Immunohistochemical Expression of Cyclin D1 and p16 in Invasive Breast Carcinoma and Its Association with Clinicopathological Parameters

Abstract

Invasive breast cancer (IBC) is a significant health concern globally, contributing to substantial morbidity and mortality among women. Dysregulated cellular proliferation, a hallmark of malignancy, involves molecular pathways modulated by proteins such as cyclin D1 and p16. Understanding their roles in IBC pathogenesis and their association with prognostic parameters is crucial for refining treatment strategies. This retrospective study included 50 female IBC patients who underwent modified radical mastectomy. Histopathological evaluation and immunohistochemical staining for cyclin D1 and p16 were conducted. Associations between protein expression and clinicopathological parameters were analyzed using statistical tests. Cyclin D1 was expressed in 76% of cases, significantly associated with lower tumor grade and lower Ki-67 proliferation index. It also correlated with luminal A/B molecular subtypes. p16 expression was observed in 48% of cases, significantly associated with higher tumor grade, higher Ki-67 index, and triple-negative/Her-2 neu–enriched subtypes. Co-expression of cyclin D1 and p16 was noted in 60% of cases. No significant association was found between protein expression and other parameters. Cyclin D1 and p16 exhibit potential as prognostic markers in IBC. Cyclin D1 expression correlates with less aggressive tumor features and luminal subtypes, suggesting a favorable prognosis and potential predictive value for CDK4/6 inhibitor therapy. Conversely, p16 expression associated with aggressive phenotypes, indicating poor prognosis. Further studies are warranted to validate these findings and explore their clinical implications. Integrating these biomarkers into clinical practice may enhance risk stratification and treatment decisions, ultimately improving outcomes for IBC patients.

Introduction

Invasive breast cancer (IBC) is one of the most frequently occurring malignancies diagnosed globally and is a leading cause of morbidity and mortality in women. The year 2022 saw 2.3 million newly diagnosed cases of IBC among females with around 670,000 breast cancer-related deaths worldwide. Moreover, the incidence is on the constant rise and it is anticipated that by the year 2040, the percentage of fatalities from IBC in the South-East Asia region will rise by 61.7%, adding to the enormity of the disease. [1, 2]

Continuous, chronic, prolonged, and dysregulated cellular proliferation is one of the fundamental characteristics of all malignant neoplasms. It is attained by alterations of molecular signaling pathways which ultimately lead to uncontrolled and sustained progression through the cell cycle [3]. Cyclin D1 and p16 are two cardinal proteins involved in key cell cycle regulation and thereby play a vital role in driving tumorigenesis.

Cyclin D1, a molecular signal protein, encoded by the gene CCND1 on chromosome 11q13.3, on binding with its partner molecule of cyclin-dependent kinase (CDK4/6), helps mediate the crucial G₁ to S phase transition in the cell cycle [4, 5]. This transition is primarily effected by cyclin D1-CDK4/6 bound complex aggregate, by causing phosphorylation of retinoblastoma protein (Rb). The phosphorylated Rb protein further undergoes dissociation from E2F1 protein (E2F transcription factor 1), thereby opening the gates for transcription of G1-S phase genes. Furthermore, the cyclin D1-CDK4/6 complex formation is stimulated by the PI3K/MAPK pathways (phosphoinositide 3-kinase/mitogen-activated protein kinase pathways), and activation of estrogen receptor (ER), which becomes an important driving factor, especially in estrogen-dependent carcinomas like IBC. [6, 7]

Moreover, the p16 protein is produced by transcription of the p16INK4a gene, which is a tumor-suppressor gene located on chromosome 9p21. Contrary to the role of cyclin D1, p16 causes a negative influence on the regulation of the cell cycle by causing blockade of the G1 to S phase transition. It binds and inhibits CDK 4/6, thereby leading to G1 phase arrest of the cell. Additionally, CDK 4/6 inhibitor therapeutic drugs including abemaciclib, palbociclib, and ribociclib have emerged as highly effective treatment options in IBC, targeting CDK4/6 proteins, and causing down-regulation of cellular proliferation [8, 9]. These intricate molecular mechanisms involved in cell cycle regulation are illustrated in Fig. 1.

Fig. 1.

