Keywords
Adiponectin - adiponectin–leptin ratio - breast cancer - leptin
Introduction
Obesity has been associated with the development of breast cancer.[1] The main underlying mechanisms that link obesity to cancer development and progression
include abnormalities of insulin resistance and the insulin-like growth factor system,
impact of adiposity on the biosynthesis and bioavailability of endogenous sex hormones,
obesity-induced low-grade chronic systemic inflammation, and alterations in the levels
of adipocyte-derived growth factors.[2]
Adiponectin, a 244-amino acid protein hormone, also known as AdipoQ, Acrp30 (adipocyte
complement-related protein of 30 kDa), apM1 (gene product of the adipose most abundant
gene transcript-1), and GBP28 (gelatin-binding protein-28), is the most abundant adipocyte-derived
factor, with insulin-sensitizing, anti-inflammatory, and antiatherogenic properties.[3] Leptin, a peptide hormone produced by the ob gene of adipocytes, increases in concert
with adiposity and has been shown to have mitogenic effects on epithelial cells and
to promote cellular proliferation, migration, and invasion in breast cancer cell lines,
properties potentially increasing breast cancer risk and progression.[4] Studies have examined and considered the ratio of these adipokines to be more important
in breast cancer than their absolute concentrations.[5]
The objectives of the study were to determine the association of serum adiponectin,
serum leptin, and adiponectin–leptin ratio (ALR) in patients with breast cancer and
matched controls, and to study their relationship with the various clinicopathological
characteristics of breast cancer.
Materials and Methods
Patients
A prospective, hospital-based case–control study was conducted on 40 patients with
a first-confirmed histopathology diagnosis of breast cancer before treatment commencement
and 40 controls comprising individuals without a history of cancer simultaneously
recruited from the health examination clinics during the same study period. Data on
the status of estrogen receptor (ER), progesterone receptor (PR), and Her-2/Neu were obtained. One control case was matched to each case by age,
menopausal status, date of enrollment (±3 months), and duration of fasting (±4 h).
Cases not histologically proven, male breast cancer patients, patients with previous
history of breast or other cancers, history of diabetes mellitus on treatment, and
subjects who reported a recent (within the previous 1–6 months) weight gain or loss
of 5% or more of their current weight were excluded from the study.
Collection of questionnaire data
The study was conducted in accordance with the Declaration of Helsinki, and the study
protocol was reviewed by the Institutional Ethics Committee. All patients gave written
informed consent and underwent a personal interview. Data were collected on sociodemographic
characteristics, menstrual and reproductive history, menopausal status, lifestyle
behavior, and medical history as well as family history of breast and other cancers.
Menopausal status was defined as last menstruation after 1 year free of menstrual
cycle and no attempt was made to distinguish between women with artificial and those
with natural menopause.
Assay principle
A 10 ml blood sample for measurement of serum adiponectin and leptin was collected
in vacutainer tubes (EDTA-treated), and all tubes were centrifuged at 4°C for collection
of serum. These were stored at −20°C until analysis. Serum adiponectin and leptin
concentrations were measured in a single run using commercially available kits (Human
ADP/Acrp30 [adiponectin] enzyme-linked immune-sorbent assay [ELISA] kit and Human
Leptin ELISA kit, Elabscience Biotechnology Co., Ltd) according to the manufacturer's
instructions. The kit is a direct ELISA for quantitative determination of adiponectin
and leptin in human serum, plasma, or other biological fluids. The detection range
of serum adiponectin kit was 0.78–50 ng/ml, and the minimum detectable dose of adiponectin
was 0.47 ng/ml. The detection range of serum leptin kit was 0.156–10 ng/ml, and the
minimum detectable dose of leptin was 0.094 ng/ml. All matched case–control blood
samples were handled identically and assayed in the same analytical run.
Statistical analysis
Mean and frequencies were used to assess the distribution of sample characteristics.
Student's t-tests and analysis of variance were used to evaluate the differences in
adipokine concentrations by categorical variables. Multiple linear regression models
were used to investigate the associations between adipokine concentrations and demographic,
reproductive, and pathological variables. For all analyses, P values were 2-sided and P < 0.05 was considered statistically significant. All statistical tests were done
using Data Analysis ToolPak (Microsoft Excel Version 2013).
