Autologous leukemia-specific T-cell-mediated lymphocytotoxicity in patients with acute myelogenous leukemia.

Short-term culture of acute myelogenous leukemia patient's remission lymphocytes with inactivated autologous leukemic blast cells plus allogeneic lymphocytes, generated effector T lymphocytes which were cytotoxic for the specific autologous blast cell in 11 of 14 patients studied. Experiments using Daudi and Molt 4 lymphoblastoid cell lines as third-party helper cell suggest that an HLA D locus incompatability is necessary to provide effective help in this system. Cold target inhibition experiments, crossover studies between pairs of patients, and experiments with allogeneic leukemic blast cells as priming stimulus suggest that the target antigen is only present on the specific autologous blast cell.

Long-term follow-up of testicular cancer patients shows no predisposition to osteoporosis.

UNLABELLED: Most patients with testis cancer are cured with treatment. However, the incidence of osteoporosis after prolonged follow-up is unknown. This study investigates the incidence of osteoporosis in 39 testis cancer patients with follow-up from 5 to 28 years. There was no increased incidence of osteoporosis. These initial data are reassuring but require further investigation. INTRODUCTION: The majority of patients with testis cancer are cured with either a unilateral orchidectomy alone or orchidectomy and chemotherapy. However, the long-term incidence of osteoporosis following treatment for testicular cancer has not been established. METHOD: This was a single-centre cross-sectional study, where bone mineral density (BMD) measurements were performed in male patients who were previously treated for testicular cancer. BMD measurements were made by dual-energy X-ray scanning (DXA) using a HOLOGIC imaging bone densitometer. The World Health Organisation criteria were used to define osteoporosis and osteopenia. Blood samples were taken from each patient at the time of the DXA scan. Statistical analyses were performed in STATA10. RESULTS: Neither orchidectomy alone nor orchidectomy and chemotherapy together predisposed to osteoporosis [p value = 0.4 (95%CI -0.1-0.8) and p value = 0.2 (95%CI -0.2-0.7), respectively]. Analysis also showed no evidence of an association between cases of osteopenia and length of follow-up (assessed by logistic regression). CONCLUSION: This work found no association between treatment for testis cancer and the development of osteoporosis. Screening the whole population of testis cancer survivors for osteoporosis in the long term is not necessary; however, targeting specific patients with risk factors may be warranted.

Prognostic factors for renal cell carcinoma.

Renal cell carcinoma is a relatively uncommon tumour with a widely varying prognosis depending on several tumour and clinical factors. This review discusses these factors and critically appraises their value both as individual markers and when they are incorporated into scoring systems/models or algorithms. Disease stage (assessed pathologically and/or clinically) and performance status have the strongest evidence as helpful individual prognostic markers but a better discrimination is obtained by combining these and adding in various other indices. Prospective validation of such integrated prognostic models will be essential.

Radiotherapy versus single-dose carboplatin in adjuvant treatment of stage I seminoma: a randomised trial.

BACKGROUND: Adjuvant radiotherapy is effective treatment for stage I seminoma, but is associated with a risk of late non-germ-cell cancer and cardiovascular events. After good results in initial studies with one injection of carboplatin, we undertook a large randomised trial to compare the approaches of radiotherapy with chemotherapy in seminoma treatment. METHODS: Between 1996 and 2001, 1477 patients from 70 hospitals in 14 countries were randomly assigned to receive radiotherapy (para-aortic strip or dog-leg field; n=904) or one injection of carboplatin (n=573; dose based on the formula 7x[glomerular filtration rate+25] mg), at two trial centres in the UK and Belgium. The primary outcome measure was the relapse-free rate, with the trial powered to exclude absolute differences in 2-year rates of more than 3%. Analysis was by intention to treat and per protocol. This trial has been assigned the International Standard Randomised Controlled Trial Number ISRCTN27163214. FINDINGS: 885 and 560 patients received radiotherapy and carboplatin, respectively. With a median follow-up of 4 years (IQR 3.0-4.9), relapse-free survival rates for radiotherapy and carboplatin were similar (96.7% [95% CI 95.3-97.7] vs 97.7% [96.0-98.6] at 2 years; 95.9% [94.4-97.1] vs 94.8% [92.5-96.4] at 3 years, respectively; hazard ratio 1.28 [90% CI 0.85-1.93], p=0.32). At 2 years' follow-up, the absolute differences in relapse-free rates (radiotherapy-chemotherapy) were -1.0% (90% CI -2.5 to 0.5) by direct comparison of proportions, and 0.9% (-0.5 to 3.0) by a hazard-ratio-based approach. Patients given carboplatin were less lethargic and less likely to take time off work than those given radiotherapy. New, second primary testicular germ-cell tumours were reported in ten patients allocated irradiation (all after para-aortic strip field) and two allocated carboplatin (5-year event rate 1.96% [95% CI 1.0-3.8] vs 0.54% [0.1-2.1], p=0.04). One seminoma-related death occurred after radiotherapy and none after carboplatin. INTERPRETATION: This trial has shown the non-inferiority of carboplatin to radiotherapy in the treatment of stage I seminoma. Although the absence of disease-related deaths and preliminary data indicating fewer second primary testicular germ-cell tumours favour carboplatin use, these findings need to be confirmed beyond 4 years' follow-up.

