Site specific genetic differences in colorectal cancer via Next-Generation-Sequencing using a multigene panel.

AIM: Next-generation sequencing (NGS) has been proposed as a comprehensive and efficient genomic profiling tool to guide personalized therapy for colorectal cancer. This study aimed to review the site-specific difference and the potential benefits of actionable mutation panel for colorectal cancer in relation to the clinicopathological features. MATERIAL AND METHODS: One hundred and six patients who underwent colorectal surgery with curative or palliative intent for histopathologically confirmed carcinoma between June 2016 and June 2018 were identified from a prospectively maintained database. Formalin-fixed, paraffin-embedded tumor tissues were analyzed for actionable variants in 11 genes via NGS (EGFR, ALK, KRAS, NRAS, KIT, BRAF, PDGFRA, ERBB2, ERBB3, ESR1, and RAF1). RESULTS: Most of the primary tumors were in the rectum (49 patients; 46.2%) followed by the right colon (32 patients; 30.1%) and left colon (25 patients; 23.5%), respectively. Of sequenced cases, 43 KRAS mutations, 7 EGFR mutations, 6 NRAS mutations, 6 BRAF mutations, 3 KIT mutations, 1 ERBB2 mutation, 1 PDGFRA mutation, and 1 RAF1 mutation were identified in 106 patients. The frequency of mutations is mostly concentrated on the right colon group. The highest drug resistance observed in all patients was against Cetuximab and Panitumumab, and the highest drug resistance was found in the right colon group (53.1%). CONCLUSIONS: The utility of actionable multigene panel revealed the value of a well-designed next-generation sequencing workflow in the practical use of clinical outcomes via the prediction of responsiveness to therapeutic agents or indications for novel treatment modalities in addition to prognosis estimate. KEY WORDS: Colorectal Cancer, Drug Resistance, Next-Generation Sequencing.

Pre-operative Neo-adjuvant Chemotherapy Related miRNAs as Key Regulators and Therapeutic Targets in Colorectal Cancer.

BACKGROUND: In colorectal cancer, the investigation of cancer pathogenesis and the determination of the relevant gene and gene pathways is particularly important to provide a basis for treatment-oriented studies. miRNAs which affect gene regulation in the molecular pathogenesis of cancer, have an active role in carcinogenesis. In the literature, miRNA expression levels have been associated with metastasis and prognosis in different cancers. OBJECTIVE: In our study, expression profiling of miRNAs involved in oncogenic and apoptotic pathways in patients with locally advanced colorectal cancer receiving neoadjuvant therapy was performed. METHODS: miRNAs were isolated from three different FFPE tissue samples taken at different times of the same patient (tumor tissue taken at the time of diagnosis, normal tissue samples, and after neoadjuvant therapy). The expression analysis of 84 miRNAs determined by PCR array (Fluidigm, USA) and mediated meta-analysis was performed comparatively to each study and non-cancerous control group. Evaluations were performed with ΔΔCT calculations. RESULTS: As a result of the miRNA PCR array study, in addition to differences were observed in miRNA expression between control and study groups. The potential biomarkers which were hsamiR- 215-5p, hsa-miR-9-59, hsa-miR-193a-5p, hsa-miR-206, hsa-miR-1, hsa-miR-96-5p have been detected for possible treatment resistance, prognosis and predispositions to cancers. CONCLUSION: In patients with colorectal cancer, miRNA expression in the tumoral regions before and after neoadjuvant therapy has represented a variable pattern. It has been shown that miRNA studies can be used to predict the clinical course and response to treatment with differences in expression levels. It has been concluded that specific miRNAs may be candidate biomarkers for colorectal cancer.

A Multicenter Study of Genotype Variation/Demographic Patterns in 2475 Individuals Including 1444 Cases With Breast Cancer in Turkey.

Objective: Breast cancer (BC) is the most common cancer type in women and may be inherited, mostly in an autosomal dominant pattern. The clinical diagnosis of BC relies on the published diagnostic criteria, and analysis of two genes, BRCA1 and BRCA2, which are strongly associated with BC, are included in these criteria. The aim of this study was to compare BC index cases with non-BC individuals in terms of genotype and diagnostic features to investigate the genotype/demographic information association. Materials and Methods: Mutational analyses for the BRCA1/BRCA2 genes was performed in 2475 individuals between 2013-2022 from collaborative centers across Turkey, of whom 1444 with BC were designated as index cases. Results: Overall, mutations were identified in 17% (421/2475), while the percentage of mutation carriers in cases of BC was similar, 16.6% (239/1444). BRCA1/BRCA2 gene mutations were detected in 17.8% (131/737) of familial cases and 12% (78/549) of sporadic cases. Mutations in BRCA1 were found in 4.9%, whereas 12% were in BRCA2 (p<0.05). Meta-analyses were performed to compare these results with other studies of Mediterranean-region populations. Conclusion: Patients with BRCA2 mutations were significantly more common than those with BRCA1 mutations. In sporadic cases, there was a lower proportion with BRCA1/BRCA2 variants, as expected, and these results were consistent with the data of Mediterranean-region populations. However, the present study, because of the large sample size, revealed more robust findings than previous studies. These findings may be helpful in facilitating the clinical management of BC for both familial and non-familial cases.

Germline landscape of BRCAs by 7-site collaborations as a BRCA consortium in Turkey.

BRCA1/2 mutations play a significant role in cancer pathogenesis and predisposition particularly in breast, ovarian and prostate cancers. Thus, germline analysis of BRCA1 and BRCA2 is essential for clinical management strategies aiming at the identification of recurrent and novel mutations that could be used as a first screening approach. We analyzed germline variants of BRCA1/2 genes for 2168 individuals who had cancer diagnosis or high risk assessment due to BRCAs related cancers, referred to 10 health care centers distributed across 7 regions covering the Turkish landscape. Overall, 68 and 157 distinct mutations were identified in BRCA1 and BRCA2, respectively. Twenty-two novel variants were reported from both genes while BRCA2 showed higher mutational heterogeneity. We herein report the collective data as BRCA Turkish consortium that confirm the molecular heterogeneity in BRCAs among Turkish population, and also as the first study presenting the both geographical, demographical and gene based landscape of all recurrent and novel mutations which some might be a founder effect in comparison to global databases. This wider perspective leads to the most accurate variant interpretations which pave the way for the more precise and efficient management affecting the clinical and molecular aspects.

The impact of rare and low-frequency genetic variants in common variable immunodeficiency (CVID).

Next Generation Sequencing (NGS) has uncovered hundreds of common and rare genetic variants involved in complex and rare diseases including immune deficiencies in both an autosomal recessive and autosomal dominant pattern. These rare variants however, cannot be classified clinically, and common variants only marginally contribute to disease susceptibility. In this study, we evaluated the multi-gene panel results of Common Variable Immunodeficiency (CVID) patients and argue that rare variants located in different genes play a more prominent role in disease susceptibility and/or etiology. We performed NGS on DNA extracted from the peripheral blood leukocytes from 103 patients using a panel of 19 CVID-related genes: CARD11, CD19, CD81, ICOS, CTLA4, CXCR4, GATA2, CR2, IRF2BP2, MOGS, MS4A1, NFKB1, NFKB2, PLCG2, TNFRSF13B, TNFRSF13C, TNFSF12, TRNT1 and TTC37. Detected variants were evaluated and classified based on their impact, pathogenicity classification and population frequency as well as the frequency within our study group. NGS revealed 112 different (a total of 227) variants with under 10% population frequency in 103 patients of which 22(19.6%) were classified as benign, 29(25.9%) were classified as likely benign, 4(3.6%) were classified as likely pathogenic and 2(1.8%) were classified as pathogenic. Moreover, 55(49.1%) of the variants were classified as variants of uncertain significance. We also observed different variant frequencies when compared to population frequency databases. Case-control data is not sufficient to unravel the genetic etiology of immune deficiencies. Thus, it is important to understand the incidence of co-occurrence of two or more rare variants to aid in illuminating their potential roles in the pathogenesis of immune deficiencies.

Current Status of Genetic Diagnosis Laboratories and Frequency of Genetic Variants Associated with Cystic Fibrosis through a Newborn-Screening Program in Turkey.

