Intravesical bacillus Calmette-Guerin versus mitomycin C for Ta and T1 bladder cancer.

BACKGROUND: Tumour recurrence following transurethral resection (TUR) for Ta and T1 bladder cancer is a major clinical problem. Intravesical administration of mitomycin C (MMC) or bacillus Calmette-Guerin (BCG) has proven prophylactic activity but both are associated with local and systemic side-effects. A systematic review was carried out to compare the efficacy of these two agents. OBJECTIVES: To undertake a systematic review and meta-analysis comparing intravesical mitomycin C and Bacillus Calmette-Guerin in terms of tumour recurrence, disease progression and overall survival in Ta and T1 bladder cancer. Treatment-related toxicities would also be evaluated. SEARCH STRATEGY: A comprehensive search of MEDLINE, EMBASE, Healthstar, Cochrane Controlled Trials Register, Cancerlit, and DARE was performed, and hand searching of relevant journals undertaken. SELECTION CRITERIA: Trials in any language were included in the meta-analysis if they were properly randomised, included medium to high risk patients with Ta or T1 bladder cancer and compared intravesical MMC versus BCG. DATA COLLECTION AND ANALYSIS: Trial eligibility, methodological quality and data extraction were assessed independently by two reviewers. Time to event analysis was evaluated using log hazard ratios, with a sensitivity analysis for subgroups according to patient's risk of recurrence. MAIN RESULTS: Twenty-five articles were identified but only seven were considered eligible. This represented 1901 evaluable patients in total, 820 randomised to MMC and 1081 to BCG. Six trials had sufficient data for meta-analysis and included 1527 patients, 693 in the mitomycin arm and 834 in the BCG arm. The weighted mean log hazard ratio (variance) for tumour recurrence for the six trials was - 0.022 (0.005). This indicated no significant difference between MMC and BCG (p = 0.76). However, the meta-analysis indicated evidence of significant heterogeneity between trials (p = 0.001). A subgroup analysis of three trials that included only high risk Ta and T1 patients indicated no heterogeneity (p = 0.25) and a log hazard ratio (variance) for recurrence of -0.371 ( 0.012). With MMC used as the control in the meta-analysis, a negative ratio is in favour of BCG and, in this case, is highly significant (p = 0.0008). The seventh trial, in abstract form only, used BCG in low doses for two arms of the trial (27 mg and 13.5mg) compared to a standard dose of mitomycin C (30mg), and reported a significantly reduced recurrent rate with BCG (27mg) compared to mitomycin C (p = 0.001). Only two trials included sufficient data to analyse disease progression and survival, representing a total of 681 patients; 338 randomised to BCG and 343 to MMC. There was no significant difference between MMC and BCG for disease progression (log hazard ratio + variance: 0.044 + 0.04, p = 0.16) or survival (-0.112 + 0.03, p = 0.50). Local toxicities (dysuria, cystitis, frequency, and haematuria) were associated with both MMC (30%) and BCG (44%). Systemic toxicities, such as chills, fever and malaise, were observed with both MMC and BCG (12% and 19%, respectively) although skin rash was more common with MMC. REVIEWER'S CONCLUSIONS: The data from the present meta-analysis indicate that tumour recurrence was significantly reduced with intravesical BCG compared to MMC only in the subgroup of patients at high risk of tumour recurrence. However, there was no difference in terms of disease progression or survival, and the decision to use either agent might be based on adverse events and cost.

Contribution of grade, vascular invasion and age to outcome in clinically localized renal cell carcinoma.

OBJECTIVE: To determine the relative prognostic importance of microvascular invasion in apparently localized renal cell carcinoma (RCC). PATIENTS AND METHODS: A retrospective clinical and pathological review was conducted of 176 consecutive patients identified from pathology records who had a nephrectomy for RCC with a median follow-up of 44 months. Vascular invasion was recorded and categorized by the level of microvascular invasion (MVI), renal vein invasion (RVI) and inferior vena cava invasion (IVCI). Tumour type, grade and size were also assessed. These variables were assessed by univariate and multivariate analysis to determine their effect on disease-free survival. RESULTS: In the univariate analysis tumour size, grade, vascular invasion and young age each predicted reduced disease-free survival. On multivariate analysis for all 176 patients, grade, vascular invasion and young age were the significant independent predictors of reduced disease-free survival. In a subgroup of 149 patients from whom those with very high risk determinants were excluded (those with grade 4 tumours and/or IVCI) most of the risk of metastasis could be accounted for by vascular invasion and young age alone (MVI vs no vascular invasion, hazard ratio 3.18, 95% confidence interval 1.29-7.84; RVI vs no vascular invasion 2.41, 0.989-5.89; and age per year 0.963, 0.94-0.992). CONCLUSIONS: Grade, vascular invasion and young age are the main independent predictors of relapse in clinically localized RCC after nephrectomy. For most patients, who do not have very high risk indicators, the main adverse predictors are vascular invasion and young age. These findings are important when selecting patients for trials of adjuvant therapy and have implications for pathological staging.

