Merkel cell polyomavirus infection and host defence in patients with Merkel cell carcinoma

Background and purpose: Merkel cell carcinoma (MCC) is a rare and often aggressive skin cancer that usually arises in elderly individuals. MCC is frequently associated with ultraviolet radiation exposure and immunosuppressive conditions. The majority of MCCs harbor Merkel cell polyomavirus (MCPyV), a newly discovered human cancer virus. Several studies have indicated its importance in MCC tumorigenesis. The aim of the thesis was to evaluate the frequency of MCPyV infection in MCC and to investigate its associations with patient and tumor characteristics, and with survival. In addition, we aimed to investigate the associations between MCPyV infection with cell cycle regulatory protein expression, platelet-derived growth factor receptor (PDGFR) family protein expression, TP53, KIT and PDGFRA mutations, and their associations with clinicopathological factors. We also determined the frequency and type of leukocytes that infiltrate MCC, and their associations with the presence of MCPyV DNA in MCC and clinicopathological factors including disease outcome. Experimental design: The study was based on a population-wide MCC patient series from Finland, with the MCCs diagnosed in 1979 to 2004, and the corresponding archival formalin-fixed paraffin-embedded tumor tissue samples. The patients were identified from the files of the Finnish Cancer Registry. MCPyV DNA was detected using polymerase chain reaction (PCR) and quantitative PCR, tumor infiltrating immune cells were identified and the expression of MCPyV large T (LT) antigen and other proteins was assessed by immunohistochemistry. Gene mutations were investigated using PCR and DNA sequencing. Protein expression was usually assessed from tissue microarray (TMA) sections, while the numbers of tumor infiltrating leukocytes were counted from full tumor tissue sections. The associations between the molecular and host response factors studied and the clinicopathological factors including survival were investigated using conventional statistical tests, such as Kaplan-Meier survival analyses and Cox s proportional hazards models. Results: We found that most (approximately 80%) MCCs harbor MCPyV DNA and that the MCPyV LT antigen was expressed in 67% of the tumors. The presence of MCPyV DNA in tumor was associated with better disease-specific (5-year survival: 75.9% vs. 41.1%, p < 0.001) and overall survival (5-year survival: 45.0% vs. 13.0%, p < 0.001) as compared to MCPyV-negative MCCs. The MCPyV DNA-positive MCCs were located more often in a limb than in a trunk or head and neck region, they had less often metastasized to regional lymph nodes at the time of the diagnosis, and less often expressed p53 and KIT than MCPyV-negative tumors (p-values < 0.05). LT antigen expression in tumor cells was associated with the female gender, location of the tumor in a limb, low cell proliferation rate, and absence of p53 expression in tumor (p-values < 0.05). Retinoblastoma protein (RB) expression was almost invariably associated with presence of MCPyV DNA and LT expression in MCC (p-values < 0.0001), whereas TP53 mutations were found exclusively in MCPyV-negative tumors (p=0.001). The presence of MCPyV DNA and LT antigen expression in tumor were independent prognostic factors for favorable overall survival in a Cox multivariable analysis, when gender and the nodal status, or the post-surgical stage were included as covariables in the analyses. No KIT or PDGFRA mutations were found in MCC. Tumors with p53 expression were associated with worse MCC-specific and overall survival as compared to p53-negative tumors, whereas tumor RB expression was associated with favorable survival. MCPyV DNA-positive MCCs contained significantly higher numbers of tumor infiltrating CD3+, CD8+, CD16+, FoxP3+, and CD68+ cells in comparison to MCPyV DNA-negative MCCs. A higher than the median number of CD3+, CD8+ and FoxP3+ T lymphocytes, and high CD8+/CD4+ and FoxP3+/CD4+ cell ratios in tumor were associated with favorable overall survival. Both a higher than the median number of intratumoral CD3+ cells and the presence of MCPyV DNA in tumor were independently associated with favorable overall survival in a Cox multivariable analysis that included also the nodal status and gender as covariables. Conclusions: MCPyV-positive and -negative MCCs differ in molecular features. They show important differences also in their clinical outcomes and associations with several clinicopathological factors, as well as in host immune response. A high number of tumor infiltrating T lymphocytes and presence of MCPyV DNA in tumor were identified as novel independent prognostic factors in MCC.Merkelinsolukarsinooma on harvinainen, ennusteeltaan melko huono ihosyöpätyyppi, jota tavataan yleisimmin vanhuksilla tai immuunipuutostiloista kärsivillä potilailla. Se syntyy useimmiten ultraviolettivalolle altistuville ihoalueille. Merkelinsolupolyoomavirus (Merkel cell polyomavirus, MCPyV) on uusi ihmisen syöpää aiheuttava virus, jolla uskotaan olevan tärkeä merkitys merkelinsolukarsinoomien synnyssä. Väitöstutkimuksessa määritettiin MCPyV-infektion yleisyys merkelinsolukarsinoomissa, sekä tutkittiin virusinfektion vaikutusta potilaan taudinkulkuun. Tutkimuksessa selvitettiin lisäksi virusinfektion yhteyttä useisiin syöpäsolujen kasvua ja jakaantumista ohjaavien valkuaisaineiden ilmentymiseen sekä eräiden syöpään liittyvän geenien (TP53, KIT ja PDGFRA) mutaatioiden esiintymiseen. Tutkimme myös merkelinsolukasvaimeen tunkeutuvien valkosolujen määrää ja tyyppejä, sekä näiden yhteyttä MCPyV-infektioon ja syövän ennusteeseen. Virusinfektion tunnistamiseksi kehitettiin virukselle spesifinen tunnistusmenetelmä (kvantitatiivinen polymeraasiketjureaktio, qPCR). Menetelmän avulla voitiin määrittää MCPyV DNA:n määrä syöpäkudoksissa. Toisessa infektion tunnistusmentelmässä tunnistettiin syöpäsoluissa ilmentyvä viruksen tuottama valkuaisaine (LT-antigeeni) immunohistokemiallisen analyysin avulla. Väitöstutkimuksen tarpeisiin kerättiin sairaaloiden patologian laitosten arkistoista koko Suomen kattava kudosaineisto, joka sisälsi maassamme vuosina 1979-2004 todetut merkelinsolukarsinoomat. Tutkimuksessa todettiin, että noin 80% kaikista merkelinsolukarsinoomista sisältää MCPyV:n DNA:ta. Virusnegatiiviset kasvaimet sisälsivät usein tunnetun kasvunrajoitegeeniin TP53:n mutaatioita toisin kuin viruspositiiviset kasvaimet, eivätkä virusnegatiiviset kasvaimet ilmentäneet solujen jakaantumista säätelevää retinoblastoomaproteiinia (RB). Useiden valkosolutyyppien määrä oli merkitsevästi suurempi MCPyV-positiivisissa kuin MCPyV-negatiivisissa syövissä. Sekä kasvaimen viruspositiivisuus että korkea T-lymfosyyttien määrä olivat itsenäisesti yhteydessä merkelinsolusyöpään sairastuneiden potilaiden keskimääräistä parempaan eloonjäämisennusteeseen. Tutkimuksen tuloksista voidaan päätellä, että MCPyV-positiiviset ja MCPyV-negatiiviset merkelinsolukarsinoomat eroavat toisistaan sekä molekyylitasolla että taudin ennusteen osalta. Merkelinsolusyövän synnyn ja kehittymisen kannalta keskeisten mekanismien tunnistaminen saattaa edistää uusien hoitomuotojen kehittämistä merkelinsolusyöpään sairastuneille potilaille

