The largest Chinese cohort study indicates homologous recombination pathway gene mutations as another major genetic risk factor for colorectal cancer with heterogeneous clinical phenotypes

The largest Chinese cohort study indicates homologous recombination pathway gene mutations as another major genetic risk factor for colorectal cancer with heterogeneous clinical phenotypes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The largest Chinese cohort study indicates homologous recombination pathway gene mutations as another major genetic risk factor for colorectal cancer with heterogeneous clinical phenotypes Yun Xu, Kai Liu, Cong Li, Minghan Li, Fangqi Liu, Xiaoyan Zhou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3174582/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Colorectal cancer (CRC) is one of the most common malignancies globally with estimated 1.87 million new cases annually. Genetic factors were associated with over 30% of CRC incidence. However, the mutations in CRC-susceptibility genes recommended by the National Comprehensive Cancer Network (NCCN) guidelines accounted for only 5-10% of CRC cases, suggesting a large proportion of CRC-susceptibility genes remain unknown. As previous works on hereditary CRC were largely designed to analyze germline mutations in patients with a single category of genetic high-risk factor, this study aims to explore the genetic mutations underlying five categories of genetic high-risk factors in clinic. Methods: From January 2015 to December 2018, 2181 patients from a cohort of 8270 consecutive CRC cases were retrospectively enrolled, covering five categories of genetic high-risk factors. Their germline mutations under each category were detected and analyzed in association with CRC susceptibility, clinical phenotypes, and prognoses. Results: In total 462 pathogenic/likely pathogenic genetic variants were detected in 19.3% CRC patients enrolled. Mutations in the mismatch repair (MMR) genes were identified in 9.1% patients, most prevalent across all high-risk groups. Mutations in homologous recombination (HR) pathway genes were detected in 6.5% patients, which were mostly penetrated in early onset, family cancer history and extra-colonic cancer risk groups. HR pathway gene mutations, including BARD1 , RAD50 and ATM , were associated with an increased risk of CRC in the cohort with an odds ratio of 2.8, 3.1 and 3.1-fold, respectively. CRC patients carrying different genetic mutations manifested heterogeneous phenotypes in clinicopathology and long-term prognoses, for which Lynch Syndrome demonstrated better prognoses than other groups, including those with HR pathway mutations. Conclusions : This largest Chinese cohort study of high-risk hereditary CRC is the first to cover five categories of genetic high-risk factors, which greatly expanded the list of CRC-susceptibility mutations. In contrast to the MMR mutations of Lynch syndrome, the study reveals for the first time at population level that carriers of mutations in the HR pathway genes are significantly susceptible to CRC, implicating HR pathway gene mutations as another major contributor for increased risk of developing CRC. Trial registration : Retrospectively registered. Colorectal cancer Cancer susceptibility Genetic risk Homologous recombination Mismatch repair deficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Colorectal cancer (CRC) is one of the most common malignancies globally with an estimated 1.87 million new cases per year, and was the second leading cause of cancer-related death in 2020 1,2 . In China, there are over 400,000 new cases of CRC each year, and the incidence of CRC increased by 2.5% per year in males and 1.5% per year in females 2,3 . The current known general risk factors contributing to CRC include age, sex, inflammatory bowel disease, and CRC-susceptibility genetic variants 2 . Genetic factors were reported to be associated with more than 30% of the incidence of CRC 4 , but due to insufficient genetic screening, the germline mutations in high-penetrance CRC-susceptibility genes were found to account for only 5-10% of all CRCs 5,6 . As a result, a large proportion of CRC-susceptibility genes remain uncovered. The empirical indications for genetic screening on CRC-susceptibility genes are based on several significant genetic risk factors in the clinic, including early onset CRC, family history of cancer, and deficient mismatch repair (dMMR) in tumor tissues by immunohistochemical (IHC) staining 7-11 . While tumor IHC manifesting dMMR is indicative of a high possibility of Lynch syndrome (LS) 12 , family history of cancer 13 and early onset cancer 8,10,14 are hallmarks of inherited cancer susceptibility. The management guidelines for the indication of genetic screening were established and recommended by National Comprehensive Cancer Network (NCCN), based on the studies of genetic analysis in the CRC patients that manifested those significant genetic risk factors. However, using present NCCN guidelines, approximately 28% of pathogenic and likely pathogenic (P/LP) variants were still missed 15 . Many other genetic risk factors associated with hereditary CRC such as multiple primary CRC, and primary hereditary cancer syndrome associated extra-colonic cancer was rarely studied 5,16 . The multiple primary cancer including synchronous and metachronous CRC, and extra-colonic cancers such as endometrial, ovarian and pancreatic cancer were commonly observed in LS and some other hereditary cancer syndromes. However, due to insufficient supporting data, these two genetic risk factors were not particularized in the current NCCN guidelines. Thus, it is necessary to include these genetic risk factors in the investigation of CRC-susceptibility genes. Although many studies were reported on germline mutations for CRC patients with genetic risk factors, those works were mostly evaluating a patient group(s) focusing on one single genetic risk factor. There lack global views on genetic abnormalities that underlie the clinical high-risk CRC, and there is limited understanding on the association between germline mutations and the clinical genetic risk factors, and on how the germline mutations contribute to the clinical characteristics and long-term outcomes for CRC patients. To address these important questions, we designed the study to investigate the germline mutations underlying the five clinical genetic risk factors. early onset CRC, family history of cancer, dMMR of tumor tissues by IHC staining, multiple primary CRC, and primary hereditary cancer syndrome associated extra-colonic cancer. To our knowledge, this is the first study that covers CRC patients with five clinical genetic risk factors. By admitting consecutive 8270 cases of CRC patients at Fudan University Shanghai Cancer Center (FUSCC) over four-year period from 2015 to 2018, we constituted the largest Chinese cohort for hereditary CRC study. Our goal is to develop a comprehensive view on the landscape of genetic abnormalities in CRC patients with high-risk factors, and understand their contributions to clinical characteristics and outcomes for those carriers. The results of the current study will lend support to expand genetic screening strategy for patients carrying respective genetic risk factor, which facilitates early detection, prevention, and treatment for hereditary CRC. Methods Definitions of Hereditary High-risk Factors and Study Population In this study, the definitions of genetic high-risk factors were defined and illustrated in the Table 1 . From January 1, 2015, to December 31, 2018, a total of 8270 CRC patients received treatment at the FUSCC. 2181 CRC patients with at least one of the genetic risk factors were retrospectively enrolled in the study. The IHC staining for MMR analysis was independently performed in the Department of Pathology at FUSCC. The majority of tumor samples were examined by IHC, although 386 tumors from patients who achieved complete response after receiving neoadjuvant therapy did not have IHC. Data including demographic information, family and medical history, pathology, and presenting symptoms were extracted from the electronic medical record. All patients were followed up as of September 30th, 2021. FUSCC Hereditary CRC Panel We designed a multiplex polymerase chain reaction (PCR) amplification-based 38 gene FUSCC-hereditary cancer panel to detect germline mutations in eligible patients. The panel included 38 genes (containing APC , ATM , ATR , AXIN2 , BARD1 , BLM , BMPR1A , BRCA1 , BRCA2 , BRIP1 , CDH1 , CDK4 , CHEK2 , CDKN2A , EPCAM , GALNT12 , GREM1 , MLH1 , MSH2 , MSH3 , MSH6 , MUTYH , NTHL1 , PALB2 , POLD1 , POLE , PIK3CA , PMS2 , PTEN , RNF43 , RPS20 , SMAD4 , STK11 , TP53 , NBN , RAD50 , RAD51C , RAD51D ), 24 of them are commonly tested in multi-gene panels mentioned in NCCN guidelines (version 1. 2021) 17 and the remaining 14 genes are frequently mutated genes detected in other CRC cohorts 6,10,18 . The gene coordinates of the coding region of each gene in hg19 are extracted from the reference genome file. The primer design uses the overlapping tile covering method by using the software "primer 3" (version 0.4.0, https://bioinfo.ut.ee/primer3-0.4.0/) to ensure that the amplicons cover the coding region to the greatest extent. A custom library was prepared and primers were designed for all 602 coding exons (915 PCR amplicons) of these 38 genes including 180-280 bp of each flanking extron. Oligos were synthesized, primer droplets were prepared and all of these droplets were pooled together to create the custom library. Genomic DNA was purified by the use of QIAamp® DNA Mini-kit (51104, QIAGEN, German) from white blood cells. A total of 20-200ng of genomic DNA was used for PCR amplification. The primer library and a template mix that included the fragmented genomic DNA and all of the components of the PCR reaction were loaded on GeneAmp® 9700 PCR (Applied Biosystem, The USA) and then amplified under the following conditions: 96°C for 3 minutes, 17 cycles of: (96°C for 30 seconds, 60°C for 4 minutes), 72°C for 4 minutes and then hold at 4°C. After amplification, the amplicons from PCR droplets were purified and quality controlled using the Qubit (Thermo Fisher Scientific, USA). PCR products were subsequently used for Illumina library preparation and sequenced using an Illumina NovaSeq 5000 platform (Illumina Inc., San Diego, CA, USA). Germline Mutation Analysis Genomic DNA was extracted from frozen peripheral lymphocytes of all enrolled patients, and the mutational spectrum was identified using the FUSCC-hereditary CRC panel. The raw data of NGS was first filtered by removing Illumina sequencing adaptor and low-quality sequences. The remaining high-quality reads were mapped to the human reference genome (GRCh37) using the BWA aligner with the BWA-MEM algorithm and default parameters. Germline