A novel EWS-CREB3L3 gene fusion in a mesenteric sclerosing epithelioid fibrosarcoma.

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.

Macrophage infiltration and genetic landscape of undifferentiated uterine sarcomas.

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.

Metastatic potential is determined early in synovial sarcoma development and reflected by tumor molecular features.

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.

Frequent mono-allelic loss associated with deficient PTEN expression in imatinib-resistant gastrointestinal stromal tumors.

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-naïve 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-naïve vs imatinib-resistant tumors (9% vs 39%, P<0.001). Neither PTEN mutations nor PTEN promoter hyper-methylation were found. By immunohistochemistry, PTEN depletion was clearly related to GIST progression. Low PTEN protein expression was common (50%) and often paralleled with total immunonegativity in imatinib-resistant tumors. The abnormal PTEN protein expression correlated with PTEN loss at the genomic level (P=0.001). In addition, the effect of small interfering RNA (siRNA) PTEN knockdown on KIT signaling was examined in GIST-T1 and GIST430 cell lines, in the absence or presence of a dual PI3K/mTOR inhibitor NVP-BEZ235, alone or in combination with imatinib. In both cell lines, siRNA silencing of PTEN resulted in the substantial upregulation of PI3K-AKT and MAPK pathways. The MAPK hyperactivation was further potentiated by NVP-BEZ235 in the imatinib-sensitive GIST-T1 cells; yet, this effect was counteracted efficiently by combined treatment. In the imatinib-resistant GIST430 cells, neither NVP-BEZ235 alone or in combination with imatinib yielded sufficient inhibition of hyper-phosphorylated MAPK and downstream intermediate S6 protein. In conclusion, depleted PTEN expression associated with mono-allelic PTEN loss occurs frequently in imatinib-resistant GIST and might serve as a biomarker for stratifying patients for optimal treatment. In vitro, the PTEN insufficiency leads to hyperactivation of AKT and MAPK pathways in tumor cells. Novel therapies targeting multiple components of the integrated KIT receptor signaling pathways in imatinib-resistant GIST warrant further studies.

Identification of a novel, recurrent MBTD1-CXorf67 fusion in low-grade endometrial stromal sarcoma.

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.

Recurrent and novel SS18-SSX fusion transcripts in synovial sarcoma: description of three new cases.

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.

Promoting role of cholecystokinin 2 receptor (CCK2R) in gastrointestinal stromal tumour pathogenesis.

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.

Frequent activation of EGFR in advanced chordomas.

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 4.3K 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.

Coactivated platelet-derived growth factor receptor {alpha} 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.

Recurrent copy number alterations in BRCA1-mutated ovarian tumors alter biological pathways.

Array CGH was used to identify recurrent copy number alterations (RCNA) characteristic of either BRCA1-related or sporadic ovarian cancer. After preprocessing, both groups of patients were modeled using a recurrent Hidden Markov Model to detect RCNA. RCNA with a probability higher than 80% were called. After removing RCNA present in both groups, the genes present in the remaining RCNA were investigated for enrichment of pathways from external databases. More RCNA were observed in the BRCA1 group, and they display more losses than gains compared to the sporadic group. When focusing on the type of RCNA, no significant difference in length was seen for the gains, but there was a statistically significant difference for the losses. In the sporadic group, a great proportion of the altered regions contain genes known to have a function in cell adhesion and complement activation, whereas the BRCA1 samples are characterized by alterations in the HOX genes, metalloproteinases, tumor suppressor genes, and the estrogen-signaling pathways. We conclude that BRCA1 ovarian tumors present a different type, number, and length of RCNA; a huge amount of the genome is lost, resulting in important genomic instability. Moreover, important biological pathways are altered differentially when compared to the sporadic group.