PDE3A Is a Highly Expressed Therapy Target in Myxoid Liposarcoma.

Liposarcomas (LPSs) are a heterogeneous group of malignancies that arise from adipose tissue. Although LPSs are among the most common soft-tissue sarcoma subtypes, precision medicine treatments are not currently available. To discover LPS-subtype-specific therapy targets, we investigated RNA sequenced transcriptomes of 131 clinical LPS tissue samples and compared the data with a transcriptome database that contained 20,218 samples from 95 healthy tissues and 106 cancerous tissue types. The identified genes were referred to the NCATS BioPlanet library with Enrichr to analyze upregulated signaling pathways. PDE3A protein expression was investigated with immunohistochemistry in 181 LPS samples, and PDE3A and SLFN12 mRNA expression with RT-qPCR were investigated in 63 LPS samples. Immunoblotting and cell viability assays were used to study LPS cell lines and their sensitivity to PDE3A modulators. We identified 97, 247, and 37 subtype-specific, highly expressed genes in dedifferentiated, myxoid, and pleomorphic LPS subtypes, respectively. Signaling pathway analysis revealed a highly activated hedgehog signaling pathway in dedifferentiated LPS, phospholipase c mediated cascade and insulin signaling in myxoid LPS, and pathways associated with cell proliferation in pleomorphic LPS. We discovered a strong association between high PDE3A expression and myxoid LPS, particularly in high-grade tumors. Moreover, myxoid LPS samples showed elevated expression levels of SLFN12 mRNA. In addition, PDE3A- and SLFN12-coexpressing LPS cell lines SA4 and GOT3 were sensitive to PDE3A modulators. Our results indicate that PDE3A modulators are promising drugs to treat myxoid LPS. Further studies are required to develop these drugs for clinical use.

Somatostatin receptor expression in Merkel cell carcinoma: correlation with clinical data.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare, high-grade neuroendocrine neoplasm (NEN) of the skin. Somatostatin receptors (SSTRs) are G protein-linked receptors that regulate cell proliferation and growth. SSTRs are expressed in many NENs; however, scant information is available on their expression in MCCs or their association with clinical parameters and patient outcomes. MATERIAL AND METHODS: This retrospective study was conducted at Helsinki University Hospital and the University of Helsinki. Using a tissue microarray, we investigated SSTR1-5 expression by immunohistochemistry in 99 MCC tissue samples. Samples were collected between 1983 and 2017 and coupled with the patients' clinical data. RESULTS: SSTR2-SSTR5 were detected in 69%, 6%, 4%, and 1% of the tumours, respectively. However, SSTR1 expression was not observed. Cytoplasmic SSTR2 positivity was associated with metastatic disease at the time of diagnosis (p = 0.009), but it did not correlate with disease-specificity or overall survival. CONCLUSION: SSTR2-5 expression was observed in MCCs. In particular, SSTR2 expression is clinically valid because it is associated with metastatic disease at the time of diagnosis and can thus serve as a prognostic marker. Moreover, SSTR2 overexpression provides a molecular basis for tumour imaging and treatment with somatostatin analogues.

Neurocan expression associates with better survival and viral positivity in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.

Patterns of Metastatic Recurrence of Genetically Confirmed Myxoid Liposarcoma.

BACKGROUND: Most sarcomas metastasize predominantly to the lungs, and chest x-ray, or computed tomography, is the most commonly used staging investigation. Myxoid liposarcomas (MLSs) are rare tumors with a tendency to metastasize to extrapulmonary loci. The aim of this study was to assess the locations of the first metastases in MLS patients, to guide the design of effective staging and follow-up imaging protocols. METHODS: Patients treated for MLS between 1987 and 2017 were identified in a prospectively maintained register. Histology of the tumors was reassessed. In addition, the presence of one of the pathognomonic gene translocations was confirmed, uniquely for a retrospective series. The surgical and oncological outcomes were reviewed. A comprehensive review of the literature was performed on the metastatic pattern of MLS, including series with 10 or more MLS patients with metastatic disease. RESULTS: A total of 32 patients with genetically confirmed MLS were identified, with a median follow-up of 7.6 years. Seven patients (22%) developed metastatic disease, five initially intra-abdominally and only one to the lungs. The comprehensive review included 14 series with 1853 patients, 348 (19%) of whom had metastases. The location of the first metastases was soft tissues in 32% of patients, intra-abdominal in 26%, pulmonary in 24%, and bone in 17%. CONCLUSIONS: MLSs metastasize often intra-abdominally and to extra-abdominal soft tissues. Thus, whole-body imaging may be indicated during the initial assessment and follow-up of these patients.