mutations were called according to the following steps ( Supplementary Figure 1 ). Single-nucleotide mutations were identified using Genome Analysis ToolKit (GATK, version 4.0) 19, 20 and Varscan (version 2.4.2) 21 ; insertions and deletions mutations were identified based on the union results of GATK and Pindel(version 0.2.5b8) 22 . The pathogenicity of the mutations reported in Clinvar 23 with at least two stars was used in this study. We used the results of InterVar 24 annotation for the unreported mutations identified in this testing. We filtered the mutations using Genome Aggregation Database (gnomAD) 25 , 1000 Genomes Project 26 , and the Exome Aggregation Consortium (ExAC) 27 . Only rare mutations (MAF <0.01% in 1000G 2015Aug, ExAC or gnomAD exome database and <0.05% in the East Asian population) were selected for mutation classification. Some splicing and the stop gain mutations classified as Class 3 were upgraded to Class 4 (likely pathogenic) 28 . Only Class 4 and Class5 (pathogenic) mutations were selected for subsequent analysis. The prevalence of P/LP variants of MMR genes and BRCA1/2 genes in the general Chinese population were adopted from recent studies 29, 30 . The prevalence of P/LP variants of other genes was re-analyzed based on the ChinaMAP reference database 31 . Statistical A nalysis Means (standard deviations) were calculated for continuous variables and percentages for categorical variables among different groups. Baseline clinical characteristics and germline mutation frequencies were compared using a two-sided Fisher exact test. Continuous variables were compared between two groups by the Wilcoxon test, and the Kruskal-Wallis H test was used to conduct comparative statistical studies on three or more groups. We used logistic regression to estimate the odds ratio for progression-free survival (PFS) and overall survival (OS) according to different risk factors. Kaplan-Meier curves were generated, and any differences in survival were evaluated with a stratified log-rank test. Hazard ratios and confidence intervals were estimated by Cox regression analysis. All statistical analyses were performed using R (version 4.0.2), Rstudio v.1.2 software, and SPSS software (version 21.0, SPSS Inc., Chicago, USA). All statistical analyses with p-value < 0.05 were considered statistically significant ( * p < 0.05, ** p < 0.01, *** p < 0.001, N.S., not significant). Results Clinical characteristics of CRC patients associated with five different genetic high-risk factors To investigate the germline mutations under all five genetic risk factors in CRC patients, we admitted 8270 consecutive CRC patients for the study over a four-year period between 2015 and 2018. We found a high proportion of patients carried genetic high-risk factors in our consecutive CRC patient cohort. A total of 2181 out of 8270 (26.4%) CRC patients enrolled at FUSCC met eligibility criteria. Early onset CRC patients accounted for 15.7% (1296/8270) of eligible patients, followed by patients with family cancer history (9.7%, 803/8270), dMMR tumors (4.8%, 401/8270), extra-colonic cancer (3.0%, 250/8270), and multiple primary CRC (2.3%, 187/8270) ( Figure 1A ). Of these enrolled patients, 72.6% (1583/2181) of patients had one genetic risk factor: 40.9% (893/2181) for early onset CRC, 20.1% (439/2181) for family history of cancer, 8.4% (183/2181) for dMMR tumors and 2.8% (61/2181) for extra-colonic cancer. Multiple primary CRC was rarely an independently observed risk factor and only present in 0.3% (7/2181) of patients ( Figure 1B ). 27.4% (652/2181) of patients had at least two genetic risk factors. Patients of different genetic risk demonstrated heterogeneous clinicopathologic characteristics. Patients with multiple primary CRC most often had early onset CRC or family cancer history. Patients with dMMR tumors were more likely to have mucinous adenocarcinoma, right-sided and poorly differentiated tumors, and a lower proportion of TNM III/IV. Patients with early onset or multiple primary CRC had a higher percentage of TNM III/IV ( Figure 1B and Supplementary Table 1 ). Prevalence of germline mutations in CRC patients with genetic high-risk To reveal the germline mutations in CRC patients carrying genetic risk factors, a hereditary cancer susceptibility gene panel consisting of 38 genes was used to test the enrolled CRC patients. The P/LP variants were detected for 32 out of 38 genes in the panel. No P/LP variants were detected for the remaining six genes, i.e., CDH1 , CDK4, GREM1 , GALNT12 , RPS20, and BMPR1A , for the CRC cohort ( Supplementary Table 2 ). P/LP variants were detected in 19.3% (421/2181) of CRC patients with at least one of the five risk factors for cancer susceptibility assessment. Specifically, 9.1% of patients (199/2181) carried a P/LP variant in the MMR pathway genes, and unexpectedly 4.2% of patients (141/2181) had a P/LP variant in the HR pathway genes. P/LP variants in APC were identified in 16 patients (0.73%, 16/2181). MLH1 (3.33%, 74/2181), MSH2 (2.83%, 63/2181) and MSH6 (2.21%, 49/2181) genes from the MMR pathway were the most frequently mutated genes in CRC patients. P/LP variants in CRC genes (NCCN guidelines) and “other genes” (not covered in the NCCN guidelines) were detected in 13.4% (335/2181) and 5.8% (126/2181) of our CRC patient cohort, respectively. P/LP with “other genes” that are not covered by the NCCN guidelines, contributed to more than 25% of all detected germline mutations ( Figure 2A ). P/LP variants with truncation or missense mutations, were the predominant variant types for majority of these genes ( Figure 2B ). P/LP variants in the moderate- and low-penetrance genes (labeled as “other genes” which are not covered in the NCCN guidelines) were detected in 3.0% of patients (65/2181) ( Figure 2C ). To determine whether carrying P/LP variants in these genes confer an increased risk of CRC in the Chinese population, we performed a control-based risk analysis, which indicates that P/LP variants in the MMR pathway genes, MLH1 , MSH2 , APC , MSH6 , TP53, and PMS2 , were associated with an increased risk for CRC, consistent with what was known previously for LS 32-34 . However unexpectedly, P/LP variants that belong to genes in the HR pathway, i.e., POLE , RAD50 , ATM, and BARD1 , were also found to be associated with an increased risk for CRC in our cohort. While P/LP variants in MLH1 , MSH2 , APC , MSH6 , and TP53 conferred a high-risk for CRC with odds ratio greater than 5, the P/LP variants in PMS2 , POLE , RAD50 , ATM, and BARD1 were associated with a moderate risk increase with odd ratios greater than 2 but smaller than 5 ( Figure 2D and Supplementary Table 3 ). These data for the first time reveal at population level that patients with germline mutations in the HR pathway genes are susceptible to CRC, suggesting mutations in HR pathway genes become another major contributor for increased risk of developing CRC. Germline mutations underlying different categories of genetic high-risk factors To understand the underlying germline mutations of different genetic high-risk factors in CRC patients, we performed an association analysis between genetic high-risk factors and the detection rates of P/LP variants in CRC or other cancer susceptibility genes. As expected, dMMR tumors were associated with the highest detection rate of P/LP variants in MMR genes. Early age of onset was associated with the second highest risk for identifying MMR gene mutations in MLH1 , MSH2, and MSH6 . Both multiple primary CRC and extra-colonic cancer were also associated with a high-risk for identifying MMR gene mutations. However unexpectedly, HR pathway gene mutations were enriched in early onset, family cancer history, and extra-colonic cancer risk groups. P/LP variants in APC , POLE , MUTYH , TP53, and AXIN2 were also significantly enriched in the early onset risk group. ( Figure 3A, B ). The prevalence of germline mutations in CRC patients was different for each genetic high-risk factor. When assessing individual high-risk factors, patients with dMMR tumors had the highest detection rate (23.5%, 43/183) of germline mutations, followed by the extra-colonic cancer group (14.8%, 9/61), multiple primary CRC (14.3%, 1/7), early onset CRC (11.4%, 102/893) and family cancer history group (9.1%, 40/439). The detection rate of germline mutations in most risk groups was greater than 10% ( Figure 3C ). dMMR tumors with at least one additional high-risk factor predicted a significantly increased probability of harboring germline mutations, with detection rates of 40%-100%. Among patients with MMR proficient (pMMR) tumors or IHC not performed, patients carrying more risk factors predicted higher mutation detection rates, ranging from 10.8% to 18.4%. Notably three pMMR CRC patients had four risk factors, but no germline mutations were detected for them ( Figure 3D-E ). The detection rate of germline mutations in patients having 1-5 risk factors, ranges from 12.3% to 100% ( Supplementary Figure 2 ). Clinical manifestations for different germline mutations To reveal the effects of germline mutations on clinical phenotype, we performed a correlation analysis between different gene mutations and clinical characteristics. Our data indicate patients carrying different germline mutations manifested heterogeneous clinicopathologic characteristics. The presence of MMR gene mutations was inversely correlated with age, serum CEA content, differentiation grade, vascular invasion, and TNM stage. On the other hand, HR gene mutations were found to be associated with the occurrence of extra-colonic cancer ( Figure 4A and Supplementary Figure 3 ). Multiple primary CRC was most frequently observed in familial adenomatous polyposis (FAP) patients, and right colon cancer was most common in LS patients. Colorectal tumors in patients with mutations in the HR genes were more often left sided ( Figure 4B ). The age of onset of CRC in LS and FAP patients was significantly younger than that of patients who have no P/LP variants detected ( Figure 4C ). LS patients with CRC had significantly fewer metastatic lymph nodes than CRC patients with HR pathway gene mutations (p=0.029). There was no statistically significant difference in the number of metastatic lymph nodes among CRC patients with other germline mutations ( Figure 4D, Supplementary Table 4 and Supplementary Table 5 ). Prognoses of CRC patients with different germline mutations and risk factors To understand the effects of germline mutations on long-term outcomes, we performed survival analysis between patients with different gene mutations. Using the cohort of CRC patients in the current study that has a median follow-up time of 53.7±24.9 months, we analyzed the correlation between the presence of germline mutations and long-term PFS and OS. For CRC patients with at least one of the five risk factors