Non-Coding RNAs and the Development of Chemoresistance to Docetaxel in Prostate Cancer: Regulatory Interactions and Approaches Based on Machine Learning Methods.

Chemotherapy based on taxane-class drugs is the gold standard for treating advanced stages of various oncological diseases. However, despite the favorable response trends, most patients eventually develop resistance to this therapy. Drug resistance is the result of a combination of different events in the tumor cells under the influence of the drug, a comprehensive understanding of which has yet to be determined. In this review, we examine the role of the major classes of non-coding RNAs in the development of chemoresistance in the case of prostate cancer, one of the most common and socially significant types of cancer in men worldwide. We will focus on recent findings from experimental studies regarding the prognostic potential of the identified non-coding RNAs. Additionally, we will explore novel approaches based on machine learning to study these regulatory molecules, including their role in the development of drug resistance.

Evaluation of the Polygenic Risk Score for Alzheimer's Disease in Russian Patients with Dementia Using a Low-Density Hydrogel Oligonucleotide Microarray.

The polygenic risk score (PRS), together with the ɛ4 allele of the APOE gene (APOE-ɛ4), has shown high potential for Alzheimer's disease (AD) risk prediction. The aim of this study was to validate the model of polygenic risk in Russian patients with dementia. A microarray-based assay was developed to identify 21 markers of polygenic risk and ɛ alleles of the APOE gene. This case-control study included 348 dementia patients and 519 cognitively normal volunteers. Cerebrospinal fluid (CSF) amyloid-ß (Aß) and tau protein levels were assessed in 57 dementia patients. PRS and APOE-ɛ4 were significant genetic risk factors for dementia. Adjusted for APOE-ɛ4, individuals with PRS corresponding to the fourth quartile had an increased risk of dementia compared to the first quartile (OR 1.85; p-value 0.002). The area under the curve (AUC) was 0.559 for the PRS model only, and the inclusion of APOE-ɛ4 improved the AUC to 0.604. PRS was positively correlated with tTau and pTau181 and inversely correlated with Aß42/Aß40 ratio. Carriers of APOE-ɛ4 had higher levels of tTau and pTau181 and lower levels of Aß42 and Aß42/Aß40. The developed assay can be part of a strategy for assessing individuals for AD risk, with the purpose of assisting primary preventive interventions.

Alteration of Blood Immune Biomarkers in MCI Patients with Different APOE Genotypes after Cognitive Training: A 1 Year Follow-Up Cohort Study.

Many studies aim to detect the early phase of dementia. One of the major ways to achieve this is to identify corresponding biomarkers, particularly immune blood biomarkers. The objective of this study was to identify such biomarkers in patients with mild cognitive impairment (MCI) in an experiment that included cognitive training. A group of patients with MCI diagnoses over the age of 65 participated in the study (n = 136). Measurements of cognitive functions (using the Mini-Mental State Examination scale and Montreal Cognitive Assessment) and determination of 27 serum biomarkers were performed twice: on the first visit and on the second visit, one year after the cognitive training. APOE genotypes were also determined. Concentrations of EGF (F = 17; p = 0.00007), Eotaxin (F = 7.17; p = 0.008), GRO (F = 13.42; p = 0.0004), IL-8 (F = 8.16; p = 0.005), MCP-1 (F = 13.46; p = 0.0001) and MDC (F = 5.93; p = 0.016) increased after the cognitive training in MCI patients. All these parameters except IL-8 demonstrated a weak correlation with other immune parameters and were poorly represented in the principal component analysis. Differences in concentrations of IP-10, FGF-2, TGFa and VEGF in patients with MCI were associated with APOE genotype. Therefore, the study identified several immune blood biomarkers that could potentially be associated with changes in cognitive function.

Suicide-Related Single Nucleotide Polymorphisms, rs4918918 and rs10903034: Association with Dementia in Older Adults.

Dementia has enormous implications for patients and the health care system. Genetic markers are promising for detecting the risk of cognitive impairment. We hypothesized that genetic variants associated with suicide risk might significantly increase the risk of cognitive decline because suicide in older adults is often a consequence of cognitive impairment. We investigated several single-nucleotide polymorphisms that were initially associated with suicide risk in dementia older adults and identified the APOE gene alleles. The study was performed with subjects over the age of 65: 112 patients with dementia and 146 healthy volunteers. The MMSE score was used to assess cognitive functions. Study participants were genotyped using real-time PCR (APOE: rs429358, rs7412; genes associated with suicide: rs9475195, rs7982251, rs2834789, rs358592, rs4918918, rs3781878, rs10903034, rs165774, rs16841143, rs11833579 rs10898553, rs7296262, rs3806263, and rs2462021). Genotype analysis revealed the significance of APOEε4, APOEε2, and rs4918918 (SORBS1) when comparing dementia and healthy control groups. The association of APOEε4, APOEε2, and rs10903034 (IFNLR1) with the overall MMSE score was indicated. The study found an association with dementia of rs4918918 (SORBS1) and rs10903034 (IFNLR1) previously associated with suicide and confirmed the association of APOEε4 and APOEε2 with dementia.

