Diabetes , Overweight and Risk of Postmenopausal Breast Cancer : A Case-Control Study in Uruguay

Obese postmenopausal women increase their risk of developing breast cancer (BC), in particular if they display an android-type pattern of adiposity, which is also associated to increased risks of diabetes mellitus, hypertension and cardiovascular disease. In order to explore the associations among anthropometry (body mass index, body composition, somatotype), some specific items of medical history (diabetes, hypertension, dislypidemias, hyperuricemia) and the risk of BC in Uruguayan women, a case-control study was carried out between 2004-2009 at our Oncology Unit. 912 women of ages between 23-69 years (367 new BC cases and 545 non hospitalized, age-matched controls with a normal mammography) were interviewed. Twenty body measurements were taken in order to calculate body composition and somatotype. Patients were queried on socio-demographics, reproductive history, family history of cancer, a brief food frequency questionnaire and on personal history of diabetes, dislypidemias, hyperuricemia, hypertension and gallbladder stones. Uni- and multivariate analyses were done, generating odds ratios (ORs) as an expression of relative risks. A personal history of diabetes was positively associated to BC risk (OR=1.64, 95% CI 1.00-2.69), being higher among postmenopausal women (OR=1.92, 95% CI 1.04-3.52). The risks of BC for diabetes in postmenopausal women with overweight combined with dislypidemia (OR=9.33, 95% CI 2.10-41.5) and high fat/muscle ratio (OR=7.81, 95% CI 2.01-30.3) were significantly high. As a conclusion, a personal history of diabetes and overweight was strongly associated to BC. The studied sample had a subset of high-risk of BC featured by postmenopausal overweight and diabetic women, who also had a personal history of hypertension and/or dyslipidemia. The present results could contribute to define new high risk groups and individuals for primary as well as for secondary prevention, since this pattern linked to the metabolic syndrome is usually not considered for BC prevention.


Introduction
At the end of XIX century, hyperglycemia and diabetes were first associated to BC, being reported among patients with cancer (Freund, 1885).In 1930, tumour samples were reported to have higher rates of glucose utilization than did normal tissues (Warburg, 1930).Since the 1950s, incidence reports have described women with BC as having higher rates of diabetes than did healthy women (Glicksman 1956, Muck 1975).
Diabetes mellitus, metabolic syndrome and BC are all more prevalent in developed societies than in developing ones, where a sedentary lifestyle and a high intake of refined carbohydrates and saturated fats are more prevalent.However, developing countries are increasingly adopting many of the lifestyle characteristics of more affluent societies.While the urbanization process continues, educational levels increase and people change their habits, as a result of which the occurrence of BC increases.This is an outcome of several of these factors diabetes and the metabolic syndrome have been associated with a state of chronic, low-grade, inflammation; inflammatory cytokines such as C-reactive protein may induce insulin resistance (Hanley et al., 2004), and C-reactive protein has been associated with risk of atherosclerotic cardiovascular disease and myocardial infarct (Wilson et al., 2006).Several studies have suggested a direct association between components of metabolic syndrome and BC risk (Xue and Michels, 2007;Vona-Davis et al., 2007;Rosato et al., 2011).Therefore, low HDL-cholesterol (Furberg et al., 2004), high blood glucose (Muti et al., 2002), high triglycerides (Potischman et al., 1991), postmenopausal overweight (World Cancer Research Fund, 2007), abdominal obesity (Connolly et al., 2002), hypertension (Soler et al., 1999), high levels of insulin (Hirose et al., 2003), C peptide (Verheus et al., 2006), and insulin-like growth factor I (IGF-I) (Campagnoli et al., 2008), have all been associated with increased BC risk.Metabolic and hormonal factors related to metabolic syndrome have also been implicated in BC prognosis (Goodwin et al., 2002;Rock et al., 2002;Pasanisi et al., 2006;Agnoli et al., 2010;Tsugane and Inoue, 2010).
Interestingly, the metabolic syndrome was also found associated with a decreased risk of incident BC in women below age 50 with high body mass index (BMI), and with an increased risk of BC mortality in women of ages above 60, according to a European study (Bjørge et al, 2010).New evidence emphasizes that metabolic syndrome increases the risk mainly in postmenopausal women (Capasso et al., 2011) and it has been found as significantly more prevalent in triple-negative BC patients as opposed to non-triple-negative patients (Maiti et al., 2010).
