The Effect of Inactivating Heterozygous Mutation in NBS1 Gene on DNA Damage and Repair Markers and Apoptosis Markers in Mice.

We studied the state of the DNA repair system and apoptosis in young mice carrying heterozygous inactivating mutation in the NBS1 gene (c.1971insT, p.Arg658Stop). In the peripheral blood cells of 4-month-old NBS1insT males, the %DNA in the comet tail was higher by 10% than in wild-type mice (wt) (p<0.05). In hepatocytes of NBS1insT mice, the proportion of γH2AX+ nuclear regions marking DNA double-strand breaks was lower by 2 times than in wt mice (p<0.05), which can be an indicator of less efficient DNA repair. In the kidney tissue of NBS1insT mice, a tendency towards the proapoptotic ratio of Bax and Bcl-2 protein markers was revealed against the background of their reduced expression. Thus, the disturbances detected NBS1insT mice in young age suggest that this model is promising for further studies of carcinogenesis.

Founder vs. non-founder BRCA1/2 pathogenic alleles: the analysis of Belarusian breast and ovarian cancer patients and review of other studies on ethnically homogenous populations.

The spectrum of BRCA1/2 mutations demonstrates significant interethnic variations. We analyzed for the first time the entire BRCA1/2 coding region in 340 Belarusian cancer patients with clinical signs of BRCA1/2-related disease, including 168 women with bilateral and/or early-onset breast cancer (BC), 104 patients with ovarian cancer and 68 subjects with multiple primary malignancies involving BC and/or OC. BRCA1/2 pathogenic alleles were detected in 98 (29%) women, with 67 (68%) of these being represented by founder alleles. Systematic comparison with other relevant studies revealed that the founder effect observed in Belarus is among the highest estimates observed worldwide. These findings are surprising, given that the population of Belarus did not experience geographic or cultural isolation throughout history.

Monitoring of the presence of EGFR-mutated DNA during EGFR-targeted therapy may assist in the prediction of treatment outcome.

The aim of our trial was to evaluate the prognostic significance of qualitative ctDNA analysis on different stages of EGFR mutated non-small cell lung cancer (NSCLC) treatment. We included 99 patients amendable for the first line treatment with either gefitinib/erlotinib (n = 87), afatinib (n = 10) or osimertinib (n = 2). Sequential qualitative analysis of ctDNA with cobas® EGFR Mutation Test v2 were performed before first dose, after 2 and 4 months of treatment, and on progression. Our analysis showed clinically significant heterogeneity of EGFR-mutated NSCLC treated with 1st line tyrosine kinase inhibitors (TKIs) in terms of progression-free and overall survival. When treated with conventional approach, i.e. monotherapy with TKIs, the patients falls into three subgroups based on ctDNA analysis before and after 2 months of treatment. Patients without detectable ctDNA at baseline (N = 32) possess the best prognosis on duration of treatment (PFS: 24.07 [16.8-31.3] and OS: 56.2 [21.8-90.7] months). Those who achieve clearance after two months of TKI (N = 42) have indistinguishably good PFS (19.0 [13.7 - 24.2]). Individuals who retain ctDNA after 2 months (N = 25) have the worst prognosis (PFS: 10.3 [7.0 - 13.5], p = 0.000). 9/25 patients did not develop ctDNA clearance at 4 months with no statistical difference in PFS from those without clearance at 2 months. Prognostic heterogeneity of EGFR-mutated NSCLC should be taken into consideration in planning further clinical trials and optimizing the outcome of patients.

CCND1 and FGFR1 gene amplifications are associated with reduced benefit from aromatase inhibitors in metastatic breast cancer.

