Diet and body constitution in relation to subgroups of breast cancer defined by tumour grade, proliferation and key cell cycle regulators

The aetiology of breast cancer is complex, but a number of risk factors have been identified. In human studies body mass index (BMI) and hip and waist circumferences have been described as positively associated with risk for developing breast cancer [1]. Animal experiments support these findings and report that obesity enhances the development of mammary tumours [2]. To our knowledge, no studies have been conducted to evaluate whether there is an eventual association between obesity and tumour characteristics in animals.

The association between dietary intake and body composition is still under debate [3, 4], and the biological link between diet and breast cancer is unclear. Dietary studies indicate that diet may contribute factors that either promote or protect against breast cancer [5]. High alcohol intake, for example, has been associated with breast cancer [1, 6]. A large number of studies have examined the link between dietary fat and breast cancer, and most studies have found no or weak associations [7–9]. A recent attempt to clarify the hypothesis that a low-fat dietary pattern can reduce breast risk was made by Prentice and coworkers [10] in a randomized trial, but the results showed that risk for breast cancer did not differ between the two groups studied. The lack of any clear associations between diet and breast cancer in epidemiological studies could result from difficulties in dietary assessment, but they may also result from the fact that breast cancer is a heterogeneous disease that may have disparate genetic associations and different aetiological bases.

Breast cancer is heterogeneous in terms of pathology parameters such as tumour type, size, grade, hormone receptor status and lymph node involvement. These subgroups are related to clinical behaviours and prognoses [11]. Tumour proliferation, as reflected by the Ki67 index, is closely associated with tumour aggressiveness, and the Ki67 index is often considered an independent prognostic marker for breast cancer [12]. Another approach to defining subgroups of breast cancer is to map different gene aberrations in breast cancer. The tumour suppressor gene p27 acts as a cyclin-dependent kinase inhibitor [13]. Other gene aberrations related to defects in cell cycle control such as overexpression of cyclin D₁ or cyclin E affect the cell cycle at the G₁/S checkpoint and may affect chromosomal stability [14, 15]. Indeed, these aberrations are common in cancer [16] and have even been proposed to be mandatory in tumour transformation processes [17]. In experimental animal studies [18] energy restriction was found to reduce mammary tumour cell proliferation via G₁ cell cycle arrest, establishing a connection between dietary intake and cell cycle regulators.

The association between fat intake and breast cancer incidence overall was studied in previous work as part of the Malmö Diet and Cancer Study (MDCS), although with a shorter follow-up period and fewer breast cancer cases [19]. The aim of the present study was to explore the association between different subgroups of breast cancer and several dietary factors and body constitution. Incident breast cancer cases from the MDCS were classified according to tumour type, grade, oestrogen receptor (ER) status, expression of p27, cyclin D₁ and cyclin E, and proliferation (Ki67).

The distribution of breast tumours revealed a predominance of ductal carcinomas (69%) as well as low-grade tumours (75% grade 1 and 2). In the TMAs, adequate tumour material was obtained in the majority of cases (91%), with a similar targeting rate for different tumour types (data not shown).

We found that low intakes of energy and total fat (especially PUFA), and high BMI were associated with more malignant breast cancer.

Some methodological issues must be addressed. Dietary assessment may be biased because of measurement error, for instance over- and under-reporting. The validity of dietary data in the MDCS has been examined using 18 days of weighed food records collected over 1 year in a subgroup of MDCS participants (126 men and 115 women) and validity was found to be high [22, 23].

Whether energy-adjusted variables should be used when analyzing any relation between dietary intake and disease is a matter of controversy among nutritional researchers [26, 27], and in this study we present results using the total, as well as energy-adjusted, intake of macronutrients.

A prevalent or subclinical breast cancer may affect anthropometric measurements and dietary habits. All data concerning objective body measurements were gathered between 0.2 and 10.4 (mean 4.3) years before breast cancer diagnosis, and the values were therefore most likely unaffected by disease. Supporting this interpretation is the lack of relation between BMI and time to diagnosis (P = 0.526, r = -0.021 [Spearman's correlation test]). Similar results were obtained for energy intake and time to diagnosis (P = 0.208, r = -0.060).

Tumour classification with regard to type and grade was performed according to current classification systems. The tissue microarray (TMA) technique used in this study is now a well documented method for high-throughput tissue screening, with two tissue cores considered a sufficient sampling amount [28, 29]. The distribution of the immunohistochemical markers was in accordance with earlier studies [16], thus validating the assessments.

The participation rate in the MDCS was about 40% of the potential participant population. The participants did have a higher incidence of breast cancer compared with the source population [21] and were most likely a selected group in terms of socioeconomic factors. Nevertheless, the distribution of histological type and grade within the incident breast tumours in this study was similar to that in other studies [11, 30]. Even if our breast cancer population were different from the background population, it would still be possible to make internal comparisons between different tumour groups in terms of dietary and anthropometric measurements.

Because several methodological factors in the MDCS may affect dietary measurements, all analyses were adjusted for diet assistant, period of data collection, diet methodology and past food habit change, as well as for total energy intake when stated. Hence, these factors ought not to have confounded dietary assessments. It was decided not to adjust the present analysis for established risk factors for breast cancer or factors that affect true dietary intake (for example socioeconomic index), because the main objective was to conduct perform a descriptive and exploratory analysis of dietary intake in different groups of breast cancer as defined by pathological and biological properties.

