POU4F3 is a Sensitive and Specific Marker of Merkel Cell Carcinoma.

Although of therapeutic importance, a single sensitive and specific immunostain to distinguish Merkel cell carcinoma (MCC) from mimics is not currently available. In addition, single tumor cells are difficult to detect in sentinel lymph node biopsy. Leveraging publicly available data sets of 9264 solid tumors and over 600,000 single-cell transcriptomes, we identified POU4F3 to be a specific marker of MCC. Analyses of Pan-Cancer RNA bulk sequencing data of 24 tumor types from Tumor Cancer Genomic Atlas (TCGA) datasets as well as non-TCGA SCLC and MCC datasets confirmed POU4F3 specificity for MCC. Single-cell RNA sequencing analyses also confirmed lack of POU4F3 expression in lung small cell carcinoma as well as a variety of normal tissues. Nuclear POU4F3 immunohistochemical expression was noted in 98.7% of 153 MCCs and in only 1.7% of mimics (3 of 180 cases, including 95 small cell carcinomas of which 55 from lung and the remainder from other sites). Three POU4F3-positive non-MCC cases were from lung (2 cases) and vagina (1 case). All 153 tested MCC cases were negative for ASCL1, a key transcriptional regulator highly expressed in SCLC. NeuroD1 was seen in a subset of MCC cases (20.9%, 32/153). POU4F3 immunostain was performed on 29 sentinel lymph nodes and strong POU4F3 nuclear expression facilitated ease of metastasis detection, even single tumor cells. Our study built on prior works shows that POU4F3 to be a sensitive and specific clinical marker of MCC.

The Role of Single Nucleotide Polymorphisms in MicroRNA Genes in Head and Neck Squamous Cell Carcinomas: Susceptibility and Prognosis.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers worldwide. The identification of molecular alterations adding to the individual risk of HNSCC development and progression is one of the most important challenges in studies on cancer genetics. MicroRNAs (miRNAs), which belong to the group of important post-transcriptional regulators of human gene expression, seem to be valuable options for consideration as key modifiers of individual cancer risk, and therefore may be helpful in predicting inter-individual differences in cancer risk, response to treatment and prognosis. METHODS: There have not been many studies focused on the relationship between miRNA variants and HNSCC published in PubMed within the last 15 years. We found and analyzed 30 reviews, meta-analyses and research papers and revealed 14 SNPs which have been reported as significant in the context of HNSCC susceptibility and/or prognosis. RESULTS: These 14 SNPs were located in 13 separate miRNAs. Among them, four were the most frequently studied (miRNA-146, -196, -149 and -499) and have been shown to have the greatest impact on the course of HNSCC. However, the presented results have been conflicting. CONCLUSIONS: It must be concluded that, despite the years of studies, there are no conclusive reports demonstrating a significant role of SNPs in miRNAs in the context of the susceptibility to HNSCC or its prognosis.

Co-Targeting of DTYMK and PARP1 as a Potential Therapeutic Approach in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with no standardized treatment for advanced disease. Based on preliminary bioinformatical analyses DTYMK and PARP1 were selected as potential therapeutic targets. High levels of both proteins were detected in uveal melanoma cells and correlated with increased tumor growth and poor prognosis. In vitro tests on MP41 (BAP1 positive) and MP46 (BAP1 negative) cancer cell lines using inhibitors pamiparib (PARP1) and Ymu1 (DTYMK) demonstrated significant cytotoxic effects. Combined treatment had synergistic effects in MP41 and additive in MP46 cell lines, reducing cell proliferation and inhibiting the mTOR signaling pathway. Furthermore, the applied inhibitors in combination decreased cell motility and migration speed, especially for BAP1-negative cell lines. Our hypothesis of the double hit into tumoral DNA metabolism as a possible therapeutic option in uveal melanoma was confirmed since combined targeting of DTYMK and PARP1 affected all tested cytophysiological parameters with the highest efficiency. Our in vitro findings provide insights into novel therapeutic avenues for managing uveal melanoma, warranting further exploration in preclinical and clinical settings.

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

The analysis of transcriptomic signature of TNBC-searching for the potential RNA-based predictive biomarkers to determine the chemotherapy sensitivity.

