Gel Droplet Embedded Culture Drug Research Articles

A PDX model combined with CD-DST assay to evaluate the antitumor properties of KRpep-2d and oxaliplatin in KRAS (G12D) mutant colorectal cancer

Patient-derived xenograft (PDX) models are more faithful in maintaining the characteristics of human tumors than cell lines and are widely used in drug development, although they have some disadvantages, including their relative low success rate, long turn-around time, and high costs. The collagen gel droplet embedded culture drug sensitivity test (CD-DST) has been used as an in-vitro drug sensitivity test for patients with cancer because of its high success rate of primary cell culture, high sensitivity, and good clinical relevance, but it is based on an in-vitro cell culture and may not simulate the tumor microenvironment accurately. This study aims to combine a PDX model with CD-DST to evaluate the efficiency of antitumor agents. KRpep-2d, a small peptide targeting KRAS (G12D), and oxaliplatin were used to verify the feasibility of this approach. Whole-exome sequencing and Sanger sequencing were first applied to test and validate the KRAS mutation status of a panel of colorectal cancer PDX tissues. One PDX model was verified to carry KRAS (G12D) mutation and was used for in-vivo and the CD-DST drug tests. We then established the PDX mouse model from the patient with the KRAS (G12D) mutation and obtained viable cancer cells derived from the same PDX model. Next, the antitumor abilities of KRpep-2d and oxaliplatin were estimated in the PDX model and the CD-DST. We found that KRpep-2d showed no significant antitumor effect on the xenograft model or on cancer cells derived from the same PDX model. In contrast, oxaliplatin showed significant inhibitory effects in both tests. In conclusion, the PDX model in combination with the CD-DST assay is a comprehensive and feasible method of evaluating the antitumor properties of compounds and could be applied for new drug discovery.

Identification of the optimal cetuximab concentration that is effective against oral squamous cell carcinoma in collagen gel droplet embedded culture drug sensitivity testing.

Anticancer drug sensitivity testing using the collagen gel droplet embedded culture drug sensitivity test (CD-DST) on oral squamous cell carcinoma (OSCC) samples beginning from 2010 has been conducted. The present study investigated the effect of adding cetuximab (Erbitux®), a molecularly targeted drug, on anticancer drug activity against clinical OSCC specimens. A total of 25 specimens were obtained from 25 patients with OSCC between October 2013 and December 2017. The present study conducted anticancer drug sensitivity testing for cisplatin (CDDP), 5-fluorouracil (5-FU), cetuximab, three-drug combination, single agent and multi drug combinations, and cetuximab addition to the aforementioned regimens using CD-DST. In addition, the optimum concentration of each drug was evaluated. The overall evaluation success rate of the CD-DST method for OSCC specimens was 84.0% (21 of 25 cases); sensitivity to anticancer drugs and cetuximab could be evaluated. The in vitro efficacy rate of a cetuximab single agent and CDDP + 5-FU (PF) at a cut-off value of 50% was similar to the known clinical response rate. However, at a cut-off value of 50%, the in vitro efficacy of PF + cetuximab was calculated to be 40%, which was higher than the clinical response rate. The CD-DST method could be used to evaluate cetuximab, a molecularly targeted drug. Furthermore, its additive effect on conventional chemotherapy could be evaluated. The CD-DST method is suitable for evaluating and selecting chemotherapy regimens, including molecularly targeted drugs. Future studies are required to generate and evaluate relevant clinical data.

In Vitro Chemosensitivity Test for Gastric Cancer Specimens Predicts Effectiveness of Oxaliplatin and 5-Fluorouracil.

Cisplatin plus 5-fluorouracil (5-FU) or S-1 is a standard therapy for gastric cancer (GC). However, cisplatin is emetic and potentially nephrotoxic. Oxaliplatin may be less toxic, but few basic data are available for this setting. Here, we evaluated oxaliplatin for GC, by testing surgical specimens. We evaluated effects of oxaliplatin and 5-FU, alone and in combination, on surgical specimens from 11 patients with GC, using collagen gel droplet embedded culture drug tests. Oxaliplatin was less efficacious than 5-FU, and its synergistic effect was less in tumors highly sensitive to 5-FU than in those with low sensitivity. Tumor differentiation and drug sensitivity were not correlated. Although oxaliplatin monotherapy had little effect on GC, we saw a limited synergistic effect of oxaliplatin with 5-FU in 5-FU-sensitive patients. Collagen gel droplet embedded culture drug tests may predict this synergistic effect, and help select candidates for this or other regimens.

