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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.

Purpose: Merkel cell carcinoma (MCC) is rare skin cancer that is often associated with Merkel cell polyomavirus (MCPyV) infection. Polyomaviruses repress tumor suppressor proteins, thus influencing cell-cycle progression, but the effect of MCPyV on the key cell-cycle regulating proteins is poorly understood.Experimental Design: We evaluated expression of the MCPyV large T-antigen (LTA), Ki-67, and the key putative tumor suppressor proteins, the retinoblastoma protein (RB and phospho-RB) and p53, and their regulatory proteins (cyclin D1, cyclin E, p16, p21, p27, and MDM2) by using immunohistochemistry from tumors of 91 MCC patients identified from a population-based nationwide cohort. Tumor MCPyV DNA was measured by using quantitative PCR, and TP53 mutations were identified with sequencing.Results: MCPyV LTA expression was strongly associated with presence of MCPyV DNA in tumor, and it was almost invariably associated with tumor RB expression (P < 0.0001 for both comparisons). Both MCC LTA and RB expression were strongly associated with favorable MCC-specific and overall survival in univariable analyses (P ≤ 0.01 for all four analyses). Presence of MCPyV LTA was also associated with the female gender, the intermediate type of tumor histology, location of the tumor in a limb, cell proliferation rate, and absence of p53 expression. TP53 mutations were detected only in MCPyV DNA–negative tumors.Conclusions: MCPyV DNA–positive MCC has several clinical and molecular features that differ from MCPyV DNA-negative cancers. MCPyV-associated MCCs express RB, but may not harbor TP53 mutations. These findings provide further support that MCPyV causes the majority of MCCs. Clin Cancer Res; 17(14); 4806–13. ©2011 AACR.

Supplementary Data 3 from Merkel Cell Polyomavirus Infection, Large T Antigen, Retinoblastoma Protein and Outcome in Merkel Cell Carcinoma

Purpose: Merkel cell carcinoma (MCC) is a rare skin cancer that often harbors Merkel cell polyomavirus (MCPyV) DNA. The clinical importance of intratumoral immune cells and their associations with MCPyV infection are poorly understood.Experimental Design: We identified T lymphocytes (CD3-positive cells), T-cell subsets (CD4, CD8, and FoxP3-positive cells), natural killer cells (small CD16-positive cells), and macrophages (CD68 and CD163-positive cells) in tumors of 116 individuals diagnosed with MCC in Finland from 1979 to 2004 using immunohistochemistry and detected MCPyV DNA with quantitative PCR. The associations between immune cell counts, MCPyV DNA, patient and tumor characteristics, and patient outcome were examined.Results: MCPyV DNA-positive cancers contained higher numbers of CD3+, CD8+, CD16+, FoxP3+, and CD68+ cells as compared with MCPyV DNA-negative carcinomas (all P values < 0.05). High intratumoral numbers of CD3+, CD8+, or FoxP3+ cells, and high CD8+/CD4+ or FoxP3+/CD4+ ratios, were significantly associated with favorable overall survival. Individuals with a high tumor CD3+ count had metastases less often and survived longer, irrespective of the tumor MCPyV status. Tumor CD3+ count and MCPyV DNA status had independent influence on survival in a Cox multivariable model that also included presence of locoregional metastases at diagnosis and gender as covariates.Conclusions: High intratumoral T-lymphocyte counts are associated with favorable survival in MCC. Although the numbers of T cells are generally higher in MCPyV-positive than in MCPyV-negative MCC, high intratumoral T-cell counts are also associated with favorable survival in MCPyV-negative MCC. Clin Cancer Res; 18(10); 2872–81. ©2012 AACR.

Supplementary Data 2 from Merkel Cell Polyomavirus Infection, Large T Antigen, Retinoblastoma Protein and Outcome in Merkel Cell Carcinoma

PDF file, 24KB, Associations of tumor infiltrating CD16+ natural killer cells, CD68+ macrophages, and CD163+ macrophages with patient and tumor characteristics.

PDF file, 19KB, Antibodies and conditions in immunohistochemical stainings.

Supplementary Data 1 from Merkel Cell Polyomavirus Infection, Large T Antigen, Retinoblastoma Protein and Outcome in Merkel Cell Carcinoma

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.