Merkel cell polyomavirus is a passenger virus in both poroma and porocarcinoma

Background Merkel cell polyomavirus (MCPyV) has been studied in several malignant and nonmalignant tissues. However, only in Merkel cell carcinoma (MCC) has the connection to tumorigenesis been established. Previously, eccrine porocarcinoma samples were shown to express MCPyV in the majority of samples. We aimed to examine MCPyV in porocarcinoma and poroma samples using MCC as the reference material. Methods We analyzed 17 porocarcinoma and 50 poroma samples for the presence of MCPyV using LT antigen immunostaining and DNA detection methods. In addition, 180 MCC samples served as controls. Results MCPyV LT antigen immunostaining was detected in 10% of poroma and 18% of porocarcinoma samples; on the other hand, it was present in 65% of MCC samples. MCPyV DNA was detected in only 10% of poroma and porocarcinoma samples compared with 96% of MCC samples. The viral DNA copy number in all MCPyV DNA-positive MCCs was at least 25 times higher than that in porocarcinoma or poroma samples with the highest MCPyV DNA-to-PTPRG ratio. Conclusions The low number of viral DNA copies in poroma and porocarcinoma samples, together with the negative LT expression of MCPyV DNA-positive tumors, indicates that MCPyV is simply a passenger virus rather than an oncogenic driver of porocarcinoma.Peer reviewe

Oncogenic Merkel Cell Polyomavirus T Antigen Truncating Mutations are Mediated by APOBEC3 Activity in Merkel Cell Carcinoma

Peer reviewe

Activation of Oncogenic and Immune-Response Pathways Is Linked to Disease-Specific Survival in Merkel Cell Carcinoma

