Yasuhiro Torikoshi

High expression of ubiquitin carboxy-terminal hydrolase-L1 and -L3 mRNA predicts early recurrence in patients with invasive breast cancer

The present study investigated the mRNA expression level of ubiquitin c‐terminal hydrolase (UCH)‐L1 and ‐L3 in breast cancer tissue and aimed to elucidate its association with tumor characteristics and patient prognosis. UCH‐L1 and UCH‐L3 mRNA levels in invasive breast cancer (n = 100) were determined by a real‐time polymerase chain reaction (PCR) assay and their relationship with various clinicopathological characteristics of breast tumors as well as patient prognosis were studied. UCH‐L3 mRNA level was significantly upregulated in breast cancer tissue compared to adjacent normal breast tissue (P < 0.005), and UHC‐L1 mRNA level also showed a non‐significant increase in tumor tissue compared to adjacent normal breast tissue. Both UCH‐L1 and UCH‐L3 mRNA levels were significantly higher in high histological grade tumors than in low histological grade tumors (P < 0.001 and P < 0.005, respectively). High UCH‐L1 mRNA level was significantly associated with negative estrogen receptor status (P < 0.05) and negative progesterone receptor status (P < 0.05). Patients with both UCH‐L1 and UCH‐L3 mRNA high tumors showed a significantly poorer prognosis than those in the UCH‐L1 or UCH‐L3 mRNA low group (P < 0.005). These observations that UCH‐L3 mRNA level is upregulated in breast cancer tissue, and breast tumors with both UCH‐L1 and UCH‐L3 mRNA high expression are associated with a poor prognosis, suggest the possible involvement of UCH‐L1 and UCH‐L3 in the pathogenesis and progression of breast cancer. (Cancer Sci 2006; 97: 523– 529)

Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells

IntroductionPaclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth state, respectively, predict sensitivity to paclitaxel.MethodsCell viability assays and DNA and chromatin morphology analyses were performed in human breast cancer cell lines to evaluate sensitivity to paclitaxel and the cell cycle response to paclitaxel. We then examined the specific activities of CDK1 and CDK2 in these cell lines and in xenograft models of human breast cancer before and after paclitaxel treatment. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system.ResultsIn the cell lines, biological response to paclitaxel in vitro did not accurately predict sensitivity to paclitaxel in vivo. Among the breast cancer xenograft tumors, however, tumors with significantly increased CDK1 specific activity after paclitaxel treatment were sensitive to paclitaxel in vivo, whereas tumors without such an increase were resistant to paclitaxel in vivo. Baseline CDK2 specific activity was higher in tumors that were sensitive to paclitaxel than in tumors that were resistant to paclitaxel.ConclusionsThe change in CDK1 specific activity of xenograft tumors after paclitaxel treatment and the CDK2 specific activity before paclitaxel treatment are both associated with the drug sensitivity in vivo. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting paclitaxel sensitivity.

Recurrence risk score based on the specific activity of CDK1 and CDK2 predicts response to neoadjuvant paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide in breast cancers

BACKGROUND We established the cell cycle profiling (C2P) assay for specific activity (SA; activity/expression) of cyclin-dependent kinases (CDKs). C2P risk score (C2P-RS) based on CDK1 and CDK2 SAs was significantly associated with relapse in breast cancer (BC). This study was conducted to investigate the predictive value of C2P-RS for neoadjuvant chemotherapy (NAC). PATIENTS AND METHODS Among 124 eligible patients, 122 were treated with weekly paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide (P-FEC) and 2 were treated with paclitaxel monotherapy. C2P-RS was determined via C2P using frozen biopsy samples before NAC. RESULTS Negative estrogen receptor (ER), negative progesterone receptor (PR), positive human epidermal growth factor receptor 2 (HER2), high Ki-67 expression and intermediate + high C2P-RS were significantly associated with high pathological complete response (pCR) rates compared with positive ER (30% versus 9%), positive PR (25% versus 6%), negative HER2 (34% versus 11%), low Ki-67 expression (24% versus 7%) or low C2P-RS (24% versus 9%), respectively. The combination of C2P-RS and Ki-67 had a stronger impact on pCR than each parameter alone, and a multivariate analysis showed that the combination was an independent predictor of pCR (odds ratio 3.3, 95% confidence interval 1.1-9.5). CONCLUSIONS C2P-RS was significantly associated with pCR after P-FEC and may be a useful predictor for chemotherapy in BCs.

Novel Functional Assay for Spindle-Assembly Checkpoint by Cyclin-Dependent Kinase Activity to Predict Taxane Chemosensitivity in Breast Tumor Patient

Taxanes are among the drugs most commonly used for preoperative chemotherapy for breast cancer. Taxanes induce mitotic arrest and subsequent apoptosis. The spindle-assembly checkpoint (SAC) is known to be activated during mitosis, along with cyclin-dependent kinase-1 (CDK1), and is required for taxane-induced cell death. We hypothesized that CDK1 activity predicts response to taxane-containing chemotherapy. This study included breast cancer patients who received preoperative chemotherapy— taxane-containing treatment followed by anthracycline-based treatment—and then underwent surgery. Before starting taxane-containing chemotherapy, patients underwent fine-needle aspiration biopsy, and the biopsy samples were incubated in paclitaxel solution to measure CDK activity. Clinical were evaluated after taxane therapy, and pathological resposes were evaluated after completion of all preoperative chemotherapy. Thirty five patients were eligible for analysis of clinical response to taxane-containing therapy. Twenty-six patients had taxane-sensitive and 9 taxane-resistant tumors. Using a cut-off of CDK activity determined by the ROC analysis, patients were classified into SAC function and dysfunction groups. Univariate logistic regression analysis with clinicopathologic parameters showed that only CDK-based SAC functionality was significantly correlated with clinical response (P =0.017). No significant correlation was observed between SAC functionality and pathologic response. CDK-based SAC functionality significantly predicted clinical response (P =.0072, overall agreement = 71.4%), and this is a unique mechanism-based marker for predicting taxane chemosensitivity. Further, large prospective study is needed to determine CDK-based SAC functionality could be developed as a predictive biomarker.

