OBJECTIVETo investigate the expression difference of DNA mismatch repair gene hMLH1 and hMSH2 between schistosomiasis-associated colorectal cancer and sporadic colorectal cancer.

METHODClinical and pathological data of colorectal cancer patients receiving operations in Zhejiang Cancer Hospital between January 2008 and December 2010 were retrospectively analyzed. Patients were divided into schistosomiasis group(n=80) and sporadic group (n=258) according to the preoperative history and pathologic results. Pathological specimens were collected and tissue chips were made to analyze the expression of hMLH1 and hMSH2 by immunohistochemistr.

RESULTSCompared with sporadic group, older age [(62.2 ± 9.6) year vs. (57.2 ± 11.7) year, P=0.000)], lower platelet level [(197.0 ± 59.6) × 10(9)/L vs. (217.0 ± 84.3) × 10(9)/L, P=0.02] and lower WBC level [(5.9 ± 1.9) × 10(9)/L vs. (6.6 ± 2.8) × 10(9)/L, P=0.02] were found in schistosomiasis group. Ratio of low differentiation-undifferentiation tumor was significantly higher in schistosomiasis group [44.2% (34/77) vs. 4.9% (12/247), P<0.05]. Lower positive rate of hMLH1 expression [77.5% (62/80) vs. 98.1% (253/258), P=0.000] and hMSH2 expression [75.0% (60/80) vs. 95.3% (246/258), P=0.000] was found in schistosomiasis group compared with sporadic group. Concurrent schistosomiasis was one of the risk factors of hMLH1/hMSH2 deficiency (RR: 0.913, 95% CI: 0.836-0.997, P=0.043), but not an independent factor (RR: 0.951, 95% CI: 0.867-1.043, P=0.286).

CONCLUSIONSchistosomiasis is associated with lower positive expression of hMLH1 and hMSH2, which indicates that hMLH1/hMSH2 deficiency may be a potential mechanism of schistosomiasis inducing carcinogenesis of colorectal cancer.

Adaptor Proteins, Signal Transducing ; Colorectal Neoplasms ; DNA Mismatch Repair ; Humans ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Nuclear Proteins ; Polymerase Chain Reaction ; Schistosomiasis

Objective: To investigate the relationship between the expression of four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) and NTRK genetic fusions in colorectal cancer. Methods: The paraffin-embedded tissue blocks of 830 cases of colorectal cancer were collected at the Affiliated Drum Tower Hospital, Nanjing University Medical School, China, from 2015 to 2019. Immunohistochemical and fluorescence in situ hybridization(FISH) method were used respectively to detect the expression of mismatch repair proteins and the break-apart of NTRKs; and the relationship between the expression of mismatch repair proteins and the NTRK genetic fusions was analyzed. Results: The overall mismatch repair protein deficiency (dMMR) rate was 9.88% (82/830), the mismatch repair proteins proficiency (pMMR) rate was 90.12%(748/830). The total deficiency rate of MLH1 protein was 9.04% (75/830), hPMS2 protein deficiency rate was 9.04% (75/830), MSH2 protein deficiency rate was 2.53% (21/830), MSH6 protein deficiency rate was 4.10% (34/830), the deficiency rate of synchronous MLH1 and PMS2 were 8.67% (72/830) and the deficiency rate of synchronous MSH2 and MSH6 were 2.17% (18/830). The dMMR group was associated with tumor location, different histological subgroups, tumor differentiation, AJCC stage and N stage (P<0.05). There were six cases (7.32%) carrying NTRK fusion by FISH among the 82 cases of dMMR, but only seven cases (0.94%) carrying NTRK fusion among the 748 cases of PMMR. The NTRKs translocation by FISH in all 13 cases were further confirmed by next generation sequencing. Among the clinicopathological characteristics, only differentiation showed significant difference between NTRK fusion positive and negative groups (P<0.05). More importantly, NTRK fusion was enriched in dMMR group (7.32% vs. 0.94%). Conclusion: In dMMR colorectal cancer group, the prevalence of NTRK fusion is higher than that in pMMR group.
Colonic Neoplasms ; Colorectal Neoplasms/genetics* ; DNA Mismatch Repair/genetics* ; Humans ; In Situ Hybridization, Fluorescence ; Mismatch Repair Endonuclease PMS2/metabolism* ; MutL Protein Homolog 1/metabolism* ; MutS Homolog 2 Protein/metabolism*