Impact of in vitro chemosensitivity test-guided platinum-based adjuvant chemotherapy on the surgical outcomes of patients with p-stage IIIA non-small cell lung cancer that underwent complete resection.

The impact of in vitro chemosensitivity test-guided platinum-based adjuvant chemotherapy on the surgical outcomes of patients undergoing complete resection for locally advanced non-small cell lung cancer (NSCLC) has yet to be elucidated. In the present study, the utility of adjuvant chemotherapy based on the collagen gel droplet embedded culture drug sensitivity test (CD-DST) in patients with p (pathology)-stage IIIA NSCLC was retrospectively analyzed. A series of 39 patients that had received platinum-based adjuvant chemotherapy following complete resection between 2007 and 2012 were enrolled. Their surgical specimens were subjected to the CD-DST. The patients were subsequently classified into two groups on the basis of in vitro anti-cancer drug sensitivity data obtained using the CD-DST: The sensitive group (25 patients) were treated with regimens including one or two of the anti-cancer drug(s) that were indicated to be effective by the CD-DST, whereas the non-sensitive group (14 patients) were treated with chemotherapy regimens that did not include any CD-DST-selected anti-cancer drugs. There were no significant differences in the background characteristics of the two groups [including in respect of the pathological TN (tumor-lymph node) stage, tumor histology, epidermal growth factor receptor mutation status, the frequency of each chemotherapy regimen, and the number of administered cycles]. The 5-year disease-free survival (DFS) rate of the sensitive group was 32.3%, whereas that of the non-sensitive group was 14.3% (P=0.037). In contrast, no difference in overall survival (OS) was observed (P=0.76). Multivariate analysis revealed that adjuvant chemotherapy based on the CD-DST had a significant favorable effect on the DFS (P=0.01). Therefore, the present study has demonstrated that CD-DST data obtained from surgical specimens aid the selection of effective platinum-based adjuvant chemotherapy regimens for patients undergoing complete resection for p-stage IIIA NSCLC. The use of CD-DST-guided platinum-based regimens may have a beneficial impact on the DFS of such patients.

ABCG2 expression in colorectal adenocarcinomas may predict resistance to irinotecan.

Irinotecan is a key drug for patients with advanced and recurrent colorectal carcinoma. However, the efficacy of irinotecan is not sufficient; partly, as there is no useful marker to predict chemosensitivity to the drug. The aim of the present study was to evaluate whether the expression levels of adenosine triphosphate-binding cassette sub-family G (WHITE) member 2 (Junior blood group) (ABCG2) in primary colorectal tumors predict chemoresistance to irinotecan. Using the resected primary tumor specimens of 189 patients with colorectal cancer, the association between the immunohistochemical expression of ABCG2 protein and the results of the collagen gel droplet embedded culture drug sensitivity test, performed to evaluate the chemosensitivity to SN-38 (an active metabolite of irinotecan), was investigated. Among the 189 patients, 17 received irinotecan-based chemotherapy, and their responses and progression-free survival (PFS) were analyzed. The tumors of patients with increased ABCG2 expression accounted for 60% of the tumors examined, and were significantly more resistant to SN-38, compared with patients with low ABCG2 expression (P<0.001). In a multivariate logistic regression analysis, increased expression of ABCG2 protein was an independent and significant predictor of resistance to SN-38, increasing the risk of resistance by 12-fold. Increased expression of ABCG2 and a low sensitivity to SN-38 was significantly associated with resistance to irinotecan-based chemotherapy (P=0.01 and 0.028, respectively). The median PFS of patients with increased expression of ABCG2 was significantly shorter, compared with patients with low expression levels of ABCG2 (104 vs. 242 days; P=0.047). The increased immunohistochemical expression of ABCG2 in primary tumors may be a useful predictive biomarker of resistance to irinotecan-based chemotherapy for patients with recurrent or metastatic colorectal cancer.

