Positive Merkel Cell Carcinoma Research Articles

Epigenetic Dysregulation in Virus-Associated Neoplasms.

The oncoproteins of human tumor viruses regularly interact with the cellular epigenetic machinery. Such interactions alter the epigenome of the host cell and reprogram its gene expression pattern. Altered levels or redistribution of (cytosine-5)-DNA methyltransferases and changes in the cellular methylome were observed in Kaposi sarcoma-associated herpesvirus (KSHV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV), and human papillomavirus (HPV) associated neoplasms and cell lines. Methylation-mediated silencing of cellular promoters was also noted in Merkel cell polyomavirus (MCPyV) positive Merkel cell carcinomas, and, as discussed elsewhere, in EBV-associated malignancies and adenovirus-induced rodent tumors as well. Promoter activation also occurred, either associated with DNA hypomethylation or with the induction of euchromatic histone modifications by viral oncoproteins. It is worthy to notice that HCV infection induced large, hypomethylated blocks of cellular chromatin, although the exact molecular mechanism remains to be elucidated. In hepatoma cells expressing HBx, the oncoprotein encoded by the HBV genome, demethylation of the repetitive satellite 2 sequences was observed, due to downregulation of the de novo DNA methyltransferase DNMT3B. Tax and HBZ, the oncoproteins of human T-cell lymphotropic virus type I (HTLV-I), can both activate and silence distinct cellular promoters by interacting with cellular enzymes involved in histone modification.

31 MCPyV and the immune system: Target and modulator

Merkel cell carcinoma (MCC) is a aggressive skin cancer associated with the Merkel cell polyomavirus (MCPyV). Presence of tumor-infiltrating lymphocytes and immune competence are strongly correlated with the course of the disease. MCPyV encoded proteins should be highly effective targets for cytotoxic immune responses as they are both foreign to the host and specific for the infection. Srutinizing for T-cell responses against MCPyV proteins revealed responses against HLA-A1, -A2, -A3, -A11 and -B7 restricted epitopes derived from both Large and Small T antigen in MCC patients. Moreover, we demonstrate the processing and presentation of the oncoprotein derived peptides, as well as the functional activity of the T antigen-reactive T cells. Despite the expression of immunogenic viral proteins and the presence of reactive cytotoxic T cell, MCCs are able to evade the immune system. Infected transformed cells in general express activating NKG2D ligands such as MICA/B activating cellular immune responses. However, in none of the MCPyV positive MCC cell lines MICA/B were expressed on transcriptional or translational level. Immunohistochemistry of 54 MCC tumors further revealed that in the majority MICA was not detectable. Since MICA/B expression is frequently silenced by histone deacetylation the effect of the HDAC inhibitor SAHA was tested revealing an induction of MICA/B resulting in an boosted lysis by cytotoxic lymphocytes. Accordingly, ChIP analysis demonstrated enhanced Histone H3 acetylation of the MICA/B promoters. Thus, MCPyV derived epitopes elicit cytotoxic T-cell responses which can be rendered functional effective by induction of NKG2D Ligand expression.

Characterization of functional domains in the Merkel cell polyoma virus Large T antigen.

Merkel cell polyomavirus (MCPyV)--positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. This RB-binding domain in MCPyV-LT is--in contrast to other polyomavirus LTs (e.g., SV40)--embedded between two large MCPyV unique regions (MUR1 and MUR2). To identify elements of the MCPyV-LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for nuclear accumulation of truncated MCPyV-LT nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTO--a second protein encoded by an alternative reading frame in the MCPyV-LT mRNA--are completely dispensable for MCPyV-driven tumor cell proliferation. Notably, even MCPyV-LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying that--while binding to Vam6p is not essential--HSC-70 interaction is significantly involved in mediating MCPyV-LT function in MCC cells including growth promotion and induction of E2F target genes.

