The percentage of ALDH-pos - and flow through cells (cells passed through the flow itive cells were calculated by using the number of DAB cytometer but not sorted). labeled cells divided by the sum of the DAB labeled and the hematoxylin labeled cells ×100. Immunocytochemistry Paraffin-embedded tumor samples were deparaffinized Statistical analysis in xylene and rehydrated in graded alcohol and rinsed Data were analyzed using Prism 5.0 software (GraphPad, in 1× PBS. Antigens were retrieved by boiling samples Inc., La Jolla, CA). for 12 min in citrate buffer, pH 6 (Invitrogen). Nonspe - cific binding sites were blocked using 1  ml PBS con - Results taining 5% goat serum and 1% bovine serum albumin Patient characteristics (BSA). The sections were incubated overnight at 4  °C This trial was a phase I/II study of temsirolimus and in a humidor with monoclonal antibody to ALDH1 liposomal doxorubicin for patients with relapsed or (1:100; BD bioscience, clone 44), diluted with 1% goat refractory soft tissue and bone sarcomas. The results serum, 0.2% BSA and 0.3% Triton X-100 in PBS (pH of the phase I portion of the study have been previ- 7.4), followed by washing with PBS. Sections were then ously reported [6]. In the phase II portion of the study, incubated with peroxidase-conjugated secondary anti- a total of 18 subjects were treated at the recommended body (Jackson Immunoresearch Laboratories, USA) of phase II dose (RP2D), liposomal doxorubicin 30 mg/m / appropriate specificity. 3,3′ -diaminobenzidine (DAB, dose monthly with temsirolimus 20  mg/m /dose weekly Pierce) was used as substrate for peroxidase and coun- (Table  1). In addition, 3 subjects were treated with a terstaining was performed with modified Harris hema - higher dose of temsirolimus, 27  mg/m /dose weekly toxylin solution (Sigma). Sections were dehydrated by as part of the dose escalation phase. Of the 18 subjects passage through graded alcohol concentrations and treated at the RP2D, 15 were evaluable for response, as finally xylene. Cover slips were mounted using DPX were all of the subjects treated at the higher dose. Of the (Sigma). Completed immunostaining was visualized three unevaluable patients, one withdrew after 20 days of Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 4 of 9 Table 1 Patient characteristics Patient Age Gender Diagnosis Status Evaluable? Prior Doxo 1 19 M Mesenchymal Chondrosarcoma RR 1 N Y 2 43 F MFH RR 1 Y Y 3 39 F Leiomyosarcoma Relapse 2 Y Y 4 18 M Rhabdomyosarcoma Relapse 1 Y N 5 9 F Rhabdomyosarcoma RR 2 Y N 6 20 F Rhabdomyosarcoma Relapse 1 N Y 7 63 F Leiomyosarcoma Relapse 2 Y Y 8 21 M Rhabdomyosarcoma RR 1 Y Y 9 70 M Spindle Cell Sarcoma Relapse 2 Y N 10 59 F Leiomyosarcoma RR 1 Y Y 11 57 F Chondrosarcoma Mult Relapse Y N 12 10 M Osteosarcoma RR 1 Y Y 13 43 F Clear Cell Sarcoma Relapse 2 Y N 14 16 M Ewing Sarcoma RR 1 Y Y 15 68 F Leiomyosarcoma RR 1 N N 16 57 F Synovial Sarcoma RR 2 Y Y 17 22 F Mesenchymal Chondrosarcoma Relapse 2 Y Y 18 32 M MPNST Relapse 2 Y Y 19 20 F HGUPS RR 1 Y Y 20 21 F Epithelioid Sarcoma Relapse 1 Y Y 21 42 M Rhabdomyosarcoma Refractory Y N MFH Malignant fibrous histiocytoma, MPNST Malignant peripheral nerve sheath tumor, HGUPS High grade undifferentiated pleiomorphic sarcoma, RR refractory relapse, Patients in italics were treated at the higher dose of temsirolimus (Dose Level 5) therapy because of stomatitis, one was removed from the at the RP2D, 3 were unevaluable as described above. study after 7 days because of persistent Grade 3 elevation Out of the 15 patients treated at the RP2D who were of ALT, and one withdrew after 7 days due to clinical dete- evaluable for response, 8 terminated therapy due to rioration. Seven of the subjects treated at the RP2D were radiographic progression of disease, 5 withdrew due to male and eleven were female. Median age was 27  years clinical deterioration, 1 withdrew because of toxicity, (range 9–70). Two of the subjects treated at the higher and 1 withdrew due to a need to discontinue treatment dose were female (ages 57 and 59), and the other was a to allow surgery for an unrelated medical condition. 10  year old boy. The subjects had a number of sarcoma The median PFS for this population was 315  days types, including rhabdomyosarcoma (n = 5), leiomyo- (range 27–799), with the patients who discontinued sarcoma (n = 3), synovial sarcoma (n = 2), mesenchymal therapy for reasons other than disease progression cen- chondrosarcoma (n = 2), and a variety of others (detailed sored at the time of discontinuation (Fig.  1a). Median in Table 1). Of the 18 subjects treated at the RP2D, 1 had EFS (discontinuation for clinical deterioration consid- primary refractory disease, 3 were treated at first relapse, ered an event) was 75  days (Fig.  1a). Including the 3 6 were treated after failure of second line therapy, 6 were subjects treated at the higher dose of temsirolimus in treated at second relapse, and 2 were treated after failure the analysis, median PFS was unchanged at 315  days of third-line therapy. Two of the subjects at the higher (range 27–799), but median EFS was longer at 119 days dose level were treated during first relapse after failure of (Fig.  1b). Response rate, defined as stable disease (SD) second line therapy, and one was multiply recurrent after or better for 60 days (2 cycles) was 53% (8 of 15) at the surgeries but had no prior systemic therapy. Fourteen of RP2D and 56% (10 of 18) including the subjects treated the subjects, including 2 treated at the higher dose, had with the higher dose of temsirolimus. A waterfall plot previously received doxorubicin. of best responses, using RECIST 1.1 criteria, shows 3 patients had progressive disease (PD) at their first eval - Response to treatment uation (20%), 2 patients had a partial response (PR) as The primary endpoint of the phase II portion of this best response (13%), and the remainder had stable dis- study was PFS. Although eighteen patients were treated ease (SD) as their best response (Fig.  1c). Those who Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 5 of 9 Fig. 1 a Event‑Free Survival (EFS) and Progression‑free Survival (PFS) of the 15 patients treated at the RP2D. A Kaplan–Meier curve indicating the time from beginning of treatment to withdrawal from study (EFS) or beginning of treatment to first objective evidence of disease progression by RECIST 1.1 criteria (PFS). b EFS and PFS of the 18 patients treated at the RP2D and the dose level above. A Kaplan–Meier curve indicating the time from beginning of treatment to withdrawal from study (EFS) or beginning of treatment to first objective evidence of disease progression by RECIST (PFS). c A waterfall plot of the best responses for the 15 patients treated at R2PD Toxicities Subjects were treated for a median of 2 cycles (mean number of cycles 5.5, range 0–24). This was a heavily pretreated group of patients, with a median of 2 prior lines of systemic therapy (range 0–6). Despite this bur- den of prior therapy, treatment was reasonably well tol- erated. At the RP2D (dose level 4: temsirolimus 20  mg/ m /dose weekly), we observed forty AEs of grade 3 or 4 with “possible” or greater attribution, thirty-three grade 3 and seven grade 4 (Table  2). Thrombocytopenia and hypophosphatemia were the most common severe Fig. 2 Progression‑Free Survival of subjects who had previously adverse events (grade 3 or higher) with six incidences received doxorubicin compared with the entire study population. A of each, followed by five incidences of grade 3 neutro - Kaplan–Meier curve indicating the PFS of the 11 subjects who had penia. There were three episodes each of ALT elevation previously received doxorubicin compared with the PFS of the total (all grade 3), lipase elevation (all grade 4), and vomiting population of subjects (all grade 3), two instances each of hypokalemia (grade 3) and amylase elevation (grade 3). Single incidences of grade 3 increased AST, bone infection, hypocalcemia, responded to therapy (defined as SD or better at first hyponatremia, stomatitis, white blood cell decreased, evaluation) tended to have prolonged responses, with a lymphocyte count decreased, and weight loss were median PFS for this group of 358 days (range = 75–799) reported. In the three patients treated at the higher dose and median EFS of 249 days. of temsirolimus, (dose level 5: 27  mg/m /dose weekly), As noted in Table  1, 9 of 15 subjects treated at the two incidences of grade 3 hypophosphatemia and eme- RP2D, and 2 of 3 treated at the higher dose, had pre- sis were reported, as well as single incidences of grade viously been exposed to doxorubicin (11 of 18 = 61%). 