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Background: More than 500 thousand new cases of malignant neoplasms are registered annually in the Russian Federation, of which more than 50 thousand new cases are due to hereditary forms. Improving the diagnosis of these diseases will make it possible to detect tumors at the early stages and take timely preventive and therapeutic measures. Aims: Creation of a database and development of a software for the NGS data analysis for the prevention and early diagnosis of hereditary forms of oncological diseases. Methods: The present study used 636 DNA samples obtained from cancer patients with a high hereditary risk or a burdened family history. DNA was isolated from blood lymphocytes. DNA libraries were prepared with a KAPA Target Enrichment Panel (Roche). The panel included probes for targeted enrichment of the coding region of 44 genes. NGS was performed on the MiSeq platform (Illumina). Results: We identified 65 pathogenic/ probably pathogenic nucleotide sequence variants in 96 patients in the ATM, BLM, BRCA1, BRCA2, CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH3, MSH6, MUTYH, PALB2, TP53 genes. We also identified 2858 nucleotide sequence variants of unknown clinical significance. Conclusions: We have created a local database that contains both genetic variants and clinical and anamnestic data. The database contains 4763 nucleotide sequence variants at the moment, among which 2522 are unique variants identified in a single patient.

The treatment of patients with advanced non-small-cell lung cancer (NSCLC) in recent years has been increasingly guided by biomarker testing. Testing has centered on driver genetic alterations involving the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangements. The presence of these mutations is predictive of response to targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs) and ALK TKIs. However, there are substantial challenges for the implementation of biomarker testing, particularly in emerging countries. Understanding the barriers to testing in NSCLC will be key to improving molecular testing rates worldwide and patient outcomes as a result. In this article, we review EGFR mutations and ALK rearrangements as predictive biomarkers for NSCLC, discuss a selection of appropriate tests and review the literature with respect to the global uptake of EGFR and ALK testing. To help improve testing rates and unify procedures, we review our experiences with biomarker testing in China, South Korea, Russia, Turkey, Brazil, Argentina and Mexico, and propose a set of recommendations that pathologists from emerging countries can apply to assist with the diagnosis of NSCLC.

Background. During the past 20 years, some large international studies have been conducted that evaluated the effectiveness of treatment programs for children with medulloblastoma. At the same time, in the standard risk group, fairly high rates of 5-year overall survival (OS) and event-free survival (EFS) were achieved, which amounted to 85% and 80%, respectively. At the present time some risk-adaptive therapeutic programs are developed according to molecular-biological features of tumor cells and possibility of chemotherapy and craniospinal radiation (CSI) therapy dose reduction. Materials and methods. From 2008 to 2018 fifty one pediatric patients with primary diagnosed medulloblastoma in the age group 318 years were included in trial, 38 in standard risk group, 13 in high risk group (without metastatic disease). Treatment program consisted of surgical removal of the primary tumor site with subsequent chemotherapy (with high-dose cyclophosphamide or thiophosphamide) and radiation therapy (with CSI of 23.4 Gy or 36 Gy, depending on the risk group). In order to detect morphological and molecular biological distinctive features of tumor cells, the following criteria were evaluated: histological variant, molecular subgroup, methyltransferase status by DNMT and MGMT proteins expression, presence of C-MYC/N-MYC gene amplification, Iso17q and TP53 gene mutation. Results. As a result of this study, sufficiently high rates of overall survival and progression/relapse-free survival (PRFS) were achieved in standard and high-risk groups patients, which amounted to 76.08.8% and 83.310.8% with median follow-up 62.96.2 months and 52.27.8 months, respectively. There was revealed patients group in the age 37 years with 100% PRFS and median follow-up 66.98.9 months. At the same time, morphological and molecular biological factors of an unfavorable outcome of the disease were absent in the tumor samples (large cell anaplastic histology, C-MYC/N-MYC gene amplification, Iso17q and TP53 gene mutation). We have also achieved 100% PRFS in patients with desmoplastic tumor histology and in patients, who were treated with thiphosphamide-based chemotherapy regimen. Molecular-biological characteristics analysis of tumor cells showed a negative effect on PRFS of DNMT-positive status (Score 4, by 3 markers) and presence of N-MYC gene amplification (SHH molecular subgroup). Conclusion. There was identified a group of patients aged 3 to 7 years, for whom the possibility for reducing of CSR dose down to 18 Gy opens. Understanding of tumor cells methyltransferase status creates the prerequisites for using of epigenetic demethylating therapy. It is necessary more observations to assess the effect of the chemotherapy regimen with high-dose thiophosphamide on the PRFS.