Neoadjuvant chemotherapy is the foundation treatment for triple-negative breast cancer (TNBC) and frequently results in pathological complete response (pCR). However, there are large differences in clinical response and survival after neoadjuvant chemotherapy of TNBC patients. The aim was to identify genes whose expression significantly associates with the efficacy of neoadjuvant chemotherapy in patients with TNBC. Transcriptomes of 46 formalin-fixed paraffin-embedded (FFPE) tumor samples from TNBC patients were analyzed by RNA-seq by comparing 26 TNBCs with pCR versus 20 TNBCs with pathological partial remission (pPR). Subsequently, we narrowed down the list of genes to those that strongly correlated with drug sensitivity of 63 breast cancer cell lines based on Dependency Map Consortium data re-analysis. Furthermore, the list of genes was limited to those presenting specific expression in breast tumor cells as revealed in three large published single-cell RNA-seq breast cancer datasets. Finally, we analyzed which of the selected genes were significantly associated with overall survival (OS) in TNBC TCGA dataset. A total of 105 genes were significantly differentially expressed in comparison between pPR versus pCR. As revealed by PLSR analysis in breast cancer cell lines, out of 105 deregulated genes, 42 were associated with sensitivity to docetaxel, doxorubicin, paclitaxel, and/or cyclophosphamide. We found that 24 out of 42 sensitivity-associated genes displayed intermediate or strong expression in breast malignant cells using single-cell RNAseq re-analysis. Finally, 10 out of 24 genes were significantly associated with overall survival in TNBC TCGA dataset. Our RNA-seq-based findings suggest that there might be transcriptomic signature consisted of 24 genes specifically expressed in tumor malignant cells for predicting neoadjuvant response in FFPE samples from TNBC patients prior to treatment initiation. Additionally, nine out of 24 genes were potential survival predictors in TNBC. This group of 24 genes should be further investigated for its potential to be translated into a predictive test(s).

Epigenetic Findings in Twins with Esophageal Atresia.

Esophageal atresia (EA) is the most common malformation of the upper gastrointestinal tract. The estimated incidence of EA is 1 in 3500 births. EA is more frequently observed in boys and in twins. The exact cause of isolated EA remains unknown; a multifactorial etiology, including epigenetic gene expression modifications, is considered. The study included six pairs of twins (three pairs of monozygotic twins and three pairs of dizygotic twins) in which one child was born with EA as an isolated defect, while the other twin was healthy. DNA samples were obtained from the blood and esophageal tissue of the child with EA as well as from the blood of the healthy twin. The reduced representation bisulfite sequencing (RRBS) technique was employed for a whole-genome methylation analysis. The analyses focused on comparing the CpG island methylation profiles between patients with EA and their healthy siblings. Hypermethylation in the promoters of 219 genes and hypomethylation in the promoters of 78 genes were observed. A pathway enrichment analysis revealed the statistically significant differences in methylation profile of 10 hypermethylated genes in the Rho GTPase pathway, previously undescribed in the field of EA (ARHGAP36, ARHGAP4, ARHGAP6, ARHGEF6, ARHGEF9, FGD1, GDI1, MCF2, OCRL, and STARD8).

Histologic margin status is a predictor of relapse in lentigo maligna melanoma.

BACKGROUND: Most surgical margins for lentigo maligna melanomas reported in the literature are clinical and not histologic. OBJECTIVES: We sought to determine whether histologic margin status is an independent predictor of progression. METHODS: Clinicopathologic information of 268 invasive lentigo maligna melanomas diagnosed from 1990-2019 were analyzed. Statistical analyses were performed using Cox proportional hazards model and Boruta method. RESULTS: A total of 75% of the lesions were located on the head and neck. The range of follow-up for all patients was 0 to 31.8 years (median, 10.2 years). Time to local recurrence ranges from 0 to 20 years (median, 3 years). Progression developed in 54 (20.1%) of 268 patients. Local recurrence was seen only in 36 (13.4%), both local recurrence and subsequent metastasis in 7 (2.6%), and only metastasis in 11 (4.1%) of 268 patients. Histologic margin status (positive and close/<3 mm) and tumor site (head and neck location) significantly correlated with worse progression-free survival. LIMITATIONS: Single institution and retrospective study. CONCLUSIONS: Histologic margin status is the strongest predictor of progression for lentigo maligna melanoma. Patients with positive or close/<3 mm histologic margins should consider a re-excision due to the increased risk of relapse.

Expression of Genes Associated With Epithelial-Mesenchymal Transition in Merkel Cell Polyomavirus-Negative Merkel Cell Carcinoma.

Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-ß signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.

Common inherited variants of PDCD1, CD274 and HAVCR2 genes differentially modulate the risk and prognosis of adenocarcinoma and squamous cell carcinoma.

BACKGROUND: To investigate the association between single nucleotide polymorphisms (SNPs) of PDCD1, CD274, and HAVCR2 genes with the risk and outcomes of non-small cell lung cancer (NSCLC) subtypes: squamous cell lung cancer (LUSC) and lung adenocarcinoma (LUAD). METHODS: TaqMan SNP genotyping assays or polymerase chain reaction-restriction fragment length polymorphism methods were used to determine genotypes of: PDCD1: rs36084323, rs7421861, rs11568821, rs2227981, rs10204525; CD274: rs822335, rs10815225, rs17718883, rs2297136, rs4742098, rs4143815; HAVCR2: rs10057302, rs1036199. Among 383 NSCLC patients, 112 were diagnosed with LUAD and 116 with LUSC. The control group consisted of 433 unrelated, cancer-free subjects. RESULTS: A CC genotype of rs4143815 and GG genotype of rs4742098 were associated with two times higher risk of developing LUSC (CC vs. GG + GC, OR = 2.31; 95% CI = 1.32, 4.06; P = 0.003; GG vs. AA + AG, OR = 2.26; 95% CI = 1.17, 4.36; P = 0.016, respectively). Moreover, rs4143815 was an independent predictor of the age at diagnosis of LUAD. The carriers of C allele were diagnosed 4.81 years later (95% CI = 1.47, 8.15; P = 0.006) than patients with the GG genotype. The rs10057302 CA genotype was an independent predictor of overall survival in LUSC (adjusted HR = 0.13; 95% CI = 0.02, 0.93; P = 0.043). NSCLC carriers of rs11568821 T allele had almost double the risk of death (adjusted HR = 2.05; 95% CI = 1.28, 3.29; P = 0.003) compared to carriers of CC genotype. CONCLUSIONS: Our results provided additional evidence that SNPs of genes for PD-1, PD-L1 and TIM-3 differentially modulate the risk and prognosis of LUSC and LUAD.

Molecular profiling of primary uveal melanoma: results of a Polish cohort.

There is no published data regarding the molecular alterations of Polish patients with primary uveal melanoma. We performed whole exome sequencing of 20 primary uveal melanomas (UMs), 10 metastasizing and 10 non-metastasizing cases to identify significant molecular alterations. We detected mutations and copy number variants in the BAP1 gene in 50% (10 cases) of the cases. GNA11 mutations were detected in 50% (10 cases) including nine p.Q209L and one p.R183C. GNAQ mutations gene were detected in 40% (8 cases) and all were p.Q209P. SF3B1, EIF1AX, PLCB4 , and PALB2 mutations were detected in one case each. Genetic aberrations of FBXW7 were detected in 55% of cases, with copy number loss of 10 and missense mutation in one. Gain or loss of copy number was observed in 60%, 60%, and 10% of cases in MYC, MLH1 , and CDKN2A genes, respectively. BAP1 and GNAQ tumor suppressor genes are more often mutated in UM with metastasis, while GNA11 mutations are more frequently detected in non-metastasizing tumors. MYC copy gain was present twice as frequently (80% versus 40%) in cases with versus those without metastases. BAP1 mutation correlated with worse overall survival; while GNA11 mutation and CDKN2A loss correlated with better and worse progression-free survival, respectively. We have confirmed BAP1 prognostic potential and documented frequent MYC amplification in metastasizing cases. Although GNA11 mutation and CDKN2A loss significantly correlated with progression-free survival in our study, our sample size is small. The prognostic significance of GNAQ/GNA11 mutation and CDKN2A loss would require further investigation.

Machine learning models demonstrate that clinicopathologic variables are comparable to gene expression prognostic signature in predicting survival in uveal melanoma.

