Keigo Gohda

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.

Circulating tumor cells as a prognostic factor in patients with small cell lung cancer

The detection of circulating tumor cells (CTCs) in peripheral blood is currently an important field of study. Detection of CTCs by the OBP-401 assay (TelomeScan®) has previously been reported to be useful in the diagnosis, prognosis and evaluation of therapeutic efficacy in breast and gastric cancer. The aim of the present study was to evaluate the OBP-401 assay as a novel method of detecting CTCs of small cell lung cancer (SCLC) patients and to evaluate whether CTC count is associated with prognosis. Prospectively, 30 consecutively diagnosed SCLC patients who had commenced chemotherapy or chemoradiotherapy were enrolled as subjects of the current study. Peripheral blood specimens were collected from the SCLC patients prior to and following the initiation of treatment and the viable CTCs were detected in the specimens following incubation with a telomerase-specific, replication-selective, oncolytic adenoviral agent, which was carrying the green fluorescent protein gene. CTCs were detected in 29 patients (96%). The group of 21 patients with a CTC count of <2 cells/7.5 ml prior to treatment (baseline) had a significantly longer median survival time than the group of eight patients with a CTC count of ≥2 cells/7.5 ml prior to treatment (14.8 and 3.9 months, respectively; P=0.007). The results of a multivariate analysis showed that the baseline CTC count was an independent prognostic factor for survival time (hazard ratio, 3.91; P=0.026). Among the patients that achieved a partial response to treatment, patients who had a CTC count of <2 cells/7.5 ml following two cycles of chemotherapy tended to have a longer median progression-free survival compared with patients who had a CTC count of ≥2 cell/7.5 ml (8.3 and 3.8 months, respectively; P=0.07). Therefore, CTCs may be detected via OBP-401 assay in SCLC patients and the CTC count prior to treatment appears to be a strong prognostic factor.

Prognostic impact of detecting viable circulating tumour cells in gastric cancer patients using a telomerase-specific viral agent: a prospective study

BackgroundThe identification of circulating tumour cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression, and measure treatment effects in various malignancies. However, clinical relevance of CTCs is controversial. We attempted to detect viable CTCs in the peripheral blood of gastric cancer patients using a telomerase-specific viral agent.MethodsWe took a 7.5-ml blood sample from 65 treatment-negative gastric cancer patients before surgery and 10 healthy volunteers. We detected viable CTCs in the blood samples after incubating them with a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene (OBP-401). GFP-positive CTCs were defined as having a diameter of at least 7.735u2009μm; this threshold was determined by receiver operating characteristic curve analysis. GFP-positive cells were counted under a fluorescence microscope.ResultsThere was a significant difference in overall survival among the patients with 0–4 and those with ≥5 GFP-positive CTCs in the stage I–IV disease group and stage II–IV advanced disease group. The number of GFP-positive CTCs was not related to cancer stage. Among the pathological findings, the number of GFP-positive CTCs was only significantly related to venous invasion, although there were trends towards more GFP-positive CTCs with disease progression (tumour depth, lymph node metastasis, distant metastasis, lymphatic invasion, and histological type).ConclusionsThere was a significant relationship between the number of GFP-positive CTCs and overall survival in the patients with gastric cancer. The detection of CTCs using OBP-401 may be useful for prognostic evaluation.Trial registrationUniversity Hospital Medical Information Network in Japan, UMIN000002018.

Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study

The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

Detection and preliminary evaluation of circulating tumor cells in the peripheral blood of patients with eight types of cancer using a telomerase-specific adenovirus

