ANTICANCER RESEARCH 26: 1657-1668 (2006) Expression of p27KIP1, p21WAF1 and p53 does Not Correlate with Prognosis in Node-negative Invasive Ductal Carcinoma of the Breast HELEN P. KOUREA1, ANGELOS K. KOUTRAS2, VASSILIKI ZOLOTA1, IRENE GRIMANI3, EVANGELOS TZORACOELEFTHERAKIS4, DIMITRIOS KOUKOURAS4, GEORGE FOUNTZILAS5, HELEN GOGAS6, KYRIAKOS KYRIAKOU7, ADAMOS ADAMOU7 and HARALAMBOS P. KALOFONOS2 Departments of 1Pathology, 2Internal Medicine and 4Surgery, University Hospital of Patras, Patras; 3Department of Biostatistics, Hellenic Cooperative Oncology Group Data Office, Athens; 5Aristotle University of Thessaloniki School of Medicine, Thessaloniki; 6First Department of Medicine, University of Athens, Athens, Greece; 7The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus Abstract. The expressions of p27Kip1 (p27) and p21Waf1 (p21) generally has a favorable outcome even without adjuvant cyclin-dependent kinase inhibitors and p53 were examined in a therapy, 30% of the patients will die of their disease without series of 170 node-negative breast carcinomas (NNBCs) to adjuvant treatment (7). Since the classic clinicopathological evaluate their prognostic significance. Low nuclear (p27TN) parameters are often not adequate to identify these patients, and cytoplasmic (p27TC) p27 expressions were noted in 66% and the benefit of adjuvant therapy in this setting is debatable and 81% of NNBCs, respectively. p21 and p53 overexpressions (7), additional information provided by novel prognostic were detected in 56% and 26%, respectively. Low p27TN was factors will assist in the fine-tuning of the prognostication significantly associated with high grade (p=0.001), age ≤50 process and the treatment decision-making. years (p=0.01), negative hormone receptors (p<0.001), low Factors involved in cell cycle control have been shown to be p27TC (p<0.001) and p53 overexpression (p=0.02). Low involved in carcinogenesis. The cell cycle progression (reviewed p27TC was associated with negative hormone receptors by Cordon-Cardo, 8) occurs via the enzymatic function of (p<0.001). p53 overexpression was associated with high grade protein kinases, the cyclin-dependent kinases (CDKs), which (p<0.001) and negative hormone receptors (p<0.001). p21 are activated after complexing with appropriate cyclins, the overexpression, although not correlated with the examined regulatory units of the holoenzymes. Each phase of the cell parameters, was associated with increased disease-free survival cycle shows a characteristic profile of cyclin-CDK complex in univariate analysis. In multivariate analysis, p27TN, p27TC, expression. Specific inhibitors, the cyclin-dependent kinase p21 and p53 were not associated with disease-free survival or inhibitors (CKIs), inactivate each complex. The CKIs are overall survival. These findings argue against the prognostic classified into two groups: the INK4 family, which includes value of p27, p21 and p53 in NNBC. p15INK4A, p16INK4B, p18INK4C and p19INK4D and the CIP/KIP family, which includes p21WAF1/Cip1 (p21), p27Kip1 (p27) and Although breast carcinoma patients are stratified into p57Kip2. The INK4 family members inhibit CDK4/6 while the different prognostic groups with the aid of classic CIP/KIP members are universal CKIs affecting all cyclin-CDK clinicopathological parameters such as the lymph node status complexes (9). (1), tumor size (1, 2) grade (3-5) and histological type (6), the The first checkpoint in the progression of the cell cycle outcome of patients in each stratum is often quite divergent. occurs at the G1-S transition, which is modulated by the In the node-negative breast carcinoma (NNBC) group, that function of the cyclin D/CDK and cyclin E/CDK2 complexes. At this point, p27, p21 and p53 proteins have principal regulatory effects (8). p27 was first identified as an inhibitor of cyclin E/CDK2 (10), the complex directly involved in the G1-S Correspondence to: Helen P. Kourea, MD, Parodos Volineou 5, #7, Patras 26442, Greece. Tel: +2610-438-985, +2610-999-650, Fax: transition (11), thus arresting G1-S progression. In addition, +2610-991-810, e-mail: [email protected] low levels of p27 activate cyclin D/CDK complexes, promoting G1-S progression. Therefore, the stoichiometry of p27 binding Key Words: Breast carcinoma, p27, p21, p53, prognosis. to these complexes is crucial for S-phase progression (12). 