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.

Multicomponent Coculture System of Cancer Cells and Two Types of Stromal Cells for In Vitro Evaluation of Anticancer Drugs.

In vitro evaluation of anticancer drugs using cancer cells has long been performed for the development of novel drugs and the selection of effective drugs for different patients. Recent studies have suggested that tumor stromal cells affect the drug sensitivity of cancer cells; however, most conventional culture systems for drug evaluation lack stromal cells. In this study, we fabricated a multicomponent coculture system that takes account of cancer-stroma interactions for drug evaluation. In this system, small-cell and nonsmall-cell lung cancer cells embedded in collagen gel were cocultured with two types of stromal cells, including stromal fibroblasts and proinflammatory cytokine-secreting monocytes, thus recreating the in vivo cancer microenvironment. Cancer drug sensitivity was significantly altered by the presence of stromal cells. Fibroblasts induced resistance of cancer cells to anticancer drugs. Monocytes induced the upregulation of thymidine phosphorylase in cancer cells, promoting the conversion of an anticancer prodrug to a cytotoxic drug, and consequently enhanced the sensitivity of cancer cells to the anticancer prodrug. These results clearly show the importance of incorporating stromal cells into culture systems for drug evaluation. Our system will help to improve the accuracy of in vitro drug evaluation and provide useful information for the in vitro recreation of cancer microenvironments.

Stratified phase II trial to establish the usefulness of the collagen gel droplet embedded culture-drug sensitivity test (CD-DST) for advanced gastric cancer.

BACKGROUND: We conducted a multicenter phase II trial to assess the suitability of three types of chemotherapy (docetaxel plus S-1, irinotecan plus S-1, or S-1 alone) for patients with advanced gastric cancer by means of the collagen gel droplet embedded culture-drug sensitivity test (CD-DST). To our knowledge, this is the first multicenter clinical trial that has employed CD-DST to choose anticancer agents for the treatment of advanced gastric cancer. METHODS: Subjects (n = 64) were patients with advanced or recurrent gastric cancer. Patients were allocated to one of the treatment regimens on the basis of CD-DST results. Outcome of the patients was compared between the groups deemed chemosensitive or chemoresistant by the CD-DST. RESULTS: Thirty-three patients showed high sensitivity (T/C ratio <60 %) to at least one type of anticancer agent (sensitive group), and 31 showed low sensitivity (T/C ratio ≥60 %) to all agents (resistant group). Specifically, the 1-year survival rate was significantly higher in the sensitive group (78.5 %; 95 % CI, 67.2-94.7 %) than in the resistant group (54.7 %; 95 % CI, 38.7-74.3 %; P = 0.019), whereas time to progression (TTP) was significantly longer in the sensitive group (59.8 %; 95 % CI, 48.2-81.7 %) than in the resistant group (30.0 %; 95 % CI 13.6-46.4 %; P = 0.023). Median survival time was also significantly longer in the sensitive group (15.5 months; 95 % CI, 12.8-18.2) than in the resistant group (12.5 months; 95 % CI, 10.2-14.9; P = 0.038). CONCLUSIONS: CD-DST predicts the outcome of patients undergoing chemotherapy for advanced gastric cancer, presumably through evaluating chemosensitivity.

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).

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Expression of Stat3 and Notch1 is associated with cisplatin resistance in head and neck squamous cell carcinoma.

Cisplatin is the most important chemotherapeutic agent involved in treatment of head and neck squamous cell carcinoma (HNSCC), but cisplatin resistance in HNSCC is still a serious problem in clinic. The reasons why patients fail chemotherapy are unclear. We examined 25 HNSCC patients who were all tested for cisplatin sensitivity by CD-DST (collagen gel droplet embedded culture-drug sensitivity) method and expression of Stat3 and Notch1. We found that high expression levels of Stat3 and Notch1 were closely associated with cisplatin resistance respectively (P=0.014, P=0.000). In addition, cisplatin resistance of HNSCC was decreased after inhibition of Stat3 or Notch signaling in vitro. Our results provide first evidence that both high Stat3 and Notch1 expression are associated with cisplatin resistance in HNSCC patients, supporting the hypothesis that co-activation of Stat3 and Notch1 by their crosstalk induces the reprogrammed survival pathways in HNSCC responding to chemotherapy.

