[Detection of free tumor-related DNA in the serum of breast cancer patients].

OBJECTIVE: To study the APC and E-cadherin gene promoter hypermethylation as tumor marker and to investigate the correlation of free tumor-related DNA in serum and tumor tissue with clinicopathological parameters. Their feasibility in early diagnosis, predicting therapeutic effect and monitoring recurrence was evaluated. METHODS: 84 cases with operated breast cancer were recruited from March 2002 to August 2002 at Beijing Cancer Hospital. Aberrant methylation of E-cadherin and APC genes was detected in tumor tissues, adjacent normal tissues and peripheral blood serum by methylation-specific PCR (MSP). 10 cases with benign breast diseases were selected as control group. RESULTS: The positive rate of promoter hypermethylation of E-cadherin and APC genes in tumor tissues was 52.4% and 45.2%, in the paired serum was 33.3% and 31.0%, respectively. Aberrant methylation of free DNA in serum presented the same alteration in tumor tissues. E-cadherin and APC hypermethylation in serum and tumor samples significantly correlated each other (E-cadherin P < 0.001; APC P = 0.002). The sensitivity of detection of free DNA methylation of E-cadherin and APC genes in serum was 63.6% and 63.2%, respectively. The specificity was 100% and 95.7%, respectively. There was no correlation for the aberrant methylation in cancer tissues and serum with the clinicopathological parameters of patients including age, tumor staging, tumor size, histological type and receptor. None of the aberrant methylation was found in adjacent normal tissues and control group serum. CONCLUSION: The same aberrant methylation in cancer tissues and serum, not correlating with tumor staging, can be detected in about one third of breast cancer patients. The aberrant methylation in serum can disappear after operation. The results imply that this approach may be feasible for early diagnosis, evaluation of therapeutic effects and monitoring recurrence of breast cancers.

[Promoter hypermethylation of the p16 gene in pre- and post-gastrectomy plasma of patients with gastric adenocarcinoma].

OBJECTIVE: To detect promoter hypermethylation of the p16 gene in pre- and post-operative plasma, matched cancer tissues and para-tumor non-cancerous tissues of patients with gastric adenocarcinoma for evaluating the effectiveness of therapeutic intervention. METHODS: Primary tumor tissues and para-tumor tissues and preoperative plasma samples of 84 patients with gastric adenocarcinoma were collected, and 14-21 days' post-operative plasma of 30 of the 84 patients who underwent curative gastrectomy was available. Plasma of 15 healthy people was also collected as control. After sodium-bisulfite treatment, extracted DNA was amplified for p16 promoter hypermethylation by methylation-specific polymerase chain reaction (MSP). The PCR products were detected by both gel-ethidium bromide electrophoresis and high performance liquid chromatogram (HPLC). RESULTS: Among the samples from 84 patients, p16 hypermethylation was detected in 26 (31.0%) cancer tissues and 2 (0.02%) para-tumor non-cancerous tissues and 12 (14.3%) preoperative plasma, while plasma of the healthy people was negative. In all positive plasma, the paired primary tumor tissues were also confirmed to be methylated. As far as samples from 30 patients with post-operative plasma were concerned, 14 cancer tissues and 6 preoperative plasma samples were positive, and only 1 of 6 post-operative plasma remained hypermethylated. The results detected by HPLC exactly matched those by gel-ethidium bromide electrophoresis. CONCLUSION: Promoter hypermethylation of the p16 gene detected in plasma consists with that in primary cancer tissues of patients with gastric adenocarcinoma. The alteration of status of hypermethylation of p16 in post-operative plasma is considered the consequences of surgical intervention. HPLC can be used as an efficient tool in detecting the product of MSP.

[Detection of p53 gene mutation in the plasma of gastric cancer patients].

OBJECTIVE: To investigated p53 gene mutation in plasma of gastric cancer patients. METHODS: DNA extracted from plasma and matched tumor and tumor-adjacent normal tissues of 96 gastric cancer patients, and DNA from 20 healthy people were studied. Exons 5, 6, 7, and 8 of p53 were amplified by PCR. The mutation status was analyzed by denaturing high-performance liquid chromatography (DHPLC), followed by direct sequencing of cases with aberrant chromatographic patterns. RESULTS: Heterozygous mutations of p53 gene were detected in 19.9% (19/96) of primary tumor tissues and 5.2% (5/96) of corresponding plasma. All p53 gene mutations detected in plasma DNA consisted with mutations in the matched primary tumor samples. Neither the tumor-adjacent gastric mucosa tissues nor control plasma from healthy volunteers showed p53 gene mutation. No correlation was found between p53 mutation status and clinicopathological features of gastric cancer patients. CONCLUSION: p53 gene mutation in plasma can be detected in tissues and plasma of gastric cancer patients, which could be applied in screening and surveillance of this disease.

