Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147795/1/his13758.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147795/2/his13758_am.pd

Molecular profiling of ETS and non‐ETS aberrations in prostate cancer patients from northern India

BACKGROUNDMolecular stratification of prostate cancer (PCa) based on genetic aberrations including ETS or RAF gene‐rearrangements, PTEN deletion, and SPINK1 over‐expression show clear prognostic and diagnostic utility. Gene rearrangements involving ETS transcription factors are frequent pathogenetic somatic events observed in PCa. Incidence of ETS rearrangements in Caucasian PCa patients has been reported, however, occurrence in Indian population is largely unknown. The aim of this study was to determine the prevalence of the ETS and RAF kinase gene rearrangements, SPINK1 over‐expression, and PTEN deletion in this cohort.METHODSIn this multi‐center study, formalin‐fixed paraffin embedded (FFPE) PCa specimens (n = 121) were procured from four major medical institutions in India. The tissues were sectioned and molecular profiling was done using immunohistochemistry (IHC), RNA in situ hybridization (RNA‐ISH) and/or fluorescence in situ hybridization (FISH).RESULTSERG over‐expression was detected in 48.9% (46/94) PCa specimens by IHC, which was confirmed in a subset of cases by FISH. Among other ETS family members, while ETV1 transcript was detected in one case by RNA‐ISH, no alteration in ETV4 was observed. SPINK1 over‐expression was observed in 12.5% (12/96) and PTEN deletion in 21.52% (17/79) of the total PCa cases. Interestingly, PTEN deletion was found in 30% of the ERG‐positive cases (P = 0.017) but in only one case with SPINK1 over‐expression (P = 0.67). BRAF and RAF1 gene rearrangements were detected in ∼1% and ∼4.5% of the PCa cases, respectively.CONCLUSIONSThis is the first report on comprehensive molecular profiling of the major spectrum of the causal aberrations in Indian men with PCa. Our findings suggest that ETS gene rearrangement and SPINK1 over‐expression patterns in North Indian population largely resembled those observed in Caucasian population but differed from Japanese and Chinese PCa patients. The molecular profiling data presented in this study could help in clinical decision‐making for the pursuit of surgery, diagnosis, and in selection of therapeutic intervention. Prostate 75:1051–1062, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111808/1/pros22989.pd

The MD Anderson prostate cancer patient-derived xenograft series (MDA PCa PDX) captures the molecular landscape of prostate cancer and facilitates marker-driven therapy development

BACKGROUND: Advances in prostate cancer (PCa) lag behind other tumor types partly due to the paucity of models reflecting key milestones in PCa progression. OBJECTIVE: To develop clinically relevant PCa models. DESIGN: Since 1996 we have generated clinically annotated patient-derived xenografts (PDXs) (the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical PCa. RESULTS: We studied two cell line-derived xenografts and the first 80 PDXs derived from 47 human PCa donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (AR, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the SPOPL gene in PDXs derived from 7 human donors out of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of PCa. SPOPL deletions are found in 7% of TCGA PCas, which suggests that our cohort is a reliable platform for targeted drug development. CONCLUSIONS: The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of PCas progressing under novel treatments and enables optimization of PCa-specific, marker-driven therapy

Neurofilament is Superior to Cytokeratin 20 in Supporting Cutaneous Origin for Neuroendocrine Carcinoma

Background: Merkel cell carcinoma (MCC) is a rare and aggressive primary cutaneous neuroendocrine carcinoma. Because MCC cannot be reliably distinguished morphologically from small cell carcinomas from other sites, immunohistochemistry is required to confirm cutaneous origin. Expression of cytokeratin-20 (CK20), especially in a paranuclear, dot-like pattern, is commonly used to confirm the diagnosis of MCC, but is negative in 5-10% of cases. Diagnostic challenges also arise in the setting of metastatic neuroendocrine carcinoma of unknown primary. Design: Neurofilament and CK20 expression was evaluated in 53 MCC specimens from 37 unique patients, including 8 previously characterized CK20-negative MCC tumors. Twelve cases had results from previously performed Merkel cell polyomavirus (MCPyV) immunohistochemistry. Neurofilament and CK20 expression was also assessed in 60 non-cutaneous neuroendocrine carcinomas (NEC) with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2), or other sites (7). CK20 and neurofilament immunohistochemistry was scored as either negative or positive (with or without paranuclear dot staining). Results: Neurofilament expression was observed in 40/53 (75.5%) MCC cases, including 6/8 (75.0%) CK20-negative MCC cases. Neurofilament was expressed in 9/9 (100%) MCPyV-positive tumors and 2/3 (66.7%) MCPyV-negative tumors. Neurofilament expression was observed in 2 non-cutaneous NEC cases (one sinonasal and one lung); the specificity of neurofilament for MCC versus non-cutaneous NEC was 96.7%. CK20 expression was observed in 25/60 (41.7%) NEC cases. Specificity of CK20 immunostaining for MCC versus noncutaneous NEC was 58.3%; specificity was only slightly improved (65.0%) by considering only cases with paranuclear dot-like CK20 staining to be positive. Because CK20 expression was a controlled variable in our MCC cohort, sensitivity of CK20 staining for MCC was not calculated. Conclusions: Neurofilament expression has superior specificity to CK20 expression in distinguishing MCC from non-cutaneous NEC. Neurofilament is a useful diagnostic marker in the majority of CK20- negative MCC cases and may be expressed in MCPyV-negative MCC tumors. Limitations of neurofilament immunohistochemistry include predicted lower sensitivity than CK20 and subtle staining in some tumors. However, our findings indicate neurofilament is a useful addition to diagnostic panels for excluding non-cutaneous NEC

