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Endometrial stromal tumors: immunohistochemical and molecular analysis of potential targets of tyrosine kinase inhibitors

Endometrial stromal tumors: immunohistochemical and molecular analysis of potential targets of... Background: The systemic treatment of malignant endometrial stromal tumors (EST) is not well established. A few reports describe objective responses to imatinib, which suggest a novel therapeutic strategy for these tumors. Due to these facts, we aimed to perform a retrospective analysis of possible molecular targets of tyrosine kinase inhibitors (TKI) in EST: KIT, PDGFRA and EGFR. Methods: 52 endometrial stromal sarcomas and 13 undifferentiated endometrial sarcomas were examined and reviewed. Mutational analysis were performed for exons 9, 11, 13, and 17 of the KIT gene, exons 12 and 18 of the PDGFRA gene and exons 18, 19, 20 and 21 of the EGFR gene. The incidence and distribution of the KIT, PDGFRA, and EGFR expression were examined by immunohistochemistry, and EGFR amplification was assessed by fluorescence in situ hybridization. Results: No mutations in KIT, PDGFRA and EGFR genes were detected. Overexpression of KIT, PDGFRA, EGFR, was detected in 2 (3%), 23 (35.4%), 7 (10.8%) cases respectively, whereas amplification of EGFR gene was not found. Conclusions: Absence of significant expression, amplification and activating mutations on these tyrosine kinase receptors suggest that it is unlikely that EST can benefit from therapies such as TKI on the systemic setting. Keywords: Endometrial stromal tumors, Tyrosine kinase inhibitors, KIT, PDGFRA, EGFR, Systemic treatment Background indolent course and late recurrences, with a 5-year overall Endometrial stromal sarcoma, low grade (ESS) and undif- survival (OS) up 70%, UES is usually diagnosed at ferentiated endometrial sarcoma (UES) belong to the rare advanced stages, and has a high rate of distant metastasis group of endometrial stromal tumors (EST), which repre- and a 5-year OS ranging from 25-55% [3-6]. FIGO stage sents 15% of uterine sarcomas [1]. ESS presents a cellular [7] is the strongest prognostic factor for these malignan- background similar to the cells of normal endometrial cies [5,8]. CD10 is the most sensitive marker for ESS stroma in proliferative phase. In contrast, UES lacks spe- [9,10]. Estrogen and progesterone receptors [11] and cific differentiation and bears no histological resemblance aromatase [12] are usually expressed in ESS, and less to endometrial stroma. Tumor cells are high-grade spindle commonly in UES [13-15]. The rearrangement t(7;17) to polygonal-shaped, with marked nuclear pleomorphism (p15;q21), which results in JAZF1/JJAZ1 gene fusion, is and high mitotic activity. Necrosis and vascular invasion the cytogenetic hallmark of ESS [16], although other are commonly seen [2]. While ESS is characterized by translocations have been reported [17-19]. In contrast, UES is characterized by a complex karyotype [20,21]. Recently, the t(10;17)(q22;p13) that results in YWHAE- FAM22A/B gene fusion with oncogenic properties was * Correspondence: edealava@usal.es Equal contributors reported in a subset of UES [22], which is associated to Centro de Investigación del Cáncer-IBMCC USAL-CSIC, Salamanca, Spain the expression of Cyclin D1 [23,24] and β-catenin [23]; Full list of author information is available at the end of the article © 2013 Sardinha et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 2 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 this supports the recent sub-classification of UES [15,25]. microarray (TMA) construction, representative areas of Surgery is the standard treatment, and includes total hys- tumor were selected on hematoxylin and eosin (H&E) sec- terectomy and bilateral salpingo-oophorectomy. However, tion and marked on the paraffin block. For each sample due the rarity of these tumors, distinct clinical behavior, were obtained two cylinders of 1 mm diameter and placed and lack of randomized studies including both categories, in a recipient block using a tissue microarrayer (Manual an appropriate systemic treatment of these malignancies Tissue Array; Beecher Instruments Inc. Sun Prairie, was not been yet established. Wisconsin, USA). In total two TMAs were constructed Molecular targets of tyrosine kinase inhibitors (TKI) according to previously described [44]. such as imatinib mesylate (Glivec , STI-571, Novartis, The tyrosine kinase receptors (TKR) evaluated were KIT, Switzerland), gefitinib (Iressa , AstraZeneca, Macclesfield, PDGFRA and EGFR, and to confirm diagnosis the expres- UK) and erlotinib (Tarceva , OSI-Pharmaceuticals, New sion of two markers most commonly used on ESS [45,46], York, NY), which includes PDGFRA/B, KIT, C-ABL and CD10 and Calponin were assessed in each tumor. Immu- EGFR, were reported to be expressed in ESS and UES by nohistochemistry (IHC) was performed in 3 μmsections. immunohistochemistry [26-40], although without presence KIT, PDGFRA, CD10 and Calponin immunostaining was of any activating mutations [36-39,41,42]. Interestingly, a performed using a Discovery Ventana automated few reports described objective responses with imatinib in immunostainer (Ventana Medical Systems, Tucson, Ari- patients who express at least one TKI target [36,37,43]. zona, USA). Heat-induced antigen retrieval was done with Another report described a unique case of UES with Tris-EDTA buffer (pH 8.0) for KIT, PDGFRA and EGFR expression and EGFR amplification which tempor- Calponin and with citrate buffer (pH 6.0) for CD10. Sec- arily responded to imatinib [42]. Based on these findings, tions were incubated with the primary antibodies Calponin an extensive evaluation of the molecular targets of TKI (clone CALP; Dako, Carpinteria, CA, USA), KIT (poly- on EST was carried out to identify a novel therapeutic clonal; Dako, Carpinteria, CA, USA), PDGFRA (polyclonal; strategy for these malignancies. In the present study we Thermo Fisher Scientific, Fremont, CA, USA) and CD10 analyzed the gene status and protein expression of KIT, (clone 56C6; Novocastra Laboratoires, Newcastle upon PDGFRA, and EGFR in a large series of ESS and UES to Tyne, UK) at 1:100 dilution. Universal secondary evaluate their distribution among the distinct subgroups biotinylated antibody (Discovery Universal Secondary and correlate the immunohistochemical expression with Antibody, Ventana Medical Systems, Tucson, Arizona, mutational status. USA) was used and developed for detection using the DAB MAP system (Ventana Medical Systems, Tucson, Arizona, Material and methods USA). For EGFR analysis expression, a ready-to-use mono- Patient selection and study design clonal antibody (clone 2.1E1; Gennova Scientific, Seville, A series of 75 EST was retrieved from Spanish centers Spain) was used. Briefly, paraffin-embedded sections were associated to Spanish Sarcoma Group (GEIS) and from deparaffinized in xylene and rehydrated in downgraded al- the Pathology Departments of Complejo Hospitalario cohols and distilled water. Antigen retrieval was performed Universitario de Badajoz, Badajoz, Spain and Hospital do with Proteinase K solution (Dako, Carpinteria, CA, USA). Espírito Santo E.P.E, Évora, Portugal, and sent to Tumor The primary antibody was detected using a secondary