3 hypokalemia, neutropenia, and abdominal pain, and PFS among the subjects previously treated with doxo- a single incidence of grade 4 anorexia. One patient died rubicin was 108  days (range 27–358) and EFS was while on study, attributed to progression of disease, not 49  days, both substantially shorter than the popula- study drug. tion as a whole, though because of the small sam- Weight loss can be a surrogate for overall health in ple size the difference was not statistically significant patients undergoing anti-cancer therapy, and mTOR (Fig.  2). Response rate in this group was also worse inhibition has the potential to alter the metabolism of than the population as a whole (4 of 11 = 36%). For both normal and cancer cells. We therefore investigated the group of patients with prior doxorubicin exposure changes in body weight during treatment. Of 15 evalu- who responded to therapy, however, response was pro- able subjects treated at the RP2D, three (21%) never lost longed, with median PFS of 315  days (range = 160– weight (treated for 2, 4, and 6 cycles). Seven subjects lost 358), comparable to the PFS of responders within the weight during the course of therapy (50%), but lost < 10% entire study population. Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 6 of 9 Table 2 Adverse events Group Toxicity Grade 3 Grade 4 Total Dose level Hematologic Thrombocytopenia 3 3 6 4 Neutropenia 5 – 5 4 Lymphocyte count decreased 1 – 1 4 White blood cell decreased 1 – 1 4 Gastrointestinal Lipase increased – 3 3 4 ALT increased 3 – 3 4 Vomiting 3 – 3 4, 5 Serum Amylase increased 2 – 2 4 Abdominal pain 1 – 1 5 Anorexia – 1 1 5 AST increased 1 – 1 4 Stomatitis 1 – 1 4 Weight loss 1 – 1 4 Metabolic Hypophosphatemia 6 – 6 4, 5 Hypokalemia 2 – 2 4, 5 Hypocalcemia 1 – 1 4 Hyponatremia 1 – 1 4 Other Bone infection 1 – 1 4 Only adverse events (AEs) of Grade ≥ 3 and with attribution of “Possible” or above reported. ALT alanine aminotransferase, AST aspartate aminotransferase, ANC absolute neutrophil count Another surrogate for overall heath is performance sta- tus. All 15 evaluable subjects treated at the RP2D started treatment with an ECOG performance status of 0 or 1. Of these, only 5 (33%) had a worsening of performance sta- tus of two or more levels during treatment: one patient’s performance status declined from 0 to 2, two from 1 to 3, and two from 0 to 3. In each of these cases, the fall in per- formance status was temporally associated with tumor growth, suggesting that disease progression, rather than toxicity of therapy, was responsible for worsening perfor- mance status. Pharmacodynamics Inhibition of mTOR We evaluated mTOR signaling by immunohistochemical analysis of phosphorylated S6 kinase (pS6K; reflecting target of rapamycin complex 1 [TORC1] signaling) and Fig. 3 Spaghetti plot of patient weights during treatment. Each line represents an individual patient and the number of cycles of therapy phosphorylated AKT (pAKT; reflecting TORC2 signal - is indicated on the Y axis ing) using archived tumor biopsy samples at the time of diagnosis (baseline). In addition, subjects underwent an optional biopsy at week 4 of therapy, and for some sub- jects at study entry (1 subject) and at the end of the study of their starting body weight, and 3 of these regained (4 subjects). Single cells suspensions were made from this weight from their minimum while continuing on treat- biopsy material, and cells were isolated based on ALDH ment. The remaining 5 (29%) lost 10% or more of their expression and analyzed by immunocytochemistry for initial body weight, but even among this group, one pS6K and pAKT staining. Total S6K and AKT stain- regained weight from her minimum while continuing on ing served as an internal control. Out of the 12 subjects treatment (Fig. 3). treated at the RP2D who had evaluable biopsies at week Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 7 of 9 Fig. 4 Correlation between mTOR inhibition and response to therapy. Cells obtained from a core biopsy at week 4 were stained for either pS6K (a) or pAKT (b), and compared with staining from the diagnostic biopsy. Inhibition of phosphorylation was compared with response or nonresponse to treatment high 4, 8 (67%) were concordant for TORC1 inhibition and our hypothesis that eliminating the ALDH population response, and 9 were concordant for TORC2 inhibition is imperative to curing the disease. and response (Fig.  4). Though not statistically significant because of the small sample size (p-values of 0.61 and Discussion 0.13 respectively), pAKT inhibition has a positive predic- The survival rate for several sarcomas has plateaued in tive value (PPV) and negative predictive value (NPV) of recent years despite attempts to intensify chemotherapy. 71.3% and 74.7% respectively. Particularly, the survival rates for sarcoma patients who One illustrative case is subject 16. This was a 43-year- present with metastatic disease or who have relapsed old woman with metastatic clear cell sarcoma who was disease have not seen significant improvement in dec - treated at 3rd relapse. Her only prior therapy had been ades [7, 8]. One possible explanation for this may be the surgery. She underwent biopsy at the time of treatment existence of small subpopulations of sarcoma cells that high initiation, and 29% of her ALDH cells stained for are resistant to conventional chemotherapy and are able pS6K and 100% stained for pAKT. In addition, 43% of to cause recurrences and metastases. We have previously low her ALDH cells stained for pS6K and 43% stained for shown that high expression of ALDH can act as a marker high pAKT. At week 4, no ALDH cells stained for pS6K for Ewing sarcoma cells that demonstrate “stem cell-like” low or for pAKT, and only 8% of her A LDH cells stained properties including resistance to conventional chemo- for pS6K, with 27% staining for pAKT. This significant therapy. Similar findings have been shown with other reduction in mTOR signaling correlated with a prolonged sarcoma histologies [3–5]. We have shown in preclinical high period of progression-free survival (752 days). testing that ALDH cells are resistant to chemotherapy agents commonly used to treat sarcomas, such as doxo- Targeting stem cells rubicin, and that inhibition of the mTOR pathway with The rationale for this study was our in  vitro observa - agents such as rapamycin can overcome this chemore- high tion that mTOR inhibition increases the sensitivity of sistance seen in the ALDH cells. sarcoma stem cells to chemotherapy and our hypothesis Clinical trials in sarcoma with single agent mTOR that eliminating CSC would translate into improved sur- inhibitors have shown modest efficacy at best [9, 10]. vival. Three patients had evaluable paired tumor biopsy While a degree of cytotoxicity from mTOR inhibition can samples to assess the effect of the treatment on the per - be seen, mTOR inhibition combined with conventional high centage of ALDH cells. Patient 8 and 11 each had a chemotherapeutic agents has yielded more promising rhabdomyosarcoma that did not respond to the tem- results [11]. The mechanism by which mTOR inhibition sirolimus/liposomal doxorubicin therapy, and had biop- enhances the efficacy of chemotherapy, however, has not sies at weeks 4 and 8 that demonstrated an increase in been fully elucidated. Our study was designed to evaluate high ALDH percentage, from 2 to 43.9% and 0.6 to 52.3% the ability of mTOR inhibition to overcome the chemore- respectively. Patient 16, however, who had a metastatic sistance of relapsed sarcomas and in particular the resist- high Clear Cell