Koichi Takagi

Circulating tumor cells as a surrogate marker for determining response to chemotherapy in Japanese patients with metastatic colorectal cancer

The purpose of this study was to investigate the potential of circulating tumor cells (CTC) as a surrogate marker of the clinical outcome in metastatic colorectal cancer (mCRC) patients in order to identify Japanese patients responsive to oxaliplatin‐based chemotherapy. Between January 2007 and April 2008, 64 patients with mCRC were enrolled in this prospective study. The treatment regimen was oxaliplatin‐based chemotherapy. Collection of CTC from whole blood was performed at baseline and at 2 and 8–12 weeks after initiation of chemotherapy. Isolation and enumeration of CTC was performed using immunomagnetics. Patients with ≥3 CTC at baseline and at 2 and 8–12 weeks had a shorter median progression‐free survival (8.5, 7.3 and 1.9 months, respectively) than those with <3 CTC (9.7, 10.4 and 9.1 months, respectively) (log‐rank test: P = 0.047, P < 0.001 and P < 0.001, respectively). Patients with ≥3 CTC at 2 and 8–12 weeks had a shorter median overall survival (10.2 and 4.1 months, respectively) than those with <3 CTC (29.1 and 29.1 months, respectively) (P < 0.001 and P = 0.001, respectively). A spurious early rise in carcinoembryonic antigen level was observed in 11 patients showing a partial response. In contrast, no rise in early CTC level was observed among responders. Our data support the clinical utility of CTC enumeration in improving our ability to accurately assess treatment benefit and in expediting the identification of effective treatment regimens for individual Japanese patients. (Cancer Sci 2011; 102: 1188–1192)

A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti-EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-theart measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

Coagulo-fibrinolytic activity as a predictor of efficacy in bevacizumab-combined chemotherapy in metastatic colorectal cancer patients.

533 Background: The combination of bevacizumab (BV) and chemotherapy in the first-line and second-line treatment of metastatic colorectal cancer (mCRC) has been shown to improve survival. Bevacizumab is a recombinant, humanized monoclonal antibody against vascular endothelial growth factor. However, the relationship between coagulo-fibrinolytic activity factors and treatment efficacy remains to be clarified. The aim of this study was to determine potential coagulo-fibrinolytic activity markers impacting survival. METHODS Among 119 consecutive patients included in the study, 85 received first-line FOLFOX4 plus BV 5 mg/kg and 34 received second-line FOLFIRI plus BV 5 mg/kg until progression of disease or unmanageable toxicity occurred. Coagulo-fibrinolytic activity factors, including D-dimer, thrombin antithrombin complex (TAT) and carbohydrate antigen 125 (CA125) encoded by the MUC16 mucin gene were evaluated as candidate predictors of outcome. RESULTS In first-line treatment, overall response, median progression-free survival (PFS) and two-year survival rate were 61.9%, 518 days and 67.3%, respectively. In second-line treatment, overall response, median PFS and median overall survival (OS) were 23.5%, 248 days and 651 days, respectively. The outcomes of the univariate analysis were as follows: normal D-dimer and CA125 levels at baseline were associated with better PFS and OS in first-line treatment; normal TAT and CA125 levels at baseline were associated with better PFS and OS in second-line treatment. According to the results of the multivariate analysis, normal D-dimer level was associated with longer PFS in first-line treatment, and only CA125 level at baseline was an independent predictor of both PFS and OS in second-line treatment. CONCLUSIONS The results suggest that coagulo-fibrinolytic activity factors such as TAT, D-dimer or CA125 may be useful predictors of outcome in mCRC patients receiving BV in combination with chemotherapy. No significant financial relationships to disclose.

Abstract 4149: Genetic analysis of circulating tumor cells using 3D immunostain and FISH

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Circulating tumor cells (CTCs) offer a non-invasive approach to characterize metastatic tumor cells. However, the low recovery rates of CTCs by methods performed in most clinical laboratory have limited their usefulness. Recent studies have suggested that discordance may exist between Her-2 status of a patients CTC and that of primary breast tumor. To investigate Her-2 status in detail, we developed a new technique of the FISH analysis of CTCs. Most CTCs enrichment techniques are anti-EpCAM antibody based. However, some tumor cells express low or no EpCAM. So, we adopted the strategy independent of EpCAM antigenecity in order to reliably analyze genes even with very few CTCs. Material and methods: The method for Her-2 analysis that we established was based on three-dimensional multi-color imaging. Briefly, CTCs of tumor patients were concentrated by negative selection from peripheral blood by using antibodies against WBC, RBC, and platelets. The concentrated CTCs were labeled by fluorescent monoclonal antibodies against pan-cytokeratin and CD45. The specimens were then hybridized with FISH probes and were mounted in DAPI with antifade reagent. The preparations were screened for a series of Z-axis optical sections with a confocal microscope and 3D images were generated using Fluoview software. To inspect the availability of our method, we analyzed the peripheral blood or effusion specimens of patients with gastrointestinal tumors. This study was approved by the Institutional Review Board of Japanese Foundation for Cancer Research. Results: CTCs (Cytokeratin+/CD45-/DAPI+) were easily discriminated from remaining hematopoietic cells (CD45+). And these immunocytochemical staining had no crossover on FISH signal by the advantage of confocal imaging. In addition, three-dimensional imaging reconstruction enabled counting of DNA probe spots that differed in the Z plane but were at the same XY position in the cell. Recovery rates of tumor cells spiked into normal blood averaged 79%. By the analysis of the several cases of peripheral blood and the colon fiber biopsy derived from the same patients, it was suggested that the expression of EpCAM in CTCs was distinctly lower than it in the primary tumor site. Conclusions: Because our method does not depend on expression of EpCAM, even the CTCs that express little or no EpCAM can be identified. In addition, the three-dimensional imaging with high spacial/wavelength resolution made it possible to analyze Her-2 gene status of CTCs easily and accurately. Using this methods, we are currently working on the investigation of Her-2 state in CTCs derived from patients with gastrointestinal tumor and its relation of Her-2 status of primary tumor and metastases. 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 4149. doi:10.1158/1538-7445.AM2011-4149