Expression of focal TTF‐1 expression in a case of CK7/CK20‐positive Merkel cell carcinoma

Expression of focal <scp>TTF</scp>‐1 expression in a case of <scp>CK7</scp>/<scp>CK20</scp>‐positive Merkel cell carcinoma

C‐terminal deletions of Merkel cell polyomavirus large T‐antigen, a highly specific surrogate marker for virally induced malignancy

In 67-100% of cutaneous Merkel cell carcinomas (MCC) the Merkel cell polyomavirus (MCPyV) integrates into the host genome. Mutations and deletions truncating the C-terminal helicase domain of the T-antigen (TAg) protein have been detected in these MCCs, but not in healthy skin specimens. C-terminal deletions of the TAg nucleic acid sequences are characteristic for about 38% of these cases. While the association of MCPyV with MCC has been proven, it is unknown whether MCPyV may play a similar role in other tumor entities. We describe in detail the development and validation of a novel Merkel cell polyomavirus TAg C-terminus deletion assay (MCPyV ΔC-TAg). The triplex real-time PCR quantifies the N- and C-terminal part of the MCPyV TAg gene and the cellular β-globin gene. By comparing the copy numbers of the N- and C-terminal part, deletions of the MCPyV TAg C-terminus are rapidly identified. MCPyV ΔC-TAg was used to assess the physical state of MCPyV TAg in a large series of 55 MCCs, 15 cutaneous lymphomas and 47 forehead smears of healthy individuals. Neither DNA positivity nor viral load was able to discriminate MCCs from the other different types of samples. However, deleted TAg C-terminus sequences were detected only in MCPyV positive MCCs (39%). Consequently, detection of deleted C-terminal TAg sequences appears to be a highly specific surrogate marker for virally induced malignancy and should be used to support novel assumed MCPyV-tumor associations. The study further supports the notion that MCPyV does not play a role in cutaneous lymphoma pathogenesis.

Rapid rational drug targeting of Merkel cell polyomavirus (MCV)-positive Merkel cell carcinoma (MCC) using the survivin inhibitor YM155.

8577 Background: MCC is an aggressive, chemoresistant skin cancer causing more deaths each year than chronic myelogenous leukemia. We discovered a new virus, Merkel cell polyomavirus (MCV), clonally integrated into ~80% of primary and metastatic MCC in 2008. To find therapeutic targets for this cancer, we examined cellular genes perturbed by MCV infection. Methods: Digital transcriptome subtraction was used to discover MCV and also to reveal survivin gene (BIRC5) upregulation in virus-positive tumors. MCV T antigen knockdown studies in seven MCC lines and large T (LT) transduction into BJ fibroblasts were used to confirm this. Drug screening was performed in vitro using Cell-Titer Glo assays in a two stage analysis. In vivo screening used an MKL-1 (MCV+) MCC NOD-SCIDg mouse xenograft model with a single three-week treatment round. Results: MCV large T oncoprotein induces survivin transcription through retinoblastoma protein sequestration by the LT LXCXE motif. MCV T antigen knockdown results in nonapoptotic MCC cell death and loss of survivin expression. YM155, a phase II survivin transcription inhibitor, causes MCV+ MCC cell necroptosis associated with autophagy at 1-12 nM EC50. Of 1359 other drugs from LOPAC and NCI Oncology Set II libraries, only bortezomib had in vitro potency comparable to YM155. In MKL-1 xenograft studies, mice were treated with saline, bortezomib or YM155 for three weeks using standard dosings. Bortezomib did not significantly improve mouse survival (33%) over saline (24%) during treatment. In contrast, all YM155-treated mice survived (100%, p&lt;0.001) the 3 week treatment period. Tumors resumed growth once YM155 treatment was stopped suggesting that YM155 is cytostatic in vivo rather than cytotoxic. Conclusions: Survivin expression is induced by MCV LT and is critical to MCV+ MCC survival. A survivin inhibitor, YM155 was nontoxic to mice and cytostatic for MCV+ MCC xenografts. Using genomic technologies, in less than four years, the primary viral cause for most MCC was discovered, new diagnostic tests developed and a promising rational drug candidate identified. A cooperative group trial (E1611) for YM155 and bortezomib in MCC patients is currently planned.

