Predictive value of MTT assay as an in vitro chemosensitivity testing for gastric cancer:One institution's experience

AIM:To investigate the predictive clinical value of in vitro 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay for directing chemosensitivity in patients with gastric cancer. METHODS:Results of a total of 353 consecutive patients with gastric cancer treated with MTT-directed chemotherapy or physician’s empirical chemotherapy from July 1997 to April 2003 were reviewed and analyzed retrospectively. RESULTS:The overall 5-year survival rate of MTT- sensitive group (MSG) and control group (CG) was 47.5% and 45.1%, respectively. The results of subgroup analysis with Cox proportional-hazards model were favorable for the MSG-sensitive group. However, no statistically significant difference in survival rate was observed between the two groups. CONCLUSION:Individualized chemotherapy based on in vitro MTT assay is beneficial, but needs to be confirmed by further randomized controlled trials.

Impact of MTT based tumor chemosensitivity assay in vitro

Objective： The aim of the study was to evaluate the impact of the chemosensitivity assay in vitro and establish a standard process of measuring the anti-cancer drug sensitivity with MTT assay. Methods： Some influencing factors of MTT assay in studying the sensitivity of human peripheral blood mononuclear cells （PBMC） to anti-cancer drugs were observed, including red blood cells, platelets, three different kinds of DMSO and different concentrations of MTT. Meanwhile the stability of tumor drug-coated plate was monitored. Results： The red blood cells and platelets may affect the results at a certain range of concentration. Analytical pure DMSO, both imported and domestic reagents showed the same color with MI3-, and the A values of the reaction were dependent on MTT dose. The stability of the freeze-drying drug-coated plates was superior to non freeze-drying ones. Conclusion： To make clear and definite all kinds of influencing factors might contribute to a kind of standard MTT assay for drug sensitivity test in vitro.

Application value of ATP based bioluminescence tumor chemosensitivity assay in the chemotherapy for hydrothorax caused by non-small cell lung cancer

Objective： The aim of the study was to investigate the clinical value and application of ATP based bioluminescence tumor chemosensitivity assay （ATP-TCA） in the chemotherapy for hydrothorax caused by non-small cell lung cancer （NSCLC）. Methods： Hydrothorax specimens from 120 NSCLC patients were analyzed by ATP-TCA and the most sensitive chemotherapeutic drugs were used in NSCLC patients （treatment group）. At the same time, 56 NSCLC patients with hydrethorax were admitted in our Hospital （Department of Oncology, The No. 2 People＇s Hospital of Yibin, China） and given chemotherapy without guidance of the ATP-TCA （control group）. Before the third chemotherapeutic cycle, clinical outcomes were analyzed in the two groups. Results： Effective rate of hydrothorax in treatment group was 67%, while 46% in control group （P 〈 0.05）. In refractory hydrothorax patients, they were 69% and 40% （P 〈 0.05）, respectively.In vitro results correlated well with clinical outcomes （P 〈 0.01）. Conclusion： Effective rate of chemotherapy for hydrothorax in NSCLC is higher in treatment group than that in control group. ATP-TCA is especially helpful for refractory hydrothorax.

Comparative analysis of ATP-based tumor chemosensitivity assay-directed chemotherapy versus physician-decided chemotherapy in platinum-resistant recurrent ovarian cancer

Objective The aim of the study was to evaluate the role of ATP-based tumor chemosensitivity assay(ATP-TCA) in patients with platinum-resistant recurrent ovarian cancer(PRROC).Methods A total of 43 patients with PRROC who underwent chemotherapy based on the results of ATPTCA in the Cancer Hospital,Chinese Academy of Medical Sciences were included in the present study.As controls,we selected another 43 patients with PRROC who were treated at the physician's discretion within the same time period and had the same clinical characteristics as the patients in the ATP-TCA group.Logrank test and Cox proportional hazards model were adopted for analysis.Results A total of 86 patients were retrospectively analyzed in the present study.Patients were routinely monitored to evaluate the rate of progression-free survival(PFS).The median follow-up time was 13 months.The PFS for the ATP-TCA and control groups was 5 and 3 months,respectively(P = 0.027).Multivariate analysis showed that the type of treatment was an independent prognostic factor for PFS(P = 0.040;HR:0.623;95% CI:0.313–0.973).Subgroup analysis showed that among patients with a treatmentfree interval(TFI) of ≥ 3 months(n = 50),those in the ATP-TCA group had longer PFS than those in the control group(7 vs 4 months,P = 0.010).Meanwhile,the median PFS of patients who underwent ≤ 2 prior chemotherapy regimens(PCR,n = 52) in the ATP-TCA and control groups was 6 months and 4 months,respectively(P = 0.025).Conclusion ATP-TCA-directed chemotherapy might improve the PFS in PRROC.In particular,the survival benefit from ATP-TCA is higher in patients with a TFI of ≥ 3 months or treated with ≤ 2 PCR.

