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Supplementary Table 1 from Coactivated Platelet-Derived Growth Factor Receptor α and Epidermal Growth Factor Receptor Are Potential Therapeutic Targets in Intimal Sarcoma

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease. Cancer Res; 70(18); 7304–14. ©2010 AACR.

Supplementary Table 2 from Coactivated Platelet-Derived Growth Factor Receptor α and Epidermal Growth Factor Receptor Are Potential Therapeutic Targets in Intimal Sarcoma

Supplementary Figure 2 from Coactivated Platelet-Derived Growth Factor Receptor α and Epidermal Growth Factor Receptor Are Potential Therapeutic Targets in Intimal Sarcoma

Supplementary Figure Legends 1-2 from Coactivated Platelet-Derived Growth Factor Receptor α and Epidermal Growth Factor Receptor Are Potential Therapeutic Targets in Intimal Sarcoma

Supplementary Table 3 from Coactivated Platelet-Derived Growth Factor Receptor α and Epidermal Growth Factor Receptor Are Potential Therapeutic Targets in Intimal Sarcoma

Supplementary Figure 1 from Coactivated Platelet-Derived Growth Factor Receptor α and Epidermal Growth Factor Receptor Are Potential Therapeutic Targets in Intimal Sarcoma

Sclerosing epithelioid fibrosarcoma (SEF) is a rare, malignant fibroblastic neoplasm, morphologically composed of cords, nests or sheets of monotonous epithelioid cells within a collagenous matrix. It has been recently characterized by recurrent pathogenic EWS-CREB3L1/2 or FUS-CREB3L2 fusions and common MUC4 protein expression by immunohistochemistry. Typically SEF occur in middle-aged adults and rarely have been reported within the abdominal cavity. Here we report an 18-year-old man with intraabdominal tumor and multiple disseminated liver metastases, presenting pure SEF histologic and immunophenotypic features. Fluorescence in situ hybridization analysis showed unbalanced rearrangement of Ewing sarcoma breakpoint region 1 (EWSR1) gene. Genomic profiling by array CGH, followed by RT-PCR and sequencing analysis, revealed a previously not reported EWSR1 translocation partner, cAMP-responsive element-binding protein 3-like 3 (CREB3L3). The novel EWSR1-CREB3L3 fusion further extends the range of fusion types involving EWSR1 that are characteristic for SEF.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare, malignant fibroblastic neoplasm, morphologically composed of cords, nests or sheets of monotonous epithelioid cells within a collagenous matrix. It has been recently characterized by recurrent pathogenic EWS-CREB3L1/2 or FUS-CREB3L2 fusions and common MUC4 protein expression by immunohistochemistry. Typically SEF occur in middle-aged adults and rarely have been reported within the abdominal cavity. Here we report an 18-year-old man with intraabdominal tumor and multiple disseminated liver metastases, presenting pure SEF histologic and immunophenotypic features. Fluorescence in situ hybridization analysis showed unbalanced rearrangement of Ewing sarcoma breakpoint region 1 (EWSR1) gene. Genomic profiling by array CGH, followed by RT-PCR and sequencing analysis, revealed a previously not reported EWSR1 translocation partner, cAMP-responsive element-binding protein 3-like 3 (CREB3L3). The novel EWSR1-CREB3L3 fusion further extends the range of fusion types involving EWSR1 that are characteristic for SEF.