in our study (n=2181), the 5-year PFS and OS were 71.0% and 79.8%, respectively. For CRC patients with germline mutations, the 5-year PFS and OS (77.1% and 83.1%, respectively) were significantly higher than those of patients without germline mutations, which were 69·8% (c 2 = 9.976, p = 0.002) and 78.3% (c 2 = 5.591, p = 0.018), respectively ( Figure 5A-B ). The 5-year PFS of LS patients was 84·8%, which was significantly higher than that of HR gene mutation carriers (70.5%, c 2 = 9.971, p = 0.002) and FAP patients (50%, c 2 = 12.478, p < 0.001), but was comparable to CRC patients with other mutations (76.4%, c 2 = 2.045, p = 0.153). We observed a similar pattern in the 5-year OS. The 5-year OS of LS patients was 89·4%, which was higher than that of HR gene mutation carriers (81.2%, c 2 = 7.201, p = 0.007) and FAP patients (60.2%, c 2 = 16.676, p < 0.001), but was comparable to CRC patients with other mutations (81.7%, c 2 = 3.252, p = 0.071) ( Figure 5C-D ). The correlations between genetic risk factors and long-term OS and PFS rates indicated that the dMMR risk factor was associated with a better prognosis compared with other risk factors, and early age of onset predicted a poorer prognostic outcome ( Figure 5E, Supplementary Table 6 and Supplementary Table 7 ). Discussions Although genetic testing is more accessible nowadays, the coverage of genetic screening for CRC patients remains insufficient. This has led to high proportion of germline mutation carriers that remain unidentified, which also hinders the implementation of precision treatment and cancer prevention 35, 36 . This is the first study that enrolled high-risk hereditary CRC patients covering all categories of genetic high-risk factors for genetic testing. Through large-scale germline mutations screening, a high proportion of germline mutations were identified in high-risk hereditary CRC patients. The Each category of genetic high-risk factor is underlain by distinct germline mutations, in which LS and HR gene mutations were registered as the most frequent. Besides, we first discovered some HR gene mutation contributing to increasing cancer risk. In addition, patients carrying different germline mutations manifested heterogeneous phenotypes in clinicopathology, family cancer spectrum, cancer penetrance, and long-term prognoses. Our results showed that up to 20% of germline mutation carriers are detected in CRC patients with at least one genetic risk factors. Previous studies have demonstrated that the prevalence of CRC-susceptibility genes among unselected patients ranges from 3-10% 6, 37-39 . Thus, the criteria for patient enrollment in our study significantly improved the detection rates of germline mutation carriers. The inclusion of the rare genetic risk factors, i.e., multiple primary CRC and extra-colonic cancer, increased the probability of detecting germline mutations by more than two folds. Therefore, the genetic risk factor of multiple primary CRC and extra-colonic cancer should be considered as an independent indication for genetic testing. Looking into the detection rate for each category of genetic high-risk factors, we found that Tumor IHC manifesting dMMR alone predicted a close to 20% probability of having LS while any one additional risk factor increases the probability of LS to more than 40%. Even though family cancer history is a key indicator of whether a CRC patient harbors germline mutations, the proportion of germline mutation carriers with family cancer history is similar to that of early onset. In clinical practice, family cancer history and significant phenotypes, such as several adenomatous or hamartomatous polyps, are indications for doctors to recommend genetic testing. However, other risk factors that may predict the presence of germline mutations have not been systematically studied. In this study, we found MMR gene mutation was enriched in all risk groups, which indicated these risk factors are significantly associated with LS. In addition, as other gene mutations were frequently detected, genetic risk factors including early onset CRC, family cancer history and extracolonic CRC may be associated with other hereditary cancer syndromes. These results indicate that mutations in some cancer susceptibility genes may lead to overlapping phenotypes of various hereditary cancer syndromes. Our study also provides strong evidence for supplementing the guidelines for hereditary CRC genetic screening. In our cohort, more than 25% of all CRC patients had at least one of these five risk factors. dMMR tumors alone or in combination with one or more risk factors predicted a high probability (>20%) of harboring P/LP variants. We therefore highly recommend that patients with dMMR tumors have germline testing for CRC susceptibility genes, particularly the corresponding MMR genes. For patients with pMMR tumors or IHC not performed, early onset CRC, family cancer history and multiple primary CRC predicted a high probability (40%) of harboring P/LP variants. Therefore, germline testing is highly recommended for these groups of patients. CRC patients with extra-colonic cancer, early onset CRC, family cancer history or multiple primary CRC alone appeared to have a relatively low probability of carrying P/LP variants (<20%). However, genetic testing may still be recommended on an individualized basis depending on personal and family history ( Supplementary Figure 4 ). Thus, genetic screening is recommended for patients carrying the any one of the five categories of genetic risks. In the current study, we not only describe the landscape of germline mutation in those CRC patients with genetic risk factors, but also evaluated whether the frequently mutated genes increased the risk of CRC. The results showed that mutation in MMR genes, namely the LS, is the most prevalent hereditary CRC in China. Unexpectedly, the HR gene mutations were the second most frequently detected genetic abnormality in CRC patients. These findings not only expand our knowledge of germline mutations in hereditary CRC, but may also become the base for developing potentially differential treatment on the group of CRC patients carrying HR gene mutations. Mutations in HR pathway genes are frequently associated with ovarian, breast and pancreatic cancer 40 . In our study, the control-based analysis revealed that germline mutations in RAD50 , ATM, and BARD1 were associated with a moderately increased risk of CRC. However, although a previous study reported that BRCA1/2 mutations increased risk of CRC, we were unable to identify an association between BRCA1/2 mutations and CRC susceptibility 38 , which might be attributed to the fact that our screening strategy cannot effectively enrich patients with BRCA1/2 mutations. In addition, the large rearrangement was found in 11% of all BRCA1/2 mutation carriers 41 . Our sequencing panel cannot detect the large rearrangement of target genes, resulting in the prevalence of BRCA1/2 mutation carriers being underestimated. The pathogenicity and penetration of the HR gene mutations observed in current study have profound implications that warrant further investigation. In particular, the HR gene mutations as the second most prevalent ones in CRC patients, their carriers demonstrated resistance to first-line chemotherapy, for which targeted therapy such as PARP inhibition may be considered. The different germline mutations identified in our study have distinct impacts on the phenotype and long-term prognosis of CRC patients. In general, patients with germline mutations had improved OS and PFS as well as fewer metastatic lymph nodes. LS patients, particularly patients with MSH2 and MSH6 mutation, had better prognosis than other germline mutation carriers, which may be due to the fact that LS-associated tumors are typically dMMR and prior studies have found that dMMR tumors have a better prognosis than pMMR tumors when matched stage for stage 42 . Consistent with previous studies 43, 44 , our results demonstrated that LS patients have a significant family history of LS-associated cancer, early onset cancer, and a propensity for multiple primary CRC. Patients with P/LP variants in HR pathway genes tended to have a higher proportion of elevated serum CEA, metastatic lymph nodes and cancer nodules, and a lower proportion of multiple primary CRC. Compared to patients without germline mutations, HR pathway gene mutation carriers had worse PFS and OS rates, possibly due to the lack of targeted therapies for HR gene mutations in CRC 45 . FAP patients are readily identifiable due to their notable phenotype of multiple colonic adenomas 46 . Our results illustrated that FAP patients had the highest penetrance, the highest proportion of cancer nodules, BRAF V600E somatic mutation, multiple primary CRC and extra-colonic cancer, as well as the worst OS and PFS. There are several limitations to this study. First, our study may have underestimated the detection rates for germline mutations, as our PCR-based sequencing panel is not designed to detect large rearrangements 47-49 . Second, the cancer susceptibility assessment in this study was performed in the selected CRC population with genetic risks, while CRC-patients outside the five categories of genetic risk factors may potentially carry additional germline mutations that are not covered in our study. Third, biallelic somatic alterations were not analyzed, which may contribute to deviations in the molecular and clinical analysis, despite the strict filtering criteria. Last, we did not differentiate monoallelic/biallelic mutations in genes associated with autosomal recessive CRC syndromes. Conclusions In conclusion, this largest Chinese cohort study of high-risk hereditary CRC was designed as the first of its kind to cover five categories of genetic high-risk factors. A greatly expanded list of germline mutations were detected from the cohort, which underlie each category with distinct mutation rates and prevalence. CRC patients carrying different germline mutations manifested heterogeneous phenotypes in clinicopathology and long-term prognoses. In contrast to the MMR gene mutations of the LS, the study reveals for the first time at population level that carriers of germline mutations in the HR pathway genes are significantly susceptible to CRC, implicating HR pathway gene mutations as another major contributor for increased risk of developing CRC. Abbreviations CRC: Colorectal cancer; dMMR: deficient mismatch repair; FAP: familial adenomatous polyposis; FUSCC: Fudan University Shanghai Cancer Center; HR: homologous recombination; IHC: immunohistochemical; LS: Lynch syndrome; MMR: mismatch repair; OS: overall survival; PCR: polymerase chain reaction; PFS: progression-free survival; P/LP: Pathogenic/likely pathogenic; pMMR: mismatch repair proficient. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the Fudan University Shanghai Cancer Center, Shanghai, China. The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from patients for genomic analysis. Consent for publication Written informed consent for publication was obtained from all participants. Availability of data and materials The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA004231) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/browse/HRA004231. Data are available from the corresponding author upon reasonable request. Competing interests The authors declared no conflicts of interest to this work. Funding This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101). The funding source played no role in research design and collection, analysis, report writing, and the decision to submit articles for publication. Authors' contributions Yun Xu, Kai Liu and Cong Li contributed equally to our study, including study concept and design, sample collection, data analysis and drafting of the manuscript. Fangqi Liu, Minghan Li, Xiaoyan Zhou, and Menghong Sun participated in the sample collection, library preparation and statistical analysis. Megha Ranganathan revised the manuscript. Liying Zhang, Sheng Wang, Xin Hu and Ye Xu supervised the whole study and revised the manuscript. All the authors read and approved the final manuscript. Acknowledgements We would like to thank Professor Xuan Li from the Institute of Plant Physiology and Ecology at the Chinese Academy of Science for his invaluable assistance in reviewing and editing our manuscript. 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Bonadona V, Bonaïti B, Olschwang S, Grandjouan S, Huiart L, Longy M, Guimbaud R, Buecher B, Bignon YJ, Caron O, Colas C, Noguès C, Lejeune-Dumoulin S, Olivier-Faivre L, Polycarpe-Osaer F, Nguyen TD, Desseigne F, Saurin JC, Berthet P, Leroux D, Duffour J, Manouvrier S, Frébourg T, Sobol H, Lasset C, Bonaïti-Pellié C; French Cancer Genetics Network. Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA. 2011; 305: 2304-10. Hanna D, Chopra N, Hochhauser D, Khan K. The role of PARP inhibitors in gastrointestinal cancers. Crit Rev Oncol Hematol. 2022; 171: 103621. Byrne RM, Tsikitis VL. Colorectal polyposis and inherited colorectal cancer syndromes. Ann Gastroenterol. 2018; 31: 24-34. Rhees J, Arnold M, Boland CR. Inversion of exons 1-7 of the MSH2 gene is a frequent cause of unexplained Lynch syndrome in one local population. Fam Cancer. 2014; 13: 219-25. Mork ME, Rodriguez A, Taggart MW, Rodriguez-Bigas MA, Lynch PM, Bannon SA, You YN, Vilar E. Identification of MSH2 inversion of exons 1-7 in clinical evaluation of families with suspected Lynch syndrome. Fam Cancer. 2017; 16: 357-61. Liu Q, Hesson LB, Nunez AC, Packham D, Williams R, Ward RL, Sloane MA. A cryptic paracentric inversion of MSH2 exons 2-6 causes Lynch syndrome. Carcinogenesis. 2016; 37: 10-7. Table Table 1. The definitions of genetic high-risk factor. Genetic high-risk factor Illustration Early onset Diagnosed before age of 50 dMMR Tumor IHC manifesting dMMR Multiple primary CRC Synchronous and/or metachronous CRC at any age Primary hereditary cancer syndrome associated extra-colonic cancer Cancer associated with hereditary CRC: (1) Extra-colonic cancer including upper gastrointestinal: gastric, small bowel, and gastro-esophageal junction; (2) Gynecologic: uterine and ovarian; (3) Urogenital: bladder, renal, prostate; (4) Breast, hepatobiliary, pancreatic; (5) Hematolymphoid, neurologic and soft tissue Family history of cancer CRC and cancer associated with hereditary CRC susceptibility syndromes in first- and/or second-degree relatives at any age. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.pdf Supplementary Figure Legends Supplementary Figure 1. Flow chart of mutation calling and classification. Supplementary Figure 2. Relationship between risk factors and germline mutation. Supplementary Figure 3. Comparison of clinical outcomes between respective genotypes. Supplementary Figure 4. Establishment of genetic testing recommendations for patients carrying respective genetic risks. SupplementaryTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3174582","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":219216406,"identity":"68cf5799-3b1b-448a-b007-9c81ba7117d2","order_by":0,"name":"Yun Xu","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Xu","suffix":""},{"id":219216407,"identity":"49a8c7ea-be2a-44f1-a882-097bcff05b70","order_by":1,"name":"Kai Liu","email":"","orcid":"","institution":"Fudan University Shanghai Cancer 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08:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3174582/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3174582/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":40376933,"identity":"f24221a5-f237-4c7d-a73b-29b78d50fb65","added_by":"auto","created_at":"2023-07-21 13:35:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":325814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the study and sample enrollment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the study. †Samples from patients with complete response after neoadjuvant therapy were not tested with IHC. (B) The upset plot illustrated enrolled samples' distribution and basic clinical characteristics.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/67321253cd6115958133d6f0.png"},{"id":40374864,"identity":"fe3b152a-894c-48e0-9dfc-f10acd3e1138","added_by":"auto","created_at":"2023-07-21 13:19:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGermline mutation spectrum of 2,181 CRC patients with high genetic risk.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Landscape of P/LP germline mutations identified in CRC patients (n = 421), the 20 genes mentioned in NCCN guidelines are shown in the top part, and the other 12 genes are shown in the bottom part, #Heterozygous mutation. (B) According to mutation classification, count the frequency of mutations. (C) Overall detection rate of germline mutation and major gene pathways in high-risk patients (n=2,181). (D) Forest plot displayed the odds ratio of CRC susceptibility genes.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/814c8b63b94a31d991d79210.png"},{"id":40376035,"identity":"9dfa477e-a5d0-4547-9070-d7fb941a37e3","added_by":"auto","created_at":"2023-07-21 13:27:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":595981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGermline mutation spectrum and prevalence with different genetic risks.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Landscape of P/LP germline mutation identified in patients with five genetic risk groups. The red background indicated the highest number of patients with the mutation. (B) Sankey plot illustrated the correlation between gene mutations and genetic risk factors. (C) Overall detection rate of germline mutation and major gene pathways in high-risk patients (n=2181). The upset plot illustrated the enrolled sample’ distribution and germline mutation rate. (D) Relationship between dMMR tumors only or with at least one additional risk factor and the detection rate of germline mutation. (E) The bar chart shows the average detection rate of germline mutation in patients with pMMR and other risk factors.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/d593f30bd1b3aa87bba4fd7f.png"},{"id":40374862,"identity":"e5db4e2b-d780-4855-a169-95087424bc30","added_by":"auto","created_at":"2023-07-21 13:19:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":331145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical outcomes of patients carrying respective germline mutations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Correlation analysis between mutation types and clinical outcomes. (B) Comparison of the proportion of CRC primary location in respective germline mutations carriers. (C) Age of onset distribution in individual germline mutations carriers. (D) Comparison of the number of metastasized lymph nodes observed in respective germline mutation carriers.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/bb3a10a6cfb069d9ca7ebf06.png"},{"id":40374868,"identity":"2ace04f9-1df9-47c5-9910-54a552647721","added_by":"auto","created_at":"2023-07-21 13:19:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":258495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival analysis of respective molecular subtype.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Overall survival curves for CRC patients carrying germline mutation. (B) Recurrence-free survival curves for CRC patients carrying germline mutation. (C) Overall survival curves for CRC of respective germline mutation status. (D) Recurrence-free survival curves for CRC of respective germline mutation status. (E) Risk ratios analysis of risk factors for survival.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/1bf82aac3ff3c565562f6aae.png"},{"id":40385691,"identity":"406d5dea-b304-4922-aa4c-1e670418cd4a","added_by":"auto","created_at":"2023-07-21 14:44:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2003062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/4ed25c19-5714-45ca-93df-95617dcc37c5.pdf"},{"id":40376036,"identity":"e38484dc-b1cb-43bb-986f-942b1ad7df83","added_by":"auto","created_at":"2023-07-21 13:27:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2004854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure Legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. \u0026nbsp;Flow chart of mutation calling and classification.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Relationship between risk factors and germline mutation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Comparison of clinical outcomes between respective genotypes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. Establishment of genetic testing recommendations for patients carrying respective genetic risks.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/4933c6bf1621c5a6c6409d43.pdf"},{"id":40374866,"identity":"987c188f-6896-4aa6-b6c2-1a80052f6233","added_by":"auto","created_at":"2023-07-21 13:19:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":105030,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3174582/v1/e9a96e6c3d29568a556ce7d0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The largest Chinese cohort study indicates homologous recombination pathway gene mutations as another major genetic risk factor for colorectal cancer with heterogeneous clinical phenotypes","fulltext":[{"header":"Background","content":"\u003cp\u003eColorectal cancer (CRC) is one of the most common malignancies globally with an estimated 1.87 million new cases per year, and was the second leading cause of cancer-related death in 2020\u003csup\u003e1,2\u003c/sup\u003e. In China, there are over 400,000 new cases of CRC each year, and the incidence of CRC increased by 2.5% per year in males and 1.5% per year in females\u003csup\u003e2,3\u003c/sup\u003e. The current\u0026nbsp;known general\u0026nbsp;risk factors contributing to CRC include age, sex, inflammatory bowel disease, and CRC-susceptibility genetic variants\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp;Genetic factors were\u0026nbsp;reported to be\u0026nbsp;associated with more than 30% of the\u0026nbsp;incidence\u0026nbsp;of CRC\u003csup\u003e4\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003ebut due to insufficient genetic screening,\u0026nbsp;the germline mutations in high-penetrance CRC-susceptibility genes were found to account for only 5-10% of all CRCs\u003csup\u003e5,6\u003c/sup\u003e\u003csup\u003e.