Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.

Background: The SARS-CoV-2 pandemic introduced a global distraction effect in cancer patients' care. The aim of this study was to explore the effect of the pandemic on the largest molecular diagnostics center for cancer patients and high-risk individuals in Serbia.Research design and methods: EGFR, KRAS/NRAS, BRAF, and BRCA1/2 mutation testing were performed by qPCR and NGS. NGS was used for panel testing of hereditary breast/ovarian cancer and cancers associated with Lynch syndrome. The analytical output during the state of emergency (SoE) was compared to the period before and after the outbreak using one-way ANOVA. Statistical significance was set at p < 0.05.Results: A 38% reduction in the number of analysis was detected during the SoE. After the SoE, a 19% reduction was noted compared to SoE and 50% compared to the period before the SoE (p = 0.038). Three of the 48 scheduled appointments for pretest genetic counseling were carried out during the SoE, but the number of NGS tests increased by 50%.Conclusions: The SARS-CoV-2 pandemic had a profound negative effect on the diagnostic output of our centralized molecular diagnostics center. The only positive effect was shortening of waiting lists for hereditary cancer patients and high-risk individuals.

Expression levels of genes involved in cell adhesion and motility correlate with poor clinicopathological features of epithelial ovarian cancer.

PURPOSE: Changes in the expression levels of genes involved in cancer cell adhesion and motility have been reported to have an important role in tumor progression. In this study, we aimed to investigate the clinical significance of ITGAV and CALD1 gene expression in epithelial ovarian cancer (EOC), the most lethal gynecological malignancy. METHODS: Reverse transcription quantitative polymerase chain reaction was used to evaluate ITGAV and CALD1 expression levels in 47 EOC and 19 benign formalin-fixed paraffin-embedded samples. We used Spearman's test to determine the association between ITGAV and CALD1 expression and Wilcoxon test to compare expression levels between malignant and benign ovarian tumor specimens as well as to determine their association with clinicopathological characteristics of EOC. Survival analysis was done by the Kaplan-Meier method and the log-rank test. P ≤ 0.05 was considered statistically significant. RESULTS: CALD1 and ITGAV showed significantly lower expression in EOC than in benign ovarian samples (p<0.001). Furthermore, CALD1 was significantly lower expressed in high-grade tumors (p=0.037) while there was a trend for a lower expression of ITGAV in tumors with high histological grade (p=0.043), in tumors with ascites (p=0.055), and in tumors of patients who relapsed (p=0.083). We also found a significant positive association between ITGAV and CALD1 expression (ρ=0.640, p<0.001) in EOC samples. Kaplan-Meier analysis showed no significant impact of ITGAV and CALD1 expression levels on overall survival of EOC patients (p=0.149 and p=0.430, respectively). CONCLUSION: Our findings indicate that CALD1 and ITGAV gene expression levels correlate with poor clinicopathological features of the EOC.

EGFR mutation testing from liquid biopsy of non-small cell lung cancer at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Resistance to tyrosine kinase inhibitors (TKIs) in lung cancer often occurs, so mutation testing from liquid biopsy is the method of choice as a minimally invasive approach that quickly provides information for additional therapeutic options. The purpose of this study was to assess the success rate and usefulness of EGFR testing from liquid biopsy at the Institute for Oncology and Radiology of Serbia (IORS). METHODS: EGFR mutation testing was performed by real-time qPCR in 4750 tumor samples using the Cobas® EGFR Mutation Test v2. EGFR testing from 104 liquid biopsy samples was used to track the resistance on first-line EGFR-TKIs as well as for initial testing of 124 patients without tissue biopsies. RESULTS: Liquid biopsy samples were tested in cases with inadequate material for DNA isolation or without tissue biopsy at diagnosis. Nine mutated samples were detected (7.3 %) with a 99.2 % testing success rate. Testing liquid biopsy samples of patients who progressed on EGFR-TKIs showed an accordance rate of 67% with driver mutations, and 49% of mutated patients had the T790M mutation which rendered them eligible for third-generation EGFR-TKIs. An additional 5 patients tested EGFR wild type from plasma after progression were rebiopsied and 3 of them had the T790M mutation. CONCLUSIONS: EGFR mutation testing from liquid biopsy has been successfully implemented in Serbia and has proven invaluable for detecting molecular resistance mechanisms to EGFR-TKIs and as an alternative sample source for patients with scarce biopsy material or without any at all.