Results
The baseline demographic characteristics and reproductive variables of cases and controls
are summarized in [Table 1]. The sociocultural factors unique to the study population are an early age at marriage
and first full-term birth, the absence of cases of habitual smoking and alcohol consumption
and almost nonexistent oral contraceptive use and hormone replacement therapy. The
clinicopathologic characteristics of cases included in the study are summarized in
[Table 2]. The low cancer literacy among Indian women and reluctance to seek immediate medical
attention is reflected in the prolonged duration of symptoms (mean ± standard deviation:
8.27 ± 13.17 months).
Table 1
Demographic characteristics and reproductive variables of cases and controls
|
Characteristic
|
Cases
|
Controls
|
|
Number of patients
|
40
|
40
|
|
Age, mean (range)
|
52 (25-75)
|
50 (30-70)
|
|
Education, n (%)
|
|
No formal education
|
21 (52.5)
|
15 (37.5)
|
|
Primary school
|
4 (10)
|
6 (15)
|
|
Secondary school
|
13 (32.5)
|
16 (40)
|
|
Graduate
|
1 (2.5)
|
1 (2.5)
|
|
Postgraduate
|
1 (2.5)
|
2 (5)
|
|
Family history of breast cancer/first
|
5 (12.5)
|
2 (5)
|
|
degree relative with breast cancer, n (%)
|
|
Supplementation of vitamins/
|
5 (12.5)
|
8 (20)
|
|
minerals, n (%)
|
|
Alcohol consumption, n (%)
|
0
|
0
|
|
Tobacco, n (%)
|
|
Smoker
|
0
|
0
|
|
Gutka chewer
|
1 (2.5)
|
0
|
|
Physical activity, n (%)
|
|
Mild
|
32 (80)
|
29 (72.5)
|
|
Moderate
|
3 (7.5)
|
5 (12.5)
|
|
Vigorous
|
5 (12.5)
|
6 (15)
|
|
Oral contraceptive pill use, n (%)
|
1 (2.5)
|
0
|
|
Hormone replacement therapy, n (%)
|
4 (10)
|
0
|
|
Age at menarche (years), n (%)
|
|
<12
|
11 (27.5)
|
9 (22.5)
|
|
13-14
|
24 (60)
|
28 (70)
|
|
>15
|
5 (12.5)
|
3 (7.5)
|
|
Parity, n (%)
|
|
Nulliparous status
|
3 (7.5)
|
1 (2.5)
|
|
1
|
1 (2.5)
|
4 (10)
|
|
2-3
|
23 (57.5)
|
25 (62.5)
|
|
>4
|
13 (32.5)
|
10 (25)
|
|
Age at first full term birth (years), n (%)
|
|
15-17
|
15 (40.5)
|
13 (33.3)
|
|
18-20
|
12 (32.4)
|
10 (25.6)
|
|
21-25
|
9 (24.3)
|
11 (28.2)
|
|
>25
|
1 (2.7)
|
5 (12.8)
|
|
Breast feeding, n (%)
|
31 (77.5)
|
35 (87.5)
|
|
Age at menopause (years), n (%)
|
|
<45
|
12 (38.7)
|
12 (37.5)
|
|
45-49
|
11 (35.4)
|
10 (31.2)
|
|
50-54
|
8 (25.8)
|
10 (31.2)
|
|
>55
|
0
|
0
|
Table 2
Clinicopathological characteristics of breast cancer cases
|
Characteristic
|
Cases (n=40)
|
|
Her2 – Human epidermal growth factor receptor 2; ER – Estrogen receptor; PR – Progesterone
receptor; IHC – Immunohistochemistry; FISH – Fluorescence in situ hybridization; SD
– Standard deviation
|
|
Duration of symptoms (in months),
|
8.27 (13.17) and 1-72
|
|
mean (SD) and range
|
|
History of benign breast disease, n (%)
|
4 (10)
|
|
Radiation to chest between age 10 and 30 years, n (%)
|
0
|
|
Side, n (%)
|
|
Left
|
15 (37.5)
|
|
Right
|
25 (62.5)
|
|
Location if present, n (%)
|
|
Upper outer quadrant
|
18(45)
|
|
Upper inner quadrant
|
6 (15)
|
|
Lower outer quadrant
|
5 (12.5)
|
|
Lower inner quadrant
|
2 (5)
|
|
Central
|
9 (22.5)
|
|
Description of lump, n (%)
|
|
Skin changes
|
14 (35)
|
|
Pain
|
8 (20)
|
|
Nipple discharge
|
4 (10)
|
|
Axillary lymphadenopathy
|
23 (57.5)
|
|
Stage grouping, n (%)
|
|
Stage 0-1B
|
0
|
|
Stage IIA
|
10(25)
|
|
Stage IIB
|
9 (22.5)
|
|
Stage IIIA
|
6 (15)
|
|
Stage IIIB
|
4 (10)
|
|
Stage IIIC
|
5 (12.5)
|
|
Hormonal receptor status
|
|
ER positive
|
21 (52.5)
|
|
PR positive
|
15 (37.5)
|
|
Her2/neu positive (IHC)
|
9 (22.5)
|
|
Her2/neu positive (FISH)
|
2 (5)
|
|
Grade
|
|
1
|
0
|
|
2
|
13 (32.5)
|
|
3
|
15 (37.5)
|
There was large interindividual variation for serum adipokines, with levels ranging
from 3.2 to 15 μg/ml for adiponectin and 0.215–12.15 ng/ml for leptin. Serum adiponectin
levels were reduced significantly in breast cancer patients, in comparison to controls
(P = 0.04), while serum leptin levels were increased significantly in breast cancer
patients, in comparison to controls (P = 0.03). ALR was significantly lower in breast cancer cases, in comparison to controls
(P = 0.05) [Table 3].
