This is the first observational study conducted in an Asian population that suggested a null association between pioglitazone use and breast cancer risk. The findings were consistent in the unmatched and the matched cohorts and in all models with different sets of adjusted covariates (Table 2). The finding of a null association was similarly observed in analyses conducted in patients whose diabetes was diagnosed during two different periods of time, i.e., 1999–2003 and 2004–2008 (Table 3).
Insulin resistance is an early pathophysiological change related to type 2 diabetes mellitus  and patients with type 2 diabetes mellitus are at an increased risk of breast cancer [15, 16]. Studies suggest that insulin resistance and hyperinsulinemia are important in the development of breast cancer [17, 18]. Therefore, it is hypothetically possible that breast cancer risk may be reduced by using antidiabetic drugs that improve insulin resistance. Our previous studies did show a reduction of breast cancer risk in patients who used either metformin  or rosiglitazone . However, this study did not support a beneficial effect of pioglitazone, another antidiabetic drug that also improves insulin resistance, on breast cancer risk. The discrepant findings between pioglitazone and other insulin sensitizers including metformin and rosiglitazone suggest that factors other than the improvement of insulin resistance might be responsible.
Findings from some in vitro and in vivo studies may provide evidence to support these discrepant clinical observations. In a breast cancer cell line, rosiglitazone stimulates the expression of tumor suppressor gene PTEN (phosphatase and tensin homolog, located on chromosome ten) but pioglitazone does not exert a similar effect . Another study showed that rosiglitazone exerts anti-proliferative and apoptotic actions on breast cancer cells; and induces autophagy and inhibits the invasiveness and metastasis of breast cancer cell lines . In an animal study, rosiglitazone suppresses mammary tumor growth in rats treated with the carcinogen 7,12-dimethylbenz(a)anthracene . On the other hand, although pioglitazone inhibits aromatase expression by inhibiting proinflammatory prostaglandin E2 signaling and upregulating tumor-suppressor gene BRCA1 , it does not inhibit mammary tumor growth induced by N-methyl-N-nitrosourea in Sprague-Dawley rats fed a high-fat diet . Studies also suggested that metformin and pioglitazone might have different effects on breast cancer cells. A Turkish study showed that diabetes patients with breast cancer treated with metformin had statistically significant reduction of serum level of hypoxia-inducible factor-1α (a nuclear transcription factor overexpressed in breast cancer cells and correlated with cancer metastasis and mortality), but the level did not change after treatment with pioglitazone . Taken together, these observations argued against a mechanism of breast cancer risk reduction associated with metformin and rosiglitazone merely through an improvement of insulin resistance and suggested that some other mechanisms might have traded off the beneficial effect of improvement in insulin resistance associated with pioglitazone.
After the withdrawal of rosiglitazone from the market because of a potential risk of macrovascular disease , pioglitazone is the only drug in the class of thiazolidinediones that remains in clinical use in most countries including Taiwan. The clinical trial (PROspective pioglitAzone Clinical Trial In macroVascular Events or the PROactive trial) published in 2005 that investigated the risk of cardiovascular disease comparing pioglitazone to placebo suggested a potentially higher risk of bladder cancer associated with pioglitazone use . This has raised a concern of cancer risk associated with pioglitazone use and an observational prospective follow-up study (i.e., the KPNC study) was requested by the US Food and Drug Administration to clarify the risk of cancer, especially bladder cancer. The interim analyses of the KPNC study suggested a potentially higher risk of bladder cancer in patients who had been exposed to pioglitazone for a long duration or a high cumulative dose  but a null association with female breast cancer . However, in the final report of the KPNC data, Lewis et al. showed weak linear trends in the risk of breast cancer associated with increasing cumulative dose and duration of pioglitazone use .