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.

Liposarcoma is a common soft tissue sarcoma, and is classified into four histological subtypes, each with distinct disease outcome, morphology, and genotype. Typical primary treatment in advanced liposarcoma consists of surgery, combined with chemotherapy, radiotherapy, or both. There are currently no approved targeted therapeutics or precision medicine opportunities in liposarcoma. Here we investigated 131 liposarcoma tumour samples by transcriptional profiling and 543 sarcomas by immunoprofiling and compared these data with high-throughput drug sensitivity testing in liposarcoma cell lines in vitro. The data showed that i) myxoid liposarcomas, specifically the high-grade phenotype, display high levels of PDE3A gene expression and that ii) PDE3A modulators are effective inhibitors in PDE3A and SLFN12 co-expressing soft tissue sarcoma cell lines. iii) In addition, PDE3A modulators synergize with many emerging targeted therapies and sensitize sarcoma cell lines to Bcl-2 family inhibitors. We suggest that overexpression of PDE3A and SLFN12 genes is common in myxoid liposarcomas and that this leads to sensitivity to PDE3A modulators and synergy with Bcl-2 family inhibition. Further studies are warranted to translate these observations into precision liposarcoma therapy.

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. 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.

Export Date: 15 September 2021 Correspondence Address: Fagerstedt, K.W.; Department of Pathology, Finland; email: klaus.wj.fagerstedt@helsinki.fi 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. © 2021 The authors.

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.

BackgroundRadiation-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.MethodsA search for RAASB patients was carried out in the Finnish Cancer Registry, and treatment data were collected to identify prognostic factors for survival.ResultsOverall, 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.ConclusionsWe 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.

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 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. publishedVersion

Background: Genetic alterations occurring in lung cancer are the basis for defining molecular subtypes and essential for targeted therapies. Exhaled breath condensate (EBC) is a form of non-invasive sample that, amongst components, contains DNA from pulmonary tissue. Nextgeneration sequencing (NGS) was herein used to analyze mutations in EBC from patients with lung cancer. Materials and Methods: EBC was collected from 26 patients with cancer and 20 healthy controls. Amplicon-based sequencing using Ion Ampliseq Colon and Lung Cancer gene panel v2 was applied. Results: The sequencing was successful in 17 patients and 20 controls. EBC from patients revealed 39 hotspot mutations occurring in: adenomatous polyposis coli (APC), v-raf murine sarcoma viral oncogene homolog B (BRAE), discoidin domain receptor tyrosine kinase 2 (DDR2), epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 4 (ERBB4), F-box and WD repeat domain containing 7 (FBXW7), fibroblast growth factor receptor 1 (FGER1), FGFR3 (fibroblast growth factor receptor 3), Kirsten rat sarcoma viral oncogene homolog (KRAS), mitogen-activated protein kinase kinase 1 (MAP2K1), met proto-oncogene (MET), neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tensin homolog (PTEN), ret proto-oncogene (RET), SMAD family member 4 (SMAD4), serine/threonine kinase 11 (STK11), and tumorprotein p53 (TP53) genes. EBC from controls revealed 35 hotspot mutations. The average mutant allele fraction was higher in patients than controls. Conclusion: NGS can identify mutations in EBCs from patients with lung cancer. This could provide a promising non-invasive method for the assessment of gene mutations in lung cancer.

Radiation-associated sarcoma (RAS) is a rare complication of radiation therapy (RT) to breast cancer (BC). This study explored RAS after RT to BC in a nationwide population-based material. The Finnish Cancer Registry was queried for patients with BC treated during 1953-2014 who were later diagnosed with a secondary sarcoma in 1953-2014. Registry data, patient files, and sarcoma specimens were analyzed to confirm diagnosis and location of RAS at or close to the RT target volume. A total of 132 512 patients were diagnosed with invasive BC during the study period. A subsequent sarcoma was diagnosed in 355 patients. After exclusion, 96 RAS were identified. Angiosarcoma (AS) was the most prevalent histology in 50 (52%) of 96 patients. However, the first radiation-associated AS was diagnosed in a patient treated for BC with breast-conserving surgery in 1984, and thereafter, the proportion of AS continuously increased. The 5-year sarcoma-specific survival was 75.1% for RAS treated with a curative intent. The distribution of histologic subtypes of RAS has changed during the 60 years of this registry study. The first radiation-associated AS was diagnosed in 1989, and presently, AS is the most common histologic subtype of RAS. It is possible that changes in BC treatment strategies are influencing the characteristics of RAS.

