Lee et al. BMC Cancer 2011, 11:20 http://www.biomedcentral.com/1471-2407/11/20 Cancer incidence density (CID) was calculated as the number of incident cancer events divided by 10,000 per- son-years at risk followed (years after the index date until the first cancer diagnosis before the end of 2007 or, for non-cancer subjects, date of withdrawal from NHI or the end of 2007). For comparison, we used the age-gender-adjusted incidence rates calculated from the 2007 National Cancer Registry in Taiwan. Covariates Comorbidity was measured by the Charlson comorbidity index (CCI) [26] using the diagnoses recorded in the NHI before the index date. This did not include the diagnosis of DM because subjects were DM-free before the index date. Use of any other oral anti-hyperglycemic agents (sulfonylureas, meglitinides, glitazones, and an alpha-glucosidase inhibitor) was recorded as ‘yes’ or ‘no’. Other covariates included age (20-49, 50-59, 60-69, ≥70 yrs), gender and geographical region. Statistical analysis The differences among four groups were evaluated by ANOVA (CCI score) and by chi-square (demographics). For the comparison of metformin users with those who had oral anti-hyperglycemic medications except metfor- min for total and 5 site-specific cancers, the hazard ratios (HRs) for cancer incidence were estimated by Cox proportional-hazards models. In these models, the time variable was the interval between the index date and the date of cancer ascertainment, or date of withdrawal from NHI, or December 31, 2007. The potential covari- ates included age, gender, other oral anti-hyperglycemic medication usage, CCI score and dose and duration of metformin exposure. Furthermore, gender-specific mod- els were also evaluated for total and site-specific cancers. The proportional hazards assumption was evaluated by comparing estimated log-log survival curves for all cov- ariates. The log-log survival plots, stratified by metfor- min usage status, of all models graphically showed two parallel lines, indicating no violation of the assumption. The statistics software SAS 9.1 was used for data man- agement and modeling. Results Since the non-DM subjects were sampled by matching to DM patients with metformin, the two groups have similar age, gender and geographical distribution. The majority were over 50 years old and males. More than 40% of the subjects lived in northern Taiwan and less than 5% lived in eastern Taiwan and off-shore isles. More females were in the DM without medication group (51.4%) than the others (45.1%-46.0%). The CCI median for the non-DM group indicates that more than half of them have no comorbidity with a mean of 0.25, but, for the DM groups, half experience 4 comorbidities or more. The follow-up Page 3 of 10 time for four groups ranged between 0.5-7.99 years. The diabetes without metformin group had the shortest mean follow-up time (3.52 years) among groups. Around 2% DM patients withdrew from NHI, while this was more than 4% for the non-DM. For those who were metformin users, 30% subjects used ≤500 mg/day, half used metfor- min for more than 2 years. (Table 1) Total and site-specific CIDs per 10000 person-years and 95% confidence intervals (CIs), as well as age- gender-standardized Taiwanese population cancer inci- dences, are listed in Table 2. Except for pancreatic cancer, both total and site-specific cancer standardized incidences are equivalent to the CIDs in our cohort. The total CID is 46.0 for non-DM subjects, 97.6 for DM patients without medication, 91.7 for DM patients without metformin and 44.8 for DM patients with metformin. Similar patterns were found regardless of gender. For site-specific cancers, only CRC and liver cancer reveal significant differences among groups, which seem related to gender. For women, non-metformin using DM patients have three times the CRC CID of people without DM (22.6 vs. 7.4). Yet, compared to non-metformin users, users have only one-sixth of their counterparts’ incidence (3.52 vs. 22.6). For liver cancer, it is only the difference in men among groups which achieves signifi- cance. The incidence rate for males, who have DM and do not use metformin (29.1) is three times and twice that of men without DM (8.6) and that of men with DM who use metformin (12.5), respectively. The metformin users were less likely to develop all-type cancers, CRC and liver cancer (model 1, HRs, 0.47, 0.38 and 0.53, P < 0.01) than non-metformin users (Table 3). When adjusted for age, gender, CCI score and time- dependent duration of metformin use (Model 2), great reductions in cancer risk for metformin users are seen for total cancer, esophageal, liver, and pancreatic cancer (HRs, 0.13, 0.44, 0.06, and 0.15, respectively) though eso- phageal and CRC are not significant. With additional adjustment for use of other anti-hyperglycemic medica- tion, the HR of metformin users to non-metformin users did not change substantially (Model 3). However, for CRC, the risk when metformin is used is smaller and sig- nificant (HR, 0.36, P < 0.05). No significant gender and metformin use interaction was found for total cancer and site-specific cancers except CRC (data not shown). Metformin users were categorized into three groups by average daily doses compared to DM patients who use other oral medication by gender (Figures 1 and 2). Gender differences are apparent in CRC and liver can- cer. In general, female metformin users are at a signifi- cantly lower risk of CRC for all three dosages compare to non-users (HRs, 0.05-0.17) while male users are not (HRs, 0.77-1.01). By contrast, there is an extremely low risk of liver cancer for male metformin users