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The Interplay of Proteins Encoded by CDKN1A, CDKN1B, CDKN2A and TP53 in the Prognosis of Breast Cancer

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The Interplay of Proteins Encoded by CDKN1A, CDKN1B, CDKN2A and TP53 in the Prognosis of Breast Cancer Research Article Clinics in Oncology Published: 02 Aug, 2019 The Interplay of Proteins Encoded by CDKN1A, CDKN1B, CDKN2A and TP53 in the Prognosis of Breast Cancer Valentina Robila1*, Bryce S Hatfield1, Nitai Mukhopadhyay2 and Michael O Idowu1 1Department of Pathology, VCU School of Medicine, USA 2Department of Biostatistics, VCU School of Medicine, USA Abstract The use of palbociclib has increased the spotlight on cell cycle regulators, especially on the proteins encoded by CDKN2A (p16), CDKN1A (p21), CDKN1B (p27), cyclin D1 and TP53 (p53). Cell cycle regulators and cyclin-dependent kinases show significant promise as new breast cancer biomarkers. Expression patterns of small sets of proteins in breast carcinoma were reported, and their overall role in breast cancer progression was investigated, with mixed results. The goal of our study was to simultaneously evaluate multiple cell cycle regulators in breast carcinoma. The protein expression was assessed on Tissue Microarrays (TMA) and the intensity and percentage of positive tumor cells were quantified. Statistical analysis was performed to establish patterns of protein associations, as well as correlation with prognostic factors, including metastases/recurrence and overall survival. Non-triple negative carcinoma showed significantly increased expression of cyclin D1, marginally increased expression of p21, 27, and decreased expression of p16, p53. This is in contrast to triple negative carcinoma which expressed high levels of p16 and p53. Statistically significant dual proteins associations were demonstrated, including direct correlations of expression of cyclin D1 with p21 or 27, and indirect correlations with p16 and p53. Cyclin D1 (p=0.0001) and p21 (p=0.0049) expression imparted a favorable outcome on development of metastases and/or survival, while p16 (p=0.0048) had a negative impact, particularly on non-triple negative carcinoma. The remainder of the tested proteins showed no prognostic significance. Given these proteins expression patterns, associations and prognostic impact, the results render cyclin D1, p21 and p16 as potential biomarkers for risk OPEN ACCESS stratification in breast carcinoma. *Correspondence: Keywords: Invasive breast carcinoma; Cyclin dependent kinases; p21; p16; Metastases Valentina Robila, Department of Pathology, VCU School of Medicine, Introduction 1200 E Marshall St. Gateway 6, PO Breast carcinoma is the most common cancer accounting for approximately 30% of new cancer Box 980662, Richmond, VA, USA, Tel: diagnoses and 14% of cancer deaths in women according to the 2018 statistics of the American 804-628-2999; Fax: 804-828-8733; Cancer Society [1]. While survival has improved with early diagnosis and advancement in E-mail: [email protected] treatment, breast cancer is still the second most common cause of death in women [1]. The recent Received Date: 03 Jul 2019 FDA approvals of palbociclib, ribociclib, and abemaciclib, selective inhibitors of Cyclin Dependent Accepted Date: 29 Jul 2019 Kinase 4 (CDK4) and CDK6, for treatment of hormone positive advanced breast cancer has brought Published Date: 02 Aug 2019 renewed focus on cell cycle regulators, especially CDK Inhibitors (CDKI). A critical function of Citation: CDKs is phosphorylation of Retinoblastoma protein (Rb) leading to progression from G1 to S phase Robila V, Hatfield BS, Mukhopadhyay of the cell cycle, while CDK inhibitors are believed to prevent this progression [2]. Conversely, N, Idowu MO. The Interplay of Proteins inactivation of CDKI leads to unopposed CDK4/6-Cyclin D1 complex formation and progression Encoded by CDKN1A, CDKN1B, from G1 to S phase of the cell cycle [2,3]. The CDK inhibitor proteins can be divided into 2 families: INK4A INKB CDKN2A and TP53 in the Prognosis 1) the INK4 family and 2) the CIP/KIP family. The INK4 family comprises of p16 , p15 , INKC INKD-4 of Breast Cancer. Clin Oncol. 2019; 4: p18 and p19 these bind to CDK4/6 and inhibit its kinase activity, leading to cell cycle CIP1 KIP1 KIP2 1644. arrest in G1 phase. The CIP/KIP family on the other hand consists of p21 , p27 and p57 ; these bind to a broader spectrum of cyclin/CDK complexed already formed (including cyclin D/ Copyright © 2019 Valentina Robila. CDK4/6 complex) and are therefore able to inhibit cell cycle progression [4]. p21 was shown to This is an open access article allow for repair of damaged DNA, [5] while p27, expressed in high concentrations in quiescent cells, distributed under the Creative decreases during entry into the cell cycle and promotes tumorigenesis [6,7]. Commons Attribution License, which permits unrestricted use, distribution, In Estrogen Receptor (ER) positive breast cancer, the prognostic significance of cyclin D1 and reproduction in any medium, over expression is still unclear. Some studies show cyclin D1 expression is associated with a poor prognosis and anti estrogen resistance, while others claim it is associated with improved