Background: Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine carcinoma of the skin with a poor prognosis. Improving the prognosis of MCC by means of targeted therapies requires further understanding of the mechanisms that drive tumor progression. In this study, we aimed to identify the genes, processes, and pathways that play the most crucial roles in determining MCC outcomes. Methods: We investigated transcriptomes generated by RNA sequencing of formalin-fixed paraffin-embedded tissue samples of 102 MCC patients and identified the genes that were upregulated among survivors and in patients who died from MCC. We subsequently cross-referenced these genes with online databases to investigate the functions and pathways they represent. We further investigated differential gene expression based on viral status in patients who died from MCC. Results: We found several novel genes associated with MCC-specific survival. Genes upregulated in patients who died from MCC were most notably associated with angiogenesis and the PI3K-Akt and MAPK pathways; their expression predominantly had no association with viral status in patients who died from MCC. Genes upregulated among survivors were largely associated with antigen presentation and immune response. Conclusion: This outcome-based discrepancy in gene expression suggests that these pathways and processes likely play crucial roles in determining MCC outcomes.Peer reviewe

LRIG1 is a positive prognostic marker in Merkel cell carcinoma and Merkel cell carcinoma expresses epithelial stem cell markers

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine malignancy of the skin. The cell of origin of MCC is thus far unknown and proposed cells of origin include Merkel cells, pro-/pre- or pre-B cells, epithelial stem cells, and dermal stem cells. In this study, we aimed to shed further light on the possibility that a subset of MCC tumors arise from epithelial stem cells of the skin by examining the expression of hair follicle and epidermal stem cell markers in MCC and normal human skin. We also aimed to elucidate any correlation between the expression of these markers and tumor Merkel cell polyomavirus (MCPyV) status or other clinicopathological characteristics or patient survival. Expression of CK19, SOX9, LGR5, and LRIG1 in MCC and normal human skin was studied by immunohistochemistry, and the staining patterns or intensities were statistically correlated with patient, tumor, MCPyV, and survival parameters. In a cohort of 137 cases of MCC, we observed dot-like immunoexpression of CK19 in 30 cases (22.1%) and homogeneous expression in 103 cases (75.7%). We also observed positive immunoexpression of SOX9 in 21 cases (15.3%), LGR5 in 118 cases (86.1%), and LRIG1 in 117 cases (86.0%). Immunoexpression of LRIG1 was found to correlate with better overall and MCC-specific survival. We observed frequent immunoexpression of several hair follicle and epidermal stem cell markers in MCC and found LRIG1 to be a positive prognostic marker in MCC.Peer reviewe

Activation of Oncogenic and Immune-Response Pathways Is Linked to Disease-Specific Survival in Merkel Cell Carcinoma

Background: Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine carcinoma of the skin with a poor prognosis. Improving the prognosis of MCC by means of targeted therapies requires further understanding of the mechanisms that drive tumor progression. In this study, we aimed to identify the genes, processes, and pathways that play the most crucial roles in determining MCC outcomes. Methods: We investigated transcriptomes generated by RNA sequencing of formalin-fixed paraffin-embedded tissue samples of 102 MCC patients and identified the genes that were upregulated among survivors and in patients who died from MCC. We subsequently cross-referenced these genes with online databases to investigate the functions and pathways they represent. We further investigated differential gene expression based on viral status in patients who died from MCC. Results: We found several novel genes associated with MCC-specific survival. Genes upregulated in patients who died from MCC were most notably associated with angiogenesis and the PI3K-Akt and MAPK pathways; their expression predominantly had no association with viral status in patients who died from MCC. Genes upregulated among survivors were largely associated with antigen presentation and immune response. Conclusion: This outcome-based discrepancy in gene expression suggests that these pathways and processes likely play crucial roles in determining MCC outcomes.Peer reviewe

UV-induced local immunosuppression in the tumour microenvironment of eccrine porocarcinoma and poroma

Eccrine porocarcinoma (EPC) is a rare malignant adnexal tumour of the skin. Part of EPCs develop from their benign counterpart, poroma (EP), with chronic light exposure and immunosuppression hypothesized to play a role in the malignant transformation. However, the impact of chronic light exposure on the microenvironment of EPCs and EPs has not been investigated yet. Although the clinical relevance of tumour infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) has been established in various tumours, their distribution and significance in EPCs and EPs is still poorly understood. We characterized the distribution of TILs and TLSs using CD3, CD4, CD8, CD20 immunohistochemistry in a cohort of 10 EPCs and 49 EPs. We then classified our samples using solar-elastosis grading, analyzing the influence of ultraviolet (UV) damage on TIL density. A negative correlation between UV damage and TIL density was observed (CD4 r = −0.286, p = 0.04. CD8 r = −0.305, p = 0.033). No significant difference in TIL density was found between EPCs and EPs. TLS was scarse with the presence rate 10% in EPCs and 8.3% in EPs. The results suggest that UV has an immunosuppressive effect on the microenvironment of EPCs and EPs.Peer reviewe

Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C θ, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p < 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST

Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C θ, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p p &lt; 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST.Peer reviewe