Endometrial stromal tumors include translocation-associated low- and high-grade endometrial stromal sarcomas (ESS) and highly malignant undifferentiated uterine sarcomas (UUS). UUS is considered a poorly defined group of aggressive tumors and is often seen as a diagnosis of exclusion after ESS and leiomyosarcoma (LMS) have been ruled out. We performed a comprehensive analysis of gene expression, copy number variation, point mutations, and immune cell infiltrates in the largest series to date of all major types of uterine sarcomas to shed light on the biology of UUS and to identify potential novel therapeutic targets. We show that UUS tumors have a distinct molecular profile from LMS and ESS. Gene expression and immunohistochemical analyses revealed the presence of high numbers of tumor-associated macrophages (TAMs) in UUS, which makes UUS patients suitable candidates for therapies targeting TAMs. Our results show a high genomic instability of UUS and downregulation of several TP53-mediated tumor suppressor genes, such as NDN, CDH11, and NDRG4. Moreover, we demonstrate that UUS carry somatic mutations in several oncogenes and tumor suppressor genes implicated in RAS/PI3K/AKT/mTOR, ERBB3, and Hedgehog signaling.

Insufficiency of phosphatase and tensin homolog (PTEN) occurs in numerous tumor types and has been implicated as a resistance mechanism to receptor tyrosine kinase-targeted therapies in human cancer. In this study, we have performed a comprehensive molecular and immunohistochemical characterization of PTEN in 58 imatinib-naive and 54 imatinib-treated gastrointestinal stromal tumors (GISTs). The findings were correlated with clinicopathological data. At the genomic level, PTEN was affected mainly by mono-allelic loss, which was significantly less frequent in imatinib-naive vs imatinib-resistant tumors (9% vs 39%, P

Introduction Synovial sarcoma (SynSa) is an aggressive mesenchymal tumor, comprising approximately 10% of all soft tissue sarcomas. Over half of SynSa patients develop metastasis or local recurrence, but the underlying molecular mechanisms of the aggressive clinical behavior remain poorly characterized. Materials and methods Sixty-four frozen tumor specimens from 54 SynSa patients were subjected to array comparative genomic hybridization (aCGH) and gene expression profiling. The examined set of tumor specimens included 16 primary tumors from untreated patients who did not develop metastasis/local recurrence (SynSa1 group), 26 primary tumors from untreated patients who developed metastases or local recurrence during follow-up (SynSa2 group), and 22 metachronous metastatic/recurrent SynSa tumors (SynSa3 group). Results AURKA and KIF18A, which play important roles in various mitotic events, were the two most up-regulated genes in SynSa2 and SynSa3 groups compared to the SynSa1 group. Expression profiles of SynSa2 and SynSa3 tumors did not show any significant differences. Analysis of genomic index (GI) based on aCGH profiles demonstrated that the SynSa1 group consisted of tumors with significantly less complex genomes compared to SynSa2 and SynSa3 groups. There was no significant difference in genome complexity between SynSa2 and SynSa3 tumors. Conclusions Primary SynSa tumors from patients who develop metastases or local recurrence share common molecular features with metastatic/recurrent tumors. Presented data suggest that the aggressive clinical SynSa behavior is determined early in tumorigenesis and might be related to impaired regulation of mitotic mechanisms. This article is part of a Directed Issue entitled: Rare Cancers.

The cholecystokinin 2 receptor (CCK2R/CCKBR) is expressed in gastrointestinal stromal tumours (GISTs). We sought to investigate the role of CCK2R in GIST pathogenesis. Molecular characterization of CCK2R was performed on a heterogeneous cohort of 50 GISTs. In addition, CCK2R expression was evaluated by immunohistochemistry (IHC), using tissue microarray (TMA) containing 292 GISTs, two cases of hyperplasia of interstitial Cajal's cells (ICC) and six gastric microscopic GISTs. Mono-allelic loss of the CCK2R/11p15 allele was identified in 13.7% of GISTs, having no impact on the level of CCK2R transcript expression. No CCK2R mutations were found. The CCK2Ri4sv, CCK2R splice variant with retention of intron 4 was detected in six of 20 tumours analysed. Wild-type CCK2R transcripts were commonly expressed (57.1% of cases) and this expression was highly correlated with gastric primary site of GISTs (p < 0.001). At the protein level, expression of CCK2R in incidental ICC hyperplasia and early stages of gastric GIST development was documented, and its gastric association was confirmed on GIST-TMA by IHC. To explore the in vivo effect of CCK2R activation on tumour growth, gastrin versus placebo was administered intraperitoneally in nude mice carrying human GIST xenografts. The tumour volume was followed for 10 weeks. The effect of this stimulation on tumour cell proliferation/apoptosis was assessed by IHC and KIT/PKC-θ signalling was evaluated by western blotting (WB). In vivo experiments showed a two-fold increase in the volume of tumours which were exposed to gastrin in comparison with non-exposed controls (p = 0.03), with a significant increase in mitotic activity (p = 0.04) and Ki-67 proliferation index (p = 0.008). By WB, gastrin stimulation resulted in hyper-activation of KIT and PKC-θ kinases, and in evident PI3K-AKT pathway over-activation. Our results indicate a promoting role of CCK2R on GIST tumourigenesis, particularly in tumours of gastric origin.