Sarcoma, had biopsies at baseline and week ance seen in ALDH populations within these tumors. high 4 that demonstrated a decrease in ALDH percent- We report the phase II portion of a phase I/II trial testing age from 29.6 to 3.7%. This patient did show a response temsirolimus in combination with liposomal doxorubicin to therapy and had a 752-day progression free interval. in patients with relapsed or refractory sarcomas. Our While these numbers are small, they are supportive of patient population was heavily pretreated, but despite Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 8 of 9 this, the patients tolerated the study regimen well. While these approaches, and evidence would suggest sarcoma interpretation of our results is limited by a small number research should follow suit. Targeting the mTOR pathway of patients and comparison to historical controls, of the to treat sarcomas is already underway. Targeting ALDH evaluable patients at our RP2D, PFS was approximately directly or in combination with mTOR blockade holds three times longer with the combination of liposomal additional promise. Finally, the development of resistance doxorubicin and temsirolimus than what has been seen to mTOR inhibition has been well described [18, 19]. in similar patients treated with single agent mTOR inhib- Further studies in how this resistance develops, and tech- itor [9]. The PFS observed was also at least two times niques to prevent or overcome this resistance are neces- longer than that reported in similar patients treated with sary for the success of this treatment strategy. liposomal doxorubicin alone or combined with other conventional chemotherapy drugs [12, 13]. The response rate (stable disease or better for 2 cycles) in our study was Conclusions consistent with other studies of relapsed sarcoma com- Within the confines of this small phase I/II study with bining chemotherapy with mTOR inhibition as were the a heterogeneous patient population, the combination incidence and severity of adverse events observed [11]. of temsirolimus with liposomal doxorubicin is safe and To our knowledge, while not the first study to test mTOR well tolerated, and PFS is better than previously reported inhibition combined with chemotherapy in sarcomas, with either agent given alone. Response to treatment cor- this is the first study where the effect of the combination relates with laboratory evidence of a reduction in the high specifically on a putative CSC population was assessed. ALDH population of putative sarcoma stem cells, vali- Though further limited by the small number of patients dating the concept that targeting this specific population for whom pre- and post-treatment tumor biopsy samples of cells can improve treatment outcomes. were available for analysis, response to therapy correlated high with reduction in the ALDH population. Abbreviations Recently, Mu et  al. [14] showed in murine osteosar- CSC: cancer stem cell; mTOR: mechanistic target of rapamycin; EFS: event coma cell lines that ALDH activity is dependent on free survival; PFS: progression free survival; ALDH: aldehyde dehydrogenase; RECIST: response evaluation criteria in solid tumors; BSA: bovine serum mTOR activity. While this was a single small in  vitro albumin; PBS: phosphate buffered saline; DAB: 3,3′‑ diaminobenzidine; RP2D: study on murine cell lines, it raises the possibility that recommended phase 2 dose; AST: aspartate aminotransferase; ALT: alanine the chemosensitizing effect of mTOR inhibition seen in aminotransferase; PD: progressive disease; PR: partial response; SD: stable disease. this and other trials could be due to direct inhibition of ALDH. There is increasing evidence that, in addition to Authors’ contributions being a marker for CSCs, ALDH may play an active role MMT, CFM, BAW contributed patients to the clinical trial, participated in writing the manuscript, and contributed to the data analysis; KAT partici‑ in providing CSCs their “stemness”, particularly contrib- pated in writing the clinical trial protocol, contributed patients to the clinical uting to the chemoresistance seen in these cells. ALDH trial, and contributed to the data analysis; PS, MACH, and LCB performed is a superfamily of phase I oxidizing enzymes responsible laboratory experiments; ARC participated in writing the clinical trial protocol and performed statistical analysis; MFF participated in writing the clinical for detoxification of aldehydes [2]. ALDH is implicated in trial protocol, was the research nurse for the clinical trial, and contributed to cellular “self-protection,” including supporting antioxi- the data analysis; US was the research coordinator for the clinical trial and dant factors countering the production of reactive oxygen contributed to the data analysis; JDP conceived of the trial and participated in writing the manuscript; DML conceived of the trial, contributed patients to species [15]. It is also known to inactivate chemotherapy the clinical trial, participated in writing the manuscript, and contributed to the drugs, the most recognized being cyclophosphamide and data analysis. All authors read and approved the final manuscript. related agents, but also doxorubicin, cisplatin, temozo- Author details lemide and taxanes, which are many of the cornerstones Division of Pediatric Oncology, Sidney Kimmel Comprehensive Cancer of sarcoma therapy. Furthermore, evidence supporting 2 Center, Johns Hopkins University, Baltimore, MD, USA. Division of Medical the importance of ALDH expression in the process of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. Present Address: Sylvester Comprehensive metastasis is emerging in several solid tumors includ- Cancer Center, University of Miami, Miami, FL, USA. Present Address: Dana ing osteosarcoma, where the ALDH inhibitor disulfiram 5 Farber Cancer Institute, Boston, MA, USA. Present Address: Akan Biosciences, appears to inhibit metastatic disease [16, 17]. Gaithersburg, MD, USA. Present Address: Columbia University College of Phy‑ sicians and Surgeons, New York, NY, USA. Department of Pediatrics, Albert While the mTOR pathway plays several roles in can- Einstein College of Medicine, Children’s Hospital at Montefiore, 3411 Wayne cer cell biology, including resistance to apoptosis and Ave., Room 910, Bronx, NY 10467, USA. metabolic reprogramming, this association with ALDH Acknowledgements expression and the emerging evidence of a functional Not applicable. role for ALDH highlights a potential new target for overcoming chemoresistance. The fields of breast and Competing interests The authors declare that they have no competing interests. colon cancer research, among others, are exploring Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 9 of 9 Availability of data and materials 8. Reed DR, Hayashi M, Wagner L, Binitie O, Steppan DA, Brohl AS, Shino‑ Data sharing is not applicable to this article as no datasets were generated or hara ET, Bridge JA, Loeb DM, Borinstein SC, et al. Treatment pathway analysed during the current study. of bone sarcoma in children, adolescents, and young adults. Cancer. 2017;123(12):2206–18. Consent for publication 9. Chawla SP, Staddon AP, Baker LH, Schuetze SM, Tolcher AW, D’Amato GZ, Not applicable. Blay JY, Mita MM, Sankhala KK, Berk L, et al. Phase II study of the mamma‑ lian target of rapamycin inhibitor ridaforolimus in patients with advanced Ethics approval and consent to participate bone and soft tissue sarcomas. J Clin Oncol. 2012;30(1):78–84. The clinical trial reported herein was approved by the Institutional Review 10. Okuno S, Bailey H, Mahoney MR, Adkins D, Maples W, Fitch T, Ettinger D, Board of the Johns Hopkins Hospital and all patients (or their legal guardians) Erlichman C, Sarkaria JN. A phase 2 study of temsirolimus (CCI‑779) in signed informed consent according to institutional standards. patients with soft tissue sarcomas: a study of the Mayo phase 2 consor‑ tium (P2C). Cancer. 