In the present study, we aimed at clarifying the effect of pioglitazone on breast cancer and therefore the balance of other potential confounders including the use of other antidiabetic drugs is important for an unbiased estimate. Although ever users and never users of pioglitazone differed significantly in the distribution of potential confounders in the unmatched cohort, they were well balanced in the matched cohort (Table 1). Because the results of a null association were consistent in different models in both the unmatched cohort and the matched cohort (Table 2), the conclusion should be robust and not affected by potential confounders.
We did not simultaneously investigate the effects of other antidiabetic drugs because no other antidiabetic drugs (except rosiglitazone that has been withdrawn from the market in many countries) had ever experienced such a great public health concern. The restrictions imposed by regulatory authorities after the publication of the interim analysis of the KPNC in 2011  on the use of pioglitazone because of its potential risk of bladder cancer have caused tremendous psychological impacts not only to the physicians who would be reluctant to prescribe the drug but also to the patients who might not have adhered to taking the drug even when they had been prescribed pioglitazone. Therefore, the time frame to be considered in study design for an investigation on pioglitazone effect should be cautious and would surely be different as for other antidiabetic drugs.
The patients were enrolled from 1999 to 2005 and followed up until 2011. This database seemed to be too old. However, the study period was deliberately selected to reduce potential biases based on the following considerations. First, this time frame would avoid unidentifiable biases resulting from the impacts of the publication of the interim analysis of the KPNC study in 2011  and the restriction of pioglitazone use imposed by regulatory authorities since then. Second, the Bureau of the NHI started to promote the use of ICD-10-CM in Taiwan since 2012 and therefore a potential bias resulting from a mixture of two disease coding systems might have happened if the follow-up ended after 2012.
It was also deemed inappropriate to investigate too many drugs and too many different cancers in a single study especially when pioglitazone was the target drug to be investigated because of the following reasons. First, as previously mentioned, the time frame for studying pioglitazone should be carefully restricted so that the findings would not be biased. The restriction on the use of pioglitazone would also affect the prescription and the adherence of other antidiabetic drugs and these behavior changes might have caused unexpected biases. Second, different antidiabetic drugs have different indications, contraindications and side effects and different cancers have different risk factors. It would be complicated to balance different sets of confounders. Third, cancer screening programs are evolving and different for different cancers. It may not be possible to simultaneously consider the impacts of these different screening programs when too many cancers are investigated in one single study.
Breast cancer screening programs have been conducted in either the USA  or in France  throughout the study periods of the clinical trials [1,2,3] and the observational study of the KPNC conducted in the USA [4, 6] and the observational study conducted in France . These breast cancer screening programs can lead to detection bias. However, none of the early studies investigating the risk of breast cancer associated with pioglitazone use have addressed the potential impacts of breast cancer screening programs.
In Taiwan, breast cancer screening programs have evolved from hospital-based project (1995–1998), to community-based projects (1999–2001 and 2002–2004) and finally to nationwide programs (phase I since July 2004, phase II since November 2009 and phase III since 2010) . The phase I nationwide biennial breast screening program by mammography was implemented for females aged 50–69 years since 2004. In 2009, the phase II screening program has been extended to females aged 45–69 years and further expanded in 2010 to women aged 40–44 years who have a second degree relative with breast cancer in the phase III program . The phase III screening program has been continuously conducted ever since 2010. Therefore, a fixed starting date of follow-up after 2009 would be less impacted by the sequential changes in the different waves of screening programs. Furthermore, we have considered the “potential detection bias” in our modeling (Table 1). The presence of “benign breast conditions” may lead to detection bias and use of estrogen may be an important risk factor of breast cancer . These had not been considered in previous studies, but we have carefully addressed these potential confounders (Table 1) in our analyses.
Although the median follow-up duration of 2.8 years in our present study was relatively short, this was comparable to the median duration of pioglitazone exposure of 2.8 years in the final report of the US KPNC study (study period 1997–2012)  and was longer than the 1.5 years in the French study (study period 2006–2009) . Because pioglitazone is not a first-line antidiabetic drug, even though the study period was longer than 10 years in the KPNC study, the median exposure time of pioglitazone was only 2.8 years . It is surely justified to conduct additional studies with longer durations of follow-up or larger sample sizes to elucidate the effect of pioglitazone on breast cancer.
