Aphidicolin decreases ex vivo resistance to cytosine arabinoside in childhood acute leukaemia.

In order to assess the effect of the DNA polymerase inhibitor aphidicolin on resistance to cytosine arabinoside, blast cells from 15 children with ALL and 9 with AML were exposed to a range of concentrations of ara-C +/- aphidicolin. Cell survival was measured using the MTT assay. Aphidicolin significantly increased sensitivity to ara-C in blast cells from both ALL (p=0.001) and AML (p<0.01). The median fold increase (sensitisation ratio) for ALL was 3.4 (range 1.2-13.6) compared to 12.4-fold (range 6.0-148) for AML blasts (p=0.005). There was a striking relationship between increasing ara-C resistance and increasing effect of aphidicolin in AML (p<0.001) but not ALL (p>0.05). These remarkable results suggest that aphidicolin should be considered for future clinical trials as a modulator of ara-C resistance, particularly in AML.

Ex vivo effects of the dual topoisomerase inhibitor tafluposide (F 11782) on cells isolated from fresh tumor samples taken from patients with cancer.

Tafluposide (F 11782), a novel epipodophylloid with a unique mechanism of interaction with both topoisomerase I and II, has shown outstanding antitumor activity in vivo against a panel of experimental human tumor xenografts. The aim of this study was to evaluate its cytotoxicity against fresh tumor cells taken from patients. Cells derived from bone marrow, peripheral blood, malignant effusions or solid biopsies from 84 patients with either hematological or solid tumors were exposed continuously to 0.8-100 nuM tafluposide for 48 h, 96 h or 7 days. Cell survival was measured using an MTT assay or the ATP assay and LC(50) values (drug concentration required for 50% cell kill) were calculated. Tafluposide showed significant cytotoxicity against cells derived from either hematological or solid tumors, with a marked inter-patient variation. There was no significant difference between the effect of tafluposide in samples from untreated or previously treated patients (p>0.05 for all cancer types). Whilst tafluposide appeared to show weak (p<0.05) cross-resistance with the topoisomerase II inhibitor etoposide in acute myeloid leukemia (AML), there did not appear to be any correlation with the effect of the topoisomerase I inhibitor topotecan (p>0.05) in either hematological or solid malignancies. True synergism was identified when combining tafluposide with cisplatin in ovarian cancer [combination index (CI)=0.14, 0.79] and with etoposide in AML (CI=0.49, 0.63 and 0.78). Our results suggest that tafluposide is a strong candidate for inclusion in clinical trials, particularly in hematological malignancies.

The use of the MTT assay to study drug resistance in fresh tumour samples.

We have found the short-term MTT assay to be a simple, reproducible chemosensitivity technique, suitable for use throughout the time course of disease. We now have 12 years' experience of using this method in a variety of tumour types, both haematological and solid malignancies. Tumour cells are isolated from bone marrow, malignant effusions or solid biopsies and subjected to drug exposure for 48-96 h. Cell survival is measured by re-incubation in MTT for 4 h. We have found a significant correlation of in vitro results with in vivo outcome for acute myeloid leukaemia (AML) and for ovarian cancer (both p < 0.0001) with an assay sensitivity of 98% for AML and 81% for ovarian cancer. Furthermore, the 5-year survival of ovarian cancer patients treated with a drug found sensitive in vitro is significantly higher than that for patients treated with a drug found resistant in vitro (p = 0.033). We have correlated assay results with drug resistance markers. For example, expression of the newly described half transporter BCRP is related to daunorubicin resistance (p < 0.05). The MTT assay is also suitable for screening for modulation of drug resistance. We have found that the DNA polymerase inhibitor aphidicolin markedly increases in vitro sensitivity to the platinum drugs in ovarian cancer and cytosine arabinoside in AML in the majority of patients. The greatest effect was seen for patients deemed resistant in vitro to these agents. We have identified novel drug combinations which demonstrate significant synergism using this methodology and have also used it to study the emergence of drug resistance in cell line models with a view to its prevention. In conclusion, we have found the MTT assay to be a simple, repeatable, adaptable technique which produces accurate information to help the clinician select suitable treatment for individual cases.

Assessment of the classical MDR phenotype in epithelial ovarian carcinoma using primary cultures: a feasibility study.

