Stephanie E Weissinger

A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma.

Spindle cell melanoma and desmoplastic melanoma differ clinically in prognosis and therapeutic implications; however, because of partially overlapping histopathological features, diagnostic distinction of spindle cell from desmoplastic melanoma is not always straightforward. A direct comparison of diagnostic and therapeutic biomarkers has not been performed. Meta-review of the literature discloses key clinicopathological differences between spindle cell and desmoplastic melanoma, including immunophenotypes. Using 50 biomarkers available in routine diagnostics, we examined 38 archival cases (n=16 spindle, 18 desmoplastic, 4 mixed spindle/desmoplastic melanoma). S100 remains as the most reliable routine marker to reach the diagnosis of melanoma in spindle cell and desmoplastic melanoma. We identified nine distinctly labeling markers with spindle cell melanoma showing positivity for laminin, p75, HMB45, c-kit, and MelanA, and desmoplastic melanoma preferentially labeling with collagen IV, trichrome, CD68, and MDM2. On the basis of comparisons of test performance measures, MelanA and trichrome were used to devise a 94% sensitive diagnostic algorithm for the distinction of desmoplastic from spindle cell melanoma. Gene amplification and expression status was assessed for a set of potentially drugable targets (HER2, EGFR, MET, MDM2, TP53, ALK, MYC, FLI-1, and KIT). Fluorescent in situ hybridizations did not reveal a significant number of gene aberrations/rearrangements; however, protein overexpression for at least one of these markers was identified in 35 of 38 cases (92%). In addition, we found BRAF mutations in 31% of spindle cell and 5% of desmoplastic melanoma, with an overall mutation frequency of 16% (n=6/38). We present the first comprehensive screening study of diagnostic and therapeutic biomarkers in spindle cell and desmoplastic melanoma. The devised algorithm allows diagnostic distinction of desmoplastic from spindle cell melanoma when routine histology is not decisive.

Loss of ATM accelerates pancreatic cancer formation and epithelial-mesenchymal transition.

Pancreatic ductal adenocarcinoma (PDAC) is associated with accumulation of particular oncogenic mutations and recent genetic sequencing studies have identified ataxia telangiectasia-mutated (ATM) mutations in PDAC cohorts. Here we report that conditional deletion of ATM in a mouse model of PDAC induces a greater number of proliferative precursor lesions coupled with a pronounced fibrotic reaction. ATM-targeted mice display altered TGFβ-superfamily signalling and enhanced epithelial-to-mesenchymal transition (EMT) coupled with shortened survival. Notably, our mouse model recapitulates many features of more aggressive human PDAC subtypes. Particularly, we report that low expression of ATM predicts EMT, a gene signature specific for Bmp4 signalling and poor prognosis in human PDAC. Our data suggest an intimate link between ATM expression and pancreatic cancer progression in mice and men.

A nuclear circularity-based classifier for diagnostic distinction of desmoplastic from spindle cell melanoma in digitized histological images

Context: Distinction of spindle cell melanoma (SM) and desmoplastic melanoma (DM) is clinically important due to differences in metastatic rate and prognosis; however, histological distinction is not always straightforward. During a routine review of cases, we noted differences in nuclear circularity between SM and DM. Aim: The primary aim in our study was to determine whether these differences in nuclear circularity, when assessed using a basic ImageJ-based threshold extraction, can serve as a diagnostic classifier to distinguish DM from SM. Settings and Design: Our retrospective analysis of an established patient cohort (SM n = 9, DM n = 9) was employed to determine discriminatory power. Subjects and Methods: Regions of interest (total n = 108; 6 images per case) were selected from scanned H and E-stained histological sections, and nuclear circularity was extracted and quantified by computational image analysis using open source tools (plugins for ImageJ). Statistical Analysis: Using analysis of variance, t-tests, and Fisher′s exact tests, we compared extracted quantitative shape measures; statistical significance was defined as P < 0.05. Results: Classifying circularity values into four shape categories (spindled, elongated, oval, round) demonstrated significant differences in the spindled and round categories. Paradoxically, DM contained more spindled nuclei than SM (P = 0.011) and SM contained more round nuclei than DM (P = 0.026). Performance assessment using a combined shape-classification of the round and spindled fractions showed 88.9% accuracy and a Youden index of 0.77. Conclusions: Spindle cell melanoma and DM differ significantly in their nuclear morphology with respect to fractions of round and spindled nuclei. Our study demonstrates that quantifying nuclear circularity can be used as an adjunct diagnostic tool for distinction of DM and SM.