Background Microbial ecosystems that inhabit the human gut form central component of our physiology and metabolism, regulating and modulating both health and disease. Changes or disturbances in the composition and activity of this gut microbiota can result in altered immunity, inflammation, and even cancer. Aim To compare the composition and diversity of gut microbiota in stool samples from patient groups based on the site of neoplasm in the gastrointestinal tract (GIT) and to assess the possible contribution of the bacterial composition to tumorigenesis. Methods We studied gut microbiota by16S RNA gene sequencing from stool DNA of 83 patients, who were diagnosed with different GIT neoplasms, and 13 healthy individuals. Results As compared to healthy individuals, stools of patients with stomach neoplasms had elevated levels of Enterobacteriaceae, and those with rectal neoplasms had lower levels of Bifidobacteriaceae. Lower abundance of Lactobacillaceae was seen in patients with colon neoplasms. Abundance of Lactobacillaceae was higher in stools of GIT patients sampled after cancer treatment compared to samples collected before start of any treatment. In addition to site-specific differences, higher abundances of Ruminococcus, Subdoligranulum and lower abundances of Lachnoclostridium and Oscillibacter were observed in overall GIT neoplasms as compared to healthy controls Conclusion Our study demonstrates that the alterations in gut microbiota vary according to the site of GIT neoplasm. The observed lower abundance of two common families, Lactobacillaceae and Bifidobacteriaceae, and the increased abundance of Enterobacteriaceae could provide indicators of compromised gut health and potentially facilitate GIT disease monitoring. Electronic supplementary material The online version of this article (10.1007/s10620-018-5190-5) contains supplementary material, which is available to authorized users.

AIM To study cancer hotspot mutations by next-generation sequencing (NGS) in stool DNA from patients with different gastrointestinal tract (GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients diagnosed with various gastric and colorectal neoplasms, including benign tumors, and from 14 healthy controls. DNA was isolated from stools by using the PSP (R) Spin Stool DNA Plus Kit. For each sample, 20 ng of DNA was used to construct sequencing libraries using the Ion AmpliSeq Cancer Hotspot Panel v2 or Ion AmpliSeq Colon and Lung Cancer panel v2. Sequencing was performed on Ion PGM. Torrent Suite Software v.5.2.2 was used for variant calling and data analysis. RESULTS NGS was successful in assaying 72 GIT samples and 13 healthy controls, with success rates of the assay being 78% for stomach neoplasia and 87% for colorectal tumors. In stool specimens from patients with gastric neoplasia, five hotspot mutations were found in APC, CDKN2A and EGFR genes, in addition to seven novel mutations. From colorectal patients, 20 mutations were detected in AKT1, APC, ERBB2, FBXW7, KIT, KRAS, NRAS, SMARCB1, SMO, STK11 and TP53. Healthy controls did not exhibit any hotspot mutations, except for two novel ones. APC and TP53 were the most frequently mutated genes in colorectal neoplasms, with five mutations, followed by KRAS with two mutations. APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions. CONCLUSION Our results show that in addition to colorectal neoplasms, mutations can also be assayed from stool specimens of patients with gastric neoplasms.

The two-stage induced-membrane technique for treatment of large bone defects has become popular among orthopedic surgeons. In the first operation, the bone defect is filled with poly(methyl methacrylate) (PMMA), which is intended to produce a membrane around the implant. In the second operation, PMMA is replaced with autograft or allograft bone. Bioactive glasses (BAGs) are bone substitutes with bone-stimulating and angiogenetic properties. The aim of our study was to evaluate the inductive vascular capacity of BAG-S53P4 and poly(lactide-co-glycolide) (PLGA)-coated BAG-S53P4 for potential use as bone substitutes in a single-stage induced-membrane technique. Sintered porous rods of BAG-S53P4, PLGA-coated BAG-S53P4 and PMMA were implanted in the femur of 36 rabbits for 2, 4 and 8 weeks. The expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF) in the induced membranes of implanted materials was analyzed with real-time quantitative polymerase chain reaction and compared with histology. Both uncoated BAG-S53P4 and PLGA-coated BAG-S53P4 increase expression of VEGF and TNF, resulting in higher amounts of capillary beds, compared with the lower expression of VEGF and less capillary beads observed for negative control and PMMA samples. A significantly higher expression of VEGF was observed for PLGA-coated BAG-S53P4 than for PMMA at 8 weeks (p

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. Electronic supplementary material The online version of this article (10.1186/s12885-019-5652-y) contains supplementary material, which is available to authorized users.