Transcriptomic analyses reveal three distinct molecular subgroups of Merkel cell carcinoma with differing prognoses.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine malignancy with a poor prognosis and an unknown cell of origin. Proffered cells of origin include epithelial stem cells of the hair follicle or interfollicular epidermis, dermal stem cells and pro/pre- or pre-B cells. MCC has also been proposed to have more than one cell of origin and indeed to represent more than one type of carcinoma, currently grouped together due to phenotypic similarities. We explored the heterogeneous nature of MCC by studying the most variably expressed genes with the goal of identifying gene expression patterns that are either clinically relevant or have implications regarding the cell(s) of origin. We performed RNA sequencing on primary tumor samples from 102 patients and identified the top 200 most variably expressed genes. These genes and the tumor samples were hierarchically clustered based on their expression. The functions of three gene clusters exhibiting clearly divergent expression between samples were studied by cross-referencing the lists of genes with online databases. High expression of a gene cluster related to embryonic developmental processes and low expression of a gene cluster related to neuroendocrine processes distinguished Merkel cell polyomavirus (MCPyV)-negative tumors from MCPyV-positive tumors. Furthermore, two prognostically relevant subgroups of MCPyV-positive MCC were identified based on dichotomic expression of genes related to epidermal structures and processes. We identified three distinct molecular subgroups of MCC with prognostic relevance. We propose that the dichotomic expression of epidermis-related genes might reflect both an epidermal and a nonepidermal origin for MCPyV-positive MCC.

PROX1 transcription factor controls rhabdomyosarcoma growth, stemness, myogenic properties and therapeutic targets.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, alternative RMS therapies are urgently needed. Here we show that the prospero-related homeobox 1 (PROX1) transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and transformed the RD cell transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, paralog-specific FGFR inhibition did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and induced apoptosis. Our findings uncover the critical role of PROX1 in RMS and offer insights into the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in clinical phase I/II trials in other cancer types, our findings provide an alternative option for RMS treatment.

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.

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.

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.

Visual Counting and Automated Image-analytic Assessment of Ki-67 and their Prognostic Value in Synovial Sarcoma.

Background: Ki-67 is a widely used proliferation marker reflecting prognosis in various tumors. However, visual assessment and scoring of Ki-67 suffers from marked inter-observer and intra-observer variability. We aimed to assess the concordance of manual counting and automated image-analytic scoring methods for Ki-67 in synovial sarcoma. Patients and Methods: Tissue microarrays from 34 patients with synovial sarcoma were immunostained for Ki-67 and scored both visually and with 3DHistech QuantCenter. Results: The automated assessment of Ki-67 expression was in good agreement with the visually counted Ki-67 (r Pearson =0.96, p<0.001). In a Cox regression model automated [hazard ratio (HR)=1.047, p=0.024], but not visual (HR=1.063, p=0.053) assessment method associated high Ki-67 scores with worse overall survival. Conclusion: The automated Ki-67 assessment method appears to be comparable to the visual method in synovial sarcoma and had a significant association to overall survival.

Radiation-associated angiosarcoma of the breast: analysis of diagnostic tools in a registry-based population.

BACKGROUND: Radiation-associated angiosarcoma of the breast (RAASB) is a serious late consequence caused by breast cancer treatment. Initial symptoms are often inconspicuous, thus contributing to diagnostic delay. Most previous studies of the diagnostic aspects of RAASB are case reports. PURPOSE: To perform a complete review of the imaging findings and biopsy methods in a nationwide RAASB cohort. MATERIAL AND METHODS: RAASB patients were identified from a national cancer registry and additional patients were included from our hospital. All available information from imaging (mammogram [MGR], ultrasound [US], magnetic resonance imaging [MRI], and computed tomography [CT]) and biopsies was reviewed. The sensitivity of imaging and biopsy methods for detection of RAASB was calculated. RESULTS: Fifty-eight patients with RAASB were found. Fourteen MGR, 30 US, 24 MRI, and 25 CT studies were available for evaluation. The sensitivity of MGR, US, MRI, and CT for detection of RAASB was 43%, 50%, 92%, and 84%, respectively. Superior sensitivity was demonstrated for punch biopsy (84%) and incisional biopsy (93%) compared to fine-needle aspiration cytology (0%) and core needle biopsy (18%). CONCLUSION: MRI and CT have comparable sensitivity for detection of RAASB, while MGR and US are unreliable. However, negative findings in MRI or CT must be interpreted with caution. Punch biopsy and incisional biopsy are the preferred biopsy methods.

GNEN-1: a spontaneously immortalized cell line from gastric neuroendocrine neoplasia.