Lynch syndrome (LS), an autosomal dominantly inherited disease previously known as hereditary non-polyposis colorectal cancer (HNPCC), leads to a high risk of colorectal cancer (CRC) as well as malignancy at certain sites including endometrium, ovary, stomach, and small bowel (Hampel et al., 2008; Lynch et al., 2009). Clinically, LS is considered the most common hereditary CRC-predisposing syndrome, accounting for about 3% of all CRC cases (Popat et al., 2005). LS is associated with mutations of DNA mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, PMS2, and EPCAM (Ligtenberg et al., 2009; Lynch et al., 2009), which can trigger a high frequency of replication errors in both microsatellite regions and repetitive sequences in the coding regions of various cancer-related genes. Immunohistochemistry (IHC) tests followed by genetic analysis of these mutations play a significant role in diagnosis, treatment determination, and therapeutic response prediction of LS (Lynch et al., 2009; Alex et al., 2017; Ryan et al., 2017). Here, we report substitution of one base-pair in exon 1 of MLH3 (c.1397C>A) and a frameshift mutation in exon 19 of MLH1 (c.2250_2251ins AA) in a 43-year-old Chinese male with an LS pedigree.
Adult ; Asian People/genetics* ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis/genetics* ; Exons ; Female ; Frameshift Mutation ; Germ-Line Mutation ; Humans ; Male ; MutL Protein Homolog 1/genetics* ; MutL Proteins/genetics* ; Pedigree

OBJECTIVETo illustrate the role of methylation level of hMLH1 gene promoter in different stages of gastric carcinogenesis by methylation-specific PCR (MSP) detection of samples from paracancerous tissue and gastric cancer tissue.

METHODSMethylation status of hMLH1 gene promoter of 40 patients undergoing radical stomach cancer operation in the Tumor Research Institute of China Medical University between January 2006 and August 2006 was detected by MSP. For each patient, 2 samples were chosen from the cancer site, paracancerous tissues of 1 cm, 3 cm, 5 cm away from the cancer site, separately. One sample was used in pathology examination, and the other in methylation detection.

RESULTSPositive rates of hMLH1 gene promoter methylation in the paracancerous tissues of 1 cm, 3 cm, 5 cm away from the cancer site were 10%(4/40), 12.5%(5/40) and 2.5%(1/40) respectively, which were significantly lower than 32.5%(13/40) in cancer site(all P<0.05). Pathological examination showed precancerous lesions in 23 samples of paracancerous 1 cm and 3 cm tissues and normal tissues in 24 samples of paracancerous 5 cm tissues. Positive rates of hMLH1 gene promoter methylation in the cancer site, paracancerous tissue and normal gastric tissue were 32.5%(13/40), 8.7%(2/23) and 0(0/24) (P<0.01). For cancer tissue penetrated the gastric serosa, 8 out of 14 tissue samples were positive methylation (57.1%), which was significantly higher compared with 5 out of 26 tissue samples without penetration of gastric serosa(19.2%). Positive rate of hMLH1 gene promoter methylation in tissue samples with 7 or more of metastatic lymphatic node number was 61.5%(8/13), which was higher compared to that with less than 7(5/27, 18.5%) (P<0.05). No significant differences of positive rate of hMLH1 gene promoter methylation were found between different tumor gross types, tumor grow pattern, tumor differentiation degree, patient age and sex(all P>0.05).

CONCLUSIONHypermethylation of hMLH1 gene promoter may be associated with the carcinogenesis stages and progression of human gastric cancer.

Adaptor Proteins, Signal Transducing ; Cell Transformation, Neoplastic ; China ; DNA Methylation ; Disease Progression ; Humans ; MutL Protein Homolog 1 ; Nuclear Proteins ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Stomach Neoplasms

OBJECTIVETo gain an insight into the large intragenic hMSH2 and hMLH1 deletions in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) families.

METHODThe large intragenic hMSH2 and hMLH1 deletions in 17 probands of HNPCC families were detected with multiplex ligation-dependent probe Three large intragenic hMSH2 deletions of examplification (MLPA) and GeneMapper techniques.

RESULTSon 8, exon 1-6, and exon 1-7 were found in three families respectively, and no hMLH1 deletion was found. The deletions accounted for 19% of the total hMSH2 and hMLHI germline pathogenic mutations.