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.

Aims Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters. Methods and results Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death. Conclusions ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment.

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.

Background: Osteosarcomas (OS) in the craniomaxillofacial (CMF) region are typically diagnosed at later age than long-bone OS, but they are reported to have better 5-year survival. Curative treatment warrants wide surgical resection, which is often not possible in the CMF region. The purpose of this article is to present a nationwide series of CMF in Finland to discuss the role of surgery. Patients and methods: All 21 CMF OS patients managed in Finland from 1992 to 2009 were included. The mean age was 40 years (range 15e72). Data on patient and tumor characteristics, treatment modalities, and survival were recorded. All patients had a minimum follow-up of 5 years or until death. Results: OS was evenly represented in the mandible and maxillary bones, which together constituted 76% of all sites. Surgery with curative intent was carried out in 20 patients. Clear margins were achieved in only five cases. Eight (40%) of these 20 patients died due to OS, and their average survival time was 1.3 years. Seven (35%) out of the 20 patients received radiotherapy due to close/intralesional surgical margins, and four of them did not develop recurrences during the follow-up. Conclusions: The results suggest that postoperative radiotherapy may alter the prognosis in CMF OS, particularly in cases with close or intralesional margins. This may increase the survival rates achieved by prompt action in performing radical surgery. (C) 2019 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer in need for novel therapies. Here we show that the PROX1 transcription factor, which is essential for normal myoblast differentiation, is highly expressed in RMS tumors. We demonstrate that PROX1 is needed for RMS cell stemness and growth in vitro, and for RMS tumor formation in mouse xenograft models. In addition, we unveil that PROX1 is an essential for myogenic properties in RMS. PROX1 depletion reprogrammed the RMS transcriptome to resemble benign mesenchymal stem cells and repressed many of the previously identified RMS effector transcripts and myogenic genes. By using proximity labeling and mass spectrometry, we found that PROX1 interacts with the NuRD and CoREST complexes containing class I HDACs. Our studies reveal a major role of PROX1-HDAC interaction in RMS and give insights that inhibiting this interaction could be a promising therapeutic approach.

Background and objectives Desmoid tumors are soft-tissue tumors originating from myofibroblasts with a tendency to recur after surgery. High expression of proliferation markers is associated with shortened progression-free and/or overall survival in many neoplasms, including soft-tissue sarcomas. We investigated the prognostic role of cyclin A and Ki67 in desmoid tumors by immunohistochemistry. Methods The study included 76 patients with desmoid tumor operated at Helsinki University Hospital between 1987 and 2011. A tissue micro array (TMA) was constructed and the TMA sections were immunostained with cyclin A and Ki67 antibodies. A computer-assisted image analysis was performed. Results Cyclin A expression was evaluable in 74 and Ki67 in 70 patients. Cyclin A immunopositivity varied from 0% to 9.9%, with a mean of 1.9%. Cyclin A expression correlated significantly with Ki67. Cyclin A expression was associated with recurrence-free survival (HR 1.9, 95% CI = 1.1-3.2, P = .02), as were positive margin (HR 6.0, 95% CI = 1.6-22.5, P = .008) and extremity location (HR 5.3, 95% CI = 1.7-16.8, P = 0.005). Ki67 immunopositivity varied from 0.33% to 13.8%, with a mean of 4.6%, but had no significant prognostic impact (HR 1.1, P = .2). Conclusions Our study indicates that cyclin A may be a new prognostic biomarker in surgically treated desmoid tumors.

