Toward Minimal Residual Disease-Directed Therapy in Melanoma.

Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.

Plocabulin, a Novel Tubulin Inhibitor, Has Potent Antitumour Activity in Patient-Derived Xenograft Models of Soft Tissue Sarcoma.

A clinically relevant subset of patients with soft tissue sarcoma presents with either locally advanced or upfront metastatic disease, or will develop distant metastases over time, despite successful treatment of their primary tumour. The currently available systemic agents to treat such advanced cases only provide modest disease control and are not active in all histological subtypes. Thus, there is an unmet need for novel and more efficacious agents to improve the outcome of this rare disease. In the current preclinical in vivo study, we evaluated plocabulin, a novel tubulin inhibitor, in five distinct histological subtypes of soft tissue sarcoma: dedifferentiated liposarcoma, leiomyosarcoma, undifferentiated sarcoma, intimal sarcoma and CIC-rearranged sarcoma. The efficacy was tested in seven patient-derived xenograft models, which were generated by the engraftment of tumour fragments from patients directly into nude mice. The treatment lasted 22 days, and the efficacy of the drug was assessed and compared to the doxorubicin and vehicle groups by volumetric analysis, histopathology and immunohistochemistry. We observed tumour volume control in all the tested histological subtypes. Additionally, in three sarcoma subtypes, extensive central necrosis, associated with significant tumour regression, was seen. This histological response is explained by the drug's vascular-disruptive properties, reflected by a decreased total vascular area in the xenografts. Our results demonstrate the in vivo efficacy of plocabulin in the preclinical models of soft tissue sarcoma and corroborate the findings of our previous study, which demonstrated similar vascular-disruptive effects in gastrointestinal stromal tumours-another subtype of soft tissue sarcoma. Our data provide a convincing rationale for further clinical exploration of plocabulin in soft tissue sarcomas.

Correlation of Immunological and Molecular Profiles with Response to Crizotinib in Alveolar Soft Part Sarcoma: An Exploratory Study Related to the EORTC 90101 "CREATE" Trial.

Alveolar soft part sarcoma (ASPS) is a rare subtype of soft tissue sarcoma characterized by an unbalanced translocation, resulting in ASPSCR1-TFE3 fusion that transcriptionally upregulates MET expression. The European Organization for Research and Treatment of Cancer (EORTC) 90101 "CREATE" phase II trial evaluated the MET inhibitor crizotinib in ASPS patients, achieving only limited antitumor activity. We performed a comprehensive molecular analysis of ASPS tissue samples collected in this trial to identify potential biomarkers correlating with treatment outcome. A tissue microarray containing 47 ASPS cases was used for the characterization of the tumor microenvironment using multiplex immunofluorescence. DNA isolated from 34 available tumor samples was analyzed to detect recurrent gene copy number alterations (CNAs) and mutations by low-coverage whole-genome sequencing and whole-exome sequencing. Pathway enrichment analysis was used to identify diseased-associated pathways in ASPS sarcomagenesis. Kaplan-Meier estimates, Cox regression, and the Fisher's exact test were used to correlate histopathological and molecular findings with clinical data related to crizotinib treatment, aiming to identify potential factors associated with patient outcome. Tumor microenvironment characterization showed the presence of PD-L1 and CTLA-4 in 10 and 2 tumors, respectively, and the absence of PD-1 in all specimens. Apart from CD68, other immunological markers were rarely expressed, suggesting a low level of tumor-infiltrating lymphocytes in ASPS. By CNA analysis, we detected a number of broad and focal alterations. The most common alteration was the loss of chromosomal region 1p36.32 in 44% of cases. The loss of chromosomal regions 1p36.32, 1p33, 1p22.2, and 8p was associated with shorter progression-free survival. Using whole-exome sequencing, 13 cancer-associated genes were found to be mutated in at least three cases. Pathway enrichment analysis identified genetic alterations in NOTCH signaling, chromatin organization, and SUMOylation pathways. NOTCH4 intracellular domain dysregulation was associated with poor outcome, while inactivation of the beta-catenin/TCF complex correlated with improved outcome in patients receiving crizotinib. ASPS is characterized by molecular heterogeneity. We identify genetic aberrations potentially predictive of treatment outcome during crizotinib therapy and provide additional insights into the biology of ASPS, paving the way to improve treatment approaches for this extremely rare malignancy.

