1 BACKGROUND

Colorectal cancer (CRC) is a common cancer worldwide. As of 2018, CRC was the second leading cause of cancer death affecting women (9.5%) and the third affecting men (10.9%) [1]. The etiology of CRC is both genetic and environmental. Accordingly, inherited susceptibility accounts for nearly one-third of CRC predispositions [2, 3], playing a crucial role in CRC risk [4, 5]. In developed countries, the average lifetime risk of CRC is approximately 2%–5% [6]. However, the lifetime risk of CRC increases up to 20% when the patient has a familial history of CRC and reaches 80%–100% in patients with hereditary CRC syndromes [4, 7, 8]. The hereditary risk factors of CRC are becoming a hot research topic globally.

Generally, hereditary CRC syndromes are divided into three subgroups: polyposis syndromes, nonpolyposis syndromes, and other syndromes related to CRC [9, 10]. The main type of polyposis syndrome is familial adenomatous polyposis (FAP), which is an autosomal-dominant CRC syndrome that is characterized by the formation of hundreds to thousands of adenomatous colorectal polyps in early adolescence. Attenuated familial adenomatous polyposis (AFAP) is a less-severe form of the disease showing fewer polyps. Other polyposis syndromes have also been defined, such as mutY DNA glycosylase (MUTYH)-associated polypos is (MAP). Above CRC hereditary syndromes are caused by corresponding mutations [APC regulator of the WNT signaling pathway (APC) in FAP/AFAP and MUTYH in MAP]. Another main subgroup is Lynch syndrome (LS), which is also called hereditary non-polyposis colorectal cancer (HNPCC). It is a common form of inherited CRC and accounts for 2.0%–5.5% of the overall CRC burden [4, 11, 12]. LS is an autosomal-dominant inherited disease that increases the risk of many types of cancer, such as endometrial cancer, urinary tract cancer, gastric cancer, and especially CRC [13, 14]. LS is related to mismatch repair (MMR) genes [MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, and PMS1 homolog 2 (PMS2)] and large deletions of 3′ end of epithelial cell adhesion molecule (EPCAM). Our gene testing panel also included PMS1 and MLH3, but we found no solid evidence to prove that germline mutations of PMS1 and MLH3 could cause a predisposition to LS [15-17]. Therefore, we removed these two genes from our analysis to avoid potential disputes. The third subgroup is comprised of juvenile polyposis syndrome (JPS), Peutz-Jeghers syndrome (PJS), phosphatase and tensin homolog (PTEN)-hamartoma tumor syndrome (PHTS) [18], and oligodontia-colorectal cancer syndrome (ODCRCS) [19], and is related to specific genes [bone morphogenetic protein receptor type 1A (BMPR1A) and SMAD family member 4 (SMAD4) in JPS, serine/threonine kinase 11 (STK11) in PJS, PTEN in PHTS, and axin 2 (AXIN2) in ODCRCS]. Previous studies have evaluated the prevalence of hereditary CRC syndromes mainly in Western countries, which were confined to several populations with specific syndromes, such as LS or MAP, and sequenced using a gene testing panel with limited genes for hereditary CRC [20-22]. Because these studies either emphasized restrictive CRC syndromes or lacked sufficient number of samples, a larger cohort for the prevalence assessment of inherited cancer susceptibility among CRC patients is needed.

Moreover, limited data, focusing on only MMR genes, have been published about germline hereditary susceptibility in Chinese CRC patients [23]. In our present research, we collected tumor samples from CRC patients diagnosed at the Sun Yat-sen University Cancer Center (SYSUCC; Guangzhou, Guangdong, China), who were recommended to undergo a hereditary CRC 14-gene screening. These 14 genes are associated with the syndromes and phenotypes of both nonpolyposis syndrome (LS) and some of the polyposis syndromes mentioned above. The current study mainly aimed to assess the prevalence of gene mutations related to cancer susceptibility among Chinese patients with CRC, to find the differences between Chinese and Western patients, and to explore the phenotype-genotype correlation. This comprehensive germline mutation assessment of Chinese patients with CRC may provide evidence support for clinical practice, facilitate primary prevention, and increase the health benefits of patients and their families.