Molecular characterisation of the t(1;15)(p22;q22) translocation in the prostate cancer cell line LNCaP.

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation.

Leukemia-induced alterations of serum glycosyltransferase enzymes.

Studies on blood group A and H glycosyltransferase enzymes in 54 patients with acute myeloid leukemia were carried out on serum derived from blood samples taken prior to treatment, and in 16 cases, further tests were performed during clinical remission and at the time of relapse. The enzyme assay procedures, using low-molecular-weight compounds as sugar acceptors and radioactive nucleotide sugars as the donor substrates, have been described by Chester et al. (Eur. J. Biochem., 69:583, 1976). Abnormally low values of H enzyme (expressed as percentage of radioactive sugar incorporated into product; (that is, 1 to 3%) were observed in practically all presentation sera, but the values reverted to normal levels (3 to 15%) at the time of clinical remission and then became low once more with the development of drug resistance and clinical relapse. A enzyme levels measured in presentation sera which had demonstrated abnormal H enzyme were mostly within the normal range. In 2 of 5 A1 patients; sera and in all of three A2 patients increases in enzyme levels were observed in remission as compared with presentation serum samples. The depression of biosynthetic enzymes in acute leukemic sera could not be accounted for on the basis of competitive inhibitors or catabolic enzymes. It is proposed that changes of serum glycosyltransferase enzymes reflect alterations in a leukemic cell population and that knowledge of these changes may be of value in prognosis in acute leukemia.

Randomized trial of bleomycin, etoposide, and cisplatin compared with bleomycin, etoposide, and carboplatin in good-prognosis metastatic nonseminomatous germ cell cancer: a Multiinstitutional Medical Research Council/European Organization for Research and Treatment of Cancer Trial.

PURPOSE: This prospective randomized multicenter trial was designed to evaluate the efficacy of carboplatin plus etoposide and bleomycin (CEB) versus cisplatin plus etoposide and bleomycin (BEP) in first-line chemotherapy of patients with good-risk nonseminomatous germ cell tumors. PATIENTS AND METHODS: Between September 1989 and May 1993, a total of 598 patients with good-risk nonseminomatous germ cell tumors were randomized to receive four cycles of either BEP or CEB. In each cycle, the etoposide dose was 120 mg/m2 on days 1, 2, and 3, and the bleomycin dose was 30 U on day 2. BEP patients received cisplatin at 20 mg/m2/d on days 1 to 5 or 50 mg/m2 on days 1 and 2. For CEB patients, the carboplatin dose was calculated from the glomerular filtration rate to achieve a serum concentration x time of 5 mg/mL x minutes. Chemotherapy was recycled at 21-day intervals to a total of four cycles. RESULTS: Of patients assessable for response, 253 of 268 (94.4%) of those allocated to receive BEP achieved a complete response, compared with 227 of 260 (87.3%) allocated to receive CEB (P = .009). There were 30 treatment failures in the 300 patients allocated to BEP and 79 in the 298 allocated to CEB (log-rank chi 2 = 26.9; P < .001), which led to failure-free rates at 1 year of 91% (95% confidence interval [CI], 88% to 94%) and 77% (95% CI, 72% to 82%), respectively. There were 10 deaths in patients allocated to BEP and 27 in patients allocated to CEB (log-rank chi 2 = 8.77; P = .003), which led to 3-year survival rates of 97% (95% CI, 95% to 99%) and 90% (95% CI, 86% to 94%), respectively. CONCLUSION: With these drug doses and schedules, combination chemotherapy based on carboplatin was inferior to that based on cisplatin. This BEP regimen that contains moderate doses of etoposide and bleomycin is effective in the treatment of patients with good-prognosis metastatic nonseminoma.

