Characteristics of MMR protein expression in colorectal cancer and MMR gene variations in Vietnamese patients with Lynch syndrome

Study design and population

This descriptive study was conducted among 218 patients diagnosed with colorectal carcinoma at the Center for Nuclear Medicine and Oncology, Bach Mai Hospital, Hanoi, Vietnam from January 2022 to June 2023. We conducted this study according to the STROBE guideline, please see Supplementary material for details.

This study was conducted in accordance with the Declaration of Helsinki (1964) and was approved by the Ethics Committee of Hanoi Medical University (Approval number 837/GCN-HĐĐĐNCYSH-ĐHYHN). After genetic counseling, all participants provided written consent for the anonymous reuse of their genomic data in this study. The genomic data were anonymized and aggregated for the cohort’s genetic analysis. Referring clinicians or participant interviews provided detailed information on personal and family cancer histories.

Data collection

The clinical and laboratory testing of patients with colorectal cancer were gathered. Immunohistochemical testing to evaluate the expression status of MMR proteins was conducted using Ventana Medical Systems (AZ, USA) and test kits VENTANA MMR RxDx panel (catalog number: MLH1: 760-5091, MSH2: 760-5093, MSH6: 760-5092, PMS2: 760-5094) on formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissue samples after biopsy or surgery.

Cases with a loss of MMR protein expression continued to undergo genetic testing to identify gene variants related to Lynch Syndrome (gene panel includes MLH1, MSH2, MSH6, PMS2, EPCAM, APC, MUTYH) using next-generation sequencing (NGS). The genetic variations examined included point mutations, deletions and short insertions (less than 10 nucleotides) in the coding region and the region adjacent to the intron (−10/+10 nucleotides from the exon) of the investigated genes. Variant detection cases were further confirmed using Sanger sequencing.

IHC assessment process

IHC was performed using the BenchMark Ultra (Roche Diagnostics, Mannheim, Germany) automated immunostainer. The slides were counterstained with haematoxylin. The following antibodies were used: anti MLH1 (clone M1, ready to use concentration: 1 μg/mL; catalog number 760-5091, Roche/Ventana), anti PMS2 (clone A16-4, ready to use concentration: 1 μg/mL; catalog number 760-5094, Roche/Ventana), anti-MSH2 (clone G219-1129, ready to use concentration: 20 μg/mL; catalog number 760-5093, Roche/Ventana) and anti-MSH6 (clone SP93, ready to use concentration: 1 μg/mL; catalog number 760-5092, Roche/Ventana). Normal colonic crypt epithelium adjacent to the tumor, lymphoid cells, and stromal cells were used as internal positive controls. Additionally, slide-positive controls are routinely employed in our IHC laboratory.

Sample preparation and sequencing

Each participant provided either a 2 mL peripheral blood sample or a buccal swab sample. Genomic DNA was extracted from blood samples using the GeneJet Whole Blood Genomic DNA Purification MiniKit (catalog number: K0781, ThermoFisher, USA). DNA fragmentation and library preparation were performed using the NEBNext Ultra II FS DNA Library Prep Kit (catalog number: E7435S, New England Biolabs, USA) following the manufacturer’s instructions. The libraries were pooled and hybridized with pre-designed probes targeting seven specific genes (MLH1, MSH2, MSH6, PMS2, EPCAM, APC, MUTYH) (Integrated DNA Technologies, USA). Massive parallel sequencing was conducted using the NextSeq 500/550 High Output Kits v2 (150 cycles) on the Illumina NextSeq 550 system (Illumina, USA), with a minimum target coverage of 100×.

Variant calling and analysis

Quality control and data processing were carried out as previously described [11]. In brief, paired-end reads were aligned to the human reference genome (GRCh38) using the Burrows–Wheeler Aligner (BWA) version 0.7.12 [12]. The resulting alignment was used to compute the coverage depth of the targeted regions, and variant calling was performed using GATK 3.8.0.0 [13]. Variants were annotated using the dbSNP (https://www.ncbi.nlm.nih.gov/snp) [14], ClinVar (https://www.ncbi.nlm.nih.gov/clinvar) [15], and LOVD (https://www.lovd.nl/3.0/home) [16] databases and their molecular impacts were evaluated using the Ensemble Variant Effect Predictor 110 [17].

Variants were classified according to the guidelines of The American College of Medical Genetics and Genomics (ACMG) [18]. The ACMG employs a five-tier classification system: “pathogenic,” “likely pathogenic,” “variant of uncertain significance,” “likely benign,” and “benign,” based on existing literature, databases, and computational tools.