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 chemosensitivity of primary cultured malignant melanoma cells using the collagen gel droplet-embedded culture drug sensitivity test.

Malignant melanoma usually shows resistance to a standard chemotherapy regimen. A useful in vitro method to evaluate individual chemosensitivity is required to select effective anti-cancer drugs. This study aimed to establish in vitro tumor response testing for malignant melanoma. We determined the chemosensitivity of primary cultured melanoma cells using the collagen gel droplet-embedded culture drug sensitivity test (CD-DST). Nineteen tests were carried out for 15 cases of malignant melanoma. Primary cultured melanoma cells in collagen gel droplets were exposed to anti-cancer drugs, including cisplatin, adriamycin, dacarbazine, nimustine and vincristine. After a 7-day incubation in a serum-free medium, living melanoma cells in a collagen droplet were detected by image analysis after staining with Neutral red reagent. In vitro drug exposure conditions were determined to reproduce the value of the plasma area under the time-drug concentration curve in vivo. The rate of evaluation of the primary culture of melanoma cells was 78.9% (15/19 tests). The chemosensitivity of cisplatin, adriamycin, dacarbazine, nimustine and vincristine was 15, 62, 0, 0 and 62%, respectively. Dacarbazine was not suitable for CD-DST due to its prodrug characteristics. The CD-DST method was able to evaluate the chemosensitivity of malignant melanoma to anti-cancer drugs in vitro. This method can also be applied to estimate the efficacy of newly developed anti-cancer drugs in vitro.

In vitro-chemosensitivity test using the collagen gel droplet embedded culture drug test (CD-DST) for malignant pleural mesothelioma: possibility of clinical application.

PURPOSE: An in vitro-chemosensitivity test using the collagen gel droplet embedded culture drug test (CD-DST), established by Kobayashi et al. (Jpn J Cancer Res 2001; 92: 203-10), has been widely used on various tumors. This study retrospectively evaluated its possibility of clinical application to patients with malignant pleural mesothelioma (MPM). PATIENTS AND METHODS: CD-DST using 26 fresh specimens obtained by biopsy or surgery on MPM patients investigated in vitro responses to cisplatin (CDDP), carboplatin (CBDCA), doxorubicin (ADR), etoposide (VP-16), 5-fluoruracil (5-FU), gemcitabine (GEM), vinorelbine (VNR), irinotecan (SN-38), and docetaxel (TXT). Correlations between CD-DST data and clinical effects were then assessed for some MPM patients undergoing chemotherapy. RESULTS: The rate of in vitro sensitivity to each chemoagent (N=tested number) was 35% for CDDP (N=23), 14% for CBDCA (N=21), 7% for ADR (N=15), 15% for VP-16 (N=13), 0% for 5-FU (N=15), 45% for GEM (N=11), 25% for VNR (N=8), 40% for SN-38 (N=5), and 44% for TXT (N=9). No difference was observed between CD-DST data of each chemoagent and histological type. Of these MPM patients, 14 clinical effects on 13 patients who underwent chemotherapy for primary or recurrent disease were reviewed in comparison with CD-DST data of each chemoagent. Among 10 chemotherapies including in vitro-sensitive chemoagents, 3 led to partial response (PR), and 7 resulted in four stable diseases (SDs) and 3 to progressive diseases (PDs). In contrast, among 4 chemotherapies using in vitro-resistant chemoagents, SD and PD were observed in 1 and 3, respectively. In regard to the clinical response rate, CD-DST sensitivity, specificity, and accuracy in the 14 examined chemotherapies were respectively 100%, 36%, and 50%, and in regard to the disease control rate, they were 88%, 60%, and 71%. CD-DST data for the chemoagents were to a limited extent significantly correlated with the disease control status of chemotherapy (p=0.052). CONCLUSION: Although the number of tested MPM specimens was small, CD-DST data obtained by biopsy or surgical-fresh specimens of MPM marginally correlated to the disease control effect of chemotherapy for this disease. Therefore CD-DST may possibly be applied to selecting the chemotherapy regimen for MPM. To determine the possibility of a clinical application of this test to MPM, a prospective clinical study of a greater number of patients will be necessary.