Bank of the Cancer Research Centre – Salamanca, Spain. antibody- horseradish peroxidase polymer conjugate (Dako ™ ™ The selection of patients was made according to the REAL EnVision Detection System; Dako, Carpinteria, following inclusion criteria - previous diagnosis of EST CA, USA), and all incubations were done with the Dako (any histological grade) and availability of histological Autostainer Plus system (Dako, Carpinteria, CA, USA). All material sufficient to perform the study. After receiving sections were counterstained with hematoxylin, upgraded and encoding the samples, cases were reviewed and alcohols, and xylene, mounted, and analyzed by standard subclassified by 1 co-author (EA) based on the current light microscopy. WHO classification [2]. The study was approved by the Ethics Committee of Hospital Germans Trias i Pujol Immunohistochemical evaluation (Spain), and was conducted in accordance with the The expression of CD10 and Calponin was considered Declaration of Helsinki and Spanish regulative law for positive when over 1% of the tumor cells showed cyto- Tumor Banks. plasmic expression. The TKR expression was detected in the cytoplasm of tumor cells, and each case was Tissue microarray and immunohistochemistry interpreted for immunoreactivity using a 0 to 3 semi- 65 samples were considered valid and included in the quantitative scoring system for both the intensity of stain study. Ten cases were discarded because of a sample too and the percentage of positive cells, as previously small to perform the proposed study or non-representative reported [47]. The multiplicative index of intensity and sample or diagnosis different than EST. Before tissue labeling was considered for statistic analysis and the Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 3 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 expression was defined as weak and/or focal for a multi- EGFR plicative index 1–3, moderate local or diffuse for a PCR amplification of exons 18, 19, 20 and 21 of the multiplicative index 4–6 and intense and diffuse if the EGFR gene, which encompass most of the EGFR muta- multiplicative index was >6. The IHC analysis was tions [51,52], was carried out using previously described scored by the same co-author who performed the histo- primers [53,54] (Table 1). Using 4 μl of DNA, PCR was logic review (EA). performed in a reaction volume of 15 μl containing 1X PCR buffer (Applied Biosystems, Carlsbad, CA, USA), 2.5 mM MgCl2 (Applied Biosystems, Carlsbad, CA, Molecular analysis of tyrosine kinase receptors USA), 0.17 mM of each primer, 200 mM of each dNTP DNA extraction (GeneAmp dNTP Blend, 10 mM, Applied Biosystems, Ten serial 5 μm sections were cut and transferred into Carlsbad, CA, USA) and 2 U of Taq DNA polymerase 15 ml conical centrifugation tubes and deparaffinized in (AmpliTaq Gold DNA Polymerase, Applied Biosystems, xylene (two times for 5 minutes) and absolute ethanol Carlsbad, CA, USA). (two times for 5 minutes) at 4000 rpm, followed by The conditions for PCR were 95°C for 12 min, 40 - drying of the samples at room temperature. Subse- cycles of 30 sec at 95°C, 45 sec at 65°C and 1 min at quently, DNA extractions were performed according to 72°C, followed by a cycle of 10 min at 72°C. After QIAamp DNA Mini Kit protocol (Qiagen, Hilden, visualization of amplified products by gel electrophoresis Germany), and then quantified by spectrophotometry on a 2% agarose, these were purified with USB (SmartSpec Plus Spectrophotometer, Bio-Rad Laboratories ExoSAP-IT (Affymetrix, Inc., Cleveland, Ohio, USA). Inc., CA, USA). Subsequently the purified products were precipitated and labeled with GenomeLab DTCS Quick Start Kit KIT and PDGFRA (Beckman Coulter Inc, Fullerton, CA, USA) according to According to previous studies [48-50], exons 9, 11, 13, the manufacturer’s instructions and were sequenced and 17 of KIT gene and exons 12 and 18 of PDGFRA using GenomeLab GeXP Genetic Analysis System gene were amplified in order to identify possible muta- (Beckman Coulter Inc, Fullerton, CA, USA). As de- tions. Amplification of GAPDH gene was performed to scribed above, sequencing was performed in both direc- confirm integrity and quality of extracted DNA. PCR tions, and the same PCR primers were used. amplification of KIT and PDGFRA was performed using The sequences were analyzed using Genome Lab 1–5 μl of genomic DNA, 1X PCR buffer + 2 mM MgCl2 Genetic Analysis System v10.0.30 (Beckman Coulter Inc, (Roche Applied Science, Mannheim, Germany), 0.15 mM Fullerton, CA, USA), and compared with the reference of each primer, 200 μM of each dNTP (GeneAmp sequence ENSG00000146648. dNTP Blend, 10 mM, ABI, Carlsbad, CA, USA), and 1.5 U of Taq DNA polymerase (Roche Applied Science, Fluorescence in situ hybridization - EGFR Mannheim, Germany) in a total volume of 25 μl. The The ploidy status of EGFR in each tumor was assessed by PCR conditions were 94°C for 10 min, 40 cycles of fluorescence in situ hybridization (FISH) on previously 1 min at 94°C, 1min30sec to 56°C (KIT) or 65°C constructed TMAs. Gene amplification was determined (PDGFRA) and 1 min at 72°C, followed by a cycle of by using an EGFR/CEN-7 FISH Probe Mix (Y5500, Dako, 10 min at 72°C. The amplified products were visualized Carpinteria, CA, USA) containing Texas Red-labeled on agarose gel 2%. DNA probe covering the full EGFR region and a mixture Prior to sequence analysis, KIT and PDGFRA PCR of fluorescein-labeled PNA probes targeted at the centro- products were first purified using a QIAquick PCR meric region of chromosome 7 (Chr7). Purification Technology Kit (Qiagen, Hilden, Germany). Table 1 EGFR oligonucleotide sequences for PCR analysis Direct sequencing of PCR products was performed using W Exon Specific primers PCR product ABI PRISM BigDye Terminator Cycle Sequencing Kit 0 0 Exon 18 F:5 - GCT GAG GTG ACC CTT GTC TC -3 225 bp in an ABI Prism 3100 Genetic Analyzer (Applied 0 0 R: 5 - CTC CCC ACC AGA CCA TGA -3 Biosystems, Carlsbad, CA, USA) according to the manu- 0 0 facturer instructions. Sequencing primers were the same Exon 19 F: 5 - CAT GTG GCA CCA TCT CAC A -3 230 bp 0 0 as those used for PCR, and both strands (forward and R: 5 - CAG CTG CCA GAC ATG AGA A -3 reverse) were sequenced. 0 0 Exon 20 F: 5 - CAT TCA TGC GTC TTC ACC TG -3 377 bp All sequences obtained were visualized and analyzed in 0 0 R: 5 - CAT ATC CCC ATG GCA AAC TC -3 the program Sequence Scanner Software v1.0 (Applied 0 0 Exon 21 F: 5 - GCT CAG AGC CTG GCA TGA A -3 348 bp Biosystems, Carlsbad, CA, USA), based on the reference 0 0 R: 5 - CAT CCT CCC CTG CAT GTG T -3 sequences: KIT - ENSG00000157404 and PDGFRA - ENSG00000134853. F: forward primer; R: reverse primer. Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 4 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 The FISH was performed using the Histology FISH codon 824 (V824V) and 1 case had a double silent base Accessory Kit (Dako, Carpinteria, CA, USA), according substitution at codons 824 and 838 (GTC>GTT and to the manufacturer’s instructions. GGC>GGT; V824V and G838G respectively) both in Hybridization signals were visualized using fluorescence exon 18. There were no somatic mutations in the genes microscope equipped with a IAI monochrome progressive KIT and PDGFRA. scan (IAI Company, Taiwan) and run by image analysis soft- EGFR mutational analysis was performed only in cases ware Cytovision (Leica Microsystems, Wetzlar, Germany). with expression of EGFR, and in which DNA was avail- 20 tumour nuclei/case were scored, and the tumor cells in able for molecular studies. Two cases without EGFR which the signals of EGFR and CEP7 were increased equally expression were included as control. There were no mu- were classified as polysomy 7 and those for which there was tations in EGFR exons 18, 19, 20 and 21. In 6 of 7 cases a double signal for EGFR or CEP7 were considered dip- silent base substitution were identified at codon 787 loid. To assess gene amplification, we calculated the (CAG>CAA; Q787Q), and in one of these cases were ratio of EGFR to CEP7 and evaluated in accordance identified another silent base substitution at codon 790 with the criteria of FISH scoring system of Colorado (ACG>ACA; T790T) both in exon 20. Group [55]. Evaluation of the ploidy status of EGFR was performed by a single pathologist (EA). EGFR amplification by FISH EGFR gene amplification analysis was performed in all Statistical analysis cases included in the study (65 cases), except in one The association between the expression of TKR and which was considered not evaluable. No amplification of ploidy of EGFR with different histological types of EST, EGFR gene was observed. Numerous cases (58/64, as well the mutational status of TKR were assessed. All 90.6%) were diploid (1–2 copies), and few cases (6/64, statistical analyses were performed based on contingency 9.4%) were polyploid (3–4 copies), and the distribution tables, with SPSS software v18.0 statistics (Chicago, was similar in both histological types. Illinois). Results Discussion Classification of endometrial stromal tumors Systemic therapy in EST has a marginal efficacy. Low From 65 EST cases, 80% (52/65) were diagnosed as ESS, grade ESS can achieve control with hormonal treat- and 20% (13/65) were UES. As expected, the majority of ments (progestins, aromatase inhibitors) and high grade ESS presented expression of CD10, 51.9% (27/52), which undifferentiated uterine sarcomas are included in clin- was lower in UES cases (23.1%; 3/13). ical trials together with leiomyosarcomas. Tanner et al. reported efficacy of docetaxel and gemcitabine or Analysis of expression of tyrosine kinase receptors adriamycin for advanced cases of high grade endomet- The expression of KIT, PDGFRA and EGFR was evalu- rial tumors [56]. Indeed, the indication of systemic ated in all cases of EST, and an example of the pattern treatment of EST is controversial. Hormonal treatment of expression of these markers is shown in Figure 1. can depend on hormonal receptor status and is as- KIT stromal expression was found in only 2 out of 65 sociated to light side-effects and occasionally indi- cases, and it was weak and/or focal. In contrast, cated. Responses were reported in a few ESS cases PDGFRA showed positive stromal expression in 35.4% treated with progestin or aromatase inhibitors [57-64], of cases (23/65), mostly weak and/or focal; its distribu- but prospective larger trials are needed. Chemotherapy tion was similar in both histological subtypes. Interest- showed no apparent benefit in ESS [34,65-67]. In the ingly, expression was not intense and/or diffuse in any case of UES some objective and partial responses were case. For EGFR, a minority of cases were classified as observed [68-70]. In a retrospective study including positive, representing 10.8% (7/65). only 21 patients was described a response rate of 62% with gemcitabine/docetaxel and doxorubicin-based reg- Mutational status of tyrosine kinase receptors imens in first-line, whereas in second- or additional The molecular study was performed in 62 cases. Sequen- chemotherapy for progressive disease, the response rate cing of exons 9, 11, 13 and 17 of KIT only revealed the was around 19% [56]. On the other hand, radiotherapy presence of a silent base substitution (ATC>ATT) in seems to have a significant effect on local-regional con- exon 17 at codon 798 (I798I) in two cases (ESS). The trol rate with a low impact on survival improvement PDGFRA analysis showed the presence of several silent [3,4,71-74]. Due to these facts several reports address mutations, a base substitution (CCA>CCG) in exon 12 the need to explore the role of TKR in these malignan- at codon 567 (P567P) in all cases, 32.3% (21/65) showed cies. The possible clinical efficacy of imatinib in the a silent base substitution (GTC>GTT) in exon 18 at treatment of EST and the contribution of its TKR- Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 5 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 Figure 1 Immunohistochemical expression of tyrosine kinase receptors in endometrial stromal tumors. Endometrial stromal sarcoma, low grade (A) and undifferentiated endometrial sarcoma (B). Negative expression of KIT in ESS (C) and very focal expression in UES (D). Weak and/or focal PDGFRA and EGFR expression in ESS (E, G) and UES (F,H). target in disease progression are the key questions to KIT and PDGFRA cytoplasmic expression was ob- solve [34]. served in 3% and 35.4% respectively. In the litera- Previous reports showed the presence at least one ture, KIT expression has been reported to be quite TKR in EST, but available data just refer a few case re- variable (0-100%) (Table 2) and this might be due ports and small series, which makes it difficult to draw to differences in the choice of antibody clones, tis- conclusions. Based on this, we performed an extensive sue pretreatment, sensitivity of immunohistochemical evaluation of molecular and immunohistochemical ex- procedures, sample selection and use of whole tissue pression of TKR KIT, PDGFRA and EGFR in a large sections or TMA sections, which was also suggested series of ESS and UES. in previous studies [75,76]. On the other hand, Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 6 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 Table 2 TKR expression on endometrial stromal tumors Author KIT (%) PDGFRA (%) EGFR (%) ESS UES ESS UES ESS UES Oliva, Young et al. [79] 0/8 (0) Hornick and Fletcher [80] 0/10 (0) Winter, Seidman et al. [81] 0/1 (0) Wang, Felix et al.[26] 3/11 (27) 2/3 (67) Rushing, Shajahan et al.[27] 2/2 (100) Klein and Kurman [28] 1/10 (10) 0/2 (0) Leath, Straughn et al.[29] 3/3 (100) Caudell, Deavers et al.[35] 0/8 (0) 1/4 (25) Moinfar, Gogg-Kamerer et al.[40] 14/20 (70) 3/3 (100) Salvatierra, Tarrats et al.[36] 1/1 (100) Nakayama, Mitsuhashi et al.[76] 0/5 (0) Liegl, Gully et al.[38] 0/37 (0) 22/37 (59) Adams, Hickson et al.[39] 0/8 (0) 7/8 (88) Mitsuhashi, Nakayama et al. [42] 0/1 (0) 1/1(100) Zafrakas, Theodoridis et al.[31] 2/2 (100) 1/2 (50) Martin, Ramesh et al.[32] 1/1 (100) Trojan, Montemurro et al.[37] 0/1 (0) 1/1 (100) Koivisto-Korander, Butzow et al.[33] 2/9 (22) Cheng, Yang et al.[34] 1/12 (8) 4/12 (33) Cossu-Rocca, Contini et al.[77] 0/23 (0) 1/5 (20) 15/23 (65) 4/5 (80) 10/23 (43) 4/5 (80) Park, Kim et al.