In Vitro Drug Sensitivity Tests to Predict Molecular Target Drug Responses in Surgically Resected Lung Cancer.

BackgroundEpidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors have dramatically changed the strategy of medical treatment of lung cancer. Patients should be screened for the presence of the EGFR mutation or echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene prior to chemotherapy to predict their clinical response. The succinate dehydrogenase inhibition (SDI) test and collagen gel droplet embedded culture drug sensitivity test (CD-DST) are established in vitro drug sensitivity tests, which may predict the sensitivity of patients to cytotoxic anticancer drugs. We applied in vitro drug sensitivity tests for cyclopedic prediction of clinical responses to different molecular targeting drugs.MethodsThe growth inhibitory effects of erlotinib and crizotinib were confirmed for lung cancer cell lines using SDI and CD-DST. The sensitivity of 35 cases of surgically resected lung cancer to erlotinib was examined using SDI or CD-DST, and compared with EGFR mutation status.ResultsHCC827 (Exon19: E746-A750 del) and H3122 (EML4-ALK) cells were inhibited by lower concentrations of erlotinib and crizotinib, respectively than A549, H460, and H1975 (L858R+T790M) cells were. The viability of the surgically resected lung cancer was 60.0 ± 9.8 and 86.8 ± 13.9% in EGFR-mutants vs. wild types in the SDI (p = 0.0003). The cell viability was 33.5 ± 21.2 and 79.0 ± 18.6% in EGFR mutants vs. wild-type cases (p = 0.026) in CD-DST.ConclusionsIn vitro drug sensitivity evaluated by either SDI or CD-DST correlated with EGFR gene status. Therefore, SDI and CD-DST may be useful predictors of potential clinical responses to the molecular anticancer drugs, cyclopedically.

Ribonucleotide reductase M2 is a promising molecular target for the treatment of oral squamous cell carcinoma.

In our previous study, ribonucleotide reductase M2 (RRM2) was identified as a cancer-related gene commonly overexpressed in human oral squamous cell carcinoma (OSCC) cell lines. Herein, we attempted to determine whether targeting RRM2 may be a plausible therapeutic approach for the treatment of patients with OSCC. First, we examined the expression levels of RRM2 in human OSCC cell lines and tissues. Overexpression of RRM2 in OSCC was confirmed by western blot analysis. Subsequently, we investigated the effects of a synthetic small interfering RNA specific for RRM2 and gemcitabine (GEM), an inhibitor of RRM2 enzymatic activity, on the growth of human OSCC cell lines and primary cultured cells. Targeting RRM2 by RNA interference almost completely suppressed the expression of RRM2 and markedly suppressed the growth of both types of cells by >54.8%. GEM also reduced the growth rate of these cells by >83.0%. Finally, we evaluated the antitumor effects of GEM, cisplatin (CDDP), 5-fluorouracil (5-FU), and docetaxel (DOC) against OSCC cells using the collagen gel droplet embedded culture drug sensitivity test. OSCC cells were more sensitive to GEM and DOC than to CDDP and 5-FU, regardless of the expression level of RRM2 mRNA. These results suggested that RRM2 supported the growth of human OSCC cells and that targeting of RRM2, e.g., via GEM treatment, may be a promising therapeutic strategy for OSCC.

Anticancer drug sensitivity test on squamous cell carcinoma cell lines derived from human oral cancers: Optimal contact concentrations of CDDP and 5-FU using the CD-DST

The collagen gel droplet embedded culture drug sensitivity test (CD-DST) is an anticancer drug sensitivity test that uses a method of three-dimensional culture of very small samples, and it is suited to primary culture of human cancer cells. It is a useful method for oral squamous cell carcinoma (OSCC), in which the cancer tissues needed for testing are in limited supply. However, as the optimal contact concentrations of anticancer drugs for OSCC treatment have yet to be established, CD-DST for OSCC is currently carried out by applying the optimal contact concentrations for stomach cancer.