CONCLUSIONSThe incidence of large intragenic mismatch repair (MMR) genes deletions is relatively higher in Chinese families, and hMSH2 deletions may be more common. It is necessary to detect the large intragenic MMR genes deletions in the molecular detection of HNPCC.

Adaptor Proteins, Signal Transducing ; genetics ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; Female ; Gene Deletion ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Nuclear Proteins ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Pedigree

OBJECTIVETo gain an insight into large genomic deletions in mismatch repair genes MSH2 and MLH1 in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) patients in order to improve genetic detections of HNPCC kindreds.

METHODSFourteen peripheral blood DNA samples were obtained from 14 unrelated HNPCC patients, and fluorescent labeled quantitative multiplex PCR was used to detect large genomic deletions in MSH2 and MLH1 genes.

RESULTSOne of the fourteen probands, a man, was found to have MSH2 exon 1-7 deletions. His cancer-distressed son was also found to have the mutations. Additionally, three normal members of the family had the same mutations.

CONCLUSIONLarge genomic deletions which mainly present to MSH2 account for 20% of general pathological sequence changes of MSH2 and MLH1 genes in Chinese HNPCC patients, and large genomic detections of mismatch repair genes should be included in the regular genetic detections of Chinese HNPCC kindreds.

Adaptor Proteins, Signal Transducing ; genetics ; Asian Continental Ancestry Group ; genetics ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis ; ethnology ; genetics ; pathology ; Female ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Nuclear Proteins ; genetics ; Pedigree ; Polymerase Chain Reaction ; Sequence Deletion

OBJECTIVETo investigate the frequency of large fragment aberrations of MSH2 and MLH1 genes from Chinese colorectal cancer (CRC) patients with family history.

METHODSSixteen exons of MSH2, nineteen exons of MLH1 and seven DNA sequences from the other genes of the samples were screened and checked by multiplex ligation dependent probe amplification (MLPA). First, the methodology was confirmed by testing the positive and negative control samples. Then, 32 CRC or hereditary nonpolyposis colorectal cancer (HNPCC) patients with family history and 20 cases of sporadic CRC were applied to investigate for the large fragment aberrations of MSH2 and MLH1 genes.

RESULTSThe genomic DNA fragment deletions of all positive controls were identified and verified by MLPA. Three cases of 32 familial (hereditary) CRC/HNPCC were detected and identified to be the germline heterozygous deletions of MSH2 gene, of which exons 1-7 were deleted from patient No.3, exon 11 from No.25 and exons 2-6 from No.11. However, no genomic DNA fragment aberration of either MSH2 or MLH1 gene was uncovered from 20 sporadic CRC.

CONCLUSIONLarge DNA fragment aberrations of MSH2 gene was a frequent cause of Chinese HNPCC and CRC patients with family history, and the identification of those aberrations should be included in the regular genetic analysis for CRC/HNPCC patients.

Adaptor Proteins, Signal Transducing ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis ; ethnology ; genetics ; DNA Mutational Analysis ; Humans ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Mutation ; Nuclear Proteins ; genetics

OBJECTIVETo identify the MLH1 and MSH2 gene mutation in two hereditary nonpolyposis colorectal cancer (HNPCC) families.

METHODSPolymerase chain reaction and DNA sequencing were used to screen for MLH1 and MSH2 gene mutation, and PCR-restriction fragment length polymorphism and DNA sequencing were performed to confirm the mutation.

RESULTSBy DNA sequencing, a novel mutation of c.243_244insA located at the exon 3 of MLH1 gene was detected in family A, while c.1215_1218dupCCGA mutation located at the exon 7 of MSH2 gene was detected in family B. These two mutations can cause the formation of premature proteins.

CONCLUSIONThe novel mutations c.243_244insA in MLH1 gene and c.1215_1218dupCCGA in MSH2 gene were the disease-causing mutations in the two HNPCC families.

Adaptor Proteins, Signal Transducing ; genetics ; Asian Continental Ancestry Group ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; Female ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Mutation ; Nuclear Proteins ; genetics ; Pedigree ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

OBJECTIVESTo determine the germ-line mutations of hMSH2 and hMLH1 genes in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) families' probands or in patients fulfilling different clinical criteria or guidelines; to clarify the nature and distribution of the mutations; to evaluate the sensitivity of different clinical criteria in mutation prediction.