[78] 32/39 (82.1) 28/39 (71.8) 0/39 (0) Present series (2013) 1/52 (2) 1/13 (7.7) 18/52 (34.6) 5/13 (38.5) 6/52 (11.5) 1/13 (7.7) cytoplasmic PDGFRA expression in ESS, although lacks activating mutations on its related genes. Further- mostly weak and/or focal, lies within the range more, the consistent expression of KIT and PDGFR reported in previous studies [34,37-39,77,78]. receptors and their ligands was seen in a phase II trial in In our series, mutational analysis of hot spots of KIT women with ovarian cancer, together with the absence and PDGFRA genes did not reveal any somatic muta- of mutations in these genes and with lack of response to tion, which is consistent with previous studies imatinib [86]. In other two phase II clinical trials in lung [36-39,41,42,77]. Constitutive activation via autocrine/ cancer [87] and uterine carcinosarcomas [88], the high paracrine stimulation of the receptor by its ligand was expression of KIT did not correlate with response to observed in several human cancers and is related with imatinib. These clinical findings support the hypothesis their tumorigenesis [82,83]. Probably, expression of that overexpression of these TKR does not confer sensi- related ligands of KIT and PDGFRA, which were not tivity to imatinib, which was previously suggested for evaluated in this series, might be the reason for expres- other uterine sarcomas [89]. sion of both receptors in these tumors, although more However, the expression of at least one of imatinib- studies are needed. Second, the work of Kang et al. dem- target (KIT, PDGFRA and PDGFRB) without evidence of onstrated that relative KIT expression ratio was fivefold activating mutations was reported in EST patients who higher in cases with KIT mutation, than in GISTs lack- responded to imatinib treatment [36,37,43]. Tumor ing KIT mutation and the mutation status of KIT and shrinkage was the main indicator of objective response PDGFRA was directly related to the different expression accompanied by stable disease in two cases [36,37]. One levels of activated KIT and PDGFRA [84]. Furthermore, explanation for these results may be due to the autocrine/ with exception of GISTs, the presence of overexpression paracrine signaling of PDGFRB. In dermatofibrosarcoma of KIT and PDGFRA in human cancers was not corre- protuberans (DFSP), t(17;22)(q22;q13) results in a lated with presence of activating mutations [85]. These COL1A1-PDGFB gene fusion. This cytogenetic abnormal- facts support our results, once that KIT and PDGFRA ity is essential for pathogenesis of the disease and respon- expression observed was mostly weak and/or focal and sible by constitutive activation of PDGFRB. Imatinib Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 7 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 inhibits the activity of the dysregulated PDGFRB [90], results in a 14-3-3 fusion protein with oncogenic proper- decreases enzymatic activity in DFSP cells and inhibits ties in a subgroup of UES [22], and the nuclear expres- their ability to divide and grow [91] and induces apoptosis sion of β-catenin, a member of Wnt and E-cadherin in tumor cells [92], which may have effects on decreasing signaling pathways in EST need to be better explored, tumor size [93]. In fact, an effect of imatinib on tumor since they can represent an entry point for targeted shrinkage was observed in patients with DFSP [94,95], and therapeutic strategies. therefore imatinib was approved for unresectable, recur- Competing interests rent, and/or metastatic DFSP in adults. However, in ESS Support for this study was obtained from Novartis. and UES cases reported as responsive to imatinib, PDGFRB was only determined in one case reported by Authors’ contributions RS carried out the molecular genetic studies, performed the sequence Trojan et al. [37], which makes difficult to draw any alignment and data analyses and drafted the manuscript. TH and SF carried conclusions. out the TMA construction and immunohistochemistry assays. TH carried out Concerning EGFR expression, our study reveals lower fluorescent hybridization assay and participated in the sequence alignment. FT, AV, MCG, AA, NH, JS, JO, LG and RR provided the samples for the study. cytoplasmic expression of EGFR in EST cases (around CB and EA conceived of the study, and participated in its design and 10.8%), in contrast with the results obtained by other coordination and helped to draft the manuscript. All authors read and authors [40,77] (Table 2). This contradiction may be a approved the final manuscript. consequence of the same variables indicated for the Acknowledgements immunoexpression of KIT. Since i) a correlation We would like to thank Professor Russell Alpizar-Jara (Department of between an increased EGFR copy number and gefitinib Mathematics, University of Évora, Portugal) for his statistical advice. was proposed in non-small-cell lung cancer (NSCLC) Author details [96] and ii) immunohistochemistry is not a reliable ap- 1 2 Pathology Department, Hospital Espírito Santo E.P.E, Évora, Portugal. Centro proach for this determination, we decided to evaluate 3 de Investigación del Cáncer-IBMCC USAL-CSIC, Salamanca, Spain. Pathology EGFR amplification status in our series by FISH. The Department, USP-Institut Universitari Dexeus, Barcelona, Spain. Pathology Department, Hospital de Bellvitge, Barcelona, Spain. Pathology Department, EGFR/Chr7 ratio was below 2, which is indicative of Hospital Universitari Germans Trias i Pujol, Badalona, Spain. Pathology non-amplification, and only a small percentage of the Department, Hospital Universitario Central de Asturias, Oviedo, Spain. cases presented polysomy. Previous evaluation of EGFR Anatomical Pathology Department, Hospital Universitario de la Laguna, Canarias, Spain. Pathology Department, Complejo Hospitalario Universitario amplification was only performed in a UES case which de Badajoz, Badajoz, Spain. Pathology Department, Hospital Clinic de presented a temporary response to imatinib. This case 10 Barcelona, Barcelona, Spain. Pathology Department, Hospital do Espírito presented a low-level amplification (mean ratio 2.9) Santo E.P.E, Évora, Portugal. Pathology Department, Hospital Son Espases, Palma de Mallorca, Spain. Medical Oncology Service, Catalan Institute of according to the evaluation criteria of the authors [42]. Oncology - Hospital Germans Trias i Pujol, Badalona, Spain. Although no consensus has been reached on how to assess the presence and extent of EGFR status Received: 13 December 2012 Accepted: 4 March 2013 Published: 7 March 2013 dysregulation in solid tumors by FISH analysis [97], it seems likely that gene amplification found in NSCLC References and glioblastoma is an uncommon event in EST. EGFR 1. Koivisto-Korander R, Butzow R, Koivisto AM, Leminen A: Clinical outcome mutational status also did not show any somatic muta- and prognostic factors in 100 cases of uterine sarcoma: experience in Helsinki University Central Hospital 1990–2001. Gynecol Oncol 2008, tion in exons 18–21. In fact, all these data confirm that 111:74–81. it is unlikely that EGFR activation could play a role in 2. WHO: Pathology and genetics of tumours of the breast and female genital tumorigenesis of EST. organs. Lyon: IARC Press; 2003. 3. 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J Clin Pathol 2009, 62:314–24. doi:10.1186/2045-3329-3-3 Cite this article as: Sardinha et al.: Endometrial stromal tumors: immunohistochemical and molecular analysis of potential targets of tyrosine kinase inhibitors. Clinical Sarcoma Research 2013 3:3. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Sarcoma Research Springer Journals