PURPOSE: Endocrine therapy is a mainstay for the treatment of hormone receptor-positive breast cancer (BC); however, only a fraction of patients experience a pronounced response to antagonists of estrogen signaling. There is a need to identify predictors for efficacy of this treatment. METHODS: This study included 138 patients with newly diagnosed metastatic BC, who received upfront endocrine therapy. Archival biopsy specimens were tested for CCND1 and FGFR1 gene amplification and mRNA expression by PCR-based methods. RESULTS: CCND1 and FGFR1 amplification was detected in 24 (17.9%) and 28 (20.9%) of 134 evaluable cases, respectively; 9 carcinomas had concurrent alterations of these two genes. Presence of amplification in at least one locus was more common in tumors of higher grade (p = 0.018) and was associated with higher Ki-67 proliferation index (p = 0.036). CCND1 gene amplification was associated with shorter progression-free survival (PFS) in patients receiving aromatase inhibitors (AI) [16.0 months vs. 32.4 months, HR = 3.16 (95% CI 1.26-7.93), p = 0.014]. FGFR1 status did not significantly affect PFS of AI-treated women; however, objective response to AI was observed less frequently in FGFR1-amplified BC as compared to cases with normal FGFR1 copy number [2/15 (13.3%) vs. 22/46 (47.8%), p = 0.031]. Meanwhile, CCND1/FGFR1 gene status did not influence the outcome of tamoxifen-treated patients. CONCLUSION: Presence of CCND1 and/or FGFR1 amplification is associated with worse outcomes of AI therapy in patients with metastatic BC.

[Clinical and laboratory features of hereditary pheochromocytoma and paraganglioma].

The widespread introduction of genetic testing in recent years has made it possible to determine that more than a third of cases of pheochromocytomas and paragangliomas (PPPGs) are caused by germline mutations. Despite the variety of catecholamine-producing tumors manifestations, there is a sufficient number of clinical and laboratory landmarks that suggest a hereditary genesis of the disease and even a specific syndrome. These include a family history, age of patient, presence of concomitant conditions, and symptoms of the disease. Considering that each of the mutations is associated with certain diseases that often determine tactics of treatment and examination of a patient, e.g. high risk of various malignancies. Awareness of the practitioner on the peculiarities of the course of family forms of PPPGs will allow improving the tactics of managing these patients.The article provides up-to-date information on the prevalence of hereditary PPPGs. The modern views on the pathogenesis of the disease induced by different mutations are presented. The main hereditary syndromes associated with PPPGs are described, including multiple endocrine neoplasia syndrome type 2A and 2B, type 1 neurofibromatosis, von Hippel-Lindau syndrome, hereditary paraganglioma syndrome, as well as clinical and laboratory features of the tumor in these conditions. The main positions on the necessity of genetic screening in patients with PPPGs are given.

[Stereotactic photodynamic therapy for recurrent glioblastoma. Case report and literature review]. / Stereotaksicheskaya fotodinamicheskaya terapiya v lechenii retsidiva glioblastomy. Sluchai iz praktiki i obzor literatury.

We report a patient with recurrent glioblastoma in eloquent brain area. Stereotactic fluorescence biospectroscopy and stereotactic photodynamic therapy of tumor in opercular area of the left frontal lobe under neurophysiological monitoring were carried out. Literature data on this issue were analyzed.

[Epidemiology and diagnosis of mutations in the ALK gene in patients with non-small cell lung cancer in the Moscow region]. / Epidemiologiya i diagnostika mutatsii v gene ALK u patsientov s nemelkokletochnym rakom legkogo v Moskovskoi oblasti.

AIM OF STUDY: To determine a diagnostic algorithm for detecting translocation of the ALK gene and its frequency in the Moscow region. MATERIALS AND METHODS: During the priod between 2014 and 2018 (inclusive), 488 patients without activating mutations in the EGFR gene in the Moscow region were tested. To detect translocation of the ALK gene, fluorescence in situ hybridization (FISH) methods, an immunohistochemical method, and, in some cases, a polymerase chain reaction were used. RESULTS: Revealed ALK gene rearrangement in a population of patients with lung adenocarcinoma amounted to an average of 7.6% of cases. With this, the main method that we used was immunohistochemical method, applicable in more than 80% of cases. The use of other methods for verification of abnormalities in the ALK gene was found necessary in rare cases (3.3%). CONCLUSIONS: Using the algorithm presented in the article, it was possible to detect ALK gene rearrangement in a population of patients with lung adenocarcinoma in the Moscow region in an average of 7.6% of cases.

PCR-based detection of EGFR, ALK, KRAS and BRAF mutations in Russian patients with lung adenocarcinoma: a single-center experience.

