Biological and Clinical Sciences Research Journal
ISSN: 2708-2261
DOI: https://doi.org/10.47264/bcsrj0201003
Biol. Clin.
Sci. Res. J., Volume, 2021: e003
Original Research Article
ASSESSMENT OF CONTROVERSIAL RISK FACTORS IN
DEVELOPMENT OF BREAST CANCER: A STUDY FROM LOCAL POPULATION
MALIK
A1, HAFEEZ K2, NAZAR W3, NAEEM M4, ALI
I1, ALI Q1, AHMED1, MUJTABA Z1,
RANA MA5, *HAFEEZ MM1
1Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore-Pakistan
2Nawaz Sharif Medical College, Gujrat-Pakistan
3Respiratory Reg., Cavan
General Hospital, Ireland
4Paediatric Medicine, Al Aleem
Medical college, Gulab devi hospital, Lahore
5Intensive Care Unit, Bahria
International Hospital Lahore-Pakistan
Corresponding
author email: mansoorhafeez140@gmail.com
Abstract: Breast cancer is the most common cause of death
worldwide in women. Several predisposing risk factors have been
identified making its incidence constantly rising. The aim of the current study
was to analyze thyroid hormone, vitamin D, and 8-hydroxydeoxyguanosine (8-OHdG)
as risk factors in the onset of breast cancer. In the present case and control
study, a total of two hundred and seventy-four (n=274) participants were
included after taken the informed consent individually. The participants were
further stratified into two groups. Group A consisted of one hundred and
thirty-seven age and sex-matched healthy individuals presented at OPD whereas
group B had one hundred and thirty-seven diagnosed cases of breast cancer.
Serum vitamin D, thyroid hormone, and 8-OHdG were measured by commercially
available ELISA kits by using the blood samples drawn from the study
population. Data were analyzed with SPSS V21.0 and a p-value less than 0.05
considered statistically significant. Cases and control were comparable in
terms of age, BMI, and gender distribution. Vitamin D deficiency was present in
78.4% of cases and 63.5% of control. (OR 3.83 95% CI 1.38 -10.5) 8-OHdG was
present in 56.8% and 11.1% in cases and control respectively (P 0.001).
Similarly, MDA was found positive in 67.6% of cases and 21.6% of control (P
0.001). Vitamin D and thyroid hormone deficiency and elevated levels of
reactive oxygen species (ROS) are supposed to associate with increased risk in
the development of breast cancer.
Keywords:
Breast Cancer,
Vitamin D deficiency, Reactive Oxygen Species, 8-hydroxydeoxyguanosine,
thyroid hormone
Introduction
Breast cancer
is a malignant tumor that has developed from undifferentiated cells of the
breast tissue. This tissue can develop rare benign conditions known as angiosarcomas or phyllodes
tumors. In most of the cases breast tumors are malignant and spread to the
other tissues such as bones (Zülch, 2013). Breast cancer is the most commonly
occurring cancer in women, comprising almost one third of all malignancies in
females (Feng et al.,
2018). According to World Health
organization (WHO), breast cancer is diagnosed in 1.5 million women every year
and the incidence rate is on rise globally (Silva et al.,
2019). In USA, the most common cancer in
women is breast cancer (excluding skin cancers) and it is 2nd
leading cause of mortality among women after lung cancer. In 2016, 124 new
female breast cancer cases were reported per 100,000 women and 20/100,000 die
because of it (Pellegriti et al.,
2013). Pakistan has highest age
standardized incidence rate (ASIR) of breast cancer, affecting 23% of females (DeSantis et al.,
2017). Estimated prevalence in 2012 was
119710, causing mortality of 16,232 patients (Begum, 2018). Age, reproductive factors, personal
or family history of breast disease, genetic pre-disposition and environmental
factors have been associated with an increased risk for the development of
female breast cancer (Sarwar and Saqib, 2017). At presentation lump in breast is
painless and nipple retraction of occasionally with breast skin changes. The
need of the day is to recognize the causative factors and sort out the ways to
minimize them. Oxidative stress is the emerging risk factor not only in breast
cancer but also proved to be contributory factor in almost all types of
cancers. Oxidative stress can be broadly
defined as an imbalance between oxidants and antioxidants in favor of the
oxidants, potentially leading to damage. If the levels of reactive oxygen species
are high and overcome the antioxidant defense mechanisms of the human body,
oxidative damage can occur to lipids, proteins, or directly to DNA. Reactive
Oxygen Species are produced by both enzymatic and non-enzymatic systems within
eukaryotic cells and play important roles in cellular physiology and pathophysiology (Majeed
et al., 2014). Although physiological concentrations are crucial
for ensuring cell survival, ROS overproduction is detrimental to cells, and
considered as key-factors for the development of several diseases, such as
neurodegenerative diseases, cardiovascular disorders, and cancer. Cancer cells
are usually submitted to higher ROS levels that further stimulate malignant
phenotype through stimulus to sustained proliferation, death evasion,
angiogenesis, invasiveness, and metastasis (Sun
et al., 2017). Breast cancer cells have been shown to be
susceptible to oxidative damage and have high levels of oxidative stress,
including protein damage, DNA damage, and lipid peroxidation
(Qi et al., 2015). However, less attention has been given to the
development of redox system-targeted strategies for
breast cancer therapy. Other known factors are vitamin D and Thyroid hormones.
