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ORIGINAL ARTICLE
Ahead of print publication  

p16 promoter methylation, expression, and its association with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 subtype of breast carcinoma


1 Department of Pathology, UCMS and GTB Hospital, University of Delhi, Delhi, India
2 Division of Molecular Oncology, National Institute of Cancer Prevention and Research, Noida, Uttar Pradesh, India
3 Department of Surgery, UCMS and GTB Hospital, University of Delhi, Delhi, India
4 Division of Molecular Cytology, National Institute of Cancer Prevention and Research, Noida, Uttar Pradesh, India

Correspondence Address:
Suresh Hedau,
Division of Molecular Oncology, National Institute of Cancer Prevention and Research, I - 7, Sector - 39, Noida - 201 301, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_472_18

 > Abstract 


Objectives: The purpose of the study is to investigate p16 protein expression and promoter methylation of p16 gene and their association with molecular subtypes based on parameter such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2).
Materials and Methods: A total of 114 breast cancer tissue biopsies were collected for methylation-specific polymerase chain reaction (MSP) and immunohistochemical (IHC) analysis.
Results: Seven tissue microarrays were constructed. p16 protein expression was studied in 114 cases, of which 35/114 (30.7%) cases showed strong expression and the majority of them had ER-positive tumor (57.6%), and it was statistically significant (P < 0.0074). Similarly, p16 expression was reduced in the majority of PR-negative tumors (83.9%) and the association was statistically significant (P = 0.0026). p16 methylation was studied in 114 cases and was positive in 71.0% cases.
Conclusion: High p16 protein expression was associated with ER-positive, PR-negative, and HER2-negative tumors which is associated with poor prognosis. p16 protein expression may be used as a prognostic indicator to predict treatment response to hormonal therapy.

Keywords: Breast cancer, estrogen receptor, human epidermal growth factor receptor 2/neu, p16, progesterone receptor



How to cite this URL:
Goyal A, Sahu RK, Kumar M, Sharma S, Qayyum S, Kaur N, Singh UR, Mehrotra R, Hedau S. p16 promoter methylation, expression, and its association with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 subtype of breast carcinoma. J Can Res Ther [Epub ahead of print] [cited 2019 Aug 17]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=263536




 > Introduction Top


Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females worldwide.[1] With urbanization and changes in lifestyle, there is increasing incidence of carcinoma of the breast, and it is estimated that every year, 144,937 women are diagnosed with breast cancer and approximately 70,218 deaths are due to this cancer.[2] The incidence of breast cancer is higher in urban India, especially the metropolitan cities where it is now the leading female cancer and is the second most common cancer after cervix.[2]

The most important biomarker in breast cancer is estrogen receptor (ER) expression as it gives information about the sensitivity of breast cancer to tamoxifen. Several studies have shown that ER-negative cases achieve pathological complete response with neoadjuvant chemotherapy compared with ER-positive cases.[3] Elledge et al. have shown that in metastatic breast cancer, the tumors observed ER, and progesterone receptor (PR) positivity showed a better response to tamoxifen than tumors showing ER positivity but lacking (PR) expression.[4] Human epidermal growth factor receptor 2 positive cases have more chances of relapse and shorter survival rate in response to tamoxifen. However, the advent of anti-HER2 therapies like monoclonal antibody trastuzumab targeted at HER2, it needs to be incorporated in the testing panel as patients are highly benefited. Patients expressing truncated cytoplasmic HER2 are poorly responsive to trastuzumab but may be responsive to tyrosine kinase inhibitor – lapatinib.[3]

The p16 protein is an inhibitor of cyclin-dependent kinase that blocks the G1/S phase of the cell cycle by inhibiting cyclin D-CDK4/6 complex formation through direct binding with cyclin-dependent kinase (CDK) 4/6.[5] The p16-CDK4/6 complex inhibits pRb phosphorylation, resulting in G1 arrest.[6] The tumor suppressor p16INK4a gene is a major target in carcinogenesis, and downregulation of the p16 protein has been reported in many malignancies.[7] Expression of p16 has been reported in benign breast lesions such as fibroadenoma and in breast carcinoma.[8] Some reports have also indicated that strong p16 expression is associated with poor prognostic parameters.[9],[10] Therefore, the pattern of p16 expression is variable, and it complicates the elucidation of the role of p16INK4a in breast tumors.