In contrast to other countries with predominantly white populations, Russian smoking-related lung cancers (LC) are mainly squamous cell carcinomas and approximately half lung adenocarcinomas (AdCa) are not related to tobacco consumption. Given that smoking significantly influences the probability of presence of actionable mutations in LC, one would expect that Russian lung AdCa patients would differ from other white populations in distribution of EGFR, ALK, KRAS and BRAF mutations. Herein, 2,336 consecutive lung AdCa cases, including 1,203 patients with known smoking status, were subjected to sequential testing for the above mutations. One quarter of lung AdCa patients carried either EGFR or ALK mutation with combined prevalence of 42% in those who had never smoked but only 8% in smokers. There was only a moderate difference in KRAS mutation frequency between ever- and never-smokers in EGFR/ALK-negative cases (31% vs. 23%), and this was mainly attributed to increased prevalence of G12C substitution in the former group. The occurrence of BRAF V600E mutation was 1.7% and 4% in EGFR/ALK/KRAS mutation-negative ever- and never-smokers, respectively. ALK testing of 470 EGFR-mutated tumors revealed only 1 (0.2%) instance of translocation. Similarly, KRAS testing identified 1 (1.25%) mutation in 80 EGFR-mutated AdCa and none in 48 ALK-rearranged AdCa. Therefore, concurrent actionable mutations in lung adenocarcinoma are exceptionally rare and sequential gene testing can be regarded as a reliable option.

[Gemistocytic astrocytomas]. / Gemistotsitarnye astrotsitomy.

Gemistocytic astrocytomas (GA) are a variant of diffuse astrocytomas GII (WHO, 2016). Like all diffuse astrocytomas, GA recur with time, which is often accompanied by malignant degeneretion into the anaplastic astrocytoma GIII or to the secondary glioblastoma GIV. However, the progression-free survival and overall survival in patients with GA is less than in patients with diffuse astrocytomas. Given that this group of patients, according to the WHO classification (2016), is classified as GII, patients with GA usually do not receive comprehensive treatment. We have conducted a thorough analysis of research on this problem for the period from 1956 to 2017. Differences in the histological pattern, immunohistochemical and molecular-genetic profiles, survival of patients with GA and diffuse astrocytomas GII are shown there. A clinical case of a patient with transformation of a diffuse astrocytoma in GA (GIII) and then into a secondary glioblastoma is presented.

Spectrum of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies.

Distribution of cancer-predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.E1309Dfs*4 [n = 5] and p.Q1062fs* [n = 3]), while 6/26 (23%) mutations were novel (p.K73Nfs*6, p.S254Hfs*12, p.S1072Kfs*9, p.E1547Kfs*11, p.L1564X and p.C1263Wfs*22). Biallelic mutations in MUTYH gene were detected in 3/12 (25%) remaining subjects with polyposis and in 6/90 (6.7%) patients with colorectal cancer (CRC) carrying KRAS p.G12C substitution, but not in 231 early-onset CRC cases negative for KRAS p.G12C allele. In addition to known European founder alleles p.Y179C and p.G396D, this study revealed a recurrent character of MUTYH p.R245H germ-line mutation. Besides that, 3 novel pathogenic MUTYH alleles (p.L111P, p.R245S and p.Q293X) were found. Targeted next-generation sequencing of 7 APC/MUTYH mutation-negative DNA samples identified novel potentially pathogenic POLD1 variant (p.L460R) in 1 patient and known low-penetrant cancer-associated allele CHEK2 p.I157T in 3 patients. The analysis of 1120 healthy subjects revealed 15 heterozygous carriers of recurrent MUTYH mutations, thus the expected incidence of MUTYH-associated polyposis in Russia is likely to be 1:23 000.

[Specific features of drug sensitivity of hereditary cancers].