Does human papillomavirus play a role in the development of bladder transitional cell carcinoma? A comparison of PCR and immunohistochemical analysis.

AIM: To investigate the role of human papillomavirus (HPV) in the development of bladder transitional cell carcinoma (TCC). METHODS: Seventy eight paraffin wax embedded TCC samples were tested for the presence of HPV by two methods. First, immunohistochemistry was carried out using a polyclonal antibody capable of detecting the capsid protein of all known papillomaviruses. The second method was a consensus GP5+/6+ primer mediated polymerase chain reaction (PCR) technique, with the products analysed by both agarose gel electrophoresis and an enzyme immunoassay using type specific oligonucleotide probes for 10 different mucosal genotypes. To exclude false negative results because of the poor quality of DNA extracted from paraffin wax embedded samples, the series was extended to include 20 further blocks for which the corresponding snap frozen unfixed tissue was available. RESULTS: The two methods produced contrasting results, with 47 of the 78 samples positive for HPV antigen and none positive for HPV DNA. HPV DNA was not detected in the 20 additional paraffin wax embedded TCCs or in the 20 paired unfixed samples. In contrast, HPV DNA was amplified by PCR from all six of the paraffin wax embedded cervical carcinoma and anogenital wart control samples. CONCLUSION: The disparity between the two sets of results is probably caused by false positives resulting from the non-specificity of the polyclonal antibody used for immunohistochemistry. These results suggest that HPV is unlikely to play an aetiological role in the development of bladder TCC.

The frequency of major histocompatibility complex antigen abnormalities in urological tumours and their correction by gene transfection or cytokine stimulation.

There is increasing evidence that loss of major histocompatibility complex (MHC) antigens from tumors may be a factor in escape from immune surveillance. In an attempt to quantify this phenomenon in bladder tumors, frozen sections were stained immunochemically and cell lines were tested in a radiobinding assay before and after treatment with interferon gamma (IFN gamma) and after attempts to correct the defect by normal human leukocyte antigen (HLA) gene transfection. Study of 68 tumor sections with W6/32 antibody against monomorphic class I demonstrated that 42% had reduced or absent staining compared with the intensity of stromal staining. Ten percent of cases had complete absence with W6/32, all of which were also negative for beta 2-microglobulin (beta 2-m) expression. Use of polymorphic antibodies for A2, A3, Bw4, and Bw6 increased this frequency of defects to 73%. Study of 21 tumor cell lines with W6/32 demonstrated negative staining in five (23%) that could not be induced by IFN gamma and reduced staining in three (14%) that could be increased by IFN gamma, the remainder showing normal levels unaffected by IFN gamma. An additional six (28%) failed to express class II in response to IFN gamma, leading to an overall incidence of abnormality of 65%. In no case did cotransfection of one cell line with a defect in one case transfection of beta 2-m gene into a class I negative line of fully assembled MHC class I antigens. It is concluded that the majority of tumor cells demonstrate some form of MHC class I and II defects.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-biopsy rise in serum PSA. A potential tool for the dynamic evaluation of prostate cancer/prostatic intraepithelial neoplasia (PIN).

OBJECTIVES: The biochemical response of prostatic cells to needle core biopsies is known to manifest as a rise in serum PSA. The aim of this study is to evaluate the PSA response to mechanical trauma in prostate cancer patients, when compared to benign controls. MATERIALS AND METHODS: 50 consecutive patients undergoing transrectal ultrasound guided prostatic needle biopsies had their total serum PSA measured thirty minutes after the procedure. Change from the baseline PSA was estimated and correlated to histology. RESULTS: Data was analysed in 48 patients (mean age 68 years; range 55-87 years). Histology showed benign, cancer and PIN results in 24, 19 and 5 patients respectively. The highest rise in post biopsy PSA was observed in the PIN group. A significant difference in the rise in serum PSA was noted between controls and the cancer group. CONCLUSION: Post biopsy PSA response differs significantly between benign and malignant prostatic tissue. PIN causes an excessive in PSA values on mechanical stimulation. This small study indicates that the biopsy model may help us to assess the dynamics of prostate cancer.