2 MATERIALS AND METHODS

3 RESULTS

3.2 Mutational profiles of patients with CRC under 70 years

Out of the 526 patients, 77 (14.6%) had at least one mutation in one of the 12 genes listed in Figure 1A, covering seven diseases related to hereditary CRC. In total, 77 mutations were identified in 77 patients consisting exclusively of pathogenic mutations and likely pathogenic variants. Among the 526 patients, MLH1 was the most frequently mutated (n = 34, 6.5%), followed by MSH2 (n = 27, 5.1%), MSH6 (n = 5, 1.0%), and PMS2 (n = 4, 0.8%). These four genes are commonly associated with LS. Mutations in EPCAM, PTEN, BMPR1A, and SMAD4 were not detected in our cohort. Apart from LS-related genes, APC mutations were detected in 4 (0.8%) patients, and all mutations were associated with non-Lynch diseases accounted for 1.4% of all patients. Interestingly, no individuals with two or more mutations or likely pathogenic variants were found in our cohort. In addition, frameshift and nonsense mutations were the most common alterations (both with a frequency of 37.3%), followed by missense mutations (13.0%), splicing mutations (6.5%), and deletion mutations (5.2%). Among all 12 genes, only the MLH1 gene covered five mutational types.

A total of 262 VUSs were detected in 191 (36.3%) of the 526 patients (Supplementary Figure S1). The genes with high VUS frequencies were MUTYH (n = 50), MSH6 (n = 41), and MLH1 (n = 32). Among all 262 VUSs, 115 (43.9%) occurred in one of the MMR genes associated with LS. Sixteen patients with deficient MMR carried VUS in MMR genes that corresponded to their abnormal MMR protein levels, suggesting that these mutations may display potential pathogenicity (Supplementary Table S1).

The top five mutated genes in the old group were MLH1, MSH2, MSH6, PMS2, and AXIN2, while in the young group, the order was MLH1, MSH2, PMS2, MSH6, and APC; notably, the frequency of APC mutations was much higher in the young group than in the old group (1.5% vs. 0%, Figure 1B and 1C). Nonsense mutation was the most common mutation in the young group (40.4%), whereas frameshift mutation was the most common mutation in the old group (43.3%). Nevertheless, there was no significant difference in the distribution of mutation types between the two groups (P = 0.247, Figure 2A). The young group had a higher gene mutation rate than the old group (18.0% vs. 11.3%, P = 0.036, Figure 2B).

3.3 Comparison of mutation frequency between the present Chinese cohort and Western cohorts

To compare the mutation prevalence between Chinese and Western cohorts, we collected mutation data of 3411 patients from six published studies [31-36] (Supplementary Table S2), which were all diagnosed with inherited or family-related colorectal cancers. With the mutation statuses of four genes (MLH1, MSH2, MSH6, and PMS2) available in all six datasets, we performed a comparison of MMR gene mutation distributions (two-tailed Fisher's exact test) between our study subjects and those from published studies. As shown in Figure 3, the mutation fractions of the four genes were largely different between our study subjects and those reported by Bonadona et al. [31], Møller et al. [33], Moreira et al. [34], and Sjursen et al. [35] (all P < 0.01). In contrast to the majority of Western cohorts, patients with MSH6 and PMS2 mutations, which play less important roles in the hereditary risks of LS, accounted for a higher proportion (38.5%) in our Chinese dataset.

3.4 Associations between molecular features and clinicopathological characteristics

Correlation analyses were performed to explore the associations between mutated genes and clinicopathological features of the 526 patients, in which some clinicopathological features (personal history of cancer, family history of cancer, and TNM stage) were reclassified into two categories (Figure 4). Among all patients, those with MLH1 or MSH2 mutations had higher frequencies of personal history of cancer (MLH1: 20.6% vs. 8.7%; MSH2: 25.9% vs. 8.6%), family history of cancer (MLH1: 73.5% vs. 48.4%; MSH2: 70.4% vs. 48.9%), and right side primary cancer (MLH1: 73.5% vs. 29.3%; MSH2: 56.0% vs. 31.0%) than patients without these gene mutations. The proportion of stage I/II disease was higher in patients with MLH1 mutations than in those without MLH1 mutations (70.6% vs. 50.7%), and the rate of polyps was higher in patients with APC mutations than in those with wild-type APC (75.0% vs. 17.4%). Detailed reclassified data are provided in Supplementary Table S3. In addition, we explored the prognostic values of the mutations, but none of them had a significant ability to predict OS (Supplementary Figure S2). By the way, we only kept MLH1 and MSH2 in further analyses, excluding other genes, because patients with corresponding mutations were too low to support the survival analysis.