Association of epigenetic inactivation of the WRN gene with anticancer drug sensitivity in cervical cancer cells

The Werner (WRN) gene codes for a DNA helicase that contributes to genomic stability and has been identified as the gene responsible for progeria. Recent studies have shown reduced WRN expression due to aberrant DNA hypermethylation in cancer cells. Furthermore, WRN expression is thought to affect sensitivity to DNA topoisomerase I inhibitors in cancer therapy. In this study, we examined the relationship between aberrant DNA hypermethylation of WRN and the sensitivity of cervical cancer cells to anticancer drugs. DNA was extracted from samples from 22 patients with primary cervical cancer and 6 human cervical cancer-derived cell lines. Aberrant DNA hypermethylation was analyzed by methylation-specific PCR. WRN expression in cultured cells before and after addition of 5-aza-2-deoxycytidine, a demethylating agent, was examined using RT-PCR. The sensitivity of cells to anticancer drugs was determined using a collagen gel droplet embedded culture drug sensitivity test (CD-DST). siRNA against WRN was transfected into a cervical cancer-derived cell line with high WRN expression. Changes in drug sensitivity after silencing WRN were determined by CD-DST. Aberrant DNA hypermethylation and decreased expression of WRN were detected in 7/21 cases of primary cervical cancer and in two cervical cancer-derived cell lines. These two cell lines showed high sensitivity to CPT-11, a topoisomerase I inhibitor, but became resistant to CPT-11 after treatment with 5-aza-2-deoxycytidine. Transfection of siRNA against WRN increased the sensitivity of the cells to CPT-11. Aberrant DNA hypermethylation of WRN also increased the sensitivity of cervical cancer cells to CPT-11. Therefore, epigenetic inactivation of this gene may be a biomarker for selection of drugs for the treatment of cervical cancer. This is the first report to show a relationship between the methylation of the WRN gene and sensitivity to CPT-11 in gynecological cancers.

Abstract 1355: Utility of primary cultured cells in personalized chemotherapy for angiosarcoma of the oral cavity

Abstract Current treatments for major malignant tumors are based on evidences accumulated over the world and clinical trials with molecular target drugs that are expected to improve their prognosis are ongoing. In contrast, rare malignant tumors have no standard therapies and few evidences for effective drugs. Moreover, epidemiology, etiology, pathogenesis and molecular mechanisms are unknown because published data are based on a few case series and experimental materials can be used in vitro do not exist. Thus we have to manage a treatment case by case when facing a rare tumor. One of the useful methods to evaluate drug efficacy is the collagen gel droplet embedded culture drug sensitivity test (CD-DST), but a resected tumor tissue as an examination materials is limited. Primary cultured cells are thought to be an alternative material for various analyses. Angiosarcoma is a rare soft-tissue sarcoma of endothelial cell origin showing a poor prognosis. A 72-year-old man who has large masses on upper and lower gingiva came to our department and a palliative excision was performed. He also had multiple nodules in both lungs and cervical lymph nodes. Histopathological diagnosis was angiosarcoma because the immunohistochemical stainings showed that vimentin, CD31 and vW factor were positive. We attempted to culture the excised tumor specimen in vitro and obtained primary cultured cells with stable proliferation, and confirmed their in vivo tumorigenicity in athymic nude mice. First, both primary tumor tissues and cultured cells were assessed their sensitivity against 8 anti-tumor drugs by CD-DST. We evaluated their growth inhibitory effects as the ratio of total volume of colonies in the treated group to that in the untreated group (T/C%). The most effective drug, gemcitabine, had 20.9 T/C% in tumor tissues and 35.4 T/C% in cultured cells, and the second, docetaxel, had 32.2 T/C% and 40.2 T/C%, respectively. The other drugs showed the similar sensitivity between both types of samples. Next, we determined the gene expression profiles of both samples and a normal gingival tissue by microarray analysis. The data showed that 2,498 genes in primary tumor tissues and 2,893 genes in primary cultured cells had more than 5-fold change compared to control. Of these, 1,627 genes were common, and then we found the overexpression of 170 cancer-related genes including Aurora kinase A (AURKA) among these genes. Finally, we investigated the effect of a selective AURKA inhibitor MLN8237 on the growth of primary cultured angiosarcoma cells in vitro, and then observed the growth inhibitory effect of MLN8237 against these cells. These results suggest that primary cultured tumor cells may be useful for predicting the efficacies of various anti-tumor drugs and developing novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1355. doi:1538-7445.AM2012-1355

Differences in chemosensitivity between primary and paired metastatic lung cancer tissues: In vitro analysis based on the collagen gel droplet embedded culture drug test (CD-DST).