Increased expression of EZH2 in Merkel cell carcinoma is associated with disease progression and poorer prognosis.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that affects tumorigenesis by epigenetic gene silencing. Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that has a high risk of disease progression with nodal and distant metastases. Here, we evaluated EZH2 expression by immunohistochemistry in a cohort of 85 MCC tumors (29 primary tumors, 41 lymph node metastases, 13 in-transit metastases, and 2 distant metastases) with clinical follow-up. We show strong/moderate EZH2 expression in 54% of tumors. Importantly, weak expression of EZH2 in the primary tumor, but not nodal metastases, correlated with improved prognosis compared to moderate/strong EZH2 expression (5-year MCC-specific survival of 68% versus 22%, respectively, P=.024). In addition, EZH2 was expressed at higher levels in nodal metastases compared to primary tumors (P=.005). Our data demonstrate that EZH2 has prognostic value and may play an oncogenic role in MCC

Loss of p16 expression and copy number changes of CDKN2A in a spectrum of spitzoid melanocytic lesions.

Spitzoid melanocytic lesions, including Spitz nevi (benign), spitzoid melanoma (malignant), and borderline atypical Spitz tumors (ASTs), frequently present challenges for accurate diagnosis and prognosis. Evaluation for loss of the tumor suppressor p16, encoded by CDKN2A gene on chromosome 9p21.3, has been proposed to be useful for evaluation of spitzoid melanocytic lesions. However, reports on the utility of p16 immunohistochemistry for spitzoid lesions have been conflicting, and few studies have directly compared p16 immunohistochemistry with fluorescence in situ hybridization (FISH) for CDKN2A genomic status. We analyzed a spectrum of benign (n=24), borderline (n=27), and malignant (n=19) spitzoid lesions for p16 protein expression by immunohistochemistry and CDKN2A copy number by FISH. Immunohistochemistry was evaluated by 2 scoring methods: H score and 2-tiered score (positive or negative for p16 loss). By immunohistochemistry, loss of p16 expression was not observed in Spitz nevi (0/24) but was seen in ASTs (7/27; 26%) and spitzoid melanomas (3/19; 16%). By H score, p16 expression was significantly higher in Spitz nevi relative to ASTs or spitzoid melanomas. Similarly, copy number aberrations of CDKN2A by FISH were absent in Spitz nevi but were found in 2 (9.5%) of 21 ASTs and 4 (33%) of 12 spitzoid melanomas. Our findings from this large cohort suggest that p16 aberrations are highly specific for borderline and malignant spitzoid neoplasms relative to Spitz nevi. Similar to ASTs, p16 loss in spitzoid melanomas may occur in the presence or absence of genomic CDKN2A loss

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma.

AIM: Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distinguished morphologically from small-cell neuroendocrine carcinomas (SmCC) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node (SLN) metastases of MCC. Cytokeratin 20 (CK20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK20 in MCC compared with SmCC from other sites. METHODS AND RESULTS: We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK20-negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK20-negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus-positive tumours and five of 10 (50%) virus-negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK20 expression was also assessed in 61 extracutaneous SmCC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK20 for MCC versus non-cutaneous SmCC was 96.7% and 59.0%, respectively. CONCLUSIONS: Neurofilament has superior specificity to CK20 in distinguishing MCC from non-cutaneous SmCC. Neurofilament is frequently expressed in CK20- and virus-negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non-cutaneous SmCC

A novel RNA in situ hybridization approach highly sensitive for detection of Merkel cell polyomavirus

Background: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCC tumors. Detection of MCPyV in MCC tumors has diagnostic utility, and potentially prognostic and therapeutic implications. We investigated whether RNAscope, a novel RNA in situ hybridization (ISH) method for detection of RNA transcripts in tissues at the single-cell/single-molecule level, is useful for MCPyV detection. Methods: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMAs) and full tissue sections encompassing 91 MCC tumors from 75 patients, 14 carcinomas of other types, and various normal tissues. qPCR and immunohistochemistry (IHC) for MCPyV were performed on 58 and 88 cases, respectively. qPCR was also performed on 18 cases of normal skin to establish background levels of MCPyV in non-tumor tissues. Results: RNA-ISH demonstrated the presence of MCPyV in 46.2% (42/91) of cases. A total of 58 samples had data from all the three detection modalities (qPCR, RNA-ISH and IHC). RNA-ISH demonstrated 100% concordance with qPCR results, whereas IHC had a slightly lower concordance (96.6%). RNA-ISH demonstrated higher agreement than IHC among TMA cores from the same case, and between TMA cores and matched full tissue sections. Of samples with background inflammatory cells, 5 of 37 (15.6%) showed moderate nonspecific staining of lymphocytes by IHC, whereas RNA-ISH lacked background. Conclusions: RNA-ISH is comparably sensitive to qPCR for detection of MCPyV, and allows for correlation with tissue morphology. Our findings support RNA-ISH as a highly effective method for MCPyV detection in tumors

CDK7 inhibition suppresses Castration-Resistant Prostate Cancer through MED1 inactivation.

Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by Androgen Receptor (AR). First-line CRPC treatments typically target AR-signaling, but are rapidly bypassed, resulting in only a modest survival benefit with the anti-androgens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional co-activator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at super-enhancer sites, and is essential for AR-mediated transcription. Additionally, a CDK7 specific inhibitor THZ1 blunts AR-dependent neoplastic growth by blocking AR/MED1 co-recruitment genome-wide, as well as reverses the hyper-phosphorylated MED1 associated enzalutamide resistant phenotype. In vivo, THZ1 induces tumor regression of AR amplified castration-resistant human prostate cancer in xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC