Endometrial Cancer-Adjacent Tissues Express Higher Levels of Cancer-Promoting Genes than the Matched Tumors.

Molecular alterations in tumor-adjacent tissues have recently been recognized in some types of cancer. This phenomenon has not been studied in endometrial cancer. We aimed to analyze the expression of genes associated with cancer progression and metabolism in primary endometrial cancer samples and the matched tumor-adjacent tissues and in the samples of endometria from cancer-free patients with uterine leiomyomas. Paired samples of tumor-adjacent tissues and primary tumors from 49 patients with endometrial cancer (EC), samples of endometrium from 25 patients with leiomyomas of the uterus, and 4 endometrial cancer cell lines were examined by the RT-qPCR, for MYC, NR5A2, CXCR2, HMGA2, LIN28A, OCT4A, OCT4B, OCT4B1, TWIST1, STK11, SNAI1, and miR-205-5p expression. The expression levels of MYC, NR5A2, SNAI1, TWIST1, and STK11 were significantly higher in tumor-adjacent tissues than in the matched EC samples, and this difference was not influenced by the content of cancer cells in cancer-adjacent tissues. The expression of MYC, NR5A2, and SNAI1 was also higher in EC-adjacent tissues than in samples from cancer-free patients. In addition, the expression of MYC and CXCR2 in the tumor related to non-endometrioid adenocarcinoma and reduced the risk of recurrence, respectively, and higher NR5A2 expression in tumor-adjacent tissue increased the risk of death. In conclusion, tissues proximal to EC present higher levels of some cancer-promoting genes than the matched tumors. Malignant tumor-adjacent tissues carry a diagnostic potential and emerge as new promising target of anticancer therapy.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

Tumor and Cerebrospinal Fluid microRNAs in Primary Central Nervous System Lymphomas.

Primary central nervous system lymphoma (PCNSL) is a rare, highly aggressive, extranodal form of non-Hodgkin lymphoma, predominantly diagnosed as primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL). Fast and precise diagnosis of PCNSL is critical yet challenging. microRNAs, important regulators in physiology and pathology are potential biomarkers. In 131 patients with CNS DLBCL and with non-malignant brain lesions (n-ML), miR-21, miR-19b and miR-92a, miR-155, miR-196b, miR-let-7b, miR-125b, and miR-9 were examined by RT-qPCR in brain biopsy samples (formalin-fixed paraffin-embedded tissues, FFPET; CNS DLBCL, n = 52; n-ML, n = 42) and cerebrospinal fluid samples (CSF; CNS DLBCL, n = 30; n-ML, n = 23) taken for routine diagnosis. FFPET samples were split into study and validation sets. Significantly higher CSF levels of miR-21, miR-19b, and miR-92a were identified in PCNSL but not in n-ML, and differentiated PCNSL from n-ML with 63.33% sensitivity and 80.77% specificity. In FFPETs, miR-155 and miR-196b were significantly overexpressed and miR-let-7b, miR-125b, and miR-9 were downregulated in PCNSL as compared to n-ML. Combined miR-155 and miR-let-7b expression levels in FFPETs discriminated PCNSL and n-ML with a 97% accuracy. In conclusion, tissue miR-155, miR-196b, miR-9, miR-125b, and miR-let-7b expression profiles differentiate PCNSL from n-ML. PCNSL CSFs and the relevant biopsy samples are characterized by specific, different microRNA profiles. A logistic regression model is proposed to discriminate between PCNSL and non-malignant brain lesions. None of the examined microRNAs influenced overall survival of PCNSL patients. Further ongoing developments involve next generation sequencing-based profiling of biopsy and CSF samples.

Modifying impact of RET gene haplotypes on medullary thyroid carcinoma clinical course.

The clinical course of medullary thyroid carcinoma (MTC) associated with the MEN2A syndrome as well as of sporadic MTC shows considerable heterogeneity. The disease picture varies not only between the same RET proto-oncogene mutation carriers but also among sporadic MTC patients with no RET germinal mutations, which suggests the involvement of additional modulators of the disease. However, genetic factors responsible for this heterogeneity of the MTC clinical course still remain unknown. The aim of this study was to determine if polymorphic variants or specific haplotypes of the RET gene may modify the MTC clinical course. We genotyped the following loci: c.73+9277T>C, c.135G>A, c.1296A>G, c.2071G>A, c.2307T>C, c.2508C>T and c.2712C>G in 142 MTC patients and controls. We demonstrated considerable differences in the genotypes distribution within c.73+9277T>C, c.135G>A and c.2307T>C loci Our results show that the c.73+9277T variant associated with a decreased activity of the MCS+9.7 RET enhancer is rare in hereditary MTC patients with primary hyperparathyroidism, and thus, may influence the MTC clinical picture. The decreased activity of the RET promoter enhancer reduces RET expression level and may counterbalance the activating mutation in this gene. Frequent co-occurrence of the c.73+9277T allele with p.E768D, p.Y791F, p.V804M or p.R844Q RET mutations may be associated with their attenuation and milder clinical picture of the disease. Haplotypes analysis showed that C-G-A-G-T-(C)-C (c.73+9277T>C - c.135G>A - c.1296A>G - c.2071G>A - c.2307T>G - (c.2508C>T) - c.2712C>G) alleles combination predisposes to pheochromocytomas and primary hyperparathyroidism. We consider that RET haplotypes defining may become an auxiliary diagnostic tool in MTC patients.

Analysis of Newly Identified and Rare Synonymous Genetic Variants in the RET Gene in Patients with Medullary Thyroid Carcinoma in Polish Population.