We developed a detection method for circulating tumor cells (CTCs) using the telomerase-specific adenovirus OBP-401. This recombinant virus has a telomerase promoter at the 5′-end of the viral genome and GFP at the 3′-end. To date, CTC enumeration using OBP-401 has shown prognostic impact for gastric and small cell lung cancer patients. In the present study, peripheral blood samples from patients with eight types of cancer, including some cancers previously untested with OBP-401 (i.e., esophagus, pancreas, and prostate cancers) were subjected to this method in order to evaluate its versatility. It was recently discovered that some white blood cells (WBCs) false-positively react with OBP-401. Although anti-CD45 antibodies can absorb these adverse cells from peripheral blood, the simplicity of the OBP-401 method would be diminished by the introduction of antibody treatment. Therefore, we evaluated another approach to minimize the false positivity of WBCs. Seven anti-CD antibodies were employed to stain the species of WBCs that false-positively reacted with OBP-401. We revealed that the false-positively reacted WBCs were monocytes in the peripheral blood of both healthy subjects and cancer patients. Based on a size distribution analysis of the GFP-positive monocytes, the size criterion for CTCs using OBP-401 was defined to be a cellular diameter >8.4 μm. In total, 43% of 86 cancer patients examined in the present study were CTC-positive using this definition. CTCs were enumerated from peripheral blood samples collected from patients with each of the eight types of cancer; the detectability of CTCs for esophagus, pancreas and prostate cancers by the OBP-401 method was confirmed for the first time in the present study. However, no clear correlation between CTC positivity and the clinical characteristics of patients with any type of cancer was observed because of the small number of patients with each type of cancer. An additional clinical study will be conducted to confirm the clinical meaning of CTCs enumerated by OBP-401.

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.

Long-term prognostic impact of circulating tumour cells in gastric cancer patients

AIM To analyse the long-term prognostic impact of circulating tumour cells (CTCs) in gastric cancer patients who underwent surgery. METHODS A 7.5-mL peripheral vein blood sample was obtained from each patient with treatment-negative gastric adenocarcinoma before surgery. OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein gene, was used to label CTCs. Correlations between the number of CTCs and clinical end points were evaluated. RESULTS The median follow-up period of the surviving patients with gastric cancer was 60 mo. The CTC number tended to increase concomitantly with disease progression. The overall survival of patients with more than five CTCs in 7.5-mL of peripheral blood was lower than that of patients with five or less CTCs, although the difference was not significant (P = 0.183). A significant difference in relapse-free survival was found between patients with more than five and those with five or less CTCs (P = 0.034). CONCLUSION A lower number of CTCs was correlated with higher relapse-free survival rates in patients. Detection of CTCs using OBP-401 may be useful for predicting prognosis in gastric cancer.

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.

Abstract 3730: Akt activity is suppressed by doxorubicin through downregulation of ErbB receptors and its enhanced activity is associated with higher sensitivity to doxorubicin in breast cancer

Anthracyclines are the backbone of breast cancer therapy and benefit patients in both the adjuvant and metastatic setting whereas they are effective for 50-60% of breast cancer patients and cause substantial morbidity and occasional life-threatening toxic effects. For these reasons, it has been eagerly anticipated to develop diagnostic markers which can predict efficacy of the treatment. HER2 amplification or overexpression of its protein (HER2 positivity) has been shown to be associated with relative benefit from anthracycline-based chemotherapy (Gennari A. et al., J. Natl. Cancer Inst, 100, 14-20, 2008). However, HER2 is positive in 15-25% of breast tumor tissues and no clear biological mechanism between HER2 positivity and action of anthracyclines has been reported. In this study, we have shown that doxorubicin exerts its anti-tumor activity through inhibiting ErbBs-Akt signal pathway. Promoted activity of Akt was associated with not only HER2 but EGFR overexpression and responsiveness to doxorubicin in human breast cancer cell lines. As well as doxorubicin, PI3 kinase inhibitor caused growth inhibition and apoptosis in cells with promoted Akt activity more significantly than cells with weakly activated Akt. Doxorubicin-induced cytotoxic stress induced internalization and degradation of ErbBs followed by inactivation of Akt. This was confirmed with the observation that doxorubicin caused decrease in the amount of immunocomplex formed between HER3 and p85, PI3 kinase regulatory subunit. These data indicate that cancer cells with ErbBs overexpression and promoted Akt activity are dependent on activities of these factors for their growth and survival, and doxorubicin inhibits the ErbBs-Akt signal pathway through inducing downregulation of ErbB receptors. Furthermore, we found that highly phosphorylated Akt in tumors corresponds to improved recurrence-free survival in breast cancer patients treated with anthracycline-based adjuvant chemotherapy relative to weakly phosphorylated Akt. Taken together, we provide mechanistic insight into molecular basis of correlation between HER2 positivity and sensitivity to anthracyclines, and propose a new possibility that Akt activity is a useful predictor of response to anthracycline-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3730.

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 AbstractsnnAbstract #6065 nnBackground: 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.nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6065.