Breast cancer remains the most frequently diagnosed malignancy and one of the leading causes of cancer-related mortality among women worldwide [1]. Although substantial improvements in screening and treatment have enhanced survival, nearly one-third of patients with invasive carcinoma eventually develop metastasis [2]. This underscores the importance of identifying precise prognostic markers capable of predicting tumor behavior and guiding therapeutic decisions. Breast cancer is a highly heterogeneous disease characterised by distinct molecular subtypes, each demonstrating unique biological behaviour and treatment responses [3]. Standard prognostic indicators—estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 are well established in clinical practice, yet their ability to predict outcomes remains imperfect, especially in aggressive subtypes such as triple-negative breast cancer (TNBC) [4,5]. p16, encoded by the CDKN2A gene, is a cyclin-dependent kinase inhibitor that regulates the G1/S checkpoint through inhibition of the cyclin D CDK4/6 complex [6]. While p16 is typically expressed at low levels in normal tissues, both overexpression and loss have been documented in malignancies including melanoma, cervical carcinoma, hepatocellular carcinoma, and esophageal cancer [7]. In HPV-related cancers, p16 overexpression serves as a reliable biomarker; however, its role in breast cancer remains controversial. Some studies report that p16 overexpression reflects a compensatory mechanism due to retinoblastoma (Rb) pathway inactivation, whereas others associate p16 loss with carcinogenesis via unrestricted proliferation [8,9]. Given these conflicting findings, evaluating p16 expression in breast cancer offers potential insight into tumor aggressiveness and prognosis. This study assesses p16 expression in invasive breast carcinoma and examines its association with Ki-67 proliferation index, tumor grade, and clinicopathological characteristics to further elucidate its biological and clinical relevance

Study Design and Setting This prospective cross-sectional study was conducted in a tertiary care academic hospital between January 2021 and December 2023. Institutional ethics approval was obtained, and all patient data were anonymised in accordance with ethical standards. Participants Fifty consecutive patients with histologically confirmed invasive breast carcinoma were included. Inclusion criteria: patients with complete pathological and immunohistochemical profiles. Exclusion criteria: (1) incomplete records, (2) absence of IHC results, (3) patients who achieved complete pathological response after neoadjuvant chemotherapy (NACT), and (4) individuals with known HPV infection to avoid p16 confounding. Immunohistochemistry (IHC) Formalin-fixed, paraffin-embedded tumor blocks were stained for p16. Expression was classified as positive when >10%of tumor cells showed nuclear or cytoplasmic staining, consistent with previous literature [8]. The Ki-67 index was calculated manually in hotspot areas and categorized as high when ≥14%, in line with international guidelines [5]. ER, PR, and HER2 were interpreted using ASCO/CAP criteria to categorize tumors into luminal A, luminal B, HER2-enriched, and TNBC. Histopathological Evaluation Tumors were graded using the Nottingham Histologic Score (NHS), integrating tubule formation, pleomorphism, and mitotic index [3]. Clinical variables including tumor mass, lymphovascular space invasion (LVSI), and lymph node involvement were extracted from histopathology reports. Statistical Analysis Categorical variables were compared using chi-square tests. Continuous variables (tumor mass, Ki-67 index) were evaluated using t-tests and Spearman’s correlation. Significance was set at p<0.05.

The mean age of participants was 48.86 ± 9.65 years. A majority of tumors were left-sided (64%), and 52% underwent upfront surgery while 48% were operated post-NACT. Modified radical mastectomy was the most common surgical procedure (80%). LVSI was present in 70% of patients, and lymph node involvement was identified in 32% with an additional 54% having 1–5 nodes involved. Tumor grades included 62% grade I, 28% grade II, and 10% grade III. IHC subtypes included luminal A (26%), luminal B (28%), HER2-enriched (18%), and TNBC (24%). p16 expression showed a trend toward association with larger tumors (102.57 vs. 56.99 g; p=0.054). Lymph node involvement was higher among p16-positive cases but was not statistically significant. No association was found between LVSI and p16 (p=0.746). The most significant finding was the strong association between p16 positivity and Ki-67 proliferation index (56.59 vs. 15.94; p<0.001). p16 expression also correlated significantly with higher tumor grade (p=0.008). Table 1. Clinicopathological Characteristics (n=50) Variable Category n (%) Age Mean ± SD 48.86 ± 9.65 Laterality Left / Right 32 (64%) / 18 (36%) LVSI Present 25 (70%) Lymph Nodes 0 / 1–5 / >5 16 (32%) / 27 (54%) / 7 (14%) Tumor Grade I / II / III 31 / 14 / 5 IHC Subtype Luminal A / B / HER2 / TNBC 13 / 14 / 9 / 12 Table 2. Associations Between p16 and Key Variables Variable p16-Negative p16-Positive p-value Tumor Mass (mean) 56.99 102.57 0.054 Ki-67 (%) 15.94 56.59 <0.001 Grade III (%) 20% 80% 0.008 TNBC positivity (%) 14.3% 85.7% <0.001 TNBC demonstrated the highest rate of p16 expression (85.7%), consistent with biological patterns of Rb pathway dysfunction reported in prior studies [1,7].