Correlation between the clonogenic initial slope and the response of polykaryon-forming units: the behavior of strains defective in XRCC5 and ATM and the heritability of small variations in radioresponse.

The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose-response curve and the clonogenic initial slope. This suggests that DNA lesions expressed in clonogens are also important in PFU. Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays. These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity. We propose that this results from the absence of cytokinesis-related loss of DNA fragments. Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1). Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant. In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.

Intravesical Bacillus Calmette-Guerin in Ta and T1 Bladder Cancer.

BACKGROUND: Intravesical therapy with Bacillus Calmette-Guerin (BCG) aims to reduce the incidence of tumour recurrence following transurethral resection (TUR) for patients with superficial bladder cancer. OBJECTIVES: The objective of this review was to compare the incidence of tumour recurrence after the standard therapy of transurethral resection versus transurethral resection plus intravesical Bacillus Calmette-Guerin. SEARCH STRATEGY: We searched the Cochrane Controlled Trials Register (March 2000), Medline (February, 2000), EMBASE (February, 2000), Cancerlit (February, 2000), Healthstar (February, 2000), Database of Abstracts of Reviews of Effectiveness (February, 2000) and the Bath Information Data Service. The Proceedings of the American Society Clinical Oncology was hand searched (1996 - 1999). SELECTION CRITERIA: Randomised or quasi-randomised trials of transurethral resection alone versus transurethral resection plus intravesical Bacillus Calmette-Guerin. Patients with Ta and T1 bladder cancer of medium or high risk of tumour recurrence, were eligible for inclusion. DATA COLLECTION AND ANALYSIS: Four reviewers assessed trial quality and two abstracted the data independently. The Peto odds ratios and log hazard ratios were determined to compare the number of patients with disease recurrence at 12 months and the rate of recurrence, respectively. MAIN RESULTS: Six randomised trials were included involving 585 eligible patients. There were significantly fewer patients with disease recurrence at 12 months in the BCG plus TUR group compared to those that received TUR alone (odds ratio 0.30, CI 0.21, 0.43). The overall log hazard ratio for recurrence (-0.83, variance 0.02) indicated a significant benefit of BCG treatment in reducing tumour recurrence. Toxicities associated with BCG consisted mainly of cystitis (67%), haematuria (23%), fever (25%) and urinary frequency (71%). No BCG-induced deaths were reported. REVIEWER'S CONCLUSIONS: In patients with medium/high risk Ta or T1 bladder cancer, immunotherapy with intravesical BCG following TUR appears to provide a significant advantage over TUR alone in delaying tumour recurrence.

Apoptosis is a mode of cell death in the polykaryon-forming unit assay.

In the polykaryon-forming unit (PFU) assay, which defines cell survival as the ability to form a cytochalasin-induced polykaryon of predetermined ploidy, the mode of PFU deletion is not known. Incubation of L5178Y-S PFU in cytochalasin resulted in polyploidy (> or =32C) and most polykaryons (>75%) ultimately underwent apoptosis, detected using chromatin condensation and externalised phosphatidylserine. However, large polykaryons carrying terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)-labelled DNA strand breaks were not observed, presumably due to rapid loss of DNA. Gamma irradiation of PFU prior to cytochalasin exposure caused a reduction in the frequency of highly polyploid cells (>16C), consistent with either a supra-induction of apoptosis or a reduction in the ability of PFU to reach high ploidies. We conclude that L5178Y-S PFU are deleted by apoptosis.

Characterization of highly radiosensitive cell lines from a human ovarian small-cell cancer.

Cells were obtained at paracentesis from a patient with a rapidly growing ovarian tumor. A monolayer cell line (V7S), a xenograft tumor line (V7), and subsequently a xenograft-derived monolayer cell line (V7M) were established. Histological and immunohistochemical studies of the original tumor, xenograft, and cell lines provided a diagnosis of small-cell carcinoma of the ovary-which is consistent with the clinical course of the patient. V7S and V7M had a predominantly hypodiploid karyotype with a small tetraploid population. The V7M, which has been in long-term culture, also showed a nonrandom translocation involving chromosomes 1 and 14 and monosomy of X. Radiobiologically, V7S, V7, and V7M showed marked radiosensitivity with surviving fractions at 2 Gy, measured by clonogenic assay, of between 0.022 and 0.147. Split-dose experiments provided evidence that this radiosensitivity was not due to an inability in cellular repair. In vivo data from the xenograft (V7) revealed a highly radiosensitive tumor, corroborating the in vitro studies.