Comprehensive targeted next-generation sequencing approach in the molecular diagnosis of gastrointestinal stromal tumor.

Mutational analysis guides therapeutic decision making in patients with advanced-stage gastrointestinal stromal tumors (GISTs). We evaluated three targeted next-generation sequencing (NGS) assays, consecutively used over 4 years in our laboratory for mutational analysis of 162 primary GISTs: Agilent GIST MASTR, Illumina TruSight 26 and an in-house developed 96 gene panels. In addition, we investigated the feasibility of a more comprehensive approach by adding targeted RNA sequencing (Archer FusionPlex, 11 genes) in an attempt to reduce the number of Wild Type GISTs. We found KIT or PDGFRA mutations in 149 out of 162 GISTs (92.0%). Challenging KIT exon 11 alterations were initially missed by different assays in seven GISTs and typically represented deletions at the KIT intron 10-exon 11 boundary or large insertions/deletions (>24 base pairs). Comprehensive analysis led to the additional identification of driver alterations in 8/162 GISTs (4.9%): apart from BRAF and SDHA mutations (one case each), we found five GISTs harboring somatic neurofibromatosis type 1 (NF1) alterations (3.1%) and one case with an in-frame TRIM4-BRAF fusion not reported in GIST before. Eventually, no driver alteration was found in two out of 162 GISTs (1.2%) and three samples (1.9%) failed analysis. Our study shows that a comprehensive targeted NGS approach is feasible for routine mutational analysis of GIST, thereby substantially reducing the number of Wild Type GISTs, and highlights the need to optimize assays for challenging KIT exon 11 alterations.

Myxoinflammatory fibroblastic sarcoma: an immunohistochemical and molecular genetic study of 73 cases.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare, low-grade soft tissue neoplasm preferentially arising in the extremities of young to middle-aged adults characterized histologically by a variegated appearance and absence of a distinctive immunophenotype. Herein we have evaluated a series of 73 cases of MIFS to define potential features and markers that may facilitate diagnosis. An immunohistochemical study with a large panel of antibodies showed strong positivity of the tumor cells for bcl-1 (94.5%), FXIIIa (89%), CD10 (80%), and D2-40 (56%). FISH and array comparative genomic hybridization (aCGH) were performed in a large subset of cases to investigate the utility for detecting the TGFBR3 and OGA t(1;10) rearrangement and BRAF abnormalities. Using a combination of FISH and/or aCGH, t(1;10) was detected in only 3 of 54 cases (5.5%). The aCGH study also demonstrated amplification of VGLL3 on chromosome 3 that was detected in 8 of 20 cases (40%). BRAF alterations were observed by FISH in 4 of 70 cases (5.7%) and correlated with gain of chromosome 3p12 (VGLL3). A novel fusion transcript involving exon 6 of ZNF335 and exon 10 of BRAF was identified in one case. Demonstration of amplification of VGLL3 on chromosome 3 in combination with expression of bcl-1 and FXIIIa may help support the diagnosis, however, due to their low specificity these markers are not sufficient for a definitive diagnosis in the absence of the appropriate clinical-pathological context. Until a more robust genetic or immunohistochemical signature is identified, the diagnosis of MIFS rests on its characteristic clinicopathological features.

PDGFRB gain-of-function mutations in sporadic infantile myofibromatosis.