2011;117(15):3468–75. Funding 11. Martin‑Liberal J, Lopez‑Pousa A, Martinez‑ Trufero J, Martin‑Broto J, This study was approved and funded by the National Comprehensive Cancer Cubedo R, Lavernia J, Redondo A, Lopez‑Martin JA, Mulet ‑Margalef N, Network (NCCN) from general research support from Pfizer, Inc. We are also Sanjuan X, et al. Phase II study of Gemcitabine Plus Sirolimus in previously grateful for the support of The Heather Brooke Foundation. Niether funding treated patients with advanced soft‑tissue sarcoma: a Spanish Group agency played any role in the design of the study, in the collection, analysis, or for Research on Sarcomas (GEIS) Study. Target Oncol. 2017. https ://doi. interpretation of the data, nor in the writing of the manuscript.org/10.1007/s1152 3‑017‑0539‑9. 12. De Sanctis R, Bertuzzi A, Basso U, Comandone A, Marchetti S, Marrari A, Colombo P, Lutman RF, Giordano L, Santoro A. Non‑pegylated liposomal Publisher’s Note doxorubicin plus ifosfamide in metastatic soft tissue sarcoma: results Springer Nature remains neutral with regard to jurisdictional claims in pub‑ from a phase‑II trial. Anticancer Res. 2015;35(1):543–7. lished maps and institutional affiliations. 13. Poveda A, Lopez‑Pousa A, Martin J, Del Muro JG, Bernabe R, Casado A, Balana C, Sanmartin O, Menendez MD, Escudero P, et al. Phase II Clini‑ Received: 16 May 2018 Accepted: 12 August 2018 cal Trial With Pegylated Liposomal Doxorubicin (CAELYX(R)/Doxil(R)) and Quality of Life Evaluation (EORTC QLQ‑ C30) in adult patients with advanced soft tissue sarcomas: a study of the Spanish Group for Research in Sarcomas (GEIS). Sarcoma. 2005;9(3–4):127–32. 14. Mu X, Isaac C, Schott T, Huard J, Weiss K. Rapamycin inhibits ALDH References activity, resistance to oxidative stress, and metastatic potential in murine 1. Wang T, Shigdar S, Gantier MP, Hou Y, Wang L, Li Y, Shamaileh HA, Yin W, osteosarcoma cells. Sarcoma. 2013;2013:480713. Zhou SF, Zhao X, et al. Cancer stem cell targeted therapy: progress amid 15. Raha D, Wilson TR, Peng J, Peterson D, Yue P, Evangelista M, Wilson C, controversies. Oncotarget. 2015;6(42):44191–206. Merchant M, Settleman J. The cancer stem cell marker aldehyde dehydro‑ 2. Tomita H, Tanaka K, Tanaka T, Hara A. Aldehyde dehydrogenase 1A1 in genase is required to maintain a drug‑tolerant tumor cell subpopulation. stem cells and cancer. Oncotarget. 2016;7(10):11018–32. Cancer Res. 2014;74(13):3579–90. 3. Awad O, Yustein JT, Shah P, Gul N, Katuri V, O’Neill A, Kong Y, Brown ML, 16. Cho HJ, Lee TS, Park JB, Park KK, Choe JY, Sin DI, Park YY, Moon YS, Lee KG, Toretsky JA, Loeb DM. High ALDH activity identifies chemotherapy‑ Yeo JH, et al. Disulfiram suppresses invasive ability of osteosarcoma cells resistant Ewing’s sarcoma stem cells that retain sensitivity to EWS‑FLI1 via the inhibition of MMP‑2 and MMP ‑9 expression. J Biochem Mol Biol. inhibition. PLoS ONE. 2010;5(11):e13943. 2007;40(6):1069–76. 4. Canter RJ, Ames E, Mac S, Grossenbacher SK, Chen M, Li CS, Borys D, 17. Greco N, Schott T, Mu X, Rothenberg A, Voigt C, McGough RL 3rd, Smith RC, Tellez J, Sayers TJ, et al. Anti‑proliferative but not anti‑angio ‑ Goodman M, Huard J, Weiss KR. ALDH activity correlates with metastatic genic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue potential in primary sarcomas of bone. J Cancer Ther. 2014;5(4):331–8. sarcoma. BMC Cancer. 2014;14:756. 18. Fujishita T, Kojima Y, Kajino‑Sakamoto R, Taketo MM, Aoki M. Tumor 5. Wang M, Xiao J, Jiang J, Qin R. CD133 and ALDH may be the microenvironment confers mTOR inhibitor resistance in invasive intestinal molecular markers of cholangiocarcinoma stem cells. Int J Cancer. adenocarcinoma. Oncogene. 2017;36(46):6480–9. 2011;128(8):1996–7. 19. Koh KX, Tan GH, Hui Low SH, Mohd Omar MF, Han MJ, Iacopetta B, Soo 6. Thornton KA, Chen AR, Trucco MM, Shah P, Wilky BA, Gul N, Carrera‑ R, Beloueche‑Babari M, Bhattacharya B, Soong R. Acquired resistance to Haro MA, Ferreira MF, Shafique U, Powell JD, et al. A dose ‑finding PI3 K/mTOR inhibition is associated with mitochondrial DNA mutation study of temsirolimus and liposomal doxorubicin for patients with and glycolysis. Oncotarget. 2017;8(66):110133–44. recurrent and refractory bone and soft tissue sarcoma. Int J Cancer. 2013;133(4):997–1005. 7. Comandone A, Petrelli F, Boglione A, Barni S. Salvage therapy in advanced adult soft tissue sarcoma: a systematic review and meta‑analysis of rand‑ omized trials. Oncologist. 2017;22(12):1518–27. Ready to submit your research ? 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A phase II study of temsirolimus and liposomal doxorubicin for patients with recurrent and refractory bone and soft tissue sarcomas

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Copyright © 2018 by The Author(s)
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Biomedicine; Cancer Research; Oncology; Surgical Oncology
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2045-3329
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10.1186/s13569-018-0107-9
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Abstract

Background: Relapsed and refractory sarcomas continue to have poor survival rates. The cancer stem cell (CSC) theory provides a tractable explanation for the observation that recurrences occur despite dramatic responses to upfront chemotherapy. Preclinical studies demonstrated that inhibition of the mechanistic target of rapamycin (mTOR) sensitizes the CSC population to chemotherapy. Methods: Here we present the results of the Phase II portion of a Phase I/II clinical trial that aimed to overcome the chemoresistance of sarcoma CSC by combining the mTOR inhibitor temsirolimus (20 mg/m weekly) with the chemo‑ therapeutic agent liposomal doxorubicin (30 mg/m monthly). Results: Fifteen patients with relapsed/refractory sarcoma were evaluable at this recommended Phase 2 dose level. The median progression free survival was 315 days (range 27–799). Response rate, defined as stable disease or better for 60 days, was 53%. Nine of the patients had been previously treated with doxorubicin. Therapy was well tolerated. In a small number of patients, pre‑ and post‑ treatment tumor biopsies were available for assessment of ALDH expres‑ sion as a marker of CSCs and showed a correlation between response and decreased ALDH expression. We also found a correlation between biopsy‑ proven inhibition of mTOR and response. Conclusions: Our study adds to the literature supporting the addition of mTOR inhibition to chemotherapy agents for the treatment of sarcomas, and proposes that a mechanism by which mTOR inhibition enhances the efficacy of chemotherapy may be through sensitizing the chemoresistant CSC population. Further study, ideally with pre‑ and post‑ therapy assessment of ALDH expression in tumor cells, is warranted. Trial registration The trial was registered on clinicaltrials.gov (NCT00949325) on 30 July 2009. http://www.edito rialm anage r.com/csrj/defau lt.aspx Keywords: mTOR, Cancer stem cell, Chemoresistance, Sarcoma, Aldehyde dehydrogenase *Correspondence: dloeb@montefiore.org Department of Pediatrics, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, 3411 Wayne Ave., Room 910, Bronx, NY 10467, USA Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 2 of 9 published the results of the dose finding portion of this Background study [6] and are reporting here the results of the Phase Therapeutic advances in the treatment of localized high- II portion of the clinical trial, including pharmacody- grade sarcomas have dramatically improved survival for namic data regarding mTOR inhibition and targeting of these patients over the past two decades, but patients high ALDH sarcoma cells. with recurrent or refractory disease continue to have a dismal prognosis. Although many high grade sarcomas Methods respond well to initial therapy, recurrence is common and Patient eligibility requirements is usually fatal. The cancer stem cell (CSC) hypothesis Eligible patients were at least 1  year of age, had a histo- provides an explanation for the discrepancy between ini- logically confirmed diagnosis of sarcoma that was either tial treatment response and overall survival. This model recurrent or refractory to conventional therapy, and had proposes that a small subpopulation of chemotherapy- measurable