Background and purpose - Soft-tissue sarcoma (STS) is rare, with challenging individualized treatment, so diagnostics and treatment should be centralized. Historical controls are sometimes used for investigation of whether new diagnostic or therapeutic tools affect patient outcome. However, as yet unknown factors may affect the outcome. We investigated prognostic factors and prognosis in 2 nationwide cohorts of patients diagnosed with a local STS during the periods 1998-2001 and 2005-2010, with special interest in finding factors lying behind possible improvement of prognosis. Patients and methods - 2 cohorts of patients with STS of the extremities or trunk diagnosed during the periods 1998-2001 and 2005-2010 were retrieved from the nationwide Finnish Cancer Registry. Detailed information was gathered from patient files. Results - Compared to first cohort, a larger proportion of patients with inadequate surgery in the second cohort received radiation therapy, and both the local control rate and the sarcoma-specific survival rate improved in the second cohort. For sarcoma-specific survival, cohort (HR =0.6, 95% CI: 0.5-0.9), age, depth, grade, and margin were significant factors in multivariate analysis. For local control, cohort (HR =0.6, 95% CI: 0.5-0.9), age, and margin were significant in multivariate analysis. Interpretation - Known prognostic factors including type of treatment did not entirely explain the secular trend of continuous improvement in prognosis in STS. This illustrates the danger of using historical controls for investigation of whether new diagnostic or therapeutic tools have an effect on patient outcome.

// Omar Youssef 1 , Aija Knuuttila 2 , Paivi Piirila 3 , Tom Bohling 4 , Virinder Sarhadi 1, * , Sakari Knuutila 1, * 1 Faculty of Medicine, Department of Pathology, University of Helsinki, Helsinki, Finland 2 Department of Pulmonary Medicine, University of Helsinki and Helsinki University Hospital, Heart and Lung Center, Helsinki, Finland 3 Unit of Clinical Physiology, HUS-Medical Imaging Center, Helsinki University Hospital and Helsinki University, Helsinki, Finland 4 Department of Pathology, University of Helsinki and HUSLAB, Helsinki University Hospital, Helsinki, Finland * These authors have contributed equally to this work Correspondence to: Sakari Knuutila, email: sakari.knuutila@helsinki.fi Keywords: exhaled breath condensate, mutations, healthy individuals, next generation sequencing Received: December 10, 2016 Accepted: January 16, 2017 Published: February 09, 2017 ABSTRACT Exhaled breath condensate (EBC) is a non-invasive source that can be used for studying different genetic alterations occurring in lung tissue. However, the low yield of DNA available from EBC has hampered the more detailed mutation analysis by conventional methods. We applied the more sensitive amplicon-based next generation sequencing (NGS) to identify cancer related mutations in DNA isolated from EBC. In order to apply any method for the purpose of mutation screening in cancer patients, it is important to clarify the incidence of these mutations in healthy individuals. Therefore, we studied mutations in hotspot regions of 22 cancer genes of 20 healthy, mainly non-smoker individuals, using AmpliSeq colon and lung cancer panel and sequenced on Ion PGM. In 15 individuals, we detected 35 missense mutations in TP53, KRAS, NRAS, SMAD4, MET, CTNNB1, PTEN, BRAF, DDR2, EGFR, PIK3CA, NOTCH1, FBXW7, FGFR3, and ERBB2: these have been earlier reported in different tumor tissues. Additionally, 106 novel mutations not reported previously were also detected. One healthy non-smoker subject had a KRAS G12D mutation in EBC DNA. Our results demonstrate that DNA from EBC of healthy subjects can reveal mutations that could represent very early neoplastic changes or alternatively a normal process of apoptosis eliminating damaged cells with mutations or altered genetic material. Further assessment is needed to determine if NGS analysis of EBC could be a screening method for high risk individuals such as smokers, where it could be applied in the early diagnosis of lung cancer and monitoring treatment efficacy.