Positive Effect of Cognitive Training in Older Adults with Different APOE Genotypes and COVID-19 History: A 1-Year Follow-Up Cohort Study.

(1) Background: Older people suffer from cognitive decline; several risk factors contribute to greater cognitive decline. We used acquired (COVID-19 infection) and non-modifiable (presence of APOE rs429358 and rs7412 polymorphisms) factors to study the progression of subjective cognitive impairment while observing patients for one year. Cognitive training was used as a protective factor. (2) Methods: Two groups of subjects over the age of 65 participated in the study: group with subjective cognitive decline receiving cognitive training and individuals who did not complain of cognitive decline without receiving cognitive training (comparison group). On the first visit, the concentration of antibodies to COVID-19 and APOE genotype was measured. At the first and last point (1 year later) the Mini-Mental State Examination scale and the Hospital Anxiety and Depression Scale were performed. (3) Results: COVID-19 infection did not affect cognitive function. A significant role of cognitive training in improving cognitive functions was revealed. Older adults with APOE-ε4 genotype showed no positive effect of cognitive training. (4) Conclusions: Future research should focus on cognitive dysfunction after COVID-19 in long-term follow-up. Attention to the factors discussed in our article, but not limited to them, are useful for a personalized approach to maintaining the cognitive health of older adults.

Platinum-based chemotherapy for pancreatic cancer: impact of mutations in the homologous recombination repair and Fanconi anemia genes.

Background: Mutations in homologous recombination (HR) and Fanconi anemia (FA) genes may predispose to pancreatic cancer (PC) and enable the prediction of sensitivity to platinum-based chemotherapy. FOLFIRINOX is a standard treatment option for non-selected PC patients and could be effective due to undiagnosed DNA repair deficiency. Here, we aimed to determine the frequency of mutations in genes involved in the HR and FA pathways, evaluate their clinical implications, and determine the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) of PC patients treated with platinum. Methods: We performed targeted DNA sequencing of 30 genes (ABRAXAS1, ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDKN2A, CHEK1, CHEK2, FANCC, FANCF, FANCG, FANCI, FANCL, FANCM, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, RAD52, RAD54B, RBBP8, RINT1, SLX4, and XRCC2) for 543 PC patients. Results: In BRCA/PALB2-mutated patients with advanced PC (33 patients, 6.1%), the PFS and OS were higher for first-line platinum therapy than for non-platinum therapy [PFS: HR = 0.28, 95% confidence interval (CI) = 0.10-0.81, p = 0.02; OS: HR = 0.31, 95% CI = 0.08-1.16, p = 0.08]. Among 93 patients (17.1%) with mutations in other HR/FA genes, no statistically significant difference in PFS and OS was observed between first-line platinum therapy and non-platinum therapy (PFS: HR = 0.83, 95% CI = 0.43-1.62, p = 0.59; OS: HR = 0.58, 95% CI = 0.28-1.22, p = 0.15). For patients with early PC, no prognostic value was observed for BRCA1/2, PALB2, or other HR/FA genes mutations. Moreover, a personal history of breast, ovarian, pancreatic, or prostate cancer was identified as the only independent predictor of the risk of BRCA/PALB2 mutations (HR = 5.83, 95% CI = 2.16-15.73, p < 0.01). Conclusion: Mutations in the BRCA1/2 and PALB2 genes increase the sensitivity of PC to platinum agents. Thus, alterations in these genes in PC patients must be determined prior to anticancer therapy.

The SDHD:p.H102R Variant Is Frequent in Russian Patients with Head and Neck Paragangliomas and Associated with Loss of 11p15.5 Region and Hypermethylation of H19-DMR.

