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Tumor-Specific T Cells in Human Merkel Cell Carcinomas: A Possible Role for Tregs and T-Cell Exhaustion in Reducing T-Cell Responses
Abstract and Figures
Merkel cell carcinomas (MCC) are rare but highly malignant skin cancers associated with a novel polyomavirus. MCC tumors were infiltrated by T cells, including effector, central memory and regulatory T cells. Infiltrating T cells showed markedly reduced activation as evidenced by reduced expression of CD69 and CD25. Treatment of MCC tumors in vitro with IL-2 and IL-15 led to T cell activation, proliferation, enhanced cytokine production and loss of viable tumor cells from cultures. Expanded tumor-infiltrating lymphocytes showed TCR repertoire skewing and upregulation of CD137. MCC tumors implanted into immunodeficient mice failed to grow unless human T cells in the tumor grafts were depleted with denileukin diftitox, suggesting tumor-specific T cells capable of controlling tumor growth were present in MCC. Both CD4(+) and CD8(+) FOXP3(+) regulatory T cells were frequent in MCC. 50% of non-activated T cells in MCC expressed PD-1, a marker of T-cell exhaustion, and PD-L1 and PD-L2 were expressed by a subset of tumor dendritic cells and macrophages. In summary, we observed tumor-specific T cells with suppressed activity in MCC tumors. Agents that stimulate T cell activity, block Treg function or inhibit PD-1 signaling may be effective in the treatment of this highly malignant skin cancer.Journal of Investigative Dermatology accepted article preview online, 18 February 2013; doi:10.1038/jid.2013.75.
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Supplementary resources (5)
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... For instance, factors such as nutrition restrictions, hypoxia, and immunosuppressive cells and signals render the TME immunosuppressive [59,60]. T cell exhaustion is an important immune escape mechanism in MCC: a significant proportion of TILs express PD-1 and TIM-3, which are not only regarded as the exhaustion markers, but actually promote T-cell dysfunction [52,61]. Activation and proliferation of T cells are inhibited through PD-1 signaling. ...
... On the basis of the observations that MCCs are immunogenic and TILs expedite the activation and exhaustion markers PD-1 on the one hand and PD-L1 on tumor and stromal cells on the other [61,72,73], it was postulated early on that this tumor entity should be well susceptible to immune checkpoint inhibitor (ICI) therapy, especially antibodies that block the PD-1/PD-L1 signaling pathway. However, the economic viability of introducing a therapy in a rare type of cancer is relatively limited; thus, it took some time for this hypothesis to be put to the test. ...
Merkel cell carcinoma (MCC) is a rare skin cancer characterized by neuroendocrine differentiation. Its carcinogenesis is based either on the integration of the Merkel cell polyomavirus or on ultraviolet (UV) mutagenesis, both of which lead to high immunogenicity either through the expression of viral proteins or neoantigens. Despite this immunogenicity resulting from viral or UV-associated carcinogenesis, it exhibits highly aggressive behavior. However, owing to the rarity of MCC and the lack of epidemiologic registries with detailed clinical data, there is some uncertainty regarding the spontaneous course of the disease. Historically, advanced MCC patients were treated with conventional cytotoxic chemotherapy yielding a median response duration of only 3 months. Starting in 2017, four programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors—avelumab, pembrolizumab, nivolumab (utilized in both neoadjuvant and adjuvant settings), and retifanlimab—have demonstrated efficacy in treating patients with disseminated MCC on the basis of prospective clinical trials. However, generating clinical evidence for rare cancers, such as MCC, is challenging owing to difficulties in conducting large-scale trials, resulting in small sample sizes and therefore lacking statistical power. Thus, to comprehensively understand the available clinical evidence on various immunotherapy approaches for MCC, we also delve into the epidemiology and immune biology of this cancer. Nevertheless, while randomized studies directly comparing immune checkpoint inhibitors and chemotherapy in MCC are lacking, immunotherapy shows response rates comparable to those previously reported with chemotherapy but with more enduring responses. Notably, adjuvant nivolumab has proven superiority to the standard-of-care therapy (observation) in the adjuvant setting.