Intricate molecular mechanisms involved in cell cycle regulation

IBC traditionally has been prognosticated by numerous clinicopathological parameters like the size of the tumor, Modified Bloom-Richardson (MBR) grade, axillary lymph node (ALN) metastasis, American Joint Committee on Cancer (AJCC) pTNM (pathological tumor, node, metastasis) staging, lympho-vascular invasion (LVI), perineural invasion (PNI), Ki-67 (MIB-1) labeling index, hormonal receptor status (estrogen and progesterone receptor), human epidermal growth factor receptor 2 (Her-2 neu) status, and surrogate molecular classification. Also, ER and Her-2neu expression status help predict the response from anti-estrogen therapy (including selective estrogen receptor modulators (SERMs) and aromatase inhibitors) and ant-Her2 drugs (including trastuzumab and pertuzumab) respectively. [10, 11]

Breast surgical oncology is an ever-evolving practice, and with the advent of new biomarkers, it becomes imperative to assess their expression, their role in the pathogenesis of this complex disease, and also establish their association with the already recognized vital parameters. In recent studies, cyclin D1 and p16 have proven to be important prognostic markers in IBC. Additionally, with the development and approval of CDK 4/6 inhibitor drugs for IBC, cyclin D1 serves to be a crucial predictive biomarker.

However, there are only a handful of studies in internationally, and even lesser in the Indian setting which have explored this intriguing, yet crucial aspect, and especially the expression of p16 in IBC cases. Moreover, there have been contradictory results as well regarding their association with the conventional prognostic factors. Therefore, the present study evaluates the immunohistochemical expression of cyclin D1 and p16 and assesses their association with various other clinicopathological parameters routinely assessed in histopathological reporting of IBC.

Materials and Methods

Selection of Study Participants

The present study was a retrospective study, conducted in the Department of Pathology of a tertiary care teaching hospital in northern India. All the patients who underwent modified radical mastectomy (MRM) for IBC, over a period of 1 year, were considered for inclusion in the study cohort.

Moreover, patients with a history of neoadjuvant chemotherapy (NACT), pre-operative radiotherapy, or hormonal therapy were excluded from the study as they can alter the immunohistochemical expression of ER, PR, Her-2 neu, cyclin D1, and p16 in IBC patients. Additionally, cases with benign and/or in situ lesions on extensive sampling and histopathological examination of the surgical specimen were also excluded.

A total of 50 female patients diagnosed with IBC-NST (invasive breast carcinoma of no special type) and fulfilling the said criteria were retrospectively included in the study cohort after attaining informed written consent.

Collection, Preparation, and Evaluation of Histopathological Sections

The oriented surgical specimens of MRM were received in the histopathology unit of the department in 10% neutral-buffered formalin and were grossly examined. All the relevant identification and clinical details mentioned on the histopathology requisition form were noted. Extensive sampling was done and representative sections were taken from the specimen.

After routine processing, 5 microns thick sections were cut from formalin-fixed paraffin-embedded (FFPE) tissue blocks and were stained with hematoxylin and eosin stain.

The histopathological sections were evaluated by skilled histopathologists and Modified Bloom-Richardson grading [12] and pTNM staging as per the eighth edition of AJCC cancer staging system [13] was done. The tumor sections were also examined for LVI and PNI.

Application of Immunohistochemistry and Its Interpretation

Two- to 3-micron-thick tumor sections were taken on poly-L-lysine-coated slides and routine immunohistochemistry (IHC) was performed for hormonal receptor status. Human estrogen receptor (ER antibody), SP1 clone which is a high-affinity rabbit monoclonal antibody directed against an epitope of the C-terminus of the ER protein, and progesterone receptor antibody (PR), clone 16, a mouse monoclonal antibody directed against the human progesterone receptor molecule were applied for determination of ER and PR status. The interpretation of ER and PR immunostaining was conducted following the latest ASCO/CAP guidelines. According to these guidelines, a positive result is indicated when 1–100% of tumor cell nuclei exhibit staining, regardless of the staining intensity. < 1% staining in tumor cells was considered as negative interpretation for ER and PR status. [14]

HER2/neu (IHC042) antibody was used to determine Her-2 neu status. IHC result of 3 + (complete, intense circumferential membranous staining in > 10% of tumor cells) was interpreted as Her-2 neu positive, while IHC result of 0 (no staining observed or incomplete faint/barely perceptible membrane staining within ≤ 10% of invasive tumor cells)/1 + (incomplete faint membrane staining and within > 10% of invasive tumor cells) was diagnosed as Her-2 neu negative. Cases with 2 + Her-2 neu staining (weak to moderate complete membrane staining observed in > 10% of invasive tumor cells) were considered equivocal and were further confirmed to be Her-2 neu positive or negative depending on the amplification status on dual-probe fluorescent ISH (in situ hybridization). [15]

Ki-67 (MIB-1) immunostaining was done for the evaluation of proliferation index using Ki-67 (clone SP6, rabbit monoclonal antibody). The characterization was done into tumors with low (< 14%) and high (≥ 14%) Ki-67 labeling index.