PURPOSE: Since molecular assays are not accessible to all uveal melanoma patients, we aim to identify cost-effective prognostic tool in risk stratification using machine learning models based on routine histologic and clinical variables. EXPERIMENTAL DESIGN: We identified important prognostic parameters in a discovery cohort of 164 enucleated primary uveal melanomas from 164 patients without prior therapies. We then validated the prognostic prediction of top important parameters identified in the discovery cohort using 80 uveal melanomas from the Tumor Cancer Genome Atlas database with available gene expression prognostic signature (GEPS). The performance of three different survival analysis models (Cox proportional hazards (CPH), random survival forest (RSF), and survival gradient boosting (SGB)) was compared against GEPS using receiver operating curves (ROC). RESULTS: In all three selection methods, BAP1 status, nucleoli size, age, mitotic rate per 1 mm2, and ciliary body infiltration were identified as significant overall survival (OS) predictors; and BAP1 status, nucleoli size, largest basal tumor diameter, tumor-infiltrating lymphocyte density, and tumor-associated macrophage density were identified as significant progression-free survival (PFS) predictors. ROC plots for the median survival time point showed that significant parameters in SGB studied model can predict OS better than GEPS. For PFS, SGB model performed similarly to GEPS. The time-dependent area under the curve (AUC) showed SGB model performing better than GEPS in predicting OS and metastatic risk. CONCLUSIONS: Our study shows that routine histologic and clinical variables are adequate for patient risk stratification in comparison with not readily accessible GEPS.

Machine-learning-based Analysis Identifies miRNA Expression Profile for Diagnosis and Prediction of Colorectal Cancer: A Preliminary Study.

BACKGROUND: The stage of colorectal cancer (CRC) at the day of diagnosis has the greatest influence on survival rate. Thus, for CRC, which is mainly identified as advanced disease, non-invasive, molecular blood or stool tests could boost the diagnosis and lower mortality. Evaluation of miRNA expression levels in serum of patients diagnosed with CRC is a potential tool in early screening. Screening can be supported by machine learning (ML) as a tool for developing a cancer risk predictive model based on genetic data. MATERIALS AND METHODS: miRNA was isolated from the serum of 8 patients diagnosed with CRC and 10 patients from a control group matched for age and sex. The expression of 179 miRNAs was determined using a serum/plasma panel (Exiqon). Determinations were conducted using real-time PCR technique on an Applied Biosystems QuantStudio3 device in 96-well plates. A predictive model was developed through the Azure Machine Learning platform. RESULTS: A wide panel of 29 up-regulated miRNAs in CRC were identified and divided into two subgroups: 1) miRNAs with significantly higher serum level in cancer patients vs. controls (24 miRNAs) and 2) miRNAs detected only in cancer patients and not in controls (5 miRNAs). Re-analysis of published miRNA profiles of CRC tumours or CRC exosomes revealed that only 2 out of 29 miRNAs were up-regulated in all datasets including ours (miR-34a and miR-25-3p). CONCLUSION: Our research suggests the potential role of overexpressed miRNAs as diagnostic or prognostic biomarkers among CRC patients. Such clustering of miRNAs may be a potential direction for discovering new diagnostic panels of cancer (including CRC), especially using ML. The low correspondence between deregulation of miRNAs in serum and tumour tissue revealed in our study confirms previously published reports.

Differential Signals From TNFα-Treated and Untreated Embryos in Uterine Tissues and Splenic CD4+ T Lymphocytes During Preimplantation Pregnancy in Mice.

The main aim of this study was to examine if a female mouse body in preimplantation pregnancy can distinguish between embryos of normal and impaired biological quality in the local and peripheral compartments. Normal (control group) and TNFα (tumor necrosis factor-α)-treated embryos (experimental group) at the morula stage were non-surgically transferred into the uteri of CD-1 strain [Crl:CD1(Icr)] female murine recipients. Twenty-four hours after the embryo transfer, females were euthanised, and uteri and spleens were dissected. In uterine tissues (local compartment), we assessed the expression of 84 genes comprising nine signal transduction pathways, using a modified RT2 Profiler PCR Array. In the spleen (peripheral compartment), we determined the proteome of splenic CD4+ lymphocytes using 2D protein electrophoresis with subsequent protein identification by mass spectrometry. Sample clustering and differential gene expression analyses within individual signal transduction pathways revealed differential expression of genes in the uteri of females after transplantation of normal vs. TNFα-treated embryos. The most affected signal transduction cascade was the NFKB (Nuclear factor NF-kappa-B) pathway, where 87.5% of the examined genes were significantly differentially expressed. Proteomic analysis of splenic CD4+ T lymphocytes revealed significant differential expression of 8 out of 132 protein spots. Identified proteins were classified as proteins influenced by cell stress, proteins engaged in the regulation of cytoskeleton stabilization and cell motility, and proteins having immunomodulatory function. These results support the hypothesis that even before embryo implantation, the body of pregnant female mice can sense the biological quality of an embryo both at the local and peripheral level.