Accelerated cisplatin-based chemotherapy for advanced bladder cancer.

The toxicity and efficacy of accelerated cisplatin, vincristine and methotrexate was assessed in patients with advanced urothelial carcinoma. 30 consecutive patients were entered into a phase II trial and treated with cisplatin, vincristine and methotrexate given every 10 days (MOPq10) for four cycles, followed by two further cycles at 21 day intervals. Five complete responses and 14 partial responses were observed (overall response rate 63%; 95% confidence interval 45-78%). The median progression-free survival was 7.5 months (range 1.8-28) and the median overall survival 10.5 months (range 2.36). Toxicity was moderate with no treatment-related deaths. It is concluded that although the overall survival is still disappointing, the toxicity is less with the protocol than reported with methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) or escalated M-VAC (E-MVAC) and the time on treatment is shorter. MOPq10 provided palliative benefit to two-thirds of patients with advanced transitional cell carcinoma including those in their eighth decade.

Epidermal growth factor-induced protection of tumour cell susceptibility to cytolysis.

Using radiobinding, transfection and colorimetric assays, the biological significance of epidermal growth factor (EGF) and its receptor on established human tumour cell lines was investigated. The intensity of class I major histocompatibility antigen (MHC) and EGF receptor (EGFR) expression on 20 tumour cell lines was investigated and showed no direct correlation (coefficient of correlation r = 0.43 and P = 0.06). furthermore, transfection of the beta 2-microglobulin gene into a class I negative bladder tumour cell line, resulting in the re-expression of fully assembled cell surface class I antigens, did not result in alteration of EGFR expression. However, there was an inverse correlation between the intensity of EGFR expression and the stimulatory response of cells to exogenously added EGF. The per cent inhibitions of cell proliferation by EGF at 100 ng/ml for A431 (highest EGFR expressor) and Scaber (lowest EGFR expressor) were 37 and -7%, respectively. The results also showed that cell lines isolated from testis tumours positive for epithelial markers (using pan keratin antibody LP34 as an epithelial marker), expressed significantly lower EGFR levels than cell lines from bladder tumours. The expression of EGFR receptor was not modulated by interferons (IFN-alpha and -gamma and only a minor effect with IFN-beta) or active supernatant containing a mixture of cytokines. Whilst the pretreatment of tumour cells with IFNs resulted in a significant increase in the susceptibility of tumour cells to interleukin-2-activated peripheral blood mononuclear cells, EGF treatment resulted in their protection. Thus, the per cent killing at an effector:target ratio of 20:1 for untreated cells and EGF (100 ng/ml), IFN-alpha (1000 U/ml), -beta (2000 U/ml) and -gamma (100 U/ml) were 53%, 33% (P = 0.004), 64% (P = 0.004), 69% (P = 0.001) and 66% (P = 0.001), respectively. These results indicate the complex interactions between EGF and EGFR and their relevance in modifying tumour cell behaviour. The hypothesis that the resistance to cytolysis of tumour cells induced by EGF stimulation may be a factor in the accelerated tumour growth seen in patients after traumatic tissue damage is discussed.

Phase I study comparing continuous infusion of recombinant interleukin-2 by subcutaneous or intravenous administration.

22 cancer patients entered a randomised phase Ib trial comparing the effects of low-dose recombinant interleukin-2 (300 micrograms/m2, approximately equivalent to 6.4 x 10(6) cetus units or 38 x 10(6) U per day) given continuously by intravenous or subcutaneous infusion. At 48 h after two 5-day courses, median lymphocyte levels (x 10(9)/l) were 6.0 (387% increase) in the subcutaneous arm (n = 9) and 5.9 (369% increase) in the intravenous arm (n = 8). Liver and renal toxicity were similar in the two groups. One minor response lasting 4 months occurred in 12 renal cancer/melanoma patients receiving subcutaneous treatment and one durable complete remission continuing at 30 months and one minor response lasting 10 months occurred in 6 renal cancer/melanoma patients receiving intravenous treatment.

Paired tumour infiltrating lymphocyte (TIL) and tumour cell line from bladder cancer: a new approach to study tumour immunology in vitro.