This study demonstrates that p16 overexpression is strongly associated with aggressive pathological features in invasive breast carcinoma. The significant correlation between p16 positivity and elevated Ki-67 index reflects heightened proliferative activity and aligns with the findings of Kim et al. and Sanli et al., who identified similar patterns in their respective cohorts [3,4]. These data suggest that p16 may serve as a surrogate marker of tumor proliferation. A robust association between p16 expression and higher tumor grade further supports its prognostic significance. Previous studies report that high p16 levels may indicate dedifferentiation and genomic instability [8]. Although the correlation between p16 and lymph node involvement in our study did not reach statistical significance, the observed trend is comparable to earlier results by Samman et al., who identified a meaningful relationship between p16 positivity and nodal metastasis. The high prevalence of p16 expression among TNBC cases in our cohort is particularly noteworthy. TNBC is characterized by poor differentiation, high mitotic activity, and lack of targeted therapies. Studies by Rakha et al. and Nielsen et al. have demonstrated that TNBC frequently exhibits basal-like features including retinoblastoma pathway abnormalities [1,7]. Loss of Rb function triggers compensatory overexpression of p16, explaining the paradox of elevated p16 in biologically aggressive tumors despite its tumor-suppressor role [6]. These findings reinforce the concept that p16 overexpression may represent cellular response to Rb pathway dysregulation rather than an effective inhibitory mechanism. Given the absence of hormone receptor targets in TNBC, markers such as p16 may improve prognostic stratification and guide identification of candidates for intensified therapy or clinical trials involving CDK4/6 pathway inhibitors.

This study demonstrates that p16 overexpression in invasive breast carcinoma is significantly associated with a higher Ki-67 proliferation index, poorer tumor differentiation, and the triple-negative breast cancer (TNBC) phenotype. These findings highlight p16 as a potential marker of tumor aggressiveness and suggest its value in refining prognostic evaluation. Although associations with tumor mass and lymph node involvement did not reach statistical significance, observable trends indicate that p16 expression may still correlate with more advanced pathological features. The integration of p16 status with established markers such as Ki-67 and tumor grade may enhance risk stratification, particularly in high-risk subtypes such as TNBC where therapeutic options remain limited. Further large-scale, multi-institutional studies are warranted to better understand the mechanisms underlying p16 dysregulation in breast cancer and to explore its potential role as both a prognostic and predictive biomarker. Acknowledgements: NIL Conflicts of interest: NIL Funding: NIL

1. Rakha EA, Reis-Filho JS, Baehner F, Dabbs DJ, Decker T, Eusebi V, et al. Breast cancer prognostic classification in the molecular era: the role of histological grade. Breast Cancer Res. 2010;12(4):207. 2. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl J Med. 2010;363(20):1938-48. 3. Kim JH, Yoon SY, Kim CN, Park SY, Lee JE, Lee JH, et al. p16INK4A expression is frequently increased in basal-like and decreased in luminal-like breast cancer. J Korean Surg Soc. 2016;89(3):129-35. 4. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell. 2002;2(2):103-12. 5. Dowsett M, Nielsen TO, A’Hern R, Bartlett J, Coombes RC, Cuzick J, et al. Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. J Natl Cancer Inst. 2011;103(22):1656-64. 6. Sanli O, Dobruch J, Knowles MA, Burger M, Alemozaffar M, Nielsen ME, et al. p16 expression and its relation to Ki-67 and histological grade in invasive breast carcinoma. Int J Breast Cancer. 2019;2019:5821927. 7. Nielsen TO, Leung SCY, Rimm DL, Dodson A, Acs B, Badve S, et al. Molecular classification of breast cancer. Nat Rev ClinOncol. 2020;17(12):735-50. 8. Geradts J, Kratzke RA, Niehans GA, Lincoln CE. Immunohistochemical detection of the cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) product p16INK4A in archival human solid tumors: correlation with retinoblastoma protein expression. Cancer Res. 1995;55(24):6006-11. 9. Liu Y, Li J, Wang X, Zhang Y, Wang Z, Wang Y, et al. p16INK4a overexpression and survival in breast cancer: a meta-analysis of 28 studies comprising 6,942 patients. Oncotarget. 2015;6(18):15789-99.