P5-13-02: Prediction of Dasatinib Sensitivity of Breast Cancer Based on a Novel Tyrosine Kinase-Activity Profiling Assay.

Background: Receptor tyrosine kinases and other membrane-associated tyrosine kinases are frequently overexpressed, activated, or mutated in cancer cells and cause aberrant signal transduction, which leads to cellular proliferation, angiogenesis, metastasis, and antiapoptosis. Blockage of these signals is considered an efficient strategy in cancer therapy, and to date, several tyrosine kinase inhibitors (TKIs) and antibody drugs targeting such kinases have been developed and are routinely used in clinics. However, the efficacy rates of the drugs are, unfortunately, limited. To provide optimum care for individual patients, an accurate prediction method for drug efficacy is now strongly demanded. Recent studies have shown that alteration of multiple kinases is involved in both primary and acquired drug resistance. Advantages of multi-targeted TKIs have also been reported. These findings indicate the need for a comprehensive evaluation of multiple kinases for predicting drug response. To achieve this, we developed a novel method of comprehensively profiling kinase activity. Materials and methods: We characterized the membranous tyrosine kinases of breast cancer cell lines using a newly established profiling assay. Briefly, crude membrane fractions were prepared and directly subjected to the assay with nonspecific substrate (Poly(Glu 4 -Tyr)). The profile of the kinases in the crude membrane fraction was obtained by inhibiting the total activity with 13 selected kinds of adenosine triphosphate antagonists, independently. The residual activity (RA) of membranous kinases was defined as the percentage of activity with/without TKI. Results: Nineteen breast cancer cell lines were classified as “sensitive” (n = 6) and “resistant” (n = 13) to dasatinib according to the definition in the publication ( Cancer Res., 67 2226–38. 2007 ). The RA of tyrosine kinases targeted by different types of TKIs was determined by our assay, and we found that two RAs (src inhibitor 1 and PP1) showed statistically significant differences between the “sensitive” and “resistant” groups (p=0.017, and p=0.002, respectively, Student9s t-test). The RA of the two TKIs were also significant predictors for dasatinib sensitivity in a receiver operating characteristic curve analysis (area under the curve: 0.846 for src inhibitor 1 and 0.910 for PP1). Since the major target of dasatinib are src family kinases, protein expressions of the src family of the cell lines were quantified by a Western blotting and compared between the groups. We found that the protein expression is not a statistically significant predictor of dasatinib sensitivity. Conclusion: We have shown that a comprehensive tyrosine kinase activity profiling assay of cancer cells can predict dasatinib sensitivity. We plan to validate this assay prospectively in patients with breast cancer who receive dasatinib. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-02.

Ratio of cyclin-dependent kinase 1 (CDK1) activity to CDK2 activity after ex vivo paclitaxel treatment predicts response to paclitaxel in human breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #6065 Background: Increased CDK1 activity and reduced CDK2 activity reflect paclitaxel sensitivity in vitro. We have developed a novel assay that measures CDK1 and CDK2 activity in small tissue samples (“C2P assay”). The aim of this study was to determine whether the CDK1/2 ratio after ex vivo paclitaxel treatment correlates with response to paclitaxel in human breast cancer. Material and Methods: This study included 38 patients with primary breast cancer. The median age was 50 years. Twenty-five patients (66%) had stage II, 12 (32%) stage III, and 1 (3%) stage IV. Thirty-three patients (87%) had invasive ductal and four (11%) invasive lobular cancer: 1 patient (3%) had mucinous disease. Histologic grade was II in 33 patients (87%) and III in 5 patients (13%). Twenty-one patients (55%) had ER-positive and 11 (29%) HER2-positive disease. All patients received preoperative chemotherapy - paclitaxel (80 mg/m2, weekly for 12 weeks) followed by 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) (500/75/500 mg/m2, every 3 weeks for 4 cycles) in 34 patients (89%) and paclitaxel alone in 4 patients (11%). Tumor tissues obtained by core needle biopsy before chemotherapy were treated ex vivo with paclitaxel (100 nM) for 24 h, and then had CDK1/2 activity measured using the C2P assay. Clinical responses were evaluated with magnetic resonance imaging after paclitaxel treatment, and tumors that showed equal and more than 80 % shrinkage were defined as responders. The cutoff value for distinguishing between high and low CDK1/2 ratio was identified as 5.2 to best separate the responders and non-responders to paclitaxel. Results: Of the 23 tumors with a high CDK1/2 ratio, 18 responded to paclitaxel. On the other hand, of the 15 tumors with a low CDK1/2 ratio, 10 did not respond to paclitaxel (positive predictive value, 78%; negative predictive value, 67%; p = 0.006). Of the 34 patients who were treated with paclitaxel followed by FEC and underwent surgery, 9 (26%) had a pathologic complete response (pCR). Eight of 21 tumors in the high CDK1/2 ratio group but only 1 of 13 tumors in the low CDK1/2 ratio group had a pCR (positive predictive value, 38%; negative predictive value, 92%; p = 0.05). Discussion: The CDK1/2 ratio is significantly associated with response to paclitaxel and also with pCR after treatment with paclitaxel followed by FEC. The CDK1/2 ratio after ex vivo paclitaxel treatment might be a useful test to predict paclitaxel sensitivity in breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6065.