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).

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Mucoepidermoid carcinoma of the thymus treated by multimodality therapy: a case report.

A case report of mucoepidermoid carcinoma of the thymus, aggressively treated by multimodality therapy including surgery, radiotherapy, chemothermotherapy, and systemic chemotherapy is presented. The patient, a 53-year-old man, underwent potentially complete resection for an anterior mediastinal tumor, histologically diagnosed as a mucoepidermoid carcinoma of the thymus with Masaoka stage II disease. However, because of local recurrences in the left chest wall and pleura, re-resection was twice performed 4 years and 5 months, and 5 years and 7 months after the initial surgery, in combination with intrathoracic chemothermotherapy and irradiation. Seven years and 1 month after the initial operation, in vitro chemosensitive test based-chemotherapy using vinorelbine for pleural disease was performed, resulting in maintenance of good quality of life (QOL) due to dramatic decrease in pleural effusion. He died of tumor progression, 7 years and 9 months after the initial treatment. Although the clinical aspects of thymic mucoepidermoid carcinoma are little known, it is assumed that such aggressive therapeutic multimodalities as repeated surgical resection, irradiation and chemothermotherapy, and chemotherapy based on in vitro chemosensitivity tests contributed to long-term survival for this unusual disease.

Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells.

Angiogenesis is a complex process involving an ECM and vascular endothelial cells (EC), and is regulated by various angiogenic factors including VEGF. The ability to form a capillary/tube-like network is a specialized function of EC. Therefore, in vitro angiogenesis was assessed by a capillary/tube-like network formation assay. There are three angiogenic parameters: capillary length, number of capillaries, and relative capillary area per field. We evaluated capillary length per field in the assay. VEGF promoted capillary/tube-like network formation of EC in a type I collagen gel matrix in vitro. Moreover, we demonstrated the involvement of ILK in a VEGF signaling pathway mediating capillary/tube-like network formation of EC using dominant-negative, kinase deficient ILK. This is a straightforward assay to monitor responses of human vascular endothelial cells.

Collagen gel droplet culture method to examine in vitro chemosensitivity.

For effective cancer chemotherapy, chemosensitivity testing of anticancer drugs should be performed using fresh surgical specimens obtained from the cancer. We have developed a new in vitro chemosensitivity test named the collagen gel droplet embedded culture drug sensitivity test (CD-DST). The CD-DST method consists of a collagen gel droplet embedded culture step, exposure and washout of anticancer drug, a serum-free culture step, and evaluation of anticancer effect by image analysis. This method has many advantages including a high success rate for primary culture, the need for only a small number of cells for the test, easy quantification of the anticancer effects without contamination with fibroblasts by using an image analysis system, a good correlation between in vitro and in vivo results, and simplicity and speed. The CD-DST method can be performed in the laboratory using a system kit Primaster.

Predicting the chemosensitivity of ovarian and uterine cancers with the collagen gel droplet culture drug-sensitivity test.

We investigated the utility of the collagen gel droplet culture drug-sensitivity test (CD-DST) for predicting the response of gynecological cancers to chemotherapy. Eighty-three cancer patients were enrolled in this study: 26 ovarian, 29 cervical and 31 endometrial cancers. The CD-DST was performed at various concentrations of drugs. We calculated the T/C ratio, where T is the total volume of the treated culture and C is the total volume of the control culture, and a T/C ratio of 50% or less was defined as sensitive in vitro. The efficacy rate (%) was defined as the number of cultures with a T/C ratio of 50% or less, divided by the total number of evaluable cultures. True-positive cases were defined as clinical responders (complete+partial responses) and true-negative cases were defined as clinical non-responders. The overall tumor evaluation rate was found to be 79.1%. The appropriate drug concentrations were selected as 1.0 microg/ml for cisplatin, 20.0 microg/ml for carboplatin, 1.0 microg/ml for paclitaxel and 0.1 microg/ml for docetaxel by the linear regression equations. The in vitro sensitivity for each drug showed a significant correlation with clinical response rates (r=0.592, p=0021). We therefore conclude that the CD-DST can be used to predict the response to anti-cancer drugs, and may also provide important information by contributing to the development of new chemotherapy regimens.

