The dual role of the X-linked FoxP3 gene in human cancers

The FoxP3 (forkhead box P3) gene is an X-linked gene that is submitted to inactivation. It is an essential transcription factor in CD4(+)CD25(+)FoxP3 regulatory T cells, which are therapeutic targets in disseminated cutaneous melanoma. Moreover, FoxP3 is an important tumor suppressor gene in carcinomas and has putative cancer suppressor gene function in cutaneous melanoma as well. Therefore understanding the structure and function of the FoxP3 gene is crucial to gaining insight into the biology of melanoma to better develop immunotherapeutics and future therapeutic strategies

Diagnostic yield of NanoString nCounter FusionPlex profiling in soft tissue tumors

Diagnostic histopathology of soft tissue tumors can be troublesome as many entities are quite rare and have overlapping morphologic features. Many soft tissue tumors harbor tumor-defining gene translocations, which may provide an important ancillary tool for tumor diagnosis. The NanoString nCounter platform enables multiplex detection of pre-defined gene fusion transcripts in formalin-fixed and paraffin-embedded tissue. A cohort of 104 soft tissue tumors representing 20 different histological types was analyzed for the expression of 174 unique gene fusion transcripts. A tumor-defining gene fusion transcript was detected in 60 cases (58%). Sensitivity and specificity of the NanoString assay calculated against the result of an alternative molecular method were 85% and 100%, respectively. Highest diagnostic coverage was obtained for Ewing sarcoma, synovial sarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. For these tumor types, the NanoString assay is a rapid, cost-effective, sensitive, and specific ancillary screening tool for molecular diagnosis. For other sarcomas, additional molecular testing may be required when a translocation transcript is not identified with the current 174 gene fusion panel

Angiosarcomatous transdifferentiation of metastatic melanoma

Melanoma is known to show considerable variation in its histopathological presentation. In exceptional cases, heterologous or divergent differentiation (metaplastic melanoma) can be observed. We report a case of a 69-year-old man who was diagnosed with nodular melanoma on the right upper leg. One year later, the patient presented with an inguinal lymph node metastasis and a lymph node dissection was carried out. In two out of five positive lymph nodes, an angiosarcomatous component was found next to a conventional melanoma component. Shortly after, the patient developed two in-transit metastases in which again an angiosarcomatous component was seen. The vascular component stained positive for ERG and CD31 and negative for melanocytic markers (Mart-1, S100, SOX-10), while the conventional melanoma had an opposite staining pattern. Molecular analysis on both components showed an identical mutation in the NRAS gene, which in our opinion proves the divergent differentiation. To the best of our knowledge, this is the first case report describing angiosarcomatous transdifferentiation of melanoma

Imaging-Based Surrogate Markers of Epidermal Growth Factor Receptor Mutation in Lung Adenocarcinoma:A Local Perspective

Purpose: To identify computed tomography (CT) features of epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma in Canadian population and whether imaging-based surrogate markers of EGFR mutation in our population were similar to those found in the Asian population. Materials and Methods: Pretreatment CT scans of 223 patients with adenocarcinoma of the lung (112 with EGFR mutation and 111 without mutation) were retrospectively assessed for 20 specific CT features by 2 radiologists, who were blinded to the status of EGFR mutation. Univariate and multivariate logistic regression analyses as well as areas under the receiver operating characteristic curve were performed to discriminate characteristics of EGFR-activating mutation features. Results: Epidermal growth factor receptor mutation-positive adenocarcinomas were more frequently found in female (P <.03), less than 20 pack-year smoking history (P <.001), smaller tumor (P <.01), spiculated margins (P <.05), without centrilobular emphysema (P <.001), and without lymphadenopathy (P <.05), similarly to the Asian population. Multivariate logistic regression analyses of combined clinical and radiological features identified less than 20 pack-year smoking history, smaller tumor diameter, fine or coarse spiculations, noncentral location of the tumor, and lack of centrilobular emphysema and pleural attachment as the strongest independent prognostic factors for the presence of an EGFR mutation. These combined features improved prognostic ability area under the curve to 0.879, compared to 0.788 for clinical features only. Conclusion: Several CT findings may help predict the presence of an activating mutation in EGFR in lung adenocarcinomas in our Canadian population. Combining clinical and radiological features improves prognostic ability to determine the EGFR mutation status compared to clinical features alone

L1CAM Expression is Related to Non-Endometrioid Histology, and Prognostic for Poor Outcome in Endometrioid Endometrial Carcinoma

The majority of endometrial carcinomas are classified as Type I endometrioid endometrial carcinomas (EECs) and have a good prognosis. Type II non-endometrioid endometrial carcinomas (NEECs) have a significant worse outcome. Yet, 20 % of the EECs are associated with an unexplained poor outcome. The aim of this study was to determine if L1CAM expression, a recently reported biomarker for aggressive tumor behavior in endometrial carcinoma, was associated with clinicopathological features of EECs. A total of 103 patients diagnosed as EEC at the Radboud University Medical Centre, based on the pathology report were selected. L1CAM status of these tumors was determined, and histologic slides were reviewed by two expert pathologists. L1CAM-positivity was observed in 17 % (18/103). Review of the diagnostic slides revealed that 11 out of these 18 L1CAM-positive tumors (61 %) contained a serous- or mixed carcinoma component that was not initially mentioned in the pathology report. L1CAM-expression was associated with advanced age, poor tumor grade, and lymphovascular space invasion. A worse five year progression free survival rate was observed for patients with L1CAM-positive tumors (55.6 % for the L1CAM-positive group, compared to 83.3 % for the L1CAM-negative group P = 0.01). L1CAM expression carries prognostic value for histologically classified EEC and supports the identification of tumors with a NEEC component. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12253-016-0047-8) contains supplementary material, which is available to authorized users

Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material

Background: Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. Methods: Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. Results: The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5-10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. Conclusions: We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers

GLI2-Mediated Melanoma Invasion and Metastasis

Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference = −130.9, 95% CI = −96.2 to −165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. Conclusion GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical stud

Clinical, histopathological and molecular features of dedifferentiated melanomas:An EORTC Melanoma Group Retrospective Analysis

PURPOSE: Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases.PATIENTS AND METHODS: A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out.RESULTS: Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome.CONCLUSIONS: Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications.</p