Infantile myofibromatosis is one of the most prevalent soft tissue tumors of infancy and childhood. Multifocal nodules with visceral lesions are associated with a poor prognosis. A few familial cases have been linked to mutations in various genes including PDGFRB. In this study, we sequenced PDGFRB, which encodes a receptor tyrosine kinase, in 16 cases of myofibromatosis or solitary myofibroma. Mutations in the coding sequence of PDGFRB were identified in 6 out of 8 patients with the sporadic multicentric form of the disease and in 1 out of 8 patients with isolated myofibroma. Two patients had the same mutation in multiple separated lesions. By contrast, a third patient had three different PDGFRB mutations in the three nodules analyzed. Mutations were located in the transmembrane, juxtamembrane and kinase domains of the receptor. We showed that these mutations activated receptor signaling in the absence of ligand and transformed fibroblasts. In one case, a weakly-activating germline variant was associated with a stronger somatic mutation, suggesting a two-hit model for familial myofibromatosis. Furthermore, the mutant receptors were sensitive to the tyrosine kinase inhibitor imatinib, except D850V, which was inhibited by dasatinib and ponatinib, suggesting a targeted therapy for severe myofibromatosis. In conclusion, we identified gain-of-function PDGFRB mutations in the majority of multifocal infantile myofibromatosis cases, shedding light on the mechanism of disease development, which is reminiscent of multifocal venous malformations induced by TIE2 mutations. Our results provide a genetic test to facilitate diagnosis, and preclinical data for development of molecular therapies.

Assessment of the platelet-derived growth factor receptor alpha antibody olaratumab in a panel of patient-derived soft tissue sarcoma xenografts.

BACKGROUND: Soft tissue sarcoma (STS) comprises a family of rare, heterogeneous tumors of mesenchymal origin. Single-agent doxorubicin remains the first-line standard-of-care treatment for advanced and inoperable STS, but response rates are only around 15%. In 2016, phase Ib/II clinical trial results reported an overall survival benefit of 11.8 months when combining doxorubicin and the platelet-derived growth factor receptor alpha (PDGFRA)-directed antibody olaratumab compared to doxorubicin alone, without providing a scientific rationale for such unprecedented therapeutic effect. We decided to evaluate the efficacy of olaratumab in a panel of STS patient-derived xenografts (PDX). METHODS: NMRI nu/nu mice were bilaterally transplanted with tumor tissue of patient-derived xenograft models expressing PDGFRA, including models of leiomyosarcoma (UZLX-STS22), malignant peripheral nerve sheath tumor (UZLX-STS39), myxofibrosarcoma (UZLX-STS59) and undifferentiated pleomorphic sarcoma (UZLX-STS84). Mice were randomly divided into four different treatment groups: (1) control, (2) doxorubicin (3 mg/kg once weekly), (3) anti-PDGFRA [olaratumab (60 mg/kg twice weekly) + mouse anti-PDGFRA antibody 1E10 (20 mg/kg twice weekly)] and (4) the combination of doxorubicin and anti-PDGFRA (same dose/schedule as in the single treatment arms). Tumor volume, histopathology and Western blotting were used to assess treatment efficacy. RESULTS: Anti-PDGFRA treatment as a single agent did not reduce tumor growth and did not result in significant anti-proliferative or pro-apoptotic activity. Combining doxorubicin and anti-PDGFRA did not reduce tumor burden, though a mild inhibition of proliferation was observed in UZLX-STS39 and -STS59. A pro-apoptotic effect was observed in all models except UZLX-STS22. Antitumor effects on histology were not significantly different comparing doxorubicin and the combination treatment. Moreover, anti-PDGFRA treatment, both as a single agent as well as combined with doxorubicin, did not result in inhibition of the downstream MAPK and PI3K/AKT signaling pathways. CONCLUSIONS: We were not able to demonstrate significant antitumor effects of anti-PDGFRA treatment in selected STS PDX models, neither alone nor in combination with doxorubicin. This is in line with the very recent results of the phase III clinical trial NCT02451943 ANNOUNCE, which did not confirm the clinical benefit of olaratumab in combination with doxorubicin over single agent doxorubicin.

