Correlation between obesity and prognostic/predictive parameters with emphasis on the importance of lymph node metastases in patients with invasive breast carcinoma

Introduction

Obesity is a major public health problem in many developed regions in the world, but also in European developing countries including Romania where obesity rates in the adult population have increased in the last decades [1, 2]. Breast carcinoma is the most frequent form of cancer in women worldwide, as it represents 23% of all cancers globally, with 1.38 million newly diagnosed cases/year and 28% of all forms of cancer in Europe [3]. In Romania, breast cancer is a major public health issue, as the incidence has increased steadily during the last decades, from 25/100 000 women in 1988 to 40.14/100 000 women in 1996, 50.56/100 000 women in 2006 and 61.1/100 000 in 2008, and this incidence continues to increase according to the published studies [4]. The rate of death due to breast cancer has decreased recently by 20% in 15 European countries (for example, it decreased by 45% in Iceland), but has remained stationary or even increased in Central and East European countries, and it increased by 17% in Romania [5]. This is due to numerous factors including the socio-economic level of the population, the lack of information, the lack of a national screening program, the lack of specialized medical centers (interdisciplinary units, with few exceptions), but is also due to the increased incidence of obesity.
Obesity has been associated in various studies with an increased risk of breast cancer (but also other non cancer-related deaths), especially in postmenopausal patients, in whom breast cancer is the most common form of cancer in Romania [6, 7]. Furthermore, obesity on breast cancer diagnosis has been associated with a higher risk of recurrence and inferior survival, including breast cancer-specific survival [8]. Management decisions and survival evaluation in breast carcinomas are based on various prognostic and predictive factors for response to targeted therapies (such as age, tumor size, histological type and grade, molecular status, presence of axillary node metastases). Thus, understanding the relation between host-related factors, such as obesity, and prognostic/predictive factors of the primary breast carcinoma, may have important clinical and prognostic implications.
One of the most important prognostic factors in breast carcinoma is the axillary lymph node status, i.e. the presence or absence of axillary metastases [9, 10, 11]. Disease-free survival and overall survival decrease proportionally with the increase of the number of positive axillary lymph nodes [9]. The number of positive axillary nodes is a strong prognostic factor in breast cancer, but this parameter is affected by variability in nodal staging technique yielding varying numbers of excised nodes. The nodal ratio of positive to excised nodes is an alternative that could address this variability, and previous studies have demonstrated that nodal ratio is a stronger predictor of disease-free survival than total positive nodes excised [12, 13]. Moreover, the presence of extracapsular extension has been demonstrated to have an impact on survival, and to be associated with higher risk of both mortality and recurrence of disease being recommended the consideration of extracapsular extension in perspectives to be validated and included in the oncologic staging [14, 15].
The aim of this study is, firstly, to evaluate the association between clinical-pathological parameters and obesity, and, secondly, to evaluate whether obese women experience more advanced disease with a higher axillary lymph node ratio and higher stage at the time of diagnosis, as very few papers have addressed this issue so far in the literature [16]. We used body mass index (BMI) as an index and further correlated it with the presence of axillary lymph node metastases (the number of involved lymph nodes, the lymph node ratio (LNR), the presence and diameter of the extracapsular extension).

Material and methods

Patient selection

We retrospectively reviewed all the medical records of the patients with invasive breast carcinomas (IBC) from our database consecutively diagnosed between 2012 and 2015 on core biopsy, in whom sentinel lymph node biopsy (SNB) and/or axillary lymph node dissection (ALND) was performed in association with quadrantectomy/mastectomy. These cases originated in a population that had not been previously screened for breast carcinoma because a national screening program concerning this disease is not yet available in Romania. Oncological treatment including chemotherapy (doxorubicin, cyclophosphamide, and paclitaxel or docetaxel), radiotherapy and/or hormonal therapy (tamoxifen or aromatase inhibitors) was standardized for all patients and in accordance with the multidisciplinary tumor board consensus. Patients were divided into a normal weight (NW) group (BMI < 25 kg/m2), overweight (OW) (BMI 25-29.9 kg/m2) and obesity (OB) group (BMI ≥ 30 kg/m2) based on the WHO criteria [17]. Details regarding the height and weight to calculate the body index mass (BMI) were retrieved from electronic registration databases. The BMI was calculated as weight divided by the square of height.

