الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Breast cancer (BC) is the most common cancer worldwide. Egypt is no exception, with ever increasing incidence rates. An interesting feature of this disease is the relatively young age of the affected women.
Despite the availability of mammography and prevalence of self-examination, there is still an additional benefit to be gained from additional screening methodologies. Novel approaches to early detection of breast cancer therefore merit investigation.
Hypermethylation is an epigenetic change that blocks the promoter region of a gene and results in gene silencing. In breast cancer, tumor-related genes may be silenced by hypermethylation; many hypermethylated genes have been reported, and silencing of these genes plays important roles in carcinogenesis and tumor progression. Identification of epigenetic changes and their correlation with other clinical factors could lead to improvements in cancer diagnosis and treatment.
In an effort to understand the molecular signature of BC in Egyptian population, we examined the promoter methylation status of two normally non methylated, biologically significant cancer genes, RAS association domain family member 1A (RASSF1A) and Protocadherin10 (PCDH10), and their association with different clinico-pathological parameters. Despite the fact that methylation of PCDH10 gene has been shown in breast cancer, the relationship between CpG island methylation of this gene and clinico-pathological characteristic of breast cancer patients has not been reported as yet.
We detected gene hypermethylation in serum because it is a more readily accessible bodily fluid compared with nipple aspirate or ductal lavage fluids. Obtaining peripheral blood sample does not require the presence of specialist and it is relatively painless.
In present study we investigated promoter hypermethylation of RASSF1A and PCDH10 genes in serum DNA of 80 breast cancer patients and 40 normal healthy subjects in Egyptian women. Methylation-specific Polymerase chain reaction (MSP) was used to investigate the promoter methylation status in serum DNA. Forward and reverse primers were used, which corresponded to the predicted sequence of methylated or unmethylated genomic DNA after sodium bisulfite treatment.
The frequency of promoter hypermethylation of RASSF1A was 50 of 80 (62.5%) and PCDH10 was 34 of 80 (42.5%) in serum DNA of breast cancer patients. sixty-four of 80 (80%) patients showed hypermethylation of one or both genes, while no hypermethylation of RASSF1A or PCDH10 was detected in serum DNA of healthy control subjects.
The clinical significance of promoter methylation of RASSF1A and PCDH10 genes was studied by investigating the relationship between methylation of these genes and clinicopathological characteristics of breast cancer patients.
There was insignificant association between serum RASSF1A promoter methylation and clinico-pathological features in breast cancer patients except lymph node involvement which is significantly associated with promoter hypermethylation of RASSF1A gene((P = 0.008). PCDH10 methylation was significantly associated with tumor size (P =0.019), tumor pathological grade Π (P = 0.020) and lymph node involvement (p =0.047). Furthermore, two panel genes (RASSF1A and/ or PCDH10) methylation were significantly associated with lymph node involvement (P = 0.001) and with negative Her2 (P = 0.042).
Our results also revealed that, serum levels of CA15.3 in breast cancer patients group were elevated in comparison with control group, and the difference was significant(P= 0.03). We found no correlation between methylation frequency of RASSF1A and PCDH10 with CA15.3 level (P>0.05), whereas, methylated RASSF1A and PCDH10were significantly associated.
All of the methylated RASSF1A, PCDH10, RASSFIA and/or PCDH10& serum CA15.3 were found to be diagnostic markers of breast cancer patients. To compare the diagnostic values of these markers, the ROC curve analysis was applied. The combination of methylated RASSF1Aand/orPCDH10 gave the highest sensitivity (80%), followed by methylated RASSF1A (62.5%), PCDH10 (42.5%), then CA15.3 (33%). This means that the combination of methylated RASSFIA with methylated PCDH10 can improve diagnostic sensitivity of breast cancer patients. In addition, methylated RASSF1A& PCDH10 genes were found to be significant predictors of short disease- free survival in breast cancer patients and can be used as independent prognostic parameters.