Hypermethylation of the promoter region of p16 as an epigenetic change involved in breast carcinogenesis has been reported by various studies. There has been a wide variation among the incidence reported ranging from 3.6% to 65%.[11],[12] The commonly proposed mechanisms of underexpression are methylation of the promoter region of the p16 gene, loss of heterozygosity, and small homozygous deletions.[13] p16 involvement has been documented in a number of cancers including melanoma, esophageal, bladder, head and neck, breast, brain, osteosarcoma, ovarian, renal and hematological malignancies such as acute lymphocytic leukemia.[14] Studying methylation of p16 in breast carcinoma can be useful not only in understanding the pathogenesis but also in treatment with various demethylating agents available, determining the prognosis and monitoring the disease progression.

Our data also suggest that strong expression of p16 protein may play an important role in mammary carcinogenesis in Indian sporadic cases. Therefore, the present study has been designed to analyze p16 promoter methylation and protein expression in breast cancer cases and correlate the expression of these proteins with various clinicopathological parameters, ER, PR, and HER2.


 > Materials and Methods Top


A total of 114 breast cancer cases were collected from Department of Surgery at University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi. Patients who had a cytological/histopathological diagnosis of breast malignancy were included in this study. Corresponding normal adjacent breast tissues of these patients were used as a control. The Histological typing and Bloom Richardson's grading was done by a trained pathologist. Ethical clearance was obtained from the College Ethical Committee.

DNA extraction

High molecular weight genomic DNA from breast cancer biopsies and normal controls were isolated by standard Proteinase K digestion and phenol-chloroform extraction procedure described by Hedau et al.[15]

Bisulfite modification

DNA methylation was carried out by sodium bisulfite treatment of genomic DNA. Briefly, 2 μg of genomic DNA was denatured with 0.3M NaOH for 15 min at 37°C in a final volume of 20 μl followed by manufacture's instruction (Wizard R DNA clean-up Resin, Promega, USA).

Analysis of p16 promoter methylation patterns

Methylation-specific polymerase chain reaction (PCR) distinguishes unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA, which convert unmethylated, but no methylated cytosine to uracil and subsequent PCR by use of primers designed for either methylated or unmethylated DNA.[16] Primer sequences of p16 for the unmethylated reaction were 5'– TTA TTA GAG GGT GGG GTG GAT TGT– 3' (sense) and 5'– CAA CCC CAA ACC ACA ACC ATA A– 3' (antisense) and for the methylated reaction, 5'– TTA TTA GAG GGT GGG GCG GAT CGC– 3' (sense) and 5'– GAC CCC GAA CCG CGA CCG TAA– 3' (antisense). The unmethylated product is 151 bp long, and the methylated product is 150 bp. Placental DNA treated in vitro with SssI bacterial methylase was used as a positive control for methylated genes. Ten microliters of each PCR product was loaded directly onto nondenaturing 6% polyacrylamide gels, stained with ethidium bromide and visualized under UV transilluminator.

Immunohistochemistry of p16, estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2

An immunohistochemical expression of p16, ER, PR, and HER2, 3–4 μm thick sections were deparaffinized with xylene, rehydrated through a graded alcohol series and brought to water according to manufacture's instructions. The sections were incubated with the monoclonal anti-p16 protein antibody (Mouse monoclonal anti p16, clone 16P04, USA), anti-ER protein antibody (Rabbit monoclonal anti ER, clone SP1, UK), anti-PR (Rabbit monoclonal anti PR, clone SP2, UK), and anti-HER2 (Mouse monoclonal anti-HER2, clone CB-11, USA) followed by manufacturer's instructions.