Table 3
Adipokine levels in cases and controls
|
Characteristic
|
Cases
|
Controls
|
P
|
|
SD – Standard deviation
|
|
Serum adiponectin (pg/ml), mean (SD)
|
8.69 (2.95)
|
10.15 (3.56)
|
0.04
|
|
Serum leptin (ng/ml), mean (SD)
|
7.93 (2.90)
|
6.26 (3.86)
|
0.03
|
|
Adiponectin leptin ratio, mean (SD)
|
1.91 (3.17)
|
5.76 (11.92)
|
0.05
|
Multiple linear regression analysis indicated body mass index (BMI) as the only statistically
significant independent correlate of serum adiponectin, serum leptin, and ALR. BMI
was negatively correlated to serum adiponectin levels and ALR (r = 0.33, P = 0.03; r = 0.39, P = 0.01, respectively) and positively correlated to
serum leptin levels (r = 0.34, P = 0.02). There was no correlation between receptor status (ER, PR, Her2/neu),
aggressiveness of disease in terms of tumor size, nodal metastases, stage, tumor grade,
and serum adiponectin levels, leptin levels, or ALR [Table 4].
Table 4
Multiple linear regression analysis with serum adiponectin, leptin, and adiponectin–leptin
ratio as the independent variables
|
Variable
|
Adiponectin (μg/ml)
|
Leptin (ng/ml)
|
Adiponectin leptin ratio
|
|
Correlation coefficient (r)
|
SE
|
P
|
Correlation coefficient (r)
|
SE
|
P
|
Correlation coefficient (r)
|
SE
|
P
|
|
SE – Standard error; Her2 – Human epidermal growth factor receptor 2; ER – Estrogen
receptor; PR – Progesterone receptor; BMI – Body mass index; *P value for BMI
|
|
ER status
|
0.21
|
2.91
|
0.17
|
0.200
|
2.885
|
0.211
|
0.22
|
3.12
|
0.15
|
|
PR status
|
0.15
|
2.95
|
0.33
|
0.160
|
2.907
|
0.321
|
0.25
|
3.1
|
0.11
|
|
Her2/neu receptor status
|
0.03
|
2.98
|
0.83
|
0.030
|
2.944
|
0.851
|
0.09
|
3.19
|
0.54
|
|
Grade
|
0.17
|
2.94
|
0.27
|
0.195
|
2.889
|
0.226
|
0.12
|
3.18
|
0.42
|
|
Stage
|
0.12
|
2.96
|
0.43
|
0.130
|
2.920
|
0.422
|
0.02
|
3.21
|
0.87
|
|
Tumour size
|
0.13
|
2.96
|
0.41
|
0.167
|
2.904
|
0.301
|
0.29
|
3.07
|
0.06
|
|
Nodal status
|
0.1
|
2.97
|
0.53
|
0.055
|
11.860
|
0.735
|
0.1
|
3.19
|
0.53
|
|
BMI
|
0.33
|
2.82
|
0.03*
|
0.346
|
2.763
|
0.028*
|
0.39
|
2.94
|
0.01*
|
|
Physical activity
|
0.12
|
2.96
|
0.42
|
0.133
|
2.919
|
0.411
|
0.12
|
3.18
|
0.44
|
|
Parity
|
0.09
|
2.97
|
0.57
|
0.087
|
2.934
|
0.589
|
0.2
|
3.14
|
0.2
|
Discussion
Obese women have on average a 33% higher risk of total (95% confidence interval [CI]:
21%–47%) and breast cancer-specific mortality (95% CI: 19%–50%) compared to nonobese
women.[6] Adipose tissue is now recognized as metabolically active and a source of adipose
tissue-derived hormones and cytokines (adipokines) such as leptin, adiponectin, and
inflammatory cytokines.