The treatment of patients with advanced non-small-cell lung cancer (NSCLC) in recent years has been increasingly guided by biomarker testing. Testing has centered on driver genetic alterations involving the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangements. The presence of these mutations is predictive of response to targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs) and ALK TKIs. However, there are substantial challenges for the implementation of biomarker testing, particularly in emerging countries. Understanding the barriers to testing in NSCLC will be key to improving molecular testing rates worldwide and patient outcomes as a result. In this article, we review EGFR mutations and ALK rearrangements as predictive biomarkers for NSCLC, discuss a selection of appropriate tests and review the literature with respect to the global uptake of EGFR and ALK testing. To help improve testing rates and unify procedures, we review our experiences with biomarker testing in China, South Korea, Russia, Turkey, Brazil, Argentina and Mexico, and propose a set of recommendations that pathologists from emerging countries can apply to assist with the diagnosis of NSCLC.

e21573 Background: Mutation in BRAF is a crucial biomarker for treatment choice in patient with locally advanced or metastatic melanoma. In patient with BRAF wild-type (WT) melanoma aPD1 is the preferred treatment regimen. In patients with BRAF V600 mutant melanoma combined targeted therapy (cTT) could also be chosen as adjuvant treatment, which significantly improves OS. Methods: We conducted a retrospective continuous study of all melanoma patients, who were tested for BRAF mutation by RT-PCR in 2020 (COIVD-19 pandemic year) in our institution and analyzed adjuvant therapy options they received. Patient demographics, mutational status, disease stage, treatment regimens and treatment outcomes were collected from electronic medical records. Results: Between January 2020 and December 2020, tumor samples from 297 pts were tested for BRAF mutations. The mean pts age was 58.2 years (95% CI 51.2 to 60.3) and 130 (43.7%) were male. BRAF mutations were detected in 147 (49.5%) patients. Most common mutation was V600E (140 out of 147 samples, 88.4%) and V600K (13 out of 147, 8.84%). We also found K601N, K601E, L597R and V600M mutations (in one sample each). Among 150 BRAF wild type samples 44 were also tested for NRAS mutations and in 13 (29.54%) pts NRAS mutations were detected: Q61R – in 6 (46.15%) pts, Q61L – in 2 (15.38%) pts, Q61K – in 3 (23.08%) pts, G12E – in 1 (7.69%) pt, and G12D - in 1 (7.69%) pt . Also among 150 BRAF wild type samples 56 were tested for KIT mutations and only one sample were found to harbour D186V mutation . Among BRAF-positive pts at time of the genetic test performed stage I CM had 11 (7.48%) out of 147 pts, stage II - 13 (8.84%) pts, stage III - 67 (45.57%) pts, and stage IV - 43 (29.25%) pts. In 13 (8.84%) pts stage could not be clearly identified. In most cases (32 out of 67, 47.76%) monotherapy with aPD1 was given as adjuvant treatment in stage III pts where as only 6 (8.95%) patients received cTT, 7 pts (10.41%) received IFN-alfa and 22 pts (32.84%) received no or were given with other treatment (Table). Median DFS did not differ significantly between treatment cohorts. Conclusions: In our retrospective study we found that among stage III BRAF-mutant patients monotherapy with aPD1 was the most common adjuvant treatment regimen. In some patients with stage III disease IFN-alfa is still given. More studies are needed to define best adjuvant treatment for BRAF mutant stage III melanoma pts.[Table: see text]

The aim of this study was to identify a group of patients aged 3 to 7 years for whom there is the possibility for reducing of craniospinal radiation dose (CSI). From 2008 to 2018 fifty one pediatric patients with primary diagnosed medulloblastoma in the age group 3 - 18 years were included in trial, 38 in standard risk group, 13 in high risk group. Treatment program consisted of surgical removal of the primary tumor site with subsequent radiation therapy (with CSI of 23,4 Gy or 36 Gy, depending on the risk group) and high-dose chemotherapy (with high-dose cyclophosphamide or thiophosphamide). As a result of this study, sufficiently high rates of overall survival and progression/relapse - free survival (PFS) were achieved in standard and high-risk groups patients, which amounted to 76,0 ± 8,8% and 83,3 ± 10,8% with median follow-up 62,9 ± 6,2 months and 52,2 ± 7,8 months, respectively. There was revealed patients group in the age 3 - 7 years with 100% PFS and median follow-up 66,9 ± 8,9 months. Morphological and molecular biological factors of an unfavorable outcome of the disease (large cell - anaplastic histology, MYC/MYC-N gene amplification, Iso17q and TP53 gene mutation) were absent in this tumor samples. We have also achieved 100% PFS in patients with desmoplastic tumor histology and in patients, who were treated with thiphosphamide - based chemotherapy regimen. Molecular - biological characteristics analysis of tumor cells showed a negative effect on PFS of DNMT - positive status (Score 4 and>, by 3 markers) and presence of MYC-N gene amplification (SHH molecular subgroup).