Statistical analysis

The association between p16 protein expression, p16 methylation, ER, PR, HER2 and clinicopathological parameters Chi-square/Fisher's exact test was used. Statistical analysis was done using statistical software SPSS version 20 (IBM, NY, USA).


 > Results Top


A total of 114 cases were included in the study, and all were histological/cytological confirmed cases of breast carcinoma. The age, type of the tumor, T stage, grade, lymph node status, presence or absence of an in situ component, hormonal receptors (ER/PR) status, and HER2 status were recorded in each case. The p16, ER, PR, and the HER2 expression were detected by immunohistochemistry (IHC) on tissue microarray sections. The age of the patients included in the study ranged from 24 to 80 years with the average age of 45.1 years with a standard deviation of 10.95 years.

Comparisons between p16 protein expression and different clinicopathological parameters

p16 protein expression was studied in 114 breast cancer cases, of which 13/114 (11.4%) cases were found negative, 45/114 (39.5%) cases showed weakly positive, 21/114 (18.4%) were moderately positive, and 35/114 (30.70%) were strongly positive as compared to control [Figure 1]. Of 114 cases, 64 (56.1%) cases below 50 years while 50 (43.9%) cases are above 50 years. The difference between p16 protein expression and age group, no statistical significance was observed.
Figure 1: Immunohistochemical analysis of p16 in breast carcinomas. The photographs show (a) negative control showing no detectable p16 immunoreactivity in which p16 antibody has been replaced with isotype-specific IgG with score 0, (b) weak expression with score 1–4, (c) moderate expression with score 5–8, and (d) strong expression with score 9–12; (a-d, ×200)

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Among 114 cases, 66 (57.9%) were premenopausal and 48 (42.1%) were postmenopausal women. Out of 66 premenopausal cases, 12/17 (70.6%) cases had negative p16 expression, 10/19 (52.6%) weak expression, 21/38 (55.3%) moderate, and 23/40 (57.5%) strong expression. While in 48 postmenopausal cases, 5/17 (29.4%) cases had negative p16 expression, 9/19 (47.4%) weak, 17/38 (44.7%) moderate, and 17/40 (42.5%) showed strong expression; however, the difference was not statistically significant (P > 0.6938) [Table 1].
Table 1: Comparison of p16 expression and age, menopausal status, estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status (n=114)

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ERs were studied in 114 cases, of which 77 (67.50%) were ER positive and 37 (32.50%) were ER negative [Figure 2]. p16 expression was negative in 6/13 (46.2%) cases showing ER positive and 7/13 (53.3%) cases showing ER negative while in weak expression, 37/45 (82.2%) cases were ER positive and 8/45 (17.8%) cases were ER negative, and in moderate expression, 16/21 (76.2%) cases were ER positive and 5/21 (23.8%) were ER negative. On the other hand, p16 strong expression was expressed in 18/35 (51.4%) cases with ER positive and 17/35 (48.6%) cases were ER negative. The statistical association was seen among p16 expression negative/moderate to strong expression and ER status (P < 0.0074). ER-positive tumors were significantly seen to be associated with lower tumor size (P < 0.026) [Table 1].
Figure 2: Immunohistochemical analysis of estrogen receptor in breast carcinomas. The photographs show (a) normal breast tissue, (b) negative control showing no detectable estrogen receptor immunoreactivity in which estrogen receptor antibody has been replaced with isotype-specific IgG, and (c) positive expression (a-c, ×200)

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PR was studied in 114 cases, of which 48 (42.1%) were PR positive and 66 (57.9%) were PR negative [Figure 3]. p16 expression was negative in 5/16 (31.2%) cases showing PR positive and 11/16 (68.8%) cases showing PR negative while in weak protein expression, 26/48 (54.2%) cases with PR positive and 22/48 (45.8%) cases with PR negative, and in moderate expression, 11/19 (57.9%) cases showed PR positive while 8/19 (42.1%) were PR negative. On the other hand, p16 strong expression was expressed in 5/31 (16.1%) cases with PR positive and 26/31 (83.9%) cases were PR negative. Statistically significant association was seen among p16 negative/moderate to strong protein expression and PR expression (P < 0.0026).
Figure 3: Immunohistochemical analysis of progesterone receptor in breast carcinomas. The photographs show (a) normal breast tissue, (b) negative control showing no detectable progesterone receptor immunoreactivity in which progesterone receptor antibody has been replaced with isotype-specific IgG, and (c) positive expression (a-c, ×200)