This paper reports the first example of tumour infiltrating lymphocytes (TILs) and a tumour cell line from the same individual and analyses their characteristics. The tumour cell line (CAT), derived from a patient with well-differentiated (G3pTa) TCC, has been in culture for 24 months and subcultured more than 100 times. Epithelial origin was established by electronmicroscopy and use of a range of monoclonal antibodies (Mabs) against cytokeratins. The TILs isolated from the same tumour expressed all the phenotypic characteristics of normal activated T cells and demonstrated low levels of cytotoxicity against the autologous tumour line (CAT). Comparison of cell surface molecules of these cells revealed the loss of HLA-B7, B44 and Bw6 from the CAT cells whilst maintaining HLA-A2, A3 and Bw4. Karyotypic analysis demonstrated three rearranged chromosomes (between chromosomes 4 and 11, 10 and 13, 11 and 17) on CAT cells. The potential that study of paired autologous tumour cells and TILs in culture offers for studying the role of MHC antigens in tumour rejection and the impact of different approaches to correcting the defect are reviewed.

Induction of MHC antigens by tumour cell lines in response to interferons.

The induction of major histocompatibility complex antigens by interferons (IFN) on 17 established tumour cell lines was investigated by radio binding. One bladder (Fen) and two testis lines (Tera I and Ha) lacked class I antigens and IFN-gamma failed to induce their expression. However, IFN-gamma upregulated these antigens on lines expressing low class I antigens (Tera II and EP2102) with little or no significant effect on high class I expressing lines (T24 and RT112). In one bladder line (Wil) IFN-gamma, whilst failing to alter monomorphic class I, upregulated polymorphic HLA-A2 and A3 antigens. None of the 17 lines expressed class II antigens, but could all be induced by IFN-gamma except T24, TccSup, Tera II and Lan lines. This defect was not due to the absence of IFN-gamma receptor, since under the same conditions intracellular adhesion molecule 1 was upregulated. IFN-alpha, whilst failing to have any effect on class II, induced class I antigens. IFN-beta showed no activity on either class I or II antigens when used alone. However, in combination, it inhibited IFN-gamma induced class II antigens. Thus, it may be possible to study cells from fresh tumours to preselect the minority of patients who might benefit from cytokine therapy.

Immunological paradox in testicular tumours: the presence of a large number of activated T-cells despite the complete absence of MHC antigens.

Tissue sections from 22 seminoma (Se) and 10 teratoma (Te) patients were investigated for correlation between the presence of tumour infiltrating T-lymphocytes (TIL) and the expression of major histocompatibility complex (MHC) antigens using an immunoperoxidase staining technique. Complete absence of both class I and II antigens was observed in all Te and 20 out of 22 Se. The two positive Se showed only weak expression on 2% of tumour cells. Despite the absence of human leucocyte antigens (HLA) there were a large number of TIL scattered throughout the tissues in the case of Se with no predominance of CD4 or CD8 subpopulations in either group. T gamma positive cells were less than 5% of total CD3 positive cells in both Se and Te. The majority of the TIL were found to express activation markers, i.e. HLA class II antigens. Culture of tumour cell suspension with IL-2 produced passageable IL-2-dependent T cells from 10 out of 15 tumours. Studies with testis cell lines showed the complete absence of class I antigens in 2 out of 5 cases and the inability of interferon gamma (IFN gamma) to induce expression. IFN gamma also failed to induce class II antigens in three out of five of these lines. The immunological paradox of the presence of a large number of activated T-cells in testicular tumours despite the complete absence of MHC antigens remains unexplained and needs further investigation. Possible hypotheses are reviewed.

Radiotherapy after chemotherapy for metastatic seminoma--a diminishing role. MRC Testicular Tumour Working Party.