To elucidate the differences in chemosensitivity to anticancer drugs between primary and metastatic lesions in non-small cell lung cancer (NSCLC) patients, we examined the in vitro chemosensitivities of surgically resected NSCLC tissues. A total of 32 specimens were enrolled: 26 specimens of primary lesions paired with metastases in the lymph node, 3 specimens of primary lesions paired with metastases in the adrenal gland, and 3 specimens of primary lesions paired with metastases in the lung. The collagen gel droplet embedded culture drug test (CD-DST) was applied to examine the sensitivity of the tissues to anticancer drugs, including cisplatin, gemcitabine, vinorelbine, docetaxel and 5-fluorouracil. The degree of in vitro sensitivity to each anticancer drug varied between the primary and metastatic lesions. The sensitivity of the paired metastatic lesions was significantly lower than that of the primary lesions only for gemcitabine (P=0.029), vinorelbine (P=0.012), and docetaxel (P=0.009). The incidence of cases diagnosed as CD-DST-sensitive among the paired metastatic lesions was significantly lower than that for the primary lesions for vinorelbine (P=0.035) or docetaxel (P=0.022). The difference in the sensitivity to gemcitabine between the primary and paired non-lymphatic metastases was clearer than that between the primary lesion and paired lymph node metastases. The sensitivities of the paired metastatic lesions to some anticancer drugs were significantly lower than those of the primary lesions. When performing chemotherapy based on CD-DST data using primary tumors from patients with postoperative recurrence, an appropriate regimen can be selected by carefully considering these differences.

Synovial sarcoma individual chemotherapy directed by collagen gel droplet embedded culture drug sensitivity test: A case report

The collagen gel droplet embedded culture-drug sensitivity test (CD-DST) is an anticancer drug sensitivity test developed about 10 years ago. This study reports the application of this test in the choice of neoadjuvant chemotherapy for the treatment of one patient with a large synovial sarcoma in the right shank. A 28-year old man presented at our hospital with a large mass in his right shank which had a hard texture and an obscure boundary. The histopathological diagnosis of excisional biopsy specimens was synovial sarcoma with low differentiation. Theprubicin/Cisplatin (THP/CDDP) neoadjuvant chemotherapy was selected based on the results obtained from the CD-DST. After three courses, a computed tomography (CT) scan was performed which indicated that the volume of the tumor had decreased significantly. Additionally, tumor necrosis, as well as the clinical response, showed complete response. The histopathological diagnosis of wide excision specimens indicated a grade III chemotherapy response. The patient was alive and without recurrence after a follow-up of 16 months. Our results indicated that the CD-DST is a useful tool for selecting neoadjuvant chemotherapeutic drugs for patients with synovial sarcoma.

Anticancer drug sensitivity testing using an oxygen electrode apparatus

Conventional anticancer drug sensitivity testing methods, such as succinate dehydrogenase inhibition (SDI), histoculture drug-response assay (HDRA) and collagen gel droplet embedded culture drug sensitivity testing (CD-DST), all require primary culturing and are extremely complex tests that require considerable time for analysis. A major drawback of these methods is that if culturing is not performed properly, ambiguous results are produced. Therefore, we developed an oxygen electrode apparatus that uses cellular metabolism as an indicator of anticancer drug sensitivity and investigated its usefulness in 29 breast cancer patients with the following histopathological classifications: papillotubular carcinoma (n= 15); solid tubular carcinoma (n= 6); and scirrhous carcinoma (n= 8). Comparison of anticancer drug sensitivity testing results obtained using the conventional HDRA method and those obtained using the oxygen electrode apparatus showed significant reproducibility between the two methods. In addition, similar anticancer drug sensitivity testing results using the oxygen electrode apparatus were obtained for in vivo testing of nude mice transplanted with established cancer cell lines. These findings suggest that the oxygen electrode apparatus is a useful procedure in anticancer drug sensitivity testing that provides better reproducibility and that is faster, more convenient, and less expensive than other testing methods.