Array CGH was used to identify recurrentcopy number alterations (RCNA) characteristic of eitherBRCA1-related or sporadic ovarian cancer. After pre-processing, both groups of patients were modeled using arecurrent Hidden Markov Model to detect RCNA.RCNA with a probability higher than 80% were called.After removing RCNA present in both groups, the genespresent in the remaining RCNA were investigated forenrichment of pathways from external databases. MoreRCNA were observed in the BRCA1 group, and theydisplay more losses than gains compared to the sporadicgroup. When focusing on the type of RCNA, nosignificant difference in length was seen for the gains,but there was a statistically significant difference for thelosses. In the sporadic group, a great proportion of thealtered regions contain genes known to have a function incell adhesion and complement activation, whereas theBRCA1 samples are characterized by alterations in theHOX genes, metalloproteinases, tumor suppressor genes,and the estrogen-signaling pathways. We conclude thatBRCA1 ovarian tumors present a different type, number,and length of RCNA; a huge amount of the genome islost, resulting in important genomic instability. Moreover,important biological pathways are altered differentiallywhen compared to the sporadic group.Hum Mutat 30:1693–1702,2009. &2009 Wiley-Liss, Inc.KEY WORDS: array CGH; BRCA1; ovarian cancer;hidden Markov model; recurrent copy number alterations

Endometrial stromal sarcomas (ESSs) are a genetically heterogeneous group of rare uterine neoplasms that are commonly driven by recurrent gene rearrangements. In conventional low-grade ESS, JAZF1-SUZ12, PHF1-JAZF1, EPC1-PHF1 and MEAF6-PHF1, and recently described ZC3H7-BCOR chimeric fusions have been reported in > 50% of cases. Conversely, oncogenic t(10;17)(q22;p13) translocation yields YWHAE-FAM22A/B chimeric proteins that are associated with histologically high-grade and clinically more aggressive ESS. Integrating whole-transcriptome paired-end RNA sequencing with fluorescence in situ hybridization (FISH) and banding cytogenetics, we identified MBTD1 (malignant brain tumor domain-containing 1) and CXorf67 (chromosome X open reading frame 67) as the genes involved in the novel reciprocal t(X;17)(p11.2;q21.33) translocation in two independent low-grade ESS of classical histology. The presence of the MBTD1-CXorf67 fusion transcript was validated in both cases using reverse-transcription polymerase chain reaction followed by Sanger sequencing. A specific FISH assay was developed to detect the novel t(X;17) translocation in formalin-fixed paraffin-embedded material, and resulted in identification of an additional low-grade ESS case positive for the MBTD1-CXorf67 fusion among 25 uterine stromal tumors [14 ESS and 11 undifferentiated endometrial sarcomas (UESs)] that were negative for JAZF1 and YWHAE rearrangements. Gene expression profiles of seven ESS (including three with YWHAE and two with JAZF1 rearrangements) and four UES without specific chromosomal aberrations indicated clustering of tumors with MBTD1-CXorf67 fusion together with low-grade JAZF1-associated ESS. The chimeric MBTD1-CXorf67 fusion identifies yet another cytogenetically distinct subgroup of low-grade ESS and offers the opportunity to shed light on the functions of two poorly characterized genes.

