Mutational spectrum of the Turkish hereditary breast and colon cancer patients

Cancer is a global burden in the twenty-first century, affects all countries regardless of income levels. According to the GLOBOCAN data, 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012. In addition, increase of the cancer incidence and cancer-related deaths are expected in coming years. Breast cancer represents the second most common in the world and the most common cancer among women, accounts for 1.15 million new cases and 411,000 cancer deaths each year. Colorectal cancer is the third most commonly diagnosed malignancy and the fourth leading cause of cancer-related death in the world. The GLOBOCAN database showed that it accounted for 1.4 million new cases and approximately 700,000 deaths in 2012. The cell cycle is a process, which includes sequential phases and checkpoints in order to regulate cell growth and replication of DNA. There are some set of proteins involved in the cell division events, which are the products of tumor suppressor genes and oncogenes. Due to the abnormalities in tumor suppressor genes, defective tumor suppressor proteins malfunction to restrict cell cycle. On the other hand, activated oncogenes leads to uncontrolled cell cycle, which drives the cell cycle forward without controlling. The accumulation of mistakes during the malfunctioned cycle, the cells no longer respond to the signals that control cellular growth and death. These sequence of events cause to formation of cancer cells, which is also called carcinogenesis. Tumor suppressor genes manage fidelity of cell cycle replication process and controls. The tumor suppressor genes such as BRCA1 and BRCA2 are the major part of homologous recombination repair, which is an important mechanism using sister chromatid in order to process error-free repair of DNA double breaks. On the other hand, the mismatch repair mechanism (MMR), which consists of MLH1, MSH2, MSH6, PMS2 and EPCAM, is responsible for correcting single base mismatches as well as small insertion/deletion loops of unpaired bases. Alterations in the MMR system stands in a significant place in carcinogenesis, associated with hereditary nonpolyposis colorectal cancer syndrome, which is also known as Lynch syndrome. The MMR defects increases the risk of colorectal, gastric, endometrial and ovarian cancer, but mostly are associated with colorectal cancer. Besides, pathogenic variants in the MMR genes cause to microsatellite instability (MSI) in tumor tissues. MSI may occur in many cancer types but it is a significant marker for colorectal cancer patients with Lynch syndrome. The majority of cancer arise from pathogenic variants sustained over a lifelong but the cancer results from genetic alterations that are transmitted through generations considered as hereditary case. Approximately 5-10% of all cancer cases appear in this category. Considering that the incidence and mortality rates of colorectal and breast cancer, hereditary patients were sequenced using multigene panel testing in order to characterize clinical features and rare variants found in those patients. Apart from that, it is aimed to reveal recurrent pathogenic variants, candidate for founder pathogenic variants, by comparing the patient and control groups. Study subjects consists of 273 breast cancer patients, 123 colorectal cancer patients as well as 338 controls older than 65 years without personal cancer history. All the patients and controls were given informed consent form. The patients were grouped whether they have increased risk of breast or colorectal cancer according to NCCN guidelines for hereditary breast and hereditary colorectal cancer. For sequencing, three different commercial kits were used. Both Sophia DDM and GATK Variant Calling pipeline were used in order to call variants, and the found variants were filtered 20 cancer predisposing genes relevant with hereditary breast and ovarian cancer, HNPCC and intestinal polyposis syndrome. The 20 cancer predisposing genes are as follows: APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11 and TP53. The exonic and splice variants, which have allele frequency equal or smaller than 1% as well as no data in 1000 Genome population database were selected. Interpretation of the selected variants was done according to the ACMG guideline. During interpretation of patients, monoallelic MUTYH pathogenic variants were excluded. Positive rates for patients and controls with a pathogenic/likely pathogenic mutation(s) were as follows: 13.55% (n=37) for breast cancer group, 11.90% (n=15) for colorectal cancer group and 4.45% (n=15) for controls. Inconclusive results were found in 24.18% (n=66) for breast cancer patients, 30.95% (n=39) for colorectal cancer patients and 21.96% (n=74) for controls. The estimation of hereditary breast cancer showed that 72% (n=197) of breast cancer patients are in high risk group, whereas 28% (n=76) of the patients falls in non-high risk group. The average ages of high and non-high risk groups are 44.9 and 59.6, respectively. In all breast cancer patients (n=273), 43 (7.56%) pathogenic variants were found. 4.76% of pathogenic variants were found in BRCA1 and BRCA2. Pathogenic variants in moderate penetrance genes (ATM, CHEK2, PALB2, NBN) had the highest rate in breast cancer patients (5.10%). Case-control comparison revealed that pathogenic variants in CHEK2 have significant OR of 3.51 in breast cancer patients. Also, a recurrent pathogenic variant, NM_007194(CHEK2):c.1427C>T (p.Thr476Met), was found in both breast cancer patients (n=4) and controls (n=3). 83 VUS (n=75) were identified in breast cancer patients, and 72 of the VUS were unique. In both all and unique rare VUS, ATM (%all = 6.2, %unique = 5.1) and BRCA2 (%all = 4.4, %unique = 3.3) had higher rates than other genes in 273 breast cancer patients. To overcome with the size of gene products and their effects on variant numbers, standardization applied for both all rare and unique rare VUS numbers identified in breast cancer patients. It is found that CHEK2, MSH2, RAD51C and MSH6 had more VUS per amino acid than other genes. The same result was obtained in comparison between high risk and non-high risk patients. Since RAD51C had only one VUS, CHEK2, MSH2 and MSH6 are promising candidates for further studies associated with breast cancer. In colorectal cancer, 35% (n=43) of the patients were classified as high risk patients, whereas 65% (n=80) were non-high risk patients. In all colorectal cancer patients, 19 pathogenic variants were identified. 17 pathogenic variants were unique, and 3 of unique pathogenic variants were identified as novel. 5.69% of pathogenic variants were found in MLH1 and MSH2. Mismatch repair genes, MLH1 and MSH2 showed significant OR of 19.45 (p = 0.0499) and 25.11 (p = 0.0308), and associated with colorectal cancer. Pathogenic variants in moderate penetrance genes, ATM and CHEK2 did not show significant associations with colorectal cancer. No pathogenic variants were found in low penetrance genes. In both all and unique rare VUS, ATM (8,9%, n=11) had the highest rate among all 20 genes. Comparison of standardized and non-standardized VUS between all and unique rare VUS revealed that standardized CHEK2 had the highest number of VUS in colorectal cancer patients. In additionally, using Sophia DDM software, 22 and 4 copy number variants were identified in breast and colorectal cancer patients, respectively. Still, these variants need to be validated with functional studies. After all, this study is a pioneer to examine the hereditary breast and colorectal cancer patients in the Turkish population. With the gathered data, the future cancer studies will represent the Turkish population more precisely.