Combination of p53 codon 72 polymorphism and inactive p53 mutation predicts chemosensitivity to 5‐fluorouracil in colorectal cancer

There are increasing reports showing the clinical significance of the p53 polymorphism status in terms of the response to chemotherapy. We investigated whether p53 polymorphism and mutation were associated with in vitro sensitivity to 5-fluorouracil (5-FU) in patients with colorectal cancer. Chemosensitivity to 5-FU was evaluated by the collagen gel droplet embedded culture drug sensitivity test. 5-FU sensitivity of tumor cells without inactive p53 mutation in the arginine/arginine (Arg/Arg) variant was significantly higher than that of tumor cells with or without inactive p53 mutation in other variants (p = 0.022), whereas the 5-FU sensitivity of tumor cells with inactive p53 mutation in the Arg/Arg variant was significantly lower than that of tumor cells with or without inactive p53 mutation in other variants (p = 0.002). In the Arg/Arg variant, apoptotic cells induced by 5-FU treatment in patients without inactive p53 mutation were more markedly increased than those in patients with inactive p53 mutation (p = 0.037). Bax and Bcl-2 protein expressions in tumor tissue treated with 5-FU were associated with both 5-FU sensitivity and the apoptotic cell count. Our data show that the Arg/Arg genotype without inactive p53 mutation could be predictive of a more favorable response and the Arg/Arg genotype with inactive p53 mutation a less favorable response to chemotherapy using 5-FU in CRC. The combination of the p53 codon 72 polymorphism and p53 mutation status is a potential predictive marker of sensitivity to 5-FU in CRC.

Prediction of chemotherapeutic effect on postoperative recurrence by in vitro anticancer drug sensitivity testing in non-small cell lung cancer patients

Background and aims The collagen gel droplet embedded culture drug test (CD-DST), is an in vitro anticancer drug sensitivity test. The test has been used with various types of malignant tumors, but the significance of clinical application remains unknown. The aim of the present study is to evaluate the ability of this test to predict the response to chemotherapy in non-small cell lung cancer (NSCLC) patients. Methods From January 2000 through March 2007, CD-DST data using the primary tumor specimens to anticancer drugs such as cisplatin (CDDP), carboplatin (CBDCA), paclitaxel (PAC), docetaxel (TXT), gemcitabine (GEM), and vinorelbine (VNR), was successfully obtained from 382 patients that underwent a radical resection for NSCLC. Eighty-one of those patients received 1st line chemotherapy using a “new generation” of anticancer drugs for postoperative recurrence. The chemotherapy regimen consisted of a CDDP (or CBDCA)-based combination ( N = 41), non-CDDP-based combination ( N = 1) and single agent ( N = 39). The predictability of the chemotherapeutic effect by the CD-DST data was analyzed retrospectively. Results Partial response (PR) was obtained in 24 patients (response rate = 30%), stable disease (SD) in 33 (41%) and progressive disease (PD) in 24 (30%). Forty-two patients underwent chemotherapy with one or more CD-DST-sensitive drugs, 21 of whom showed PR (RR = 50%), whereas only 3 (8%) patients showed PR with chemotherapy with regimen including no CD-DST-sensitive drugs. Good predictability was obtained, with a 50% positive predictive value (PPV) for PR and a 92% negative predictive value (NPV) by CD-DST. The predictive accuracy for the response based on the CD-DST data was 70%. Interestingly, a subset analysis according to recurrence site showed that the predictive accuracy was highest (86%) for CD-DST-based chemotherapy for recurrence in the lymph nodes. Conclusions The application of the CD-DST for “new generation” anticancer drugs using surgically resected specimens of primary lesion in NSCLC patients may be clinically useful in the prediction of the response to chemotherapy for postoperative recurrence. CD-DST-oriented chemotherapy for postoperative recurrence especially in the lymph nodes may therefore be promising for the improvement of the treatment outcome.