Parameter analysis

In each patient diagnosed with invasive breast carcinoma we assessed the following parameters: age, weight, height, tumor size, histological tumor type and grade, molecular profile, presence of axillary lymph node metastases, the LNR, and presence and size of the extracapsular extension of the lymph node metastasis. The histological type was assessed according to the 2012 WHO classification, and histological grading used the Elston-Ellis system [9, 18]. In this study, the presence of extracapsular extension was defined as extension of tumor cells through the nodal capsule into the peri-nodal adipose tissue and was reported in mm (millimeters) in every case. In this study, lymph node ratio (LNR) was defined as the number of nodes involved by the tumor divided by the total number of resected lymph nodes during the surgical treatment. Patients were divided into two groups: high range of LNR (LNR over or equal to 0.2) and low LNR (LNR less than 0.2).
Hormone receptor (ER – estrogen receptor, PR – progesterone receptor), Ki67, and HER2 testing was done by immunohistochemistry according to international recommendations [19, 20, 21, 22]. Three-micrometer-thick sections were cut, dried, deparaffinized, and rehydrated following standard procedures. All the sections were subjected to heat-induced antigen retrieval in citrate buffer (pH 6). Immunohistochemical staining was performed manually, using a NovoLink Polymer Detection System (Leica). Table I reveals the monoclonal antibodies that were used for this study.
In this study, we interpreted tumor foci as ER/PR positive if expression was observed in at least 1% of the nuclei of tumor cells (in the total area of the tumor, regardless of staining intensity, with positive internal control), and ER/PR negative when less than 1% of the tumor cells were positive. Ki67 index was defined as low (when less than 14% of all tumor cells’ nuclei were positive) or high (equal to or more than 14%) by assessing the whole section and recording the overall average score. HER2 expression was scored as follows: 0 (no staining), 1+ (weak incomplete membrane positivity in at least 10% of the tumor cells), 2+ (weak/moderate complete membrane positivity in at least 10% of the tumor cells), and 3+ (strong complete membrane positivity in at least 30% of the tumor cells). For statistical analysis, 0 and 1+ HER2 scores were considered negative, and scores of 2+ (confirmed by chromogenic in situ hybridization [CISH] test) and 3+ were considered positive. In this study, CISH was performed in all cases with a 2+ score.
We used surrogate definitions of intrinsic subtypes of breast cancer according to Goldhirsch et al. [23] and defined as Luminal A cases with ER and/or PR positive, HER2 negative and Ki67 low (<14%) both in tumor and in lymph nodes; as Luminal B proliferative (HER2 negative) (Bp) cases with ER and/or PR positive expression, HER2 negative and Ki67 high; Luminal B (HER2 positive) (Bh) cases which are ER and/or PR positive, any Ki67 and HER2 positive; as HER2 enriched (H) cases with HER2 overexpressed and ER and PR absent; as triple negative (TN) cases with ER, PR and HER 2 negative [23].

Statistical analysis

Associations between BMI and other prognostic parameters (excluding unknown ones) were analyzed using the chi square test and Fisher’s test. All P values were two-tailed. It was considered statistically significant if p < 0.05. Statistical analyses were performed in Graph Pad Prism 6.1 and SPSS.

Results

The study population comprised 245 consecutive patients with invasive breast carcinoma aged between 27 and 80 years (mean age 58.29 years). Of these, 84 patients (33.07%) were OW, 72 (29.38%) OB and 91 (37.14%) NW. Mean age was 52.81 years in the NW group, 60.38 years in the OW group, and 62.8 in the OB group. However, most of the OW/OB women were aged > 40 (p = 0.0092) (Table II).
The size of the primary tumor was grouped in 0-20 mm, 21-50 mm, and over 51 mm category. In 59.2% of cases, the patients had tumors between 0-20 mm (54 NW, 59 OW and 39 OB). In the 21-50 mm group, we detected slightly more OW and OB patients than NW (28 NW, 34 OW and 30 OB). The statistical analysis revealed no significant differences between the tumor diameters in different BMI groups (p = 0.705).
We grouped our patients regarding the histological type of the tumor in no special type of invasive carcinoma (NST type) and other histological types of tumors. One hundred and ninety-eight patients (80.2%) had NST type tumors (38.4% NW, 32.3% OW, and 29.3% OB); 46 patients were diagnosed with other types of invasive breast cancer (4.08% NW; 9.39% OW and 5.31% OB). In our series, overweight patients presented a significantly higher rate of other histological types of tumors than the other BMI groups (p = 0.049).
Almost 30% of all cases (29.8% – 73 patients) had grade 1 tumor (23.3% NW, 52.1% OW and 24.7% OB), 48.2% presented grade 2 tumors (55.8% NW, 37.5% OW, and 52.1% OB) and only 22% had grade 3 tumors (38.9% NW, 31.5% OW and 29.6% OB). In this series, overweight patients had a significantly higher rate of grade 1 tumor compared to other BMI groups, while normal weight patients presented more grade 2 type tumors (p = 0.013).
We studied the influence of obesity on the axillary lymph node status, since this is still the most important prognostic factor for breast cancer survival. We grouped our cases in metastatic and non-metastatic lymph nodes highlighted by the histopathological examination after sentinel lymph node biopsy and/or axillary clearance. Over half (54.3%) of our cases had no metastatic lymph nodes, and in 45.7% macrometastasis was revealed in the axillary lymph nodes (no cases with micrometastases were documented). NW patients had significantly fewer metastatic lymph nodes (p = 0.05) than in the OW and OB groups.
There was no statistically significant difference between BMI groups according to the LNR (p = 0.66).
Out of 111 cases with macrometastasis in the axillary lymph nodes, 58 cases (52.25%) had extracapsular extension (ECE) (11.7% NW, 24.32% OW and 16.22% OB), and 54 cases were identified without extracapsular extension (Fig. 1). We found statistically significant relations between BMI groups and the presence of ECE in these series: significantly more overweight patients presented extranodal invasion, while normal weight patients were more likely to be without extracapsular extension (p = 0.04).
Out of 58 cases which presented ECE, 4 were ruled out of the study because original slides were not accessible for size measurements. In the remaining 54 cases, we measured an average 4.4 mm ECE diameter (minimum 0.1 mm, maximum 21 mm). We grouped our cases with ECE in ECE ≤ 1 mm (9.25% NW, 7.01% OW and 12.96% OB) and ECE > 1 mm groups (17.81% NW, 36.84% OW, 16.66% OB). We found no statistically significant relations between the extracapsular extension diameter and BMI groups (p = 0.1), but the high value of the odds ratio (9.53) between NW and OW groups suggests that an important proportion of OW patients had larger extracapsular extension.
We identified 173 consecutive patients with invasive breast carcinoma and a complete molecular profile, of whom 55 (31.79%) were NW, 60 (34.68%) OW, and 58 (33.52%) OB. Out of the 173 cases, 58 were Luminal A-type, 66 were Luminal B-Her 2 negative-type, 21 were of Luminal B-Her2 positive-type, 5 were of Her2 positive-type, and 23 were of triple negative-type. Statistical analysis revealed that OW patients presented more likely Luminal A type of breast cancer (p = 0.012), while normal weight patients were more frequently Luminal B-Her2 negative (Table II).