Until recently the detection of carriers of mutations in hereditary cancer genes was aimed almost exclusively to the detection of subjects-at-risk, and consequently, personalized monitoring and preventive actions. However, it was revealed several years ago that some hereditary cancers are characterized by unique biological features and, therefore, unusual spectrum of drug sensitivity. For example, BRCA1/2-associated cancers usually demonstrate somatic loss of the remaining gene allele, and, hence, tumor-specific defects of DNA repair of double-strand breaks. This mechanism determines increased sensitivity of BRCA1/2-related cancers to cisplatin, mitomycin C and PARP inhibitors. Cancers arising as a part of Lynch syndrome can be effectively treated by the modulators of immune response. Tumors in patients with tuberous sclerosis often regress after administration of mTOR inhibitors. For the time being, there is already about a dozen of drugs demonstrating specific activity towards certain categories of hereditary cancers.

[Processing of morphological material for molecular genetic tests].

Molecular genetic analysis has become a mandatory component of cancer diagnostics. Preanalytical step for DNA and RNA analysis is a complex process requiring tight interaction between surgeons, pathologists and molecular geneticists. This article discusses key aspects of handling of the tissues before DNA- and RNA-testing.

[Whole exome sequencing in oncology].

Whole exome sequencing (WES) has become a leading tool for genetic analysis right after its invention. This approach permits the detection of mutations spread within coding regions of the entire genome. For cancer patients WES is particularly effective for the search of hereditary cancer mutations and identification of somatically mutated druggable genes. Use of WES already resulted in significant advances in understanding for molecular mechanisms of cancer.

[CHEK2-associated hereditary breast cancer].

CHEK2 is classified as a moderate-penetrance gene for hereditary breast cancer (BC). In Russia, CHEK2 mutations hold second position in the list of BC-predisposing gene defects after BRCAl, and include CHEK2 1100deIC, de15395, and IVS2+lG>A gene-inactivating alleles. CHEK2-driven breast carcinomas are generally characterized by poor prognosis and low sensitivity to the conventional therapeutic regimens. CHEK2 testing needs to be incorporated into routine clinical practice owing its overt clinical significance.

[Acral lentiginous melanoma: the current state of the problem].

This review is aimed at the analysis the current state of acral lentiginous melanoma. This tumor has rather aggressive course and is the main cause of death in skin cancer patients. In Russia the incidence of melanoma during the period 2000-2010 increased from 3.18 to 3.95 cases per 100000 of population. The average annual growth rate was 1.99% and the total growth rate was 21.81%. Although skin melanoma is a visual tumor, more than one third of patients visit oncologists at advanced stages of the disease. Primary melanoma with localizations on the skin of fingers, interdigital spaces, soles, palms and nail plates is especially difficult for early diagnostics.

[Immunohistochemical and genetic profiles of melanomas with spindle cell morphology].

OBJECTIVE: to comparatively study the immunohistochemical profile and to analyze mutations in the BRAF and N-RAS genes. MATERIAL AND METHODS: The spindle cell melanomas taken from the Institute's archives were divided into 6 groups according to the results of clinical and morphological analyses and follow-up studies. Immunohistochemical examination was conducted in 58 cases, including 19 nodular spindle cell melanomas, 10 superficial spreading melanomas, 4 combined melanomas, 8 sarcoma- toid melanomas, 13 mixed desmoplastic melanomas, and 4 pure desmoplastic melanomas. RESULTS: All tumors of the spectrum in question expressed S100, SOX10, KBA.62, nestin, and cyclin D1. The rate of positive staining was 80% for MITF, 69% for PNL2, 61% for HMB45, 58% for Melan A, 36% for CD117, and 35% for SMA. The expression of HMB45 and Melan A was diffuse and marked in the groups of nodular and superficial spreading melanomas; sarcomatoid and mixed desmoplastic melanomas showed only scattered stained cells; pure desmoplastic melanomas were negative to these markers. SMA immunoexpression was observed in only sarcomatoid and desmoplastic types. Dual S100 staining showed a separate actin-positive myofibroblast-like population disappearing in more cellular zones. EMA, claudin 1, and DOG1 were negative in all cases. BRAFV expression was detected in 14% (in 2 nodular and 1 superficial spreading melanomas) and correlated with the presence of mutation. NRAS mutation was found in 1 nodular spindle cell melanoma. Desmoplastic melanomas did not harbor the above mutations. CONCLUSION: This study indicates the variant heterogeneity of spindle cell melanomas, as confirmed by clinical, morphological, immunohistochemical, and molecular examinations. The findings may be useful in the differential diagnosis of these tumors.

