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Objectives The insulin-Like Growth Factor-II (IGF-II) gene has four promoters (P1-P4), each of which produces a distinct transcript. Promoter-specific transcription is regulated through methylation. Abnormal methylation and change in promoter-specific transcription may be involved in carcinogenesis and cancer progression. This study was conducted to examine DNA methylation patterns of IGF-II promoters P2-P4 in breast cancer and their association with IGF-II expression and breast cancer survival.

Methods Fresh tumor samples were obtained from 350 breast cancer patients who underwent surgery between January 1998 and July 1999 in Turin, Italy. Follow-up information was collected after surgery through August 2006 for 302 patients. DNA was extracted and chemically modified with sodium bisulfite. Quantitative methylation specific real-time PCRs were developed to determine methylated and unmethylated DNA sequences in three IGF-II promoters. Percentages of methylated DNA in total DNA were calculated; 0.1% and 1% were used as cut-offs to classify the results into unmethylation, low methylation and high methylation. Expression of IGF-II transcripts 2-4 were measured by quantitative RT-PCR. Cox proportional hazard regression models were applied for survival analysis using lower tertile as reference after adjusting for age, TNM stage and grade.

Results IGF-II methylation was seen in 27.3% of P2, 10.9% of P3 and 19.0% of P4. The methylation of all three IGF-II promoters was increased with age. Significantly higher percentages of methylation were observed among patients >55 years compared to those <55 years. Positive correlations were shown between age, tumor size and methylation in P2 and P3, and between lymph nodes and methylation in P4. Although no significant correlation was suggested between methylation and expression, promoter 3 with lowest methyaltion rate (10.9%) did have the highest expression; P3 transcripts were about 400 times higher than P2 and P4 transcripts. Survival analysis showed that both P3 hypomethylation and high P3 expression were associated with reduced risk of disease progression; the adjusted hazard ratios (HRs) were 0.28 (95%CI: 0.09-0.89) and 0.47 (95%CI: 0.24-0.89) respectively, with significant trends (ptrend=0.037 and 0.020). Over 50% reduction in risk for disease progression was also suggested among patients with hypermethylated P4; the HR was 0.43 (95%CI: 0.18-1.03, Ptrend=0.063). Although there was slightly decreased risk for overall survival among patients with P3 or P4 hypermethylation, the differences were not statistically significant. Conclusion The study showed that methylation varied among three IGF-II promoters in breast cancer. Hypomethylation in P3 and high expression of P3 were associated with better disease free survival. Our results suggest that methylation patterns of IGF-II promoters may have clinical implications in breast cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA