Figure S1. Increased TLR9 expression in induced glioma spheres. Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs. Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth. Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs. Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site.

Suppl. Fig. S3 showing CTLA4 expression by tumor associated CD19 B cells

Suppl. Fig. S1 showing CTLA4 expression and CD86 cellular internalization by human BCL and human MM cells acquired by flow cytometry.

<p>Suppl. Fig. S2 showing improved Th1 and Th2 maturation and increased CD3 T cell activation upon CTLA4 blockade in vivo.</p>

To date, there are no inhibitors that directly and specifically target activated STAT3 and c-Myc in the clinic. Although peptide-based inhibitors can selectively block activated targets, their clinical usage is limited because of low cell penetration and/or serum stability. Here, we generated cell-penetrating acetylated (acet.) STAT3, c-Myc, and Gp130 targeting peptides by attaching phosphorothioated (PS) polymer backbone to peptides. The cell-penetrating peptides efficiently penetrated cells and inhibited activation of the intended targets and their downstream genes. Locally or systemically treating tumor-bearing mice with PS-acet.-STAT3 peptide at low concentrations effectively blocked STAT3 in vivo, resulting in significant antitumor effects in 2 human xenograft models. Moreover, PS-acet.-STAT3 peptide penetrated and activated splenic CD8+ T cells in vitro. Treating immune-competent mice bearing mouse melanoma with PS-acet.-STAT3 peptide inhibited STAT3 in tumor-infiltrating T cells, downregulating tumor-infiltrating CD4+ T regulatory cells while activating CD8+ T effector cells. Similarly, systemic injections of the cell-penetrating c-Myc and Gp130 peptides prevented pancreatic tumor growth and induced antitumor immune responses. Taken together, we have developed therapeutic peptides that effectively and specifically block challenging cancer targets, resulting in antitumor effects through both direct tumor cell killing and indirectly through antitumor immune responses.

ABSTRACTThe identification of a vaccination candidate against COVID-19 providing protecting activity against emerging SARS-COV-2 variants remains challenging. Here, we report protection activity against a spectrum of SARS-COV-2 and variants by immunization with protein-based recombinant RBD-C-tag administered with aluminum-phosphate adjuvant intramuscularly. Immunization of C57BL/6 mice with RBD-C-tag resulted in the in vivo production of IgG antibodies recognizing the immune-critical spike protein of the SARS-COV-2 virus as well as the SARS-COV-2 variants alpha (“United Kingdom”), beta (“South Africa”), gamma (“Brazil/Japan”), and delta (“India”) as well as wt-spike protein. RBD-C-tag immunization led to a desired Th1 polarization of CD4 T cells producing IFNγ. Importantly, RBD-C-tag immunization educated IgG production delivers antibodies that exert neutralizing activity against the highly transmissible SARS-COV-2 virus strains “Washington”, “South Africa” (beta), and “India” (delta) as determined by conservative infection protection experiments in vitro. Hence, the protein-based recombinant RBD-C-tag is considered a promising vaccination candidate against COVID-19 and a broad range of emerging SARS-COV-2 virus variants.

Vaccination efficacy is enhanced by targeting the antigen-presenting cell compartment. Here, we show that S1-Fc antigen delivery targeting the FcγR + antigen-presenting cell compartment elicits anti-SARS-CoV-2 S1-antigen specific IgG production in vivo exerting biologically functional and protective activity against live virus infection, assessed in a stringent experimental virus challenge assay in vitro . The S1-domain of the SARS-CoV-2 spike protein was genetically fused to a human immunoglobulin Fc moiety, which contributes to mediate S1-Fc cellular internalization by FcγR + antigen-presenting cells. Immediately upon administration intramuscularly, our novel vaccine candidate recombinant rS1-Fc homes to lymph nodes in vivo where FcγR + antigen-presenting cells reside. Seroconversion is achieved as early as day 7, mounting considerably increased levels of anti-S1 IgGs in vivo . Interestingly, immunization at elevated doses with non-expiring S1-Fc encoding dsDNA favors the education of a desired antigen-specific adaptive T cell response. However, low-dose immunization, safeguarding patient safety, using recombinant rS1-Fc, elicits a considerably elevated protection amplitude against live SARS-CoV-2 infection. Our promising findings on rS1-Fc protein immunization prompted us to further develop an affordable and safe product for delivery to our communities in need for COVID-19 vaccinations.