0250-7005/2006 $2.00+.40 1657 ANTICANCER RESEARCH 26: 1657-1668 (2006) The first CKI to be identified is p21 (reviewed by Dotto Table I. Clinical and histopathological data of patients (n=170). and Coqueret, 13, 14). p21 affects G1-S and G2-M Parameter No. of patients (%) checkpoints as well as DNA transcription, when localized in the nuclei. In addition to cell cycle control, p21 modulates Age (years) ≤50 years 85 (50) stress response and apoptosis when localized in the (median 50, range 24-92) >50 years 82 (48) cytoplasm. Following DNA damage, p21 transcription is Unknown 3 (2) activated by p53 and results in CDK inhibition (15, 16) and Surgical treatment Mastectomy 99 (58) Partial mastectomy 19 (11) transient G1 arrest (17). p53-independent mechanisms of p21 Tumor resection 35 (21) transcription are also well known (18). Unknown 17 (10) The p53 tumor suppressor protein plays a major role in the Tumor size (∆¡ª) ∆1 80 (47) G1-S transition, by arresting cells in G1. This allows for DNA ∆2 86 (51) repair to occur or, if repair is not feasible, p53 induces ∆3 1 (1) Unknown 3 (2) apoptosis (19-21). Tumor grade I 28 (16.5) Recently, studies on breast carcinoma have suggested the II 76 (45) prognostic importance of p27 (22-29), although others had III 65 (38) contradictory results (30-35). The prognostic significance of Unknown 1 (1) p53 detected by immunohistochemistry is even more Histological type Invasive ductal NOS 154 (90.5) Mucinous 6 (3.5) controversial in NNBC, with certain studies advocating its Papillary 3 (2) importance (36-39), while others are against it (40-43). The Medullary 2 (1.2) prognostic relevance of p21 expression remains to be Apocrine 2 (1.2) elucidated (31, 44-49). Metaplastic 1 (1) The aim of this work was to evaluate, retrospectively, the Tubulolobular 1 (1) Tubular 1 (1) expressions of p27, p21 and p53 by immunohistochemistry in Carcinoma in situ Absent 41 (24) a series of NNBCs in order: a) to detect protein expression Present (≤25%) 49 (29) profiles in the tumor; b) to evaluate any associations between Extensive (>25%) 37 (22) the proteins and the classic clinicopathological parameters; Unknown 43 (25) and c) to assess the prognostic significance of these proteins Hormonal receptor status Positive 103 (61) Negative 42 (25) in the group of NNBCs. Unknown 25 (15) Adjuvant treatment CT only 3 (2) Materials and Methods HT only 20 (12) RT only 2 (1.2) Patient and tissue samples. This retrospective study included 170 CT & HT 42 (25) patients with T1-T3, N0, M0 invasive ductal breast carcinoma, for CT & RT 7 (4) whom paraffin tissue blocks and clinical information were available. CT & HT &RT 17 (10) The pathology reports were reviewed for data regarding the type of RT & HT 24 (14) surgery, the greatest tumor diameter, the status of surgical margins Unknown 55 (32) and the number of retrieved axillary lymph nodes. Information Outcome NED 137 (81) regarding the status of estrogen and progesterone receptors, Relapse 31 (18) adjuvant treatment (chemotherapy, hormonal or radiation therapy) Death 20 (12) and outcome, i.e., recurrence, metastasis or death, disease-free Disease-free survival Median (mo) NRY Range: 2.10 – 187.6+ Overall survival Median (mo) NRY Range: 14.8 – 187.6+ survival (DFS) and overall survival (OS) was collected from the clinical records. Abbreviations: CT, chemotherapy; HT, hormonal therapy; NED, no For each case, representative hematoxylin and eosin-stained slides evidence of disease; NRY, not reached yet; RT, radiation therapy. were reviewed in order to assess the tumor grade, according to the Nottingham modification of the Bloom-Richardson grading system (3), the histological type and the presence/extent of an in situ component. The receptor status was examined by for all the antibodies studied. After cooling, the sections were immunohistochemistry on paraffin-embedded tissue when such incubated with 1% hydrogen peroxide for 15 minutes to block information was not available in the patient’s records. endogenous peroxidase activity and, subsequently, with 1% bovine serum albumin (BSA) diluted in Tris-buffered saline (TBS) at pH Immunohistochemical analysis. Formalin-fixed, paraffin-embedded 7.6 for 20 minutes, to block non-specific binding. The slides were 5-Ìm-thick sections were deparaffinized in xylene, rehydrated in wiped and incubated overnight at 4ÆC in a humid chamber with the graded alcohols and processed using the streptavidin-biotin- appropriately diluted primary antibody. The antibodies used were immunoperoxidase method. Briefly, the sections were submitted to anti-p53 protein (DO-7) mouse monoclonal antibody (NCL-p53- antigen retrieval by microwave oven treatment for 10 minutes in DO7; Novocastra Laboratories Ltd., UK; 1:50 dilution), anti- 0.01 mol/L citric acid buffer at pH 6.0. This procedure was followed p21WAF1 mouse monoclonal antibody (WAF1 (Ab-1); Oncogene 1658 Kourea et al: p27, p21 and p53 in Breast Carcinoma Table II. Immunoreactivity for p27TN, p27TC, p27bn, p21 and p53 in benign and neoplastic breast tissue. Score 0 Score 1+ Score 2+ Score 3+ Score 4+ p27TN (n=168) 63 (37.5%) 48 (29%) 36 (21%) 21 (12.5%) p27TC (n=160) 115 (72%) 15 (9%) 5 (3%) 25 (16%) p27bn (n=139) 17 (12%) 39 (28%) 57 (41%) 26 (19%) p53 (n=150) 111 (74%) 4 (3%) 6 (4%) 14 (9%) 15 (10%) p21TN (n=166) 46 (28%) 46 (28%) 55 (33%) 19 (11%) p21bn (n=138) 109 (79%) 24 (17%) 5 (4%) - Abbreviations: p27TN, p27 expression in tumor nuclei; p27TC, p27 expression in tumor cytoplasm; p27bn, p27 expression in benign breast tissue; p21TN, p21 expression in tumor nuclei; p21bn, p21 expression in benign breast tissue.