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HER2 expression was analyzed in 114 cases, of which 43 (37.8%) were HER2 positive and 71 (62.2%) were HER2 negative [Figure 4]. p16 expression was absent in 10/17 (58.8%) cases showing HER2 positive and 7/17 (41.2%) cases showing HER2 negative while in weak expression, 30/44 (68.2%) cases with HER2 positive and 14/44 (31.8%) cases with HER2 negative, and in moderate expression, 14/25 (56.0%) cases were HER2 positive while 11/25 (44.0%) were HER2 negative. On the other hand, HER2 strong expression was expressed in 12/28 (42.8%) cases with HER2 positive and 16/28 (57.2%) cases were HER2 negative. No statistically significant association was seen among p16 negative/moderate to strong protein expression and HER2 expression (P < 0.2078).
Figure 4: Immunohistochemical analysis of human epidermal growth factor receptor 2 in breast carcinomas. The photographs show (a) negative control showing no detectable human epidermal growth factor receptor 2 immunoreactivity in which human epidermal growth factor receptor 2 antibody has been replaced with isotype-specific IgG, (b) equivocal, and (c) positive expression (a-d, ×200)

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Similarly, no statistical association was observed between histological subtypes, grade of the tumor, tumor staging, in situ component, lymph node status, radiation therapy/cytotoxic therapy (RT/CT) status, and p16 protein expression.

Comparisons between p16 promoter methylation and different clinicopathological parameters

p16 promoter methylation was studied in 114 breast cancer tissues, of which 81/114 (71.0%) cases showed promoter methylation while 33/114 (29.0%) cases showed the absence of methylation or no methylation [Figure 5]. p16 promoter methylation was observed in 61/114 (53.3%) cases below 50 years while 53/114 (46.3%) cases are above 50 years. When we compare the age of the patients with below 50 years and above 50 years, no statistical difference was observed.
Figure 5: Representative picture showing the promoter methylation of p16 gene tumor and control samples. M is the φ × 174 Hae III digested marker. P is SSs1-treated CpG-methylated DNA used as positive control. N is negative control. U is unmethylated product (150 bp). M is methylated product (151 bp)

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Menopausal status was studied in 114 cases, 66 (57.9%) were premenopausal cases and 48 (42.1%) were postmenopausal cases. Out of 66 premenopausal cases, 25 (59.5%) cases observed p16 promoter methylation while 41 (59.5%) cases observed no methylation or absent p16 methylation. Whereas in 48 postmenopausal cases, 17 (40.5%) showing methylation and 31 (43.1%) are no methylation. No statistical significance was observed between menopausal status and p16 promoter methylation (P < 0.8956) [Table 2].
Table 2: Comparison of p16 promoter methylation and age, menopausal status, estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status (n=114)

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ER was studied in 114 cases, of which 77 (67.5%) were ER positive and 37 (32.5%) were ER negative. p16 methylation was present in 30 (73.1%) which are ER-positive cases while 47 (64.4%) of ER positive is unmethylated whereas 11 (26.9%) of ER-negative cases were p16 methylated while 26 (35.6%) of ER-negative cases are unmethylated. No statistically significant difference was observed (P > 0.4068).

PR was analyzed in 114 cases, 48 (42.1%) are PR positive and 66 (57.9%) are PR negative. p16 promoter methylation was seen in 19 (43.2%) of PR-positive cases while 29 (41.4%) of PR-positive cases are unmethylated whereas 25 (56.8%) of PR-negative cases were p16 methylated and 41 (58.6%) were unmethylated but not statistically significant (P > 1.0000). Similarly, we check the difference between p16 promoter methylation and other clinical factors such as histological subtypes, tumor staging, in situ component, lymph node, RT/CT status, and HER2; no statistical association was observed.