Furthermore, the increase in BC incidence has occurred in parallel with a steady increase in the frequency of type 2 Diabetes and metabolic syndrome (Xue and Michels, 2007).Although BC is still a major public health issue in developed societies, its incidence has been also rising in several developing countries over the past few years (Bray et al., 2004).International data (Ferlay et al., 2010) have located Uruguay among those with the highest rates in the world.Moreover, the capital city, Montevideo, has displayed the highest incidence rate for a city (Parkin et al., 2002).In fact, although this small South American country is a developing one, it shares some characteristics with developed regions, i.e. a very high level of red meat consumption, (FAO, 2010) a high human development index (50º in the world ranking according to United Nations, by factors as birth rate, infant mortality, life expectancy, literacy, among others) (United Nations, 2009) and an aged population (US Census Bureau, 2010).In other words, a developing country has shown a high occurrence of a disease which is typical of developed countries.
We and others have thoroughly studied possible links of nutrition and BC, from the dietary viewpoint (Ronco et al., 1996;1999;2002;2003;2006;2010a,b,c,d;De Stéfani et al., 1997a,b;1998) as well as from an anthropometric one (Ronco et al., 2008;2009) and other epidemiologic ones (Ronco et al., 2007;2009).In order to explore the associations among anthropometry (BMI, body composition, somatotype), some specific components of the metabolic syndrome in the medical history (diabetes, hypertension, dislypidemias, hyperuricemia) and the risk of BC in Uruguayan women, we decided to carry out the present epidemiologic case-control study, whose preliminary report was recently communicated (Ronco et al., 2010d).

Materials and Methods
The authors carried out a hospital-based case-control study on anthropometry and BC during the period between June/2004 and December/2009 at the Instituto de Radiología y Centro de Lucha Contra el Cáncer, which is a reference center of BC in Uruguay and is located at the Pereira Rossell Women's Hospital in Montevideo.It admits women coming from all the country and mainly belonging to the lowest socioeconomic strata, who pertain to the public hospital health system.Currently, around 50 diagnostic mammograms in a predominantly asymptomatic population are daily performed.
During the study period, 367 incident cases of BC up to age 69 were identified in the screening population and enrolled into the study.Cases were women with new incident and histologically diagnosed carcinomasinterviewed between 0 and 10 days after their diagnosis-.Potential cases with ages 70 or higher were not taken into account, regarding the lack of healthy controls to match them and also to reduce a possible recall bias.Their recruitment was performed in patients with mamographically BI-RADS 4 (suspicious of malignancy) and 5 (highly suspicious of cancer) lesions (Varas et al., 1992;Feig, 1999), and a positive cytologic (on site) study, which was furtherly histologically confirmed, according to its high correlation with histopathology (Jaumandreu et al., 2001).Initially, no information on cancer stage was collected through the study period.
Since BC cases were interviewed and measured very soon, they have not experienced any post-diagnostic or treatment-induced weight change.Although women do not participate formally in a screening program, cancers are usually diagnosed at early stages (ca.10% carcinoma in situ).In the same time period and in the same institution, 559 healthy women with a negative diagnostic mammogram (BI-RADS categories 1 [completely negative]-2 [only with findings not associated with pathology, e.g.benign calcifications and/or axillary lymphnodes]) (American College of Radiology, 1998) performed the same day of the interview, were randomly selected as controls.They were frequency-matched by age (± 5 years) to cases, being mandatory requirements for the controls not to be hospitalized at the moment of the interview and not being afflicted by a cancer.Most women of ages under 30 were examined only with ultrasonography, unless findings required also mammography due to the high density of breasts at those ages.Normal aged controls were relatively unfrequent in consulting at the Institute, and it was difficult to find completely normal mammographies in the older women.After excluding 12 women who had had a previous cancer in the past and 2 who rejected the interview, a final number of 545 controls were recruited DOI:http://dx.doi.org/10.7314/APJCP.2012.13.1.139 Diabetes, Overweight and Risk of Postmenopausal Breast Cancer (response rate 97.5%).Therefore, 912 women consulting for a mammography at our Center were included in the study.Interviews and measurements were performed by an only trained nurse, who was blinded regarding the objectives of the study, previously trained and periodically supervised during the study period.All interviews were conducted in the hospital and performed face to face, and a written consent was obtained from every interviewed subject.People affiliated to the public health system are prone to cooperate with surveys and studies, therefore a high participation is always expected.The research was approved by the ethical committee of the Hospital.
