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

Abstract

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.

Key Words: Gastric cancer, Chemosensitivity testing, Chemotherapy, MTT assay, Survival rate

INTRODUCTION

Gastric cancer ranks second of all cancers and is the leading cause of cancer-related deaths worldwide[12]. The incidence of gastric cancer worldwide is reported to be especially high in Asia, South America, and Eastern Europe[2–5]. Gastric cancer patients are treated in clinical practice with various therapies, such as chemotherapy and radiation, though further improvement and progress would be required. With the development of new anti-cancer drugs, such as taxanes, CPT-11, oxaliplatin, gefitinib and S-1, significant improvements in the efficacy of chemotherapies against gastric cancer have been achieved[46–8]. However, some patients still fail to respond to chemotherapy and finally die of the critical toxicity of intensive chemotherapy[9]. Thus, new therapies and technologies are desperately needed for the treatment of gastric cancer. Advances in this area would have a major impact on the outcome of a large number of patients with this disease. Hence, chemosensitivity assay has been developed to individualize chemotherapy for gastric cancer patients[10]. 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay is a rapid and quantitative colorimetric method for determination of cell viability by measuring the anticancer drug effectiveness on human tumor cells. Several studies on advanced gastric cancer using this approach revealed that in vitro sensitivities are associated with in vivo tumor responses[11–15]. However, most of these studies were small-scale trials (< 100 patients).

In this study, MTT assay was used to predict the efficacy of individualized assay-directed chemotherapy for Chinese gastric cancer patients, and to prove whether in vitro chemosensitivities are associated with in vivo tumor responses by survival analysis.

MATERIALS AND METHODS

Patients

This was a retrospective study. The medical records of patients registered for adjuvant chemotherapy from July 1997 to April 2003 were reviewed. The criteria for case inclusion were as follows: (1) a diagnosis of histologically or cytologically proven gastric cancer, (2) without prior chemotherapy or radiotherapy, (3) adequate blood counts (hemoglobin ≥ 10 g/L, WBC count of 3000/&mgr;L, and platelets of 100 000/&mgr;L), normal renal function (creatinine clearance ≥ 60 mL/min), and normal liver function (serum transaminase level less than double the normal upper limit). Patients with esophageal cancer, small cell carcinoma, lymphoma, and squamous cell carcinoma were excluded from the study. A total of 353 eligible records of patients were collected and analyzed. The patients were divided into MTT-sensitive group (MSG) and control group (CG). One hundred and fifty-seven patients in the MSG-sensitive group were treated by chemotherapy containing at least one sensitive drug based on the MTT assay results, and 196 patients received physician’s empirical chemotherapy. The chemotherapeutic drugs used were cisplatin (CDDP), 5-fluorouracil (5-Fu), mitomycin (MMC), doxorubicin (DOX), paclitaxel (PAC) and docetaxel (DOC). The protocols of chemotherapy have been described elsewhere[61617].

MTT assay

Fresh tumor tissue obtained from the surgically resected specimens was tested within 6 h. The tumor tissue was cut into pieces (smaller than 1 mm³) and passed through No. 100 and No. 200 stainless steel meshes respectively into a complete medium containing RPMI 1640 solution, 100 &mgr;g/mL penicillin, and 100 &mgr;g/mL streptomycin, and washed twice gently with the same solution. The viable cells were assessed using trypan blue exclusion method. Cell viability was measured by MTT assay to assess the chemosensitivity of tumor cells. Cell suspension was collected into sterile 96-well flat-bottomed microtiter plates (1 × 10⁵ cells/per well) with or without chemotherapeutic agents. The drug and testing drug concentrations used were 25 &mgr;g/mL cisplatin (CDDP), 100 &mgr;g/mL -fluorouracil (5-Fu), 10 &mgr;g/mL mitomycin (MMC), 4 &mgr;g/mL doxorubicin (DOX), 100 &mgr;g/mL paclitaxel (PAC) and 30 &mgr;g/mL docetaxel (DOC). Each drug was tested in triplicate. The plates were then incubated at 37°C in a humidified atmosphere containing 50 mL/L CO₂ for 72 h. Microtiter wells containing tumor cells but no anticancer agents were used to control cell viability, in which the total number of tumor cells was equivalent to that in the test wells, and wells containing only a complete medium were used as blank controls for nonspecific dye reduction. After incubation, MTT solution was added to each well at a final concentration of 1 mg/mL per well and the plates were incubated at 37°C for another 4 h. Then the mixture containing the medium, the drug, and the unconverted MTT was removed. DMSO was added to each well to dissolve the formazan and absorbance was read at 550 nm using a spectrophotometric microplate reader (Labsystems, Finland). The inhibition rate of tumor cells for each drug with different concentrations was calculated following the formula: inhibition rate (%) = (1 - OD_{drug exposure}/OD_control) × 100. The effective anticancer activity was regarded as sensitive when the tumor inhibitory rate was greater than or equal to 70%.