Surrogate molecular classification of IBC cases into luminal A (ER positive, PR positive, HER2 negative, Ki67 proliferative index low), luminal B (ER positive, PR negative/positive, HER2 negative/positive, Ki67 proliferative index high), Her-2 neu–enriched (ER negative, PR negative, HER2 positive), and triple-negative (ER negative, PR negative, HER2 negative) categories was done based on the expressed hormone receptor status. [16]

Cyclin D1 (Bcl-1 or PRAD-1), clone EP12, a rabbit monoclonal antibody was applied for cyclin D1 immunostaining in IBC cases. A histopathologically diagnosed and confirmed case of cyclin D1–positive mantle cell lymphoma was taken for positive control. Strong, nuclear staining with cyclin D1 in IBC tumor sections was given a score of 3, moderate intensity nuclear staining was attributed a score of 2, weak nuclear staining was scored as 1, and finally absent nuclear staining was given a score of 0. The tumor sections with ≥ 10% of tumor nuclei showing moderate (2 +) or strong nuclear staining (3 +) were considered as cyclin D1–positive cases. [17]

p16 INK4a, clone BC42, a mouse monoclonal antibody, was used for p16 immunostaining in IBC cases. A histopathologically diagnosed and confirmed case of p16-positive, human papillomavirus (HPV)–dependent squamous cell carcinoma (SCC) of the cervix was taken as a positive control for checking of proper working of the antibody. High p16 immunoexpression was indicated when ≥ 70% of the tumor cells showed cytoplasmic and nuclear staining. Sections with 30–70% of the tumor cells showing cytoplasmic and nuclear positivity were graded as moderate p16 expression. Tumors showing cytoplasmic and nuclear positivity in 10–30% of tumor cells were categorized as low or abnormal positive p16 expression, and finally, tumor sections with less than 10% tumor cells taking up p16 immunostaining were classified as negative p16 expression [18]. Therefore, cases showing moderate and high p16 staining were considered as positive for p16 immunoexpression for statistical analysis.

Immunoexpression of cyclin D1 and p16 was evaluated and scored by trained histopathologists and association with other clinicopathological parameters including age of the patient, Modified Bloom-Richardson grade, tumor extension, lympho-vascular invasion, dermal lympho-vascular invasion, perineural invasion, nodal status, pathological staging, Ki-67 proliferation index, and surrogate molecular classification was established.

Statistical Analysis

The presentation of the categorical variables was done in the form of numbers and percentages (%). On the other hand, the quantitative data with normal distribution were presented as the mean ± SD and the data with non-normal distribution as median with 25th and 75th percentiles (interquartile range). The data normality was checked by using the Shapiro–Wilk test. In the cases in which the data was not normal, we used non-parametric tests. The following statistical tests were applied to the results:

• • The association of the variables that were quantitative and not normally distributed in nature was analyzed using the Mann-Whitney test and variables which were quantitative and normally distributed in nature were analyzed using the independent t-test.

• • The association of the variables which were qualitative in nature was analyzed using the chi-square test. If any cell had an expected value of less than 5, then Fisher’s exact test was used.

The data entry was done in the Microsoft EXCEL spreadsheet and the final analysis was done with the use of Statistical Package for Social Sciences (SPSS) software, IBM manufacturer, Chicago, USA, version 25.0.

For statistical significance, a p-value of less than 0.05 was considered statistically significant.

