Paul W. Harms

The Distinctive Mutational Spectra of Polyomavirus-Negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine tumor. Merkel cell polyomavirus (MCPyV) may contribute to tumorigenesis in a subset of tumors via inhibition of tumor suppressors such as retinoblastoma (RB1) by mutated viral T antigens, but the molecular pathogenesis of MCPyV-negative MCC is largely unexplored. Through our MI-ONCOSEQ precision oncology study, we performed integrative sequencing on two cases of MCPyV-negative MCC, as well as a validation cohort of 14 additional MCC cases (n = 16). In addition to previously identified mutations in TP53, RB1, and PIK3CA, we discovered activating mutations of oncogenes, including HRAS and loss-of-function mutations in PRUNE2 and NOTCH family genes in MCPyV-negative MCC. MCPyV-negative tumors also displayed high overall mutation burden (10.09 ± 2.32 mutations/Mb) and were characterized by a prominent UV-signature pattern with C > T transitions comprising 85% of mutations. In contrast, mutation burden was low in MCPyV-positive tumors (0.40 ± 0.09 mutations/Mb) and lacked a UV signature. These findings suggest a potential ontologic dichotomy in MCC, characterized by either viral-dependent or UV-dependent tumorigenic pathways.

Autopsy findings in eight patients with fatal H1N1 influenza.

A novel H1N1 influenza A virus emerged in April 2009, and rapidly reached pandemic proportions. We report a retrospective observational case study of pathologic findings in 8 patients with fatal novel H1N1 infection at the University of Michigan Health Systems (Ann Arbor) compared with 8 age-, sex-, body mass index-, and treatment-matched control subjects. Diffuse alveolar damage (DAD) in acute and organizing phases affected all patients with influenza and was accompanied by acute bronchopneumonia in 6 patients. Organizing DAD with established fibrosis was present in 1 patient with preexisting granulomatous lung disease. Only 50% of control subjects had DAD. Peripheral pulmonary vascular thrombosis occurred in 5 of 8 patients with influenza and 3 of 8 control subjects. Cytophagocytosis was seen in all influenza-related cases. The autopsy findings in our patients with novel H1N1 influenza resemble other influenza virus infections with the exception of prominent thrombosis and hemophagocytosis. The possibility of hemophagocytic syndrome should be investigated in severely ill patients with H1N1 infection.

Distinct Gene Expression Profiles of Viral- and Nonviral-Associated Merkel Cell Carcinoma Revealed by Transcriptome Analysis

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor with high mortality rates. Merkel cell polyomavirus (MCPyV), identified in the majority of MCC, may drive tumorigenesis via viral T antigens. However, mechanisms underlying pathogenesis in MCPyV-negative MCC remain poorly understood. To nominate genes contributing to pathogenesis of MCPyV-negative MCC, we performed DNA microarray analysis on 30 MCCs. MCPyV status of MCCs was determined by PCR for viral DNA and RNA. 1593 probe-sets were differentially expressed between MCPyV-negative and -positive MCC, with significant differential expression defined as at least 2-fold change in either direction and p-value of ≤ 0.05. MCPyV-negative tumors showed decreased RB1 expression, whereas MCPyV-positive tumors were enriched for immune response genes. Validation studies included immunohistochemistry demonstration of decreased RB protein expression in MCPyV-negative tumors and increased peritumoral CD8+ T lymphocytes surrounding MCPyV-positive tumors. In conclusion, our data suggest that loss of RB1 expression may play an important role in tumorigenesis of MCPyV-negative MCC. Functional and clinical validation studies are needed to determine whether this tumor suppressor pathway represents an avenue for targeted therapy.

Transcriptome Meta-Analysis of Lung Cancer Reveals Recurrent Aberrations in NRG1 and Hippo Pathway Genes

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here, we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyze 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumors without known driver mutations. In addition, we observe exon skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

Merkel Cell Polyomavirus Small T Antigen Is Oncogenic in Transgenic Mice

Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor frequently associated with clonal integration of a polyomavirus, MCPyV, and MCC tumor cells express putative polyomavirus oncoproteins small T antigen (sTAg) and truncated large T antigen (tLTAg). Here, we show robust transforming activity of sTAg in vivo in a panel of transgenic mouse models. Epithelia of pre-term sTAg-expressing embryos exhibited hyperplasia, impaired differentiation, increased proliferation and apoptosis, and activation of a DNA damage response. Epithelial transformation did not require sTAg interaction with the PP2A protein complex, a tumor suppressor in some other polyomavirus transformation models, but was strictly dependent on a recently-described sTAg domain that binds Fbxw7, the substrate-binding component of the SCF ubiquitin ligase complex. Postnatal induction of sTAg using a Cre-inducible transgene also led to epithelial transformation with development of lesions resembling squamous cell carcinoma in situ and elevated expression of Fbxw7 target proteins. Our data establish that expression of MCPyV sTAg alone is sufficient for rapid neoplastic transformation in vivo, implicating sTAg as an oncogenic driver in MCC and perhaps other human malignancies. Moreover, the loss of transforming activity following mutation of the sTAg Fbxw7 binding domain identifies this domain as crucial for in vivo transformation.