Thou the formation and release of thyroid hormone is depended on the normal
functioning of vitamin D but thyroid hormone is considered individually a risk
factor of breast cancer. 1, 25-dihydroxyvitamin
(cholecalciferol) or Vitamin D is fat-soluble vitamin
essential for calcium hemostasis and bone
development. Primary source of vitamin D is through dermal synthesis by
ultraviolet rays in sunlight. Subclinical deficiency exits in many people which
are a risk factor for osteoporosis, bone fractures, cardiovascular disease,
cancer and diabetes (Wu et al., 2016).
It possesses anti-carcinogenic
properties which include cell proliferation inhibition, invasion, metastasis
and angiogenesis and induction of apoptosis and differentiation (Lee
et al., 2017). In recent
developments, a vitamin D receptor (VDR) gene has been reported that increase
risk of breast cancer. Efforts have been directed toward identification of
vitamin D deficiency as its levels (levels ≥45 ng/mL) may protect against breast cancer (Parva et al.,
2018). The association between thyroid
disorders and breast cancer has been studied in the past with mixed results.
Most of the articles published to date have relied on studies of relatively
small sample sizes (Hernando et al.,
2020; Kleine et al., 2019; Wolf
et al., 2020).
A recent systematic review and meta-analysis that included 13
population-based studies with 24,808 participants through June 2016 found that
neither hypothyroidism nor hyperthyroidism was related to the risk of breast
cancer (Kuijpens et al.,
2005).
A study utilizing the national registry in Denmark by Sogaard and
his colleagues also examined this association without age stratification
and found a similar result for hyperthyroidism, but not for hypothyroidism (Søgaard et al.,
2016). The current study assessed the
association of reactive oxygen species, vitamin D and thyroid profile in
development of breast cancer.
Material and methods
The
current case and control study included two hundred and seventy-four
participants divided into two groups. Group A was the control group and had one
hundred thirty-seven age and sex matched healthy participants that visited the
OPD of different Hospitals of the Lahore. Group B constituted the one hundred
and thirty-seven diagnosed cases of breast cancer. All participants were
included in the study after taken the informed consent and the approval of
ethical review board of Institute of Molecular Biology and Biotechnology
(IMBB), The University of Lahore. Data was collected on specially design Proforma and 5ml of blood sample was withdraw and stored
for further analyses from different hospitals of Lahore (31.5204° N; 74.3587°
E). Commercially available ELISA kits were used to analyzed
the different variables such as vitamin D, thyroid hormone and Reactive oxygen
species. A method has already been used for screening lipid peroxidation product (MDA)
by Thiobarbituric acid reactive substances with the help of spectrophotometer. In this method we took 200 µl of sample, then 0.2ml Sodium dodecyl sulfate was added, then 20% of acetic acid by 1.5
ml was added, and lastly TBA was added about 1.5ml. After this, the solution
was centrifuged at 3000 rpm for 10 min. Two layers were formed after
centrifugation and upper organic layer was taken from test tube to check
absorbance at 532 nm. A sample size of
274 patients (1:1 ratio, 137 Cases and 137 Control) is calculated by using WHO
sample size calculator with 80% power of test and 5% level of significance. All
data was recorded in proforma and analyzed with SPSS
version 20. Qualitative data in cases and controls were presented in frequency
and percentages. Quantitative data such as age and level of vitamin D was presented
in mean and standard deviation. Odd
Ratio were calculated between cases and control. Chi
Square test and student t test/ ANNOVA were used to compare categorical and
numerical data respectively. P value
< 0.05 considered significant.