outcomes provided the original work is properly [8-12]. Among CDKIs, cyclin-dependent kinase inhibitor 2A (CDKN2A) p16 expression was cited. Remedy Publications LLC., | http://clinicsinoncology.com/ 1 2019 | Volume 4 | Article 1644 Valentina Robila, et al., Clinics in Oncology - Breast Cancer shown to play a significant role in advanced tumor stage, higher Table 1: Patients demographics. grade, increased tumor proliferation, hormone receptor negativity, Characteristic Number patients (%) metastasis, and decreased overall survival [4,13-17]. Age <50 61 (28.1) Similarly, there is strong evidence correlating the reduced or >50 156 (71.8) absent p21 expression and progression of colorectal carcinoma, Ethnicity White 101 (46.5) Hodgkin lymphoma, and breast carcinoma, which tends to be African-American 107 (49.3) expressed more often in HER2 negative than in HER2 positive tumors Other 10 (4.6) [18-22]. A p27 decreased expression has been seen in the transition Body Mass Index (BMI) <35 171 (78.8) from ductal carcinoma in situ to invasive ductal carcinoma [6]. In >=35 46 (21.1) addition, a study of African American women with breast cancer Histologic type showed a significant decrease in p27 expression coupled with c-Myc Ductal 186 (85.7) Lobular 19 (8.7) over expression and decreased overall survival in patients with ER Other 12 (5.5) negative, PR negative, triple negative, and grade 3 breast cancers by Histologic grade Immunohistochemistry (IHC) [23]. I 28 (12.9) II 65 (29.9) The emerging role and early successes of CDK4/6 inhibitors to III 95 (43.7) n/a 29 (13.3) treat advanced breast cancer has highlighted the importance of finding Hormonal status specific biomarkers that could guide patient selection and treatment. Luminal A 135 (62.2) Additionally, these early successes have led to growing interest in Luminal B 10 (4.6) applying these drugs to early stage breast cancer [24]. Given that HER2 13 (5.9) Triple negative 52 (23.9) inhibitors of CDK4/CDK6 affect the progression from G1/S or G2/M Unknown 7 (3.2) phase of the cell cycle, it stands to reason to expect the expressions Pathologic tumor stage of cell cycle protein regulators like p16, p21 and p27 to achieve and T1 83 (38.2) T2 107 (49.3) maintain tumor cells growth arrest. In the present study, we sought a T3 23 (10.5) comprehensive approach and simultaneously evaluated multiple cell T4 4 (1.8) cycle regulator proteins, including p16, p21, p27, cyclin D1 and p53, Pathologic nodal stage N0 83 (38.2) in invasive breast carcinoma. Their immunohistochemical expression N1 61 (28.1) and correlation with clinicopathologic features such as distant N2 31 (14.2) metastasis or survival were investigated. N3 18 (8.2) Unknown 24 (11) Materials and Methods Recurrence None 40 (18.4) Patient demographics Loco-regional 137 (63.1) Distant 61 (28.1) The study analyzed invasive breast carcinoma from patients Unknown 26 (11.9) diagnosed and treated at our institution between 2005 and 2013. Survival Consult cases and cases reviewed for second opinions were excluded. Alive free/ unknown disease 60 (27.6) Alive with disease 87 (40) Only cases with adequate follow-up information were included. Died of disease 70 (32.2) Clinical-pathologic and demographic information including distant metastasis and survival, were collected via patient electronic medical was inferred using single covariate logistic regression. As the records and the Cancer Registry. expressions of several biomarkers are often strongly correlated, models with multiple covariates were avoided. A type I error threshold Immunohistochemistry and scoring of 5% was used to infer statistical significance. As reviewers assigned Tissue microarrays were constructed (1.0 mm cores) using the A score and H score, Cohen’s kappa measure was calculated Beecher ATA27 Tissue microarrayer. Each case has triplicate 1 mm for each marker and in most cases, were statistically significantly cores for the TMA. Immunohistochemistry for Cyclin D1 (clone different from 0. Thus only the average score was used in all analysis. EP12, Dako), p16 (clone INK4a, Roche), p21/WAF1 (clone DCS- Only statistical significant association with both Allred and H score 60.2, Cell Marque,) and p27/KiP1 (clone SX53G8, Cell Marque), and scoring system are considered significant. Significant association p53 (clone DO-7, Dako) were performed. with only one scoring system is not considered medically significant. Two board certified pathologists independently evaluated the Association of time to event outcomes, such as overall survival, was immunohistochemically stained slides, for percentage of positive assessed using a Cox proportional hazards regression model. cells and intensity of staining, using scales of 0% to 100% and 0-3, Results respectively. The percentage and intensity scores were converted to Allred score and H score. Allred scoring system uses the sum total Characteristics of the study population of proportion score and intensity score (range 0-8), while H score All 217 patients included in the study were women, age range 27 uses the multiplication of percentage reactivity with intensity score to 92 years old, diagnosed with invasive breast carcinoma, ductal and (range 0-300) [25,26].
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