disease amenable to percutaneous image- resistant tumor cells with a “stem cell-like” phenotype is guided biopsy. Additionally, patients were required responsible for both regrowth of the tumor in its original to have an adequate performance status (ECOG ≤ 2; site and/or for metastatic dissemination [1]. Therapies Karnofsky or Lansky ≥ 60% for children), a life expec- that target cancer stem cells would therefore be expected tancy greater than 3  months, and adequate organ func- to decrease the risk of local and metastatic recurrence, tion (absolute neutrophil count ≥ 1500/µl, platelet dramatically improving the survival of patients with count ≥ 100,000/µl, total bilirubin ≤ 1.5× institutional high-risk disease. upper limit of normal, AST and ALT ≤ 2.5× institu- Cancer cells expressing high levels of aldehyde dehy- tional upper limit of normal, and creatinine ≤ 1.5× insti- drogenase (ALDH) have been shown to possess a phe- tutional upper limit of normal for age or a creatinine notype reminiscent of stem cells in numerous cancers clearance ≥ 60  ml/min/1.73  m ). Since temsirolimus can [2]. In particular, our laboratory has demonstrated that affect both lipid and glucose metabolism, patients were Ewing sarcoma cells expressing high levels of ALDH, as high required to have a fasting cholesterol ≤ 350  mg/dl, fast- determined by cell sorting using Aldefluor (ALDH ing serum triglycerides ≤ 400  mg/dl, amylase and lipase cells), exhibit a variety of stem cell properties, includ- within normal limits (unless elevations were related ing clonogenic growth in soft agar, the ability to grow to tumor involving the pancreas), and a hemoglobin as spheres under non-adherent conditions, and expres- A1c ≤ 10%. Patients were excluded if they had a history of sion of so-called “stem cell genes” such as OCT4 and high pulmonary hypertension or pneumonitis, prior therapy NANOG [3]. Most importantly, as few as 160 ALDH with an mTOR inhibitor, uncontrolled brain metasta- cells can generate a tumor in immune deficient mice, ses, a history of hypersensitivity to macrolide antibiot- as compared with 800,000 unsorted cells. Similar find - ics (because of the risk of crossreactions), or grade 3 or ings have been demonstrated in osteosarcoma and soft 4 proteinuria. The study was approved by the Johns Hop - tissue sarcomas [4, 5]. As predicted by the CSC model, high kins University Institutional Review Board and patients ALDH cells are resistant to doxorubicin and etoposide signed written informed consent according to institu- in  vitro when compared with unsorted cells. We subse- tional standards. The trial was registered with Clinical - quently demonstrated that inhibition of the mechanistic Trials.gov (Registration ID: NCT00949325). target of rapamycin (mTOR) by sirolimus increases the high sensitivity of ALDH cells to doxorubicin and causes Treatment plan synergistic cytotoxicity when unsorted cells are treated Temsirolimus and liposomal doxorubicin were admin- in  vitro [6]. Based on these findings, we conducted a istered intravenously in the outpatient clinic, and dos- Phase I/II study of the combination of liposomal doxoru- ing was based on body surface area to allow concurrent bicin (Doxil) with temsirolimus (Torisel), an intravenous enrollment of both children and adults. Temsirolimus mTOR inhibitor that is rapidly converted to sirolimus was given weekly, and liposomal doxorubicin was admin- in  vivo, for patients with recurrent and refractory bone istered every 28  days. Patients were pretreated with and soft tissue sarcomas, hypothesizing that the addition high diphenhydramine to avoid infusion-related hypersen- of temsirolimus will sensitize the A LDH population sitivity reactions. We are reporting here the results of to the liposomal doxorubicin. Encapsulating doxorubicin 18 subjects treated at the recommended phase II dose in pegylated liposomes allows improved localization of combination of liposomal doxorubicin 30  mg/m /dose drug to tumors, resulting in activity in chemotherapy- monthly with temsirolimus 20 mg/m /dose weekly. Three refractory disease. Pegylated liposomal doxorubicin is patients were treated at a higher dose of temsirolimus generally better tolerated than standard doxorubicin, (27 mg/m weekly), and they are included in some of the allowing treatment of patients who have already received analyses as well. substantial doses of anthracyclines. We have previously Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 3 of 9 Treatment efficacy using microscopy (Nikon E600), and photographed Subjects were considered evaluable for response if with digital camera (Nikon DXM1200F; ACT-1 they received therapy at least until the first scheduled software). radiologic evaluation. Event-free survival (EFS) and For immunocytochemical detection of ALDH, cells progression-free survival (PFS) were determined from were pelleted on slides using a cytospin. Cells were fixed the date of the first dose of study drug. EFS was defined with 4% paraformaldehyde for 20 min at room tempera- as the time to either documentation of disease progres- ture, washed with PBS, and blocked in PBS with normal sion or withdrawal from the study due to unacceptable goat serum for 1  h at room temperature. After block- toxicity. Subjects who withdrew from the study for rea- ing, cells were stained overnight with primary antibody sons other than toxicity or progression were censored against ALDH1 (BD Bioscience, clone 44)) at a 1∶100 for EFS on the date of withdrawal. PFS was defined as dilution in PBS with 5% normal goat serum and 0.02% the time to documentation of disease progression as Triton X-100 at 4  °C. The next day, cells were washed defined by RECIST criteria. Subjects who withdrew with PBS and stained with Alexa Fluor 555 goat anti- from the study were censored for PFS on the date of mouse antibody (Invitrogen) at 1:400 dilution. After sec- withdrawal. ondary labeling, cells were washed in PBS and mounted in Prolong Gold with DAPI. Flow cytometry Analysis of ALDH immunohistochemical staining Patients underwent core needle biopsies at study entry, Quantification of ALDH expression was performed using after 4  weeks of therapy, and at the time of progres- FRIDA [FRamework for Image Dataset Analysis; [3], a sion. Samples were digested using collagenase/dispase custom open source image analysis software package as previously described, and single cells isolated using (available at http://sourc eforg e.net/proje cts/frida jhu/)] a Ficoll gradient. These cells were then treated with for the analysis of RGB color images generated from Aldefluor (Stem Cell Technologies, Vancouver, BC) scanning of tissue microarray slides. Hue Saturation and according to the manufacturer’s instructions [6], and Brightness (HSB) segmentation ranges for DAB brown then separated by flow cytometry using FACSAria and staining and hematoxylin alone (cells not staining brown) FACSDiva software (BD Biosciences, Franklin Lakes, were defined by creating different color masks. Num - NJ) into populations with high aldehyde dehydroge- high low bers of cells were counted by using particle count filter nase expression (ALDH ), low expression (ALDH ), set with size limitation. The percentage of ALDH-pos - and flow through cells (cells passed through the flow itive cells were calculated by using the number of DAB cytometer but not sorted). labeled cells divided by the sum of the DAB labeled and the hematoxylin labeled cells ×100. Immunocytochemistry Paraffin-embedded tumor samples were deparaffinized Statistical analysis in xylene and rehydrated in graded alcohol and rinsed Data were analyzed using Prism 5.0 software (GraphPad, in 1× PBS. Antigens were retrieved by boiling samples Inc., La Jolla, CA). for 12 min in citrate buffer, pH 6 (Invitrogen). Nonspe - cific binding sites were blocked using 1  ml PBS con - Results taining 5% goat serum and 1% bovine serum albumin Patient characteristics (BSA). The sections were incubated overnight at 4  °C This trial was a phase I/II study of temsirolimus and in a humidor with monoclonal antibody to ALDH1 liposomal doxorubicin for patients with relapsed or (1:100; BD bioscience, clone 44), diluted with 1% goat refractory soft tissue and bone sarcomas. The results serum, 0.2% BSA and 0.3% Triton X-100 in PBS (pH of the phase I portion of the study have been previ- 7.4), followed by washing with PBS. Sections were then ously reported [6]. In the phase II portion of the study, incubated with peroxidase-conjugated secondary anti- a total of 18 subjects were treated at the recommended body (Jackson Immunoresearch Laboratories, USA) of phase II dose (RP2D), liposomal doxorubicin 30 mg/m / appropriate specificity. 