Results

Cyclin D1 and Its Association with Various Clinicopathological Parameters

The mean age of the study participants included in the present study was 53.46 ± 12.2 years. Out of 50 cases of IBC included in the study, 27 patients (54%) had positive expression of cyclin D1 (Fig. 2), 14 (28%) patients had a cyclin D1 immunohistochemical score of 3, and 13 (26%) cases exhibited cyclin D1 expression score of 2. Furthermore, 11 (22%) patients were scored as 1 and 12 (24%) cases were negative for cyclin D1 immunoexpression. The majority of patients with cyclin D1 expression were found to have a lower Modified Bloom-Richardson grade (i.e., grade 2) (p-value = 0.017, statistically significant) (Fig. 3). Cyclin D1 expression was also found to be significantly associated with low Ki-67 proliferation index in tumors (i.e., < 14%) (p-value = 0.002, statistically significant) (Fig. 4). Furthermore, there was a strong association observed between cyclin D1 expression in IBC cases and luminal A and luminal B molecular subtypes (p-value = 0.002, statistically significant) (Fig. 5).

Fig. 2.

Immunohistochemical grading of cyclin D1 expression. a 3 + . b 2 + . c 1 + 

Fig. 3.

Association of Modified Bloom-Richardson score with cyclin D1. ^*p-value = 0.017. ^*Fisher’s exact test

Fig. 4.

Association of Ki67 with cyclin D1. ^*p-value = 0.002. ^*Fisher’s exact test

Fig. 5.

Association of surrogate molecular classification with cyclin D1. ^*p-value = 0.002. ^*Fisher’s exact test

However, no statistically significant association (i.e., p-value < 0.05) could be established between cyclin D1 and other clinicopathological parameters evaluated including age of the patient, tumor extension, lympho-vascular invasion, dermal lympho-vascular invasion, perineural invasion, nodal status, and pathological staging (pTNM stage) (Table 1).

Table 1.

Association of clinicopathological parameters with cyclin D1 and p16 immunoexpression

Parameters	Total number of patients (N = 50)	Cyclin D1 positive (N = 38) (%)	Cyclin D1 negative (N = 12) (%)	p-value	p16 positive (N = 42) (%)	p16 negative (N = 8) (%)	p-value
Age (in years)
< 50	17	12 (70.59%)	5 (29.41%)	0.52+	14 (82.35%)	3 (17.65%)	1#
≥ 50	33	26 (78.79%)	7 (21.21%)		28 (84.85%)	5 (15.15%)
Modified Bloom-Richardson grade
Grade 2	29	26 (89.66%)	3 (10.34%)	0.017#	21 (72.41%)	8 (27.59%)	0.015#
Grade 3	21	12 (57.14%)	9 (42.86%)		21 (100%)	0 (0%)
Resected margins
Free	39	30 (76.92%)	9 (23.08%)	1#	35 (89%)	4 (10.26%)	0.059#
Involved	11	8 (72.73%)	3 (27.27%)		7 (63.64%)	4 (36.36%)
Lympho-vascular invasion
Absent	22	17 (77.27%)	5 (22.73%)	0.852++	17 (77.27%)	5 (22.73%)	0.277#
Present	28	21 (75%)	7 (25%)		25 (89.29%)	3 (10.71%)
Dermal lympho-vascular invasion
Absent	44	35 (79.55%)	9 (20.45%)	0.141#	38 (86.36%)	6 (13.64%)	0.242#
Present	6	3 (50%)	3 (50%)		4 (66.67%)	2 (33.33%)
Perineural invasion
Absent	44	34 (77.27%)	10 (22.73%)	0.621#	37 (84.09%)	7 (15.91%)	1#
Present	6	4 (66.67%)	2 (33.33%)		5 (83.33%)	1 (16.67%)
Nodal status
Free	26	19 (73.08%)	7 (26.92%)	0.614++	22 (84.62%)	4 (15.38%)	1#
Involved	24	19 (79.17%)	5 (20.83%)		20 (83.33%)	4 (16.67%)
Pathological staging
T stage
T1	9	7 (77.78%)	2 (22.22%)	0.156#	9 (100%)	0 (0%)	0.093#
T2	20	18 (90%)	2 (10%)		18 (90%)	2 (10%)
T3	11	6 (54.55%)	5 (45.45%)		9 (81.82%)	2 (18.18%)
T4	10	7 (70%)	3 (30%)		6 (60%)	4 (40%)
N stage
N0	24	18 (75%)	6 (25%)	0.894#	20 (83.33%)	4 (16.67%)	0.605#
N1	7	5 (71.43%)	2 (28.57%)		7 (100%)	0 (0%)
N2	11	8 (72.73%)	3 (27.27%)		8 (72.73%)	3 (27.27%)
N3	8	7 (87.50%)	1 (12.50%)		7 (87.50%)	1 (12.50%)
Ki-67 proliferation index
< 14%	18	18 (100%)	0 (0%)	0.002#	11 (61.11%)	7 (38.89%)	0.002#
> 14%	32	20 (62.50%)	12 (37.50%)		31 (96.88%)	1 (3.13%)
Surrogate molecular classification
Luminal A	18	18 (100%)	0 (0%)	0.002#	11 (61.11%)	7 (38.89%)	0.011#
Luminal B	10	8 (80%)	2 (20%)		9 (90%)	1 (10%)
Her2neu enriched	13	6 (46.15%)	7 (53.85%)		13 (100%)	0 (0%)
Triple negative	9	6 (66.67%)	3 (33.33%)		9 (100%)	0 (0%)

