Sara B. Peters

Merkel Cell Polyomavirus in Cutaneous Squamous Cell Carcinoma of Immunocompetent Individuals

Squamous cell carcinoma (SCC) is the second most frequently diagnosed skin cancer. It has a higher incidence in immunosuppressed individuals such as organ transplant recipients and human immunodeficiency virus (HIV) carriers. Recently, a newly described polyoma virus, Merkel cell polyomavirus (MCPyV), was found in Merkel cell carcinoma (MCC), a rare aggressive skin cancer also associated with immunosuppression. We hypothesized that MCPyV would be present in SCCs. To test for the presence of MCPyV in immunocompetent SCC patients, we used PCR primer sets directed against the large T (LT) antigen and VP1 gene of MCPyV. We detected MCPyV in 15% (26/177) of SCC DNA samples and 17% (11/63) of adjacent skin DNA samples from 21 of 58 (36%) individuals studied. We did not detect MCPyV in any matched normal blood DNA (0/57), but observed the presence of MCPyV DNA in 1 of 12 normal mouthwash DNAs. All sequenced SCC samples had a common mutation truncating the LT antigen that provides indirect evidence of viral integration. The presence of MCPyV in approximately 15% of SCCs from immunocompetent individuals warrants evaluation of MCPyV as an etiologic agent in the carcinogenesis of SCC.

PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.

Mismatch repair protein deficiency is common in sebaceous neoplasms and suggests the importance of screening for Lynch syndrome.

Abstract:The association between Lynch syndrome and sebaceous neoplasms is well characterized. The absence of expression of mismatch repair proteins (MMRPs) by immunohistochemistry (IHC) is often used in other Lynch-associated tumors to guide testing. IHC for MLH1, PMS2, MSH2, and MSH6 was performed on 36 benign and malignant sebaceous neoplasms with the absence of one or more MMRP in 38.9% of cases. Among lesions with abnormal IHC, 71.4% were missing both MSH2 and MSH6, 21.4% lacked MLH1 and PMS2, and 7.1% lacked only MSH6. Of the 10 patients with absent MMRP, 5 had gene-test confirmed Lynch syndrome, 3 had no suggestive personal or family medical history and 2 had no recorded data. Tumor-infiltrating lymphocytes in neoplasms with absent MMRP were statistically significantly greater than in those with intact MMRP (16.5 vs. 9.7, P = 0.027). MMRP deficiency is common in sebaceous neoplasms, suggesting the importance of screening for Lynch syndrome in these patients.

Phosphatase and tensin homolog immunohistochemical staining and clinical criteria for Cowden syndrome in patients with trichilemmoma or associated lesions.

Abstract:Trichilemmomas and mucocutaneous papillomatous papules are associated with Cowden syndrome (CS). Germline Phosphatase and tensin homolog (PTEN) mutations have been identified in 34% to 80% of those meeting clinical criteria for CS. PTEN expression has not been well evaluated in large numbers of trichilemmoma. We investigated clinical criteria for CS in trichilemmoma patients and studied PTEN staining to determine how often patients with trichilemmoma have CS and whether PTEN staining is useful. About 102 cases of trichilemmoma or associated lesions from 95 patients were collected. Clinical histories were reviewed to investigate the incidence of CS using International Cowden Consortium operational criteria for diagnosis of CS, version 2000. PTEN staining was performed and graded for intensity and percentage. Although 1 patient had 3 trichilemmoma or associated lesions, and 5 had 2 trichilemmoma or associated lesions, none of 95 patients met clinical criteria for a diagnosis of CS. Twelve of the 89 cases available for staining (13.5%) showed decreased PTEN. Of these, the demographic, clinical, and pathological features were not significantly different compared with PTEN intact cases. None of the cases from the 6 patients with more than 1 trichilemmoma or associated lesions showed decreased PTEN staining. We thus conclude that the likelihood of a clinical diagnosis of CS among patients with a solitary or only a few trichilemmoma is extremely low. PTEN expression is decreased in some sporadic trichilemmomas or associated lesions. This is the largest study investigating clinical history and PTEN staining in patients with trichilemmoma or associated lesions. Because none of our patients met clinical diagnostic criteria for CS, the direct correlation of PTEN in CS and sporadic trichilemmoma remains unclear.