[Cardiac intimal sarcoma: A case report of a rare tumor with peculiar histopathological findings]. / Le sarcome intimal cardiaque : tumeur rare illustrée par un cas de présentation histopathologique inhabituelle.

Primary cardiac sarcomas are rare tumors with poor prognosis. Intimal sarcoma, a mesenchymal malignant tumor described mainly in the great vessels, may rarely involve the heart. Herein we describe the case of a 70-years-old female who was found to have a left atrial mass during an investigation of a new onset dyspnea. The patient underwent surgery and the resected mass was found to be an intimal sarcoma. The objectives of this report were to describe a case of this rare disease entity and to discuss its pathological and molecular findings based on relevant literature.

Gene expression profiling of low-grade endometrial stromal sarcoma indicates fusion protein-mediated activation of the Wnt signaling pathway.

OBJECTIVE: Low-grade endometrial stromal sarcomas (LGESS) harbor chromosomal translocations that affect proteins associated with chromatin remodeling Polycomb Repressive Complex 2 (PRC2), including SUZ12, PHF1 and EPC1. Roughly half of LGESS also demonstrate nuclear accumulation of ß-catenin, which is a hallmark of Wnt signaling activation. However, the targets affected by the fusion proteins and the role of Wnt signaling in the pathogenesis of these tumors remain largely unknown. METHODS: Here we report the results of a meta-analysis of three independent gene expression profiling studies on LGESS and immunohistochemical evaluation of nuclear expression of ß-catenin and Lef1 in 112 uterine sarcoma specimens obtained from 20 LGESS and 89 LMS patients. RESULTS: Our results demonstrate that 143 out of 310 genes overexpressed in LGESS are known to be directly regulated by SUZ12. In addition, our gene expression meta-analysis shows activation of multiple genes implicated in Wnt signaling. We further emphasize the role of the Wnt signaling pathway by demonstrating concordant nuclear expression of ß-catenin and Lef1 in 7/16 LGESS. CONCLUSIONS: Based on our findings, we suggest that LGESS-specific fusion proteins disrupt the repressive function of the PRC2 complex similar to the mechanism seen in synovial sarcoma, where the SS18-SSX fusion proteins disrupt the mSWI/SNF (BAF) chromatin remodeling complex. We propose that these fusion proteins in LGESS contribute to overexpression of Wnt ligands with subsequent activation of Wnt signaling pathway and formation of an active ß-catenin/Lef1 transcriptional complex. These observations could lead to novel therapeutic approaches that focus on the Wnt pathway in LGESS.

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.

Recurrent MALAT1-GLI1 oncogenic fusion and GLI1 up-regulation define a subset of plexiform fibromyxoma.

Plexiform fibromyxomas are rare neoplasms, being officially recognized as a distinct entity among benign mesenchymal gastric tumours in the 2010 WHO Classification of Tumours of the Digestive System. Characteristically, these tumours have a multinodular/plexiform growth pattern, and histologically contain variably cellular areas of bland myofibroblastic-type spindle cells embedded in an abundant myxoid matrix, rich in capillary-type vessels. As yet, the molecular and/or genetic features of these tumours are unknown. Here we describe a recurrent translocation, t(11;12)(q11;q13), involving the long non-coding gene metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the gene glioma-associated oncogene homologue 1 (GLI1) in a subgroup of these tumours. The presence of the fusion transcript in our index case was confirmed using polymerase chain reaction (PCR) on genomic DNA, followed by Sanger sequencing. We showed that the truncated GLI1 protein is overexpressed and retains its capacity to transcriptionally activate its target genes. A specific FISH assay was developed to detect the novel MALAT1-GLI1 translocation in formalin-fixed, paraffin-embedded (FFPE) material. This resulted in the identification of two additional cases with this fusion and two cases with polysomy of the GLI1 gene. Finally, immunohistochemistry revealed that the GLI1 protein is exclusively overexpressed in those cases that harbour GLI1/12q13 genomic alterations. In conclusion, overexpression of GLI1 through a recurrent MALAT1-GLI1 translocation or GLI1 up-regulation delineates a pathogenically distinct subgroup of plexiform fibromyxomas with activation of the Sonic Hedgehog signalling pathway. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lysosomal Sequestration Determines Intracellular Imatinib Levels.