⁺Independent t-test

^#Fisher exact test

⁺⁺Chi-square test

p16 and Its Association with Various Clinicopathological Parameters

Twenty-four patients (i.e., 48% of the IBC cases) out of 50 cases showed positive p16 immunoexpression (Fig. 6). Fourteen (28%) participants showed high p16 immunoexpression, while 10 (20%) patients had a moderate p16 expression. Eighteen (36%) cases expressed low p16, whereas eight (16%) participants were negative for p16 expression. It was also observed that patients who expressed p16 tended to have higher Modified Bloom-Richardson grade (i.e., grade 3) (p-value = 0.015, statistically significant) (Fig. 7). Additionally, a strong relationship of p16 positivity was seen with high Ki67 proliferation index (i.e., ≥ 14%) (p-value = 0.002, statistically significant) (Fig. 8). Furthermore, a statistically significant association was seen between positive p16 expression and triple-negative and Her-2 neu–enriched molecular subtypes (p-value = 0.011, statistically significant) (Fig. 9).

Fig. 6.

Immunohistochemical grading of p16 expression. a 3 + . b 2 + . c 1 + 

Fig. 7.

Association of Modified Bloom-Richardson score with p16. ^*p-value = 0.015. ^*Fisher’s exact test

Fig. 8.

Association of Ki67 with p16. ^*p-value = 0.002. ^*Fisher’s exact test

Fig. 9.

Association of surrogate molecular classification with p16. ^*p-value = 0.011. ^*Fisher’s exact test

However, no statistically significant association (p-value < 0.05) was noted between p16 and other clinicopathological parameters evaluated including age of the patient, tumor extension, lympho-vascular invasion, dermal lympho-vascular invasion, perineural invasion, nodal status, and pathological staging (pTNM stage) (Table 1).

Association Between Cyclin D1 and p16 Immunoexpression

When comparing the association between expression of cyclin D1 and p16 among the IBC patients, it was observed that 30 patients (60%) expressed co-expression of cyclin D1 and p16 simultaneously. Twelve patients (24%) expressed p16 but did not show expression of cyclin D1 and eight patients (16%) had cyclin D1–positive immunoexpression while being negative for p16. However, no statistically significant association was seen when comparing the association of expression of these two antibodies in the IBC cases studied.

Discussion

Cyclin D1 and p16 are one of the defining cell cycle regulator protein molecules. Their influence on cellular proliferation and especially tumor propagation is immense. While cyclin D1 helps in mediating the G1 to S phase transition and further aids in the progression of the tumor cell cycle, p16 by blocking CDK 4/6 provides a key check on the multiplication of malignant cells.

The magnitude of the burden of IBC globally, in terms of morbidity and mortality, cannot be emphasized enough. However, the role of these two crucial biomarkers has not been explored to the fullest potential. Moreover, there have been conflicting observations regarding their association with other important prognostic parameters as well, adding to the confusion. The present study, therefore, made a sincere attempt to evaluate the expression and role of cyclin D and p16 in cases of IBC in an Indian setup.

In this study, positive cyclin D1 expression detected by IHC was seen in about 54% of IBC cases which is in concordance with that reported in literature, i.e., 34–81% [19]. Moreover, we found a significant association between cyclin D1 positivity and lower MBR grade of the tumor (i.e., grade 2). This suggests a favorable inverse association between the two, which can be attributed to the hypothesis that cyclin D1, being a harbinger of cellular proliferation, helps in enhanced maturation of the tumor cells, culminating in increased tubular differentiation, lesser nuclear pleomorphism, and mitotic count. Our results were in line with the observations of van Diest et al. [20] However, Kim et al. [21] did not observe any significant association between histologic grade and cyclin D1 expression.