Comparison of MelanA/MART-1 and HMB45 labeling in desmoplastic melanoma

To the Editor: I read with interest the article by Weissinger et al1 ‘A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma’ published in the September 2013 issue. They propose MelanA as a first and, if positive, exclusionary step in their algorithm. This appears to be based on their cluster analysis of 38 cases (16 spindle cell, 18 desmoplastic, 4 mixed). All 4 of their mixed and all 18 of their desmoplastic melanomas were negative for MelanA. Their meta-review (Supplementary Table 9) is a summary of published immunoprofiles comparing spindle cell to desmoplastic melanoma. It is based on much larger number of cases and suggests that MelanA is not quite that specific and that HMB-45 may actually be of superior discriminatory value than MelanA.

Absence of BRAF and KRAS hotspot mutations in primary mediastinal B-cell lymphoma

Th e potential of molecularly targeted therapy has been propagated to improve overall outcome in diff use large B-cell lymphoma (DLBCL) [1]. Recent whole-exome sequencing data revealed BRAF and KRAS mutations as likely drivers and potentially targetable mutations [2] in a clinically relevant fraction (4%) of patients with DLBCL [3]. Interestingly, for primary mediastinal B-cell lymphoma (PMBL), a genetically distinct variant of DLBCL [4,5], the prevalence of BRAF and KRAS hotspot mutations has not been elucidated. Given the widespread clinical availability of sensitive molecular genetic BRAF / KRAS assays, screening by genotyping might be a feasible option to identify a subset of patients with PMBL

Checkpoint inhibition for advanced mucosal melanoma

BackgroundWhereas anti-PD-1 therapy has demonstrated a significant and durable response against advanced cutaneous melanoma, conventional chemotherapies have shown only minor benefit against advanced mucosal melanoma.ObjectivesTo investigate the efficacy of anti-PD- 1 therapy in a small cohort of patients with mucosal melanoma of the head and neck.Materials & methodsWe analysed five patients with mucosal melanoma of the head and neck who received nivolumab or pembrolizumab, at an advanced stage. Expression of PD-L1 and PD-1 in all tumour samples was evaluated immunohistochemically.ResultsAll patients received at least two cycles of nivolumab or pembrolizumab. The most severe adverse events were categorised as CTCAE (common terminology criteria for adverse events) Grade 2. All patients showed progressive disease after restaging at three and six months, and no partial or complete response was observed. Immunohistochemical staining demonstrated PD-L1 expression in less than 5% of tumour cells.ConclusionSystemic therapy with either nivolumab or pembrolizumab showed no clinical response, however, tumour progression was identified in all patients using Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 and immune-related response criteria (irRC) to evaluate tumour response.

Low SOX2 expression marks a distinct subset of adenoid cystic carcinoma of the head and neck and is associated with an advanced tumor stage