 > Discussion Top


Breast cancer is the second most common cancer in the world and the leading cause of cancer-related death in women in both developed and developing countries.[1] ER, PR, and HER2 are used for prognostication and to stratify patients for appropriate targeted therapies.[17] Molecular profiling has provided evidence for this heterogeneity, and thus, there is a steady interest to identify new markers that will help in predicting prognosis and response to therapy.

Age is an important independent prognostic factor in breast cancer; younger age has been shown to be an adverse factor in general. Indian population is relatively young as compared to the aging of the western population, and also, the life expectancy of western population is more, i.e., 75–80 years while in Indian population, it is 65 years. Majority of cases (39.5%) were less than 50 years with a peak at 40–49 years and our observation is also in agreement with other studies.[18]

p16 is a cell cycle regulator, and it inhibits the activity of cyclin-dependent kinases – CDK4 and CDK6 which in turn inhibit the retinoblastoma gene. RB gene controls progression from G1 to S phase of cell cycle.[19] The alteration in p16 gene and its expression has been studied in many cancers. p16 expression has been studied as a prognostic marker and has been associated with disease-free survival.[20] Low expression of p16 has been associated with triple-negative breast cancer, aggressive behavior, higher grade, and resistance to chemotherapy.[21],[22] In our study, p16 protein expression was found positive in 30.7% of patients. Several authors showed that the p16 positivity has been reported to be quite different in different studies (26%–75%).[20],[23],[24] These different positivity rates might be due to different cutoffs taken by different studies.

The hormone receptors play an important role in regulating growth and differentiation of normal breast tissue.[25] They are established prognostic factors with positive receptor status correlating with lower rate of cell proliferation and better tumor differentiation.[26] Inverse correlation between hormone receptors and strong p16 expression might thus suggest that strong p16 expression is related to more aggressive cancer behavior. However, further larger studies are required to substantiate this.

The study showed 67.5% ER-positive cases and 42.1% PR-positive cases. About 42.1% cases were double positive and 32.5% cases were double negative for ER and PR expression. In the present study, 32.5% cases are ER-negative cases and 57.9% are PR-negative cases. The incidence of ER- or PR-negative tumors in the western population is 55%–75%, whereas in Indian studies, the ER or PR negativity is somewhat higher around 32.6%–46.1%.[27] The reason for lower incidence of HR-positive cases in India may be due to improper staining or suboptimal manual assays rather than the presence of genetic differences.[28] Another study by Kakarala et al. reported much higher incidence of ER positivity in Asian (Indian/Pakistani) women. They reported ER positivity in 71.9% cases and PR positivity in 28.1%.[29]

Navani et al.[28] in his studies re-stained manually 37 ER-negative and PR-positive tumors using Food and Drug Administration-approved automated technique and staining protocols similar to the manual assays found the majority (75.6%) of tumors were false negative.[30] However, in our study, not even a single case showing such discrepancy was found. Hereditary breast cancers are seen to have high cases of ER-negative cases and approximately 70% cases of sporadic breast cancers are ER-positive,[31] and in the present study, 67.50% cases were ER positive consistent with previous studies. HER2 is overexpressed in 20%–30% of breast cancer.[32] In the present study, 37.8% cases were seen to be HER2 positive. HER2 overexpression has been shown to be associated with partial resistance to endocrine treatment. Several other studies reported a wider range from 10% to 34%.[33],[34] This wide range of positivity is probably due to different antibodies being used having differing sensitivity and specificity. The time and methods used for fixation also vary from laboratory to laboratory. The ASCO/CAP guidelines recommend that for HER-2/neu testing fixation of the tissue in 10%, buffered formalin should be done for at least 6 h. False positivity may occur due to edge artifact, cytoplasmic positivity, overstaining due to improper antibody titration, and misinterpretation of HER-2/neu in DCIS (which is often HER-2 positive and should not be interpreted). On the other hand, false-negative results may be due to prolonged cold ischemia time, tumor heterogeneity, and use of lower concentration of antibody. Thus, CAP guidelines suggest that controls should be properly assessed, and if HER-2 is negative by IHC but tumor has characteristics associated with HER-2 positivity (such as Grade II/III tumors, weak or negative PR, and increased proliferative index), then repeat testing by in situ hybridization methods should be considered.[34]