Current Achievements and Applications of Transcriptomics in Personalized Cancer Medicine.

Over the last decades, transcriptome profiling emerged as one of the most powerful approaches in oncology, providing prognostic and predictive utility for cancer management. The development of novel technologies, such as revolutionary next-generation sequencing, enables the identification of cancer biomarkers, gene signatures, and their aberrant expression affecting oncogenesis, as well as the discovery of molecular targets for anticancer therapies. Transcriptomics contribute to a change in the holistic understanding of cancer, from histopathological and organic to molecular classifications, opening a more personalized perspective for tumor diagnostics and therapy. The further advancement on transcriptome profiling may allow standardization and cost reduction of its analysis, which will be the next step for transcriptomics to become a canon of contemporary cancer medicine.

The Regulation of Collagen Processing by miRNAs in Disease and Possible Implications for Bone Turnover.

This article describes several recent examples of miRNA governing the regulation of the gene expression involved in bone matrix construction. We present the impact of miRNA on the subsequent steps in the formation of collagen type I. Collagen type I is a main factor of mechanical bone stiffness because it constitutes 90-95% of the organic components of the bone. Therefore, the precise epigenetic regulation of collagen formation may have a significant influence on bone structure. We also describe miRNA involvement in the expression of genes, the protein products of which participate in collagen maturation in various tissues and cancer cells. We show how non-collagenous proteins in the extracellular matrix are epigenetically regulated by miRNA in bone and other tissues. We also delineate collagen mineralisation in bones by factors that depend on miRNA molecules. This review reveals the tissue variability of miRNA regulation at different levels of collagen maturation and mineralisation. The functionality of collagen mRNA regulation by miRNA, as proven in other tissues, has not yet been shown in osteoblasts. Several collagen-regulating miRNAs are co-expressed with collagen in bone. We suggest that collagen mRNA regulation by miRNA could also be potentially important in bone metabolism.

SNP-SNP Interaction in Genes Encoding PD-1/PD-L1 Axis as a Potential Risk Factor for Clear Cell Renal Cell Carcinoma.

PD-1/PD-L1 axis plays an important role in maintaining homeostasis and prevention from autoimmunity; however, in the tumor microenvironment, PD-1/PD-L1 interaction is responsible for the evasion of immune surveillance by tumor cells. We therefore hypothesized that single nucleotide polymorphisms (SNPs) in genes encoding PD-1 and PD-L1 molecules are associated with the development and outcome of renal cell carcinoma (RCC). Here we genotyped nine polymorphisms: five of PDCD1: rs36084323G>A, rs11568821G>A, rs2227981C>T, rs10204525G>A, rs7421861T>C and four of PD-L1: rs822335C>T, rs4143815G>C, rs4742098A>G, rs10815225G>C in 237 RCC patients (including 208 with clear cell RCC (ccRCC)) and 256 controls, with application of allelic discrimination method with use of TaqMan Assays. Interestingly, we found the SNP-SNP interaction between rs10815225 and rs7421861 polymorphisms associated with ccRCC risk. The rs7421861 TC genotype decreased the risk of ccRCC development compared to TT and CC genotypes in the group of rs10815225 GC + CC individuals (OR = 0.21, CI95% = 0.08; 0.54). While possessing of rs10815225 GC or CC genotype increased susceptibility to ccRCC when compared to rs10815225 GG genotype in individuals with rs7421861 TT or CC genotype (OR = 2.40, CI95% = 1.25; 4.61). In conclusion, genetic variants in PDCD1 and PD-L1 genes, especially taken together as SNP-SNP interactions, can be considered to be ccRCC risk factors.