Results
Mean age of patents
was 52.2 ± 9.1 years and 53.7 ± 10.9 years in case and control group
respectively. Majority of patients in both groups were > 50 years. (Table 1)
Table 1: Age distribution of patients
AGE DISTRIBUTION OF PATIENTS |
||||
Age |
Cases |
Control |
||
n |
% |
n |
% |
|
Age < 50 Years |
64 |
37.8 |
67 |
45.9 |
Age > 50 Years |
73 |
62.2 |
80 |
54.1 |
Total |
137 |
100.0 |
137 |
100.0 |
Mean + SD |
52.2 ± 9.1 |
53.7 ± 10.9 |
Cases and control were comparable in terms
of height, weight and BMI as p valve
is insignificant (Table 2)
Table
2: Height, weight and BMI
HEIGHT, WEIGHT & BMI |
|||||
Parameter |
Cases |
Control |
P Value |
||
Mean |
SD |
Mean |
SD |
||
Height (cm) |
153.1 |
6.3 |
155.1 |
4.7 |
0.219 |
Weight (Kg) |
58.24 |
7.4 |
56.9 |
7.2 |
0.420 |
BMI (Kg/m2) |
37.9 |
4.9 |
36.6 |
4.4 |
0.226 |
Vitamin D deficiency was present in 78.4%
of cases and 63.5% of control. (OR 3.83, CI 95) (Table 3).
Table 3: Assessment of vitamin d levels
and risk of development of breast cancer
Vitamin D Deficiency |
||
Vitamin D n (%) |
Breast Cancer |
No Breast Cancer |
Cases |
Control |
|
Deficiency (YES) |
79 (78.4) |
68 (63.5) |
Deficiency (NO) |
58 (21.6) |
69 (36.5) |
Total |
137 (100.0%) |
137 (100.0) |
Odd ratio |
3.83 |
ROS namely 8OHdG and MDA were analyzed in
cases and control. Elevated levels of 8-OHdG were present in 56.8% and 11.1% in
cases and control respectively (P 0.001). Similarly, MDA was found
positive in 67.6% case and 21.6% of control. (P= 0.001)
(Table 4)
Table 4: Assessment of levels of ROS and
risk of development of breast cancer
REACTIVE OXYGEN SPECIES (ROS) |
|||||
8OHdG |
Cases |
Control |
P-Value |
||
n |
% |
n |
% |
0.001 |
|
ELEVATED (YES) |
71 |
56.8 |
53 |
11.1 |
|
ELEVATED (NO) |
66 |
43.2 |
84 |
91.9 |
|
Total |
137 |
100.0 |
137 |
100.0 |
|
MDA |
Cases |
Control |
P-Value |
||
n |
% |
n |
% |
0.001 |
|
ELEVATED (YES) |
75 |
67.6 |
58 |
21.6 |
|
ELEVATED (NO) |
62 |
32.4 |
79 |
78.4 |
Total |
137 |
100.0 |
137 |
100.0 |
Thyroid levels
as estimated free T3 and free T4 were statistically low in the group B as
compare to the Group A (p=0.041,
p=0.035 respectively) (Table-5)
Table 5:
Estimation of thyroid profile in breast cancer patients
Variables |
Control |
Patient |
P-Value |
FT3 pmol/L |
4.72±0.24 |
3.73±0.189 |
0.041 |
TSH pmol/L |
10.11± 1.64 |
17.86±4.98 |
0.035 |
Figure-1: Levels
of free T3 and TSH in both groups
|
|
Discussion
Breast cancer has been considered as the most common type of
cancer among the women. Known and well-established risk factors for breast
cancer include age, family history, and the density of breast tissue, parity,
overweight, alcohol intake, and genetic risk factors such as BRCA mutations (Tseng et al.,
2015). Recently,
vitamin D receptor (VDR) genes were reported to increase breast cancer
risk. Several molecular breast cancer subtypes have been identified:
luminal A and B (accounting for 50%-60% of breast cancer cases), basal-like or
triple-negative (10%-20% of breast cancer cases) and human epidermal growth
factor receptor 2 (HER2)-enriched (10%-15% of cases) (Krashin et al.,
2019; Weng et al.,
2018). Vitamin
D receptor genes operated by vitamin D have important roles in the mammary
gland through regulation of calcium transport during lactation, hormone
differentiation, and milk production. Many efforts and enormous research
have been directed toward identifying vitamin D as a breast cancer risk factor
to be targeted for cancer prevention. This is because circulating vitamin D
levels (levels ≥45 ng/mL)
may protect against breast cancer (Hernando et al.,
2020; Parva et al.,
2018) and because breast cancer
chemoprevention drugs that alternate the carcinogenesis process such as
estrogen receptor modulators, tamoxifen, raloxifene, and aromatase
inhibitor have high toxicities and not effective in the aggressive estrogen
receptor–negative (ER−) breast cancers (Journy et al.,
2017; Kitahara et al.,
2019). Our study shows that vitamin D was found deficient in
78.4% case and 63.5% of control. (OR 3.83 95% CI 1.38 -10.5) Our result were comparable with Shaukat
et al where serum vitamin
D levels were significantly lower in cases (85.7%) than controls (55.8%). ORs
(95% CIs) for breast cancer risk were 7.8 (1.99-30.58) for women with vitamin D
concentrations less than 20 ng/mL. Bilinski et al
examined the association between vitamin D status and risk of breast
cancer in Australian women and they reported that 25(OH) D concentration below
75 nmol/L at diagnosis was associated with a
significantly higher risk of breast cancer (Dong
et al., 2018). Compared with
subjects with sufficient 25(OH) D concentration, the odds ratios of breast
cancer were 2.3 (95% CI = 1.3-4.3), 2.5 (95%
CI = 1.6-3.9) and 2.5 (95% CI = 1.6-3.8) for subjects
categorized as severely deficient, deficient, or insufficient vitamin D status,