Sanger sequencing

Sanger sequencing was performed to confirm all pathogenic variants identified by NGS. Primers flanking the mutation location were designed using Primer3Plus (https://www.primer3plus.com) [19] and synthesized by Integrated DNA Technologies, USA. PCR amplification was performed using the same genomic DNA samples, with Q5 High-Fidelity 2X Mastermix (New England Biolabs, USA) according to the manufacturer’s protocol. The PCR products were purified and sequenced using the Genetic Analyzer 3500xl (Applied Biosystems, USA).

Study outcomes

The primary outcome of our study was the prevalence and distribution of MMR gene variants in colorectal cancer patients.

Statistical analysis

Characteristics of the study population are presented as means and standard deviations for continuous variables. All categorical factors are presented as percentages. Using Fisher’s exact test for categorical variables. We compare clinical and laboratory characteristics among gene variant groups. Statistical analyses were performed using Stata 17.0 (StataCorp, USA).

Characteristics of the study population

Our study comprised 218 patients diagnosed with colorectal cancer, of whom 135 were men (61.9 %) and 83 were women (38.1 %) with ages ranging from 16 to 86 years old. The average age of the study participants was 59.9 and the age of diagnosis was typically over 50 years (accounting for 78.9 %). Of the 218 patients diagnosed with colorectal cancer, 32 had dMMR status. However, only 25 patients went on to undergo genetic testing. We found eight patients with pathogenic variants in the MMR gene and 17 patients with no known MMR gene variants (Figure 1).

Figure 1:

Overview of research results.

The prevalence and distribution of the MMR protein expression

In the study population, the patients with a loss of MMR expression (dMMR) accounted for 14.7 % (Figure 2).

Figure 2:

The prevalence of MMR protein expression in patients with colorectal cancer.

Among patients with dMMR, the proportion of patients with loss of MLH1 protein expression was 9.4 %; MSH2 was 3.1 %; MSH6 was 3.1 %; PMS2 was 21.9 %; MLH1/PMS2 was 46.9 %; and MSH2/MSH6 was 15.6 % (Table 1).

Here are three figures showing the results of MMR status assessment with different MMR protein loss patterns: loss of MLH1/PMS2 proteins (Figure 3), loss of MSH2/MSH6 proteins (Figure 4), and isolated loss of PMS2 (Figure 5).

Figure 3:

IHC staining images from a patient loss of expression of MLH1 and PMS2 proteins. (A) Loss of expression of MLH1 protein; (B) loss of expression of PMS2 protein; (C) normal expression of MSH2 protein; (D) normal expression of MSH6 protein.

Figure 4:

IHC staining images from a patient loss of expression of MSH2 and MSH6 proteins. (A) Normal expression of MLH1 protein; (B) normal expression of PMS2; (C) loss of expression of MSH2 protein; (D) loss of expression of MSH6. These images were captured from the same region of the tumor across four different slides and appear to be similar in certain areas.

Figure 5:

IHC staining images from a patient loss of expression of PMS2 protein alone. (A) Normal expression of MLH1 protein; (B) loss of expression of PMS2 protein; (C) normal expression of MSH2 protein; (D) normal expression of MSH6 protein.

The prevalence and distribution of MMR gene variants in dMMR patients

In 32 patients with dMMR, there was 25 patients agreed to have genetics testing. Of those, 32.0 % (8/25) patients carried pathogenic variants in MMR genes. In the group of patients with mutations in MMR genes, we detected 04 variations in the MLH1 gene, 01 variation in the MSH2 gene, 02 variations in the PMS2 gene, and 01 variation in the EPCAM gene – all classified as pathogenic variants, and causes of Lynch syndrome (Table 2). The most frequently affected gene is MLH1, with four cases, followed by PMS2 with three cases. EPCAM and MSH2 each have one case. This indicates a higher prevalence of MLH1 mutations in the studied population. The mutations include nonsense, frameshift, and missense variants. Frameshift mutations are the most common, appearing in four cases, which may suggest a significant impact on protein function leading to disease. The loss of MMR protein expression is consistent with the gene affected. For instance, MLH1 mutation result in the loss of MLH1 and PMS2 protein expression, while PMS2 mutation result in the loss of PMS2 expression. There is a strong familial component, with multiple cases reporting relatives who have had cancer, particularly colon cancer. This highlights the hereditary nature of these genetic variants and their association with cancer risk.