Toxicity

All patients who started treatment were considered assessable for toxicity. Toxicity was analyzed following the National Cancer Institute Common Toxicity Criteria (version 2.0).

Statistical analysis

All statistical analyses were done using the SAS 6.12 statistical software (SAS Institute, Cary, NC). The clinical and pathological characteristics, including gender, age, cancer stage (TNM), and histological type (differentiated versus undifferentiated type), were evaluated by Mann-Whitney’s U-test and the Kruskal-Wallis test. The overall probability was calculated using the Kaplan-Meier method for censored failure time data, and the statistical significance was analyzed by the log-rank test for comparison of survival rate between the two groups. The Cox proportional-hazards model was used to calculate the hazard ratios. P < 0.05 was considered statistically significant. All P values were two-tailed and unadjusted for potential multiple comparisons.

RESULTS

Patient characteristics

The clinical and pathological characteristics of the patients are outlined in Table 1. Between the MSG and CG arms, there was no significant difference in baseline clinical characteristics and pathological findings which were considered to be related to the prognosis of gastric cancer patients.

Table 1 Baseline clinical and pathological characteristics of the patients n (%).

Characteristic	MSG (n = 157)	CG (n = 196)
Gender
Male	104 (66.2)	137 (69.9)
Female	53 (33.8)	59 (30.1)
Age
< 60 yr	87 (55.4)	110 (56.1)
60-69 yr	54 (34.4)	67 (34.2)
70-80 yr	16 (10.2)	19 (9.7)
Median year	58	62
Range year	29-80	31-78
Cancer stage, TNM classification
IB	3 (1.9)	4 (2.0)
II	85 (54.1)	103 (52.6)
III	54 (34.4)	71 (36.2)
IV	15 (9.6)	18 (9.2)
Histologic type
Differentiated	73 (46.5)	103 (52.6)
Undifferentiated	84 (53.5)	93 (47.4)

Overall survival analysis

The overall 5-year survival rate of the patients was 47.5% and 45.1% in the MSG-sensitive group and CG group, respectively, with no statistical difference (Figure 1). The hazard ratio for deaths in the MSG-sensitive group, as compared with the CG group, was 0.92 [95% confidence interval (CI) = 0.69 to 1.23, P = 0.57].

Figure 1 Kaplan-Meier curves of overall survival probabilities for gastric cancer patients, there was no significant difference between the two groups.

Subgroup analysis

The overall survival rate of the patients was analyzed according to sex, age, cancer stage (TNM classification), and histologic type. The hazard ratio of deaths was favorable for the MSG-sensitive group (Figure 2). There were no significant interactions between the two groups and any of the variables studied.

Figure 2 Hazard ratios of deaths for the baseline characteristics among gastric cancer patients. NS: No significant difference.

Adverse events and treatment compliance

Data on the 157 patients in the MSG-sensitive group and 196 patients in the CG group were analyzed for adverse events. The main emergent adverse toxicities (grades 3 and 4) related to treatment are listed in Table 2. The severe adverse events (defined according to NCI-CTC version 2.0), including hematologic and nonhematologic toxic effects, did not occur more frequently in the MSG-sensitive group than in the CG group.

Table 2 Severe adverse effects and toxicities (NCI-CTC version 2.0).

Toxicities	MSG (n = 157)			CG (n = 196)
	Grade III (No.)	Grade IV (No.)	Grade III/IV (%)	Grade III (No.)	Grade IV (No.)	Grade III/IV (%)
Hematologic toxicity
Leukopenia	2	2	2.5	4	1	2.6
Anemia	2	0	1.3	2	2	2.0
Thrombocytopenia	0	0	0.0	1	0	0.5
Non-hematologic toxicity
Diarrhea	2	0	1.3	3	0	1.5
Stomatitis	1	0	0.6	0	0	0.0
Nausea	2	1	2.5	2	0	1.0
Vomiting	2	0	1.3	3	0	1.5
Anorexia	8	2	6.4	6	3	4.6
Fever	0	0	0.0	2	0	1.0
Rash	1	0	0.6	1	0	0.5
Elevated aminotransferase	2	0	1.3	3	1	2.0
Hyperbilirubinemia	1	0	0.6	2	0	1.0
Elevated creatinine	0	0	0.0	1	0	0.5