Based on the following additional considerations, we did not follow the patients forward from the time of drug exposure. First, pioglitazone is not a first-line antidiabetic drug and it has not been approved for clinical use in Taiwan until after 2002. If the patients were to be followed since enrollment at the time of diabetes diagnosis (from 1999 to 2008, Fig. 1) or at the time of drug exposure, the starting dates of different patients would vary remarkably throughout a long period of time and never users would surely have earlier starting dates of follow-up than ever users. This would probably introduce other unexpected bias. Second, during a long and varying starting date of follow-up, the prescription of antidiabetic drugs would be affected by the evolution of changes in treatment guidelines. Third, environmental risk factors of breast cancer and cancer diagnostic methods and screening programs should have changed at different time points of start of follow-up and these would surely introduce additional bias.
There are some clinical implications in the present study. First, together with our previous studies that do not suggest an increased risk of bladder cancer , ovarian cancer , oral cancer , kidney cancer , thyroid cancer , lung cancer  and prostate cancer  associated with pioglitazone use, the public health concern of an increased cancer risk associated with pioglitazone can be relieved and should not impede the clinical use of pioglitazone. Second, the potential benefits of pioglitazone on the improvement of lipid profile , the risk reduction of dementia [38, 39], chronic obstructive pulmonary disease , non-alcoholic fatty liver disease , stroke  and cardiovascular disease  and the usefulness of pioglitazone in the treatment of polycystic ovarian syndrome in women  suggest that some patients may gain pleiotropic benefits beyond glycemic control from the appropriate use of pioglitazone.
The present study has some other strengths. Because the database was derived from the whole population and they spanned the whole period from the beginning of the marketing of pioglitazone in 2002 in Taiwan  until the end of follow-up on December 31, 2011, the potential risk of selection bias related to sampling error could be minimized. Because the NHI covers almost the whole population of Taiwan and the database was complete and included all claim records on outpatient visits, emergency department visits and hospital admission, and we caught the diagnoses from all sources. The use of medical records would have markedly avoided self-reporting bias. Because cancer is considered a catastrophic illness by the NHI and most medical co-payments can be waived, detection bias related to different socioeconomic status might have much reduced. Furthermore, there is a low drug cost-sharing required by the NHI and patients with certain conditions such as low-income household, veterans or patients with prescription refills for chronic disease are exempted from the drug cost-sharing. The risk of detection bias would be much reduced among different social classes in Taiwan.
The study limitations included a lack of actual measurement data for potential confounders such as obesity, smoking, alcohol drinking, family history, lifestyle, dietary pattern, and genetic parameters. In addition, we did not have biochemical data such as hormonal profiles, blood glucose levels, hemoglobin A1C concentrations, insulin, C-peptide levels, or calculation of homeostasis model assessment for insulin resistance for evaluating their impacts. Another limitation is the lack of information on the pathology, grading and staging of breast cancer. Finally, we should point out that the short median follow-up time of 2.8 years and the relatively low number of breast cancer cases (Table 2: n = 35 in ever users in the unmatched cohort and the matched cohort and n = 41 in never users in the matched cohort) would potentially lead to a conclusion of null association because of lack of statistical power. Therefore, additional studies are warranted to confirm our findings.
In summary, this study supports a null association between pioglitazone use and breast cancer risk in Taiwanese female patients with type 2 diabetes mellitus. The findings of the present study together with those of our previous studies [30,31,32,33,34,35,36, 45, 46] should at least relieve the concern of a potentially higher risk of common cancers associated with pioglitazone use. Because of the small case numbers of breast cancer and the limited follow-up time, further studies are warranted to confirm our conclusion of a null association.