Major Histocompatibility Complex Genes as Therapeutic Opportunity for Immune Cold Molecular Cancer Subtypes.

Current immunotherapies are effective only in a subset of patients, likely due to several factors including defects in tumor cell antigen presentation, decreased response to immune effectors, and molecular heterogeneity of cancers. Recent molecular classifications enable the categorization of many tumor types. However, deregulation of major histocompatibility complex (MHC) gene expression is poorly characterized in the context of molecular cancer subtypes. To suppress the confounding effect of immune infiltrates on expression patterns of immunoregulators, we identified and removed genes with strong correlation to estimated immune compartment levels in each tumor type. Next, we reanalyzed a total of 13 TCGA cancer types encompassing 5651 tumors and 485 normal adjacent tissues by performing unsupervised clustering of 14 MHC genes. Subsequently, resultant clusters were statistically compared in terms of expression of other immune-related genes. Three MHC expression clusters were discovered by unsupervised clustering. We identified concordantly decreased expression of MHC genes (MHC-low) in 26 out of 55 molecular subtypes. Consequently, our study underlines the urgent need for designing strategies to enhance tumor MHC expression that could improve immune cold tumor rejection by cytotoxic T lymphocytes.

Transcriptomic Profiling for the Autophagy Pathway in Colorectal Cancer.

The role of autophagy in colorectal cancer (CRC) pathogenesis appears to be crucial. Autophagy acts both as a tumor suppressor, by removing redundant cellular material, and a tumor-promoting factor, by providing access to components necessary for growth, metabolism, and proliferation. To date, little is known about the expression of genes that play a basal role in the autophagy in CRC. In this study, we aimed to compare the expression levels of 46 genes involved in the autophagy pathway between tumor-adjacent and tumor tissue, employing large RNA sequencing (RNA-seq) and microarray datasets. Additionally, we verified our results using data on 38 CRC cell lines. Gene set enrichment analysis revealed a significant deregulation of autophagy-related gene sets in CRC. The unsupervised clustering of tumors using the mRNA levels of autophagy-related genes revealed the existence of two major clusters: microsatellite instability (MSI)-enriched and -depleted. In cluster 1 (MSI-depleted), ATG9B and LAMP1 genes were the most prominently expressed, whereas cluster 2 (MSI-enriched) was characterized by DRAM1 upregulation. CRC cell lines were also clustered according to MSI-enriched/-depleted subgroups. The moderate deregulation of autophagy-related genes in cancer tissue, as compared to adjacent tissue, suggests a prominent field cancerization or early disruption of autophagy. Genes differentiating these clusters are promising candidates for CRC targeting therapy worthy of further investigation.

Genetic testing in Poland and Ukraine: should comprehensive germline testing of BRCA1 and BRCA2 be recommended for women with breast and ovarian cancer?

PURPOSE: To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. METHODS: Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. RESULTS: We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). CONCLUSIONS: These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

In-silico analysis of Thr767Ile pathogenic variant in the MSH6 gene in family with endometrial cancer.

OBJECTIVE: To examine the mechanism of pathogenity of Thr767Ile variant on MSH6 protein. STUDY DESIGN: We describe a family diagnosed with endometrial cancer in two generations associated with variant in the MSH6 gene (p. Thr767Ile / c. 2300C>T) (rs587781462). MSH6 c. 2300C>T was associated with autosomal-dominant pattern of inheritance. MSH6 c. 2300C>T has pathogenic status in ClinVar and LOVD3 databases but it has never been described in context of hereditary endometrial cancer. We utilized a number of in-silico bioinformatic approaches using MSH6 protein sequence and structural information to assess influence of Thr767Ile on MSH6 properties. RESULTS: MSH6 Thr767 is highly conservative amino acid among various kingdoms of organisms. Thr767Ile was predicted deleterious and likely decreases affinity of MSH2-MSH6 complex to DNA but not affect interaction between MSH2 and MSH6. CONCLUSIONS: To the best of our knowledge, this is the first description of MSH6 T767I pathogenic variant that could be associated with a hereditary endometrial cancer. Bioinformatic analyses showed that T767I substitution most likely affects the MSH6 most important role, which is a DNA binding.