The present study includes a large number of comparisons, and P values should be interpreted with caution. Because no previous study has addressed the same issue using our methodology, we consider the present analyses as a first, hypothesis-generating study. Our findings need confirmation in future studies including healthy control individuals.

In this report we demonstrate an association between highly proliferative tumours and low energy intake, as well as low intakes of fat and PUFA. Several other human studies have reported that high energy intake is related to increased risk for breast cancer [31, 32], but it is still unclear whether fat has a specific impact on breast cancer risk [7, 9]. To our knowledge, no studies have reported on any association between diet and proliferation in breast cancer.

Furthermore, we found an association between low energy intake, particularly low fat intake, and overexpression of cyclin D₁. Cyclin E, however, was positively associated with fat intake, but this association did not remain significant in the dichotomized analyses. The opposite behaviours for cyclin D₁ and cyclin E are in accordance with earlier observations showing that cyclin E overexpressing tumours are low in cyclin D₁ and vice versa [33]. Experimental animal studies support our findings of a connection between dietary intake and cell cycle regulators because energy restriction has been demonstrated to reduce mammary tumour cell proliferation via G₁ cell cycle arrest, possibly through increased expression of p27 and reduced expression of cyclin D₁ [18, 34]. This may appear to be in conflict with our results, in which low energy intake was associated with cyclin D₁ overexpression, and p27 did not exhibit any connection to energy intake. However, animal models using chemically induced breast tumours cannot readily be translated into a far more complex tumour genesis in humans, which presumably are influenced by a multitude of environmental factors.

Subgroups defined by ER were not linked to dietary behaviour or body composition. Some studies indicate that high fat diets are associated with increased risk for ER-positive tumours [35–37], whereas Verreault and coworkers [38] found no association between dietary fat and ER status. A few studies suggest that different types of PUFA may have opposing influences on breast cancer risk [39, 40]. Studies concerning body constitution report an increased risk for ER-positive tumours in obese postmenopausal women who are supplied with oestrogens via conversion of androstendione to oesterone in fat tissues, which is in contrast to premenopausal women, whose primarily oestrogen source is the ovaries [41]. However, the present analyses include both premenopausal and postmenopausal women because there are only a limited number of cases in each subgroup, making further stratification into premenopausal and postmenopausal women unsuitable. This might explain the different results. Furthermore, analyses in the present study did not include healthy control individuals, which might also have contributed to the different results. However, in order to map potential differences in anthropometric risk factors in premenopausal and postmenopausal breast cancer, we performed separate analyses, which confirmed our initial results in the entire cohort. In addition, premenopausal breast tumours were characterized by high proliferation and low expression of p27 in tall women. The suppressor gene product p27 was not associated with any examined variable in the entire cohort. Few comparable human studies have addressed the relationship between p27 and dietary habits or body constitution; Daling and coworkers [42] examined the relationship between BMI and p27 but found no significant associations.

In contrast to fat, protein and carbohydrate were not significantly associated with any specific tumour characteristics. A recently published report [43] indicates an association between increased intake of carbohydrates and breast cancers with more favourable prognosis, which is consistent with the nonsignificant tendency observed in the present study.

The tumour-promoting effects of fat may be dependent on amount as well as type [44]. In the present study, the inverse relation between energy-adjusted PUFA and proliferation was most pronounced, indicating that low intake of PUFA corresponds with the occurrence of highly proliferative breast cancer. A previous study from the MDCS including 237 incident breast cancer cases found a positive association between high intake of PUFA, specifically ω 6 fatty acids, and breast cancer incidence [19]. Other studies analyzing the risk for breast cancer have indicated similar positive associations between PUFA and breast cancer incidence [19, 45]. Human studies on the association between fatty acids and breast tumour characteristics have, to our knowledge, not yet been published, and further studies are needed in order to validate our preliminary findings.

Body composition, expressed in terms of BMI, waist and hip circumference, and height, was associated with tumour grade, and slender body constitution corresponded with low-grade tumours. The association between body constitution and breast cancer stage has been reported in other studies [46, 47], and an association between obesity and high-grade tumours has also been described [42]. These observations support our findings.

In the present study body size and distribution of body fat were clearly associated with histological grade, whereas energy intake – with an emphasis on fat – appeared to influence the proliferation rate and overexpression of cell cycle regulators. The image of a slender figure with high energy and fat intake and a correspondence with a less malignant phenotype may seem controversial. However, similar to other epidemiological studies [3, 48], energy and fat intake and body constitution were shown not to be significantly associated (data not shown). Notably, the variable 'total energy' should not be confused with 'energy balance', which takes energy expenditure into account [49]. This might explain why, in this study, high energy intake and a slender figure are both related to less aggressive tumours. Physical activity was nevertheless not associated with any specific subgroup of breast cancer, which could be caused by difficulties in assessing this variable [50]. Obese individuals commonly report low-energy diets [51] as well as recent changes in dietary habits [52], making it plausible that obese participants are in reality low in energy intake.

Alcohol intake was not associated with any of the breast cancer subgroups examined, but a previous report from the MDCS identified an association between high intake of wine or total alcohol (nonsignificant) and increased breast cancer risk [6].

The above findings indicate that the strategy of subgrouping breast cancer according to proliferation, tumour grade and expression of cyclin D₁ and E might be a useful approach when studying breast cancer in relation to dietary behaviours and body constitution. Further studies are needed to improve the delineation of women at risk for developing highly malignant breast cancer and, hopefully, to contribute to novel prevention strategies.