Prediction of the initial slope of the acute clonogenic survival curve by the post-irradiation behaviour of cytochalasin-induced polykaryons.

We have investigated the behaviour of 11 lines of cultured cells in a survival assay whose endpoint is the ability of cells to become polyploid when incubated in the presence of cytochalasin B (CB). Single cells were induced by CB to form polykaryons after irradiation, and, by analogy with the colony-forming assay, the survival of polykaryon-forming units (PFUs) was defined as the fraction of cells able to achieve a given DNA content (at least 16C in most experiments). There was a radiation dose-dependent reduction in PFU survival, which, following the appearance of cells containing at least 16C DNA, was not markedly dependent upon the sampling time. In all cases, PFUs appeared to be more radioresistant than clonogens, especially at high dose. In 9/11 lines the PFU dose-response curves were exponential, while in two there was a pronounced curvature (quadratic parameter). There was a highly significant positive correlation between PFU response and the corresponding clonogenic initial slope. We suggest that in polykaryons the opportunity for the mechanical loss of DNA fragments may be reduced because the cells do not divide, and therefore that DNA damage resulting from chromosome aberrations may be less important in PFUs than in clonogens. Consequently, PFUs may express lesions not directly associated with mechanical gene loss. This assay may yield an alternative estimate for the clonogenic initial slope, and could be of use where colony-forming assays fail due to incomplete cell monodispersion.

Variation in radiosensitivity due to cell age and split-dose recovery in polykaryons induced by cytochalasin.

We have investigated the properties of an in vitro cell survival assay that uses as its endpoint the ability to form polyploid cells (polykaryons) in the presence of cytochalasin B (CB). The criterion for survival is that a polykaryon-forming unit (PFU) must reach the arbitrary DNA content of at least 16C. The age-dependence of PFU sensitivity to 137Cs irradiation was determined using V79-379A cells synchronized at mitosis. Cells assayed as PFUs demonstrated much less variation in radiosensitivity with age than did clonogens, but the changes in curve shape were qualitatively similar. In both assays mitotic cells yielded an exponential survival curve while that obtained at 5 h (mid-late S) had a marked quadratic component. Owing to the small overall variation in PFU survival with age, at doses greater than about 25 Gy the surviving fraction at 5 h was lower than in mitosis. In both V79-379A and HeLa S3 cells, PFUs demonstrated a capacity for split-dose recovery and yielded recovery ratios at 2.6 at 50 Gy in V79 and 1.5 at 20 Gy in HeLa. Since these ratios were much lower than in clonogens at the same dose, we suggest that this is consistent with an association that we have previously demonstrated between PFU response and the clonogenic initial slope. In an attempt to clarify the DNA lesions to which PFUs may be sensitive, we determined PFU response following exposure to 254-nm UV irradiation. In contrast with ionizing radiation, PFU response to UV was very similar to that of clonogens. This suggests that following UV exposure the absence of cytokinesis in polykaryons may confer less protection than in the case of ionizing radiation, possibly due to fundamental differences in the spectrum of DNA lesions produced.

The micronucleus assay: an evaluation of its use in determining radiosensitivity in vitro.

The cytokinesis-block micronucleus assay was used to measure radiosensitivity in vitro in a panel of seven cell lines. Six of these cell lines were used to study the major parameters of this assay. We observed varying sensitivities following cytochalasin-B exposure. Treatment with 1 microgram/ml cytochalasin-B for 24 h reduced cell survival in four of the six cell lines by > 60%. Cytochalasin-B concentration and post-irradiation culture time were both found to influence cell-response. In three cell lines (V39, V134 and HX142), a decrease in cytochalasin-B concentration (2-0.5 microgram/ml) resulted in an increase in the frequency of radiation-induced micronuclei per binucleate cell. In other cell lines, either the opposite (V7M, CHO-K1) or no effect (WiDr) was seen. A linear dose-response was observed between induced damage expressed as the frequency of micronuclei and radiation dose in all but one melanoma (V39) cell line. Evidence for radiation-induced division-delay, with the maximum frequency of binucleation in irradiated cultures occurring 24-48 h after that of controls, was only seen in two cell lines. Of particular note, and in contrast to some other published reports, was the lack of a general correlation between cell-response measured in the clonogenic and the cytokinesis-block micronucleus assays. Consideration of lethal lesions, determined from the clonogenic dose-response curve, with respect to micronucleus frequency showed a complex relationship, with one micronucleus per binucleate cell corresponding to a wide range of lethal lesions depending on the cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Cycle delay in irradiated cytochalasin-induced polykaryons. Does cytoskeleton status affect cell cycle checkpoints?