3,3′ -diaminobenzidine (DAB, dose monthly with temsirolimus 20  mg/m /dose weekly Pierce) was used as substrate for peroxidase and coun- (Table  1). In addition, 3 subjects were treated with a terstaining was performed with modified Harris hema - higher dose of temsirolimus, 27  mg/m /dose weekly toxylin solution (Sigma). Sections were dehydrated by as part of the dose escalation phase. Of the 18 subjects passage through graded alcohol concentrations and treated at the RP2D, 15 were evaluable for response, as finally xylene. Cover slips were mounted using DPX were all of the subjects treated at the higher dose. Of the (Sigma). Completed immunostaining was visualized three unevaluable patients, one withdrew after 20 days of Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 4 of 9 Table 1 Patient characteristics Patient Age Gender Diagnosis Status Evaluable? Prior Doxo 1 19 M Mesenchymal Chondrosarcoma RR 1 N Y 2 43 F MFH RR 1 Y Y 3 39 F Leiomyosarcoma Relapse 2 Y Y 4 18 M Rhabdomyosarcoma Relapse 1 Y N 5 9 F Rhabdomyosarcoma RR 2 Y N 6 20 F Rhabdomyosarcoma Relapse 1 N Y 7 63 F Leiomyosarcoma Relapse 2 Y Y 8 21 M Rhabdomyosarcoma RR 1 Y Y 9 70 M Spindle Cell Sarcoma Relapse 2 Y N 10 59 F Leiomyosarcoma RR 1 Y Y 11 57 F Chondrosarcoma Mult Relapse Y N 12 10 M Osteosarcoma RR 1 Y Y 13 43 F Clear Cell Sarcoma Relapse 2 Y N 14 16 M Ewing Sarcoma RR 1 Y Y 15 68 F Leiomyosarcoma RR 1 N N 16 57 F Synovial Sarcoma RR 2 Y Y 17 22 F Mesenchymal Chondrosarcoma Relapse 2 Y Y 18 32 M MPNST Relapse 2 Y Y 19 20 F HGUPS RR 1 Y Y 20 21 F Epithelioid Sarcoma Relapse 1 Y Y 21 42 M Rhabdomyosarcoma Refractory Y N MFH Malignant fibrous histiocytoma, MPNST Malignant peripheral nerve sheath tumor, HGUPS High grade undifferentiated pleiomorphic sarcoma, RR refractory relapse, Patients in italics were treated at the higher dose of temsirolimus (Dose Level 5) therapy because of stomatitis, one was removed from the at the RP2D, 3 were unevaluable as described above. study after 7 days because of persistent Grade 3 elevation Out of the 15 patients treated at the RP2D who were of ALT, and one withdrew after 7 days due to clinical dete- evaluable for response, 8 terminated therapy due to rioration. Seven of the subjects treated at the RP2D were radiographic progression of disease, 5 withdrew due to male and eleven were female. Median age was 27  years clinical deterioration, 1 withdrew because of toxicity, (range 9–70). Two of the subjects treated at the higher and 1 withdrew due to a need to discontinue treatment dose were female (ages 57 and 59), and the other was a to allow surgery for an unrelated medical condition. 10  year old boy. The subjects had a number of sarcoma The median PFS for this population was 315  days types, including rhabdomyosarcoma (n = 5), leiomyo- (range 27–799), with the patients who discontinued sarcoma (n = 3), synovial sarcoma (n = 2), mesenchymal therapy for reasons other than disease progression cen- chondrosarcoma (n = 2), and a variety of others (detailed sored at the time of discontinuation (Fig.  1a). Median in Table 1). Of the 18 subjects treated at the RP2D, 1 had EFS (discontinuation for clinical deterioration consid- primary refractory disease, 3 were treated at first relapse, ered an event) was 75  days (Fig.  1a). Including the 3 6 were treated after failure of second line therapy, 6 were subjects treated at the higher dose of temsirolimus in treated at second relapse, and 2 were treated after failure the analysis, median PFS was unchanged at 315  days of third-line therapy. Two of the subjects at the higher (range 27–799), but median EFS was longer at 119 days dose level were treated during first relapse after failure of (Fig.  1b). Response rate, defined as stable disease (SD) second line therapy, and one was multiply recurrent after or better for 60 days (2 cycles) was 53% (8 of 15) at the surgeries but had no prior systemic therapy. Fourteen of RP2D and 56% (10 of 18) including the subjects treated the subjects, including 2 treated at the higher dose, had with the higher dose of temsirolimus. A waterfall plot previously received doxorubicin. of best responses, using RECIST 1.1 criteria, shows 3 patients had progressive disease (PD) at their first eval - Response to treatment uation (20%), 2 patients had a partial response (PR) as The primary endpoint of the phase II portion of this best response (13%), and the remainder had stable dis- study was PFS. Although eighteen patients were treated ease (SD) as their best response (Fig.  1c). Those who Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 5 of 9 Fig. 1 a Event‑Free Survival (EFS) and Progression‑free Survival (PFS) of the 15 patients treated at the RP2D. A Kaplan–Meier curve indicating the time from beginning of treatment to withdrawal from study (EFS) or beginning of treatment to first objective evidence of disease progression by RECIST 1.1 criteria (PFS). b EFS and PFS of the 18 patients treated at the RP2D and the dose level above. A Kaplan–Meier curve indicating the time from beginning of treatment to withdrawal from study (EFS) or beginning of treatment to first objective evidence of disease progression by RECIST (PFS). c A waterfall plot of the best responses for the 15 patients treated at R2PD Toxicities Subjects were treated for a median of 2 cycles (mean number of cycles 5.5, range 0–24). This was a heavily pretreated group of patients, with a median of 2 prior lines of systemic therapy (range 0–6). Despite this bur- den of prior therapy, treatment was reasonably well tol- erated. At the RP2D (dose level 4: temsirolimus 20  mg/ m /dose weekly), we observed forty AEs of grade 3 or 4 with “possible” or greater attribution, thirty-three grade 3 and seven grade 4 (Table  2). Thrombocytopenia and hypophosphatemia were the most common severe Fig. 2 Progression‑Free Survival of subjects who had previously adverse events (grade 3 or higher) with six incidences received doxorubicin compared with the entire study population. A of each, followed by five incidences of grade 3 neutro - Kaplan–Meier curve indicating the PFS of the 11 subjects who had penia. There were three episodes each of ALT elevation previously received doxorubicin compared with the PFS of the total (all grade 3), lipase elevation (all grade 4), and vomiting population of subjects (all grade 3), two instances each of hypokalemia (grade 3) and amylase elevation (grade 3). Single incidences of grade 3 increased AST, bone infection, hypocalcemia, responded to therapy (defined as SD or better at first hyponatremia, stomatitis, white blood cell decreased, evaluation) tended to have prolonged responses, with a lymphocyte count decreased, and weight loss were median PFS for this group of 358 days (range = 75–799) reported. In the three patients treated at the higher dose and median EFS of 249 days. of temsirolimus, (dose level 5: 27  mg/m /dose weekly), As noted in Table  1, 9 of 15 subjects treated at the two incidences of grade 3 hypophosphatemia and eme- RP2D, and 2 of 3 treated at the higher dose, had pre- sis were reported, as well as single incidences of grade viously been exposed to doxorubicin (11 of 18 = 61%). 