\u003c/sup\u003e As a result, a large proportion of CRC-susceptibility genes\u0026nbsp;remain\u0026nbsp;uncovered.\u003c/p\u003e\n\u003cp\u003eThe empirical indications for genetic screening on CRC-susceptibility genes are based on several significant genetic risk factors in the clinic, including early onset CRC, family history of cancer, and deficient mismatch repair (dMMR) in tumor tissues by immunohistochemical (IHC) staining\u003csup\u003e7-11\u003c/sup\u003e. While tumor IHC manifesting dMMR is indicative of a high possibility of Lynch syndrome (LS)\u003csup\u003e12\u003c/sup\u003e, family history of cancer\u003csup\u003e13\u003c/sup\u003e and early onset cancer\u003csup\u003e8,10,14\u003c/sup\u003e are hallmarks of inherited cancer susceptibility. The management guidelines for the indication of genetic screening were established and recommended by National Comprehensive Cancer Network (NCCN), based on the studies of genetic analysis in the CRC patients that manifested those significant genetic risk factors. However, using present NCCN guidelines, approximately 28% of pathogenic and likely pathogenic (P/LP) variants were still missed\u003csup\u003e15\u003c/sup\u003e. Many other genetic risk factors associated with hereditary CRC such as multiple primary CRC, and primary hereditary cancer syndrome associated extra-colonic cancer was rarely studied\u003csup\u003e5,16\u003c/sup\u003e.\u0026nbsp;The multiple primary cancer including synchronous and metachronous CRC, and extra-colonic cancers such as endometrial, ovarian and pancreatic\u0026nbsp;cancer were commonly observed in LS and some other hereditary cancer syndromes. However, due to insufficient supporting data, these two\u0026nbsp;genetic risk factors were not particularized in\u0026nbsp;the current\u0026nbsp;NCCN\u0026nbsp;guidelines. Thus, it is necessary to include these genetic risk factors in the investigation of CRC-susceptibility genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough many studies were reported on germline mutations for CRC patients with genetic risk factors, those works were mostly evaluating a patient group(s) focusing on one single genetic risk factor. There lack global views on genetic abnormalities that underlie the clinical high-risk CRC, and there is limited understanding on the association between germline mutations and the clinical genetic risk factors, and on how the germline mutations contribute to the clinical characteristics and long-term outcomes for CRC patients. To address these important questions, we designed the study to investigate the germline mutations underlying the five clinical genetic risk factors. early onset CRC, family history of cancer, dMMR of tumor tissues by IHC staining, multiple primary CRC, and primary hereditary cancer syndrome associated extra-colonic cancer. To our knowledge, this is the first study that covers CRC patients with five clinical genetic risk factors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy admitting consecutive 8270 cases of CRC patients at Fudan University Shanghai Cancer Center (FUSCC) over four-year period from 2015 to 2018, we constituted the largest Chinese cohort for hereditary CRC study. Our goal is to develop a comprehensive view on the landscape of genetic abnormalities in CRC patients with high-risk factors, and understand their contributions to clinical characteristics and outcomes for those carriers. \u0026nbsp;The results of the current study will lend support to expand genetic screening strategy for patients carrying respective genetic risk factor, which facilitates early detection, prevention, and treatment for hereditary CRC.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDefinitions of Hereditary High-risk Factors and Study Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the definitions of genetic high-risk factors were defined and illustrated in the \u003cstrong\u003eTable 1\u003c/strong\u003e. From January 1, 2015, to December 31, 2018, a total of 8270 CRC patients received treatment at the FUSCC. 2181 CRC patients with at least one of the genetic risk factors were retrospectively enrolled in the study. \u003c/p\u003e\n\u003cp\u003eThe IHC staining for MMR analysis was independently performed in the Department of Pathology at FUSCC. The majority of tumor samples were examined by IHC, although 386 tumors from patients who achieved complete response after receiving neoadjuvant therapy did not have IHC. Data including demographic information, family and medical history, pathology, and presenting symptoms were extracted from the electronic medical record. All patients were followed up as of September 30th, 2021. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUSCC Hereditary CRC Panel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe designed a multiplex polymerase chain reaction (PCR) amplification-based 38 gene FUSCC-hereditary cancer panel to detect germline mutations in eligible patients. The panel included 38 genes (containing \u003cem\u003eAPC\u003c/em\u003e,\u003cem\u003e ATM\u003c/em\u003e,\u003cem\u003e ATR\u003c/em\u003e,\u003cem\u003e AXIN2\u003c/em\u003e,\u003cem\u003e BARD1\u003c/em\u003e,\u003cem\u003e BLM\u003c/em\u003e,\u003cem\u003e BMPR1A\u003c/em\u003e,\u003cem\u003e BRCA1\u003c/em\u003e,\u003cem\u003e BRCA2\u003c/em\u003e,\u003cem\u003e BRIP1\u003c/em\u003e,\u003cem\u003e CDH1\u003c/em\u003e,\u003cem\u003e CDK4\u003c/em\u003e,\u003cem\u003e CHEK2\u003c/em\u003e,\u003cem\u003e CDKN2A\u003c/em\u003e,\u003cem\u003e EPCAM\u003c/em\u003e,\u003cem\u003e GALNT12\u003c/em\u003e,\u003cem\u003e GREM1\u003c/em\u003e,\u003cem\u003e MLH1\u003c/em\u003e,\u003cem\u003e MSH2\u003c/em\u003e,\u003cem\u003e MSH3\u003c/em\u003e,\u003cem\u003e MSH6\u003c/em\u003e,\u003cem\u003e MUTYH\u003c/em\u003e,\u003cem\u003e NTHL1\u003c/em\u003e,\u003cem\u003e PALB2\u003c/em\u003e,\u003cem\u003e POLD1\u003c/em\u003e,\u003cem\u003e POLE\u003c/em\u003e,\u003cem\u003e PIK3CA\u003c/em\u003e,\u003cem\u003e PMS2\u003c/em\u003e,\u003cem\u003e PTEN\u003c/em\u003e,\u003cem\u003e RNF43\u003c/em\u003e,\u003cem\u003e RPS20\u003c/em\u003e,\u003cem\u003e SMAD4\u003c/em\u003e,\u003cem\u003e STK11\u003c/em\u003e,\u003cem\u003e TP53\u003c/em\u003e,\u003cem\u003e NBN\u003c/em\u003e, \u003cem\u003eRAD50\u003c/em\u003e,\u003cem\u003e RAD51C\u003c/em\u003e, \u003cem\u003eRAD51D\u003c/em\u003e), 24 of them are commonly tested in multi-gene panels mentioned in NCCN guidelines (version 1. 2021)\u003csup\u003e17\u003c/sup\u003e and the remaining 14 genes are frequently mutated genes detected in other CRC cohorts\u003csup\u003e6,10,18\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eThe gene coordinates of the coding region of each gene in hg19 are extracted from the reference genome file. The primer design uses the overlapping tile covering method by using the software \u0026quot;primer 3\u0026quot; (version 0.4.0, https://bioinfo.ut.ee/primer3-0.4.0/) to ensure that the amplicons cover the coding region to the greatest extent. A custom library was prepared and primers were designed for all 602 coding exons (915 PCR amplicons) of these 38 genes including 180-280 bp of each flanking extron. Oligos were synthesized, primer droplets were prepared and all of these droplets were pooled together to create the custom library.\u003c/p\u003e\n\u003cp\u003eGenomic DNA was purified by the use of QIAamp\u0026reg; DNA Mini-kit (51104, QIAGEN, German) from white blood cells. A total of 20-200ng of genomic DNA was used for PCR amplification. The primer library and a template mix that included the fragmented genomic DNA and all of the components of the PCR reaction were loaded on GeneAmp\u0026reg; 9700 PCR (Applied Biosystem, The USA) and then amplified under the following conditions: 96\u0026deg;C for 3 minutes, 17 cycles of: (96\u0026deg;C for 30 seconds, 60\u0026deg;C for 4 minutes), 72\u0026deg;C for 4 minutes and then hold at 4\u0026deg;C. After amplification, the amplicons from PCR droplets were purified and quality controlled using the Qubit (Thermo Fisher Scientific, USA). PCR products were subsequently used for Illumina library preparation and sequenced using an Illumina NovaSeq 5000 platform (Illumina Inc., San Diego, CA, USA). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGermline Mutation Analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from frozen peripheral lymphocytes of all enrolled patients, and the mutational spectrum was identified using the FUSCC-hereditary CRC panel. The raw data of NGS was first filtered by removing Illumina sequencing adaptor and low-quality sequences. The remaining high-quality reads were mapped to the human reference genome (GRCh37) using the BWA aligner with the BWA-MEM algorithm and default parameters.