Sarcomas of the uterus are derived from uterine smooth muscle (leiomyosarcomas) or from endometrial stromal cells. The latter group includes three subtypes: the low-grade (LG) and high-grade (HG) endometrial stromal sarcomas (ESS), each defined by characteristic chromosomal translocations, and undifferentiated uterine sarcoma (UUS), the most aggressive form. The majority of UUS patients present with high-stage disease and even patients with stage I tumors often die within 2 years from diagnosis. Adjuvant radio- and chemotherapy do not improve the clinical outcome. No prior comprehensive molecular analysis of all three types of endometrial stromal tumors has been reported. We performed genomic analysis (whole exome sequencing and aCGH), gene expression profiling (RNA-seq and microarrays), immunohistochemistry (IHC) and FISH on 31 endometrial stromal tumors, including 17 UUS and 14 LG and HG ESS. All ESS cases carried characteristic JAZF1, PHF1, MBTD1 or YWHAE rearrangements. Selected findings were validated by IHC, qRT-PCR and Sanger sequencing. Genomic analysis revealed that UUS are characterized by complex chromosomal aberrations. The most frequent somatic mutations (in TP53, KRAS, FBXW7, PIK3CA and ERBB3) and copy number changes (e.g. gains of 19q, 8q11.1-q24.3 and 3q26.2-q29) in UUS resemble those seen in uterine carcinosarcomas and carcinomas. UUS over-expressed numerous genes encoding M2 macrophage-specific markers, including CD163, CCL18, mannose receptor, stabilin-1, and macrophage galactose-type C-type lectin 2. Immunohistochemistry for CD163 and CD68 confirmed high tumor-associated macrophages (TAM) counts in UUS tissues compared to the ESS. In UUS, we also observed significantly higher mRNA expression for genes implicated in angiogenesis (CD34, MMP9), immunosuppression and tumor invasion (e.g. CCR2, IL10RA, IRAK3, CCL13, CXCL9, CXCL10). TAMs are associated with progression, resistance to cytotoxic therapy and metastatic spread in most human tumor types. In animal models, TAMs were shown to induce increased angiogenesis. CSF1 is a major regulator of macrophage function and is implicated in these tumor-promoting effects. Clinical trials have shown an effect of inhibitors of the CSF1 pathway in tenosynovial giant cell tumors and our data indicate that these inhibitors may be considered for the treatment of UUS. Our study also showed significantly elevated expression in UUS of two additional therapeutic targets involving macrophage pathways: CCR2 (C-C chemokine receptor type 2) that can be targeted by monoclonal antibodies, and BTK (Bruton's tyrosine kinase) that can be targeted by ibrutinib. In conclusion, our findings demonstrate abundant presence of TAMs and over-expression of TAM-associated markers in UUS compared to the other tumors derived from endometrial stroma. Moreover, our results indicate novel potential therapeutic targets for UUS patients management. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Sushama Varma, Robert T. Sweeney, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Matt van de Rijn, Maria Debiec-Rychter. Tumor associated macrophages in undifferentiated uterine sarcoma: association with angiogenesis and therapeutic implications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3182.