Abstract 557: Fluorescence in situ hybridization versus qPCR as means to detect Merkel cell polyomavirus in Merkel cell carcinoma

Abstract Background: Merkel cell polyoma virus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). The clonal integration and tumor specific mutations in the large T Antigen (LTAg) gene strongly implicate an oncogenic impact of the virus. To date the relationship between the viral presence and cancer induction, development or clinical prognosis is discussed controversial. Yet almost all studies are based on quantitative virus detection e.g. PCR or qPCR. Material and Method: In order to gain additional information about the quality of the viral presence on the single cell level we performed FISH analysis of formalin fixed and paraffin embedded (FFPE) MCCs (n= 62) on tissue micro arrays (TMA), determined the FISH pattern and correlated the results of the FISH analysis with the qPCR data. We grouped the MCCs according to different FISH evaluations and correlated them with the respective qPCR data on the basis of a determined cut-off. MCPyV FISH was established using the MKL-1 cell line which harbors integrated copies of MCPyV DNA. For MCPyV-qPCR the LT3 primer pair was used on whole tissue sections. Results: MCPyV-FISH on FFPE MKL-1 cells revealed punctate signals compatible with viral integration. The MCPyV FISH positive MCC cores (76%) mainly revealed two different signal patterns: a punctate pattern (85%) which correlated with a moderate relative viral presence and in some areas the punctate pattern was combined with a diffuse pattern (15%) indicating the episomal presence of the virus which is linked to viral replication. If a pattern mixture was seen this was associated with very high qPCR values. Comparing MCPyV FISH and qPCR data the results correlated highly with 83 % in the MCPyV positive evaluated group, whereas the negative group showed a concordance of 93 %. The mean of the qPCR values of all MCPyV positive cores differed significantly from the negative cores (p= 0.0076). The FISH signals were in some tumor areas from the same patient heterogenic in intensity, pattern and localization. Conclusion: While the qPCR using the LT3 primer pair was highly sensitive to detect MCPyV sequences in the respective MCC cases, the MCPyV FISH analysis highly concordantly revealed the quality of the viral presence, i.e. viral integration or episomal presence and morphological sublocalization in the tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 557. doi:1538-7445.AM2012-557

Activation of the PI3K/AKT Pathway in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients.

Merkel Cell Polyomavirus Infection, Large T Antigen, Retinoblastoma Protein and Outcome in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is rare skin cancer that is often associated with Merkel cell polyomavirus (MCPyV) infection. Polyomaviruses repress tumor suppressor proteins, thus influencing cell-cycle progression, but the effect of MCPyV on the key cell-cycle regulating proteins is poorly understood. We evaluated expression of the MCPyV large T-antigen (LTA), Ki-67, and the key putative tumor suppressor proteins, the retinoblastoma protein (RB and phospho-RB) and p53, and their regulatory proteins (cyclin D1, cyclin E, p16, p21, p27, and MDM2) by using immunohistochemistry from tumors of 91 MCC patients identified from a population-based nationwide cohort. Tumor MCPyV DNA was measured by using quantitative PCR, and TP53 mutations were identified with sequencing. MCPyV LTA expression was strongly associated with presence of MCPyV DNA in tumor, and it was almost invariably associated with tumor RB expression (P < 0.0001 for both comparisons). Both MCC LTA and RB expression were strongly associated with favorable MCC-specific and overall survival in univariable analyses (P ≤ 0.01 for all four analyses). Presence of MCPyV LTA was also associated with the female gender, the intermediate type of tumor histology, location of the tumor in a limb, cell proliferation rate, and absence of p53 expression. TP53 mutations were detected only in MCPyV DNA-negative tumors. MCPyV DNA-positive MCC has several clinical and molecular features that differ from MCPyV DNA-negative cancers. MCPyV-associated MCCs express RB, but may not harbor TP53 mutations. These findings provide further support that MCPyV causes the majority of MCCs.