\u003c/p\u003e\n\u003cp\u003eGermline mutations were called according to the following steps (\u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e). Single-nucleotide mutations were identified using Genome Analysis ToolKit (GATK, version 4.0)\u003csup\u003e19, 20 \u003c/sup\u003eand Varscan (version 2.4.2)\u003csup\u003e21\u003c/sup\u003e; insertions and deletions mutations were identified based on the union results of GATK and Pindel(version 0.2.5b8)\u003csup\u003e22\u003c/sup\u003e. The pathogenicity of the mutations reported in Clinvar\u003csup\u003e23\u003c/sup\u003e with at least two stars was used in this study. We used the results of InterVar\u003csup\u003e24 \u003c/sup\u003eannotation for the unreported mutations identified in this testing. We filtered the mutations using Genome Aggregation Database (gnomAD)\u003csup\u003e25\u003c/sup\u003e, 1000 Genomes Project\u003csup\u003e26\u003c/sup\u003e, and the Exome Aggregation Consortium (ExAC)\u003csup\u003e27\u003c/sup\u003e. Only rare mutations (MAF \u0026lt;0.01% in 1000G 2015Aug, ExAC or gnomAD exome database and \u0026lt;0.05% in the East Asian population) were selected for mutation classification. Some splicing and the stop gain mutations classified as Class 3 were upgraded to Class 4 (likely pathogenic)\u003csup\u003e28\u003c/sup\u003e. Only Class 4 and Class5 (pathogenic) mutations were selected for subsequent analysis. The prevalence of P/LP variants of MMR genes and \u003cem\u003eBRCA1/2\u003c/em\u003e genes in the general Chinese population were adopted from recent studies\u003csup\u003e29, 30\u003c/sup\u003e. The prevalence of P/LP variants of other genes was re-analyzed based on the ChinaMAP reference database\u003csup\u003e31\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical \u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003enalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeans (standard deviations) were calculated for continuous variables and percentages for categorical variables among different groups. Baseline clinical characteristics and germline mutation frequencies were compared using a two-sided Fisher exact test. Continuous variables were compared between two groups by the Wilcoxon test, and the Kruskal-Wallis H test was used to conduct comparative statistical studies on three or more groups. We used logistic regression to estimate the odds ratio for progression-free survival (PFS) and overall survival (OS) according to different risk factors. Kaplan-Meier curves were generated, and any differences in survival were evaluated with a stratified log-rank test. Hazard ratios and confidence intervals were estimated by Cox regression analysis. All statistical analyses were performed using R (version 4.0.2), Rstudio v.1.2 software, and SPSS software (version 21.0, SPSS Inc., Chicago, USA). All statistical analyses with p-value \u0026lt; 0.05 were considered statistically significant (\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001, N.S., not significant).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical characteristics of CRC patients associated with five different\u003c/strong\u003e\u003cstrong\u003e genetic\u003c/strong\u003e\u003cstrong\u003e high-risk factors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the germline mutations under all five genetic risk factors in CRC patients, we admitted 8270 consecutive CRC patients for the study over a four-year period between 2015 and 2018. We found a high proportion of patients carried genetic high-risk factors in our consecutive CRC patient cohort. A total of 2181 out of 8270 (26.4%) CRC patients enrolled at FUSCC met eligibility criteria. Early onset CRC patients accounted for 15.7% (1296/8270) of eligible patients, followed by patients with family cancer history (9.7%, 803/8270), dMMR tumors (4.8%, 401/8270), extra-colonic cancer (3.0%, 250/8270), and multiple primary CRC (2.3%, 187/8270) (\u003cstrong\u003eFigure 1A\u003c/strong\u003e). Of these enrolled patients, 72.6% (1583/2181) of patients had one genetic risk factor: 40.9% (893/2181) for early onset CRC, 20.1% (439/2181) for family history of cancer, 8.4% (183/2181) for dMMR tumors and 2.8% (61/2181) for extra-colonic cancer. Multiple primary CRC was rarely an independently observed risk factor and only present in 0.3% (7/2181) of patients (\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e 1B\u003c/strong\u003e). 27.4% (652/2181) of patients had at least two genetic risk factors. Patients of different genetic risk demonstrated heterogeneous clinicopathologic characteristics. Patients with multiple primary CRC most often had early onset CRC or family cancer history. Patients with dMMR tumors were more likely to have mucinous adenocarcinoma, right-sided and poorly differentiated tumors, and a lower proportion of TNM III/IV. Patients with early onset or multiple primary CRC had a higher percentage of TNM III/IV (\u003cstrong\u003eFigure 1B and \u003c/strong\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003e Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrevalence of germline mutations in CRC patients with genetic high-risk\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the germline mutations in CRC patients carrying genetic risk factors, a hereditary cancer susceptibility gene panel consisting of 38 genes was used to test the enrolled CRC patients. The P/LP variants were detected for 32 out of 38 genes in the panel. No P/LP variants were detected for the remaining six genes, i.e., \u003cem\u003eCDH1\u003c/em\u003e, \u003cem\u003eCDK4, GREM1\u003c/em\u003e, \u003cem\u003eGALNT12\u003c/em\u003e, \u003cem\u003eRPS20,\u003c/em\u003e and\u003cem\u003e BMPR1A\u003c/em\u003e, for the CRC cohort (\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003e Table 2\u003c/strong\u003e). P/LP variants were detected in 19.3% (421/2181) of CRC patients with at least one of the five risk factors for cancer susceptibility assessment. Specifically, 9.1% of patients (199/2181) carried a P/LP variant in the MMR pathway genes, and unexpectedly 4.2% of patients (141/2181) had a P/LP variant in the HR pathway genes. P/LP variants in \u003cem\u003eAPC\u003c/em\u003e were identified in 16 patients (0.73%, 16/2181). \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMLH1 \u003c/em\u003e(3.33%, 74/2181), \u003cem\u003eMSH2 \u003c/em\u003e(2.83%, 63/2181) and \u003cem\u003eMSH6 \u003c/em\u003e(2.21%, 49/2181) genes from the MMR pathway were the most frequently mutated genes in CRC patients. P/LP variants in CRC genes (NCCN guidelines) and \u0026ldquo;other genes\u0026rdquo; (not covered in the NCCN guidelines) were detected in 13.4% (335/2181) and 5.8% (126/2181) of our CRC patient cohort, respectively. P/LP with \u0026ldquo;other genes\u0026rdquo; that are not covered by the NCCN guidelines, contributed to more than 25% of all detected germline mutations (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). P/LP variants with truncation or missense mutations, were the predominant variant types for majority of these genes (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). P/LP variants in the moderate- and low-penetrance genes (labeled as \u0026ldquo;other genes\u0026rdquo; which are not covered in the NCCN guidelines) were detected in 3.0% of patients (65/2181) (\u003cstrong\u003eFigure 2C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo determine whether carrying P/LP variants in these genes confer an increased risk of CRC in the Chinese population, we performed a control-based risk analysis, which indicates that P/LP variants in the MMR pathway genes, \u003cem\u003eMLH1\u003c/em\u003e, \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003eAPC\u003c/em\u003e, \u003cem\u003eMSH6\u003c/em\u003e, \u003cem\u003eTP53, \u003c/em\u003eand \u003cem\u003ePMS2\u003c/em\u003e, were associated with an increased risk for CRC, consistent with what was known previously for LS\u003csup\u003e32-34\u003c/sup\u003e. However unexpectedly, P/LP variants that belong to genes in the HR pathway, i.e., \u003cem\u003ePOLE\u003c/em\u003e, \u003cem\u003eRAD50\u003c/em\u003e, \u003cem\u003eATM,\u003c/em\u003e and \u003cem\u003eBARD1\u003c/em\u003e, were also found to be associated with an increased risk for CRC in our cohort. While P/LP variants in \u003cem\u003eMLH1\u003c/em\u003e, \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003eAPC\u003c/em\u003e, \u003cem\u003eMSH6\u003c/em\u003e, and \u003cem\u003eTP53\u003c/em\u003e conferred a high-risk for CRC with odds ratio greater than 5, the P/LP variants in\u003cem\u003e PMS2\u003c/em\u003e, \u003cem\u003ePOLE\u003c/em\u003e, \u003cem\u003eRAD50\u003c/em\u003e, \u003cem\u003eATM,\u003c/em\u003e and \u003cem\u003eBARD1\u003c/em\u003e were associated with a moderate risk increase with odd ratios greater than 2 but smaller than 5 (\u003cstrong\u003eFigure 2D and \u003c/strong\u003e\u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e). These data for the first time reveal at population level that patients with germline mutations in the HR pathway genes are susceptible to CRC, suggesting mutations in HR pathway genes become another major contributor for increased risk of developing CRC.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eGermline mutations underlying different categories of genetic high-risk factors \u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eTo understand the underlying germline mutations of different genetic high-risk factors in CRC patients, we performed an association analysis between genetic high-risk factors and the detection rates of P/LP variants in CRC or other cancer susceptibility genes. As expected, dMMR tumors were associated with the highest detection rate of P/LP variants in MMR genes. Early age of onset was associated with the second highest risk for identifying MMR gene mutations in \u003cem\u003eMLH1\u003c/em\u003e, \u003cem\u003eMSH2, \u003c/em\u003eand \u003cem\u003eMSH6\u003c/em\u003e. Both multiple primary CRC and extra-colonic cancer were also associated with a high-risk for identifying MMR gene mutations. However unexpectedly, HR pathway gene mutations were enriched in early onset, family cancer history, and extra-colonic cancer risk groups. P/LP variants in \u003cem\u003eAPC\u003c/em\u003e,\u003cem\u003e POLE\u003c/em\u003e,\u003cem\u003e MUTYH\u003c/em\u003e,\u003cem\u003e TP53,\u003c/em\u003e and \u003cem\u003eAXIN2\u003c/em\u003e were also significantly enriched in the early onset risk group. (\u003cstrong\u003eFigure 3A, B\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThe prevalence of germline mutations in CRC patients was different for each genetic high-risk factor. When assessing individual high-risk factors, patients with dMMR tumors had the highest detection rate (23.5%, 43/183) of germline mutations, followed by the extra-colonic cancer group (14.8%, 9/61), multiple primary CRC (14.3%, 1/7), early onset CRC (11.4%, 102/893) and family cancer history group (9.1%, 40/439). The detection rate of germline mutations in most risk groups was greater than 10% (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). dMMR tumors with at least one additional high-risk factor predicted a significantly increased probability of harboring germline mutations, with detection rates of 40%-100%. Among patients with MMR proficient (pMMR) tumors or IHC not performed, patients carrying more risk factors predicted higher mutation detection rates, ranging from 10.8% to 18.4%. Notably three pMMR CRC patients had four risk factors, but no germline mutations were detected for them (\u003cstrong\u003eFigure 3D-E\u003c/strong\u003e). The detection rate of germline mutations in patients having 1-5 risk factors, ranges from 12.3% to 100% (\u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e 2\u003c/strong\u003e). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eClinical manifestations for different germline mutations \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the effects of germline mutations on clinical phenotype, we performed a correlation analysis between different gene mutations and clinical characteristics. Our data indicate patients carrying different germline mutations manifested heterogeneous clinicopathologic characteristics. The presence of MMR gene mutations was inversely correlated with age, serum CEA content, differentiation grade, vascular invasion, and TNM stage. On the other hand, HR gene mutations were found to be associated with the occurrence of extra-colonic cancer (\u003cstrong\u003eFigure 4A \u003c/strong\u003eand \u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e 3\u003c/strong\u003e). Multiple primary CRC was most frequently observed in familial adenomatous polyposis (FAP) patients, and right colon cancer was most common in LS patients. Colorectal tumors in patients with mutations in the HR genes were more often left sided (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). The age of onset of CRC in LS and FAP patients was significantly younger than that of patients who have no P/LP variants detected (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). LS patients with CRC had significantly fewer metastatic lymph nodes than CRC patients with HR pathway gene mutations (p=0.029). There was no statistically significant difference in the number of metastatic lymph nodes among CRC patients with other germline mutations (\u003cstrong\u003eFigure 4D, \u003c/strong\u003e\u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eTable 4 \u003c/strong\u003eand \u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePrognoses of CRC patients with different germline mutations and risk factors \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the effects of germline mutations on long-term outcomes, we performed survival analysis between patients with different gene mutations. Using the cohort of CRC patients in the current study that has a median follow-up time of 53.7\u0026plusmn;24.9 months, we analyzed the correlation between the presence of germline mutations and long-term PFS and OS. For CRC patients with at least one of the five risk factors in our study (n=2181), the 5-year PFS and OS were 71.0% and 79.8%, respectively. For CRC patients with germline mutations, the 5-year PFS and OS (77.1% and 83.1%, respectively) were significantly higher than those of patients without germline mutations, which were 69\u0026middot;8% (c\u003csup\u003e2 \u003c/sup\u003e= 9.976, p = 0.002) and 78.3% (c\u003csup\u003e2\u003c/sup\u003e = 5.591, p = 0.018), respectively (\u003cstrong\u003eFigure 5A-B\u003c/strong\u003e). The 5-year PFS of LS patients was 84\u0026middot;8%, which was significantly higher than that of HR gene mutation carriers (70.5%, c\u003csup\u003e2 \u003c/sup\u003e= 9.971, p = 0.002) and FAP patients (50%, c\u003csup\u003e2\u003c/sup\u003e = 12.478, p \u0026lt; 0.001), but was comparable to CRC patients with other mutations (76.4%, c\u003csup\u003e2\u003c/sup\u003e = 2.045, p = 0.153). We observed a similar pattern in the 5-year OS. The 5-year OS of LS patients was 89\u0026middot;4%, which was higher than that of HR gene mutation carriers (81.2%, c\u003csup\u003e2\u003c/sup\u003e = 7.201, p = 0.007) and FAP patients (60.2%, c\u003csup\u003e2\u003c/sup\u003e = 16.676, p \u0026lt; 0.001), but was comparable to CRC patients with other mutations (81.7%, c\u003csup\u003e2\u003c/sup\u003e = 3.252, p = 0.071) (\u003cstrong\u003eFigure 5C-D\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThe correlations between genetic risk factors and long-term OS and PFS rates indicated that the dMMR risk factor was associated with a better prognosis compared with other risk factors, and early age of onset predicted a poorer prognostic outcome (\u003cstrong\u003eFigure 5E, \u003c/strong\u003e\u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eTable\u003c/strong\u003e \u003cstrong\u003e6 \u003c/strong\u003eand\u003cstrong\u003eSupplementary \u003c/strong\u003e\u003cstrong\u003eTable 7\u003c/strong\u003e). \u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eAlthough genetic testing is more accessible nowadays, the coverage of genetic screening for CRC patients remains insufficient. This has led to high proportion of germline mutation carriers that remain unidentified, which also hinders the implementation of precision treatment and cancer prevention\u003csup\u003e35, 36\u003c/sup\u003e. This is the first study that enrolled high-risk hereditary CRC patients covering all categories of genetic high-risk factors for genetic testing. Through large-scale germline mutations screening, a high proportion of germline mutations were identified in high-risk hereditary CRC patients. The Each category of genetic high-risk factor is underlain by distinct germline mutations, in which LS and HR gene mutations were registered as the most frequent. Besides, we first discovered some HR gene mutation contributing to increasing cancer risk. In addition, patients carrying different germline mutations manifested heterogeneous phenotypes in clinicopathology, family cancer spectrum, cancer penetrance, and long-term prognoses.\u003c/p\u003e\n\u003cp\u003eOur results showed that up to 20% of germline mutation carriers are detected in CRC patients with at least one genetic risk factors. Previous studies have demonstrated that the prevalence of CRC-susceptibility genes among unselected patients ranges from 3-10%\u003csup\u003e6, 37-39\u003c/sup\u003e. Thus, the criteria for patient enrollment in our study significantly improved the detection rates of germline mutation carriers. The inclusion of the rare genetic risk factors, i.e., multiple primary CRC and extra-colonic cancer, increased the probability of detecting germline mutations by more than two folds. Therefore, the genetic risk factor of multiple primary CRC and extra-colonic cancer should be considered as an independent indication for genetic testing. \u003c/p\u003e\n\u003cp\u003eLooking into the detection rate for each category of genetic high-risk factors, we found that Tumor IHC manifesting dMMR alone predicted a close to 20% probability of having LS while any one additional risk factor increases the probability of LS to more than 40%. Even though family cancer history is a key indicator of whether a CRC patient harbors germline mutations, the proportion of germline mutation carriers with family cancer history is similar to that of early onset. In clinical practice, family cancer history and significant phenotypes, such as several adenomatous or hamartomatous polyps, are indications for doctors to recommend genetic testing. However, other risk factors that may predict the presence of germline mutations have not been systematically studied. In this study, we found MMR gene mutation was enriched in all risk groups, which indicated these risk factors are significantly associated with LS. In addition, as other gene mutations were frequently detected, genetic risk factors including early onset CRC, family cancer history and extracolonic CRC may be associated with other hereditary cancer syndromes. These results indicate that mutations in some cancer susceptibility genes may lead to overlapping phenotypes of various hereditary cancer syndromes.\u003c/p\u003e\n\u003cp\u003eOur study also provides strong evidence for supplementing the guidelines for hereditary CRC genetic screening. In our cohort, more than 25% of all CRC patients had at least one of these five risk factors. dMMR tumors alone or in combination with one or more risk factors predicted a high probability (\u0026gt;20%) of harboring P/LP variants. We therefore highly recommend that patients with dMMR tumors have germline testing for CRC susceptibility genes, particularly the corresponding MMR genes. For patients with pMMR tumors or IHC not performed, early onset CRC, family cancer history and multiple primary CRC predicted a high probability (40%) of harboring P/LP variants. Therefore, germline testing is highly recommended for these groups of patients. CRC patients with extra-colonic cancer, early onset CRC, family cancer history or multiple primary CRC alone appeared to have a relatively low probability of carrying P/LP variants (\u0026lt;20%). However, genetic testing may still be recommended on an individualized basis depending on personal and family history (\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e). Thus, genetic screening is recommended for patients carrying the any one of the five categories of genetic risks.\u003c/p\u003e\n\u003cp\u003eIn the current study, we not only describe the landscape of germline mutation in those CRC patients with genetic risk factors, but also evaluated whether the frequently mutated genes increased the risk of CRC. The results showed that mutation in MMR genes, namely the LS, is the most prevalent hereditary CRC in China. Unexpectedly, the HR gene mutations were the second most frequently detected genetic abnormality in CRC patients. These findings not only expand our knowledge of germline mutations in hereditary CRC, but may also become the base for developing potentially differential treatment on the group of CRC patients carrying HR gene mutations.\u003c/p\u003e\n\u003cp\u003eMutations in HR pathway genes are frequently associated with ovarian, breast and pancreatic cancer\u003csup\u003e40\u003c/sup\u003e. In our study, the control-based analysis revealed that germline mutations in \u003cem\u003eRAD50\u003c/em\u003e, \u003cem\u003eATM,\u003c/em\u003e and \u003cem\u003eBARD1\u003c/em\u003e were associated with a moderately increased risk of CRC. However, although a previous study reported that \u003cem\u003eBRCA1/2\u003c/em\u003e mutations increased risk of CRC, we were unable to identify an association between \u003cem\u003eBRCA1/2\u003c/em\u003e mutations and CRC susceptibility\u003csup\u003e38\u003c/sup\u003e, which might be attributed to the fact that our screening strategy cannot effectively enrich patients with \u003cem\u003eBRCA1/2\u003c/em\u003e mutations. In addition, the large rearrangement was found in 11% of all \u003cem\u003eBRCA1/2\u003c/em\u003e mutation carriers\u003csup\u003e41\u003c/sup\u003e. Our sequencing panel cannot detect the large rearrangement of target genes, resulting in the prevalence of \u003cem\u003eBRCA1/2 \u003c/em\u003emutation carriers being underestimated. The pathogenicity and penetration of the HR gene mutations observed in current study have profound implications that warrant further investigation. In particular, the HR gene mutations as the second most prevalent ones in CRC patients, their carriers demonstrated resistance to first-line chemotherapy, for which targeted therapy such as PARP inhibition may be considered. \u003c/p\u003e\n\u003cp\u003eThe different germline mutations identified in our study have distinct impacts on the phenotype and long-term prognosis of CRC patients. In general, patients with germline mutations had improved OS and PFS as well as fewer metastatic lymph nodes. LS patients, particularly patients with \u003cem\u003eMSH2 \u003c/em\u003eand \u003cem\u003eMSH6\u003c/em\u003e mutation, had better prognosis than other germline mutation carriers, which may be due to the fact that LS-associated tumors are typically dMMR and prior studies have found that dMMR tumors