This in vitro feasibility study has assessed a number of techniques and their applicability when looking at the role of multidrug resistance (MDR) in solid tumours. Fresh tumour material was obtained from 34 patients, (11 previously treated, 23 untreated) with ovarian adenocarcinoma. Doxorubicin sensitivity was measured using the MTT assay +/- the cyclosporins, Pgp expression was assessed by immunocytochemistry with the MRK-16 MoAb and flow cytometry was used to assess intracellular drug accumulation +/- PSC 833. 85% of samples showed some evidence of modest chemosensitisation by the cyclosporins (median 1.74-fold). We saw a marked variation in the number of Pgp positive cells between patients (1-87%, median 31%). 63% of samples tested showed an enhancement of DNR accumulation in the presence of PSC 833, with a median increase of 7% (sample range 0-29%). The present study highlights some of the technical difficulties encountered when working with fresh tumour material ex vivo. We conclude that screening of patients for their suitability to enter clinical trials incorporating MDR modulating agents is technically demanding, but feasible.

Cellular glutathione content, in vitro chemoresponse, and the effect of BSO modulation in samples derived from patients with advanced ovarian cancer.

OBJECTIVES: The objective was to assess the relationship between glutathione content and drug sensitivity with glutathione modulation in ovarian cancer in a pilot study using 31 samples of freshly obtained ovarian tumor material from 26 patients with advanced disease. METHODS: Processed tumor samples were screened to determine the glutathione content using an enzyme recycling assay modified for use in a 96-well plate format. Chemosensitivity testing (MTT assay) was used to assess sensitivity to cisplatin and doxorubicin and modulation using buthionine sulfoximine. Multidrug-resistance-associated protein MRP1 (putative drug-glutathione conjugate transporter) expression was also assessed. RESULTS: There was a significant increase in the tumor cell GSH levels in samples from patients who had received previous chemotherapy (9) versus those from chemotherapy-naïve patients (20), P = 0.005. In vitro chemosensitivity testing with doxorubicin and cisplatin (using LC(50) values, i.e., drug dose causing 50% reduction in cell survival relative to untreated control) failed to show a relationship with glutathione levels. Coincubation of cisplatin and doxorubicin with buthionine sulfoximine resulted in a significant increase in sensitivity to both of these drugs overall (cisplatin, P = 0.05; doxorubicin, P = 0.025), with 20 samples showing sensitization to a drug to which they were previously resistant. MRP1 expression failed to show a correlation with drug sensitivity and glutathione levels. CONCLUSIONS: Our study supports the use of glutathione modulation using agents such as buthionine sulfoximine in patients with heavily pretreated, drug-resistant ovarian cancer.

Ex vivo chemosensitivity to mitomycin C in bladder cancer and its relationship with P-glycoprotein and apoptotic factors.

BACKGROUND: Intravesical treatment with mitomycin C (MMC) leads to a complete response rate of around 40% in superficial bladder cancer (TCC). In order to determine in advance which patients will fail to respond, we describe a study assessing the feasibility of applying the ATP assay to test the chemosensitivity of samples from patients with this disease. MATERIALS AND METHODS: TURBT or biopsy samples were received from 27 patients, 23 of which were suitable for the ATP assay (16 primary tumours and 7 recurrences). RESULTS: The success rate of the assay was 91%. There was a marked variation in the effect of MMC between patients with a > 50-fold range in LC50 values (drug concentration required to kill 50% of cells) from 2.99- > 150 microM with a median value of 22.4 microM. We were unable to determine any overall correlation between chemosensitivity and tumour stage or grade or the treatment status of the patient in this small data set. P-glycoprotein status and caspase-3 levels were assessed on these samples using immunohistochemistry but there did not appear to be any relationship between either of these parameters and MMC resistance. Apoptotic counts and mitotic counts were also measured but, whilst these appeared to correlate with grade (p < 0.01), there was no overall significant relationship established with MMC chemosensitivity. CONCLUSION: This study suggests that it is possible to use the ATP assay for chemosensitivity testing in TCCs. Despite a lack of overall correlation between ex vivo MMC resistance and the conventional prognostic factors tested, further studies are warranted in a larger data set to test the ability of this technique to predict clinical outcome in this disease.