Evaluation of the individual 50% inhibitory area under the concentration curve of 5-fluorouracil based on the collagen gel droplet embedded culture drug sensitivity test in colorectal cancer

We have previously reported the 5-fluorouracil (5-FU) sensitivity of cancer cells from colorectal cancer (CRC) patients using the collagen gel droplet embedded culture-drug sensitivity test (CD-DST) under multiple drug concentrations and contact durations. Moreover, the area under the concentration curve (AUC) and growth inhibition rate (IR) were combined, resulting in the AUC-IR curve, which was approximated to the logarithmic curve. In the present study, we used the AUC-IR curve to calculate the individualized AUCIR50, the AUC value that imparts 50% growth inhibition. Individual AUCIR50 was calculated in CRC patients, and its distribution was evaluated. The cumulative distribution of individual AUCIR50 was regressed over two lines (logarithmic scale). Among the 45 resectable CRC patients, those who achieved more than the individual AUCIR50 during post-operative 5-FU-based chemotherapy demonstrated a trend towards better disease-free survival compared to those who did not achieve AUCIR50. Of the Dukes' D patients (n=10), those who achieved more than twice the individual AUCIR50 during post-operative 5-FU-based chemotherapy demonstrated significantly better survival rates (p=0.05) than those who did not. In this study, the distribution of the individual AUCIR50 suggested that approximately 6% of patients demonstrated very low 5-FU sensitivity. Therefore, the individual AUCIR50 was useful in classifying good, intermediate and poor 5-FU response. Achievement of the individual AUCIR50 may be a prerequisite for individualized 5-FU-based adjuvant chemotherapy. As well, the early achievement of twice the individual AUCIR50 may indicate an improved prognosis in Dukes' D patients. The individual AUCIR50 using CD-DST is useful in determining the individualized chemotherapy of CRC patients, thus CD-DST has the potential to facilitate the establishment of individualized chemotherapy for CRC.

Expression level of thymidylate synthase mRNA reflects 5-fluorouracil sensitivity with low dose and long duration in primary colorectal cancer

To investigate the prognostic marker for the adjuvant chemotherapy of primary colorectal carcinoma. Primary colorectal cancer tissue from 24 patients was investigated to evaluate the relationship between the mRNA expression level of several 5-fluorouracil (5-FU)-related metabolic enzymes (thymidylate synthase, TS; dihydropyrimidine dehydrogenase, DPD; and thymidine phosphorylase, TP) and chemosensitivity to two different 5-FU doses and duration (1: 5-FU concentration 1.0 microg/mL (7.68 microM), 24 h exposure and 2: 5-FU concentration 0.3 microg/mL (2.30 microM), 144 h exposure). Chemosensitivity and mRNA expression levels were measured using collagen gel droplet embedded culture drug sensitivity tests and real-time quantitative reverse transcription-polymerase chain reaction. Clinicopathological features and chemosensitivity were also compared. The TS mRNA expression level was significantly higher in the 5-FU resistant group (T/C > 60%) compared with the 5-FU sensitive group (T/C < 60%) in both 5-FU regimens (1: 5.03 +/- 0.92 vs. 1.58 +/- 0.76, p < 0.01, 2: 4.88 +/- 0.91 vs. 0.96 +/- 0.20, p < 0.001). The group with the higher TS mRNA expression level (>3.83, the average) were more resistant to both 5-FU regimens than those with lower TS mRNA (<3.83) (1: T/C = 80 vs. 66%, p = 0.11, 2: T/C = 89 vs. 64%, p < 0.005). The TS mRNA expression level inversely correlated with the sensitivity to the latter 5-FU regimen (R = 0.577, p < 0.01). There were no relationships between chemosensitivity to 5-FU and the mRNA expression level of DPD and TP and clinicopathological factors. The TS mRNA expression level might be a good marker of chemosensitivity to 5-FU in primary colorectal cancer, especially the sensitivity to low dose 5-FU with a long duration.