have a better prognosis than pMMR tumors when matched stage for stage\u003csup\u003e42\u003c/sup\u003e. Consistent with previous studies\u003csup\u003e43, 44\u003c/sup\u003e, our results demonstrated that LS patients have a significant family history of LS-associated cancer, early onset cancer, and a propensity for multiple primary CRC. Patients with P/LP variants in HR pathway genes tended to have a higher proportion of elevated serum CEA, metastatic lymph nodes and cancer nodules, and a lower proportion of multiple primary CRC. Compared to patients without germline mutations, HR pathway gene mutation carriers had worse PFS and OS rates, possibly due to the lack of targeted therapies for HR gene mutations in CRC\u003csup\u003e45\u003c/sup\u003e. FAP patients are readily identifiable due to their notable phenotype of multiple colonic adenomas\u003csup\u003e46\u003c/sup\u003e. Our results illustrated that FAP patients had the highest penetrance, the highest proportion of cancer nodules, \u003cem\u003eBRAF V600E\u003c/em\u003e somatic mutation, multiple primary CRC and extra-colonic cancer, as well as the worst OS and PFS. \u003c/p\u003e\n\u003cp\u003eThere are several limitations to this study. First, our study may have underestimated the detection rates for germline mutations, as our PCR-based sequencing panel is not designed to detect large rearrangements\u003csup\u003e47-49\u003c/sup\u003e. Second, the cancer susceptibility assessment in this study was performed in the selected CRC population with genetic risks, while CRC-patients outside the five categories of genetic risk factors may potentially carry additional germline mutations that are not covered in our study. Third, biallelic somatic alterations were not analyzed, which may contribute to deviations in the molecular and clinical analysis, despite the strict filtering criteria. Last, we did not differentiate monoallelic/biallelic mutations in genes associated with autosomal recessive CRC syndromes. \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this largest Chinese cohort study of high-risk hereditary CRC was designed as the first of its kind to cover five categories of genetic high-risk factors. A greatly expanded list of germline mutations were detected from the cohort, which underlie each category with distinct mutation rates and prevalence. CRC patients carrying different germline mutations manifested heterogeneous phenotypes in clinicopathology and long-term prognoses. In contrast to the MMR gene mutations of the LS, the study reveals for the first time at population level that carriers of germline mutations in the HR pathway genes are significantly susceptible to CRC, implicating HR pathway gene mutations as another major contributor for increased risk of developing CRC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRC: Colorectal cancer; dMMR: deficient mismatch repair; FAP: familial adenomatous polyposis; FUSCC: Fudan University Shanghai Cancer Center; HR: homologous recombination; IHC: immunohistochemical; LS: Lynch syndrome; MMR: mismatch repair; OS: overall survival; PCR: polymerase chain reaction; PFS: progression-free survival; \u0026nbsp;P/LP: Pathogenic/likely pathogenic; pMMR: mismatch repair proficient.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Fudan University Shanghai Cancer Center, Shanghai, China. The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from patients for genomic analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from all participants.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA004231) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/browse/HRA004231. Data are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflicts of interest to this work. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101). The funding source played no role in research design and collection, analysis, report writing, and the decision to submit articles for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYun Xu, Kai Liu and Cong Li contributed equally to our study, including study concept and design, sample collection, data analysis and drafting of the manuscript. Fangqi Liu, Minghan Li, Xiaoyan Zhou, and Menghong Sun participated in the sample collection, library preparation and statistical analysis. Megha Ranganathan revised the manuscript. Liying Zhang, Sheng Wang, Xin Hu and Ye Xu supervised the whole study and revised the manuscript. All the authors read and approved the final manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Professor Xuan Li from the Institute of Plant Physiology and Ecology at the Chinese Academy of Science for his invaluable assistance in reviewing and editing our manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209-49.\u003c/li\u003e\n\u003cli\u003eGBD 2017 Colorectal Cancer Collaborators. The global, regional, and national burden of colorectal cancer and its attributable risk factors in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. 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Carcinogenesis. 2016; 37: 10-7.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe definitions of genetic high-risk factor.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenetic high-risk factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIllustration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.5%\" valign=\"top\"\u003e\n \u003cp\u003eEarly onset\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.5%\" valign=\"top\"\u003e\n \u003cp\u003eDiagnosed before age of 50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.5%\" valign=\"top\"\u003e\n \u003cp\u003edMMR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.5%\" valign=\"top\"\u003e\n \u003cp\u003eTumor\u0026nbsp;IHC manifesting\u0026nbsp;dMMR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.5%\" valign=\"top\"\u003e\n \u003cp\u003eMultiple primary CRC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.5%\" valign=\"top\"\u003e\n \u003cp\u003eSynchronous and/or metachronous CRC at any age\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.5%\" valign=\"top\"\u003e\n \u003cp\u003ePrimary\u0026nbsp;hereditary cancer syndrome associated\u0026nbsp;extra-colonic cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.5%\" valign=\"top\"\u003e\n \u003cp\u003eCancer associated with hereditary\u0026nbsp;CRC:\u003c/p\u003e\n \u003cp\u003e(1) Extra-colonic cancer including upper gastrointestinal: gastric, small bowel, and gastro-esophageal junction;\u003c/p\u003e\n \u003cp\u003e(2) Gynecologic: uterine and ovarian;\u003c/p\u003e\n \u003cp\u003e(3) Urogenital: bladder, renal, prostate;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(4) Breast, hepatobiliary, pancreatic;\u003c/p\u003e\n \u003cp\u003e(5) Hematolymphoid, neurologic and soft tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.5%\" valign=\"top\"\u003e\n \u003cp\u003eFamily history of cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"62.5%\" valign=\"top\"\u003e\n \u003cp\u003eCRC and cancer associated with hereditary CRC susceptibility syndromes in first- and/or second-degree relatives at any age.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colorectal cancer, Cancer susceptibility, Genetic risk, Homologous recombination, Mismatch repair deficiency","lastPublishedDoi":"10.21203/rs.3.rs-3174582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3174582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Colorectal cancer (CRC) is one of the most common malignancies globally with estimated 1.87 million new cases annually. Genetic factors were associated with over 30% of CRC incidence. However, the mutations in CRC-susceptibility genes recommended by the National Comprehensive Cancer Network (NCCN) guidelines accounted for only 5-10% of CRC cases, suggesting a large proportion of CRC-susceptibility genes remain unknown. As previous works on hereditary CRC were largely designed to analyze germline mutations in patients with a single category of genetic high-risk factor, this study aims to explore the genetic mutations underlying five categories of genetic high-risk factors in clinic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eFrom January 2015 to December 2018, 2181 patients from a cohort of 8270 consecutive CRC cases were retrospectively enrolled, covering five categories of genetic high-risk factors. Their germline mutations under each category were detected and analyzed in association with CRC susceptibility, clinical phenotypes, and prognoses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eIn total 462 pathogenic/likely pathogenic genetic variants were detected in 19.3% CRC patients enrolled. Mutations in the mismatch repair (MMR) genes were identified in 9.1% patients, most prevalent across all high-risk groups. Mutations in homologous recombination (HR) pathway genes were detected in 6.5% patients, which were mostly penetrated in early onset, family cancer history and extra-colonic cancer risk groups. HR pathway gene mutations, including \u003cem\u003eBARD1\u003c/em\u003e, \u003cem\u003eRAD50\u003c/em\u003e and \u003cem\u003eATM\u003c/em\u003e, were associated with an increased risk of CRC in the cohort with an odds ratio of 2.8, 3.1 and 3.1-fold, respectively. CRC patients carrying different genetic mutations manifested heterogeneous phenotypes in clinicopathology and long-term prognoses, for which Lynch Syndrome demonstrated better prognoses than other groups, including those with HR pathway mutations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This largest Chinese cohort study of high-risk hereditary CRC is the first to cover five categories of genetic high-risk factors, which greatly expanded the list of CRC-susceptibility mutations. In contrast to the MMR mutations of Lynch syndrome, the study reveals for the first time at population level that carriers of mutations in the HR pathway genes are significantly susceptible to CRC, implicating HR pathway gene mutations as another major contributor for increased risk of developing CRC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration\u003c/strong\u003e: Retrospectively registered.\u003c/p\u003e","manuscriptTitle":"The largest Chinese cohort study indicates homologous recombination pathway gene mutations as another major genetic risk factor for colorectal cancer with heterogeneous clinical phenotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-21 13:19:21","doi":"10.21203/rs.3.rs-3174582/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af4dbde5-e81a-49c2-897e-0e1c08e10256","owner":[],"postedDate":"July 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-21T14:44:36+00:00","versionOfRecord":[],"versionCreatedAt":"2023-07-21 13:19:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3174582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3174582","identity":"rs-3174582","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}