In a retrospective study, data from 302 patients with metastatic testicular seminoma treated with chemotherapy between 1978 and 1990 in 10 European centres were analysed to evaluate the role, if any, of postchemotherapy treatment with irradiation. The primary endpoint of this study was the progression-free survival rate after chemotherapy with or without additional radiotherapy. This was related to the type of primary chemotherapy, sites and sizes of pre- and postchemotherapy masses, the extent of surgical resection after chemotherapy and the use of radiotherapy. 174 patients had residual disease at the end of chemotherapy. The most important prognostic factors for progression were the presence of any visceral metastases or raised LDH prechemotherapy, and the presence of residual disease at visceral sites after chemotherapy. Approximately half the patients with residual masses underwent postchemotherapy radiotherapy, with selection based predominantly on institutional practice. In patients receiving platinum-based chemotherapy, no significant difference was detected in progression-free survival whether or not radiotherapy was employed. Patients receiving BEP (bleomycin, etoposide and cisplatin) had a progression-free survival rate of 88% (95% CI, 80-96%) uninfluenced by postchemotherapy radiotherapy. In patients with residual masses confined to the abdomen after platinum-based chemotherapy, the absolute benefit to radiotherapy was estimated to be 2.3%. The potential benefit of postchemotherapy radiotherapy is minimal, and so it is concluded that the use of adjuvant radiotherapy to residual masses after platinum-based chemotherapy for metastatic seminoma is unnecessary.

Prognostic factors for patients with advanced seminoma treated with platinum-based chemotherapy.

Prognostic factors for 3-year progression-free survival (PFS) were defined in 286 patients with advanced seminoma treated with cisplatin-based chemotherapy at 10 European oncology units (no prior treatment: 236; prior radiotherapy: 50). Previously irradiated patients displayed a 69% PFS as compared to 87% in those presenting with advanced seminoma at the time of diagnosis (P = 0.009). In the univariate analysis, the extent and site of disease before chemotherapy and the level of serum LDH (< 2.0 versus > or = 2.0 x upper limit of normal) correlated with PFS in previously non-irradiated patients, but not in patients with prior radiotherapy. The multivariate analysis was, therefore, restricted to previously non-irradiated patients. The presence of non-pulmonary visceral metastases and a serum LDH level of > or = 2 x normal (N) proved to be independent prognostic factors. Based on these variables, two prognostic models were constructed and validated in an external data set of 166 comparable patients. For clinical use, Model 2 is recommended. The good-prognosis group comprises non-irradiated patients with stage II seminoma and any LDH level at presentation, or stage III and IV patients (with lung metastases only) whose serum LDH level is < 2 x N. These patients display a 94% 3-year PFS. The poor prognosis group includes all other patients with a 56% PFS. With this prognostic model, individualisation of the therapeutic approach may be considered in patients with advanced seminoma and a high risk of chemotherapy-related toxicity.

A new approach for measurement of cytotoxicity using colorimetric assay.

Using in vitro established tumour cell lines attempts were made to assess the suitability of tetrazolium salt reduction (MTT) assay to replace the conventional radioactive base techniques for measuring cell proliferation and cell killing. The optimum conditions for MTT loading time, concentration of MTT and the time for colour development were found to be 4 h, 5 mg/ml and 30 min respectively, conditions which were used for subsequent experiments. Analysis of the correlation between increasing cell numbers and optical densities (OD) showed a direct relationship with correlation of coefficient values of r > 0.98 and 10,000 cells/well was found to provide an accurate ODs for a wide variety of cell types. The accuracy of replicate readings of the assay was investigated by setting a wide range of cell numbers and the variation among seven replicates was calculated and found to be less that 6% of the mean values. The reproducibility of the assay for two cell lines was tested using the lines on four different occasions. The ODs for Jar and Fen cell lines were 0.80 +/- 0.01, 0.82 +/- 0.02, 0.90 +/- 0.02, 0.79 +/- 0.05 and 0.56 +/- 0.01, 0.58 +/- 0.03, 0.60 +/- 0.02 and 0.61 +/- 0.02 respectively giving maximum variation of less than 11% of mean on repeated testings. Comparison between the results of MTT with 3H-Tdr or 51Cr release assays showed a high degree of correlation over a wide range of cell numbers and cell types. The r values between the results of MTT with 3H-Tdr (for cell number ranging from 1.8 to 60 x 10(3)/well) or 51Cr release assays (for E/T ratios of between 5:1 and 40:1) were 0.89 (p = 0.001) and 0.96 (p < 0.03) respectively. These results demonstrate that it is possible to use the MTT assay interchangeably with radioactive base techniques to measure cell proliferation and cytotoxicity. The ease of its execution, safety and its suitability for analysing as few as 3000 cells makes this method a serious contender for replacing the conventional radioactive techniques.