Abstract 2717: Detection of Merkel cell polyomavirus in chronic lymphocytic leukemia cells by fluorescence in situ hybridization (FISH)

Abstract Background: Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of Merkel cell carcinomas (MCC). A number of previous studies have shown that MCC patients are at a significantly increased risk to develop chronic lymphocytic leukemia (CLL) and vice versa. Until recently, clonal integration and truncating mutations of the Large T antigen (LTAg) of MCPyV were restricted to MCC. We have recently reported the presence of the MCPyV in highly purified tumor cells of CLL (n = 19/70, 27.1%) (Blood. 2010 Sep 3). Of these, six revealed a novel 246 bp deletion in the helicase gene of the LTAg. The presence of MCPyV was confirmed by immunohistochemistry. Material and Method: Here we aimed to determine the presence of MCPyV by fluorescence in situ hybridization (FISH) analysis in CLL cells in order to evaluate whether MCPyV was integrated or episomal. For this purpose we performed FISH analysis as previously described (Int J Cancer. 2005, 115:419-428) using the MCPyV genome as FISH probe. We tested 2 of the previously reported MCPyV positive CLL cases (EDTA decalcified bone marrow trephines) and MCPyV-positive MCC (n = 5). In addition, we tested MCPyV-negative tumors, e.g. breast and colon cancers. All tissues were formalin-fixed and paraffin-embedded. Results: Specific MCPyV DNA by FISH analysis was detected in the nuclei of MCPyV-positive CLL and MCC cells. In contrast to particularly punctate FISH signals in MCC indicating viral integration, the nuclear FISH signals of the CLL cases revealed especially granular signals, indicative for integrated MCPyV DNA together with transcribed viral RNA (Int J Cancer. 2008, 122:2656-2664). No signals were obtained by MCPyV FISH in breast or colon cancer specimens. Conclusion: The specific detection of MCPyV in CLL cells further supports our previous report of a possible involvement of MCPyV in a significant subset of CLL. The specific but rather granular nuclear FISH signals in MCPyV positive CLL cells needs further study to evaluate if it is also generated by co-existing episomal virus copies, by viral RNA or by the decalcification process. In addition, more CLL cases will be analyzed for the presence of MCPyV. 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 2717. doi:10.1158/1538-7445.AM2011-2717

Immunological detection of viral large T antigen identifies a subset of Merkel cell carcinoma tumors with higher viral abundance and better clinical outcome

Immunological detection of viral large T antigen identifies a subset of Merkel cell carcinoma tumors with higher viral abundance and better clinical outcome

FC28 Characterization of the small T Antigen in Merkel cell-associated polyomavirus in Merkel cell carcinogenesis

Background Merkel cell carcinoma (MCC) is a highly aggressive skin malignancy affecting neuroectodermal cells. Recently it was discovered that MCC have an infectious origin and a novel polyomavirus, the Merkel cell associated polyomavirus (MCPyV), was identified in MCC. MCPyV expresses two ‘early’ proteins, namely the Large T (LT) and Small t (St) antigens [1,2]. Hypothesis Based on the research data on other polyomaviruses, it is suggestive that LT and St antigens are involved in viral replication and host cell transformation. However the LT antigen is found truncated in MCC losing helicase activity and putatively inactivating p53 tumor suppressor binding capacity. Therefore, in this study, we propose to investigate the role of the intact St antigen in MCC carcinogenesis. Aim We planned to express and purify St protein. Polyclonal antibody will be developed against specific St oligopeptide and will be used in the verification of the produced St protein. Methods The full St gene was PCR amplified using DNA extracted from a MCPyV positive MCC case. Prokariotic expression system including pEXP5-CT/TOPO vector and BL21 Star(tm)(λDE3)pLysS E. coli cells (Invitrogen Co.) was utilized. Results Small t protein was successfully expressed and verified with His-Tag antibody and St specific antibody. Conclusion The expressed St protein will be purified and utilized in further assays to find the cellular protein binding partners. These experiments intended to reveal the interference of St antigen with cell regulatory proteins. This information can lead to understanding of MCC carcinogenesis and to developing better therapy to combat this malignant cancer. Key Words: MCC, MCPyV, small t antigen protein expression Funding: NIAID: ‘Molecular Basis of Infectious Disease’ (1 T32 AI055449-01A2) Co-Investigator (i.e. Mentor), 2005-2010.