Both the extracellular matrix (ECM) and DNA epigenetic regulation are critical for maintaining stem cell phenotype and cancer progression. Whether and how ECM regulates epigenetic alterations to influence cancer stem cells (CSCs) remain to be explored. Here we report that ECM through laminin-integrin α6 upregulates ten-eleven translocation enzyme 3 (TET3) dioxygenase. TET3 in turn mediates DNA cytosine 5′-hydroxymethylation (5hmC) and upregulates genes critical for maintenance of glioma stem cells (GSCs). Activating integrin α6-FAK pathway increases STAT3 activity, TET3 expression and 5hmC levels in GSCs. Moreover, targeting STAT3 disrupts integrin α6-FAK signaling and inhibits TET3 + GSC maturation in vivo. STAT3 directly regulates TET3 expression and the two proteins are co-localized with 5hmC in GSC clusters. 5hmC is upregulated by STAT3 at the promoters of several tumorigenic genes, including c-Myc, known to be critical for GSCs. In vivo silencing of TET3 in GSC-enriched tumors reduces 5hmC accumulation and expression of the GSC critical genes, leading to tumor growth inhibition. TET3 expression and 5hmC accumulation also co-segregate with integrin α6 in patient malignant glioma. Thus, ECM- integrin α6-STAT3-TET3 axis regulates hydroxymethylation of genes important for GSCs, thereby increasing GSC tumorigenicity and resistance to therapies.

Despite their well-recognized success in the clinic, antibodies generally do not penetrate cellular membranes to target intracellular molecules, many of which underlie incurable diseases. Here we show that covalently conjugating phosphorothioated DNA oligonucleotides to antibodies enabled their efficient cellular internalization. Antibody cell penetration was partially mediated by membrane potential alteration. Moreover, without an antigen to bind, intracellular levels of the modified antibodies underwent cellular clearance, which involved efflux and lysosomal degradation, enabling detection of intended intracellular molecules as tested in fibroblasts, tumor cells, and T cells. This target-dependent cellular retention of modified antibodies extended to in vivo studies. Both local and systemic administrations of low doses of modified antibodies effectively inhibited intracellular targets, such as transcription factors Myc, interferon regulatory factor 4, and tyrosine-protein kinase SRC, and expression of their downstream genes in tumors, resulting in tumor cell apoptosis and tumor growth inhibition. This simple modification enables the use of antibodies to detect and modulate intracellular molecules in both cultured living cells and in whole animals, forming the foundation for a new paradigm for antibody-based research, diagnostics, and therapeutics.

Abstract CTL–associated antigen 4 (CTLA4) is a well-established immune checkpoint for antitumor immune responses. The protumorigenic function of CTLA4 is believed to be limited to T-cell inhibition by countering the activity of the T-cell costimulating receptor CD28. However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including via CTLA4 overexpression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3 activation and expression of genes critical for cancer immunosuppression and tumor growth and survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas silencing or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in the absence of T cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3 activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4–Tyk2–STAT3 signal pathway was also active in tumor-associated nonmalignant B cells in mouse models of melanoma and lymphoma. Overall, our results show how CTLA4-induced immune suppression occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B-cell lymphoma proliferation and survival. Cancer Res; 77(18); 5118–28. ©2017 AACR.

Understanding supports for cancer stem–like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem–like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG–siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reducedGSC alongwith glioma growth. Our findings identify TLR9 as a functionalmarker for GSC and a target for the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 5218–28. 2014 AACR.

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte-associated antigen 4 (CTLA4(apt)) allows gene silencing in exhausted CD8⁺ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8⁺ T cells in the tumor milieu; therefore, CTLA4(apt) fused to a STAT3-targeting siRNA (CTLA4(apt)-STAT3 siRNA) resulted in internalization into tumor-associated CD8⁺ T cells and silencing of STAT3, which activated tumor antigen-specific T cells in murine models. Both local and systemic administration of CTLA4(apt)-STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4(apt)-STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4(apt)-STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4(apt)-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis.

Abstract Understanding supports for cancer stem–like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem–like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG–siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reduced GSC along with glioma growth. Our findings identify TLR9 as a functional marker for GSC and a target for the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 5218–28. ©2014 AACR.