The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression.

Co-segregation of Freiberg's infraction with a familial translocation t(5;7)(p13.3;p22.2) ascertained by a child with cri du chat syndrome and brachydactyly type A1B.

The identification of chromosomal breakpoints in association with human abnormal phenotypes can enable elucidation of gene function. We report on epiphyseal aseptic necrosis of the lesser head of the second metatarsal bone, known as Freiberg's infraction (FI), in two female carriers of the apparently balanced t(5;7)(p13.3;p22.2) ascertained by a 16-year-old girl with cri-du-chat syndrome and unusual skeletal features in association with an unbalanced translocation der(5) t(5;7)(p13.3;p22.2). Mapping of the chromosome breakpoints using fluorescent in situ hybridization (FISH) narrowed them to the coding sequence of ADAMTS12 on chromosome 5p13.3 and SDK1 on 7p22.2. In addition, several skeletal abnormalities classified as brachydactyly type A1B (BDA1B) were present in the proband and in both carriers of t(5;7)(p13.3;p22.2), suggesting a potential role of ADAMTS12 in the development of the BDA1B observed in this family.

14-3-3 fusion oncogenes in high-grade endometrial stromal sarcoma.

14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development and progression of cancer. We report a transforming 14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoprotein results from a t(10;17) genomic rearrangement, leading to fusion between 14-3-3ε (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE-FAM22 fusion oncoproteins was demonstrated by immunoblot in t(10;17)-bearing frozen tumor and cell line samples. YWHAE-FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t(10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE-FAM22 maintains a structurally and functionally intact 14-3-3ε (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE-FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE-FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n = 827). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions.

Identification of an MLL4-GPS2 fusion as an oncogenic driver of undifferentiated spindle cell sarcoma in a child.

Undifferentiated spindle cell sarcoma (UDS) is a poorly defined or understood entity, essentially a waste-basket for cases failing to fulfill criteria for better-established diagnoses based on combined histology, immunohistochemistry, and tumor genetic assays. We identified a novel chromosomal translocation t(17;19)(p13;q13) in a pediatric UDS and have characterized this alteration to show rearrangement of the MLL4 and GPS2 genes, resulting in an in-frame fusion gene MLL4-GPS2, the expression of which promotes anchorage-independent growth. MLL4 was previously reported to be similarly rearranged in hepatocellular carcinomas, notably those positive for hepatitis B virus. Isolated reports of individual rearrangements of GPS2 in a prostate carcinoma cell line and in glioblastoma multiforme, each with different partner genes, recently emerged from high-throughput sequencing studies but have not been further evaluated for biological effect.

Angiomatoid fibrous histiocytoma - case series with emphasis on a late fibrotic variant.