Background: We aimed to assess the connection between chronic inflammatory disorders (CIDs) and Merkel cell carcinoma (MCC). Methods: Merkel cell carcinoma cases diagnosed in 1978–2009 were extracted from the Finnish Cancer Registry and controls from the Population Registry. Information on reimbursed CIDs was linked to clinicopathological data including Merkel cell polyomavirus (MCV) status by qPCR and immunohistochemistry for the large T antigen of MCV (LTA), Ki-67 and tumour-infiltrating lymphocytes. Results: Chronic inflammatory disorders increased the risk of MCC significantly (odds ratio (OR) 1.39, 95% confidence interval (CI) 1.03–1.88), specifically connective tissue/systemic diseases (OR 1.75, 95% CI 1.09–1.80) and diabetic conditions (OR 1.51, 95% CI 1.03–2.22). Chronic inflammatory disorders associated with larger tumour diameter (P=0.02) and higher Ki-67 expression (P=0.005). The expression of LTA was seen significantly more often in the absence of CIDs (P=0.05). Conclusions: Patients with CID are at significantly higher risk for aggressive MCC. Merkel cell polyomavirus positivity is more common in MCC patients unafflicted by CID.

Background The primary neuroendocrine skin cancer, Merkel cell carcinoma (MCC), has a well-known predilection to metastasize systemically. However, the experience of systemic metastases in MCC is mainly disseminated through case reports due to the rarity of MCC. Purpose To elucidate the frequency and locations of systemic metastasis in MCC by reviewing the imaging of patients with metastatic MCC in a national cohort. Material and Methods Patients with diagnosed metastatic MCC by imaging studies in Finland during 1999–2012 were included in this study. We reviewed their imaging studies to evaluate the most frequent sites for systemic metastasis and determined the latency between the primary tumor diagnosis and systemic metastasis. The material includes 30 MCC patients with complete imaging series and 187 examinations, of which 102 (54%) were CT images. Results The mean latency from the primary tumor diagnosis to systemic metastasis was 2.1 years and the mean latency between the radiologic diagnosis of the metastases and death was 299 days. Metastases were recorded in several organ systems in most of the cases, and at least two separate metastatic sites in 63% of the cases. Metastatic spread was noted in 60% of the cases in distant lymph nodes. Liver and lungs were the most affected solid organs. Conclusion Systemic metastasis in MCC has no predilection site, basically every organ system can be involved. Most of the systemic metastases were recorded during the first two years after the MCC diagnosis.

English summary

Clear cell papillary renal cell carcinoma (CCPRCC) is a recently recognised neoplasm with a broad spectrum of morphological characteristics, thus representing a challenging differential diagnosis, especially with the low malignant potential multicystic renal cell neoplasms and clear cell renal cell carcinoma. We selected 14 cases of CCPRCC with a wide spectrum of morphological features diagnosed on morphology and CK7 immunoreactivity and analysed them using a panel of immunohistochemical markers, focusing on 34 beta E12 and related CKs 1,5,10 and 14 and several molecular analyses such as fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH), VHL methylation, VHL and TCEB1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Twelve of 13 (92%) CCPRCC tumours were positive for 34 beta E12. One tumour without 3p alteration by FISH revealed VHL mutation and 3p deletion at aCGH; thus, it was re-classified as clear cell RCC. We concluded that: (1) immunohistochemical expression of CK7 is necessary for diagnostic purposes, but may not be sufficient to identify CCPRCC, while 34 beta E12, in part due to the presence of CK14 antigen expression, can be extremely useful for the recognition of this tumour; and (2) further molecular analysis of chromosome 3p should be considered to support of CCPRCC diagnosis, when FISH analysis does not evidence the common loss of chromosome 3p.

Malignant tumours are the foremost complications of immunosuppressive treatment. They are a major challenge for organ transplant recipients and their treating physicians. This paper reviews the aetiology and current treatment of an unusual neuroendocrine skin cancer, Merkel cell carcinoma (MCC), caused by a Merkel cell polyomavirus infection. MCC occurs more frequently than expected in immunosuppressed subjects, especially in organ transplant recipients. The current literature comprises reports of 79 organ transplant recipients with MCC. The risk of MCC in organ transplant recipients is increased up to 66-182-fold compared with the general population. In addition to the increased risk of developing MCC, immunosuppressed individuals have poorer MCC-specific survival. The aim of this review article is to familiarize organ transplant doctors with this unique and clinically challenging skin cancer, and to provide recent data on the diagnosis and current treatment recommendations for an immunosuppressed population.