... CLA, which guides memory T cells back to the skin, has been associated with higher total T cell infiltration and better clinical outcomes in MCC. 61 In contrast, we also identified a CD39 + CD103 + CD8 T cell population that is transcriptionally distinct and inversely correlated with patient response to pembrolizumab. CD103, a resident memory T cell marker that also mediates cell binding to epithelial and endothelial cells, has also been implicated as a marker for tumor reactivity in TILs 19,62,63 and in blood 64 in the context of other tumor types. ...
Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39⁺CLA⁺ CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39⁺CD103⁺ CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells.
... We also identified a cluster of CD8+ T cells that amplified during the RespD376 and showed both exhaustion and proliferation phenotypes. This was consistent with the previous finding that infiltrating T cells were usually characterized by an exhausted phenotype [33,34]. The impact of exhausted phenotype T cell effectors on patients may be greater than the activity of their CD8+ T cells expanded in vitro, leading to the gradual depletion of these T cells used for immunotherapy and loss of their therapeutic efficacy. ...
Purpose
Merkel cell carcinoma (MCC) is a neuroendocrine carcinoma originating in the skin. Studies are needed to determine the mechanisms of immune escape in patients with MCC, and malignant cell conditions that promote immune evasion.
Methods
We used Single-cell RNA sequencing (scRNA-seq) to determine cellular features associated with MCC disease trajectory. A longitudinal multi-omics study was performed using scRNA-seq data of peripheral blood harvested from four-time points. Six major cell types and fifteen cell subgroups were identified and confirmed their presence by expression of characteristic markers. The expression patterns and specific changes of different cells at different time points were investigated. Subsequently, bulk RNA data was used to validate key findings.
Results
The dynamic characteristics of the cells were identified during the critical period between benign improvement and acquisition of resistance. Combined with the results of the validation cohort, the resistance program expressed in the relapse stage is mainly associated with T cell exhaustion and immune cell crosstalk disorder. Coinciding with immune escape, we also identified a decrease non-classical monocytes and an expansion of classical monocytes with features of high inflammation and immune deficiency.
Conclusion
Changes in cellular status, such as depletion of T cells and dysregulation of B cell proliferation and differentiation, may lead to drug resistance in MCC patients. Meanwhile, the widespread decreased antigen presentation ability and immune disorders caused by deletion of MHC class II gene expression should not be ignored.
... Because LT can reinitiate DNA replication off of the integrated viral origin (7), leading to replication fork collision and DNA fragmentation, the nascent cancer cell survives because another, independent mutation is present in the LT gene to truncate its C-terminal helicase domain preventing LT-dependent DNA replication (7,14). It is unknown which mutation comes first, LT gene truncation (7) or virus integration (11), but both are required, together with loss of effective cytotoxic T lymphocyte responses against early viral antigens (15,16), for emergence of this virus-driven cancer (8). ...
Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.
Zusammenfassung
Das Merkel-Zell-Karzinom (MCC) stellt einen seltenen, jedoch hochaggressiven und rasch expandierenden malignen Hauttumor dar. Die periokuläre Region ist in etwa 10% der Fälle betroffen. Die aktuelle Therapieempfehlung des resektablen, nicht metastasierten MCC umfasst die chirurgische Totalexzision, allerdings sind bei Diagnosestellung bereits häufig Lymphknoten- oder Fernmetastasen vorhanden. Seit der Erstzulassung einer Immun-Checkpoint-Inhibitor-Therapie mit Avelumab für das metastasierte MCC im Jahr 2016 hat sich das mittlere Überleben im Vergleich zur zytostatischen Therapie erheblich gebessert bei gleichzeitig seltenerem Auftreten schwerwiegender therapieassoziierter unerwünschter Ereignisse. Weitere Immun-Checkpoint-Inhibitoren mit ersten vielversprechenden Ergebnissen sind derzeit noch in der klinischen Erprobung. Eine interdisziplinäre Betreuung an einem spezialisierten Zentrum mit Vorstellung in einem Tumorboard ist bei Patienten MCC aufgrund der komplexen Diagnostik, Therapie und Prognoseabschätzung essenziell.
Zusammenfassung
Das Merkelzellkarzinom (MCC) ist ein seltener, aggressiver Hauttumor mit epithelialer und neuroendokriner Differenzierung, dessen Inzidenz in den letzten Jahrzehnten deutlich zugenommen hat. Risikofaktoren sind fortgeschrittenes Lebensalter, heller Hauttyp, UV‐Exposition und Immunsuppression. Pathogenetisch wird ein durch das Merkelzell‐Polyomavirus (MCPyV) hervorgerufener Typ von einem UV‐induzierten Typ mit hoher Tumormutationslast unterschieden.