BACKGROUND: Angiomatoid fibrous histiocytoma (AFH) is a rare, low-grade malignant, subcutaneous neoplasm in children or young adults. METHODS: AFHs in different disease stages were studied histologically, in part, also immunohistologically, and by fluorescence in situ hybridization. RESULTS: Depending on the degree of fibrosclerosis, nine AFH were divided into the following categories: classic type (n = 3): well-defined subcutaneous lesions composed of multinodular spindle to epithelioid (histiocytoid) cells surrounding a pseudoangiomatous space filled with blood. Peripherally, there is a fibrous pseudocapsule and an inflammatory infiltrate. Early sclerotic type (n = 4): the fibrous capsule extends more to the inner circle of the lesion, focally replacing the cellular neoplastic component and pseudoangiomatous spaces. Late sclerotic type (n = 2): the architecture of AFH with its zonal arrangement of an outer fibrous and inner cellular component is largely replaced by fibrosis occluding the pseudovascular space in the center of the lesion. Immunohistochemistry was available in 5/9 cases with positivity for EMA (5/5), desmin (3/5), caldesmon (1/2), and CD99 (2/5). One of two cases tested displayed EWSR1 rearrangement. CONCLUSION: Late-stage AFH may present with marked fibrosis obscuring the real nature of the lesion and may easily be misinterpreted by the unwary as a harmless fibrotic condition.

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.

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 CUX1 as the recurrent chromosomal band 7q22 target gene in human uterine leiomyoma.

Uterine leiomyomas are benign solid tumors of mesenchymal origin which occur with an estimated incidence of up to 77% of all women of reproductive age. The majority of these tumors remains symptomless, but in about a quarter of cases they cause leiomyoma-associated symptoms including chronic pelvic pain, menorrhagia-induced anemia, and impaired fertility. As a consequence, they are the most common indication for pre-menopausal hysterectomy in the USA and Japan and annually translate into a multibillion dollar healthcare problem. Approximately 40% of these neoplasms present with recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 and/or 10q22. Using positional cloning strategies, we and others previously identified HMGA1, HMGA2, RAD51L1, MORF, and, more recently, NCOA1 as primary target (fusion) genes associated with tumor initiation in four of these distinct cytogenetic subgroups. Despite the fact that the del(7)(q22) subgroup is the largest among leiomyomas, and was first described more than twenty years ago, the 7q22 leiomyoma target gene still awaits unequivocal identification. We here describe a positional cloning effort from two independent uterine leiomyomas, containing respectively a pericentric and a paracentric chromosomal inversion, both affecting band 7q22. We found that both chromosomal inversions target the cut-like homeobox 1 (CUX1) gene on chromosomal band 7q22.1 in a way which is functionally equivalent to the more frequently observed del(7q) cases, and which is compatible with a mono-allelic knock-out scenario, similar as was previously described for the cytogenetic subgroup showing chromosome 14q involvement.

Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a mesenchymal neoplasm displaying variable morphologic and clinical features. To identify pathogenetically important genetic rearrangements, 44 SFTs were analyzed using a variety of techniques. Chromosome banding and fluorescence in situ hybridization (FISH) showed recurrent breakpoints in 12q13, clustering near the NAB2 and STAT6 genes, and single nucleotide polymorphism array analysis disclosed frequent deletions affecting STAT6. Quantitative real-time PCR revealed high expression levels of the 5'-end of NAB2 and the 3'-end of STAT6, which at deep sequencing of enriched DNA corresponded to NAB2/STAT6 fusions. Subsequent reverse-transcriptase PCR (RT-PCR) analysis identified a NAB2/STAT6 fusion in 37/41 cases, confirming that this fusion gene underlies the pathogenesis of SFT. The hypothesis that the NAB2/STAT6 fusions will result in altered properties of the transcriptional co-repressor NAB2--a key regulator of the early growth response 1 (EGR1) transcription factor - was corroborated by global gene expression analysis; SFTs showed deregulated expression of EGR1 target genes, as well as of other, developmentally important genes. We also identified several nonrandom secondary changes, notably loss of material from 13q and 14q. As neither chromosome banding nor FISH analysis identify more than a minor fraction of the fusion-positive cases, and because multiple primer combinations are required to identify all possible fusion transcripts by RT-PCR, alternative diagnostic markers might instead be found among deregulated genes identified at global gene expression analysis. Indeed, using immunohistochemistry on tissue microarrays, the top up-regulated gene, GRIA2, was found to be differentially expressed also at the protein level.