Clinical evaluation of chemosensitivity testing for patients with unresectable non-small cell lung cancer (NSCLC) using collagen gel droplet embedded culture drug sensitivity test (CD-DST)

In the present study, we prospectively evaluated the clinical feasibility and efficacy of collagen gel droplet embedded culture drug sensitivity test (CD-DST) in unresectable non-small cell lung cancer (NSCLC) without previous treatment. Eighty patients with unresectable NSCLC, aged less than 81 years old, PS 0-1, and with evaluable tumor lesions, entered the study. If the patient had CD-DST active drugs, more than three cycles of chemotherapy containing these drugs were administered. If the patient did not have CD-DST active drugs, the patient could choose any treatment including best supportive care. Of the 80 patients in this study, CD-DST yielded results successfully in 49 patients (61.3%). CD-DST active drugs were present in 22 patients, and significantly more female patients had in vitro active anticancer agents than male (P=0.0008). All of the patients with CD-DST active agents received chemotherapy including these agents. In these patients, the response rate was 72.7%, and median survival was 15.0 months. In the patients without CD-DST active agents, 11 patients received standard, empirical chemotherapy. In these patients, response rate was 0%, and median survival was 6.0 months. The results show that CD-DST is capable of selecting the responders and the respective optimal regimens, and also delineating the patients less likely benefit from treatment.

Bcl-2 overexpression enhances in vitro sensitivity against docetaxel in non-small cell lung cancer.

Taxanes (docetaxel and paclitaxel) as well as cisplatin (CDDP) are key chemotherapeutic agents in the treatment of non-small cell lung cancer (NSCLC). Although some indicators of taxane resistance, such as beta-tubulin mutations, P-glycoprotein (P-gp) and Bcl-2, have been reported in malignant cells, the mechanisms of taxane resistance in NSCLCs have yet to be fully elucidated. We evaluated in vitro chemosensitivity to docetaxel (DOC) and CDDP in 87 surgically-resected specimens of NSCLC by collagen gel-droplet embedded culture drug sensitivity test (CD-DST). Bcl-2 and P-gp expression in these specimens were also investigated by immunohistochemistry. We examined the association between Bcl-2 and P-gp expression and in vitro chemosensitivity to DOC and CDDP. Out of the 87 NSCLCs that were examined, Bcl-2 and P-gp were expressed in 32 (36.8%) and 28 (32.2%) of the tumors, respectively. Positive Bcl-2 expression was significantly associated with enhanced DOC sensitivity in NSCLCs (p=0.007) while no apparent association was observed between DOC sensitivity and P-gp expression. Interestingly, although DOC, but not CDDP has been reported to be a substrate of P-gp, P-gp expression was significantly inversely correlated with CDDP sensitivity in pulmonary adenocarcinomas (p=0.03). Positive Bcl-2 expression may be a promising indicator in determining in vitro taxane sensitivity in NSCLCs. On the other hand, positive P-gp expression may be an indicator of enhanced in vitro resistance to CDDP in pulmonary adenocarcinomas.

Usefulness of collagen gel droplet embedded culture drug sensitivity testing in ovarian cancer

Due to the emergence of new anticancer agents for the treatment of ovarian cancer, methods to determine which agents will be most effective in individual patients are required. In order to investigate the potential for tailor-made chemotherapy, the drug sensitivities of various ovarian cancers were examined using collagen gel droplet embedded culture drug sensitivity testing (CD-DST), and the results were correlated with clinical outcomes. Sensitivities to paclitaxel, cisplatin, doxorubicin, etoposide, and SN-38, which is an active metabolite of irinotecan, were examined. Eight out of 22 samples failed to grow colonies and thus, their cell sensitivities could not be determined. Out of the 14 cases from which CD-DST results were obtained, seven patients then received chemotherapy aimed at inducing remission, while four received adjuvant, and three did not receive any chemotherapy. Three of the four tumors subsequently treated with adjuvant chemotherapy showed sensitivity to TXL and CDDP on CD-DST analysis, while one did not. None of these patients experienced recurrent disease from 24 to 36 months. Five of the seven tumors subsequently treated with chemotherapy aimed at inducing remission showed sensitivity to the relevant anticancer agents upon CD-DST analysis, while two did not. Among the five cases that showed tumor cell sensitivity, three experienced complete responses, one achieved a partial response and one had progressive disease. For the remaining two cases that demonstrated tumor cell resistance, one had stable disease and one had progressive disease following chemotherapy. Thus, six out of the seven cases (85.7%) that received chemotherapy aimed at inducing remission had clinical outcomes in keeping with the results of CD-DST. In conclusion, CD-DST results reflect clinical outcomes and may be a useful means by which to select drugs to which ovarian cancer cells are chemosensitive.