The questionnaire included the following sections: 1) Socio-demographic variables; 2) Menstrual-repro¬ductive events (age at menarche, age at first live birth, number of children, months of breastfeeding, menopausal status (pre/post).Menopausal status was defined a priori: if according to the subject (aged >=45) menstruations have ceased at least for 6 months, excluding pregnancy, she was classified as postmenopausal; 3) History of cancers in first and second degree relatives; 4) Physical exercise (yes/no), frequency, duration and intensity; 5) Self reported weight at age 18; 6) A short food frequency questionnaire; 7) Queries on personal history of components of metabolic syndrome and others: diabetes, hypertension, dislypidemia, hyperuricemia, bile lithiasis.Hormonal replacement therapy was not asked, because it is not usually prescribed to postmenopausal women who belong to the studied subpopulation.Physical exercise was queried on activities out of the job time, even recreational or competitive, 5 years prior the interview.This assessment, whose method was not validated, was performed only as an exploratory tool in the studied group, whose restricted incomes limit their time and access to sport institutions.The analyses were focused mainly on the components of metabolic syndrome.The ER status was not among the variables examined in this study.
Anthropometric equipment included a height scale and headboard, a weighing scale, a vernier caliper, a flexible plastic tape, and a skinfold caliper.The same mechanical scale was used along the whole study period, with a weekly calibration.Subjects were weighed wearing minimal clothing.For body measurements a plastic centimeter at intervals of 0.5 cm (for circumferences), a vernier caliper (for diameters) and a digital caliper (for skinfolds) were used.Regarding these latter, if two consecutive measurements were similar, the obtained value was registered as valid.If both were different, a third one was taken and the median value was then registered.Measurements were performed according to Carter's Instruction Manual (Carter, 2002).

Body composition
Anthropometric data were used to quantify body size  (Faulkner 1968), according to the anatomic four compartments method of De Rose (1984).

Statistic analysis
Calculations of mean ± standard deviation for each variable were made.Based on the measurements done, the following calculations were made: Body Mass Index (BMI, kg/m 2 ), BMI at age 18 (kg/m 2 ), BMI difference (current-18 yrs), weight difference (id.) and waist-to-hip ratio.Somatotype variables were categorized in tertiles, according to the controls distribution.Crude and adjusted Odds Ratios (OR)s and 95% Confidence Intervals (CI) s were calculated by unconditional logistic regression (Breslow and Day, 1980) .Potential confounders were included in the multivariate analysis.ORs were calculated including age, residence, age at menarche, parity, age at first live birth, months of breastfeeding, use of oral contraceptives, BMI, menopausal status, family history of BC, and intake of beef, tomatoes and oranges.For analysis purposes, stratifications by menopausal status (pre-/post-), by levels of body mass index (normoweight, overweight-obese) and by endomorphism [low-mid (≤7.6), high (≥7.7)]were also done.All the calculations were performed with the software STATA ® (Release 10, College Station, Texas, USA 1999).

Results
Regarding the general features of the study population and taking into account some lack of older controls (≥60 yr) when data entry was finished for this analysis (December 2009), a very homogeneous population is described (Table 1).Age -as a consequence of the matching design-, sociodemographic and lifestyle variables were very similar in both series.Besides, menstrual and reproductive variables displayed some differences related to the age at first live birth, number of live births, and number of months of breastfeeding.Cases showed a higher percentage of participants with family history of BC among first-degree relatives compared with controls (16.9% vs. 9.5%, p = 0.02).In the studied population, the physical activity in leisure time was not different for both cases and controls.
Mean values of the anthropometric parameters showed that although height and weight were rather similar, as were waist and hip circumferences, significant differences between cases and controls were found for several skinfold thickness parameters (subscapular, tricipital, bicipital, supraspinal)(Table 2).Among the calculations, only Endomorphism was significantly different (p=0.002) and Ectomorphism was borderline associated (p=0.057),but fat fraction, muscle fraction, and the other parameters did not show significant differences.
In Table 3 the relative frequency of medical items associated to metabolic syndrome are presented by age groups.All of the analyzed items displayed an increasing trend along the ages (p for trend <0.001).Overweight/ obesity was the most frequent pathology, involving 3 of each 4 subjects of ages 50 or more.Hypertension was also a very frequent pathology, involving 60% of women at ages 60 or more.