Head and neck paragangliomas (HNPGLs) are rare neuroendocrine neoplasms derived from the parasympathetic paraganglia of the head and neck. At least 30% of HNPGLs are linked to germline mutations, predominantly in SDHx genes. In this study, we analyzed an extended cohort of Russian patients with HNPGLs using whole-exome sequencing and found a highly frequent missense variant p.H102R in the SDHD gene. We determined this variant in 34% of the SDHD mutation carriers. This variant was associated with somatic loss of the gene wild-type allele. Data from the B allele frequency method and microsatellite and microdeletion analysis indicated evident LOH at the 11p15.5 region and potential loss of the whole of chromosome 11. We found hypermethylation of H19-DMR in all tumors, whereas differential methylation of KvDMR was mostly retained. These findings do not support the paternal transmission of SDHD:p.H102R but are in agreement with the Hensen model. Using targeted sequencing, we also studied the variant frequency in a control cohort; we found SDHD:p.H102R in 1.9% of cases, allowing us to classify this variant as pathogenic. The immunohistochemistry of SDHB showed that the SDHD:p.H102R mutation, even in combination with wild-type allele loss, does not always lead to SDH deficiency. The obtained results demonstrate the frequent variant associated with HNPGLs in a Russian population and support its pathogenicity. Our findings help with understanding the mechanism of tumorigenesis and are also important for the development of cost-effective genetic screening programs.

The classification of bacterial survival strategies in the presence of antimicrobials.

The survival of bacteria under antibiotic therapy varies in nature and is based on the bacterial ability to employ a wide range of fundamentally different resistance mechanisms. This great diversity requires a disambiguation of the term 'resistance' and the development of a more precise classification of bacterial survival strategies during contact with antibiotics. The absence of a unified definition for the terms 'resistance', 'tolerance' and 'persistence' further aggravates the imperfections of the current classification system. This review suggests a number of original classification criteria that will take into account (1) the bacterial ability to replicate in the presence of antimicrobial agents, (2) existing evolutionary stability of a trait within a species, and (3) the presence or absence of specialized genes that determine the ability of a microorganism to decrease its own metabolism or switch it completely off. This review describes potential advantages of the suggested classification system, which include a better understanding of the relationship between bacterial survival in the presence of antibiotics and molecular mechanisms of cellular metabolism suppression, the opportunity to pinpoint targets to identify a true bacterial resistance profile. The true resistance profile in turn, could be used to develop effective diagnostic and antimicrobial therapy methods, while taking into consideration specific bacterial survival mechanisms.

TYMS 3'-UTR Polymorphism: A Novel Association with FOLFIRINOX-Induced Neurotoxicity in Pancreatic Cancer Patients.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy that has the worst 5-year survival rate of all of the common malignant tumors. Surgery, chemotherapy, and/or chemoradiation remain the main tactics for PDAC treatment. The efficacy of chemotherapy is often compromised because of the substantial risk of severe toxicities. In our study, we focused on identification of polymorphisms in the genes involved in drug metabolism, DNA repair and replication that are associated with inter-individual differences in drug-induced toxicities. Using the microarray, we genotyped 12 polymorphisms in the DPYD, XPC, GSTP1, MTHFR, ERCC1, UGT1A1, and TYMS genes in 78 PDAC patients treated with FOLFIRINOX. It was found that the TYMS rs11280056 polymorphism (6 bp-deletion in TYMS 3'-UTR) predicted grade 1-2 neurotoxicity (p = 0.0072 and p = 0.0019, according to co-dominant (CDM) and recessive model (RM), respectively). It is the first report on the association between TYMS rs11280056 and peripheral neuropathy. We also found that PDAC patients carrying the GSTP1 rs1695 GG genotype had a decreased risk for grade 3-4 hematological toxicity as compared to those with the AA or AG genotypes (p = 0.032 and p = 0.014, CDM and RM, respectively). Due to relatively high p-values, we consider that the impact of GSTP1 rs1695 requires further investigation in a larger sample size.

Microarray-based analysis of the BRAF V600 mutations in circulating tumor DNA in melanoma patients.

BACKGROUND: Circulating tumor DNA (ctDNA) holds great potential for cancer therapy and can provide diagnostic and prognostic information before and during treatment. METHODS: Plasma DNA samples from 97 melanoma patients, 20 healthy donors and 3 patients with benign skin tumors were analyzed by microarray analysis and droplet digital PCR (ddPCR). RESULTS: A microarray for simultaneous detection of six BRAF V600 mutations in ctDNA has been developed. The method allows the detection of 0.05% mutated DNA from WT DNA background. For paired samples (pre-surgery plasma and tumor tissue) isolated from 74 patients, the concordance of genotypes between tumor DNA and ctDNA was 65% (48/74). BRAF mutations in ctDNA were detected in 27/50 patients with BRAF-positive tumors and in 3/24 patients with BRAF wild-type tumors. The presence of ctDNA BRAF mutations in 23 plasma samples from melanoma patients undergoing therapy correlated significantly with tumor progression (P=0.005). The increase in cell-free DNA levels measured by ddPCR also correlated with disease progression (P=0.02). The concordance of results obtained by microarray identification of BRAF mutations and those obtained by ddPCR was 91%. CONCLUSION: The novel microarray-based approach can be a useful non-invasive tool for accurate identification of ctDNA BRAF mutations to monitor disease progression.