Klinisch präsentiert sich das MCC als meist schmerzloser, schnell wachsender, rötlich‐violetter Tumor mit glänzender Oberfläche, der bevorzugt im Kopf‐Hals‐Bereich und an den distalen Extremitäten lokalisiert ist. Eine sichere Diagnose kann nur anhand histologischer und immunhistochemischer Merkmale gestellt werden. Bei Erstdiagnose weisen 20%–26% der Patienten lokoregionäre Metastasen und 8%–14% Fernmetastasen auf, weshalb eine Ausbreitungsdiagnostik unabdingbar ist. Bei fehlenden klinischen Hinweisen auf Metastasen wird eine Sentinel‐Lymphknotenbiopsie empfohlen.
Wesentliche Säulen der Therapie sind die Operation, die adjuvante oder palliative Strahlentherapie und in fortgeschrittenen inoperablen Stadien die medikamentöse Tumortherapie. Die Einführung von Immuncheckpoint‐Inhibitoren führte zu einem Paradigmenwechsel, da sich hiermit ein wesentlich langfristigeres Ansprechen und bessere Überlebensraten als mit Chemotherapie erreichen lassen. Zur Therapie des metastasierten MCC ist in Deutschland der PD‐L1‐Inhibitor Avelumab zugelassen, aber auch die PD‐1‐Antikörper Pembrolizumab und Nivolumab werden mit Erfolg eingesetzt. Adjuvante und neoadjuvante Therapiekonzepte, Immunkombinationstherapien und zielgerichtete Therapien als Monotherapie oder in Kombination mit Immuncheckpoint‐Inhibitoren befinden sich in klinischer Prüfung.
Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with epithelial and neuroendocrine differentiation, the incidence of which has increased substantially during the last decades. Risk factors include advanced age, fair skin type, UV exposure, and immunosuppression. Pathogenetically, a type caused by the Merkel cell polyomavirus is distinguished from a UV‐induced type with a high tumor mutational burden.
Clinically, MCC presents as a mostly painless, rapidly growing, reddish‐violet tumor with a shiny surface, which is preferentially localized in the head‐neck region and at the distal extremities. A reliable diagnosis can only be made based on histological and immunohistochemical features. At initial diagnosis, 20–26% of patients show locoregional metastases and 8–14% distant metastases, making staging examinations indispensable. If there is no clinical evidence of metastases, a sentinel lymph node biopsy is recommended.
Essential columns of therapy are surgery, adjuvant or palliative radiotherapy and, in advanced inoperable stages, medicamentous tumor therapy. The introduction of immune checkpoint inhibitors has led to a paradigm shift, as they provide a considerably longer duration of response and better survival rates than chemotherapy. The PD‐L1 inhibitor avelumab is approved for treatment of metastatic MCC in Germany, but the PD‐1 antibodies pembrolizumab and nivolumab are also used with success. Adjuvant and neoadjuvant treatment concepts, immune combination therapies and targeted therapies as monotherapy or in combination with immune checkpoint inhibitors are in the clinical trial phase.
Purpose:
Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer. Although essentially all MCCs are antigenic through viral antigens or high tumor mutation burden, MCC has a response rate of only ~50% to PD-(L)1 blockade suggesting barriers to T cell responses. Prior studies of MCC immunobiology have focused on CD8 T-cell infiltration and their exhaustion status, while the role of innate immunity, particularly myeloid cells, in MCC remains underexplored.
Experimental design:
We utilized single cell transcriptomics from 9 MCC patients and multiplex-immunohistochemistry staining of 54 patients' pre-immunotherapy tumors, to identify myeloid cells and evaluate association with immunotherapy response.
Results:
Single cell transcriptomics identified tumor-associated macrophages (TAMs) as the dominant myeloid component within MCC tumors. These TAMs express an immunosuppressive gene signature characteristic of monocytic myeloid derived suppressor cells and importantly express several targetable immune checkpoint molecules, including PD-L1 and LILRB receptors, that are not present on tumor cells. Analysis of 54 pre-immunotherapy tumor samples showed that a subset of TAMs (CD163+, CD14+, S100A8+) selectively infiltrated tumors that had significant CD8 T-cells. Indeed, higher TAM prevalence was associated with resistance to PD-1 blockade. While spatial interactions between TAMs and CD8 T cells were not associated with response, myeloid transcriptomic data showed evidence for cytokine signaling and expression of LILRB receptors, suggesting potential immunosuppressive mechanisms.