Discovery of the Biginelli hybrids as novel caspase-9 activators in apoptotic machines: Lipophilicity, molecular docking study, influence on angiogenesis gene and miR-21 expression levels.

In order to investigate potential therapeutically agents, novel products of Biginelli reaction (4a-l) were synthesized and exposed to cytotoxic and caspase activities, angiogenesis, cell cycle distribution, gene and microRNA expression levels, lipophilicity assessment and docking study. Among the twelve novel compounds (4a-l) evaluated for the cytotoxic activity, five of them (4c, 4d, 4f, 4k and 4l) that showed excellent activity on the tested cell lines (HeLa, LS174 and A549) were selected for further evaluation. Interestingly, compound 4f has up to three times higher selectivity index (SI) towards cancer cells than cisplatin (on HeLa, LS174 and A549 SI = 18.2, 13.5 and 11.2, respectively). The obtained results from cell cycle distribution and caspase activity indicate that tested compounds (4c, 4d, 4f, 4k and 4l) promoted caspase-9 activation, implicated in the intrinsic pathway of apoptosis. Lipophilicity of 4a-l was determinate by using reversed-phase high-performance liquid chromatography.

Germline mutations in cancer susceptibility genes in high grade serous ovarian cancer in Serbia.

Clinical criteria for genetic testing of genes other than BRCA1/2 in epithelial ovarian cancer (EOC) still do not exist. We assessed the frequency and predictors of deleterious mutations in 19 cancer predisposition genes in high-grade serous ovarian cancer (HGSOC) in Serbia. Next-generation sequencing was used to identify germline mutations in the whole coding regions of a gene panel. Patients' characteristics and sequencing data were summarized with descriptive statistics and compared using chi-square test. Among 131 HGSOC patients, 23 had BRCA1 (17.6%) while 5 had BRCA2 (3.8%) mutation. In addition, 9 (6.9%) pathogenic mutations were detected in other genes including BRIP1 (n = 2;1.5%), CHEK2 (n = 2;1.5%), NBN (n = 3;2.3%) and RAD51C (n = 2;1.5%). Factors that predicted for BRCA1/2 mutations were: breast and ovarian cancers in the same patient (p = 0.031), young age of EOC (p = 0.029), menstrual status (p = 0.004) and family history of cancer (p < 0.0001). However, these factors did not predict for mutations in other cancer susceptibility genes. Applying established referral criteria for genetic testing in Serbia will help identify BRCA1/2 mutation carriers but will not help identify mutations in other cancer susceptibility genes. Until better predictors emerge we should be performing wider genetic testing of EOC in order to identify all mutation carriers.

TP53 and DNA-repair gene polymorphisms genotyping as a low-cost lung adenocarcinoma screening tool.

AIM: TP53 and DNA repair polymorphisms have been proposed as cancer risk factors. This study evaluated the usability of TP53 Arg72Pro single-nucleotide polymorphism, X RCC1 Arg399Gln and RAD51 G135C as a low-cost lung adenocarcinoma screening tool. PATIENTS AND METHODS: This case-control study included 78 atients with lung adenocarcinoma and 79 healthy matched controls. TP53, XRCC1 and RAD51 genotyping was done by PCR followed by restriction length polymorphism. Descriptive analyses included genotype and allelic frequencies and deviations of the frequencies from those expected under Hardy-Weinberg equilibrium were assessed using the χ2 test. The OR and 95% CIs were calculated as an estimate of relative risk, with significance set at p value <0.05. RESULTS: The TP53 codon 72 Pro allele and the XRCC1 codon 399 Arg allele in a homozygous state were associated with lung adenocarcinoma (p=0.037; OR (95% CI) 2.42 (1.10 to 5.31)), that is, p=0.037; OR (95% CI) 2.16 (1.08 to 4.33), respectively. Also, carriers of the TP53 codon 72 Pro allele and the XRCC1 codon 399 ArgArg genotype older than 50 showed an even higher risk of developing lung adenocarcinoma (p=0.03 in both cases). CONCLUSIONS: The TP53 codon 72 Arg allele and XRCC1 codon 399 Gln allele are likely to have a protective effect against lung adenocarcinoma, especially in individuals older than 50 years of age. XRCC1 and TP53 genotyping might be a useful low-cost tool for evaluating individual lung cancer risk, leading to earlier detection and management of this disease.