Melanoma arising in a Giant congenital melanocytic nevus: two case reports.

BACKGROUND: A giant congenital melanocytic nevus (GCMN) is found in 0.1% of live-born infants. If present, the lesion has a chance of about 6% to develop into malignant melanoma. Both children and adults can be affected by malignant melanoma arising in a giant congenital nevus. Up to 95% of GCMNs harbor NRAS mutations, and mutations in the BRAF, MC1R, TP53, and GNAQ genes have also been described. The individualization of therapy is required, but diagnostic and prognostic criteria remain controversial. CASE PRESENTATIONS: We report two cases: 1) melanoma arising in a giant congenital nevus during the first month of life complicated with neurocutaneous melanosis (NCM), and 2) melanoma arising in a giant congenital nevus during the first 6 months of life. Pathology, immunohistochemistry, and genetic analyses of tumor tissue were performed. The first case revealed only a non-pathogenic P72R polymorphism of the TP53 gene in the homozygote condition. For the second case, a Q61K mutation was detected in the NRAS gene. CONCLUSION: Malignant melanoma associated with GCMN is rare and therefore poorly understood. Outcomes have been linked to the stage at diagnosis, but no additional pathological prognostic factors have been identified. The most frequent genetic event in giant CMNs is NRAS mutations, which was discovered in one of our cases. To accumulate evidence to improve disease prognosis and outcomes, children with congenital melanocytic nevus should be included in a systemic follow-up study from birth.

Metformin increases antitumor activity of MEK inhibitor binimetinib in 2D and 3D models of human metastatic melanoma cells.

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.

Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis.

Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.

Autophagy inhibitors chloroquine and LY294002 enhance temozolomide cytotoxicity on cutaneous melanoma cell lines in vitro.

Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.

The \textit{BRAF} V600E mutation in single- institution study of Russian melanoma patients.

BACKGROUND: The activating mutation BRAF V600E is considered to be a diagnostic cutaneous melanoma (CM) marker important for prognosis and targeted therapy. OBJECTIVE: The aim of this study was to determine the frequency of the V600E mutation in CM patients in Russia and to estimate the influence of the BRAF gene mutation status on prognosis and clinical outcome. METHODS: To ensure mutation detection in FFPE tissue, interlaboratory validation was performed using three different methods: allele-specific hybridisation on a biochip, allele-specific real-time PCR and, in some cases, direct sequencing. RESULTS: Mutation V600E was detected in 49% of patients. The age of disease manifestation was significantly lower in mutated (MT) BRAF patients, and the median age difference between the wild-type (WT) and MT BRAF groups (P= 0.002) was 10 years. A tumour thickness more than 1 mm was also more frequently observed in the MT BRAF group (P= 0.059). Patients from the MT BRAF group were more likely to have ulceration compared to the WT group (P= 0.088). No statistically significant differences were found between the relapse-free, progression-free or overall survival of CM patients in the MT BRAF and WT BRAF groups. CONCLUSIONS: The data obtained show that the V600E BRAF mutation occurred in about half of melanoma patients; it was associated with earlier manifestation of melanoma and likely with more aggressive clinical features.

Sensitive genotyping of somatic mutations in the EGFR, KRAS, PIK3CA, BRAF genes from NSCLC patients using hydrogel biochips.

Targeted inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR gene and key effectors of the EGFR-signaling pathway (KRAS, BRAF, PIK3CA) are associated with sensitivity to these drugs. We developed a highly sensitive LUNG CANCER (LC)-biochip approach for the detection of the most common EGFR, KRAS, PIK3CA, and BRAF gene mutations. The locked nucleic acid clamp PCR technique was used to increase the sensitivity of the assay, then allele-specific hybridization of a fluorescently labeled target on a biochip was performed. To prove the feasibility of the approach, clinical samples from 112 patients with NSCLC were analyzed. A total of 14 EGFR (12.5%) mutations, 21 (18.8%) KRAS mutations, 12 (10.7%) PIK3CA mutations, and 1 BRAF mutation (0.9%) were found. We compared the results with those from direct sequencing. We detected 50 different mutations by the LC-biochip assay and only 33 of them were found by direct sequencing. To demonstrate that the LC-biochip assay did not give false-positive results, the 17 specimens with discordant results were subjected to locked nucleic acid clamp PCR followed by sequencing. The results of this analysis were identical to the results obtained by the LC-biochip assay indicating that the biochip-based assay was both accurate and reliable. This approach was able to detect approximately 0.5% of mutated alleles in wild-type DNA background. The biochip-based assay is a reliable and inexpensive method for the identification of NSCLC patients, who may respond to a specific targeted therapy.