Chemosensitivity testing predicts survival in ovarian cancer.

The aim of this study was to assess the use of the MTT assay for chemosensitity testing to identify drug resistance and predict survival in patients with advanced ovarian cancer. Samples of ascitic fluid and/or solid biopsies were taken from 120 patients with FIGO stage III or IV ovarian adenocarcinoma at presentation. Cells were exposed for 48 hours to four concentrations of clinically relevant drugs including platinums, anthracyclines and alkylating agents. Cell survival was measured using the 3-4,5-dimethyl-2, 5-diphenyl tetrazolium bromide (MTT) assay allowing patients to be grouped as "sensitive" or "resistant" in vitro. Clinical data including age, residual disease, histological grade, treatment, response after initial treatment and overall survival were collected. There was a highly significant (p<0.0001) correlation of in vitro sensitivity with in vivo response in the patients who completed their therapy, with an 83% positive predictive accuracy for resistance. This translated in the longer term to an increased survival for the patients found to be sensitive in vitro to their therapy with a 5-year survival rate of 24% compared to 12% for the resistant group (p=0.033). These results suggest that MTT chemonsensitivity testing can predict response in ovarian cancer leading to the prospect of increased survival in this devastating disease by customising therapy to individual patients.

Circumvention of ara-C resistance by aphidicolin in blast cells from patients with AML.

Treatment failure in AML is often attributed to P-glycoprotein-associated multidrug resistance. However, the importance of increased DNA repair in resistant cells is becoming more apparent. In order to investigate the ability of the DNA repair inhibitor aphidicolin to modulate drug resistance, we continually exposed blasts cells, isolated from 22 patients with AML, to a variety of agents +/- 15 microM aphidicolin for 48 hours. Cell survival was measured using the MTT assay. Overall, there was no significant effect of aphidicolin on sensitivity to daunorubicin, doxorubicin, etoposide or fludarabine. However, there was a marked increase in sensitivity to ara-C with a median 4.75-fold increase overall (range 0.8-80-fold;P< 0.005). The effect of aphidicolin was significantly greater in blast cells found resistant in vitro to ara-C (8.9-fold compared to 2.12-fold, P< 0.01). This observation was further validated by the correlation between ara-C LC(50)and extent of modulation effect (P< 0.05). Cells isolated from 10 cord blood samples were also tested in order to establish the haematological toxicity of combining ara-C and aphidicolin. The therapeutic index (LC(50)normal cells/tumour cells) for ara-C + aphidicolin was higher than that for ara-C alone suggesting no increased myelotoxicity for the combination. Increased cytotoxicity without increased haematotoxicity makes the combination of ara-C plus aphidicolin ideal for inclusion in future clinical trials.

Chemosensitivity in childhood brain tumours in vitro: evidence of differential sensitivity to lomustine (CCNU) and vincristine.

The aim of this study was to examine the range of sensitivity of a panel of short-term cultures derived from different types of malignant childhood brain tumours including medulloblastoma, ependymoma and glioblastoma multiforme to three cytotoxic drugs, lomustine (CCNU), vincristine (VCR) and procarbazine (PCB). Sensitivity was assessed using a modification of the dimethylthiazolyl-2,5-diphenyl tetrazolium bromide (MTT) assay. Short-term cell lines derived from ependymomas were considerably more resistant to VCR than other types of childhood brain tumours, while cultures derived from supratentorial primitive neuroectodermal tumour (PNET) displayed marked sensitivity to both lomustine and VCR. Cultures from ependymomas, medulloblastoma and astrocytic gliomas had similar sensitivity to lomustine and PCB as cultures derived from adult malignant astrocytoma.

Comparison of P-glycoprotein expression and function with in vitro sensitivity to anthracyclines in AML.

The importance of P-glycoprotein (P-gp) in AML has been well documented. Resistance to the anthracyclines can be overcome by several agents including Cyclosporin A (CsA), PSC833 and GF120918. We describe an investigation into the expression, using MRK16 and UIC2, and function of P-gp using daunorubicin with and without modulators by flow cytometric analysis on previously frozen blast cells from 27 patients with primary or secondary AML. We compared this with the in vitro chemosensitivity, using the MTT assay, of fresh blast cells from the same patients. Whilst we found a correlation between P-gp function using CsA and GF120918 and expression using MRK16 (p < 0.05) and (p < 0.02) respectively, we were unable to find any overall correlation between expression and function of P-gp with either in vitro sensitivity to the anthracyclines, previous treatment, or 1 degree or 2 degrees disease. However it was possible to identify individual patients whose cells exhibited P-gp expression and function teamed with in vitro resistance to, and modification of, the anthracyclines. Furthermore, it is possible to identify which modulator had the greatest effect. The fact that we obtained higher indications of resistance reversal using the MTT assay along with finding P-gp expression and function in patients sensitive to the anthracyclines, suggests studies of P-gp should be teemed with chemosensitivity testing to identify specific patients who will benefit.

In vitro sensitivity to the liposomal preparation, DaunoXome in CLL.

In order to determine the efficacy of liposomal encapsulated daunorubicin (DaunoXome; DNX) in chronic Iymphocytic leukaemia, the sensitivities of cells from 10 patients with this disease were assessed and compared with that of free drug using the MTT assay. There was a marked variation in effect between patients for both drug preparations. Despite this, there was a small but significant enhancement of cytotoxicity afforded by the liposomal preparation (median 2.8-fold, p < 0.01). The cells from 2 of the patients appeared to be resistant to free daunorubicin. However, incubation for 96 h in DNX appeared to circumvent this resistance. This increase in sensitivity for resistant cells could not be demonstrated in an MDR positive cell line (K562AR) suggesting that another mechanism of resistance may be involved. We conclude that it is feasible to assess sensitivity to DNX using the MTT assay in fresh cells from patients with CLL using a continuous drug exposure of 96 h. Furthermore, DNX appears more effective than free drug in this disease.

Chemosensitivity testing of fresh and continuous tumor cell cultures using lactate dehydrogenase.

We have examined the use of the LDH (lactate dehydrogenase) assay for chemosensitivity testing in established and primary cultures of sarcoma, leukaemia and ovarian cancer in parallel with the MTT assay. The method we describe is rapid, sensitive and ideal for 96-well plate assays using adherent or suspension cultures. Excellent agreement between the two methods was observed (r = 0.936) using a variety of antitumour agents, with some notable exceptions. In the Bax (human synovial sarcoma) cell line MTT colour production by control cells was very low, thus MTT-->formazan production could not be relied upon as a definitive end point equating with cell number. In contrast, colour production of control cells using the LDH assay was significantly greater and all cultures tested were suitable for titration of chemosensitivity. There was a discrepancy between IC50 values obtained either by cell counting or MTT in the HTB88 (human leiomyosarcoma) line treated with 5-FU (59.9 microM vs > 200 microM, respectively). However, cell counting agreed well with the LDH assay (IC50 47.3 microM). Whilst the MTT assay remains a reliable method for chemosensitivity testing, the LDH assay may prove more appropriate in certain experimental settings.