Barbara Dewaele

Activity of Dasatinib, a Dual SRC/ABL Kinase Inhibitor, and IPI-504, a Heat Shock Protein 90 Inhibitor, against Gastrointestinal Stromal Tumor–Associated PDGFRAD842V Mutation

Purpose: Activating mutations in platelet-derived growth factor receptor-α (PDGFRA) have been reported in ∼5% to 10% of patients with gastrointestinal stromal tumors (GIST). Imatinib efficiently inhibits the juxtamembrane PDGFRA mutations, whereas many tyrosine kinase domain activation loop PDGFRA mutations confer primary resistance to imatinib. In this study, we compared the efficacy of second-line tyrosine kinase inhibitors such as dasatinib, sorafenib, and nilotinib against two GIST-related PDGFRA mutants, PDGFRAD842V and PDGFRAΔDIM842-844. In addition, we sought to investigate the inhibitory effect of the heat shock protein 90 inhibitor, IPI-504, on these mutants. Experimental Design: Primary imatinib-resistant tumor cells and cell lines expressing imatinib-resistant PDGFRAD842V or imatinib-sensitive PDGFRAΔDIM842-844 mutants were treated with different concentrations of dasatinib, sorafenib, nilotinib, and IPI-504. The effect of treatment on proliferation, survival, and signaling was determined. Results: All inhibitors tested exhibited a high efficacy toward the PDGFRAΔDIM842-844 mutant. In contrast, ex vivo and in vitro assays revealed that only dasatinib potently inhibited the PDGFRAD842V isoform with an IC50 value of 62 nmol/L. Sorafenib and nilotinib were significantly less efficacious against this mutation, inhibiting the PDGFRA kinase activity at >1,000 and >5,000 nmol/L, and suppressing the proliferation of the cells expressing the PDGFRAD842V mutant with an IC50 value of 239 and 1,310 nmol/L, respectively. IPI-504 treatment potently inhibited PDGFRA kinase activity by inducing the degradation of PDGFRAD842V and PDGFRAΔDIM842-844 at 256 and 182 nmol/L, respectively. Conclusions: Treatment with dasatinib or the heat shock protein 90 inhibitor IPI-504 may provide a therapeutic alternative for GIST patients whose tumors carry the imatinib-resistant PDGFRAD842V mutant isoform.

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.

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.

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.

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 Cajals 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. Copyright

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.

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.

Single-Center Experience with Intimal Sarcoma, an Ultra-Orphan, Commonly Fatal Mesenchymal Malignancy

Background: Intimal sarcoma is a rare malignancy that, clinically and radiographically, often mimics pulmonary embolism. The intravascular tumor tends to disseminate rapidly and metastases can be present at first diagnosis. Methods: We reviewed all cases of intimal sarcoma that were diagnosed, treated and followed at the University Hospitals Leuven between April 2006 and April 2016. Results: We identified 13 patients with a median age of 51 years. In 6 patients initial findings were suggestive of thromboembolic disease. Platelet-derived growth factor receptor α (PDGFRA) amplification was the most prevalent molecular finding, present in 11 patients. The MDM2 gene was amplified in 9 cases, and the EGFR gene in 3 patients. The median overall survival was 13 months. 11 patients underwent surgery. In 5 cases with inoperable and/or metastatic disease chemotherapy was given. Treatment with imatinib was initiated in 4 patients. Conclusions: Intimal sarcoma is an extremely rare and aggressive malignancy that has a very poor prognosis. Mimicking thromboembolic disease, diagnosis and treatment can be delayed. Surgery is the mainstay of treatment but is seldom curative. The disease is highly resistant to cytotoxic and targeted treatment. Given the fact that intimal sarcoma commonly expresses more than 1 molecular target, combination therapy might be an option, although toxicity may be a limitation.

Uterus: Low-grade endometrial stromal sarcoma with t(X;17)(p11.2;q21.33) MBTD1/CXorf6

Short communication on t(X;17)(p11.2;q21.33) MBTD1-CXorf67 in low-grade endometrial stromal sarcoma.

Highly sensitive assays are mandatory for the differential diagnosis of patients presenting with symptoms of mast cell activation: diagnostic work-up of 38 patients

Mastocytosis is a heterogeneous disease caused by excessive mast cell (MC) proliferation. Diagnosis of systemic mastocytosis (SM) is based on the presence of major and minor criteria defined by the World Health Organization. Symptoms of MC activation can also occur in patients without SM or without allergic or inflammatory disease. These MC activation syndromes (MCAS) can be divided into primary (monoclonal) MCAS (MMAS) vs. secondary and idiopathic MCAS. In this single center study, the diagnostic work-up of 38 patients with a clinical suspicion of SM and/or with elevated basic tryptase levels is presented. Clinical symptoms, biochemical parameters, results of bone marrow investigation, flow cytometric immunophenotyping, and molecular analysis were retrospectively reviewed. Twenty-three patients were found to have a monoclonal MC disorder of which 19 were diagnosed with SM and 4 with MMAS. In 13/19 SM patients, multifocal MC infiltrates in the bone marrow were found (major criterion), while in 6 the diagnosis was based on the presence of ≥3 minor criteria. Flow cytometric analysis of bone marrow showed CD25 expression of MCs in all patients with SM and MMAS (range: 0.002–0.3% of cells). In bone marrow, the KIT D816V mutation was detected in all SM patients but in only 2 patients with MMAS (range: 0.007–9% mutated cells). Basic tryptase elevation was demonstrated in 16/19 patients with SM but also in 9/19 patients without SM. Our study reveals the heterogeneity of primary MC disorders and the importance of sensitive assays in patients suspected of having SM.