3 hypokalemia, neutropenia, and abdominal pain, and PFS among the subjects previously treated with doxo- a single incidence of grade 4 anorexia. One patient died rubicin was 108  days (range 27–358) and EFS was while on study, attributed to progression of disease, not 49  days, both substantially shorter than the popula- study drug. tion as a whole, though because of the small sam- Weight loss can be a surrogate for overall health in ple size the difference was not statistically significant patients undergoing anti-cancer therapy, and mTOR (Fig.  2). Response rate in this group was also worse inhibition has the potential to alter the metabolism of than the population as a whole (4 of 11 = 36%). For both normal and cancer cells. We therefore investigated the group of patients with prior doxorubicin exposure changes in body weight during treatment. Of 15 evalu- who responded to therapy, however, response was pro- able subjects treated at the RP2D, three (21%) never lost longed, with median PFS of 315  days (range = 160– weight (treated for 2, 4, and 6 cycles). Seven subjects lost 358), comparable to the PFS of responders within the weight during the course of therapy (50%), but lost < 10% entire study population. Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 6 of 9 Table 2 Adverse events Group Toxicity Grade 3 Grade 4 Total Dose level Hematologic Thrombocytopenia 3 3 6 4 Neutropenia 5 – 5 4 Lymphocyte count decreased 1 – 1 4 White blood cell decreased 1 – 1 4 Gastrointestinal Lipase increased – 3 3 4 ALT increased 3 – 3 4 Vomiting 3 – 3 4, 5 Serum Amylase increased 2 – 2 4 Abdominal pain 1 – 1 5 Anorexia – 1 1 5 AST increased 1 – 1 4 Stomatitis 1 – 1 4 Weight loss 1 – 1 4 Metabolic Hypophosphatemia 6 – 6 4, 5 Hypokalemia 2 – 2 4, 5 Hypocalcemia 1 – 1 4 Hyponatremia 1 – 1 4 Other Bone infection 1 – 1 4 Only adverse events (AEs) of Grade ≥ 3 and with attribution of “Possible” or above reported. ALT alanine aminotransferase, AST aspartate aminotransferase, ANC absolute neutrophil count Another surrogate for overall heath is performance sta- tus. All 15 evaluable subjects treated at the RP2D started treatment with an ECOG performance status of 0 or 1. Of these, only 5 (33%) had a worsening of performance sta- tus of two or more levels during treatment: one patient’s performance status declined from 0 to 2, two from 1 to 3, and two from 0 to 3. In each of these cases, the fall in per- formance status was temporally associated with tumor growth, suggesting that disease progression, rather than toxicity of therapy, was responsible for worsening perfor- mance status. Pharmacodynamics Inhibition of mTOR We evaluated mTOR signaling by immunohistochemical analysis of phosphorylated S6 kinase (pS6K; reflecting target of rapamycin complex 1 [TORC1] signaling) and Fig. 3 Spaghetti plot of patient weights during treatment. Each line represents an individual patient and the number of cycles of therapy phosphorylated AKT (pAKT; reflecting TORC2 signal - is indicated on the Y axis ing) using archived tumor biopsy samples at the time of diagnosis (baseline). In addition, subjects underwent an optional biopsy at week 4 of therapy, and for some sub- jects at study entry (1 subject) and at the end of the study of their starting body weight, and 3 of these regained (4 subjects). Single cells suspensions were made from this weight from their minimum while continuing on treat- biopsy material, and cells were isolated based on ALDH ment. The remaining 5 (29%) lost 10% or more of their expression and analyzed by immunocytochemistry for initial body weight, but even among this group, one pS6K and pAKT staining. Total S6K and AKT stain- regained weight from her minimum while continuing on ing served as an internal control. Out of the 12 subjects treatment (Fig. 3). treated at the RP2D who had evaluable biopsies at week Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 7 of 9 Fig. 4 Correlation between mTOR inhibition and response to therapy. Cells obtained from a core biopsy at week 4 were stained for either pS6K (a) or pAKT (b), and compared with staining from the diagnostic biopsy. Inhibition of phosphorylation was compared with response or nonresponse to treatment high 4, 8 (67%) were concordant for TORC1 inhibition and our hypothesis that eliminating the ALDH population response, and 9 were concordant for TORC2 inhibition is imperative to curing the disease. and response (Fig.  4). Though not statistically significant because of the small sample size (p-values of 0.61 and Discussion 0.13 respectively), pAKT inhibition has a positive predic- The survival rate for several sarcomas has plateaued in tive value (PPV) and negative predictive value (NPV) of recent years despite attempts to intensify chemotherapy. 71.3% and 74.7% respectively. Particularly, the survival rates for sarcoma patients who One illustrative case is subject 16. This was a 43-year- present with metastatic disease or who have relapsed old woman with metastatic clear cell sarcoma who was disease have not seen significant improvement in dec - treated at 3rd relapse. Her only prior therapy had been ades [7, 8]. One possible explanation for this may be the surgery. She underwent biopsy at the time of treatment existence of small subpopulations of sarcoma cells that high initiation, and 29% of her ALDH cells stained for are resistant to conventional chemotherapy and are able pS6K and 100% stained for pAKT. In addition, 43% of to cause recurrences and metastases. We have previously low her ALDH cells stained for pS6K and 43% stained for shown that high expression of ALDH can act as a marker high pAKT. At week 4, no ALDH cells stained for pS6K for Ewing sarcoma cells that demonstrate “stem cell-like” low or for pAKT, and only 8% of her A LDH cells stained properties including resistance to conventional chemo- for pS6K, with 27% staining for pAKT. This significant therapy. Similar findings have been shown with other reduction in mTOR signaling correlated with a prolonged sarcoma histologies [3–5]. We have shown in preclinical high period of progression-free survival (752 days). testing that ALDH cells are resistant to chemotherapy agents commonly used to treat sarcomas, such as doxo- Targeting stem cells rubicin, and that inhibition of the mTOR pathway with The rationale for this study was our in  vitro observa - agents such as rapamycin can overcome this chemore- high tion that mTOR inhibition increases the sensitivity of sistance seen in the ALDH cells. sarcoma stem cells to chemotherapy and our hypothesis Clinical trials in sarcoma with single agent mTOR that eliminating CSC would translate into improved sur- inhibitors have shown modest efficacy at best [9, 10]. vival. Three patients had evaluable paired tumor biopsy While a degree of cytotoxicity from mTOR inhibition can samples to assess the effect of the treatment on the per - be seen, mTOR inhibition combined with conventional high centage of ALDH cells. Patient 8 and 11 each had a chemotherapeutic agents has yielded more promising rhabdomyosarcoma that did not respond to the tem- results [11]. The mechanism by which mTOR inhibition sirolimus/liposomal doxorubicin therapy, and had biop- enhances the efficacy of chemotherapy, however, has not sies at weeks 4 and 8 that demonstrated an increase in been fully elucidated. Our study was designed to evaluate high ALDH percentage, from 2 to 43.9% and 0.6 to 52.3% the ability of mTOR inhibition to overcome the chemore- respectively. Patient 16, however, who had a metastatic sistance of relapsed sarcomas and in particular the resist- high Clear Cell Sarcoma, had biopsies at baseline and week ance seen in ALDH populations within these tumors. high 4 that demonstrated a decrease in ALDH percent- We report the phase II portion of a phase I/II trial testing age from 29.6 to 3.7%. This patient did show a response temsirolimus in combination with liposomal doxorubicin to therapy and had a 752-day progression free interval. in patients with relapsed or refractory sarcomas. Our While these numbers are small, they are supportive of patient population was heavily pretreated, but despite Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 8 of 9 this, the patients tolerated the study regimen well. While these approaches, and evidence would suggest sarcoma interpretation of our results is limited by a small number research should follow suit. Targeting the mTOR pathway of patients and comparison to historical controls, of the to treat sarcomas is already