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Abstract

Background: The systemic treatment of malignant endometrial stromal tumors (EST) is not well established. A few reports describe objective responses to imatinib, which suggest a novel therapeutic strategy for these tumors. Due to these facts, we aimed to perform a retrospective analysis of possible molecular targets of tyrosine kinase inhibitors (TKI) in EST: KIT, PDGFRA and EGFR. Methods: 52 endometrial stromal sarcomas and 13 undifferentiated endometrial sarcomas were examined and reviewed. Mutational analysis were performed for exons 9, 11, 13, and 17 of the KIT gene, exons 12 and 18 of the PDGFRA gene and exons 18, 19, 20 and 21 of the EGFR gene. The incidence and distribution of the KIT, PDGFRA, and EGFR expression were examined by immunohistochemistry, and EGFR amplification was assessed by fluorescence in situ hybridization. Results: No mutations in KIT, PDGFRA and EGFR genes were detected. Overexpression of KIT, PDGFRA, EGFR, was detected in 2 (3%), 23 (35.4%), 7 (10.8%) cases respectively, whereas amplification of EGFR gene was not found. Conclusions: Absence of significant expression, amplification and activating mutations on these tyrosine kinase receptors suggest that it is unlikely that EST can benefit from therapies such as TKI on the systemic setting. Keywords: Endometrial stromal tumors, Tyrosine kinase inhibitors, KIT, PDGFRA, EGFR, Systemic treatment Background indolent course and late recurrences, with a 5-year overall Endometrial stromal sarcoma, low grade (ESS) and undif- survival (OS) up 70%, UES is usually diagnosed at ferentiated endometrial sarcoma (UES) belong to the rare advanced stages, and has a high rate of distant metastasis group of endometrial stromal tumors (EST), which repre- and a 5-year OS ranging from 25-55% [3-6]. FIGO stage sents 15% of uterine sarcomas [1]. ESS presents a cellular [7] is the strongest prognostic factor for these malignan- background similar to the cells of normal endometrial cies [5,8]. CD10 is the most sensitive marker for ESS stroma in proliferative phase. In contrast, UES lacks spe- [9,10]. Estrogen and progesterone receptors [11] and cific differentiation and bears no histological resemblance aromatase [12] are usually expressed in ESS, and less to endometrial stroma. Tumor cells are high-grade spindle commonly in UES [13-15]. The rearrangement t(7;17) to polygonal-shaped, with marked nuclear pleomorphism (p15;q21), which results in JAZF1/JJAZ1 gene fusion, is and high mitotic activity. Necrosis and vascular invasion the cytogenetic hallmark of ESS [16], although other are commonly seen [2]. While ESS is characterized by translocations have been reported [17-19]. In contrast, UES is characterized by a complex karyotype [20,21]. Recently, the t(10;17)(q22;p13) that results in YWHAE- FAM22A/B gene fusion with oncogenic properties was * Correspondence: edealava@usal.es Equal contributors reported in a subset of UES [22], which is associated to Centro de Investigación del Cáncer-IBMCC USAL-CSIC, Salamanca, Spain the expression of Cyclin D1 [23,24] and β-catenin [23]; Full list of author information is available at the end of the article © 2013 Sardinha et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 2 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 this supports the recent sub-classification of UES [15,25]. microarray (TMA) construction, representative areas of Surgery is the standard treatment, and includes total hys- tumor were selected on hematoxylin and eosin (H&E) sec- terectomy and bilateral salpingo-oophorectomy. However, tion and marked on the paraffin block. For each sample due the rarity of these tumors, distinct clinical behavior, were obtained two cylinders of 1 mm diameter and placed and lack of randomized studies including both categories, in a recipient block using a tissue microarrayer (Manual an appropriate systemic treatment of these malignancies Tissue Array; Beecher Instruments Inc. Sun Prairie, was not been yet established. Wisconsin, USA). In total two TMAs were constructed Molecular targets of tyrosine kinase inhibitors (TKI) according to previously described [44]. such as imatinib mesylate (Glivec , STI-571, Novartis, The tyrosine kinase receptors (TKR) evaluated were KIT, Switzerland), gefitinib (Iressa , AstraZeneca, Macclesfield, PDGFRA and EGFR, and to confirm diagnosis the expres- UK) and erlotinib (Tarceva , OSI-Pharmaceuticals, New sion of two markers most commonly used on ESS [45,46], York, NY), which includes PDGFRA/B, KIT, C-ABL and CD10 and Calponin were assessed in each tumor. Immu- EGFR, were reported to be expressed in ESS and UES by nohistochemistry (IHC) was performed in 3 μmsections. immunohistochemistry [26-40], although without presence KIT, PDGFRA, CD10 and Calponin immunostaining was of any activating mutations [36-39,41,42]. Interestingly, a performed using a Discovery Ventana automated few reports described objective responses with imatinib in immunostainer (Ventana Medical Systems, Tucson, Ari- patients who express at least one TKI target [36,37,43]. zona, USA). Heat-induced antigen retrieval was done with Another report described a unique case of UES with Tris-EDTA buffer (pH 8.0) for KIT, PDGFRA and EGFR expression and EGFR amplification which tempor- Calponin and with citrate buffer (pH 6.0) for CD10. Sec- arily responded to imatinib [42]. Based on these findings, tions were incubated with the primary antibodies Calponin an extensive evaluation of the molecular targets of TKI (clone CALP; Dako, Carpinteria, CA, USA), KIT (poly- on EST was carried out to identify a novel therapeutic clonal; Dako, Carpinteria, CA, USA), PDGFRA (polyclonal; strategy for these malignancies. In the present study we Thermo Fisher Scientific, Fremont, CA, USA) and CD10 analyzed the gene status and protein expression of KIT, (clone 56C6; Novocastra Laboratoires, Newcastle upon PDGFRA, and EGFR in a large series of ESS and UES to Tyne, UK) at 1:100 dilution. Universal secondary evaluate their distribution among the distinct subgroups biotinylated antibody (Discovery Universal Secondary and correlate the immunohistochemical expression with Antibody, Ventana Medical Systems, Tucson, Arizona, mutational status. USA) was used and developed for detection using the DAB MAP system (Ventana Medical Systems, Tucson, Arizona, Material and methods USA). For EGFR analysis expression, a ready-to-use mono- Patient selection and study design clonal antibody (clone 2.1E1; Gennova Scientific, Seville, A series of 75 EST was retrieved from Spanish centers Spain) was used. Briefly, paraffin-embedded sections were associated to Spanish Sarcoma Group (GEIS) and from deparaffinized in xylene and rehydrated in downgraded al- the Pathology Departments of Complejo Hospitalario cohols and distilled water. Antigen retrieval was performed Universitario de Badajoz, Badajoz, Spain and Hospital do with Proteinase K solution (Dako, Carpinteria, CA, USA). Espírito Santo E.P.E, Évora, Portugal, and sent to Tumor The primary antibody was detected using a secondary Bank of the Cancer Research Centre – Salamanca, Spain. antibody- horseradish peroxidase polymer conjugate (Dako ™ ™ The selection