R.T.D. Oliver

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.

Radiotherapy after chemotherapy for metastatic seminoma : a diminishing role

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.

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.

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.

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.

Accurate and rapid assessment of MHC antigen upregulation following cytokine stimulation

Three human tumour cell lines, A431 (cervical), Scaber (bladder) and Fen (bladder), were studied using immunohistochemical staining (IC), radiobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major histocompatibility antigen (MHC) induction in response to interferon-gamma (IFN-gamma). Induction of class II antigens by IFN-gamma was observed on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive for intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta 2 microglobulin (beta 2-m) (Mab BBM.1), while Fen cells were positive only with HC10. The IP technique demonstrated the presence of a 45 kDa band on precipitation of the Fen lysate with HC10 Mab, whereas no such band was observed when W6/32 was used. The DB technique confirmed the negative reaction with W6/32 and BBM.1 Mabs, while HC10 showed positive staining which was upregulated by IFN-gamma. Transfection of the Fen cells with the beta 2-m gene resulted in the surface expression of fully assembled class I molecules. The DB technique showed upregulation of class I antigens following IFN-gamma stimulation, while RB detected no significant increase in cell surface expression (t test; p = 0.104). The binding values for transfected Fen cells before and after IFN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectively. These results demonstrate that the DB technique facilitates an accurate assessment of cytokine induced antigens, corrected against a background of total cellular protein synthesis. The ease of execution, simplicity, non-radioactive nature and economy make it the method of choice for routine screening prior to the selection of suitable patients for cytokine therapy.

Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC-restricted cytotoxicity.

Using colorimetric MTT assay the susceptibility of a newly established bladder epithelial cell line, Fen cells was compared with conventional target cells, i.e., K562 and Daudi and other epithelial lines for investigation of non-specific killing activity (NK/LAK) of effector cells previously activated with interleukin-2 (IL-2). The results showed that Fen cell line was more sensitive than K562 and Daudi targets and this was seen whether the effector cells were IL-2-activated or not. The percent killing of effector cells from nine normal donors against Fen, K562 and Daudi targets at effector/target (E/T) ratio of 10/1 after IL-2 activation were 63.4 +/- 7.3, 42.6 +/- 4.3 (p = 0.0001) and 38.6 +/- 5.1 (p = 0.0001) respectively. The corresponding values for inactivated effector cells at 50/1, E/T ratio were 44.8 +/- 9.0, 25.1 +/- 8.3 (p = 0.0001) and 24.4 +/- 9.4 (p = 0.0001) indicating exquisite sensitivity of Fen cells to NK/LAK killing. The susceptibility of Fen cells was found to increase by pre-treatment of target cells with interferons (IFN). Thus the percent killing of untreated, IFN-alpha (1000 U/ml), beta (2000 U/ml) and gamma (100 U/ml) treated cells were 52%, 64% (p = 0.005), 70% (p = 0.001) and 67% (p = 0.001) respectively. These results indicated that Fen cells were more susceptible to NK/LAK killing than the conventional K562 and Daudi target cells. These results and the epithelial origin of Fen cells indicate that this cell line might prove to be a more realistic system to replace the conventional approach for assessment of NK/LAK activity in patients with cancer of epithelial origin.

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.