Allele-specific imbalance mapping identifies HDAC9 as a candidate gene for cutaneous squamous cell carcinoma

More than 3.5 million nonmelanoma skin cancers were treated in 2006; of these 700,000 were cutaneous squamous cell carcinomas (cSCCs). Despite clear environmental causes for cSCC, studies also suggest genetic risk factors. A cSCC susceptibility locus, Skts5, was identified on mouse chromosome 12 by linkage analysis. The orthologous locus to Skts5 in humans maps to 7p21 and 7q31. These loci show copy number increases in ∼10% of cSCC tumors. Here, we show that an additional 15–22% of tumors exhibit copy‐neutral loss of heterozygosity. Furthermore, our previous data identified microsatellite markers on 7p21 and 7q31 that demonstrate preferential allelic imbalance (PAI) in cSCC tumors. On the basis of these results, we hypothesized that the human orthologous locus to Skts5 would house a gene important in human cSCC development and that tumors would demonstrate allele‐specific somatic alterations. To test this hypothesis, we performed quantitative genotyping of 108 single nucleotide polymorphisms (SNPs) mapping to candidate genes at human SKTS5 in paired normal and tumor DNAs. Nine SNPs in HDAC9 (rs801540, rs1178108, rs1178112, rs1726610, rs10243618, rs11764116, rs1178355, rs10269422 and rs12540872) showed PAI in tumors. These data suggest that HDAC9 variants may be selected for during cSCC tumorigenesis.

Differential mutation frequencies in metastatic cutaneous squamous cell carcinomas versus primary tumors

Exome and targeted sequencing studies have identified potential driver mutations for a variety of tumor types. Cutaneous squamous cell carcinoma (cSCC) is one of the most highly mutated cancers but typically is associated with low rates of metastasis and high survival rates. Nevertheless, metastatic cSCC is a significant health threat; up to 8800 individuals die each year of this disease.

Pancreatic panniculitis in a patient with BRCA2 mutation and metastatic pancreatic adenocarcinoma

A 63-year-old woman with BRCA2 mutation and history of ovarian cancer treated five years previously with hysterectomy and, bilateral salpingo-oophorectomy presented with painful bilateral lower extremity nodules. These lesions existed for 4 months, and occasionally broke open and drained a thick oily substance. Previously, she had an extensive evaluation including chest x-ray, bacterial and fungal tissue cultures, and several unremarkable laboratory measurements. A previous biopsy of the nodules demonstrated a granulomatous and neutrophilic panniculitis. She had previously been given the diagnosis of erythema nodosum (EN) and was treated unsuccessfully with different antibiotics, anti-inflammatories and topical steroids. On examination, multiple violaceous, tender nodules were seen on both the shins and knees of her lower extremities. A photograph was obtained at 3 months follow-up (Fig. 1). Laboratory analysis revealed a significantly elevated lipase level of 8000 U/l, normal amylase and eosinophilia. The patient denied any abdominal or systemic symptoms. A punch biopsy specimen obtained from one of her nodules displayed histopathological features that were consistent with pancreatic panniculitis (PP) including focal areas of fat necrosis with ghost-cell like outlines of adipocytes and adjacent basophilic calcium deposition (Fig. 2). Tissue cultures for bacteria, fungi, mycobacteria, Gram, Gomori methenamine-silver, acid-fast and periodic-acidSchiff stains were negative for organisms. CT scan of the abdomen and pelvis showed a 10.8 cm · 9.1 cm heterogeneous mass at the tail of the pancreas with liver metastasis.

MicroRNA expression profiling in metastatic cutaneous squamous cell carcinoma

or sinecatechin may take up to 3 to 16 weeks for complete clearing of AGWs, treatment with IM was much more rapid. This could be explained by the loss of mitochondrial respiration mediated by IM leading to cell death and a cytotoxic reaction within hours. Our histological examination 18 h after treatment confirmed this direct cytotoxic effect of IM in AGWs. So far only surgical therapies show clearance rates approaching 100%. However, only single, protruding AGWs can be treated properly with surgical therapies and cautery can lead to scarring since it is difficult to restrict the effect to the skin lesions only. All those treatments bear the risk of high relapse rates after one to three months, whereas we could show that our patients are still in remission after up to 240 days. Further clinical trials with a longer follow-up period are required to confirm this persistent remission.