of patients was made according to the REAL EnVision Detection System; Dako, Carpinteria, following inclusion criteria - previous diagnosis of EST CA, USA), and all incubations were done with the Dako (any histological grade) and availability of histological Autostainer Plus system (Dako, Carpinteria, CA, USA). All material sufficient to perform the study. After receiving sections were counterstained with hematoxylin, upgraded and encoding the samples, cases were reviewed and alcohols, and xylene, mounted, and analyzed by standard subclassified by 1 co-author (EA) based on the current light microscopy. WHO classification [2]. The study was approved by the Ethics Committee of Hospital Germans Trias i Pujol Immunohistochemical evaluation (Spain), and was conducted in accordance with the The expression of CD10 and Calponin was considered Declaration of Helsinki and Spanish regulative law for positive when over 1% of the tumor cells showed cyto- Tumor Banks. plasmic expression. The TKR expression was detected in the cytoplasm of tumor cells, and each case was Tissue microarray and immunohistochemistry interpreted for immunoreactivity using a 0 to 3 semi- 65 samples were considered valid and included in the quantitative scoring system for both the intensity of stain study. Ten cases were discarded because of a sample too and the percentage of positive cells, as previously small to perform the proposed study or non-representative reported [47]. The multiplicative index of intensity and sample or diagnosis different than EST. Before tissue labeling was considered for statistic analysis and the Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 3 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 expression was defined as weak and/or focal for a multi- EGFR plicative index 1–3, moderate local or diffuse for a PCR amplification of exons 18, 19, 20 and 21 of the multiplicative index 4–6 and intense and diffuse if the EGFR gene, which encompass most of the EGFR muta- multiplicative index was >6. The IHC analysis was tions [51,52], was carried out using previously described scored by the same co-author who performed the histo- primers [53,54] (Table 1). Using 4 μl of DNA, PCR was logic review (EA). performed in a reaction volume of 15 μl containing 1X PCR buffer (Applied Biosystems, Carlsbad, CA, USA), 2.5 mM MgCl2 (Applied Biosystems, Carlsbad, CA, Molecular analysis of tyrosine kinase receptors USA), 0.17 mM of each primer, 200 mM of each dNTP DNA extraction (GeneAmp dNTP Blend, 10 mM, Applied Biosystems, Ten serial 5 μm sections were cut and transferred into Carlsbad, CA, USA) and 2 U of Taq DNA polymerase 15 ml conical centrifugation tubes and deparaffinized in (AmpliTaq Gold DNA Polymerase, Applied Biosystems, xylene (two times for 5 minutes) and absolute ethanol Carlsbad, CA, USA). (two times for 5 minutes) at 4000 rpm, followed by The conditions for PCR were 95°C for 12 min, 40 - drying of the samples at room temperature. Subse- cycles of 30 sec at 95°C, 45 sec at 65°C and 1 min at quently, DNA extractions were performed according to 72°C, followed by a cycle of 10 min at 72°C. After QIAamp DNA Mini Kit protocol (Qiagen, Hilden, visualization of amplified products by gel electrophoresis Germany), and then quantified by spectrophotometry on a 2% agarose, these were purified with USB (SmartSpec Plus Spectrophotometer, Bio-Rad Laboratories ExoSAP-IT (Affymetrix, Inc., Cleveland, Ohio, USA). Inc., CA, USA). Subsequently the purified products were precipitated and labeled with GenomeLab DTCS Quick Start Kit KIT and PDGFRA (Beckman Coulter Inc, Fullerton, CA, USA) according to According to previous studies [48-50], exons 9, 11, 13, the manufacturer’s instructions and were sequenced and 17 of KIT gene and exons 12 and 18 of PDGFRA using GenomeLab GeXP Genetic Analysis System gene were amplified in order to identify possible muta- (Beckman Coulter Inc, Fullerton, CA, USA). As de- tions. Amplification of GAPDH gene was performed to scribed above, sequencing was performed in both direc- confirm integrity and quality of extracted DNA. PCR tions, and the same PCR primers were used. amplification of KIT and PDGFRA was performed using The sequences were analyzed using Genome Lab 1–5 μl of genomic DNA, 1X PCR buffer + 2 mM MgCl2 Genetic Analysis System v10.0.30 (Beckman Coulter Inc, (Roche Applied Science, Mannheim, Germany), 0.15 mM Fullerton, CA, USA), and compared with the reference of each primer, 200 μM of each dNTP (GeneAmp sequence ENSG00000146648. dNTP Blend, 10 mM, ABI, Carlsbad, CA, USA), and 1.5 U of Taq DNA polymerase (Roche Applied Science, Fluorescence in situ hybridization - EGFR Mannheim, Germany) in a total volume of 25 μl. The The ploidy status of EGFR in each tumor was assessed by PCR conditions were 94°C for 10 min, 40 cycles of fluorescence in situ hybridization (FISH) on previously 1 min at 94°C, 1min30sec to 56°C (KIT) or 65°C constructed TMAs. Gene amplification was determined (PDGFRA) and 1 min at 72°C, followed by a cycle of by using an EGFR/CEN-7 FISH Probe Mix (Y5500, Dako, 10 min at 72°C. The amplified products were visualized Carpinteria, CA, USA) containing Texas Red-labeled on agarose gel 2%. DNA probe covering the full EGFR region and a mixture Prior to sequence analysis, KIT and PDGFRA PCR of fluorescein-labeled PNA probes targeted at the centro- products were first purified using a QIAquick PCR meric region of chromosome 7 (Chr7). Purification Technology Kit (Qiagen, Hilden, Germany). Table 1 EGFR oligonucleotide sequences for PCR analysis Direct sequencing of PCR products was performed using W Exon Specific primers PCR product ABI PRISM BigDye Terminator Cycle Sequencing Kit 0 0 Exon 18 F:5 - GCT GAG GTG ACC CTT GTC TC -3 225 bp in an ABI Prism 3100 Genetic Analyzer (Applied 0 0 R: 5 - CTC CCC ACC AGA CCA TGA -3 Biosystems, Carlsbad, CA, USA) according to the manu- 0 0 facturer instructions. Sequencing primers were the same Exon 19 F: 5 - CAT GTG GCA CCA TCT CAC A -3 230 bp 0 0 as those used for PCR, and both strands (forward and R: 5 - CAG CTG CCA GAC ATG AGA A -3 reverse) were sequenced. 0 0 Exon 20 F: 5 - CAT TCA TGC GTC TTC ACC TG -3 377 bp All sequences obtained were visualized and analyzed in 0 0 R: 5 - CAT ATC CCC ATG GCA AAC TC -3 the program Sequence Scanner Software v1.0 (Applied 0 0 Exon 21 F: 5 - GCT CAG AGC CTG GCA TGA A -3 348 bp Biosystems, Carlsbad, CA, USA), based on the reference 0 0 R: 5 - CAT CCT CCC CTG CAT GTG T -3 sequences: KIT - ENSG00000157404 and PDGFRA - ENSG00000134853. F: forward primer; R: reverse primer. Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 4 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 The FISH was performed using the Histology FISH codon 824 (V824V) and 1 case had a double silent base Accessory Kit (Dako, Carpinteria, CA, USA), according substitution at codons 824 and 838 (GTC>GTT and to the manufacturer’s instructions. GGC>GGT; V824V and G838G respectively) both in Hybridization signals were visualized using fluorescence exon 18. There were no somatic mutations in the genes microscope equipped with a IAI monochrome progressive KIT and PDGFRA. scan (IAI Company, Taiwan) and run by image analysis soft- EGFR mutational analysis was performed only in cases ware Cytovision (Leica Microsystems, Wetzlar, Germany). with expression of EGFR, and in which DNA was avail- 20 tumour nuclei/case were scored, and the tumor cells in able for molecular studies. Two cases without EGFR which the signals of EGFR and CEP7 were increased equally expression were included as control. There were no mu- were classified as polysomy 7 and those for which there was tations in EGFR exons 18, 19, 20 and 21. In 6 of 7 cases a double signal for EGFR or CEP7 were considered dip- silent base substitution were identified at codon 787 loid. To assess gene amplification, we calculated the (CAG>CAA; Q787Q), and in one of these cases were ratio of EGFR to CEP7 and evaluated in accordance identified another silent base substitution at codon 790 with the criteria of FISH scoring system of Colorado (ACG>ACA; T790T) both in exon 20. Group [55]. Evaluation of the ploidy status of EGFR was performed by a single pathologist (EA). EGFR amplification by FISH EGFR gene amplification analysis was performed in all Statistical analysis cases included in the study (65 cases), except in one The association between the expression of TKR and which was considered not evaluable. No amplification of ploidy of EGFR with different histological types of EST, EGFR gene was observed. Numerous cases (58/64, as well the mutational status of TKR were assessed. All 90.6%) were diploid (1–2 copies), and few cases (6/64, statistical analyses were performed based on contingency 9.4%) were polyploid (3–4 copies), and the distribution tables, with SPSS software v18.0 statistics (Chicago, was similar in both histological types. Illinois). Results Discussion Classification of endometrial stromal tumors Systemic therapy in EST has a marginal efficacy. Low From 65 EST cases, 80% (52/65) were diagnosed as ESS, grade ESS can achieve control with hormonal treat- and 20% (13/65) were UES. As expected, the majority of ments (progestins, aromatase inhibitors) and high grade ESS presented expression of CD10, 51.9% (27/52), which undifferentiated uterine sarcomas are included in clin- was lower in UES cases (23.1%; 3/13). ical trials together with leiomyosarcomas. Tanner et al. reported efficacy of docetaxel and gemcitabine or Analysis of expression of tyrosine kinase receptors adriamycin for advanced cases of high grade endomet- The expression of KIT, PDGFRA and EGFR was evalu- rial tumors [56]. Indeed, the indication of systemic ated in all cases of EST, and an example of the pattern treatment of EST is controversial. Hormonal treatment of expression of these markers is shown in Figure 1. can depend on hormonal receptor status and is as- KIT stromal expression was found in only 2 out of 65 sociated to light side-effects and occasionally indi- cases, and it was weak and/or focal. In contrast, cated. Responses were reported in a few ESS cases PDGFRA showed positive stromal expression in 35.4% treated with progestin or aromatase inhibitors [57-64], of cases (23/65), mostly weak and/or focal; its distribu- but prospective larger trials are needed. Chemotherapy tion was similar in both histological subtypes. Interest- showed no apparent benefit in ESS [34,65-67]. In the ingly, expression was not intense and/or diffuse in any case of UES some objective and partial responses were case. For EGFR, a minority of cases were classified as observed [68-70]. In a retrospective study including positive, representing 10.8% (7/65). only 21 patients was described a response rate of 62% with gemcitabine/docetaxel and doxorubicin-based reg- Mutational status of tyrosine kinase receptors imens in first-line, whereas in second- or additional The molecular study was performed in 62 cases. Sequen- chemotherapy for progressive disease, the response rate cing of exons 9, 11, 13 and 17 of KIT only revealed the was around 19% [56]. On the other hand, radiotherapy presence of a silent base substitution (ATC>ATT) in seems to have a significant effect on local-regional con- exon 17 at codon 798 (I798I) in two cases (ESS). The trol rate with a low impact on survival improvement PDGFRA analysis showed the presence of several silent [3,4,71-74]. Due to these facts several reports address mutations, a base substitution (CCA>CCG) in exon 12 the need to explore the role of TKR in these malignan- at codon 567 (P567P) in all cases, 32.3% (21/65) showed cies. The possible clinical efficacy of imatinib in the a silent base substitution (GTC>GTT) in exon 18 at treatment of EST and the contribution of its TKR- Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 5 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 Figure 1 Immunohistochemical expression of tyrosine kinase receptors in endometrial stromal tumors. Endometrial stromal sarcoma, low grade (A) and undifferentiated endometrial sarcoma (B). Negative expression of KIT in ESS (C) and very focal expression in UES (D). Weak and/or focal PDGFRA and EGFR expression in ESS (E, G) and UES (F,H). target in disease progression are the key questions to KIT and PDGFRA cytoplasmic expression was ob- solve [34]. served in 3% and 35.4% respectively. In the litera- Previous reports showed the presence at least one ture, KIT expression has been reported to be quite TKR in EST, but available data just refer a few case re- variable (0-100%) (Table 2) and this might be due ports and small series, which makes it difficult to draw to differences in the choice of antibody clones, tis- conclusions. Based on this, we performed an extensive sue pretreatment, sensitivity of immunohistochemical evaluation of molecular and immunohistochemical ex- procedures, sample selection and use of whole tissue pression of TKR KIT, PDGFRA and EGFR in a large sections or TMA sections, which was also suggested series of ESS and UES. in previous studies [75,76]. On the other hand, Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 6 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 Table 2 TKR expression on endometrial stromal tumors Author KIT (%) PDGFRA (%) EGFR (%) ESS UES ESS UES ESS UES Oliva, Young et al. [79] 0/8 (0) Hornick and Fletcher [80] 0/10 (0) Winter, Seidman et al. [81] 0/1 (0) Wang, Felix et al.[26] 3/11 (27) 2/3 (67) Rushing, Shajahan et al.[27] 2/2 (100) Klein and Kurman [28] 1/10 (10) 0/2 (0) Leath, Straughn et al.[29] 3/3 (100) Caudell, Deavers et al.[35] 0/8 (0) 1/4 (25) Moinfar, Gogg-Kamerer et al.[40] 14/20 (70) 3/3 (100) Salvatierra, Tarrats et al.[36] 1/1 (100) Nakayama, Mitsuhashi et al.[76] 0/5 (0) Liegl, Gully et al.[38] 0/37 (0) 22/37 (59) Adams, Hickson et al.[39] 0/8 (0) 7/8 (88) Mitsuhashi, Nakayama et al. [42] 0/1 (0) 1/1(100) Zafrakas, Theodoridis et al.[31] 2/2 (100) 1/2 (50) Martin, Ramesh et al.[32] 1/1 (100) Trojan, Montemurro et al.[37] 0/1 (0) 1/1 (100) Koivisto-Korander, Butzow et al.[33] 2/9 (22) Cheng, Yang et al.[34] 1/12 (8) 4/12 (33) Cossu-Rocca, Contini et al.[77] 0/23 (0) 1/5 (20) 15/23 (65) 4/5 (80) 10/23 (43) 4/5 (80) Park, Kim et al.