BACKGROUND: Cystic fibrosis (CF) is the most common worldwide, life-shortening multisystem hereditary disease, with an autosomal recessive inheritance pattern caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The national newborn screening (NBS) program for CF has been initiated in Turkey since 2015. If the immunoreactive trypsinogen (IRT) is elevated (higher than 70 µg/L in the second control) and confirmed by sweat test or clinical findings, genetic testing is performed. The aims of this study are to emphasize the effect of NBS on the status of genetic diagnosis centers with the increasing numbers of molecular testing methods, and to determine the numbers and types of CFTR mutations in Turkey. METHODS: The next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) results of 1595 newborns, who were referred to Cukurova University Adana Genetic Diseases Diagnosis and Treatment Center (AGENTEM) for molecular genetic testing, were evaluated with positive CF NBS program results since 2017. RESULTS: According to the results; 560 (35.1%) of the 1595 patients carried at least 1 (one) CF-related variant, while 1035 patients (64.9%) had no mutation. Compound heterozygosity for two mutations was the most common in patients, while two detected variants were homozygote in 14 patients. A total of 161 variants were detected in 561 patients with mutations. Fifteen novel variants that have not been previously reported were found. Moreover, p.L997F was identified as the most frequent pathogenic mutation that might affect the IRT measurements used for the NBS. The distribution of mutation frequencies in our study showed a difference from those previously reported; for example, the well-known p.F508del was the third most common (n = 42 alleles), rather than the first. The most striking finding is that 313 cases had a pathogenic variant together with the V470M variant, which might have a cumulative effect on CF perpetuation. CONCLUSION: This study is the first to determine the mutational spectrum of CFTR in correlation with the NBS program in the Turkish population. NBS for CF raises issues regarding screening in diverse populations, both medical and non-medical benefits, and carrier identification. Through the lens of NBS, we focused on the integrated diagnostic algorithms and their effect on the results of genetic testing.

Integration of Liquid Biopsies into Clinical Laboratory Applications via NGS in Cancer Diagnostics.

BACKGROUND: Next Generation Sequencing is one of the latest advances in molecular testing and clinical laboratory applications. Next Generation Sequencing techniques involving liquid biopsies are emerging as important tools in cancer diagnostics and prognostics. Thus, integration of liquid biopsy studies into clinical laboratory applications has become a necessity. By virtue of liquid biopsies, determining potential treatment targets through metastasis and primary tumor sites in the right clinical context can result in a more comprehensive treatment. This also helps to overcome re-sampling difficulties which require an invasive procedure with the problem of tumor heterogeneity. As the literature involving liquid biopsies and next generation sequencing increases, the rate of laboratories with competencies and experience in this novel technology remains limited. METHODS: Next generation sequencing was performed via a comprehensive multi-gene cancer panel (Actionable In-sight Solid Tumor Panel, Qiagen) consisting of 12 solid tumor related genes (EGFR, ALK, KRAS, PIK3CA, NRAS, PDGFRA, KIT, ERBB2, ERBB3, ESR1, BRAF and RAF1) from lung cancer patients who applied or were referred to CU AGENTEM (Cukurova University Adana Genetic Diseases Diagnosis and Treatment Center) for routine genetic testing. RESULTS: A modified next generation sequencing workflow was performed with a multi-gene solid tumor panel using liquid biopsies in comparison with formalin fixed paraffin embedded samples to integrate this technique into the routine clinical laboratory applications and bioinformatics. In this study, next generation sequencing of liquid biopsies in cancer patients was integrated into cancer diagnostics. CONCLUSIONS: Liquid biopsy studies provide numerous advantages when integrated with next generation sequencing through a well-optimized workflow.

BRCA mutation characteristics in a series of index cases of breast cancer selected independent of family history.

Certain genetic predisposition factors, such as BRCA1 and BRCA2 mutations play a pivotal role in familial breast cancer development in both males and females. Due to this, the importance and necessity of genetic screening to identify mutations affecting the population is paramount. Undergoing genetic screenings allows for a more knowledgeable risk assessment for the patients and their care providers. The aim of this study was to evaluate the prevalence of BRCA1/BRCA2 mutated genes in the Turkish population among unselected patients. To identify the molecular markers, we utilized a gene panel analysis consisting of BRCA1 and BRCA2 genes, with a next generation sequencing platform (MiSeq System, Illumina). Sequencing was performed using leukocyte DNA from breast cancer patients. In-silico analysis for novel mutations was carried out using SIFT, PolyPhen2 and MutationTaster. BRCA1 and BRCA2 pathogenic variants were identified in 18 of 129 (14%) patients among the study population; of those 18 patients, seven (39%) were found in the BRCA1 gene and 11 (61%) in the BRCA2 gene. Ten of the eleven BRCA2 variants (90%) were novel mutations. Four of ten (40%) of the novel mutations were determined to be deleterious and six out of ten (60%) were identified as single nucleotide variations. Clinically significant mutations of the BRCA1/BRCA2 genes are related to an increased susceptibility for breast cancer. There is however, little known about BRCA mutations amongst the general population. Thus, it is important that patients are able to undergo genetic screenings and counseling. This also allows for greater care from health care providers and can only facilitate disease prevention which in turn can lead to a decreased cancer morbidity rate.