Background Chordomas are rare neoplasms, arising from notochordal remnants in the midline skeletal axis, for which the current treatment is limited to surgery and radiotherapy. Recent reports suggest that receptor tyrosine kinases (RTK) might be essential for the survival or proliferation of chordoma cells, providing a rationale for RTK targeted therapy. Nevertheless, the reported data are conflicting, most likely due to the assorted tumor specimens used for the studies and the heterogeneous methodological approaches. In the present study, we performed a comprehensive characterization of this rare entity using a wide range of assays in search for relevant therapeutic targets. Methods Histopathological features of 42 chordoma specimens, 21 primary and 21 advanced, were assessed by immunohistochemistry and fluorescent in situ hybridization (FISH) using PDGFRB, CSF1R, and EGFR probes. Twenty-two of these cases, for which frozen material was available (nine primary and 13 advanced tumors), were selectively analyzed using the whole-genome 4.3 K TK-CGH-array, phospho-kinase antibody array or Western immunoblotting. The study was supplemented by direct sequencing of KIT, PDGFRB, CSF1R and EGFR. Results We demonstrated that EGFR is frequently and the most significantly activated RTK in chordomas. Furthermore, concurrent to EGFR activation, the tumors commonly reveal co-activation of alternative RTK. The consistent activation of AKT, the frequent loss of the tumor suppressor PTEN allele, the recurrent activation of upstream RTK and of downstream effectors like p70S6K and mTOR, all indicate the PI3K/AKT pathway as an important mediator of transformation in chordomas. Conclusions Given the complexity of the signaling in chordomas, combined treatment regimens targeting multiple RTK and downstream effectors are likely to be the most effective in these tumors. Personalized therapy with careful selection of the patients, based on the molecular profile of the specific tumor, is anticipated.

The cholecystokinin 2 receptor (CCK2R/CCKBR) is expressed in gastrointestinal stromal tumours (GISTs). We sought to investigate the role of CCK2R in GIST pathogenesis. Molecular characterization of CCK2R was performed on a heterogeneous cohort of 50 GISTs. In addition, CCK2R expression was evaluated by immunohistochemistry (IHC), using tissue microarray (TMA) containing 292 GISTs, two cases of hyperplasia of interstitial Cajal's cells (ICC) and six gastric microscopic GISTs. Mono-allelic loss of the CCK2R/11p15 allele was identified in 13.7% of GISTs, having no impact on the level of CCK2R transcript expression. No CCK2R mutations were found. The CCK2Ri4sv, CCK2R splice variant with retention of intron 4 was detected in six of 20 tumours analysed. Wild-type CCK2R transcripts were commonly expressed (57.1% of cases) and this expression was highly correlated with gastric primary site of GISTs (p0.001). At the protein level, expression of CCK2R in incidental ICC hyperplasia and early stages of gastric GIST development was documented, and its gastric association was confirmed on GIST-TMA by IHC. To explore the in vivo effect of CCK2R activation on tumour growth, gastrin versus placebo was administered intraperitoneally in nude mice carrying human GIST xenografts. The tumour volume was followed for 10 weeks. The effect of this stimulation on tumour cell proliferation/apoptosis was assessed by IHC and KIT/PKC-θ signalling was evaluated by western blotting (WB). In vivo experiments showed a two-fold increase in the volume of tumours which were exposed to gastrin in comparison with non-exposed controls (p = 0.03), with a significant increase in mitotic activity (p = 0.04) and Ki-67 proliferation index (p = 0.008). By WB, gastrin stimulation resulted in hyper-activation of KIT and PKC-θ kinases, and in evident PI3K-AKT pathway over-activation. Our results indicate a promoting role of CCK2R on GIST tumourigenesis, particularly in tumours of gastric origin.

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease. Cancer Res; 70(18); 7304–14. ©2010 AACR.

