Maxim Leonidovich Filipenko

Epithelial ovarian cancer, primarily high-grade serous ovarian cancer, is a tumor characterized by a high probability of recurrence following primary treatment, which consists of cytoreductive surgery combined with platinum-based chemotherapy. Despite standardized therapeutic approaches, the platinum-free interval, progression-free survival, and overall survival vary substantially. Beyond somatic tumor characteristics and the completeness of cytoreduction, a significant portion of this variability may be driven by germline single-nucleotide polymorphisms (SNPs) in genes encoding proteins responsible for drug transport, detoxification, and DNA repair. The objective of this review is to systematize data on the molecular mechanisms of platinum sensitivity and resistance in serous ovarian cancer, with an emphasis on the role of germline structural DNA variants (SNPs), discuss the limitations of existing data, and outline directions for future research. A literature search was performed in PubMed, Scopus and Google Scholar using the following keywords: “high-grade serous ovarian cancer”, “platinum”, “carboplatin”, “cisplatin”, “polymorphism”, “SNP”, “pharmacogenetics”, “progression-free survival”, and “platinum resistance”. Priority was given to studies with a sample size of ≥100 ovarian cancer patients and those with independent validation or a meta-analysis. A number of studies with smaller sample sizes were included due to the particular significance of their results and are specified in the text. The most reproducible associations with platinum resistance are demonstrated by SNPs in genes involved in nucleotide excision repair pathways (ERCC1, ERCC5), platinum transport and excretion (ABCB1), and specific signaling pathways (STAT3, FGFR4). Many candidate associations remain controversial owing to variations in the definition of platinum resistance, small sample sizes, heterogeneity in treatment regimens, and interethnic differences in allele frequencies and effects.

Atypical endometrial hyperplasia and endometrial adenocarcinoma are successive stages of malignant transformation of the uterine epithelium. Usually genomic instability and tumor mutation burden progressively increase during carcinogenesis. In case of endometrial cancer, the presence of a mutation in the exonuclease domain of the POLE gene and/or a DNA mismatch repair deficiency (dMMR) leads to genomic instability at early stages of tumor development. This paper describes a clinical case of a 49-year-old female patient with synchronous presence of several stages of the endometrium malignant transformation: glandular polyp without signs of atypia, atypical hyperplasia and highly differentiated endometrioid adenocarcinoma. In all substrates, the POLE p.Pro286Arg mutation was detected. In the glandular polyp sample, it was detected with a minimal variant allele frequency (1%). At the same time, in the tissue of atypical hyperplasia and adenocarcinoma, this mutation led to an explosive accumulation of oncogenic genetic variants. This observation indicates role of mutation in the POLE gene as an early driver of carcinogenesis and emphasizes the importance of its determination in atypical endometrial proliferations.

Introduction.Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine metabolism caused by pathogenic variants of the PAH gene. Allelic heterogeneity of the gene and the need for differential diagnosis of forms of hyperphenylalaninemia require molecular confirmation. Sanger sequencing remains valuable as an accessible and reliable method for variant verification and family analysis.The objectiveof the study was to develop and validate a unified protocol for PCR and Sanger sequencing of all coding exons of PAH with extended coverage of exon -intron boundaries.Materials and methods. A panel of 31 primers was developed to amplify 13 PAH exons, including overlapping amplicons for exon 13. Standardized PCR conditions (60 °C) were used. Validation was performed on five DNA samples, including a family trio. Sequencing was performed on an Applied Biosystems 3730xl.Results. The developed primer panel ensured reproducible and highly specific amplification of all target regions of the expected size. Sequencingdemonstrated high-quality chromatograms and complete coverage of the analyzed fragments, including overlapping regions. In the family trio, two pathogenic missense variants of the PAH gene in a state of compound heterozygosity were identified in the proband: c.728G>A (p.Arg243Gln) in exon 7 and c.1238G>C (p.Arg413Pro) in exon 12. Family analysis showed that the c.728G>A variant was paternally inherited, and c.1238G>C was maternally inherited. Additionally, a synonymous substitution c.735G>A was identified in the paternal allele. The established genotype fully corresponded to the clinical diagnosis of classical PKU and confirmed the diagnostic suitability of the developed approach.Conclusions. The proposed protocol is a reproducible, accessible and clinically informative method for molecular diagnostics of PKU and can be used both as a standalone tool and for validation of results obtained by other approaches.Keywords: phenylketonuria, hyperphenylalaninemia, exome sequencing, DNA mutation analysis.