[Experience with the use of gefitinib in patients with inoperable non-squamous cell lung cancer with activating EGFR mutations].

Discovery of activating EGFR mutations led to dramatic modification of treatment schemes for nonsquamous lung cancer. 70 patients with activating EGFR mutations were treated by gefitinib being either a part of prospective phase II trial (n = 25) or, subsequently, subjected to routine clinical management (n = 45). Objective response rate approached to 32.7%. Median time to disease progression was 14 months, and median overall survival was 26.1 months. Subgroup analysis revealed statistically longer time to disease progression (p < 0,0001) and overall survival (p = 0,001) in latter vs. former group, despite the lower rate of objective response (22% vs 48%). Possible explanations include more relaxed standards for routine gefitinib use, i.e. inclusion of the patients with non-measurable tumor lumps, continuation of gefitinib uptake upon slow disease progression, and increasing availability and quality of radiosurgery for brain metastases.

[Assessment of applicability of archived cytological lung cancer specimens for molecular genetic analysis].

Molecular genetic analysis of lung tumors is often essential for the proper choice of therapy. EGFR mutation is a well-known marker of sensitivity to gefitinib, erlotinib and afatinib; ALK-translocations make tumor sensitive to several ALK inhibitors; low intratumoral expression of DNA repair genes (ERCC1, BRCA1, etc.) may increase the therapeutic index of platinum-based drugs. Usually these markers are evaluated using formalin-fixed paraffin-embedded tumor tissues. The goal of this work was to assess utility of archived cytological lung cancer specimens as an alternative source of material for molecular genetic testing. We analyzed paired histological and cytological lung adenocarcinoma specimens. Comparison of results within the pairs showed that cytological material can be used instead of histological material for qualitative analyses (detection of EGFR mutations or ALK-translocations); however, gene expression measurements, obtained by quantitative real-time PCR, may differ significantly in histological and cytological samples from the same patient.

Endocrine metabolic disorders in patients with breast cancer, carriers of BRCA1 gene mutations.

Two groups of breast cancer patients (53±2 years) in clinical remission receiving no specific therapy were examined: group 1, with BRCA1 gene mutations (N=11) and group 2, without mutations of this kind (N=11). The two groups did not differ by insulinemia and glycemia, insulin resistance index, blood levels of thyrotropic hormone, sex hormone-binding globulin, insulin-like growth factor-1, triglycerides, or lipoproteins. In group 1, blood estradiol level was higher. Intensive glucose-induced generation of reactive oxygen species in these patients was associated with a decrease of cholesterolemia, of the C-peptide/insulin proportion, and a trend to higher urinary excretion of 4-hydroxyestrone, one of the most genotoxic catecholestrogens. BRCA1 gene mutations in breast cancer patients were associated with signs of estrogenization and a pro-genotoxic shift in the estrogen and glucose system, which could modulate the disease course and requires correction.

Hereditary breast-ovarian cancer syndrome in Russia.

Hereditary breast-ovarian cancer syndrome contributes to as much as 5-7% of breast cancer (BC) and 10-15% of ovarian cancer (OC) incidence. Mutations in the "canonical" genesBRCA1andBRCA2occur in 20-30% of affected pedigrees. In addition toBRCA1andBRCA2 mutations, germ-line lesions in theCHEK2,NBS1, andPALB2genes also contribute to familial BC clustering. The epidemiology of hereditary breast-ovarian cancer in Russia has some specific features. The impact of the "founder" effect is surprisingly remarkable: a single mutation,BRCA15382insC, accounts for the vast majority ofBRCA1defects across the country. In addition, there are two other recurrentBRCA1alleles:BRCA14153delA andBRCA1185delAG. BesidesBRCA1, in Russia breast cancer is often caused by germ-line alterations in theCHEK2andNBS1genes. In contrast toBRCA1andBRCA2, theCHEK2andNBS1heterozygosity does not significantly increase the OC risk. Several Russian breast cancer clinics recently started to investigate the efficacy of cisplatin in the therapy ofBRCA1-related cancers; initial results show a unique sensitivity ofBRCA1-associated tumours to this compound.