Abstract 5352: A high percentage of Merkel cell carcinomas is biologically associated with Merkel cell polyomavirus

Abstract Merkel cell polyomavirus (MCPyV) DNA has been detected by PCR in 75-100% of Merkel cell carcinomas (MCC), an aggressive neuroendocrine skin cancer. MCPyV is a 5.4 kb DNA virus that expresses tumor (T) antigen in tumor tissues. The aim of this study was to analyze which subset of MCC is biologically associated with MCPyV using various viral detection methods. Genomic DNA isolated from paraffin-embedded tissue of 61 MCC was analyzed by MCPyV-specific PCR. Using a 5kb MCPyV DNA probe, the physical status of MCPyV was determined by fluorescence in situ hybridization (FISH) using tyramide signal amplification and ApoTome microscopy. Expression of large T antigen was assessed by immunohistochemistry (IHC) using monoclonal antibody CM2B4 (kindly provided by Y. Chang). A total of 44 of 61 (72%) MCC were positive for MCPyV by PCR. 34 of these 44 cases (77%) were also positive for MCPyV by FISH and 28 (64%) showed strong nuclear large T antigen expression by IHC. In contrast, none of the 17 PCR-negative tumors showed FISH signals and only 1 case showed a weak immunostaining for large T antigen. FISH analysis showed in 16 cases (47%) tumor nuclei with a single punctate signal indicating viral integration. In 8 of these tumors also areas were observed with a granular pattern with &amp;gt;1 nuclear signals varying significantly in size and intensity, indicating the presence of episomal viral DNA and/or RNA. In the remaining 18 cases (53%) an exclusive granular FISH pattern was observed. Our data indicate that a subset of MCC is caused by MCPyV as determined by PCR, FISH and IHC analysis (46% is positive with all 3 detection methods). FISH and IHC showed a strong positive and negative concordance. FISH signal evaluation furthermore suggests viral integration in half of the positive MCC and a non-clonal viral persistence in the remaining positive cases. Further studies have to validate these findings and elucidate the molecular mechanisms underlying MCV-positive and -negative MCC development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5352.

Frequent Expression of Glypican-3 in Merkel Cell Carcinoma

The pathologic diagnosis of Merkel cell carcinoma (MCC) is mainly based on routine morphology and a characteristic cytokeratin 20 (CK20) immunohistochemical staining pattern. However, about 10% to 15% of MCCs are reportedly CK20 negative and about 10% to 20% of MCCs have only focal CK20 positivity. We aimed to study glypican-3 (GPC3) expression in MCC and to compare it with CK20 expression in MCC. Immunohistochemical expression of GPC-3, CK20, and CD23 were studied in 55 cases of MCC and 21 cases of noncutaneous small cell neuroendocrine carcinoma (SCNC), including 10 cases of pulmonary SCNC and 11 cases of nonpulmonary SCNC. The results of GPC3 expression in these tumors were then compared with that of CK20. Of the 55 cases, 39 were positive for GPC3 (70.9%). Seventy-seven percent of the positive MCCs (30/39) showed GPC3 immunoreactivity in > or = 30% tumor cells. Of the 55 cases, 53 were positive for CK20 (96%); 49 of them showed CK20 immunoreactivity in > or = 30% tumor cells. All MCC cases were either positive for both GPC3 and CK20 (37 cases) or at least 1 of the markers (18 cases, 16 CK20 only and 2 GPC3 only). In addition, we studied GPC3 and CK20 expression in 10 cases of pulmonary SCNC and 11 cases of nonpulmonary SCNC. Three of ten pulmonary SCNC and 5 of 11 nonpulmonary SCNC expressed GPC3. All pulmonary SCNC and 9 of 11 nonpulmonary SCNC were negative for CK20. All cases of MCC and SCNC were negative for CD23. In conclusion, GPC3 is frequently expressed in SCNC of various origins, in particular in MCC, which, in combination with CK20, may represent another useful marker in the diagnosis of MCC.

Outcomes in Sentinel Lymph Node Positive or Clinically Positive Merkel Cell Carcinoma Treated With Nodal Irradiation Alone

Outcomes in Sentinel Lymph Node Positive or Clinically Positive Merkel Cell Carcinoma Treated With Nodal Irradiation Alone