Introduction The transcription factor SOX2 has been identified as a lineage survival oncogene in squamous cell carcinoma and copy number gain is a common event in several human malignancies including head and neck cancer. However, the regulation and function of SOX2 during carcinogenesis as well as its prognostic value appears to be highly context dependent. As an example, high SOX2 expression in lung squamous cell carcinoma (SCC) is related to a favorable prognosis, while it is associated with poor outcome in lung adenocarcinoma. More recently, higher SOX2 levels and improved survival was also reported for head and neck SCC (HNSCC), and silencing of SOX2 expression in HNSCC cell lines revealed a mesenchymal-like phenotype with prominent vimentin expression. So far, SOX2 expression and its clinical relevance for other head and neck cancers, such as adenoid cystic carcinoma (HNACC) have not been sufficiently investigated. Material and methods SOX2, vimentin and E-cadherin expression was assessed by immunohistochemical staining on serial sections from formalin fixed and paraffin embedded tissue samples of a patient cohort (n = 45) with primary ACC and correlated with patient and tumor characteristics as well as survival. Results High SOX2 expression was found in 14 (31%) primary tumor specimens and was significantly correlated with a N0 lymph node status (p = 0.04), while low SOX2 expression was correlated with a solid growth pattern (p = 0.031). Of the 45 patients, 27 tumor samples resembled an EMT-like phenotype, as assessed by high vimentin and low E-cadherin levels. However, in HNACC SOX2 levels were neither correlated with vimentin nor with E-cadherin expression, further supporting a context dependent regulation and function of SOX2 in distinct tumor entities. Conclusion The absence of SOX2 was predominantly found in solid HNACC, which are characterized by a more aggressive phenotype in ACC. However, the underlying molecular mechanisms of SOX2 regulation and function in distinct HNACC subgroups remain to be fully elucidated.

Performance Testing of RREB1, MYB, and CCND1 Fluorescence In Situ Hybridization in Spindle-Cell and Desmoplastic Melanoma Argues for a Two-Step Test Algorithm.

Background. Diagnostic confirmation of spindle-cell melanoma (SM) or desmoplastic melanoma (DM) as a melanoma can be challenging. In conventional melanoma (CM), a recently established fluorescence in situ hybridization (FISH) assay for RREB1, MYB, CCND1 can be helpful. Here, we determined the presence of RREB1, MYB, and CCND1 abnormalities in an SM/DM/mixed cohort. Methods. We assembled 49 cases and performed 3 separate hybridizations for RREB1/MYB/CCND1. We assessed clinical utility in diagnostically challenging cases and performed a cost and turnaround time analysis. Results. With regard to the diagnosis of melanoma, the FISH assay is 76% sensitive (n = 31/41 true positives melanomas) and 88% specific (n = 1/8 false positive desmoplastic nevi). The prevalence of abnormalities in DM is lower (12/19 cases, 63%; P = .03) than in SM (15/18 cases, 83%; P = .27), mixed (4 of 4 cases), or the reported sensitivity in CM (345/411 cases, 84%). The implied genetic differences in DM result in a higher false negative rate in DM (37%). Despite these limitations, when restricted to diagnostically challenging cases (n = 23), the FISH assay and, in particular, RREB1 was able to confirm melanoma in 70% (n = 16/23). Individual probe sensitivities (RREB1 > MYB > CCND1) and a cost and turnaround time analysis argues for a 2-step test algorithm that reduces the economic impact of FISH testing considerably (~55%; n = 69 vs 123 hybridizations). Conclusion. We propose a step-by-step genetic testing algorithm to support the diagnosis of melanoma in the setting of SM/DM and show that FISH testing is useful in diagnostically challenging cases.

Silencer of Cytokine Signaling 1 gene is not hypermethylated in diffuse large B-cell lymphoma.

Keywords: suppressor of cytokine signaling 1; diffuse-large B-cell lymphoma; biomarker; silencing; epigenetic

Biopanel identifies expression status of targetable proteins in sinonasal melanoma

BACKGROUND Advanced stage at presentation, lack of BRAF mutations and overall rarity pose unique challenges to the therapy and trial design in sinonasal melanoma. METHODS Here, we assessed the expression status of 12 proteins in two independent cohorts of sinonasal melanoma (n = 20). RESULTS Each case showed expression of at least one protein (KIT, TP53, MYC, HER2, EGFR, MET, VEGFR, BRAF V600E and/or MDM2), whereas lack of ALK, FLI1 and PDGFRα expression underscores differences to cutaneous melanoma. Comparison of marker frequencies to a metareview of the literature indicates that MYC, HER2, EGFR and MET had not been previously assessed. CONCLUSION Expression of at least one potentially targetable protein per case illustrates proteome pathway profiling as one starting point for marker stratified trial design.