Table 4 shows the crude and adjusted odds ratios (ORs) of BC for each analyzed component among postmenopausal women.Slight changes took place when comparing both series of ORs, indicating that neither family history of cancer nor menstrual-reproductive variables modified substantially the risks.A personal history of diabetes was positively associated with the risk of BC (OR=1.92,95% CI 1.04-3.52),while a high endomorphism (≥7.7) was also positively associated with the risk of the disease (OR=1.76,95% CI 1.14-2.71).Surprisingly, a history of any type of dislypidemia was negatively associated with BC in this sample (OR=0.59,95% CI 0.38-0.94).
The crude and adjusted risks of BC for Diabetes, stratified by menopausal status are shown in Table 5. Statistical significant increased risks, not modified after adjustment for potential confounders were found only among postmenopausal women (adjusted OR= 1.92, 95% CI 1.04-3.52).This estimate was similar to those found in strata of postmenopausal women, as hypertension (OR=1.93),dislypidemia (OR=2.29)and high fat/muscle ratio (OR=2.01),although they not reach statistical significance.On the other hand, diabetes was strongly associated with BC risk in overweight postmenopausal women (OR=5.42,95% CI 1.77-16.6),but lacked of association among obese ones (OR=0.84).
Finally, Table 6 displays the relative risks of BC among postmenopausal women for diabetes combined with other components of metabolic syndrome (hypertension, dislypidemia, overweight, high fat/muscle ratio).All estimates of these combinations, being crude or adjusted, were statistically significant increases in risk.The highest risks for diabetes were found for postmenopausal women with overweight and dislypidemia (OR=9.33 and OR=10.6,crude and adjusted respectively), followed by those having a high fat/muscle ratio (adjusted OR=7.85).

Discussion
Our study gives support to the positive association of diabetes to the risk of postmenopausal BC, almost doubling it (OR=1.92).Other components of the metabolic syndrome, as hypertension and dislypidemia also displayed positive associations, slightly changing the estimates or not.BMI, on the contrary, showed that overweight (OR=5.42)was determinant -but not obesity (OR=0.84)-to the risk increase for diabetes among postmenopausal women.The analyses revealed that among obese women the frequency of diabetes is so high that this latter does not discriminate cases and controls in the studied population.
A recent review on diabetes, metabolic syndrome and BC (Xue and Michels, 2007) reported that among all casecontrol studies in which the association between diabetes and risk of BC was addressed, results suggested that BC patients were more likely to have a history of diabetes, with odds ratios ranging from 1.10 to 2.15.On the other hand, among cohort studies, results indicated that women with a history of diabetes were more likely to develop BC, with hazard ratios ranging from 1.10 to 2.06.In both type of studies, around the half showed statistically significant increases in risk.Our results concerning diabetes reveal similar associations to the existing literature, from which diabetes is being considered as a probable independent risk factor (Grote et al., 2010).The history of diabetes seems to have enough statistical strength by itself, roughly doubling the risk whichever the analysis was.Taking into account different studied strata, it was also clear that the disease concerned mainly to postmenopausal women and specifically those bearing an overweight.
Almost a decade ago, a case-control study conducted in Italy reported an increased prevalence of type 2 diabetes mellitus, hypertension, and dislypidemia (abnormal serum cholesterol and/or triglycerides level) among BC cases compared with women with benign breast pathology or women with no breast pathology (Sinagra et al., 2002).Other study reported that the risk of postmenopausal BC was significantly increased for women with metabolic syndrome (OR = 1.75, 95% CI 1.37-2.22),for three or more components of metabolic syndrome (Rosato et al., 2011).
Regarding the BC risk, the worst possible scenario for the subset of our postmenopausal women with overweight is associated with the history of blood hypertension and any of the components of dislypidemia.Also considering the analysis of fat fraction, the estimates were higher for those women with high fat/muscle ratio (≥1.5 times), remarking that not only the amount but also the proportion of fat is important regarding the risks.