CUMULATIVE RESULTS OF CHEMOSENSITIVITY TEST USING MTT ASSAY IN DOUBLE-LAYER AGAROSE

Objective To investigate cumulative results of chemosensitivity test using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl （MTT） assay in double-layer agarose. Methods A total of 2 491 patients with different kinds of cancers were enrolled in the study, in which 18 kinds of different anticancer drugs were used. A computer soft was used to get charts. ResultsThe total evaluability rate was 82.7% （2 060/2 491）. Among all agents, the efficiency rates of 5-Fu, MMC, DDP, BLM and CBP were higher than the efficiency rates of others. The response rate range of different cancer in vitro sensitivity by using MTT assay in double layer agarose were from 9.2% （biliary duct） to 37.5% （malignant lymphoma）. For colon and rectum cancer, 5-Fu, DDP, MMC and BLM were more sensitive than other anti-tumor agents. For breast carcinomas, ACTD and DDP were more sensitive. For gastric cancer, 5-Fu, DDP and BLM were more sensitive. For leukemia, VM-26 and HHRT were more sensitive. ACM was more sensitive to kidney and MXT and BLM were more sensitive to pancreas cancer. For Lung cancer, DDP and EPI were more sensitive. Mean true positive rate, mean true negative rate, mean sensitivity, mean specificity and mean accuracy were 44% , 92% , 72% , 77% , and 76% , respectively. Conclusion Chemosensitivity tesing using the MTT assay in a double layer agarose was a very useful reference to chem- therapy.

The feasibility of antitumor drugs chemosensitivity testing by flow cytometry

Objective: To investigate the feasibility of chemosensitivity testing of antitumor drugs by flow cytometry in clinical applications so as to provide experimental and theoretical basis for the establishment of a novel antitumor drugs sensitivity testing and the screening of particular antitumor drugs. Methods: Detect the apoptosis rate of 12 cases of Molt-4 cell line, 57 cases of fresh clinical gastrointestinal tumor cells by Sub-G1 and Annexin V assay of flow cytometry under the effects of antitumor drugs at different times and the outcomes were compared with the ones of the MTT (3-(4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) assay. Results: The lethality of drugs on Molt-4 cell, clinical gastrointestinal tumor cells had a positive correlation with the acting time of antidrugs by employing Annexin V, Sub-G1 and MTT assay. Drug-incurring maximum lethality of Annexin V assay was higher than MTT colorimetric assay, that of Sub-G1 was lower than MTT assay, the virtual times of Annexin V and Sub-G1 assay were obviously earlier than that of MTT colorimetric assay. Conclusion: Annexin V and Sub-G1 assay of flow cytometry can be taken as potent protocols testing anti-tumor drug chemosensitivity. Annexin V assay is featured by more sensitive, concise, reliable compared with the classical chemosensitivity testing assay of MTT colorimetric assay and it possesses clinical applied value.

Cell Survival Assays for Individualised Chemotherapy in Primary Glioma Cultures—Colourmetric or Luminescent?

In vitro chemosensitivity testing of short term primary glioma cultures derived from brain biopsies is still in the research phase and has not yet found a place in clinical use. The main reasons for this slow progression are the small amounts of tissue available and the lack of a suitably sensitive assay capable of use in the clinical setting. This study examines whether the MTS and ATP cell survival assays, which determine cytotoxicity via colorimetric and luminescence analysis respectively, could potentially fulfill this role. Primary glioma cultures were tested for chemosensitivity using the MTS and ATP assays and were found to be generally sensitive to cisplatin and paclitaxel but relatively resistant to carmustine and etoposide. For both assays, LD50 values lay in the range 2 - 130 μg/ml but in the vast majority of cases, those obtained by the ATP assay were markedly lower those obtained by the MTS assay. Moreover, at cell numbers less than 2000 in the cases of paclitaxel and carmustine and less than 4500 in the case of cisplatin, these drugs were generally indicated as ineffective against the glioma cultures tested by the MTS assay but effective against these cultures by the ATP assay. These data clearly demonstrate that the ATP assay is more sensitive when estimating small cell numbers generated by primary glioma cultures from brain biopsies and more reliably detects higher kill rates by anticancer drugs. This study also supports the feasibility of using the ATP assay for chemosensitivity testing in a clinical setting.

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

AIM:To determine whether expression of certain enzymes related to 5-fluorouracil(5-FU)metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma(CCA).METHODS:The histoculture drug response assay(HDRA)was performed using surgically resected CCA tissues.Tumor cell viability was determined morphologically with hematoxylin and eosin-and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues.The mRNA expression of thymidine phosphorylase(TP),orotate phosphoribosyl transferase(OPRT),thymidylate synthase(TS),and dihydropyrimidine dehydrogenase(DPD)was determined with realtime reverse transcriptase-polymerase chain reaction.The levels of gene expression and the sensitivity to 5-FU were evaluated.RESULTS:Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital,Khon Kaen University from 2007 to 2009.HDRA was used to determine the response of these CCA tissues to 5-FU.Based on the dose-response curve,200μg/mL 5-FU was selected as the test concentration.The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU.When the relationship between TP,OPRT,TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined,only OPRT mRNA expression was significantly correlated with the response to 5-FU.The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group(0.41±0.25 vs 0.22±0.12,P<0.05).CONCLUSION:OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA.Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.