Introduction Endometrial stromal tumors (EST) are the second most prevalent uterine sarcomas. EST are divided into three histosubtypes: benign endometrial stromal nodules (ESN), low-grade endometrial stromal sarcomas (ESS) and the most malignant undifferentiated stromal sarcomas (UES). Moreover, due to clinicopathological features intermediate between classical low-grade ESS and UES, some tumors have been described in the literature as “high-grade ESS”. The histological distinction between these three subtypes has important prognostic implications. The molecular biology of these tumors remains poorly understood because of their low incidence. Patients and Methods In the present study, we performed gene expression profiling of 22 EST using microarray, RNA-Seq and qRT-PCR techniques. Our cohort included 10 classical low-grade ESS (8 with ESS-associated gene fusions), 8 UES and 4 tumors which were classified as high-grade ESS based on the presence of YWHAE-FAM22A/B fusion and histological features. Results Using gene expression microrarrays, we have identified 81 entities differentially expressed in analyzed samples (fold change > 2, p < 0.005). Hierarchical clustering analysis revealed that high-grade ESS formed a separate sub-cluster with a distinct molecular profile as compared with the low-grade ESS and UES cases. Low-grade ESS cases formed 2 distinct sub-clusters, each containing 5 cases, one of which presented gene expression profile partially corresponding to high-grade ESS cases. GO analysis of genes strongly up-regulated in UES specimens showed over-representation of immune response (GO:0006955) and immune system process (GO:0002376) pathways (MARCO, MYO1F, FCN1, GPR183, HLA-DPB1, FCGR2B, NCF1, CCL18, CD28, PTAFR, CCL13, STK4, CD14, NCF4 and CTLA4 genes) (p < 0.05). UES cases were also characterized by significant down-regulation of WT1 tumor suppressor gene as compared to the low- and high-grade ESS. Moreover, both high-grade ESS and UES cases were characterized by significantly lower expression level of PEG3 gene, which has been reported as a tumor suppressor gene. All low-grade ESS cases presented significant over-expression of estrogen receptor ESR1 gene. Microarray results were validated by RNA-Seq and qRT-PCR experiments. Conclusions This study provides gene expression profiles that distinguish between EST subtypes and sheds light on the biology of these tumors. Our results point to immune system involvement in the pathogenesis of UES and support the notion that the current EST classification should discriminate high-grade ESS as a distinct subtype. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Julio Finalet-Ferreiro, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Maria Debiec-Rychter. Gene expression profiling distinguishes between endometrial stromal tumors subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2014-4698

Introduction. Synovial sarcoma (SynSa) is an aggressive type of tumor, comprising approximately 10% of soft tissue sarcomas. Over half of SynSa patients develop metastases or local recurrence, but the molecular mechanism of aggressive SynSa outcome remains poorly characterized. Methods.Sixty five frozen tumor specimens from 54 SynSa patients [34 males; median age 37 years (range 5-86 years)] were subjected to aCGH and gene expression profiling. Examined set of tumor specimens included 16 primary tumors from untreated patients who did not develop metastasis/local recurrence (“SynSa1 group;" median follow-up 76 months), 26 primary tumors from untreated patients who developed metastases or local recurrence during follow-up (“SynSa2 group;" median follow-up 29 months), and 23 metastatic/recurrent SynSa tumors (“SynSa3 group;" median follow-up 46 months). For 8 patients primary tumor specimen was paired with metastatic/recurrent tumor specimens. Median follow-up for all patients was 40 months (range 3-218 months). Results. AURKA and KIF18A, which play important roles in various mitotic events, were the two most up-regulated genes in SynSa2 and SynSa3 groups compared to the SynSa1 group (corr p = 4.48e-6 and corr p = 8.65e-6, respectively). Moreover, we identified 125 genes differentially expressed between SynSa1 and SynSa2 groups (t test unpaired, corr p value cut-off = 0.05, fold change ≥ 2), 38 of which were common with recently published CINSARC (Complexity Index in Sarcomas) prognostic signature (Ref. 1, 2). Comparison of expression profiles between SynSa2 and SynSa3 groups did not show any significant differences. Analysis of genomic index (GI) based on aCGH profiles demonstrated that the SynSa1 group consisted of tumors with significantly less complex genomes compared to SynSa2 and SynSa3 groups (p = 0.021 and p = 0.020, respectively). The median GI in SynSa1, SynSa2 and SynSa3 groups was 9 (range 0-49), 16 (range 0-123) and 28 (range 0-130), respectively. Of note, there was no statistically significant difference in genome complexity between SynSa2 and SynSa3 groups, which suggests that progressive disease is not associated with accumulation of secondary genomic rearrangements/copy number changes. Conclusions. Primary SynSa tumors from patients who develop metastases or local recurrence share common molecular features with metastatic/recurrent SynSa. Presented data confirm that aggressive SynSa behavior is determined early in SynSa development and may be related to impaired mitotic regulatory mechanism. Citation Format: Joanna Przybyl, Raf Sciot, Agnieszka Wozniak, Patrick Schöffski, Vanessa Vanspauwen, Ignace Samson, Janusz A. Siedlecki, Piotr Rutkowski, Maria Debiec-Rychter. Metastatic potential is determined early in synovial sarcoma development and reflected by tumor molecular features. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2013-19

