Kelly G. Paulson

Association of Merkel Cell Polyomavirus–Specific Antibodies With Merkel Cell Carcinoma

Background Merkel cell polyomavirus (MCPyV) has been detected in approximately 75% of patients with the rare skin cancer Merkel cell carcinoma. We investigated the prevalence of antibodies against MCPyV in the general population and the association between these antibodies and Merkel cell carcinoma. Methods Multiplex antibody-binding assays were used to assess levels of antibodies against polyomaviruses in plasma. MCPyV VP1 antibody levels were determined in plasma from 41 patients with Merkel cell carcinoma and 76 matched control subjects. MCPyV DNA was detected in tumor tissue specimens by quantitative polymerase chain reaction. Seroprevalence of polyomavirus-specific antibodies was determined in 451 control subjects. MCPyV strain–specific antibody recognition was investigated by replacing coding sequences from MCPyV strain 350 with those from MCPyV strain w162. Results We found that 36 (88%) of 41 patients with Merkel cell carcinoma carried antibodies against VP1 from MCPyV w162 compared with 40 (53%) of the 76 control subjects (odds ratio adjusted for age and sex = 6.6, 95% confidence interval [CI] = 2.3 to 18.8). MCPyV DNA was detectable in 24 (77%) of the 31 Merkel cell carcinoma tumors available, with 22 (92%) of these 24 patients also carrying antibodies against MCPyV. Among 451 control subjects from the general population, prevalence of antibodies against human polyomaviruses was 92% (95% CI = 89% to 94%) for BK virus, 45% (95% CI = 40% to 50%) for JC virus, 98% (95% CI = 96% to 99%) for WU polyomavirus, 90% (95% CI = 87% to 93%) for KI polyomavirus, and 59% (95% CI = 55% to 64%) for MCPyV. Few case patients had reactivity against MCPyV strain 350; however, indistinguishable reactivities were found with VP1 from strain 350 carrying a double mutation (residues 288 and 316) and VP1 from strain w162. Conclusion Infection with MCPyV is common in the general population. MCPyV, but not other human polyomaviruses, appears to be associated with Merkel cell carcinoma.

Antibodies to Merkel Cell Polyomavirus T Antigen Oncoproteins Reflect Tumor Burden in Merkel Cell Carcinoma Patients

Merkel cell polyomavirus (MCPyV) is a common infectious agent that is likely involved in the etiology of most Merkel cell carcinomas (MCC). Serum antibodies recognizing the MCPyV capsid protein VP1 are detectable at high titer in nearly all MCC patients and remain stable over time. Although antibodies to the viral capsid indicate prior MCPyV infection, they provide limited clinical insight into MCC because they are also detected in more than half of the general population. We investigated whether antibodies recognizing MCPyV large and small tumor-associated antigens (T-Ag) would be more specifically associated with MCC. Among 530 population control subjects, these antibodies were present in only 0.9% and were of low titer. In contrast, among 205 MCC cases, 40.5% had serum IgG antibodies that recognize a portion of T-Ag shared between small and large T-Ags. Among cases, titers of T-Ag antibodies fell rapidly (∼8-fold per year) in patients whose cancer did not recur, whereas they rose rapidly in those with progressive disease. Importantly, in several patients who developed metastases, the rise in T-Ag titer preceded clinical detection of disease spread. These results suggest that antibodies recognizing T-Ag are relatively specifically associated with MCC, do not effectively protect against disease progression, and may serve as a clinically useful indicator of disease status.

Merkel polyomavirus-specific T cells fluctuate with merkel cell carcinoma burden and express therapeutically targetable PD-1 and Tim-3 exhaustion markers.

Purpose: The persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins in Merkel cell carcinoma (MCC) provides a unique opportunity to characterize immune evasion mechanisms in human cancer. We isolated MCPyV-specific T cells and determined their frequency and functional status. Experimental Design: Multiparameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n = 7) and blood (n = 18) of patients with MCC and control subjects (n = 10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n = 35) and immunohistochemistry (n = 13). Results: MCPyV-specific CD8 T cells were detected directly ex vivo from the blood samples of 7 out of 11 (64%) patients with MCPyV-positive tumors. In contrast, 0 of 10 control subjects had detectable levels of these cells in their blood (P < 0.01). MCPyV-specific T cells in serial blood specimens increased with MCC disease progression and decreased with effective therapy. MCPyV-specific CD8 T cells and MCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared with T cells specific to other human viruses (P < 0.01). PD-L1 was present in 9 of 13 (69%) MCCs and its expression was correlated with CD8-lymphocyte infiltration. Conclusions: MCC-targeting T cells expand with tumor burden and express high levels of immune checkpoint receptors PD-1 and Tim-3. Reversal of these inhibitory pathways is therefore a promising therapeutic approach for this virus-driven cancer. Clin Cancer Res; 19(19); 5351–60. ©2013 AACR.

Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

Purpose: Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. Experimental Design: Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402–restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. Results: We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402–restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. Conclusions: Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients. Clin Cancer Res; 17(21); 6671–80. ©2011 AACR.

Array-CGH Reveals Recurrent Genomic Changes in Merkel Cell Carcinoma Including Amplification of L-Myc

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with poorly characterized genetics. We performed high resolution comparative genomic hybridization on 25 MCC specimens using a high-density oligonucleotide microarray. Tumors frequently carried extra copies of chromosomes 1, 3q, 5p, and 6 and lost chromosomes 3p, 4, 5q, 7, 10, and 13. MCC tumors with less genomic aberration were associated with improved survival (P=0.04). Tumors from 13 of 22 MCC patients had detectable Merkel cell polyomavirus DNA, and these tumors had fewer genomic deletions. Three regions of genomic alteration were of particular interest: a deletion of 5q12-21 occurred in 26% of tumors, a deletion of 13q14-21 was recurrent in 26% of tumors and contains the well-characterized tumor suppressor RB1, and a previously unreported focal amplification at 1p34 was present in 39% of tumors and centers on L-Myc (MYCL1). L-Myc is related to the c-Myc proto-oncogene, has transforming activity, and is amplified in the closely related small cell lung cancer. Normal skin showed no L-Myc expression, whereas 4/4 MCC specimens tested expressed L-Myc RNA in relative proportion to the DNA copy number gain. These findings suggest several genes that may contribute to MCC pathogenesis, most notably L-Myc.

Systemic Immune Suppression Predicts Diminished Merkel Cell Carcinoma–Specific Survival Independent of Stage

Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy linked to a contributory virus (Merkel cell polyomavirus/MCPyV). Multiple epidemiologic studies have established an increased incidence of MCC among persons with systemic immune suppression. Several forms of immune suppression are associated with increased MCC incidence, including hematologic malignancies, HIV/AIDS, and immunosuppressive medications for autoimmune disease or transplant. Indeed, immune suppressed persons represent approximately 10% of the MCC patients, a significant over-representation relative to the general population. We hypothesized that immune suppressed patients may have a poorer MCC-specific prognosis and examined a cohort of 471 patients with a combined follow-up of 1427 years (median 2.1 years). Immune suppressed persons (n=41) demonstrated reduced MCC-specific survival (40% at 3 years) compared to persons with no known systemic immune suppression (n=430; 74% MCC-specific survival at 3 years). By competing risk regression analysis, immune suppression was a stage-independent predictor of worsened MCC-specific survival (hazard ratio 3.8, p < 0.01). Immune-suppressed persons thus have both an increased chance of developing MCC and poorer MCC-specific survival. It may be appropriate to follow these higher-risk individuals more closely, and, when clinically feasible, there may be benefit of diminishing iatrogenic systemic immune suppression.Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy linked to a contributory virus (Merkel cell polyomavirus). Multiple epidemiologic studies have established an increased incidence of MCC among persons with systemic immune suppression. Several forms of immune suppression are associated with increased MCC incidence, including hematologic malignancies, HIV/AIDS, and immunosuppressive medications for autoimmune disease or transplant. Indeed, immune-suppressed individuals represent ∼10% of MCC patients, a significant overrepresentation relative to the general population. We hypothesized that immune-suppressed patients may have a poorer MCC-specific prognosis and examined a cohort of 471 patients with a combined follow-up of 1,427 years (median 2.1 years). Immune-suppressed patients (n=41) demonstrated reduced MCC-specific survival (40% at 3 years) compared with patients with no known systemic immune suppression (n=430; 74% MCC-specific survival at 3 years). By competing risk regression analysis, immune suppression was a stage-independent predictor of worsened MCC-specific survival (hazard ratio 3.8, P<0.01). Thus, immune-suppressed individuals have both an increased chance of developing MCC and poorer MCC-specific survival. It may be appropriate to follow these higher-risk individuals more closely, and, when clinically feasible, there may be a benefit of diminishing iatrogenic systemic immune suppression.

Relationships among primary tumor size, number of involved nodes, and survival for 8044 cases of Merkel cell carcinoma

BACKGROUND The effects of primary tumor size on nodal involvement and of number of involved nodes on survival have not, to our knowledge, been examined in a national database of Merkel cell carcinoma (MCC). OBJECTIVE We sought to analyze a retrospective cohort of patients with MCC from the largest US national database to assess the relationships between these clinical parameters and survival. METHODS A total of 8044 MCC cases in the National Cancer Data Base were analyzed. RESULTS There was a 14% risk of regional nodal involvement for 0.5-cm tumors that increased to 25% for 1.7-cm (median-sized) tumors and to more than 36% for tumors 6 cm or larger. The number of involved nodes was strongly predictive of survival (0 nodes, 76% 5-year relative survival; 1 node, 50%; 2 nodes, 47%; 3-5 nodes, 42%; and ≥6 nodes, 24%; P < .0001 for trend). Younger and/or male patients were more likely to undergo pathological nodal evaluation. LIMITATIONS The National Cancer Data Base does not capture disease-specific survival. Hence, relative survival was calculated by comparing overall survival with age- and sex-matched US population data. CONCLUSION Pathologic nodal evaluation should be considered even for patients with small primary MCC tumors. The number of involved nodes is strongly predictive of survival and may help improve prognostic accuracy and management.

Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

ABSTRACT Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region.

Effect of Host, Tumor, Diagnostic, and Treatment Variables on Outcomes in a Large Cohort With Merkel Cell Carcinoma

IMPORTANCE Merkel cell carcinoma (MCC) is a rare, aggressive, neuroendocrine-derived skin cancer with high rates of recurrence and associated mortality. Few published studies have used comprehensive patient data and long-term follow-up to examine factors that predict MCC outcomes. OBJECTIVE To characterize MCC in a large defined-population cohort and analyze predictors of disease recurrence and survival. SETTING, DESIGN, AND PARTICIPANTS Retrospective cohort study of 218 patients with MCC from the cancer registry of Kaiser Permanente Northern California, a large integrated health care delivery system. Patients were diagnosed as having MCC and followed up from January 1, 1995, through December 31, 2009. We examined host (age, sex, race, and immunosuppression), tumor (anatomic site, size, and extent), diagnostic (results of imaging and pathologic nodal evaluation), and treatment (surgery, radiation therapy, and chemotherapy) variables for their association with MCC outcomes. EXPOSURE Host, tumor, diagnostic, and treatment factors. MAIN OUTCOMES AND MEASURES Recurrence (locoregional and distant) of MCC and patient survival (overall and MCC specific). RESULTS We estimated adjusted hazard ratios (AHRs) and 95% CIs for outcomes using Cox proportional hazards regression models. After adjustment for host, tumor, diagnostic, and treatment variables, tumor extent (categorized as local, regional, and distant) remained significantly associated with all outcomes. Immunosuppression was associated with higher MCC-specific mortality (AHR, 4.9 [95% CI, 1.7-14.4]), and an unknown primary site was associated with a lower risk for distant metastasis (0.1 [0.0-0.7]) and improved survival (0.4 [0.2-0.9]). Pathological nodal evaluation was associated with a lower risk for metastasis (AHR, 0.2 [95% CI, 0.0-1.0]) and improved survival. Radiation treatment was associated with a decreased risk for locoregional recurrence (AHR, 0.3 [95% CI, 0.1-0.6]), whereas chemotherapy was not associated with any alteration in outcomes. CONCLUSIONS AND RELEVANCE Tumor site and extent, results of pathologic nodal evaluation, and the presence of radiation treatment were associated with MCC recurrence. Immunosuppression, tumor extent, and results of pathologic nodal evaluation were associated with MCC-specific survival, whereas chemotherapy was not associated with any outcomes. Our findings may help to inform diagnostic and therapeutic management of MCCs.

Regression of metastatic Merkel cell carcinoma following transfer of polyomavirus-specific T cells and therapies capable of re-inducing HLA class-I

Chapuis, Afanasiev, and colleagues show that the combined regimen of local tumor-targeted preconditioning and systemic immune therapies elicited a durable complete response in two of three lesions with a prolonged period without development of additional distant metastasis. Merkel cell carcinoma (MCC) is an aggressive skin cancer that typically requires the persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins that can serve as ideal immunotherapeutic targets. Several immune evasion mechanisms are active in MCC, including downregulation of HLA class-I expression on tumor cells and dysfunctional endogenous MCPyV-specific CD8 T-cell responses. To overcome these obstacles, we combined local and systemic immune therapies in a 67-year-old man, who developed metastatic MCPyV-expressing MCC. Intralesional IFN-β-1b or targeted single-dose radiation was administered as a preconditioning strategy to reverse the downregulation of HLA-I expression noted in his tumors and to facilitate the subsequent recognition of tumor cells by T cells. This was followed by the adoptive transfer of ex vivo expanded polyclonal, polyomavirus-specific T cells as a source of reactive antitumor immunity. The combined regimen was well tolerated and led to persistent upregulation of HLA-I expression in the tumor and a durable complete response in two of three metastatic lesions. Relative to historical controls, the patient experienced a prolonged period without development of additional distant metastases (535 days compared with historic median of 200 days; 95% confidence interval, 154–260 days). The transferred CD8+ T cells preferentially accumulated in the tumor tissue, remained detectable and functional for more than 200 days, persisted with an effector phenotype, and exhibited evidence of recent in vivo activation and proliferation. The combination of local and systemic immune stimulatory therapies was well tolerated and may be a promising approach to overcome immune evasion in virus-driven cancers. Cancer Immunol Res; 2(1); 27–36. ©2013 AACR.