[78] 32/39 (82.1) 28/39 (71.8) 0/39 (0) Present series (2013) 1/52 (2) 1/13 (7.7) 18/52 (34.6) 5/13 (38.5) 6/52 (11.5) 1/13 (7.7) cytoplasmic PDGFRA expression in ESS, although lacks activating mutations on its related genes. Further- mostly weak and/or focal, lies within the range more, the consistent expression of KIT and PDGFR reported in previous studies [34,37-39,77,78]. receptors and their ligands was seen in a phase II trial in In our series, mutational analysis of hot spots of KIT women with ovarian cancer, together with the absence and PDGFRA genes did not reveal any somatic muta- of mutations in these genes and with lack of response to tion, which is consistent with previous studies imatinib [86]. In other two phase II clinical trials in lung [36-39,41,42,77]. Constitutive activation via autocrine/ cancer [87] and uterine carcinosarcomas [88], the high paracrine stimulation of the receptor by its ligand was expression of KIT did not correlate with response to observed in several human cancers and is related with imatinib. These clinical findings support the hypothesis their tumorigenesis [82,83]. Probably, expression of that overexpression of these TKR does not confer sensi- related ligands of KIT and PDGFRA, which were not tivity to imatinib, which was previously suggested for evaluated in this series, might be the reason for expres- other uterine sarcomas [89]. sion of both receptors in these tumors, although more However, the expression of at least one of imatinib- studies are needed. Second, the work of Kang et al. dem- target (KIT, PDGFRA and PDGFRB) without evidence of onstrated that relative KIT expression ratio was fivefold activating mutations was reported in EST patients who higher in cases with KIT mutation, than in GISTs lack- responded to imatinib treatment [36,37,43]. Tumor ing KIT mutation and the mutation status of KIT and shrinkage was the main indicator of objective response PDGFRA was directly related to the different expression accompanied by stable disease in two cases [36,37]. One levels of activated KIT and PDGFRA [84]. Furthermore, explanation for these results may be due to the autocrine/ with exception of GISTs, the presence of overexpression paracrine signaling of PDGFRB. In dermatofibrosarcoma of KIT and PDGFRA in human cancers was not corre- protuberans (DFSP), t(17;22)(q22;q13) results in a lated with presence of activating mutations [85]. These COL1A1-PDGFB gene fusion. This cytogenetic abnormal- facts support our results, once that KIT and PDGFRA ity is essential for pathogenesis of the disease and respon- expression observed was mostly weak and/or focal and sible by constitutive activation of PDGFRB. Imatinib Sardinha et al. Clinical Sarcoma Research 2013, 3:3 Page 7 of 10 http://www.clinicalsarcomaresearch.com/content/3/1/3 inhibits the activity of the dysregulated PDGFRB [90], results in a 14-3-3 fusion protein with oncogenic proper- decreases enzymatic activity in DFSP cells and inhibits ties in a subgroup of UES [22], and the nuclear expres- their ability to divide and grow [91] and induces apoptosis sion of β-catenin, a member of Wnt and E-cadherin in tumor cells [92], which may have effects on decreasing signaling pathways in EST need to be better explored, tumor size [93]. In fact, an effect of imatinib on tumor since they can represent an entry point for targeted shrinkage was observed in patients with DFSP [94,95], and therapeutic strategies. therefore imatinib was approved for unresectable, recur- Competing interests rent, and/or metastatic DFSP in adults. However, in ESS Support for this study was obtained from Novartis. and UES cases reported as responsive to imatinib, PDGFRB was only determined in one case reported by Authors’ contributions RS carried out the molecular genetic studies, performed the sequence Trojan et al. [37], which makes difficult to draw any alignment and data analyses and drafted the manuscript. TH and SF carried conclusions. out the TMA construction and immunohistochemistry assays. TH carried out Concerning EGFR expression, our study reveals lower fluorescent hybridization assay and participated in the sequence alignment. FT, AV, MCG, AA, NH, JS, JO, LG and RR provided the samples for the study. cytoplasmic expression of EGFR in EST cases (around CB and EA conceived of the study, and participated in its design and 10.8%), in contrast with the results obtained by other coordination and helped to draft the manuscript. All authors read and authors [40,77] (Table 2). This contradiction may be a approved the final manuscript. consequence of the same variables indicated for the Acknowledgements immunoexpression of KIT. Since i) a correlation We would like to thank Professor Russell Alpizar-Jara (Department of between an increased EGFR copy number and gefitinib Mathematics, University of Évora, Portugal) for his statistical advice. was proposed in non-small-cell lung cancer (NSCLC) Author details [96] and ii) immunohistochemistry is not a reliable ap- 1 2 Pathology Department, Hospital Espírito Santo E.P.E, Évora, Portugal. Centro proach for this determination, we decided to evaluate 3 de Investigación del Cáncer-IBMCC USAL-CSIC, Salamanca, Spain. Pathology EGFR amplification status in our series by FISH. The Department, USP-Institut Universitari Dexeus, Barcelona, Spain. Pathology Department, Hospital de Bellvitge, Barcelona, Spain. Pathology Department, EGFR/Chr7 ratio was below 2, which is indicative of Hospital Universitari Germans Trias i Pujol, Badalona, Spain. Pathology non-amplification, and only a small percentage of the Department, Hospital Universitario Central de Asturias, Oviedo, Spain. cases presented polysomy. Previous evaluation of EGFR Anatomical Pathology Department, Hospital Universitario de la Laguna, Canarias, Spain. Pathology Department, Complejo Hospitalario Universitario amplification was only performed in a UES case which de Badajoz, Badajoz, Spain. Pathology Department, Hospital Clinic de presented a temporary response to imatinib. This case 10 Barcelona, Barcelona, Spain. Pathology Department, Hospital do Espírito presented a low-level amplification (mean ratio 2.9) Santo E.P.E, Évora, Portugal. Pathology Department, Hospital Son Espases, Palma de Mallorca, Spain. Medical Oncology Service, Catalan Institute of according to the evaluation criteria of the authors [42]. Oncology - Hospital Germans Trias i Pujol, Badalona, Spain. Although no consensus has been reached on how to assess the presence and extent of EGFR status Received: 13 December 2012 Accepted: 4 March 2013 Published: 7 March 2013 dysregulation in solid tumors by FISH analysis [97], it seems likely that gene amplification found in NSCLC References and glioblastoma is an uncommon event in EST. EGFR 1. Koivisto-Korander R, Butzow R, Koivisto AM, Leminen A: Clinical outcome mutational status also did not show any somatic muta- and prognostic factors in 100 cases of uterine sarcoma: experience in Helsinki University Central Hospital 1990–2001. Gynecol Oncol 2008, tion in exons 18–21. In fact, all these data confirm that 111:74–81. it is unlikely that EGFR activation could play a role in 2. WHO: Pathology and genetics of tumours of the breast and female genital tumorigenesis of EST. organs. Lyon: IARC Press; 2003. 3. 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J Clin Pathol 2009, 62:314–24. doi:10.1186/2045-3329-3-3 Cite this article as: Sardinha et al.: Endometrial stromal tumors: immunohistochemical and molecular analysis of potential targets of tyrosine kinase inhibitors. Clinical Sarcoma Research 2013 3:3. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Clinical Sarcoma ResearchSpringer Journals

Published: Mar 7, 2013

References