Hair Follicle Nevus With Features of Comedo Nevus: An Expanding Spectrum.

Hair follicle nevus (HFN) is a rare hamartomatous lesion of the folliculosebaceous unit, with or without admixed fibroadipose or muscular tissue. It typically has a congenital presentation in the preauricular area of infants and is frequently confused with an accessory tragus. Acquired tumors with similar histopathologic features have been described infrequently during adolescence and adult life. We report yet another unique presentation of this unusual lesion in a 4-year-old girl who had a long-standing tumor of the nasal columella that started growing rapidly after trauma. Histopathologic examination revealed increased numbers of hair follicles, some of which were associated with diminutive sebaceous glands, with no associated central cystic structure. In addition, the infundibula of the follicles were dilated and filled with keratinous debris. Although these hamartomas are common in the head and neck region, to our knowledge, this is the first report of a HFN at this anatomic location. In addition, this tumor has an overall architecture of a HFN but is accompanied by features of a comedo nevus. We also present a review of the literature and summarize the current diagnostic criteria for HFN.

Abstract 933: The expression of PRMT5 methyltransferase mediates cell survival and metastatic phenotype in malignant melanoma

Post-translational modification of proteins is involved at all levels of cellular regulation. The PRMT5 enzyme is a type II arginine methyltransferase that catalyzes the transfer of a methyl group to two of three guanidino nitrogen atoms within the arginine molecule. This enzyme has been shown to methylate histone H3 at arginine 8 (H3R8) and H4R3 to trigger silencing of tumor suppressor genes. In addition, PRMT5 has been shown to interact with p53, TRAIL receptor, and the CDK4 complex to regulate the cell cycle or apoptosis. Although prior studies have provided insight into these mechanistic features of the PRMT enzymes, most data are derived from a limited panel of cell lines. PRMT5 over-expression has recently been shown to influence progression of leukemia and lymphoma. However, few, if any publications exist which document the role of PRMT enzymes in melanoma. We hypothesized that PRMT5 expression promotes metastasis and contributes to reduced immunologic recognition of melanoma cells. Immunoblot analysis and confocal microscopy revealed that PRMT5 is expressed in a panel of melanoma cell lines regardless of the mutational status of B-Raf, NRas, or p53. siRNA-mediated inhibition of PRMT5 led to apoptosis and restored CXCL10 chemokine expression. PRMT5 expression was next determined by immunohistochemical (IHC) analysis of formalin-fixed samples from patients with melanoma (n=56 primary; n=20 metastases) or benign nevi (n=24). The specimens were obtained as a commercially-available tissue microarray, or procured as de-identified primary patient samples (IRB #20100071; P.I. Lesinski; OSU IRB # 2002H0089; co-PI: Peters). PRMT5 expression was significantly increased in the nucleus of melanoma cells as compared to normal epidermis or benign nevi (p=0.001). The level of cytoplasmic PRMT5 expression was also elevated in melanoma lesions, however its distribution in benign tissues was bimodal, and expressed in a subset of benign samples. Notably, nuclear PRMT5 expression increased as melanoma cells invaded the dermis or in melanoma lesions exhibiting pagetoid spread. Finally, we have identified a specific inhibitor of PRMT5 arginine methyltransferase activity from a small molecule library. This lead compound (BLL-1) blocked the dimethylation of arginine 3 on histone H4 (H4R3me2s). A series of titration experiments revealed reduction of both H4R3me2s and proliferation rate. Treatment of multiple melanoma cell lines with BLL-1 (24 hr) led to significantly increased apoptosis. These data represent the first report of PRMT5 and its inhibition in melanoma. Together, this suggests PRMT5 inhibition as a potential therapeutic strategy by virtue of its ability to promote apoptosis and restore immunomodulatory gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2011-933