The most common somatic mutations in solid tumors affect the TP53 gene, leading to the formation of the tumor-specific p53 antigen through overexpression of the wild-type antigen (WT-p53) or the generation of mutant-specific neoepitopes (MUT-p53). Thus, the p53 protein is a promising target for immunotherapy. This article summarizes clinical and immunological data on p53-targeting vaccine strategies. Vaccination against WT-p53 is shown to reproducibly induce p53-specific T-cell responses and has a favorable safety profile; however, its clinical efficacy as a monotherapy is limited. Key challenges include central immune tolerance to WT-p53 epitopes, low density of p53 peptide-HLA complexes on the tumor cell surface, an immunosuppressive tumor microenvironment, and HLA (HLA – human leukocyte antigens) heterogeneity. For mutant p53, specific immunogenic epitopes have been validated, yet phase II clinical trials of vaccines targeting frequent p53 mutations are still lacking. Future prospects for enhancing the efficacy of p53-targeted vaccines lie in combination strategies (e.g., with immune checkpoint inhibitors, adjuvants), precise patient stratification, and for mutant p53, the application of TCR-based (TCR – T-cell receptor) technologies and the development of personalized neoantigen selection platforms.

Bacterial poly(A) polymerases (PAPs) play an important role in RNA metabolism but remain poorly characterized outside Gammaproteobacteria. Here, we cloned and biochemically characterized the first PAP from Alphaproteobacteria, specifically from Marinobacter lipolyticus (Mli PAP). Using homology-based screening against E. coli PAP-1, we identified Mli PAP, sharing 54.8% sequence identity with its E. coli counterpart. The enzyme was expressed in E. coli but formed insoluble inclusion bodies; the active enzyme was purified as a fusion protein with the DsbA protein and used for functional assays. Mli PAP exhibited optimal activity at 30 °C and similar thermostability to E. coli PAP-1. ATP was the preferred substrate, with K_m comparable to E. coli PAP-1 (1.61 mM and 1.70 mM, respectively), and Mg²⁺ (10 mM) was identified as the optimal cofactor. Mli PAP displayed salt-dependent activity, with the most effective polyadenylation in KCl and inhibition by NaCl and ammonium salts, contrasting with the halophilic nature of its host. This study provides the first functional insights into PAPs from Alphaproteobacteria, broadening the understanding of PAP diversity and biochemical properties, as well as the potential applications of PAPs in biotechnology.

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF–mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF–mCherry did not bind Cu²⁺-charged IMAC resin, while moderate binding to Ni²⁺-charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production.

Введение. Опухоли, содержащие мутацию в кодоне 600 гена BRAF, демонстрируют чувствительность к ингибиторам BRAF-киназы. Молекулярно-генетическое тестирование данного гена является неотъемлемым компонентом диагностических мероприятий, применяемых в отношении меланом кожи, карцином толстой кишки, легкого, щитовидной железы, а также некоторых других разновидностей опухолей. Частота мутаций BRAF, наблюдаемая у российских пациентов с меланомой кожи, по данным различных публикаций, колеблется от 28 до 60 % — это указывает на высокую вероятность межлабораторных вариаций в процедуре тестирования. Цель. Оценка качества тестирования мутаций BRAF в различных лабораториях. Материалы и методы. Для организации контроля качества определения BRAF-статуса в меланомах была подготовлена панель из 15 образцов опухолей, которые продемонстрировали идентичные результаты молекулярно-генетического анализа в двух референсных лабораториях Межрегиональной общественной организации молекулярных генетиков в онкологии и онкогематологии (МОО ОМГО). Результаты. Панель была предложена 13 лабораториям, которые выразили предварительный интерес к участию в данном проекте. 10 лабораторий подтвердили согласие принять образцы в работу, четыре из которых продемонстрировали удовлетворительные результаты тестирования: одна лаборатория правильно определила статус BRAF во всех предоставленных образцах и три лаборатории ошиблись в одном из тестов. Не было зафиксировано ни одного ложноположительного результата. К основным причинам неудовлетворительных итогов относились несоблюдение заранее оговоренного двухнедельного срока тестирования (две лаборатории), три и более ложноотрицательных результата (три лаборатории) и отказ принять в работу более половины образцов вследствие недоступности процедуры диссекции опухолевых клеток (одна лаборатория). Выводы. По итогам данной инициативы, можно сделать вывод о наличии существенных межлабораторных вариаций в отношении качества молекулярно-генетических исследований. Необходимы дальнейшие усилия, которые позволят снизить риск ошибок при тестировании биологического материала на предмет наличия мутаций.

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry.

The endothelium, as the inner layer of the vascular wall, is in constant contact with blood components, so that leukocytes have the ability to adhere to endotheliocytes and penetrate to the subendothelial space. When studying heterogenic vascular samples containing endothelial cells or pathological processes related to inflammation within the endothelium, it may be necessary to distinguish DNA by endothelial and leukocyte origin, which is possible due to its specific epigenetic modifications. To identify CpG loci that could serve as markers for endothelial cells, we searched for their distinctive stable methylated or demethylated states by applying marginal filtering (selecting CpG loci with methylation Beta values closer to 0 and 1) to the microarray data and identified 47 CpG loci with relatively stable methylation/demethylation status that differentiate endothelial (HUVEC, HCMEC, HPAEC, HPMEC, and LSEC) DNA from leukocyte (granulocytes, monocytes, and lymphocytes) DNA. In addition, we compared CpG loci with high and low levels of DNA methylation between different types of endothelial cells and leukocytes. We believe that the obtained data will hopefully facilitate further studies on endothelial dysfunction.