[In vitro chemosensitivity test for hepatocellular carcinoma using collagen-gel droplet embedded cultures].

This study evaluated an in vitro assay for chemosensitivity test using a collagen-gel droplet-embedded culture drug sensitivity test (CD-DST) for hepatocellular carcinoma (HCC). In 25 patients with HCC, in vitro chemosensitivity to 5-fluorouracil (5-FU), epirubicin (EPI), and cisplatin (CDDP) was examined by CD-DST, and 5-FU, EPI, and paclitaxel (PTX) were examined in 38 patients with breast cancer. Successful rates of chemosensitive evaluation by CD-DST were 64% for HCC and 79% for breast cancers. Although chemosensitivities of breast cancer were 5-FU 23.1%, EPI 83.3%, and PTX 67.7%, only one HCC sample was sensitive to EPI. Growth rates of HCC for 7 days of culture were significantly lower than those of breast cancers (1.04 vs 3.61). The culture methods for HCC in CD-DST should be improved to estimate accurate results.

Development of a new in vitro chemosensitivity test using collagen gel droplet embedded culture and image analysis for clinical usefulness.

The results of chemotherapy are not fully satisfactory in many cases, particularly solid cancers. Therefore, it seems useful if the effective anticancer drugs can be selected for each patient using the screening methods. In such a background, we developed a new anticancer-drug sensitivity testing method that overcame several defects in the existing method, which is the collagen gel droplet embedded culture drug sensitivity test (CD-DST) and satisfies the following requirements: a high success rate, ability to assay biopsy specimens, and quantification of the anticancer effects without contamination with fibroblasts. Under the biological exposure condition, the sensitivities of the cancers were similar to their clinical response rate and there was a statistically significant correlation between clinically reported response rates and the response rates obtained by CD-DST (P < 0.01). Furthermore, the true positive rate was 79.8%, and the true negative rate was 88.8%. Sensitivity and specificity were 88.2% and 80.6%, respectively, resulting in an overall predictive accuracy of 84.1% (154/183). The CD-DST not only shows high predictive accuracy for humans, but because of the high correlation between the results of in vitro and nude mice assays, if it also is practical as a pre-clinical screening, it can easily provide these predictions.

Prediction of chemotherapeutic response by collagen gel droplet embedded culture-drug sensitivity test in human breast cancers.

Collagen gel droplet embedded culture-drug sensitivity test (CD-DST) is the newly developed in vitro chemosensitivity test that has several advantages over the conventional ones. The aim of the present study is to examine the clinical usefulness of this test in the prediction of response to chemotherapy in breast cancer patients. Seventy patients with primary (n = 45) or locally recurrent (n = 25) breast cancers were recruited, and each patient underwent tumor biopsy before chemotherapy. The biopsy specimens were used for CD-DST and immunohistological examination of 6 biological markers (P-gp, erbB2, p53, BCL2, MIB1 and ER-alpha). As chemotherapy, cyclophosphamide 600 mg/m(2) plus epirubicin 60 mg/m(2) q3w (CE, n = 28) or docetaxel 60 mg/m(2) q3w (DOC, n = 42) was given. Interpretable results using the CD-DST assay were obtained from 84.3% (59/70) of tumor specimens studied. Of the 18 tumors diagnosed as CE sensitive by CD-DST, 15 (83.3%) exhibited a response to CE therapy and none of the 5 tumors diagnosed as CE resistant by CD-DST exhibited a response to CE therapy. Of the 14 tumors diagnosed as DOC sensitive by CD-DST, 13 (92.9%) exhibited a response to DOC therapy and only one of the 22 tumors diagnosed as DOC resistant by CD-DST exhibited a response to DOC therapy. P-gp expression was found to exhibit a significant (p < 0.05) association with the resistance to CE therapy but not to DOC therapy. Diagnostic accuracy (72.7%) achieved by P-gp was lower than that (87.0%) achieved by CD-DST in CE therapy. Expressions of other biological markers (erbB2, p53, BCL2, MIB1 and ER-alpha) were not significantly associated with response to CE or DOC therapy. These results demonstrate that CD-DST can predict the response to CE and DOC therapy with a high accuracy in breast cancer patients and seems to be superior to the conventional predictors.