Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker

Abstract

BackgroundSquamous cell carcinoma of esophagus (SCCE) occurs at a high incidence rate in certain parts of the world. This feature necessitates that different aspects of the disease and in particular genetic characteristics be investigated in such regions. In addition, such investigations might lead to achievement of molecular markers helpful for early detection, successful treatment and follow up of the disease. Adenomatous Polyposis Coli (APC) promoter hypermethylation has been shown to be a suitable marker for both serum and solid tumors of adenocarcinoma of esophagus. We investigated the status of APC promoter hypermethylation in Iranian patients, compared the results with the former studies, and evaluated its applicability as a candidate molecular marker by examining association between survival of SCCE patients and APC promoter methylation.MethodsFor evaluating the status of APC promoter hypermethylation and its association with SCCE, a qualitative methylation specific PCR (MSP) was used. DNA was extracted and digested with an appropriate restriction enzyme, treated with sodium bisulfite in agarose beads and amplified in two-step PCR reaction by applying either methylated or unmethylated promoter specific primers. Universally methylated DNA and methylase treated blood DNA of healthy donors were used as positive controls as well. Survival of patients was followed up for two years after treatment and survival rate of patients with methylated APC promoter was compared with that of unmethylated patients.ResultsAssessment of APC promoter methylation revealed that normal tissues were unmethylated, while twenty out of forty five (44.4%) tumor tissues were hypermethylated either in one or both alleles of APC. Among the tissues in which methylation was detected, seven were hypermethylated in both alleles while the other thirteen were hypermethylated in one of the two alleles of APC. Analyzing two-year survival rate of patients with respect to promoter hypermethylation showed a lower rate of survival for patients with methylated APC promoter following their treatment. Further investigation into the association between promoter hypermethylation and tumor differentiation status indicated that patients with well differentiated tumors were more likely to develop promoter hypermethylation.ConclusionObserving similar level of APC promoter hypermethylation in patients with SCCE in this high risk region and comparing it with other parts of the world could support the hypothesis that a common molecular mechanism might be involved in tumorigenesis of SCCE. In addition, the higher rate of two-year survival for patients with unmethylated APC promoter as well as its relationship with tumor differentiation would suggest that this tumor suppressor could be an appropriate candidate molecular marker for evaluating tumor malignancy and predicting survival of patients subsequent to treatment.

Genome–Wide Hypomethylation and Specific Tumor-Related Gene Hypermethylation are Associated with Esophageal Squamous Cell Carcinoma Outcome

“…Previous studies investigating ESCC primaries demonstrated that APC promoter hypermethylation is a prognostic biomarker. However, the frequency of APC promoter hypermethylation was shown to be greater than 40%, 21,26,38 which is higher than the frequency detected in our study (19%). This discrepancy may be because of differences in the patient selection criteria, because in our cohort, all the advanced T4 cases treated with neoadjuvant chemotherapy were excluded.…”

“…Several of these regions have been described to be hypermethylated [21][22][23][24] and correlated with disease outcome to some extent in ESCC. 25,26 RASSF1, RARB, and RUNX3 gene hypermethylations have been shown to positively correlate with ESCC progression. [22][23][24]27 However, they have not been assessed in combination for ESCC primary tumor progression and overall disease outcome.…”

Genomic and Epigenomic Aberrations in Esophageal Squamous Cell Carcinoma and Implications for Patients

“…For example, CDKN2A , CDKN2B, 42,43 and TFF1 48 are hyper-methylated during early stages of esophageal carcinogenesis, and might therefore serve as biomarkers for early diagnosis of ESCC. In addition, hypermethylation of the APC 49 and FHIT 50 promoters was significantly associated with reduced survival times of patients with ESCC, so these might be prognostic factors. Plasma samples of patients suspected of having ESCC might be collected and analyzed for these hypermethylation events.…”

Alpha-1 Antitrypsin Deficient Squamous Cell Carcinoma of Esophagus in the Azeri Population of Iran

“…17 Iran is located in the so-called Asian esophageal cancer belt, where reports indicate the highest incidence of squamous cell carcinoma of the esophagus (SCCE) in certain areas of the country. Although recent reports [18][19][20] indicate attempts to recognize the molecular etiology of SCCE as well as achieve suitable tumor markers for this type of cancer from this region of the world, such efforts have so far been inconclusive and further efforts are therefore required.…”