Gain-of-function germline mutations of the RET proto-oncogene are responsible for initiation of carcinogenesis within the thyroid gland and development of hereditary form of medullary thyroid carcinoma and MEN2 syndrome. Genotype-phenotype correlations are established for most RET mutations, but the importance of the synonymous changes in this gene remains debatable. We aimed to analyze RET gene variants in Polish population. Genetic testing for the RET gene variants was performed with standard methods in 585 people aged 1-85, including 448 patients with medullary thyroid carcinoma and 131 of their first- and second-degree relatives, as well as six patients suspected of MTC/MEN2. Besides the most frequent synonymous changes, p.Leu769Leu, p.Ser836Ser, and p.Ser904Ser, four rare changes-c.1827C>T (p.Cys609Cys), c.2364C>T (p.Ile788Ile), c.2418C>T (p.Tyr806Tyr), and c.2673G>A (p.Ser891Ser)-were found in the RET gene, in the Polish population. Two of the rare changes, p.Cys609Cys and p.Ile788Ile, had not been previously described. The frequency of molecular synonymous variants in the general population was evaluated by testing 400 anonymous blood samples of neonates. Our findings may contribute to a better understanding of the genetic diversity of the RET gene and the involvement of synonymous variants in this diversity.

Changes in plasma miR-9, miR-16, miR-205 and miR-486 levels after non-small cell lung cancer resection.

PURPOSE: The majority of non-small cell lung cancer (NSCLC) patients presents with an advanced-stage disease and, consequently, exhibits a poor overall survival rate. We aimed to assess changes in plasma miR-9, miR-16, miR-205 and miR-486 levels and their potential as biomarkers for the diagnosis and monitoring of NSCLC patients. METHODS: Plasma was collected from 50 healthy donors and from NSCLC patients before surgery (n = 61), 1 month after surgery (n = 37) and 1 year after surgery (n = 14). microRNA levels were quantified using qRT-PCR. RESULTS: We found in NSCLC patients before treatment, both with squamous cell carcinoma (SQCC) and adenocarcinoma (ADC), significantly higher plasma miR-16 and miR-486 levels than in healthy individuals. Pre-treatment miR-205 concentrations were found to be significantly higher in SQCC than in ADC patients, and only SQCC patients presented significantly higher circulating miR-205 levels than healthy donors. SQCC plasma miR-9 levels were not different from normal control levels, but in ADC they were found to be significantly decreased. A combination of plasma miR-16, miR-205 and miR-486 measurements was found to discriminate NSCLC patients from healthy persons, with a specificity of 95% and a sensitivity of 80%. Following tumor resection, we found that the miR-9 and miR-205 levels significantly decreased, even below the normal level, whereas the increased miR-486 level persisted up to one year after surgery, and the miR-16 level decreased to normal. After tumor resection, none of the miR levels tested was found to relate to recurrence. CONCLUSIONS: Our data indicate that miR-9, miR-16, miR-205 and miR-486 may serve as NSCLC biomarkers. The observed cancer-related pre- and post-operative changes in their plasma levels may not only reflect the presence of a primary cancer, but also of a systemic response to cancer.

miR-7 expression in serous ovarian carcinomas.

BACKGROUND/AIM: miR-7 has recently been linked to cancer. Some miR-7 targets, including B-cell lymphoma 2 (BCL2) and epidermal growth factor receptor (EGFR), are involved in ovarian cancer (OC) pathogenesis. The majority of OCs display TP53 mutations, which are critically important for OC development. We aimed to study the expression level of miR-7 and of two of its postulated target genes, BCL2 and EGFR, in serous ovarian carcinomas of different TP53 status and tumour grade. MATERIALS AND METHODS: Gene and miR expression was assessed by real-time reverse transcription polymerase chain reaction in 45 clinical samples of low- (G1+G2) and high- (G3) grade primary serous OC with wild-type (wt) or mutated TP53, as well as in three OC cell lines, each representing a different TP53 status. The results obtained in patients with OC were analysed against their disease-free survival (DFS). RESULTS: In high-grade OC with TP53 mutations, the level of miR-7 expression significantly exceeded (by several fold) that in wtTP53 cancer (p<0.01). Within the wtTP53 tumour series, the level of miR-7 expression was significantly higher (by over 10-fold) in high-grade than in low-grade OC (p<0.01). miR-7 expression was not found to influence DFS. The differences in miR-7 expression depending on TP53 status found in clinical OC samples were not observed in OC cell lines. miR-7 overexpression correlated with diminished BCL2 expression, but there was no relationship between miR-7 and EGFR expression, neither in tumour samples nor in the cell lines. CONCLUSION: There is a link between miR-7 expression and TP53 status and tumour grade in serous OC. Molecular mechanisms of these relationships need to be elucidated. Of the two postulated miR-7 target genes we examined, BCL2, but not EGFR, seems to be a possible miR-7 target in OC.

The frequency of selected polymorphic variants of the RET gene in patients with medullary thyroid carcinoma and in the general population of central Poland.

The object of this work was to compare the frequency of three polymorphic changes in the RET proto-oncogene: L769L, S836S, and S904S in patients with medullary thyroid carcinoma (MTC; n = 246) and in the general population (n = 420 for single-nucleotide polymorphism [SNP] L769L and S904S; n = 411 for SNP 836). We tried to investigate how the harbored SNPs affect the age at onset of sporadic medullary thyroid carcinoma (sMTC) and MTC in carriers of known pathogenic mutations at codons 634 and 791 of the RET gene. A statistically significant difference was found in the frequency of the heterozygous change L769L in patients with sMTC (48.3%) and in unaffected individuals (39.5%). The presence of the polymorphic change L769L in the RET gene predisposes to the development of sMTC and also lowers the age of onset of MTC in carriers of the homozygous polymorphic variant L769L. The presence of this polymorphic change in MTC patients carrying, at the same time, the RET codon 634 mutation lowers the age of onset of MTC in this group.

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples.

MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 microg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.