Following exposure of CHO-K1 cells to 137Cs irradiation at doses up to 20Gy, a delay in G2 was observed to occur in cells permitted to divide normally, while cells induced to become giants by means of cytochalasin B demonstrated a minimal delay in the transition 2C-8C suggesting that the inhibition of cytokinesis results in modification of one or more cell cycle checkpoints. We postulate that this may occur as a consequence of damage tolerance, or by a feedback loop resulting from the reorganisation of the cytoskeleton that precludes cytokinesis.

The prognostic significance of hyperploid cells in squamous carcinoma of the uterine cervix treated with radiotherapy.

Smears were obtained from patients undergoing radiotherapy for Stage I-IV squamous cell carcinoma of the cervix. The presence of cells with large or multiple nuclei in smears taken during or not more than 1 month following therapy was found to be associated with a significantly increased risk of early relapse.

The survival of cytochalasin-induced polykaryons following exposure to cytotoxic agents.

Using CHO-K1, HeLa S3 and two Walker lines (WR and WS) differentially sensitive to cis-diamminedichloroplatinum(II) (cisplatin), the survival after exposure to cisplatin, mitomycin C, vinblastine, vincristine or cytosine arabinoside has been determined either of clonogens or of cells rendered polyploid by post-exposure incubation in the presence of cytochalasin B (CB). It is suggested that the inhibition of cytokinesis by CB permits an assessment to be made of the fraction of damage whose expression is cell division-related, possibly including that resulting from a loss or malsegregation of genetic material. It was found that the response of polykaryons in comparison to clonogens was both agent- and cell line-dependent. After cisplatin exposure, polykaryon survival (defined as the ability to accumulate at least 16C DNA) declined exponentially with dose and was qualitatively, and to some extent quantitatively, similar to that observed previously after irradiation. In HeLa S3, giant cells induced by 10-20Gy irradiation in the absence of CB exhibited a radiation dose-dependent reduction in the relative frequency of highly polyploid cells which was similar to that observed in CB-induced polykaryons.

Flow cytometric studies of the survival of cytochalasin-induced polyploidy in Chinese hamster ovary cells.

A method has been developed to estimate the post-irradiation survival of cytochalasin B-induced polyploidization of adherent Chinese hamster ovary cell using the flow cytometer. After exposure to radiation, surviving cells are allowed to become polyploid in the presence of cytochalasin, and are detached using trypsin, fixed by the addition of glutaraldehyde and stained using mithramycin. DNA content distributions are polymodal, and the absolute number of cells per culture in any given ploidy class is estimated by reference to a non-fluorescent bead internal standard, detected using forward scatter. Post-irradiation survival is defined as the ability to reach a given DNA content, and is reduced exponentially with dose. A bioassay to determine optimum cytochalasin concentrations can be derived from the relative size of the 2C (G0/G1) peak in the DNA content distribution. At culture densities greater than about 8 x 10(4) cell/cm2 the relative number of cells reaching at least 16C is reduced, but this inhibition is partially reversible by an increase in the medium glucose concentration, but not by the use of cytochalasin D or dihydro B.

The survival of cytochalasin-induced multinucleation following irradiation of Chinese hamster ovary cells.

Chinese hamster ovary cells were cultured for up to 280 hr in medium containing 1.75 mcg/ml cytochalasin B. The distribution of the number of nuclei per cell in unirradiated cultures on the 6th day was unimodal with some cells containing 27 or more nuclei. The DNA content distribution was in contrast polymodal with the means of the two terminal major peaks occurring at approximately 40 and 80 units of DNA content (antimodes at 29 and 58 units), where 1 unit is the content of untreated G1 cells. Irradiation (gamma, 137-Cs) at doses up to 10 Gy caused an exponential reduction in the proportion of plated cells able to reach high nucleus- or DNA-contents. The reduction due to 5 Gy was stable at least up to 280 hr in culture. The accumulation of total DNA in the culture was well-fitted by a Gompertz function, with little further increase after 230 hr when the average DNA content per cell reached about 90 units.