From 2008 to 2018 forty nine patients with de novo metastatic medulloblastoma were included in trials. Median age is 7.8 years (range, 4 – 17). Children underwent adjuvant therapy arm A (like-SJMB03, n = 14) from 2008 to 2014: craniospinal radiation (CSI) 36Gy + local radiation to the tumor bed – up to 54Gy (daily fraction 1.8Gy), 4 cycles (vincristine/HD-cyclophosphamide/cisplatin) with auto stem cell rescue; arm B (n = 19) from 2014 to 2018 – induction chemotherapy (vincristine/HD-cyclophosphamide/cisplatin/etoposide +/- HD-methotrexate), CSI 24.4 (without C-MYC/N-MYC/Iso17q), other 36Gy + local - up to 54Gy, 2 cycles thiophosphamide/carboplatin +/- 5-azacitidine, with auto stem cell transplantation; arm C (n = 16) – individual treatment program (including CSI 23.4-36Gy and HD-chemotherapy). R1 status was revealed in 24 patients (49%), M2/3 in 41 (83.7%), large-cell anaplastic histological variant in 8 (16.3%), MGMT protein expression in 23 (69.7%), iso17q in 20 (48.8%), N-MYC amplification in 5 (13.5%), C-MYC amplification in 2 (4.3%). Disease-free survival (DFS) was 38.5 ± 13.5% in arm A, 65.6 ± 19.9% in B, 28.6 ± 13.8% in C (p = 0.008), a median follow-up 54.7 ± 10.2 months (A), 24.3 ± 4.2 (B), 11.9 ± 3.4 (C). DFS was 16.7 ± 14.6% in group with MGMT-positive tumor; 23.2 ± 13.8% - with iso17q-positive tumor; 33.3 ± 25.5% - with C-MYC/N-MYC-positive tumor; with CSI 23.4 Gy 73.3 ± 11.4% (a median follow-up 24.5 ± 6.4 months), 90.9 ± 8.7% (arm B, a median follow-up 28.3 ± 6.2 months), with CSI 36 Gy 29.8 ± 10.8% (a median follow-up 32.7 ± 5.7 months), p = 0.320. 9 events were revealed in arm A, 6 in B, 11 in C (p = 0.055), relapses in 12 cases, progression in 7. Treatment related mortality was 16.3% (8 children, 5 - due to septic complications, 3 – other toxicity). Chemotherapy intensification could help us to reduce CSI dose down to 23.4 Gy in some patients with metastatic disease and C-MYC/N-MYC/Iso17q - negative tumor.

e15549 Background: Role of metastasectomy in pts with mBRAF metastatic CRC is still controversial. We performed analysis of prospective multicentric database of pts with mBRAF mCRC to evaluate the efficacy of metastasectomy in such group of pts in the real clinical practice. Methods: We analyzed a database of pts with mCRC in 7 cancer clinics in Russia and chose pts with metastasectomy with different mutational status. The primary endpoints were disease free survival (DFS) and overall survival (OS), which were calculated from the time of metastasectomy. Analysis was performed with the SPSS v.20 software package. Results: The study included 126 pts: 26 pts with mBRAF, 57 pts with mRAS and 43 pts with wtRAS/BRAF. Pts with mBRAF more often had synchronous metastases (50%/19,3/11,6%, p

Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ∼5% and 15%–20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ∼10-fold increase in the detection sensitivity for mutant BRAF and NRAS genes was achieved.

The aim of this study was to estimate treatment toxicity and event-free survival (EFS) according to therapeutic program, MYC/MYC-N gene amplification and MGMT/DNMT (1, 3a, 3b) proteins expression in tumor cells. From 2016 to 2018 twenty four patients were included in trial. Children underwent adjuvant therapy: craniospinal radiation (CSI) or local radiation therapy (RT) to the relapsed site up to 23.4Gy with 5-azacytidine, 2 cycles methotrexate/5-azacytidine/cisplatin/etoposide, 3 cycles 5-azacytidine/temozolomide - for relapsed group (arm A, n = 5); for patients with de novo medulloblastoma: arm B, n = 11 – vincristine/cyclophosphamide/cisplatin/etoposide (OPEC) - based induction, CSI 36Gy + local RT to the tumor bed up to 54Gy with 5-azacytidine, 1 cycle OPEC and 2 cycles thiophosphamide/carboplatin with auto stem cell transplantation (auto-SCT); arm C, n = 8 – cyclophosphamide/cisplatin - based induction, CSI 23.4 Gy followed by 2 cycles 5-azacytidine/thiophosphamide/carboplatin with auto-SCT, local RT with 5-azacytidine. The combination of 5-azacytidine with local RT or temozolomide was safety and tolerability. Arm C was discontinued due to severe gastrointestinal grade 3/4 toxicity, hemorrhagic syndrome after combination of 5-azacytidine with thiophosphamide/carboplatin. EFS was 0% in arm A, 53.0 ± 15.5%, 50.0 ± 17.7% in arms B and C, a median follow-up 8.8 ± 1.1 months (arm A), 18.8 ± 2.5 months (arm B), 25.0 ± 4.4 months (arm C). Addition of 5-azacytidine to RT or chemotherapy did not improve EFS of patients with MYC/MYC-N gene amplification positive tumor. There was not determined any prognostic significance of MGMT/DNMT (1, 3a, 3b) proteins expression in this cohort.

