A Novel Association Between ABO Histo-Blood Group And Tumor Mutational Burden In Colorectal Cancer Patients

Background: Colorectal cancer(CRC)is cancer that develops in the colon and/or the rectum.Colorectal cancer is the third-most commonly diagnosed cancer in males and the secondmost common in females.It was estimated from the International Agency for Research on Cancer in 2018,globally,CRC refers as the second leading cause of cancer deaths.Colorectal cancer caused by various environmental and genetic factors through promoting the acquisition of hallmark behaviours of cancer such as genome instability,mutations,and inactivation of DNA repair mechanisms in colon epithelial cells.Moreover,losing genomic and epigenomic stability accelerates the accumulation of mutations and epigenetic alterations in tumor suppressor genes and oncogenes.The process of colorectal cancer formation is driven by accumulation of mutations and epigenetics alterations takes 10-15 years to occur before it becomes symptomatic.In CRC the most frequently mutated genes were APC,TP53,KRAS,PIK3 CA,FBXW7,SMAD4,TCF7L2 and NRAS.Importantly,an association between mutational load and clinical benefit was recently observed,the relation between mutations number and prognosis across multiple type of cancer showed a higher number of mutations was associated with improved overall survival,additionally,an obvious trend toward decreasing hazard ratio(HR)of death with increasing TMB across variety of cancer types.In colorectal cancer,TMB-high is associated with better treatment outcome in stage III or high-risk stage II of colorectal cancer.The ABO blood group is by far the most important among human blood group systems.The ABO histo-blood group consists of two antigens(A and B antigens)and four blood types(A,B,AB,and O type).ABO blood group antigens are expressed on the surface of red blood cells and several other tissue types,including cells from the gastrointestinal tract.It was reported that 23 primary adenocarcinomas of the distal colon(comprising the descending colon,sigmoid colon and rectum)tested in the IP assay,22 expressed Y hapten and 19 expressed H type-2 antigen.All of 5 primary adenocarcinomas of the proximal colon(comprising the caecum,ascending and transverse colon)were positive for both Y hapten and H type-2 antigen.Clinically more than 60 years ago the role of ABO blood group in cancer biology has been intensely studied by several investigators,and recognised that ABO antigens are associated with the risk of developing several types of tumors,namely pancreatic,colorectal cancer,and gastric cancers.It was reported individuals with blood group A had a greater risk of developing rectal cancer than individuals with blood group O,however,AB blood type is a favorable prognostic factor for individuals with colon cancer.Thence,is there a relationship between ABO Histo-blood group and tumor mutational burden(TMB)in patients with colorectal cancer,and how far this relationship have a certain value for guiding the early diagnosis of colorectal cancer.Recent studies have shown an association between ABO Histo-Blood group and the risk of developing multiple type of cancers,colorectal(CRC),gastric,pancreatic,breast,ovarian and nasopharyngeal cancers showed increasing risk for A blood group compared with non-A.However,the association between the ABO HistoBlood group and tumor mutation burden(TMB)has not been rigorously evaluated in the colorectal cancers.We,therefore,sought to examine whether the ABO blood group associated with more mutations burden in colorectal cancer patients.In this study targeted next generation sequencing(NGS)techniques were applied,we used our lab recently designed multiplex PCR-based library panels(968 Hotspot panel and 48 Genes panel)with targeted genes for sequencing the somatic mutations in colorectal cancer,amplicons were sequenced on Illumina Hiseq X-TEN platform(pair-end,2 × 150 bp)using ligated and barcoded adaptors,then a raw data is obtained in FSATQ file format,this step followed by multiple bioinformatics steps to call the mutations in tumor and paracancerous samples,then Snp Eff was used to annotate the variants in each tumor and paracancerous samples to get the information about genomic region and mutation consequences.Our study showed the average number of mutations in patients with blood type A is higher(higher TMB)compare with others blood type(B,O,AB).Mutations count with O-blood type was significantly lower compared with A-blood type(P ≤ 0.05).In addition,other tumor parameters such as tumor size and tumor stage may have a relation to the ABO-blood types which can be useful to predict and use in management options.Tumor size with A-blood type was significantly smaller compared with B-blood type tumor size(P ≤ 0.05)in colorectal cancer patients.Our result can be useful to predict and use in colorectal cancer management options.Purpose and aim of the study: ABO blood group considers important genetic factors that can be associated with colorectal cancer.A preliminary association between the ABO blood group and mutations in colorectal cancer-related genes investigated and the average number of mutations in patients was calculated.Our main study goals are:(1)Detect mutations types,mutations number and mutated genes by second-generation sequencing “targeted sequencing “technology in colorectal cancer patient resected tumor and para-tumor tissue.(2)Observe the gene mutations frequency in(CRC)tumor samples,the gene mutations frequency in different ABO-blood group types,and in different tumor anatomical location.(3)To examine the possible Association between ABO Histo-blood group and tumor mutational burden in colorectal cancer patients.