DISCUSSION

Conventional chemo-/radio-therapy for gastric cancer is limited to improve the treatment outcomes and quality of survival/life of human patients[1819]. Physicians’ empirical choice of chemotherapeutic regimen for gastric cancer is based on the data obtained from clinical trials[20]. However, even the same gastric cancer behaves so differently that the response rate of cancers to the chemotherapeutic agents varies. These variations are partly contributed to the failure of treatment of gastric cancer patients. The effectiveness of current chemotherapies for cancer is limited mainly due to its heterogeneity[21]. To overcome this problem, selecting a sensitive chemotherapeutic regimen in vitro for individual gastric cancer patients appears to be an attractive way. Chemosensitivity testing is an ex vivo means of determining the cytotoxic and/or cytostatic, or apoptosis-inducing effect of anticancer drugs. The most common in vitro assays include MTT and ATP-TCA assays, etc[22]. These assays have been successfully used in the assay-guided chemotherapy for certain cancers, including breast, ovarian, melanoma and colorectal cancers[23–25]. MTT assay has been most widely used in different cancers, and is sensitive, accurate, and efficient in the in vitro evaluation of anticancer or immunological agents prior to preclinical and clinical testing. Some research groups have used MTT assay to guide individual adjuvant chemotherapy for gastric cancer[10], showing that the therapy based on the chemosensitivity testing can improve the clinical outcomes of cancer patients. In the present study, based on the criteria for chemosensitivity in vitro, we predicted and evaluated the efficacy of chemotherapy for 353 gastric cancer patients according to the result of MTT assay. The overall survival rate of the patients, treated with chemotherapy regimen containing at least one sensitive agent, was higher in the MSG-sensitive group than in the CG group treated with the physicians’ empirical therapy. The hazard ratio of most subgroups was favorable for the MSG-sensitive group as demonstrated in Cox proportional-hazards mode. However, no significant difference between the two groups was observed. These results indicate that MTT assay can lead to additional clinical outcomes as compared with physicians’ empirical therapy. The real benefit of MTT assay for chemosensitivity testing is to predict which agent is useful or not useful. Although the results of this retrospective study are not all concordant with previous studies[11122226], and do not definitively support the clinical values of MTT assay in detecting chemosensitivity to the adjuvant chemotherapy for patients with gastric cancer, they support chemosensitivity testing in patients with gastric cancer. Since the frequency of toxicity in patients is not reported in previous studies[111226], we compared the frequency of severe toxicity of grades 3 and 4 between the two groups, showing that chemotherapy regimen based on MTT assay could not reduce its the adverse effect and toxicity.

At present, although some studies have shown a potential clinical benefit of chemotherapy for patients with drug-sensitive cancer[2227], chemosensitivity testing has not been widely accepted by physicians. Meanwhile, prediction of chemosensitivity in clinical practice is a challenge because in vitro chemosensitivity testing systems have not considered the pharmacokinetic and pharmacodynamic variables affecting drug action in vivo. Because of varied pharmacogenetic make-ups of cancer patients, leading to interpatient variations in drug half-life, volume of distribution, types of metabolites formed, and route of elimination, dependence on in vitro and in vivo results is often not a straightforward process[28]. Ultimately, individualized chemotherapy based on cellular and genetic characteristics of cancer patients may be on the horizon[29–31]. The potential clinical benefits of individualized chemotherapy based on chemosensitivity testing need to be confirmed by further randomized controlled trials in comparison with standard chemotherapy.

COMMENTS

Background

Since cancer patients with histologically similar tumors respond differently to standard drug treatment, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) chemosensitivity assay is performed to provide predictive information to help physicians choose sensitive chemotherapeutic agents for eliminating potentially ineffective agents used in chemotherapeutic regimens for each cancer patients.

Research frontiers

At present, several new chemosensitivity assays, such as histoculture drug response assay (HDRA), collagen gel-droplet-embedded culture drug sensitivity test (CD-DST) and fluorometric microculture cytotoxicity assay (FMCA), are used in selection of an appropriate chemotherapeutic drug, showing the predictive value of chemotherapy for cancer patients.

Innovations and breakthroughs

There is no evidence for the clinical benefits of MTT chemosensitivity assay. The present study evaluated the clinical usefulness of MTT chemosensitivity assay in gastric cancer patients, and showed no significant differences in clinical outcomes between the MTT-sensitive group (MSG) and the control group (CG), indicating that the potential value of MTT assay for patients with gastric cancer is limited.

Applications

Although some studies have shown a potential clinical benefit for patients with drug-sensitive cancer, MTT assay of chemosensitivity is not widely accepted by physicians because there is no sufficient evidence obtained in the clinical setting. The potential clinical benefits of individualized chemotherapy based on chemosensitivity assay for gastric cancer patients need to be confirmed by further randomized controlled trials in comparison with standard chemotherapy.

Terminology

MTT assay is a laboratory test and a standard colorimetric assay for measuring cellular proliferation. Yellow MTT is reduced to purple formazan in the mitochondria of living cells. A solution (usually dimethyl sulfoxide) is added to dissolve the insoluble purple formazan products into a colored solution. The absorbance of this colored solution can be quantified at a certain wavelength with a spectrophotometer.

Peer review

This is an interesting report on the predictive value of MTT assay as an in vitro chemosensitivity testing for gastric cancer patients. Individualized chemotherapy based on in vitro MTT assay has clinical benefit, but needs to be confirmed by further randomized controlled trials.