Mixed neuroendocrine-non-neuroendocrine neoplasms (MINEN) are rare tumors that consist of at least 30% of both neuroendocrine and non-neuroendocrine components. The data concerning the pathogenesis of MINEN suggest a monoclonal origin. We describe a spontaneously immortalized cell line derived from gastric MINEN called GNEN-1. Primary tumor consisted of components of high-grade neuroendocrine carcinoma and adenocarcinoma. The GNEN-1 cell line was initiated from metastatic tumor cells of peritoneal fluid and expresses a purely neuroendocrine phenotype. The GNEN-1 cell line grows as monolayers and has retained the neuroendocrine phenotype with positivity for chromogranin A in immunohistochemistry. Electron microscopy showed cytoplasmic dense core granules and axon hillocks. The karyotype revealed alterations typical of both adenocarcinoma and neuroendocrine carcinoma such as trisomy 7 and 8. GNEN-1 cells were also positive for stanniocalcin-1, a marker of poor prognosis in gastric carcinomas. Expression of several markers related to neuroendocrine tumors was found. There have been only a few studies on the pathogenesis of MINEN and management of the disease due to the rarity of this tumor type. Here we describe for the first time an immortalized cell line derived from mixed gastric NEN. The GNEN-1 line offers a tool for future research on gastric NEN.

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.

ALK is frequently phosphorylated in Merkel cell carcinoma and associates with longer survival.

Merkel cell carcinoma (MCC) is a rare skin cancer with only limited therapeutic options for advanced disease. We previously showed that oncogene ALK is frequently expressed at the RNA level in MCC and further that ALK positivity by immunohistochemistry is frequent and correlates strongly with Merkel cell polyomavirus (MCPyV) positivity. In this study, we investigated whether ALK receptor is active in MCC tumor samples and MCC cell lines, and whether ALK would be a prospective treatment target in MCC. We utilized tissue microarrays constructed from 136 primary MCC tumor samples as well as nine previously established MCC cell lines to determine the presence of ALK and phosphorylated ALK (p-ALK) via immunohistochemistry. Almost half of the analyzed MCC tumors displayed ALK phosphorylation (47.8%). Analysis of MCC tumor samples revealed that the presence of p-ALK correlated to MCPyV positivity, younger age, nonexistence of metastases at diagnosis and ultimately to better MCC-specific survival. In contrast to MCC tumor samples only two out of nine MCC cell lines showed only low ALK phosphorylation by immunohistochemistry. Our study reveals clear disparity in ALK activity between patient derived tumors and cell line samples and therefore, more advanced disease models such as xenografts are necessary to resolve whether ALK is a useful treatment target in MCC.

Malignant Eccrine Porocarcinoma in Finland During 2007 to 2017.

Malignant eccrine porocarcinoma is a rare skin adnexal cancer arising from the sweat glands. Little is known about the epidemiology and incidence of eccrine porocarcinoma. This registry-based study examined the epidemiology and incidence data for eccrine porocarcinoma from the Finnish Cancer Registry. The study included all persons diagnosed with eccrine porocarcinoma in 2007 to 2017. There were 69 cases in the study period; 34 (49%) male and 35 (51%) female patients. Mean age at diagnosis was 75.5 years. Incidence for men was 0.06 per 100,000 person-years and for women 0.04 per 100,000 person-years adjusted for age according to the World Standard Population. Incidence increased with age. There was one eccrine porocarcinoma-specific death among the 69 patients. The incidence of eccrine porocarcinoma in Finland is therefore low. The mean age at time of diagnosis and the location of eccrine porocarcinoma are consistent with previous reports. The survival of patients with eccrine porocarcinoma is high.

Treatment and Prognosis of Radiation-Associated Breast Angiosarcoma in a Nationwide Population.

BACKGROUND: Radiation-associated angiosarcoma of the breast (RAASB) is an aggressive malignancy that is increasing in incidence. Only a few previous population-based studies have reported the results of RAASB treatment. METHODS: A search for RAASB patients was carried out in the Finnish Cancer Registry, and treatment data were collected to identify prognostic factors for survival. RESULTS: Overall, 50 RAASB patients were identified. The median follow-up time was 5.4 years (range 0.4-15.6), and the 5-year overall survival rate was 69%. Forty-seven (94%) patients were operated on with curative intent. Among these patients, the 5-year local recurrence-free survival, distant recurrence-free survival, and overall survival rates were 62%, 75%, and 74%, respectively. A larger planned surgical margin was associated with improved survival. CONCLUSIONS: We found that the majority of RAASB patients were eligible for radical surgical management in this population-based analysis. With radical surgery, the prognosis is relatively good.

CDKN2A copy number and p16 expression in malignant pleural mesothelioma in relation to asbestos exposure.

