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[Background] C2P is an assay measuring specific activities (SA; kinase activity compensated by its protein expression) of cyclin-dependent kinases CDK1 and CDK2 by examining a small piece of fresh-frozen tumor tissue. We reported previously that the C2P risk score given by CDK1SA and CDK2SA is a potent prognostic factor in node-negative breast cancer patients. Likewise, a uPA/PAI-1 ELISA test (FEMTELLE®, American Diagnostica Inc. Stamford, CT) quantitatively determines uPA (urokinase-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor type-1) antigen levels in tumor tissue extracts, to identify patients with high or low risk of disease recurrence of node-negative breast cancer patients. These cancer biomarkers are recommended by the ASCO at the highest level of evidence (LOE-1) for therapy decision making in node-negative breast cancer patients. From the biological point of view, the above two assays can be placed into two categories: tumor cell proliferation for C2P and tumor cell invasion for uPA/PAI-1. This fact led us to examine the concept of combination of the two assays to better select breast cancer patients at risk. [Results] Fifty-nine cases of frozen primary breast cancer tissues were subjected to the C2P assay in a blinded manner. Twenty-one cases (40%) were categorized into “high risk”, 7 cases (14%) into “intermediate risk”, and 24 cases (46%) into “low risk”. Seven cases were judged as “not informative”. The uPA/PAI-1 results and clinical information (26: recurrent cases, 30: non-recurrent cases, 1: stage IV, 2: unknown) were provided by the TUM tumor bank. uPA/PAI-1 risk categories of 36 cases (61%) were classified “high” and 23 cases (39%) categorized “low”. C2P and uPA/PAI-1 showed statistically significant correlation to histological grading (Pearson correlation coefficient; 0.45 and 0.40, respectively). No significant correlation was observed between C2P and uPA/PAI-1. By Kaplan-Meier analysis for disease-free survival, in cases treated with endocrine therapy only, both C2P and uPA/PAI-1 showed a reproducible trend to the respective claimed performances as prognostic factors. In the combination analysis of the two parameters, where low/low was judged as “low” and the others as “high”, 11 cases (24%) were categorized into “low” and 34 cases (76%) into “high”. The sensitivity and negative predictive value for disease recurrence were 90% (19/20) and 91% (10/11), respectively. Strong statistical significance was observed between the risk categories by the log-rank test; p=0.0089, and also by Cox proportional hazards regression analysis; HR=9.18, p=0.032. By multivariate analysis, also including tumor size and nodal status, the CP2 uPA/PAI-1 combination evolved as a significant, statistically independent parameter (HR: 6.51, p Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-08.

Background: We have reported that breast tumors with a high ratio of cyclin-dependent kinase (CDK) 2 to CDK 1 are associated with high tumor cell proliferation and poor clinical outcome in Japanese. In addition, we have developed risk score based on CDK2/1 ratio (CDK-RS), and have demonstrated a high risk group showed a significantly poor prognosis in Hollanders (van Nes, et al: Br J C 2009: 100: 494). The aim of the present study is to evaluate the correlation of CDK-RS with response to neoadjuvant paclitaxel followed by fluorouracil + epirubicin + cyclophosphamide (FEC) in breast cancers. Material and Methods: Of 131 primary breast cancer patients (age: 25-73y, mean: 51.7y), 126 were treated with paclitaxel (80 mg/m2, weekly) for 12 cycles followed by FEC (500/75/500 mg/m2, q3w) for four cycles and 5 were treated with paclitaxel-monotherapy (5 — 29 cycles) in the NAC or primary chemotherapy setting. Frozen tumor tissues were obtained from core needle biopsy before NAC, and CDK-RS was determined by the Cell Cycle Profiling (C2P) assay as previously reported. Clinical response was evaluated with MRI before NAC and after paclitaxel and FEC. Patients were classified into responders showing ≥80 % in reduction rates and non-responders showing < 80 %. Pathological CR (pCR) was defined as no residual invasive foci and no axillary lymph node metastasis. Results: Patients characteristics were as follows: menopausal status: pre-47%, post-53%; Stage: II 68%, III 27%, IV 7%; Tumor size (cm): ≥5 cm 73%, > 5 cm 27%; histologic grade (HG): I 16%, II 60%, III 24%; ER: (+) 56%, (-) 44%; PR: (+) 39%, (-) 61%; HER2: (+) 28%, (-) 72%. Of 131 patients, 22 (18%) attained pCR but 100 (82%) did not pCR (9 were excluded from evaluation of pathologic response because of stage IV diseases, no operations, and not available pathologic examination). In CDK-RS, 47% of 131 tumors were classified into high, 17% intermediate, and 37% low. In combination with high + intermediate risk score groups, that group had a tendency to show high HG (grade 2 or 3) (87% vs 78%), ER negativity (49% vs 35%), and PR negativity (65% vs 54%) compared with a low risk group, but their differences were not statistically significant. Tumors in the high + intermediate group were significantly more likely to show clinical response after the completion of not only paclitaxel (52% vs 27%, p=0.006) but also FEC (75% vs 52%, p=0.011) as well as to show pCR (24% vs 9%, p=0.037) compared with a low risk score group. In univariate analysis, CDK-RS showed a significant correlation with pCR (high + intermediate vs low, Odds ratio 4.03, 95%CI 1.03 — 10.3, p=0.045). Discussion: CDK-RS in tumor samples before NAC is significantly associated with clinical and pathological response to paclitaxel followed by FEC. Thus CDK-RS seems to be a novel and useful predictive factor for paclitaxel — FEC in breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-29.

Background: We established original methods enabling simultaneous analysis of protein expressions and kinase activities of the CDK (cyclin-dependent kinase) molecules in lysate of tumor tissue in a clinical setting (C2P technology, Ishihara et al: Biochim Biophys Acta. 1741; 226-233, 2005). The clinical utility of the technology was first evaluated in breast cancer, and combination analysis of CDK1 and CDK2 activity was shown to be a significant prognostic indicator for relapse (Kim et al: Ann Oncol. 19; 68-72, 2009). The objective of our study is to evaluate the efficacy of CDK1 and CDK2 activity as a prognostic marker in human renal cell carcinoma (RCC).Methods: Surgical specimens were obtained from 125 patients with RCC without metastasis. These patients were selected randomly for this study. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system. The system to measure the CDK specific activity (SA) is named C2Ps (Sysmex, Kobe, Japan). We then examined the specific activities of CDK1 and CDK2 and calculated CDK2/CDK1 ratio in RCC. Also, risk score (RS) was examined as described in previous study (JGH van Nes et al: Br J Cancer. 100; 494-500, 2009). Cut off value was calculated by ROC analysis.Results: 125 cases were tested, though 34 cases were excluded of low sample quality (25 cases) and of assay failure (9 cases). 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow up of 36 months (1-109M), tumor with low CDK2/CDK1 ratio showed significantly better 5-year progression free survival (PFS) than those with high CDK2/CDK1 ratio (88.7% vs 54.7%, P=0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, patients with tumors classified as low RS showed better PFS than patients with tumors with high RS (88.7% vs 54.7%, P=0.0141).Conclusion: CDK1 specific activity of tumors and the CDK2 specific activity are both associated with recurrence and prognosis. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting cancer recurrence and prognosis in RCC after surgery and has potential for use as a routine laboratory test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2011-299