Unprotected exposure to UVB radiation from the sun and the resulting DNA damage are thought to be responsible for physiological changes in the skin and for a variety of skin cancers, including basal cell and squamous cell carcinoma and malignant melanoma. Although the mutagenic effects of UVB have been well documented and studied mechanistically, there is only limited information as to whether UV light may also be responsible for inducing epigenetic changes in the genome of exposed cells. DNA methylation is a stable epigenetic modification involved in gene control. To study the effects of UVB radiation on DNA methylation, we repeatedly exposed normal human keratinocytes to a UVB light source. After a recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) method in combination with high-resolution microarrays. Bioinformatics analysis revealed only a limited number of possible differences between UVB-exposed and control cells. However, these minor apparent changes could not be independently confirmed by bisulfite sequencing-based approaches. This study reveals that UVB irradiation of keratinocytes has no recognizable global effect on DNA methylation patterns and suggests that changes in DNA methylation, as observed in skin cancers, are not immediate consequences of human exposure to solar UVB irradiation.

Raw sequence reads for bisulfite sequencing data shown in Figure 4

Environmental chemicals and radiation have often been implicated in producing alterations of the epigenome thus potentially contributing to cancer and other diseases. Ionizing radiation, released during accidents at nuclear power plants or after atomic bomb explosions, is a potentially serious health threat for the exposed human population. This type of high-energy radiation causes DNA damage including single- and double-strand breaks and induces chromosomal rearrangements and mutations, but it is not known if ionizing radiation directly induces changes in the epigenome of irradiated cells. We treated normal human fibroblasts and normal human bronchial epithelial cells with different doses of γ-radiation emitted from a cesium 137 (137Cs) radiation source. After a seven-day recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) in combination with high-resolution microarrays. Bioinformatics analysis revealed only a small number of potential methylation changes with low fold-difference ratios in the irradiated cells. These minor methylation differences seen on the microarrays could not be verified by COBRA (combined bisulfite restriction analysis) or bisulfite sequencing of selected target loci. Our study shows that acute γ-radiation treatment of two types of human cells had no appreciable direct effect on DNA cytosine methylation patterns in exposed cells.

'Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

TO THE EDITOR The molecular mechanisms of malignant melanoma, especially epigenetic silencing, are little understood. In particular, RAS/RAF kinases and changes in p16 and the PTEN state have attracted interest (see the introduction to the Supplementary material). In our study, epigenetic silencing of the tumor suppressor genes PTEN, p16, and RASSF1A and mutations of BRAF and NRAS were analyzed in 619 samples of 230 cutaneous melanomas (median tumor thickness, 1.90mm) from 230 patients. All sections were reevaluated to confirm diagnoses and known histological prognosis parameters. In addition, local dermal actinic aging was scored semi-quantitatively as previously described (Helmbold et al., 2006Helmbold P. Lautenschlager C. Marsch W. Nayak R.C. Detection of a physiological juvenile phase and the central role of pericytes in human dermal microvascular aging.J Invest Dermatol. 2006; 126: 1419-1421Google Scholar). Mutations of codon 600 of BRAF and codon 61 of NRAS were analyzed. Promoter methylation of PTEN, p16, and RASSF1A was investigated by methylation-specific PCR (Schagdarsurengin et al., 2006Schagdarsurengin U. Gimm O. Dralle H. Hoang-Vu C. Dammann R. CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma.Thyroid. 2006; 16: 633-642Google Scholar). Mutation and methylation analyses were confirmed in different regions of the same tumor (mean: 2.59±0.98 Figure 1). Expression of PTEN was studied immunohistologically in selected corresponding paraffin sections. Intercorrelations between the investigated molecular and clinicopathological features were studied. Prospective follow-up data were used for survival analysis. Details of all methods are provided in the Supplementary material (Material and Methods, Supplementary Tables S1 and S2). Download .pdf (.38 MB) Help with pdf files Supplementary material In 147 of 221 (67%) melanomas, a BRAF mutation was detected. Single-nucleotide substitutions at position 1,799 (T>A) were detected more frequently compared with two nucleotide changes (GT>AA) at positions 1,798 and 1,799 (52 and 22%, respectively). There was a negative correlation between the types of BRAF mutation (P=0.001) as well as between BRAF (T>A) and NRAS (A>T) mutations (P=0.037). Ulcerated tumors exhibited a higher frequency of BRAF mutations compared with nonulcerated melanomas (75 and 58%, respectively; P=0.006). We found a strong negative correlation between BRAF (T>A) mutations and dermal actinic aging (P=0.004). (For further details see Supplementary materials: Results, Supplementary Figure S1 and Supplementary Table S3). Methylation of RASSF1A, p16, or PTEN was detected in 25 of 217 (12%), 46 of 213 (22%), or 120 of 200 (60%) melanomas, respectively. There was no correlation between the investigated mutations and/or promoter hypermethylations. Positive correlations were found between PTEN hypermethylation and the degree of actinic aging as well as the TANS (trunk, upper arm, neck, and scalp) location of the tumor (P=0.011 and P=0.043, respectively). Twelve selected samples were investigated for correlation between PTEN methylation and immunohistological PTEN expression. In the subgroup of melanomas with an unmethylated PTEN promoter, +++ (strong), ++ (equivalent to endothelial cells), + (low), or - (no) PTEN expression was found in 3 of 6, 2 of 6, 0 of 6, or 1 of 6 tumors, respectively. In contrast, in the hypermethylated subgroup, the corresponding frequencies were 0 of 6 (+++ PTEN), 0 of 6 (++ PTEN), 1 of 6 (low PTEN), or 5 of 6 (- PTEN). Further details are given in the Supplementary material: Results, Supplementary Figures S2 and S3 and Supplementary Table S3. Kaplan–Meier estimations showed that patients with tumors that are methylated for PTEN have an increased risk of dying compared with patients without such methylation (P=0.035) (Figure 1). This was confirmed by multivariate Cox regression analysis (Table 1). Survival analyses showed an additional prognostic influence of tumor thickness, tumor surface diameter, mitotic index, and age on survival, and tumor thickness and ulceration on recurrence-free survival (Figure 1, Table 1; Supplementary material: Results, Supplementary Figure S4, Supplementary Tables S4 and S5).Table 1Multivariate Cox regression analysis of overall survival and recurrence-free survivalOdds ratio95% CIP-valuenOverall survival176 Tumor diameter2.541.56–4.130.000 Tumor thickness2.011.22–3.290.006 PTEN methylation1.751.12–2.730.014 Mitotic index1.681.08–2.600.020 Age1.541.00–2.390.052Recurrence-free survival223 Tumor thickness4.292.43–7.580.000 Ulceration1.841.09–3.090.023CI, confidence interval; n, number of cases; P, significance level. Open table in a new tab CI, confidence interval; n, number of cases; P, significance level. The most interesting result of our study is that PTEN methylation acts as an independent prognostic parameter, and to our knowledge this has not previously been reported. However, PTEN methylation as a prognostic marker was not superior to the strongest traditional markers (tumor thickness and ulceration). Thus, it cannot act as a single survival parameter for clinical purposes. The observed frequency of PTEN methylation was consistent with other epigenetic data and PTEN expression analyses (Zhou et al., 2000Zhou X.P. Gimm O. Hampel H. Niemann T. Walker M.J. Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation.Am J Pathol. 2000; 157: 1123-1128Google Scholar; Mikhail et al., 2005Mikhail M. Velazquez E. Shapiro R. Berman R. Pavlick A. Sorhaindo L. et al.PTEN expression in melanoma: relationship with patient survival, Bcl-2 expression, and proliferation.Clin Cancer Res. 2005; 11: 5153-5157Google Scholar; Mirmohammadsadegh et al., 2006Mirmohammadsadegh A. Marini A. Nambiar S. Hassan M. Tannapfel A. Ruzicka T. et al.Epigenetic silencing of the PTEN gene in melanoma.Cancer Res. 2006; 66: 6546-6552Google Scholar). Our data show a direct relationship between PTEN hypermethylation and its decreased expression. The lack of expression in an unmethylated tumor could be attributed to loss of heterozygosity of PTEN, which is frequently observed in melanoma (Zhou et al., 2000Zhou X.P. Gimm O. Hampel H. Niemann T. Walker M.J. Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation.Am J Pathol. 2000; 157: 1123-1128Google Scholar). PTEN methylation is lower in the melanomas of Japanese individuals (Furuta et al., 2004Furuta J. Umebayashi Y. Miyamoto K. Kikuchi K. Otsuka F. Sugimura T. et al.Promoter methylation profiling of 30 genes in human malignant melanoma.Cancer Sci. 2004; 95: 962-968Google Scholar). This could be related to the detection of a pseudogene located on chromosome 9 (Zysman et al., 2002Zysman M.A. Chapman W.B. Bapat B. Considerations when analyzing the methylation status of PTEN tumor suppressor gene.Am J Pathol. 2002; 160: 795-800Google Scholar). However, in silico analysis with our utilized primers revealed only homology with the PTEN CpG island promoter on chromosome 10q23 (Schagdarsurengin et al., 2006Schagdarsurengin U. Gimm O. Dralle H. Hoang-Vu C. Dammann R. CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma.Thyroid. 2006; 16: 633-642Google Scholar). It has been reported that decreased PTEN expression correlated significantly with ulceration, but not with survival (Mikhail et al., 2005Mikhail M. Velazquez E. Shapiro R. Berman R. Pavlick A. Sorhaindo L. et al.PTEN expression in melanoma: relationship with patient survival, Bcl-2 expression, and proliferation.Clin Cancer Res. 2005; 11: 5153-5157Google Scholar). Interestingly, the frequency of PTEN methylation significantly increased with the degree of actinic aging and TANS location in our study. This suggests an influence of chronic UV light exposure. The frequency of BRAF mutations in our study (67%) and the absence of a link between BRAF mutation and survival are consistent with previous findings (Akslen et al., 2005Akslen L.A. Angelini S. Straume O. Bachmann I.M. Molven A. Hemminki K. et al.BRAF and NRAS mutations are frequent in nodular melanoma but are not associated with tumor cell proliferation or patient survival.J Invest Dermatol. 2005; 125: 312-317Google Scholar). In contrast to a previously postulated influence of UV light on BRAF mutagenesis (Besaratinia and Pfeifer, 2008Besaratinia A. Pfeifer G.P. Sunlight ultraviolet irradiation and BRAF V600 mutagenesis in human melanoma.Hum Mutat. 2008; 29: 983-991Google Scholar), we observed an inverse correlation between BRAF (T>A) mutation and dermal actinic aging, and no correlation between BRAF mutation and TANS tumor location. Thus, chronic UV exposition, at least, seems not to be a direct predictor of BRAF (T>A) mutation. Another difference from previous studies was the absence of synergy between the BRAF mutation and RASSF1A hypermethylation in our study. Such a synergy was previously suggested on the basis of only 17 cases (Reifenberger et al., 2004Reifenberger J. Knobbe C.B. Sterzinger A.A. Blaschke B. Schulte K.W. Ruzicka T. et al.Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas.Int J Cancer. 2004; 109: 377-384Google Scholar). In summary, our results show that mutation of BRAF and methylation of PTEN occur frequently in melanoma. PTEN methylation correlates with tumor localization or dermal actinic aging and is an independent predictor of impaired patient survival. Reinhard Dammann and Peter Helmbold received grant support from Deutsche Krebshilfe (107742). Reinhard Dammann received grant support from BMBF (FKZ 01ZZ0104) and DFG (DA552). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. It has recently been reported that integration of a Merkel cell polyomavirus (MCPyV) in receptor tyrosine phosphates type G (PTPRG) gene occurs in MCC, and that viral infections are associated with epigenetic silencing of tumor suppressor genes (TSG) in cancer. To examine whether a correlation between TSG inactivation and viral infection can be found in MCC, we investigated the promoter hypermethylation of RASSF1A, TP73, PTPRG, FHIT, and CDKN2A and the presence of MCPyV and SV40 in 98 MCC by PCR. Hypermethylation of RASSF1A was frequently found in 42 of 83 (51%) of MCC. Methylation of CDKN2A was present in 9 of 41 (22%) of MCC. Hypermethylation of TP73 (0%), PTPRG (4%), and FHIT (0%) was infrequent in MCC. Interestingly, MCPyV was found in 90 of 98 (92%) MCC, however, no SV40 signal was detected. No correlation between TSG hypermethylation and viral infection was found. Our results show frequent hypermethylation of RASSF1A and the presence of MCPyV in primary MCC, and that these events may contribute to the pathogenesis of MCC. © 2009 Wiley‐Liss, Inc.