Novel BRCA1 splice-site mutation in ovarian cancer patients of Slavic origin.

Mutations in breast cancer susceptibility gene 1 (BRCA1) lead to defects in a number of cellular pathways including DNA damage repair and transcriptional regulation, resulting in the elevated genome instability and predisposing to breast and ovarian cancers. We report a novel mutation LRG_292t1:c.4356delA,p.(Ala1453Glnfs*3) in the 12th exon of BRCA1, in the splice site region near the donor site of intron 12. It is a frameshift mutation with the termination codon generated on the third amino acid position from the site of deletion. Human Splice Finder 3.0 and MutationTaster have assessed this variation as disease causing, based on the alteration of splicing, creation of premature stop codon and other potential alterations initiated by nucleotide deletion. Among the most important alterations are frameshift and splice site changes (score of the newly created donor splice site: 0.82). c.4356delA was associated with two ovarian cancer cases in two families of Slavic origin. It was detected by next generation sequencing, and confirmed with Sanger sequencing in both cases. Because of the fact that it changes the reading frame of the protein, novel mutation c.4356delA p.(Ala1453Glnfs*3) in BRCA1 gene might be of clinical significance for hereditary ovarian cancer. Further functional as well as segregation analyses within the families are necessary for appropriate clinical classification of this variant. Since it has been detected in two ovarian cancer patients of Slavic origin, it is worth investigating founder effect of this mutation in Slavic populations.

Pharmacogenetics in cancer therapy - 8 years of experience at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Pharmacogenetics is a study of possible mechanism by which an individual's response to drugs is genetically determined by variations in their DNA sequence. The aim of pharmacogenetics is to identify the optimal drug and dose for each individual based on their genetic constitution, i.e. to individualize drug treatment. This leads to achieving the maximal therapeutic response for each patient, while reducing adverse side effects of therapy and the cost of treatment. A centralized pharmacogenetics service was formed at the Institute for Oncology and Radiology of Serbia (IORS) with the aim to provide a personalized approach to cancer treatment of Serbian patients. METHODS: Analyses of KRAS mutations in metastatic colorectal cancer, EGFR mutations in advanced non-small cell lung cancer, CYP2D6 polymorphism in breast cancer, DPD polymorphism in colorectal cancer and MTHFR polymorphism in osteosarcoma have been performed by real time polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Mutation testing analyses were successful for 1694 KRAS samples and 1821 EGFR samples, while polymorphism testing was successful for 9 CYP2D6 samples, 65 DPD samples and 35 MTHFR samples. CONCLUSIONS: Pharmacogenetic methods presented in this paper provide cancer patients in Serbia the best possible choice of treatment at the moment.

Impact of RAD51 G135C and XRCC1 Arg399Gln polymorphisms on ovarian carcinoma risk in Serbian women.

OBJECTIVE: The polymorphic variations of DNA repair genes may contribute to functional deficiencies in DNA repair processes increasing susceptibility to cancer. We aimed to investigate the impact of 135G>C RAD51 and XRCC1 Arg399Gln polymorphisms on ovarian carcinoma risk in Serbian women. METHODS: The study included 50 ovarian carcinoma samples and 78 cervical swabs of gynecologically healthy age-matched controls. RAD51 G135C and XRCC1 Arg399Gln polymorphisms were determined by PCR-RFLP. Deviations of the genotype frequencies from Hardy-Weinberg equilibrium were assessed using the χ2 test. The allele- and genotype-specific risks were estimated as odds ratios with 95% confidence intervals. RESULTS: RAD51 135C and XRCC1 Arg allele are associated with ovarian carcinoma [OR (95% CI): 2.54 (1.22-5.29) for C vs. G; 2.64 (1.53-4.55) for Arg vs. Gln]. RAD51 C exerts its effect in dominant (CC plus GC vs. GG) [OR (95% CI): 2.83 (1.21-6.62], while XRCC1Arg in dominant (ArgArg plus ArgGln vs. GlnGln) [OR (95% CI): 4.76 (1.69-13.42)] and recessive model (ArgArg vs. ArgGln plus GlnGln) [OR (95% CI): 2.21 (1.07-4.56)]. CONCLUSION: The results suggest that the RAD51 G135C and XRCC1 Arg399Gln polymorphisms could be biomarkers of susceptibility for ovarian carcinoma development. Further larger case-control study is needed to confirm our findings.