Analysis of microsatellite instability in stool DNA of patients with colorectal cancer using denaturing high performance liquid chromatography

AIM： To evaluate the usefulness of denaturing high performance liquid chromatography （DHPLC） for analyzing microsatellite instability （MSI） status in stool DNA of patients with colorectal cancer. METHODS： A total of 80 cancer tissues from patients with primary sporadic colorectal tumor （proximal cancer： 27, distal cancer： 53） and matched stool （which were employed for comparison with the tissues） were analyzed for MSI status in BAT 26. DNA samples extracted from stool were evaluated by nested polymerase chain reaction （PCR） and DHPLC for MSI analysis. RESULTS： Six cases （7.5%） of MSI were identified in BAT 26 from 80 cancer tissues. All the stool DNA samples from patients whose cancer tissue showed IVlSI also displayed MSI in BAT 26. CONCLUSION： As MSI is one of the established fecal DNA markers to screen colorectal cancer, we propose to use DHPLC for the IVlSI analysis in fecal DNA.

Germline mutation analysis of MLH1 and MSH2 in Malaysian Lynch syndrome patients

AIM: To investigate the protein expression profi le of mismatch repair (MMR) genes in suspected cases of Lynch syndrome and to characterize the associated germline mutations. METHODS: Immunohistochemical analysis of tumor samples was performed to determine the protein expression profile of MMR protein. Germline mutation screening was carried out on peripheral blood samples. The entire exon regions of MLH1 and MSH2 geneswere amplifi ed by polymerase chain reaction, screened by denaturing high performance liquid chromatography (dHPLC) and analyzed by DNA sequencing to characterize the germline mutations. RESULTS: Three out of 34 tissue samples (8.8%) and four out of 34 tissue samples (11.8%) showed loss of nuclear staining by immunohistochemistry, indicating the absence of MLH1 and MSH2 protein expression in carcinoma cells, respectively. dHPLC analysis followed by DNA sequencing showed these samples to have germline mutations of MSH2 gene. However, no deleterious mutations were identifi ed in any of the 19 exons or coding regions of MLH1 gene, but we were able to identify MLH1 promoter polymorphism, -93G > A (rs1800734), in 21 out of 34 patients (61.8%). We identified one novel mutation, transversion mutation c.2005G > C, which resulted in a missense mutation (Gly669Arg), a transversion mutation in exon 1, c.142G > T, which resulted in a nonsense mutation (Glu48Stop) and splice-site mutation, c.2006-6T > C, which was adjacent to exon 13 of MSH2 gene. CONCLUSION: Germline mutations were identified in four Malaysian Lynch syndrome patients. Immunohistochemical analysis of tumor tissue proved to be a good pre-screening test before proceeding to germline mutation analysis of DNA MMR genes.

Expression of pituitary homeobox 1 gene in human gastric carcinogenesis and its clinicopathological significance

AIM： To investigate the effect of pituitary homeobox 1 （PITX1） expression in cases of human gastric cancer on cancer differentiation and progression, and carcinogenesis. METHODS： Using polyclonal PITX1 antibodies, we studied the expression of PITX1 in normal gastric mucosa, atypical hyperplasia, intestinal metaplasia, and cancer tissue samples from 83 gastric cancer patients by immunohistochemistry. Moreover, semi-reverse transcription polymerase chain reaction （semi-RT-PCR） was performed to detect the mRNA level of PITX1 in three gastric cancer cell lines and a normal gastric epithelial cell line. Subsequently, somatic mutations of the PITX1 gene in 71 gastric cancer patients were analyzed by a combination of denaturing high performance liquid chromatography （DHPLC） and DNA sequencing. RESULTS： Immunohistochemistry showed that PITXl was strongly or moderately expressed in the parietal cells of normal gastric mucosa （100%）, while 55 （66.3%） out of 83 samples of gastric cancers showed decreased PITXl expression. Moreover, PITXl expression was reduced in 20 out of 28 cases （71.5%） of intestinal metaplasia, but in only 1 out of 9 cases （11%） of atypical hyperplasia. More importantly, PITXl expression was significantly associated with the differentiation, position and invasion depth of gastric cancers （r = -0.316, P 〈 0.01; r = 0.213, P 〈 0.05; r = -0.259, P 〈 0.05, respectively）. Similarly, levels of PITXl mRNA were significantly decreased in 2 gastric cancer cell lines, BGC-823 and SGC-7901, compared with the normal gastric epithelial cell line GES-1 （0.306 ± 0.060 vs 0.722 ± 0.102, P 〈 0.05; 0.356 ± 0.081 vs 0.722 ± 0.102, P 〈 0.05, respectively）. Nevertheless, no somatic mutation of PITX1 gene was found in 71 samples of gastric cancer by DHPLC analysis followed by sequencing. CONCLUSION： Down-regulation of PITX1 may be a frequent molecular event in gastric carcinogenesis. Aberrant levels of PITXl expression may be closely correlated with the progression and differentiation of gastric cancer,

Trial of the correlation between cytochrome oxidase CYP3A4 with the susceptibility of paclitaxel-based regimen for advanced gastric cancer

Objective: The aim of the study was to investigate the relationship between susceptibility of paclitaxel-based regimen and gene polymorphisms of cytochrome oxidase CYP3A4 for advanced gastric cancer. Methods: Peripheral venous blood sample of 53 advanced gastric cancer patients were enrolled to test the mutation of CYP3A4 gene by denaturing high performance liquid chromatography(DHPLC) and DNA sequencing. The relation between the efficacy of paclitaxel-based regimen and CYP3A4 gene polymorphisms was further analyzed. Results: DHPLC indicated that among the 53 patients, 21 cases showed biomodal type(mutation) and 32 cases were of unimodal type(wild-type). Sequencing results showed that the deletion mutation was found at the 27 th basic group of C in exon 10 of CYP3A4 gene. The response rate(RR) and disease control rate(DCR) of wild-type group were 40.6% and 84.4%, while in mutation group they were 33.3% and 85.7%, respectively, with no significances between the two groups(P > 0.05). Of all 53 cases, the median progression-free survival(PFS) was 6.5 months(95% CI: 3.576–9.424 months), and the median overall survival(OS) was 11.0 months(95% CI: 6.955–15.045 months). The median PFS and OS in wild-type group had no differences compared with those in mutation group(7.0 months vs. 7.0 months, P > 0.05; 10.0 months vs. 14.0 months, P > 0.05). Between wild-type and mutation groups, the median PFS of patients applied with oxaliplatin containing regimen and the median OS in patients applied with/without oxaliplatin had no significant differences(P > 0.05), while the median PFS in patients received non-oxaliplatin regime had statistical differences(P = 0.024). The median PFS and OS in patients receiving 3-drug or 2-drug regimes had no correlation with CYP3A4 gene polymorphisms. The adverse effects in the two groups were mild, mainly in grades 1–2. The common adverse effects were anorexia, nausea/vomiting and leucopenia. Conclusion: Deletion mutation was located in the 27 th basic group of C in exon 10 of CYP3A4 gene. Paclitaxel-based regime has a trend to prolong the OS of advanced gastric cancer with mutation type.