underway. Targeting ALDH evaluable patients at our RP2D, PFS was approximately directly or in combination with mTOR blockade holds three times longer with the combination of liposomal additional promise. Finally, the development of resistance doxorubicin and temsirolimus than what has been seen to mTOR inhibition has been well described [18, 19]. in similar patients treated with single agent mTOR inhib- Further studies in how this resistance develops, and tech- itor [9]. The PFS observed was also at least two times niques to prevent or overcome this resistance are neces- longer than that reported in similar patients treated with sary for the success of this treatment strategy. liposomal doxorubicin alone or combined with other conventional chemotherapy drugs [12, 13]. The response rate (stable disease or better for 2 cycles) in our study was Conclusions consistent with other studies of relapsed sarcoma com- Within the confines of this small phase I/II study with bining chemotherapy with mTOR inhibition as were the a heterogeneous patient population, the combination incidence and severity of adverse events observed [11]. of temsirolimus with liposomal doxorubicin is safe and To our knowledge, while not the first study to test mTOR well tolerated, and PFS is better than previously reported inhibition combined with chemotherapy in sarcomas, with either agent given alone. Response to treatment cor- this is the first study where the effect of the combination relates with laboratory evidence of a reduction in the high specifically on a putative CSC population was assessed. ALDH population of putative sarcoma stem cells, vali- Though further limited by the small number of patients dating the concept that targeting this specific population for whom pre- and post-treatment tumor biopsy samples of cells can improve treatment outcomes. were available for analysis, response to therapy correlated high with reduction in the ALDH population. Abbreviations Recently, Mu et  al. [14] showed in murine osteosar- CSC: cancer stem cell; mTOR: mechanistic target of rapamycin; EFS: event coma cell lines that ALDH activity is dependent on free survival; PFS: progression free survival; ALDH: aldehyde dehydrogenase; RECIST: response evaluation criteria in solid tumors; BSA: bovine serum mTOR activity. While this was a single small in  vitro albumin; PBS: phosphate buffered saline; DAB: 3,3′‑ diaminobenzidine; RP2D: study on murine cell lines, it raises the possibility that recommended phase 2 dose; AST: aspartate aminotransferase; ALT: alanine the chemosensitizing effect of mTOR inhibition seen in aminotransferase; PD: progressive disease; PR: partial response; SD: stable disease. this and other trials could be due to direct inhibition of ALDH. There is increasing evidence that, in addition to Authors’ contributions being a marker for CSCs, ALDH may play an active role MMT, CFM, BAW contributed patients to the clinical trial, participated in writing the manuscript, and contributed to the data analysis; KAT partici‑ in providing CSCs their “stemness”, particularly contrib- pated in writing the clinical trial protocol, contributed patients to the clinical uting to the chemoresistance seen in these cells. ALDH trial, and contributed to the data analysis; PS, MACH, and LCB performed is a superfamily of phase I oxidizing enzymes responsible laboratory experiments; ARC participated in writing the clinical trial protocol and performed statistical analysis; MFF participated in writing the clinical for detoxification of aldehydes [2]. ALDH is implicated in trial protocol, was the research nurse for the clinical trial, and contributed to cellular “self-protection,” including supporting antioxi- the data analysis; US was the research coordinator for the clinical trial and dant factors countering the production of reactive oxygen contributed to the data analysis; JDP conceived of the trial and participated in writing the manuscript; DML conceived of the trial, contributed patients to species [15]. It is also known to inactivate chemotherapy the clinical trial, participated in writing the manuscript, and contributed to the drugs, the most recognized being cyclophosphamide and data analysis. All authors read and approved the final manuscript. related agents, but also doxorubicin, cisplatin, temozo- Author details lemide and taxanes, which are many of the cornerstones Division of Pediatric Oncology, Sidney Kimmel Comprehensive Cancer of sarcoma therapy. Furthermore, evidence supporting 2 Center, Johns Hopkins University, Baltimore, MD, USA. Division of Medical the importance of ALDH expression in the process of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. Present Address: Sylvester Comprehensive metastasis is emerging in several solid tumors includ- Cancer Center, University of Miami, Miami, FL, USA. Present Address: Dana ing osteosarcoma, where the ALDH inhibitor disulfiram 5 Farber Cancer Institute, Boston, MA, USA. Present Address: Akan Biosciences, appears to inhibit metastatic disease [16, 17]. Gaithersburg, MD, USA. Present Address: Columbia University College of Phy‑ sicians and Surgeons, New York, NY, USA. Department of Pediatrics, Albert While the mTOR pathway plays several roles in can- Einstein College of Medicine, Children’s Hospital at Montefiore, 3411 Wayne cer cell biology, including resistance to apoptosis and Ave., Room 910, Bronx, NY 10467, USA. metabolic reprogramming, this association with ALDH Acknowledgements expression and the emerging evidence of a functional Not applicable. role for ALDH highlights a potential new target for overcoming chemoresistance. The fields of breast and Competing interests The authors declare that they have no competing interests. colon cancer research, among others, are exploring Trucco et al. Clin Sarcoma Res (2018) 8:21 Page 9 of 9 Availability of data and materials 8. Reed DR, Hayashi M, Wagner L, Binitie O, Steppan DA, Brohl AS, Shino‑ Data sharing is not applicable to this article as no datasets were generated or hara ET, Bridge JA, Loeb DM, Borinstein SC, et al. Treatment pathway analysed during the current study. of bone sarcoma in children, adolescents, and young adults. Cancer. 2017;123(12):2206–18. Consent for publication 9. Chawla SP, Staddon AP, Baker LH, Schuetze SM, Tolcher AW, D’Amato GZ, Not applicable. Blay JY, Mita MM, Sankhala KK, Berk L, et al. Phase II study of the mamma‑ lian target of rapamycin inhibitor ridaforolimus in patients with advanced Ethics approval and consent to participate bone and soft tissue sarcomas. J Clin Oncol. 2012;30(1):78–84. The clinical trial reported herein was approved by the Institutional Review 10. Okuno S, Bailey H, Mahoney MR, Adkins D, Maples W, Fitch T, Ettinger D, Board of the Johns Hopkins Hospital and all patients (or their legal guardians) Erlichman C, Sarkaria JN. A phase 2 study of temsirolimus (CCI‑779) in signed informed consent according to institutional standards. patients with soft tissue sarcomas: a study of the Mayo phase 2 consor‑ tium (P2C). Cancer. 2011;117(15):3468–75. Funding 11. Martin‑Liberal J, Lopez‑Pousa A, Martinez‑ Trufero J, Martin‑Broto J, This study was approved and funded by the National Comprehensive Cancer Cubedo R, Lavernia J, Redondo A, Lopez‑Martin JA, Mulet ‑Margalef N, Network (NCCN) from general research support from Pfizer, Inc. We are also Sanjuan X, et al. Phase II study of Gemcitabine Plus Sirolimus in previously grateful for the support of The Heather Brooke Foundation. Niether funding treated patients with advanced soft‑tissue sarcoma: a Spanish Group agency played any role in the design of the study, in the collection, analysis, or for Research on Sarcomas (GEIS) Study. Target Oncol. 2017. https ://doi. interpretation of the data, nor in the writing of the manuscript.org/10.1007/s1152 3‑017‑0539‑9. 12. De Sanctis R, Bertuzzi A, Basso U, Comandone A, Marchetti S, Marrari A, Colombo P, Lutman RF, Giordano L, Santoro A. 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Journal

Clinical Sarcoma ResearchSpringer Journals

Published: Nov 5, 2018

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