A 69-year-old female patient (L4) was diagnosed with colorectal cancer, histopathology showing an adenocarcinoma, immunohistochemistry staining revealing loss of MSH2-MSH6 protein expression. The patient’s medical history includes hypertension for 5 years, with no previous record of cancer in any organ. Family history includes mother’s death at 51 years old due to unknown illness, and the patient’s younger sister died at 49 years old due to colorectal cancer. Next generation sequencing test results detected a pathogenic variants on the MSH2 gene (c.2231_2235del) (p.Thr744AsnfsTer12) in a heterozygous state – Lynch syndrome – a hereditary colorectal cancer syndrome. Individuals with this gene variant are at risk for colorectal cancer, endometrial, gastric, and ovarian cancer. The patient received genetic counseling regarding the disease characteristics and cancer risks in other organs, and was advised to undergo genetic testing for family members to facilitate early screening and appropriate intervention (Figure 6).

Figure 6:

Pedigree of a patient (L4).

Characteristics of the study population

Among 218 patients diagnosed with CRC, men accounted for 61.9 % (135/218) and women for 38.1 % (83/218), so the male/female ratio was about 1.63. The incidence of colorectal cancer is higher in men than in women. The male/female ratio of studies in Vietnam ranges from 1.10 to 1.98 [20], 21]. The variation between studies may be owing to differences in sample size and inclusion criteria. Age is an important risk factor for colorectal cancer. Our study results indicated that the age of study participants ranged from 16 to 86 years; the average age was 59.9 and diagnosis typically occurred in those over 50 years, accounting for 78.9 %. Our study results are similar to other studies generated internationally and from Vietnam, revealing that the majority of colorectal cancers are detected after the age of 50 years [20], 22].

Screening methods to define high risk patients with Lynch syndrome

MSI-H/dMMR status has been linked to different features of CRC tumors, such as the primary tumor’s location, size, T stage, and spread to distant sites. Numerous retrospective studies have demonstrated a significant connection between dMMR/MSI-H status and early onset of the disease, larger tumor sizes, considerable tumor volume, primary tumor location, and advanced T stage in patients with stage I–III or I–IV CRC, including TNM stages [23]. MMR screening and MSI testing are crucial in diagnosing hereditary colorectal cancer syndrome; selecting treatment regimens for stage II/III colorectal cancer; and identifying indications for immunotherapy at the metastatic stage [24]. The proportion of dMMR patients in our study was 14.7 %, similar to the results of other studies in Vietnam (12.4 %) [25], China (13.8 %) [22], and the United States (15 %) [26].

In the typical dMMR case, immunostaining is completely absent for one of the two MMR protein heterodimers (MLH1/PMS2 or MSH2/MSH6), which represents by far the largest proportion, approximately 85 % of MSI-H/dMMR cases in the French dataset [27]. In our study, the percentage of patient with loss of MLH1 and PMS2 were 46.9 % and while the loss of MSH2 and MSH6 was 15.6 %. According to Hao et al., the most common finding was a simultaneous loss of expression of MLH1 and PMS2 (53.8 %), followed by a simultaneous loss of expression of MSH2 and MSH6 (30.8 %), loss of PMS2 expression accounts for the lowest proportion (15.4 %), and a single loss of MLH1, MSH2, MSH6 expression is not reported [25]. The majority of all non-typical findings consisted of isolated loss of PMS2 or MSH6 without loss of their respective heterodimerization partners MLH1 or MSH2 [27]. In our study, the proportion of patients with loss of MLH1 protein expression was 9.4 %; loss of MSH2 was 3.1 %; loss of MSH6 was 3.1 %; loss of PMS2 was 21.9 %. This difference may be owing to differences in study sample sizes and populations. Our study with a larger sample size observed patients with a single loss of expression of MLH1, MSH2, and MSH6. Isolated loss of PMS2 or MSH6 is typically due to germline mutations of the respective gene, and is therefore associated with Lynch syndrome [28].

The genetic testing to diagnose Lynch syndrome in dMMR patients

This is one of the studies in Vietnam using NGS techniques to analyze germline MMR gene variants and diagnose Lynch syndrome. The proportion of patients carrying mutations in our study was 32.0 % (8/25). This result is higher than in Li et al.’s study, in which the proportion was 23.5 % (154/656). This result may be because Li et al. tested both the dMMR and pMMR groups, whereas we conducted genetics testing only in the dMMR group [22].