Merkel cell polyomavirus (MCV) infection underlies most Merkel cell carcinoma (MCC), a primary neuroendocrine carcinoma of the skin. While previous research has focused on MCV-positive MCC tumors, less is known about the oncogenesis in MCV-negative tumors. In this study, we analyzed mutational status of 27 MCC tumors with known MCV status for hotspot regions of 50 cancer-related genes by targeted next-generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel. In addition to previously reported TP53 , KIT , and PIK3CA gene mutations, we found somatic mutations in the tyrosine kinase domain of the EGFR gene in a small proportion of the cells in six tumor tissues. RB1 mutations were seen only in virus negative tumors. Hotspot mutations were more frequent in MCV-negative tumors, although the difference was not statistically significant. No clear hotspot mutation profile was observed. Novel RB1 mutations were detected only in MCV-negative tumors.

cited By 0 Myxoinflammatory fibroblastic sarcoma is a soft-tissue neoplasm most frequently found in the distal extremities of middle-aged adults. Most myxoinflammatory fibroblastic sarcoma are low-grade tumors with propensity for local recurrence after incomplete removal. We report a myxoinflammatory fibroblastic sarcoma which developed in the foot of a 41-year-old male and showed an exceptionally aggressive course with metastatic spread and fatal outcome within 16 months. We managed to establish a spontaneously transformed continuous cell line, called JU-PI, from a metastatic lesion. The JU-PI cells have a sub-tetraploid karyotype including the 1;10 chromosomal translocation and amplification of the proximal end of 3p; these features are considered genetic signatures of myxoinflammatory fibroblastic sarcoma. Both the primary tumor and the JU-PI cells showed nuclear expression of the TFE3 transcription factor but TFE3-activating chromosomal rearrangements were not found. To our knowledge, JU-PI is the first established myxoinflammatory fibroblastic sarcoma cell line. JU-PI cells offer a tool for investigating the molecular oncology of myxoinflammatory fibroblastic sarcoma. © 2018, © The Author(s) 2018.

Desmoid tumors are soft-tissue tumors originating from myofibroblasts with a tendency to recur after surgery. High expression of proliferation markers is associated with shortened progression-free and/or overall survival in many neoplasms, including soft-tissue sarcomas. We investigated the prognostic role of cyclin A and Ki67 in desmoid tumors by immunohistochemistry.The study included 76 patients with desmoid tumor operated at Helsinki University Hospital between 1987 and 2011. A tissue micro array (TMA) was constructed and the TMA sections were immunostained with cyclin A and Ki67 antibodies. A computer-assisted image analysis was performed.Cyclin A expression was evaluable in 74 and Ki67 in 70 patients. Cyclin A immunopositivity varied from 0% to 9.9%, with a mean of 1.9%. Cyclin A expression correlated significantly with Ki67. Cyclin A expression was associated with recurrence-free survival (HR 1.9, 95% CI = 1.1-3.2, P = .02), as were positive margin (HR 6.0, 95% CI = 1.6-22.5, P = .008) and extremity location (HR 5.3, 95% CI = 1.7-16.8, P = 0.005). Ki67 immunopositivity varied from 0.33% to 13.8%, with a mean of 4.6%, but had no significant prognostic impact (HR 1.1, P = .2).Our study indicates that cyclin A may be a new prognostic biomarker in surgically treated desmoid tumors.

Background Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. Methods To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. Results In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. Conclusions Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms. Prostate © 2015 Wiley Periodicals, Inc.

Merkel cell polyomavirus (MCV) is frequently detectable in Merkel cell carcinoma (MCC) tumors, but the significance of MCV infection is not yet totally understood. Thus far, no key regulatory miRNA has been identified for MCC tumorigenesis. However, distinct miRNA expression profiles have been suggested for MCV-positive and MCV-negative tumors. We used microarray hybridization to identify miRNA expression differences in MCC tumor samples according to MCV status and further validated these results by quantitative reverse transcription polymerase chain reaction (qRT-PCR). When compared with MCV-negative tumors, we detected overexpression of miR-34a, miR-30a, miR-142-3p, and miR-1539 in those MCV positives. In addition, slight underexpression was detectable in MCV-positive tumors of miR-181d. We confirmed the distinct expression of miRNAs in MCV-positive and MCV-negative tumors and confirmed statistically significant underexpression of miR-34a in MCV-negative tumors by both array analysis and qRT-PCR. Neither tumor location nor development of metastases affected miRNA expression.