Background: Inactivation of PTEN occurs in numerous tumor types and has been implicated as a resistance mechanism to receptor tyrosine kinase (RTK)-targeted therapies in human cancer. The role of PTEN inactivation in GIST in the acquisition of late resistance to imatinib (IM), a RTK inhibitor, remains elusive. Aim: We investigated the incidence and the mechanisms of PTEN inactivation in a cohort of GIST specimens, and the impact of PI3K inhibition on KIT signaling in IM-sensitive vs. IM-resistant GIST cell lines. Methods: Molecular characterization of PTEN was performed on 65 (23 primary, 10 recurrent/metastatic, and 32 IM-resistant) GIST by CGH array/FISH, mutational analysis, PTEN promoter methylation and QPCR analysis. The PTEN expression and KIT downstream signaling were assessed by Western blotting (WB). The effect of PTEN knock-down by siRNA on KIT signaling was examined in KIT-positive (GIST48) and KIT-negative (KUL-5) GIST cell lines. The impact of NVP-BEZ235 (BEZ), a dual PI3K/mTOR inhibitor, was evaluated in vitro, alone and in combination with IM on IM-sensitive (GIST882, GIST882LY) and IM-resistant (GIST48, GIST48B) cell lines. GIST882LY is a sub-line with bi-allelic PTEN loss. GIST48 has an IM-resistant KIT-D820A mutation, whereas GIST48B has lost its former dependence on KIT oncoprotein. Biological consequences of PI3K inhibition were determined by WB for KIT signaling pathways and by cell proliferation and apoptosis assays. Results: On genomic level PTEN was affected mainly by mono-allelic loss (9%, 16% and 36% of primary, recurrent/metastatic and IM-resistant tumors, respectively). PTEN mutations or PTEN promoter hypermethylation were not found. By QPCR, a positive correlation was observed between low PTEN expression and its genomic loss (p Conclusions: Downregulation of PTEN occurs frequently in IM-resistant GIST in parallel with PI3K/AKT pathway hyperactivation. Inhibitors of the PI3K/AKT pathway represent a potential treatment option for GIST and additional research efforts are required to fully explore this possible treatment strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A166.

Synovial sarcoma (SS) is an aggressive type of tumor, comprising approximately 10 % of soft tissue sarcomas. Over 90 % of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation of oncogenic SS18-SSX1 or SS18-SSX2 fusions. In a typical SS18-SSX fusion transcript, exon 10 of SS18 is fused to exon 6 of SSX1/2. However, several variant fusion transcripts have been already described. In the present study, we examined the fusion transcript type in a series of 40 primary untreated SS tumor specimens using reverse transcription polymerase chain reaction and fluorescence in situ hybridization assay. We detected SS18-SSX1 transcript in 22 (55 %) patients and SS18-SSX2 transcript in 17 (42.5 %) patients, while in one patient, none of SS18-SSX1/2 fusion transcripts were identified. Among the cases under study, two tumors carried novel SS18-SSX1 and SS18-SSX2 variant translocations that were allegedly created by an alternative splicing, and in additional case, an unusual translocation variant previously described by other group was found. Our data suggest that alternative splicing may play an important role in novel fusion transcript formation, and additionally we show that it may be a recurrent event in SS. Furthermore, we describe the first case of a complex rearrangement possibly linking SS to REPS2 gene.