METHODSThe entire coding regions (35 exons including exon-intron boundaries) of hMSH2 and hMLH1 genes were directly sequenced in 24 Amsterdam criteria (AC) probands, 15 Japanese criteria (JC) probands (except AC kindreds) and 19 Bethesda guidelines (BG) patients (except two former groups). All available affected and unaffected members from families of those with mutations were screened for mutation.

RESULTSIn 16 unrelated families selected by the different clinical criteria, 17 germ-line mutations were found with 11 (64.7%) of hMLH1 and 6 (35.3%) of hMSH2. Two mutations were identified in one of the families. Among the 17 germ-line mutations, 12 had not been reported previously. A diversified mutation spectrum was found, but 6 hMLH1 mutations were found to be concentrated in the region encompassing exon 14, 15 and 16. There was a wide spectrum of mutation type including frame shift, nonsense, splice site mutation, in frame insertion or deletion and missense mutations. The mutation detection rate of hMSH2 and hMLH1 in the AC group was significantly higher than that in the JC group (12/24 vs. 3/15). On the other hand, a low mutation rate (1/19) was detected in 19 BG patients. The mutation cosegregated with disease. Besides, three different genotypes in tumors from probands of mutation-positive families were found.

CONCLUSIONShMSH2 and hMLH1 mutations in Chinese HNPCC families show a wide spectrum. It seems that hMLH1 gene is involved more frequently than hMSH2 gene in Chinese HNPCC families. Different clinical criteria predict mutations with different sensitivities. The Amsterdam Criteria are most sensitive, while Japanese Criteria are highly practical and the Bethesda Guidelines are also practical to some extent. Gene mutations cosegregate with the disease phenotype. Carriers with no symptom in HNPCC families are most vulnerable groups, follow-ups are required for this group to get early diagnosis and to prevent the development of CRCs.

Adaptor Proteins, Signal Transducing ; Carrier Proteins ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA-Binding Proteins ; Germ-Line Mutation ; Humans ; Microsatellite Repeats ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Neoplasm Proteins ; genetics ; Nuclear Proteins ; Pedigree ; Proto-Oncogene Proteins ; genetics

OBJECTIVETo study the protein expression pattern of DNA mismatch repair genes hMSH(2), hMLH(1) and the microsatellite instability (MSI) status in the tumor tissue from hereditary nonpolyposis colorectal cancer in the Chinese.

METHODSFifty-eight families fulfilling different clinical criteria including Amsterdam Criteria (AC) (22/24 families, 38 tumors), Japanese Criteria (JC) (12/15 families, 16 tumors) and Bethesda Guidelines (BG) (12/19 patients, 13 tumors) were studied. Monoclonal antibodies against hMSH(2), hMLH(1) proteins and a panel of microsatellite markers (5 loci) including BAT26, BAT25, D2S123, D5S346 and D17S250 were used for study.

RESULTSMSI-H was identified in all 22 (100%) AC tumors, with 81.8% (18/22) showing altered hMSH(2) or hMLH(1) expression; in 14/15 (93.8%) JC cancer, 1/1 (100%) JC adenoma, with 45.5% (5/11) showing altered hMSH(2) or hMLH(1) expression; and in 7/13 (53.8%) BG tumors, with 4/7 showing loss of hMSH(2) or hMLH(1) gene expression.

CONCLUSIONThe frequency of MSI-H and loss of mismatch repair protein are different in the families fulfilling different clinical criteria. Amsterdam Criteria and Japanese Criteria are the two most useful criterion systems for identifying mismatched repair defective tumors. However, Bethesda Guidelines should also be used for detecting more such tumors. The combination of immunohistochemical methods and microsatellite instability analysis is an effective strategy to detect the mismatch repair defective tumors. A close correlation does exist between hMSH(2), hMLH(1) protein expression pattern and MSI status.

Adaptor Proteins, Signal Transducing ; Base Pair Mismatch ; Carrier Proteins ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA Repair ; DNA-Binding Proteins ; Humans ; Immunohistochemistry ; Microsatellite Repeats ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Neoplasm Proteins ; genetics ; Nuclear Proteins ; Proto-Oncogene Proteins ; genetics