Conclusions:
This study further characterizes TAMs in MCC tumors and provides insights into their possible immunosuppressive mechanism. TAMs may reduce the likelihood of treatment response in MCC by counteracting the benefit of CD8 T-cell infiltration.
Cytokine driven or “bystander” proliferation of T cells occurs in vivo independently of major histocompatibility complex–T cell receptor interactions. This process may be important for supporting T cell homeostasis and facilitating T cell responses to microbial antigens, and may involve the cytokine interleukin (IL)-15. In this study, we find that IL-15Rα–deficient (IL-15Rα−/−) mice fail to undergo poly I:C or IL-15 driven bystander proliferation of CD8⁺ T cells. Surprisingly, IL-15Rα−/− CD8⁺ T cells proliferate in response to poly I:C when adoptively transferred into normal mice, and normal CD8⁺ T cells fail to proliferate in IL-15Rα−/− mice. Normal mice reconstituted with IL-15Rα−/− bone marrow cells also fail to exhibit bystander responses. Thus, CD8⁺ T cell independent IL-15Rα signals from radiation sensitive hematopoietic cells are likely required for bystander responses. Moreover, normal CD8⁺ T cells proliferate in IL-15Rα−/− mice after treatment with IL-15. Therefore, IL-15Rα signals may mediate a positive feedback loop involving the further physiological production of IL-15. These findings provide new insights into how IL-15Rα supports memory phenotype CD8⁺ T cell proliferation, and suggest novel mechanisms by which memory CD8⁺ T cells are maintained in vivo.
Background
An oncolytic herpes simplex virus engineered to replicate selectively in tumor cells and to express granulocyte–macrophage colony-stimulating factor (GM-CSF) was tested as a direct intralesional vaccination in melanoma patients. The work reported herein was performed to better characterize the effect of vaccination on local and distant antitumor immunity.
Methods
Metastatic melanoma patients with accessible lesions were enrolled in a multicenter 50-patient phase II clinical trial of an oncolytic herpesvirus encoding GM-CSF (OncovexGM-CSF). An initial priming dose of 106 pfu vaccine was given by intratumoral injection, followed by 108 pfu every 2 weeks to 24 total doses. Peripheral blood and tumor tissue were collected for analysis of effector T cells, CD4+FoxP3+ regulatory T cells (Treg), CD8+FoxP3+ suppressor T cells (Ts), and myeloid-derived suppressive cells (MDSC).
Results
Phenotypic analysis of T cells derived from tumor samples suggested distinct differences from peripheral blood T cells. There was an increase in melanoma-associated antigen recognized by T cells (MART-1)-specific T cells in tumors undergoing regression after vaccination compared with T cells derived from melanoma patients not treated with vaccine. There was also a significant decrease in Treg and Ts cells in injected lesions compared with noninjected lesions in the same and different melanoma patients. Similarly MDSC were increased in melanoma lesions but underwent a significant decrease only in vaccinated lesions.
Conclusions
Melanoma patients present with elevated levels of Tregs, Ts, and MDSC within established tumors. Direct injection of OncovexGM-CSF induces local and systemic antigen-specific T cell responses and decreases Treg, Ts, and MDSC in patients exhibiting therapeutic responses.
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.
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.
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.
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.
MCC, Merkel cell carcinoma; MCPyV, Merkel cell polyomavirus
CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.
Most treatments for patients with metastatic melanoma have a low rate of complete regression and thus overall survival in these patients is poor. We investigated the ability of adoptive cell transfer utilizing autologous tumor-infiltrating lymphocytes (TIL) to mediate durable complete regressions in heavily pretreated patients with metastatic melanoma.
Ninety-three patients with measurable metastatic melanoma were treated with the adoptive transfer of autologous TILs administered in conjunction with interleukin-2 following a lymphodepleting preparative regimen on three sequential clinical trials. Ninety-five percent of these patients had progressive disease following a prior systemic treatment. Median potential follow-up was 62 months.
Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) in the 3 trials using lymphodepleting preparative regimens (chemotherapy alone or with 2 or 12 Gy irradiation) were 49%, 52%, and 72%, respectively. Twenty of the 93 patients (22%) achieved a complete tumor regression, and 19 have ongoing complete regressions beyond 3 years. The actuarial 3- and 5-year survival rates for the entire group were 36% and 29%, respectively, but for the 20 complete responders were 100% and 93%. The likelihood of achieving a complete response was similar regardless of prior therapy. Factors associated with objective response included longer telomeres of the infused cells, the number of CD8(+)CD27(+) cells infused, and the persistence of the infused cells in the circulation at 1 month (all P(2) < 0.001).
Cell transfer therapy with autologous TILs can mediate durable complete responses in patients with metastatic melanoma and has similar efficacy irrespective of prior treatment. Clin Cancer Res; 17(13); 4550-7. ©2011 AACR.
Vascular cell adhesion molecule 1 (VCAM-1) mediates extravasation of circulating leukocytes into inflamed tissues, and presumably, plays a role in the immigration of cytotoxic effector lymphocytes into tumor metastases. Since metastases are rarely cleared by blood-borne cells from the immune system, we asked whether the tumor may escape host defense by interfering with the mechanism of effector cell extravasation. Here we show that in mice and humans, VCAM-1 expression is repressed on tumor-infiltrating vascular endothelial cells in the lungs. On lung blood vessels distant from the tumor, VCAM-1 is constitutively expressed. When melanoma and endothelioma cells were cultured on either side of a Nucleopore membrane, the expression of VCAM-1 on the endothelioma cells was inhibited and VCAM-1 gene transcription was suppressed. We propose that the downregulation of VCAM-1 is a mechanism by which vascularized melanoma and carcinoma avoid invasion by cytotoxic cells of the immune system.
The complexity of immune response-associated cells present in normal human skin was recently redefined as the skin immune system (SIS). In the present study, the exact immunophenotypes of lymphocyte subpopulations with their localizations in normal human skin were determined quantitatively. B cells were not found to be present in normal human skin. Lymphocytes were always of T-cell type, and 90% of these T cells were clustered in 1-3 rows around postcapillary venules of the papillary vascular plexus or adjacent to cutaneous appendages. In such perivascular localizations, they were found to differ from their circulating counterparts in three ways. First, skin perivascular cells were found to be approximately evenly distributed over CD4+ inducer and CD8+ suppressor-cytotoxic T-cell subsets (mean CD4/CD8 ratio: papillary layer 0.96, reticular layer 0.99). Second, within the category of CD4+ inducer T cells, most were phenotyped as CD4+, 4B4+ helper inducer T lymphocytes, whereas CD4+, 2H4+ suppressor inducer T lymphocytes were found to be relatively rare (less than 5%). Third, the majority of skin perivascular T cells were activated as they expressed HLA-DR and interleukin 2 receptors. Intraepidermal, directly subepidermal, and other ("free") lymphocytes were mostly of the CD8+ suppressor-cytotoxic T-cell subset but accounted for less than 10% of the total number of lymphocytes. Intraepidermally localized T cells accounted for less than 2% of the total number of lymphocytes present in normal skin. Our results indicate that preferential perivascular localization of activated T lymphocytes is the characteristic of normal human skin. This might be a reflection of continuous antigen recognition upon endothelial cell presentation and/or continuous T cell-mediated endothelial cell activation thereby inducing enhanced antigen clearing by the skin's endothelium.
A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen.
Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer.
We sought to describe primary MCC incidence trends, epidemiology, and predictors of survival.
The population covered by the Surveillance, Epidemiology, and End Results Program was analyzed as a prospective cohort. We measured age-adjusted incidence rates (per 100,000 person-years) and effect of age, anatomic site, and stage on survival.
Incidence was higher in males (0.34) than in females (0.17). Cases (n = 1034) occurred mostly in whites (94%), in people older than 65 years (76%), and at the head (48%). The 5-year relative survival was 75%, 59%, and 25% for localized, regional, and distant MCC, respectively. Female sex, limb presentation, localized disease, and younger age were positive predictors of survival.
The highest incidence of MCC was observed in whites, males, and in people older than 65 years. Only 49% of cases were reported as localized. Better survival was associated with limb localization, early-stage disease, younger age, and female sex.