In the eight patients diagnosed with Lynch syndrome among dMMR cases, the age of diagnosis ranged from 29 to 69 years, with two men and six women. All the patients had at least one relative (first-degree, second-degree, or third-degree) with a history of stomach cancer, colorectal cancer, rectal cancer, and premature death owing to illness (and not due to other causes such as accidents or war). This aligns with the established understanding of early onset of colorectal cancer and Lynch syndrome as described by Quach and Nguyen [29]. In a study by Sjursen et al. on 834 individuals with germline mutations in MMR genes, 221 different MMR variants were identified, including frame-shift mutations, nonsense mutations, insertions/deletions, missense mutations, and others [30]. Similarly, Pearlman et al. reported about 40 of 48 patients with MMR-deficient tumors had at least one mutation in a cancer susceptibility gene, and 37 patients had Lynch syndrome [31]. In Japan, Suzuki et al. conducted research on 119 young colorectal cancer patients (<50 years old), and eight patients were identified by the screening evaluations as candidates for germline MMR mutation analysis [32]. In Vietnam, according to research by Tran et al., which involved analyzing 17 genes in 1165 individuals across Vietnam who were either referred by physicians or self-enrolled in genetic testing, nine cases with genetic variants in the MLH1, MSH6, and PMS2 genes were identified. These mutations resulted in frameshift or nonsense mutations in the MMR genes [33]. This finding indicates that similar variants are present in the Vietnamese population as reported in the global literature. Apart from the study by Tran et al. [33], there is a notable absence of large-scale, comprehensive research in Vietnam addressing the prevalence of Lynch syndrome among colorectal cancer patients. Furthermore, Abu-Ghazaleh et al. highlighted that the interpretation of the prevalence data was hampered by poor reporting on key baseline characteristics of the patients, emphasizing the need for large, well-reported, epidemiological studies of Lynch syndrome prevalence [34]. According to Quach and Nguyen, the application of Bethesda and Amsterdam criteria for Lynch syndrome screening in 2012 was limited due to the unavailability of microsatellite instability testing methods and insufficient awareness of the disease in Vietnam [29]. While advancements have been made in testing methodologies, the general awareness of genetic diseases, particularly Lynch syndrome, is not fully established.

The association between MMR gene variants and loss protein expression

The pattern of nuclear staining depends on the specific gene involved, making it useful for predicting which gene carries a mutation. For instance, the absence of both MSH2 and MSH6 proteins in a tumor is associated with a germline mutation in MSH2 gene. In addition to subtle mutations impacting the MSH2 open reading frame, large deletions involving one or more exons are also a common cause of Lynch syndrome [35]. Our study results revealed that one patient had a mutation in the MSH2 gene, causing simultaneous loss of MSH2 and MSH6 expression; and two patients had a mutation in the PMS2 gene, causing loss of the corresponding protein PMS2 expression (Table 2). This result is consistent with previous studies reporting that loss of MSH2 expression is typically accompanied by loss of MSH6 expression and that expression and that the loss of PMS2 expression often occurs alone [31].

The patient who had lost MSH6 protein expression and carried EPCAM gene variant represents a case of a germline mutation in the EPCAM gene in Vietnamese individuals. On study in Mayo Clinic investigated the prevalence of EPCAM (TACSTD1) deletions in cases of Lynch Syndrome associated with MSH2. They found that 20–25 % of cases showed deletions suspected to affect MSH2, although no germline mutation was detected [21]. In dMMR patients, the most frequent constitutional epimutation involves hypermethylation of the MLH1 promoter on one allele, silencing gene expression across most somatic tissues, as well as MSH2 hypermethylation due to EPCAM gene mutation [36]. Families with Lynch syndrome who lack detectable sequence mutations in MMR genes should be evaluated for epigenetic mutations. Gene variants do not always correspond with loss of protein expression of the same gene and vice versa; therefore, genetic testing was highly recommended in the case of dMMR patients to confirm Lynch syndrome.

This research applies NGS technology for diagnosing Lynch syndrome in Vietnamese colorectal cancer patients with dMMR cases. We analyzed the association between MMR gene variants and loss of protein expression. Our study also has some limitations, first, selection bias may be present since only 25 out of 32 dMMR patients underwent genetic testing, potentially affecting the representativeness of our findings. Second, we did not assess the methylation status of genes, particularly the MLH1 gene. In addition, we only did genetics testing in dMMR group, so we could not examine the genetic variants in pMMR group. Future studies with larger prospective cohorts and genetic analysis of both dMMR and pMMR groups are need to further clarify the prevalence and genetic basis of Lynch syndrome in Vietnam.