(4)To demonstrate whether there is a relation between ABO Histo-blood group types and tumor mutational burden based upon patients’ pathological characteristics such as tumor size,tumor stage,and tumor location within colorectal cancer patients.Materials and Methods: The experiment workflow was focused on extracted the colonic DNA molecule from all tissues,then amplify these Colonic DNA samples by following amplicon library preparation protocol steps.DNA sequencing has been used as the gold standard for identification mutations in colorectal cancer.Next-Generation Sequencing technology has been used to perform targeted sequencing library on the total of 57 samples from 19 colorectal cancer patients include fresh tumor,para tumor,and normal tissue,samples have been collected from 2013 to 2016 to examine the genomic aspects of tissue samples.In the targeted region both cancer panels;cancer 968 Hotspot panel and cancer 48 Genes panel were used,both panels were selected because they include genes with a high incidence of colon and colorectal cancer(CRC).All targeted libraries had been sent for sequencing company,the data are grouped by molecular barcodes during the analysis,and the bases at each position are calculated to construct a new single-strand consensus sequence to detect the mutations and high-confidence mutations are selected.Different software programs had been used to analyze the type and frequency of mutations in different colorectal cancer tumor tissue and para tumor tissue specimens,we used this software to ensure the quality,accuracy of data,and easy-to-interpret results.Software such as data pre-processing: 1.Fastq files.2.ptrimmer,used to trim primer sequences.3.Use BWA to compare reads to the reference genome hg38;(bwa is used to map the reads from fastq file into reference human genome and generate the bam files.4.Use Samtools to remove reads that are aligned to multiple positions;5.Use GATK4 Mutect2 to detect mutations;6.Cellular prevalence was estimated using the Py Clone software package.After mutations detection we get an excel file with total tumor gene mutations burden,then we calculated the average mutations number in patients’ tumor samples using microsoft excel software functions.Moreover,the relation between tumor stages,tumor size,and ABO-Blood group types were examined.All the statistical analysis and plotting were conducted in R software.The P-value of ABO blood and clinical parameters was calculated by the Wilcoxon-signed rank test,and P-values ≤ 0.05 were considered statistically significant.Results: Successfully a Hotspot,and 48 Genes target libraries were constructed.The sequencing data quality result was very good.In our study,in ABO-blood group,A-blood type samples were the most affected samples in comparison with other blood types by TP53 and APC,the frequency of TP53,APC mutations were the highest in A-blood type samples(TP53 75%,APC 85.71%,KMT2 C 75%,KRAS 50% respectively)in comparison with other mutations type.Moreover,most of the variations are missense and synonymous and stop gaining.After detecting the tumor mutations number,we were able to match the tumor gene mutations number with different ABO-Blood types to demonstrate which blood type is associated with higher tumor mutation burden TMB(more tumor mutations number)among other blood types,in 19 colorectal cancer patients’ tumor tissue samples,a novel preliminary association between the ABO blood group and mutations in colorectal cancer-related genes was found,and the average number of mutations in patients with blood type A is higher compare with others blood type(B,O,AB).Mutations count with O-blood type was significantly lower compared with A-blood type(P ≤ 0.05).Mutations average number for A-blood type is(12.142),B-blood type(8.4)mutations,while O-blood type is(6).On the one hand,our finding showed,most of colorectal cancer A-blood type patients were second or third cancer stage(TNM staging T2 and T3)at diagnosis point,and before chemotherapy treatment,in addition,cancer anatomical location focused at Rectal and Sigmoid part of the large bowel,histologically classification of cancer is rectal and sigmoid tubular adenocarcinoma for A-blood type.On the other hand,O-blood type patients were observed to associate with advanced cancer stage(stage T4),three cases were found to be at Right Colonic Side(RCS)and one case with liver metastasis,while other blood type B-blood type had various stages T4 and T2 stage.A new promising finding is tumor size with A-blood type was significantly smaller compared with B-blood type tumor size(P=0.04)in colorectal cancer patients.Clinical variables control analysis showed a trend was an evident that patients with A-blood type have a higher tumor mutation burden with smaller tumor size,and earlier stage.Conclusion For the first time,our present study demonstrates a preliminary interesting relationship between ABO-blood group types and tumor mutation burden(TMB)in colorectal cancer patients.Our results showed the tumor gene mutations average number was found to be higher in patients with A-blood type while lower in non-A blood type patient.Moreover,tumor size with A-blood type was significantly smaller compared with the O-blood type and B-blood type tumor size(P≤0.05).A clear trend showed that Ablood type patients were at T2 and T3 stages,however O-blood type patients were at T4 stage,and located in rectal and sigmoid colon.Our study results may provide new information with regard to the clinical application of ABO Histo-blood group,and help health policies maker introduce(a new annually screening and monitoring protocol)for individuals who are holding the blood type which associate with higher tumor mutation burden(higher TMB)which can help diagnosis colorectal cancer at early stage before becoming symptomatic,provide better detect,prevent and treat colorectal cancer patients.Moreover,the results of the study may have a certain value for guiding the early diagnosis of CRC,and help scientists to develop new drugs for colorectal cancer.