Discussion

Obesity and overweight have an increased incidence worldwide. Almost half of European adults are overweight or obese [24], and more than two-thirds of American women are obese, with an increasing risk of postmenopausal breast cancer [25].
OB and OW are associated with different clinical, pathological, and molecular parameters suggesting a poorer prognosis in breast cancer.
Studies confirmed the association between obesity and the age of breast cancer patients. It is considered as an independent adverse prognostic factor for elderly patients [26, 27]. Our study revealed that there is a statistically significant relationship between the patients’ age and their BMI: significantly more OW patients were in the > 40 age group, while most of the patients in the < 40 group were NW (p = 0.005).
Different studies have revealed that OW and OB are associated with larger tumor diameters [16, 28, 29]. In our study, OB and OW were also associated with tumors with a diameter of 21-50 mm (p = 0.7, OR = 1.26, 1.48).
Regarding the correlation between the histological type of tumor and obesity, some studies have demonstrated a significant relation between higher BMI and NST histological type [27], while others claim that there is no connection between obesity and histological type of the tumor [29, 30]. In our series, OW and OB were associated with other histological tumor types than NST (p = 0.049).
Also, several studies showed a strong relation between OB, OW, and higher histological grade of the primary breast tumor [29, 31, 32]. Contrary to these studies, we found that OW patients had more likely grade 1 tumors (p = 0.01, OR = 0.307), while normal weight patients in our series had significantly more grade 2 and 3 tumors.
Obesity in association with the molecular profile of breast cancer is a controversial issue in the literature. While several studies indicate that OB and OW patients more likely have a TN breast cancer [33, 34], others reveal a significant relationship with ER-positive breast cancer [35]. In our series, OW was strongly associated with Luminal A molecular type (p = 0.012), which is known to have a better prognosis. These findings may be attributed in part to the increased estrogen production in peripheral fat deposits through androgen aromatization in patients with obesity.
The most important prognostic factor in breast cancer is axillary lymph node involvement. According to the literature, OB and OW are associated with a higher rate of axillary metastatic lymph nodes and higher lymph node ratio [29, 25, 28]. However, no other studies evaluating the relationship between obesity and LNR and/or ECE (the presence of ECE and the size of ECE) have been previously published to the best of our knowledge. Both LNR and ECE are important prognostic parameters as mentioned by international guidelines. This study shows that OW and OB patients have a significantly higher number of metastatic lymph nodes than NW patients (p = 0.05), and significantly more cases of extracapsular extension (p = 0.04). There was no significant relation between the BMI groups and the LNR or the size of the extracapsular extension.

Conclusions

In our series, breast cancer patients had a high prevalence of obesity and overweight, mostly over the age of 40.
OB and OW were strongly associated with larger tumor diameter, lower tumor grade, Luminal A-type tumor, higher number of metastatic lymph nodes, and presence of ECE in the metastatic lymph nodes.
Since obese women experience more advanced invasive breast carcinoma, it would be of interest to implement national scale level programs to improve ideal weight maintenance.
We thank Adrian Naznean from the Department of Foreign Language of the University of Medicine and Pharmacy of Tirgu Mures for carefully revising the text of the manuscript.

The authors declare no conflict of interest.

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Address for correspondence

Rares Georgescu
Department of Surgery
University of Medicine and Pharmacy Targu Mures
Ghoerghe Marinescu no. 1
540103 Targu Mures, Romania
e-mail: breastcancer.reas.tgm@gmail.com