3.5 Gene exclusive analysis

Many disease-causing genes in cancer showed strong exclusiveness or co-occurrences in their mutation patterns. We analyzed the distribution of these mutations in the 77 patients who had at least one mutation in one of the 12 genes. A set of genes, including MLH1, MSH2, and MSH6, were found to be mutually exclusive in CRC (P < 0.01, Figure 5A). No patients harbored coincident mutations in this set of genes. This set of genes as a group was also confirmed to be mutually exclusive in mutation profiles (P < 0.01, Figure 5B).

4 DISCUSSION

Studies on hereditary CRC in China are limited. The present study provides a comprehensive description of the hereditary genetic risks of CRC among Chinese patients. In the current study, the proportion of hereditary CRC in CRC patients under 70 years old was 14.6% (77 of 526). Patients with MLH1 or MSH2 mutations had higher frequencies of personal/family history of cancer and right side primary cancer than patients without these gene mutations. Different from Western patients, Chinese patients had a higher rate of MSH6 and PMS2 mutations (38.5%). Mutations in MLH1, MSH2, and MSH6 were found to be mutually exclusive.

The mutation landscape in our cohort represented a particular pattern in which all patients harbored sole mutation. The mutation rate of non-MMR genes was 1.3% (7 out of 526), which displays the advantage of comprehensive multi-gene panel over the confined panel in detecting CRC hereditary gene mutations. More potential patients and their families harboring germline mutations would be found and consequently seek professional counseling by genetic experts to reduce future cancer risks. Patients in the young group had a significantly higher frequency of gene mutations than those in the old group (18.0% vs. 11.3%, P < 0.05), which is consistent with clinical observations. These data indicate the necessity to perform genetic testing in young patients to identify hereditary risks.

The molecular genetic background of many inherited CRC syndromes has been clarified [5, 7, 8]. LS is a well-described hereditary cancer syndrome caused by germline mutations in DNA MMR genes (MLH1, MSH2, MSH6, and PMS2) or by a deletion in the EPCAM gene, which regulates methylation of the MSH2 promoter [14, 37, 38]. We found that 13.3% of patients in the current study harbored LS-associated gene mutations.

The MMR gene mutation frequency between Chinese and Western patients was not in complete accordance. Our data presented that Chinese patients showed a much higher proportion of mutations of MSH6 and PMS2 genes than Western patients. On one hand, this discrepancy may be attributed to racial differences. On the other hand, this phenomenon indicates the importance of the database for annotation of gene mutations that contribute to corresponding CRC syndrome. With the development and enrichment of a gene mutation database, we may detect MSH6 and PMS2 mutations which were not detectable when western studies were published (before 2017). More panel testing data are required to improve the detection rate of pathogenic and likely pathogenic mutations.

Of note, several patients in our cohort did not show related clinical phenotypes for a specific syndrome, but the corresponding mutations were detected. For instance, 2 of 5 FAP/MAP patients lacked the phenotype of diffuse colorectal polyposis, which implies that the presence of polyps is not a perfect indicator for hereditary susceptibility [20]. MMR gene mutations accounted for a large proportion (90.9%) of all gene mutations. Some mutation carriers did not meet strict criteria for assessing hereditary CRC risks (e.g., Amsterdam criteria or Bethesda guidelines), suggesting that such history and phenotypic characteristics may have limited utility in identifying hereditary mutation carriers. In this circumstance, panel-based testing may be particularly more useful than syndrome-based testing as they could detect more patients with potential cancer risks. A recent study also suggested that a threshold should be discussed at which systematic gene panel testing would be more efficient irrespective of phenotype [32]. Considering cost-effectiveness, the loose standard of gene panel testing should be encouraged with decreasing sequencing costs for CRC patients.

In our cohort, APC mutations were highly associated with the polyp history, and MLH1 and MSH2 mutations were highly associated with a personal/family history of cancer. These findings were consistent with clinical observations and previous studies [39, 40]. Patients with MLH1 mutations were prone to be within the early TNM stage. A previous study observed better clinical outcome in patients with MLH1- or MSH2-negative CRC at stage II or III compared with MLH1- or MSH2-positive patients [41], suggesting that MLH1/MSH2 mutations may provide useful prognostic information for the management of stage II and III CRC patients.

The alterations of mutually exclusive genes that affect the same pathway tend not to co-occur in the same patients. MLH1 is the most important susceptibility gene for LS. The role of MLH1 in MMR has often been described as that of a "molecular matchmaker", which involves coupling mismatch recognition with downstream steps of MMR. hMutSa, the predominant mismatch-binding factor in humans, consists of two proteins, MSH2 and MSH6. MSH2 is the second most frequently detected MMR gene. MSH6 and MSH2 work through forming heterodimers, and MSH6 appears to be the subunit responsible for mismatch recognition in hMutSa complex [42]. MLH1 and MSH2 belong to the same pathway, the “mismatch pathway”. No patient had mutations of these three genes simultaneously, suggesting that pathogenic mutation of only one of them could be enough to promote CRC tumorigenesis.

As reported, IHC testing has approximately only 62%–78% sensitivity for identifying tumors with germline mutations [43]. In the present study, IHC sensitivities were high enough for identifying MLH1, MSH2, and PMS1 mutations, but extremely low for MSH6 mutations. The low sensitivity for MSH6 mutation identification may be explained by the majority of MSH6 mutations being VUSs, which may have possible pathogenicity [44].

The major strength of the present study includes the use of a large, consecutive cohort of Chinese patients along with detailed clinicopathological data. These data were used to perform an in-depth and comprehensive examination of their associations with the corresponding gene mutation profile. The use of a clinical laboratory with extensive experience in gene panel testing and professional interpretation of germline cancer susceptibility gene variants allowed for comprehensive and rapid analysis.

We also recognized the limitations in the present study. The major limitation was the clinic-based cohort with some high-risk features recruited from a large academic center, which may limit the generalization to population-based patients. Point mutations of PTEN, BMPR1A, and SMAD4 as well as truncated mutations in EPCAM were not found in our study. One possible reason is the sample size limitation of our cohort. The other reason may be the low mutation rates of these genes in the Chinese population. In addition, although our gene panel was comprised of the majority of high-penetrance cancer susceptibility genes (i.e., MMR genes, APC, MUTYH, STK11, PTEN, BMPR1A, and SMAD4) adopted by the majority of commercially available multigene panels, other emerging cancer susceptibility genes [such as DNA polymerase delta 1, catalytic subunit/ DNA polymerase epsilon, catalytic subunit (POLD1/POLE) [45-47], BRF1 RNA polymerase III transcription initiation factor subunit (BRF1) [48], and FANCD2 and FANCI associated nuclease 1 (FAN1) [49]] also show a promising future in the area of CRC susceptibility.

In conclusion, the present study used a multigene panel testing to display the comprehensive molecular landscape of hereditary CRC susceptibility in the Chinese population and the genotype-phenotype association within hereditary syndromes. Our results also suggest the necessity of multigene panel testing in CRC patients with or without high risks for identifying patients, as well as their relatives, with CRC susceptibility gene mutations for further supervision.

FUNDING

This work was supported, in part, by the National Key Research and Development Program of China (2018YFC1313300, 2017YFC1308900), National Natural Science Foundation of China (81930065, 81872011, 81903163), Science and Technology Program of Guangdong (2019B020227002), Science and Technology Program of Guangzhou (201904020046, 201803040019, 201704020228), Guangzhou Health and Medical Collaborative Innovation Project (201704020220), Guangdong Esophageal Cancer Institute Science and Technology Program (M201905), and the Sun Yat-sen University Clinical Research 5010 Program (2018014).

AUTHOR CONTRIBUTIONS

Conception and design: QZ, Feng W

Acquisition of data: QZ, TJJ, Fang W, YNW

Analysis and interpretation of data (including statistical analysis, biostatistics, computational analysis): QZ, TJJ, Fang W, PRD, ZZP, GC, JYS, JZL, Feng W, RHX.

Writing, review, and/or revision of the manuscript: Feng W, QZ, TJJ, QNW, Fang W.

Study supervision: Feng W, QZ.

All authors read and approved the manuscript for publication.

CONFLICT OF INTEREST

none declared.