Another significant finding observed in our study was the association of lower Ki-67 proliferation index and positive cyclin D1 immunoexpression. This also highlights that cyclin D1 expression is associated with the indolent nature of the tumor and our observations were similar to those obtained by Peurala et al. [22] However, contrary to our findings, Ahlin et al. [23] demonstrated that cyclin D1 expression was associated with higher proliferation rates and mortality in ER-positive breast cancer. These conflicting results indicate that larger multicentred studies may be needed to further validate our findings on a limited sample.

In the present study, we also found a strong association between cyclin D1 expression and luminal A and B surrogate molecular subtypes, which is concordant with the findings of Ravikumar et al. [24] Estrogen receptor activation is a strong stimulator of cyclin D1-CDK4/6 axis; therefore, it points towards the fact that ER positivity in breast cancers and cyclin D1 expression go hand in hand, as was observed in this study.

Cyclin D1 expression in breast carcinoma therefore carries the potential to become one of the very important prognostic biomarkers in the work-up of breast carcinoma patients. Moreover, the US Food and Drug Administration (FDA) has approved the use of three CDK4/6 inhibitors including palbociclib, ribociclib, and abemaciclib for appropriate use in the management of hormone receptor-positive and Her-2 neu-negative advanced breast carcinoma patients [25]. It has resulted in a paradigm shift in the treatment protocols and has proven to be a boon for the patients. This makes cyclin D1 a vital predictive marker as well which can predict the response to the CDK4/6 inhibitor drugs.

In the present study, positive p16 immunoexpression was seen in 48% of the IBC cases. This is, however, slightly on the higher side than the findings of Salih MM et al. [18], wherein the authors observed p16 expression in 31.2% of the breast carcinoma cases studied. This difference can be attributed to the smaller sample size of our study and the regional and ethnic differences between the two study populations. Moreover, there is a definite lacuna in the published literature, regarding p16 expression in IBC, and thus larger studies are the need of the hour to generate more data to establish the validity of our findings.

A significant association of positive p16 expression with higher Modified Bloom-Richardson grade (grade 3) was also observed, which is in agreement with the findings of Rana et al. [26] This further corroborates with the hypothesis that p16 expression in IBC suggests a poor prognosis for the patient. This altered expression of p16 protein, in high-grade breast malignancy, can be applied as a useful prognostic parameter in the work-up of IBC patients in the future.

Moreover, a strong association of positive p16 immunostaining was observed with a higher Ki-67 proliferation index (≥ 14%) of the neoplasm. This is in alignment with the observations made by Emig et al. [27], where the authors also suggested that aberrant p16 expression in breast carcinoma was linked to higher proliferation rates.

Another interesting aspect found in the present study was a significant association between p16 expression and triple negative and Her-2 neu–enriched surrogate molecular subtypes. Similar findings were obtained in the study conducted by Shan et al. [28] These findings suggest that aberrant increased p16 expressed by the malignant cells in breast carcinomas is associated with the subtypes which inherently pose a poor prognostic outcome for the patient.

In conclusion, cyclin D1 and p16 possess a vast potential to serve as key prognostic and cyclin D1, additionally, as an important predictive biomarker as well in the clinicopathological work-up of breast carcinoma patients. However, this study has a few limitations that should be acknowledged. Firstly, the retrospective design and the small sample size of 50 patients may affect the generalizability of the findings. Furthermore, larger-scale studies may be taken up, to effectively establish robust association of these useful markers with prognostic parameters and overall survival in IBC patients.

Author Contribution

SM, SV: conceptualization, data curation, formal analysis, methodology, writing original draft; SA: data curation, methodology, formal analysis, supervision; CA: conceptualization, data curation, formal analysis, supervision, resources. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Data Availability

The data is available from the corresponding author on request.

Declarations

Ethics Approval and Consent to Participate

This study was done in accordance with the Declaration of Helsinki. It was exempt from ethical approval due to being a retrospective study. Informed consent was taken from the patient.

Consent for Publication

Informed consent was sought from patient regarding participation and publication.

Competing Interests

The authors declare no competing interests.