The aim of this study was to estimate MGMT protein expression and MGMT gene promoter methylation in medulloblastoma specimens and their prognostic significance. Fifty four patients were treated according to like-SJMB03, MTX-OPEC-based, thiophosphamide/carboplatin-based chemotherapy regimens combined with radiation therapy (average risk group - 22, high risk group - 32). MGMT protein expression and molecular subgroups were assessed by immunohistochemical method. MGMT positive specimen was defined as weak (1+), moderate (2+), strong (3+) colouring of the tumor nuclei and cytoplasm with number of positive tumor cells above 25%. MGMT gene promoter methylation was detected by methylation-specific PCR (MSP) method. MGMT positive tumor cells were revealed in 31 out of (/) 54 specimens (57.4%), 2/10 (20%) - for infants, 29/44 (65.9%) - for children (p = 0.08); MGMT2,3+ - in 13/54 (24%), 0/10 (0%) – for infants, 13/44 (29.5%) – for children (p = 0.049), MGMT gene promoter methylation - in 1/15 (6.7%). MGMT positive endotheliocytes were observed in 25/42 (59.5%). MGMT positive tumor cells and endotheliocytes were revealed in 21/27 (77.8%), in 10/12 (83.3% - for MGMT2,3+). MGMT2,3+ positive tumor cells were detected in 1/12 (8.3% - SHH), 5/12 (41.7% - Group 3), 4/10 (40% - Group 4), p = 0.137. There was not determined any significant correlation between MGMT expression and M0/M+ status, overall survival, event-free survival. These data suggest that the presence of MGMT expression in tumor cells and endotheliocytes opens the way for using demethylating therapeutic agents in patients with Group 3 and Group 4 medulloblastoma.

e15035 Background: m BRAF mCRC has the aggressive phenotype. The incidence of such mutation in Europe and the USA is around 8-14%, in Asian countries - 4-8%. The purpose of this population-based study was to determine the incidence and identifying prognostic factors in pts with mBRAF mCRC in Russia. Methods: A multicenter retrospective analysis of clinical data and treatment results of pts with mBRAF mCRC was performed. The main method for determining mutations was a PCR. The main efficacy endpoint was progression free survival (PFS) at the 1st line. Multivariate analysis was performed using Cox regression model. Results: 437 out of 8646 pts (5%) with a known mutational status had m BRAF (V600E). Clinical data were collected from 119/437 (27.2%): female - 65.5%, average age - 60 years (28-86), MSI-H -10%; the right-sided primary tumor – in 65%, left-sided – in 17%, rectum – in 18%; the primary tumor was removed in 76%; adjuvant chemotherapy was administered in 30%; lung metastases – in 15 %, liver - 45%, peritoneal metastases – in 38%; metastasectomy was performed in 13% pts. The first line was administered in 86 (72%) pts: FOLFIRI / XELIRI - 17 (20%), FOLFOX / XELOX - 50 (58%), FOLFOXIRI - 12 (14%), monotherapy of fluoropyrimidines – in 7 (8%). Bevacizumab was added to chemotherapy at 1st line in 25 (29%) patients, anti-EGFR – in 8 (9%) pts. PFS at the 1st line was 7 months: XELOX / FOLFOX - 7, FOLFOXIRI - 7, FOLFIRI / XELIRI - 6 and fluoropyrimidines - 2 months (HR 0.9, 95% CI 0.6-1.1, p = 0.3). None of the clinical or morphological factors except the presence of metastases in the retroperitoneal lymph nodes (HR 2.6, 95% CI 1.3-5.4, p = 0.006) did not have an independent negative prognostic value. Conclusions: In contrast to Western countries the incidence of mBRAF gene in the population of pts with mCRC in the Russia is low and we found a high incidence of localization of the primary tumor in the rectum. We didn’t reveal any prognostic factors except metastases in the retroperitoneal lymph nodes, and didn’t reveal any differences between the usual duplets and standard regimen for such mutation - FOLFOXIRI in term of 1st line PFS. This suggests we need a prospective randomized study to determine the optimal regimen of chemotherapy at 1st line for mBRAF mCRC pts.

e15026Background: The concordance of KRAS mutation status between the PT of CRC and synchronous metastases is 95%. However, there were reports showing that KRAS mutation status in metastases may ch...