BACKGROUND: Deletion of the CDKN2A locus is centrally involved in the development of several malignancies. In malignant pleural mesothelioma (MPM), it is one of the most frequently reported genomic alteration. MPM is strongly associated with a patients' asbestos exposure. However, the status of CDKN2A and the expression of the corresponding protein, p16, in relation to MPM patient's asbestos exposure is poorly known. Copy number alterations in 2p16, 9q33.1 and 19p13 have earlier been shown to accumulate in lung cancer in relation to asbestos exposure but their status in MPM is unclear. METHODS: We studied DNA copy numbers for CDKN2A using fluorescence in situ hybridization (FISH) and p16 expression by immunohistochemistry (IHC) in 92 MPM patients, 75 of which with known asbestos exposure status. We also studied, in MPM, copy number alterations in 2p16, 9q33.1 and 19p13 by FISH. RESULTS: We were unable to detect an association between p16 expression and pulmonary asbestos fiber count in MPM tumor cells. However, significantly more MPM patients with high pulmonary asbestos fiber count (> 1 million fibers per gram [f/g]) had stromal p16 immunoreactivity than MPM of patients with low exposure (≤ 0.5 million f/g) (51.4% vs 16.7%; p = 0.035, Chi-Square). We found that an abnormal copy number of CDKN2A in MPM tumor cells associated with a high pulmonary asbestos fiber count (p = 0.044, Fisher's Exact test, two-tailed). In contrast to our earlier findings in asbestos associated lung cancer, DNA copy number changes in 2p16, 9q33 and 19p13 were not frequent in MPM although single cases with variable copy numbers on those regions were seen. CONCLUSIONS: We found two instances where the gene locus CDKN2A or its corresponding protein expression, is associated with high asbestos exposure levels. This suggests that there may be biological differences between the mesotheliomas with high pulmonary asbestos fiber count and those with low fiber count.

(Neo)adjuvant chemotherapy and interdigitated split-course hyperfractionated radiation in high risk soft tissue sarcoma - Results from a large single-institution series.

A single-institution series using a (neo)adjuvant chemotherapy and interdigitated hyperfractionated split-course radiation therapy (CRT) treatment protocol for soft tissue sarcoma was reviewed. Our specific aims were to study recurrence rates and long-term toxicity. Between 1998 and 2016, 89 patients with non-metastatic soft tissue sarcoma were treated with surgery combined with six courses of doxorubicin and ifosfamide and hyperfractionated radiation therapy (42-60 Gy/1.5 Gy twice daily). Patients were considered being at high risk if tumour malignancy grade was high and the tumour fulfilled at least two of the following criteria: size >8 cm, presence of necrosis or vascular invasion. The mean age of the patients was 50.7 years. With a median follow-up of 5.4 years for survivors, the local control rate was 81.4%. Six (7%) patients progressed during neoadjuvant CRT. Seven (8%) patients discontinued the treatment due to toxicity. Eighty-six patients were operated and three (3%) of these developed a long-term complication. The estimated metastasis-free survival was 47.6% and overall survival 53.0% at five years. The limb-salvage rate was 93%. The limb-salvage rate, local control and complication rates were good in these patients with high risk soft tissue sarcoma. Metastases-free survival and overall survival rates were less satisfactory, reflecting the aggressive nature of these tumours.

The expression of terminal deoxynucleotidyl transferase and paired box gene 5 in Merkel cell carcinomas and its relation to the presence of Merkel cell polyomavirus DNA.

BACKGROUND: Merkel cell carcinoma (MCC) tumor samples frequently express B-lymphoid lineage markers. However, the reasons for expression of specific B-lymphoid lineage markers are still unclear. We studied the expression of TdT and PAX5 (two B-cell lymphoid lineage markers) in a large pool of MCC tissue microarray samples. METHODS: Immunoexpression and staining intensities of TdT and Pax-5 were statistically correlated with patient, tumor, Merkel cell polyomavirus (MCV), and disease-specific parameters. RESULTS: In a cohort of 117 MCC patients and their corresponding tumor samples, TdT was expressed in 37 (31.6%) samples and PAX5 in 26 (22.2%). Simultaneous immunostaining for TdT and PAX5 was observed in 13 (11.1%) samples. A statistically significant relationship was observed between MCV virus copy number and positive TdT expression (P = 0.0056). Similarly, a significant relationship was also observed between positive TdT and tumor MCV virus positivity (P = 0.000495). CONCLUSION: We observed frequent TdT and PAX5 immunoexpression in MCC tumor samples. However, simultaneous immunoexpression of these markers was scarce. TdT expression was statistically significantly associated with MCV positivity. The absence of a statistically significant association between tumor parameters and disease progression markers undermines the systemic use of these markers in clinical practice.