A high BMI in early ages is relevant to develop diabetes, and the studied population revealed high prevalence of overweight/obesity (47.1%) and high endomorphy (30.1%) just at ages 30-39, with trends parallel to age increase.After menopause, the suprarenal glands and adipocytes are major estrogen sources.If body fat is excessive there is an increased hormonal bioavailability.In addition, in the adipose tissue androgens which are gathered from the circulation are also transformed into estrogens through the action of the aromatase, in the process known as "androgen aromatization".In addition, obesity creates a doubly favorable environment for mammary carcinogenesis, since insulin requirements and androgen aromatization are stimulated and increased, the latter for producing more estrogens.Aromatization is stimulated bearing an excessive adipose mass, especially in the thighs, buttocks and abdominal-pelvic regions (gynoid obesity, sharing features with high endomorphism), with a high intake of Ω-6 polyunsaturated fatty acids (PUFAs) and having increased circulating glucocorticoids (McTernan et al., 2002) A recent study suggested that the ratio of 2/16-α-hydroxyestrogen metabolites may be a marker for lifestyle influences on estrogen metabolism associated with westernization (Falk et al., 2005).In particular, body composition was associated with 2-α-and 16-α-OHestrone levels: while thicker skinfolds were associated with higher 16-α-OH levels, (Campbell et al., 2005) an increase in lean body mass was associated with an improvement in 2/16-α-OH estrogens ratio (Campbell et al., 2007).Women who metabolize a large proportion of their estrogens via the 16-α-hydroxylation pathway could be at a higher risk of BC (Cauley et al., 2003).It has been also suggested that women at higher risk for developing BC due to low 2/16-α-OHestrogens may reduce their risk by participating in lifestyle interventions such as exercise/ calorie restriction (Westerlind and Williams, 2007).
The overabundance of insulin, called hyperinsulinemia, amplifies the bioavailability of Insulin-like Growth Factor I (IGF-I).IGF-I and insulin together have been shown to stimulate motility in human BC cell lines, an effect that could enhance migration and invasion (Macaulay, 1992;Sachdev and Yee, 2001).IGF-I signaling enhances estrogen receptor activation by inducing phosphorylation of the estrogen receptor, and IGF-I and estrogen have synergistic effects on the cell cycle signaling cascade and proliferation (Hamelers and Steenberg, 2003).Because the IGF-I system can be cross-activated by insulin, the synergic effects of IGF-I and estrogen may also play a role in the etiology of BC in the hyperinsulinemic state of type 2 diabetes (Chaudhuri et al., 1986, Guastamacchia et al., 2003).Recent experimental findings evidenced that Type 2 diabetes accelerates mammary gland development and carcinogenesis and that the insulin resistance and/or the IGF are major mediators of these effects (Novosyadlyy et al., 2010).If alterations of endogenous estrogen concentrations indeed play an important role in the association of type 2 diabetes with the risk of BC, this association is expected to be stronger for tumours that are estrogen receptor positive, which was confirmed in the Nurses Health Study (Michels et al., 2003).
As other case-control studies, our work has limitations and strengths.A major limitation is related to the sample size; it would be desirable to analyze a larger one, in order to have enough statistical power for certain results.On the other hand, both cases and controls belong to a very homogeneous base subpopulation: they were matched by age frequency and they also proceeded from the same DOI:http://dx.doi.org/10.7314/APJCP.2012.13.1.139 Diabetes, Overweight and Risk of Postmenopausal Breast Cancer healthcare system, showing similarities concerning sociodemographic variables.To be quoted also among the strengths, we selected as controls women with normal breasts according to mammography, not only without cancer; thus, if benign breast diseases had any association with the analyzed dietary items, we avoided the possibility of biasing results due to this.The inclusion of detailed anthropometric measurements and calculations as body composition and somatotype instead of using only the BMI or waist-to-hip ratio can be considered also a strength.Finally, a high participation was achieved (around 98% of the incident cases below 70 years old during the study period): selection and recall bias appear to be unlikely.Although it is not possible to avoid completely any bias, we think that results were not chance findings.Anyway, we need caution in the interpretation of results, since generalizability is limited due to the population features: they have mainly low educational level and belong to low socioeconomic strata.
In conclusion, we can state that: First, a personal history of diabetes was strongly associated with overweight and it tended to increase the risk of BC.Second, a high-risk subset of BC arose from the analyses.This subset was composed by postmenopausal, diabetic and overweight women, who in addition display a personal history of blood hypertension and/or dislypidemias and/or a high endomorphism.Finally, the results give elements with potentiality to define high-risk subjects, for primary as well as for secondary prevention, since this pathologic pattern related to the metabolic syndrome is usually not taken into account for BC prevention.As an example of this, the ages with the highest prevalence of metabolic factors fall out of the range considered for screening and early detection programmes, but those ages involve a large fraction of the diagnosed BC cases.

Table 4 . Relative Frequency and Crude and Adjusted Odds Ratios (ORs) of Breast Cancer for Each Analyzed Component Among Postmenopausal Women
Adjusted by age, residence, family history of BC 1ºdegree, age at menarche, number of live births, age at first delivery and number of breastfeeding months. *