Epigenetic changes are the changes which do not directly modify the DNA sequence but are heritable.[35],[36] One of the epigenetic changes includes methylation of DNA, which involves methylation of CpG islands in the promoter regions. Thus, the expression of the gene is altered.[37] DNA methyltransferases are required to maintain CpG island methylation. Tumors with p16 methylation can be responsive to these agents. Furthermore, promoter region methylation might be responsible for resistance to certain chemotherapeutic agents and they can thus help to predict response to chemotherapy.[38]

In our study, we found p16 promoter methylation in 71.0% (81/114) cases. All the controls from normal breast tissue were unmethylated. Methylation of p16 in breast cancer has been reported differently in different studies, ranging from 3.6% to 65%.[11],[12],[39],[40],[41],[42],[43],[44],[45],[46],[47],[48],[49] The higher incidence of promoter methylation in our study may be due to a small sample size, which might not be representative of the entire breast cohort.

In the present study, 59.5% cases of methylation were premenopausal whereas 40.5% cases of methylation were postmenopausal. Therefore, greater proportion of cases was premenopausal in methylated group than nonmethylated group, but there was no statistical significance between them. Our observations are similar to Feng Jing et al.'s findings where 58% cases of methylated p16 were premenopausal and 42% were postmenopausal but no statistical significance was observed. Our findings are consistent with most other studies where no statistically significant association was found between them [11],[39],[44] significance. Very few studies have significant with methylation and menopausal status.[40]

p16 methylation was related to ER/PR status. In both ER and PR, positivity was found to be more with methylated cases than in ER- or PR-negative cases. However, no statistical significance was found. Naqvi et al. found statistically significant association between ER-/P- positive status and methylation (P = 0.018 for ER and P = 0.000009 for PR).[39] However, other studies did not find significant association between p16 methylation and ER/PR status.[11],[41],[46] Tao et al. reported ER-negative status to be associated with methylation in postmenopausal women.[44] In our study, statistical significance was probably not found because of the small sample size. Slightly less methylated cases were HER-2/neu positive than unmethylated cases, and no statistically significant association was found between them. This is in concordance with other studies which also did not find association between the two.[41],[46]

However, studies by Naqvi et al. and Raish et al. found significant inverse association between methylation and p16 expression. Naqvi et al. found significant association among various categories of methylation and expression. They determined the protein expression by RT-PCR and Northern blotting, and thus, our results might not be comparable.[39] Raish et al. followed different cutoffs for immunoexpression interpretation, and thus, our results cannot be compared with them.[40]

Thus, we conclude that p16 expression shows significant association with histological type, ER and PR status, and triple negativity, but no association was found with other clinicopathological factors such as age, menopausal status, side, in situ component, grade, stage, lymph node status, neoadjuvant therapy, or HER-2/neu status. On the other hand, p16 methylation was found to be associated with grade of the tumor and not with other factors such as age, menopausal status, histological type, in situ component, stage, lymph node status, neoadjuvant treatment, ER/PR and HER-2/neu status, and triple negativity.


 > Conclusion Top


Higher percentage of breast carcinoma cases with strong p16 expression was found with increasing tumor grade, but this association was not statistically significant. p16 expression showed significant association with ER-positive tumor and PR-negative tumors. p16 methylation showed association with grade with more percentage of cases showing methylation as the grade increased. p16 may be involved in breast carcinogenesis in the majority of sporadic breast cancer cases. p16 protein expression may be used as a prognostic indicator to predict treatment response to hormonal therapy.

Acknowledgment

We thank Science and Engineering Research Board (SERB), UCMS and ICMR for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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