Elevated leptin levels stimulate breast tumor cell proliferation through several signal
transduction pathways and by altering cell-cycle checkpoints via upregulation of cyclin
D1 and cyclin-dependent kinase 2.[7],[8] In contrast to leptin, adiponectin levels are diminished in obesity. Adiponectin
via its antagonism of leptin reduces aromatase activity and local estrogen production,
signaling through the phosphatidylinositol-3-kinase pathway, and proliferation.[9],[10] Adiponectin blocks activation of nuclear factor-kappa B by cytokines such as transforming
growth factor-α and thus reduces subsequent production of proinflammatory adipocytokines
and insulin resistance.[11] By upregulating peroxisome proliferator-activated receptor-γ, which forms heterodimers
with the retinoid X-receptor, adiponectin promotes differentiation and apoptosis through
p53-dependent mechanisms.[12] Finally, adiponectin upregulates the tumor suppressor liver kinase B1 and increases
5' adenosine monophosphate-activated protein kinase, which in turn blocks activation
of the mammalian target of rapamycin pathway and reduces motility and angiogenesis.[13]
Serum adiponectin levels and ALR were significantly reduced whereas serum leptin levels
were increased in breast cancer cases in comparison to controls which were in agreement
with previous studies.[3],[5] The mean adiponectin levels of cases (8.69 μg/ml ± 2.95) in the present study were
comparable to other studies (2–20 μg/mL).[14],[15] However, the optimal level of adipokines for breast health and levels that should
be considered unhealthy have not been established and vary by race and assay methodology.[16]
Neither receptor status (ER, PR, and HER-2/neu receptor) nor aggressiveness of disease
(tumor size, nodal metastases, stage, and grade) had an effect on the serum adiponectin
levels, leptin levels, or ALR in cases. Some but not all studies have suggested that
breast tumors arising in women with hypoadiponectinemia may present a more aggressive
phenotype (large size of tumor, higher histological grade, and ER negativity).[17],[18],[19] Studies have found a significant association of serum adiponectin with either receptor-negative
breast cancer [18],[20] or receptor-positive breast cancer.[21] However, there are several studies which have found no significant associations
in regard to hormonal receptor status.[22],[23]
BMI was negatively correlated with serum adiponectin levels and ALR and positively
correlated with serum leptin levels and was the only statistically significant independent
correlate. In other words, the association of serum adiponectin and serum leptin levels
with breast cancer was not independent of measures of adiposity in the present study.
Limitations
The cross-sectional design of this study meant that there was a potential for selection
bias, particularly in the selection of controls. However, controls came from the same
study base as our cases and criteria for inclusion and exclusion were strictly adhered
to minimize any influence on results of the study. It has been hypothesized that adiponectin
may exert carcinogenic effects through modulation of insulin sensitivity.[3] Therefore, another limitation of this study is the lack of information on insulin
levels in cases and controls. The results of the study are based on a one-time measurement
of serum adipokine levels. However, previous studies have confirmed the stability
and reliability of a one-time measure to be high.[24] Another limitation of this study is its small sample size.
Recommendations for future work
The foremost reason why investigators have been attempting to accurately define the
link between obesity and breast cancer is understanding that it offers an opportunity
at chemoprevention. The interventions that have been tested to raise adiponectin,
particularly for overweight or insulin-resistant individuals, include, encouraging
weight loss,[25] bariatric surgery,[26] antidiabetic drugs of the thiazolidinedione class,[27] lipid-lowering drugs including statins,[28] omega-3 fatty acids,[29] and fibrates,[30] antihypertensives such as angiotensin-converting enzyme inhibitors [31] and beta blockers.[32]
Conclusion
In summary, our results suggest that low serum adiponectin levels, low ALR and high
serum leptin levels are associated with breast cancer. BMI was the only statistically
significant independent correlate of serum adiponectin, serum leptin and ALR. The
inconsistencies in data attempting to define the relationship between obesity and
breast cancer reinforce the complexity and multifactorial nature of the relationship.