respectively (Hall
et al., 2008; Wang et al., 2018). Park et al studied the association
between vitamin D and breast cancer risk among the Asian population. Examined the association between serum 25(OH) D and breast cancer
risk stratified by menopausal status and hormone receptor (HR) status of the
tumor. Women with vitamin D deficiency had 27% increased the risk for breast
cancer compared with women who have sufficient levels of serum 25(OH) D. This
association did not significantly vary by menopausal status. HR status has
significant inverse association and this association was more pronounced in
HR-negative breast cancer, particularly with patients with triple-negative
breast cancer (Davis
et al., 2009; Joo and Jetten, 2010). Few studies have shown no association
between 25(OH) D deficiency and risk breast cancer. Eliassen et al investigated whether
plasma 25(OH) D interacts with breast tumor expression of VDR and its risk of
breast cancer in women followed more than 20 years. No overall
association was observed between plasma 25(OH) D and breast cancer risk. Women
with high plasma 25(OH) D levels in the summer have a reduced breast cancer
risk. Plasma 25(OH) D may be inversely associated with risk of tumors
expressing high levels of VDR (Tosovic
et al., 2012). Neuhouser et al
in multivariate-adjusted models for colorectal cancer, the association
strengthened (OR = 4.45, 95% CI = 1.96-10.10), whereas
in multivariate-adjusted breast cancer models, associations were not
significant (OR = 1.06, 95% CI = 0.78, 1.43) (Glover
et al., 2015; Khan et al., 2016).
Our study has shown significant association with ROS and
breast cancer. ROS were more frequent in cases than control (P=0.001). Sarmiento-Salinas et al has shown triple negative breast
cancer (TBNC) have been found to have increased ROS levels, our data shows
increased ROS levels in all the TNBC cell lines studied in comparison to an ER+
breast cancer cell line or the non-tumorigenic cells (Chi et al., 2012). Other cancer types that have also
been characterized by increased oxidation levels and in which a role of ROS has
been proposed in the promotion of malignancy include prostate, gastric, and
pancreatic cancer (Lin et al., 2013). In the literature, there is
conflicting evidence regarding the use of antioxidants during cancer
progression and treatment. In normal cells or pre-cancerous lesions, ROS have
been proposed to induce DNA damage and increase oncogenic
mutations, raising the possibility that dietary supplementation with
antioxidants could suppress the initiation or progression of some types of
cancer. However, antioxidant treatment is known to suppress cancer initiation
in some contexts and increase cancer progression in others. Moreover, the use
of dietary antioxidants has not been shown to reduce cancer incidence and in
fact, antioxidant supplementation has actually increased incidence and death
from some types of cancer including lung cancer or increase the risk of
developing another type of unrelated diseases (Wang and Chen, 2013).
Clinical
studies have found a protective association between hypothyroidism and breast
cancer development; this may be due to the biological effect of T3 at the
cellular level, the interaction of T3 with TRs, or modulation of the thyrotropin receptor (TSH-R) (Lin
et al., 2015). The
antioxidant property of iodine may also play a role, especially considering the
capacity of breast tissue to transport and concentrate iodide (Chi
et al., 2016; Wu et al., 2013). To date, there is still limited
epidemiologic evidence for the link between thyroid replacement treatment and
the risk of breast cancer. In this study, we found an inverse association
between hypothyroidism and invasive breast cancer development among women (p=0.043,0.035).
Weng and his
colleagues using the Taiwanese national database that included 103,466
Asian women, found that women younger than 55 years with a history of
hyperthyroidism had a 16% higher risk of developing breast cancer compared with
those without a history of thyroid disorder (Weng et al.,
2018). A history of
hypothyroidism was also found to be associated with a 19% higher risk, without
stratification by age (Chen et al.,
2013; Kaminskyy et al.,
2013).
Conclusion
In our study age, BMI and education status did not show any
statistically significant association with breast cancer risk. In addition, our
study showed no significant association of BMI and vitamin D
deficiency. The topic of vitamin D deficiency and breast cancer risk is a
field of intense study and many aspects of it require further investigations.
Conflict
of interest
The authors declared absence of any conflict of interest.
References
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"Brain tumors: their biology and pathology," Springer-Verlag.