Extensive Survey of STAT6 Expression in a Large Series of Mesenchymal Tumors

OBJECTIVES Expression of strong nuclear STAT6 is thought to be a specific marker for solitary fibrous tumors (SFTs). Little is known about subtle expression patterns in other mesenchymal lesions. METHODS We performed immunohistochemical studies against the C-terminus of STAT6 in tissue microarrays and whole sections, comprising 2366 mesenchymal lesions. RESULTS Strong nuclear STAT6 was expressed in 285 of 2,021 tumors, including 206 of 240 SFTs, 49 of 408 well-differentiated/dedifferentiated liposarcomas, eight of 65 unclassified sarcomas, and 14 of 184 desmoid tumors, among others. Expression in SFTs was predominately limited to the nucleus. Other positive tumors typically expressed both nuclear and cytoplasmic STAT6. Complete absence of STAT6 was most common in pleomorphic liposarcoma and alveolar soft part sarcoma (60% and 72% cases negative, respectively). CONCLUSIONS Strong nuclear STAT6 is largely specific for SFTs. Physiologic low-level cytoplasmic/nuclear expression is common in mesenchymal neoplasia and is of uncertain significance.

IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis

Background Generalized pustular psoriasis (GPP) is a rare, debilitating, and often life‐threatening inflammatory disease characterized by episodic infiltration of neutrophils into the skin, pustule development, and systemic inflammation, which can manifest in the presence or absence of chronic plaque psoriasis (PV). Current treatments are unsatisfactory and warrant a better understanding of GPP pathogenesis. Objective We sought to understand better the disease mechanism of GPP to allow improved targeted therapies. Methods We performed a gene expression study on formalin‐fixed paraffin‐embedded GPP (n = 28) and PV (n = 12) lesional biopsies and healthy control (n = 20) skin. Differential gene expression was analyzed using gene ontology and enrichment analysis. Gene expression was validated with quantitative RT‐PCR and immunohistochemistry, and a potential disease mechanism was investigated using primary human cell culture. Results Compared with healthy skin, GPP lesions yielded 479 and PV 854 differentially expressed genes, respectively, with 184 upregulated in both diseases. We detected significant contributions of IL‐17A, TNF, IL‐1, IL‐36, and interferons in both diseases; although GPP lesions furnished higher IL‐1 and IL‐36 and lower IL‐17A and IFN‐&ggr; mRNA expression than PV lesions did. We detected prominent IL‐36 expression by keratinocytes proximal to neutrophilic pustules, and we show that both neutrophils and neutrophil proteases activate IL‐36. Suggesting another mechanism regulating IL‐36 activity, the protease inhibitors serpin A1 and A3, which inhibit elastase and cathepsin G, respectively, were upregulated in both diseases and inhibited activation of IL‐36. Conclusions Our data indicate sustained activation of IL‐1 and IL‐36 in GPP, inducing neutrophil chemokine expression, infiltration, and pustule formation, suggesting that the IL‐1/IL‐36 inflammatory axis is a potent driver of disease pathology in GPP. Graphical abstract Figure. No Caption available.

Host expression of PD-L1 determines efficacy of PD-L1 pathway blockade–mediated tumor regression

Programmed death-1 receptor (PD-L1, B7-H1) and programmed cell death protein 1 (PD-1) pathway blockade is a promising therapy for treating cancer. However, the mechanistic contribution of host and tumor PD-L1 and PD-1 signaling to the therapeutic efficacy of PD-L1 and PD-1 blockade remains elusive. Here, we evaluated 3 tumor-bearing mouse models that differ in their sensitivity to PD-L1 blockade and demonstrated a loss of therapeutic efficacy of PD-L1 blockade in immunodeficient mice and in PD-L1– and PD-1–deficient mice. In contrast, neither knockout nor overexpression of PD-L1 in tumor cells had an effect on PD-L1 blockade efficacy. Human and murine studies showed high levels of functional PD-L1 expression in dendritic cells and macrophages in the tumor microenvironments and draining lymph nodes. Additionally, expression of PD-L1 on dendritic cells and macrophages in ovarian cancer and melanoma patients correlated with the efficacy of treatment with either anti–PD-1 alone or in combination with anti–CTLA-4. Thus, PD-L1–expressing dendritic cells and macrophages may mechanistically shape and therapeutically predict clinical efficacy of PD-L1/PD-1 blockade.

Expression of the embryonic morphogen Nodal in cutaneous melanocytic lesions

Nodal, a potent embryonic morphogen in the transforming growth factor-β family, is a proposed key regulator of melanoma tumorigenicity. However, there has been no systematic study of Nodal expression in melanocytic lesions. We investigated Nodal expression by immunohistochemistry in 269 melanocytic lesions, including compound nevi, dysplastic nevi, congenital nevi, Spitz nevi, melanoma in situ, malignant melanoma including the variant desmoplastic melanoma, and metastatic melanoma. We found that the Nodal expression was significantly increased in malignant lesions (including melanoma in situ, malignant melanoma, and metastatic melanoma) compared with compound nevi, Spitz nevi, and dysplastic nevi. Surprisingly, congenital nevi expressed a level of Nodal comparable with malignant lesions, whereas desmoplastic melanoma showed lower expression than nondesmoplastic malignant melanoma (P<0.05). Deep melanoma (Breslow depth >1 mm) displayed a higher percentage of Nodal-positive tumor cells than did superficial melanoma (Breslow depth ≤1 mm), although there was no statistical difference in the overall staining intensity (P=0.18). Melanomas in situ showed a lower level of Nodal expression than did deep melanomas and metastatic melanomas (P<0.05). The low expression of Nodal in normal and dysplastic nevi, and its increasing expression with the progression of malignant lesions, are suggestive of a role for Nodal in melanoma progression.

Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation

Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes with CK20-positive Merkel cell carcinoma, including RB1 mutations restricted to Merkel cell polyomavirus-negative tumors. However, some CK20-negative Merkel cell carcinomas harbor mutations not previously described in Merkel cell carcinoma. Hence, CK20-negative Merkel cell carcinomas harbor diverse oncogenic drivers which may represent therapeutic targets in individual tumors.