[Clinical significance of CASP8AP2 gene methylation in childhood acute lymphoblastic leukemia].

OBJECTIVE: To study the methylation level in the promoter of caspase 8 associated protein 2 (CASP8AP2) gene between samples at diagnosis and in complete remission, and to investigate its relationship with clinical features and prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: Diagnostic DNA samples from 109 newly diagnosed children with ALL admitted from August 2007 to March 2010, and 94 ALL children in CR (complete remission) among them were collected. Bisulfite modification and MethyLight method established by our research team were used to determine the methylation level of the two key CpG sites (at -1189 and -1176) of the promoter of CASP8AP2 gene. RESULTS: The average methylation level of the two CpG sites in newly diagnosted samples was higher than that in CR samples (71.1% ± 1.7% vs 64.2% ± 21.2%) (P = 0.008). Analysis with receiver operating characteristic (ROC) curve showed that the area under curve was 0.687 (P = 0.024), indicating that the methylation level of the two CpG sites was able to predict relapse efficiently to some extent, 76.9% was chosed as a cutoff value to divide the patients into high methylation group (49 patients) and low methylation group (60 patients). The incidence of relapse in high methylation group was higher than that in low methylation group (20.4% vs 6.7%) (P = 0.044), five year relapse free survival in high methylation group was also lower than that in low methylation group (Log rank, P = 0.033). Furthermore, high methylation at new diagnosis were correlated with high level of minimal residual disease (MRD) before consolidation therapy (P = 0.011). In the 34 children with MRD ≥ 10(-4) at the end of induction remission, the relapse rate of high methylation patients was significantly higher than that of low methylation patients (8/16 vs 3/18)(P = 0.038). CONCLUSION: The abnormal hypermethylation of the two CpG sites (at -1189 and -1176) of the promoter of the CASP8AP2 gene is possibly associated with leukemogenesis in childhood ALL. The treatment outcome is more poor in patients with hypermethylation than that in patients with low methylation. The combination of the methylation level of the two CpG sites and MRD level at the end induction remission is able to predict relapse more effectively.

Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol.

ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia.

DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of -1189 and -1176 was inversely correlated with mRNA expression (Spearman correlation: -0.333, P=0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P=0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.

[Detection and significance of APC gene promoter hypermethylation in serum of breast cancer patients].

BACKGROUND & OBJECTIVE: Detection of circulating tumor markers is one of current hot spots in tumor research. Free tumor DNA may exist in the peripheral blood of malignant tumor patients. Identical DNA mutations existing in both peripheral serum and primary tumor are found in many kinds of malignant tumors. This study used adenomatous polyposis coli (APC) gene promoter hypermethylation as a tumor marker to investigate the correlations of free tumor DNA to primary tumor and clinicopathologic features of breast cancer. METHODS: The methylation status of APC gene in tumor tissue, paracancer normal tissue, and paired peripheral serum from 84 patients with breast cancer and 10 patients with benign breast diseases were detected by methylation-specific polymerase chain reaction (MSP). RESULTS: The detection rate of APC gene promoter hypermethylation was 45.2% in tumor tissues and 31.0% in paired peripheral sera. APC gene hypermethylation in peripheral serum was significantly correlated to that in tumor tissue (r=0.977, P=0.002). The sensitivity of detecting APC gene hypermethylation in peripheral serum was 68.4%; the specificity was 97.8%. The aberrant methylation of APC gene in tumor tissue and peripheral serum had no correlation to patients' age, tumor stage, tumor size, histological type, and receptor status (P>0.05). No aberrant methylation of APC gene was found in the serum samples from healthy control and the patients without gene methylation in tumor tissue. CONCLUSION: The aberrant tumor gene methylation in peripheral serum of breast cancer patients is significantly correlated to that in primary tumor.