Cisplatin-DNA adduct formation in patients treated with cisplatin-based chemoradiation: lack of correlation between normal tissues and primary tumor.

PURPOSE: In this study, the formation of cisplatin-DNA adducts after concurrent cisplatin-radiation and the relationship between adduct-formation in primary tumor tissue and normal tissue were investigated. METHODS: Three intravenous cisplatin-regimens, given concurrently with radiation, were studied: daily low-dose (6 mg/m(2)) cisplatin, weekly 40 mg/m(2), three-weekly 100 mg/m(2). A (32)P-postlabeling technique was used to quantify adducts in normal tissue [white blood cells (WBC) and buccal cells] and tumor. RESULTS: Normal tissue samples for adduct determination were obtained from 63 patients and tumor biopsies from 23 of these patients. Linear relationships and high correlations were observed between the levels of two guanosine- and adenosine-guanosine-adducts in normal and tumor tissue. Adduct levels in tumors were two to five times higher than those in WBC (P<0.001). No significant correlations were found between adduct levels in normal tissues and primary tumor biopsies, nor between WBC and buccal cells. CONCLUSIONS: In concurrent chemoradiotherapy schedules, cisplatin adduct levels in tumors were significantly higher than in normal tissues (WBC). No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies. This lack of correlation may, to some extent, explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as a predictive test in anticancer platinum therapy.

Rapid fluorescence-based assay for radiosensitivity and chemosensitivity testing in mammalian cells in vitro.

An efficient and rapid cytotoxicity assay has been developed, particularly for radiobiological studies, utilizing 96-well microtiter plates. Several days after treatment, cell numbers per well were measured by fluorescent intensity using an automatic reader after staining with the DNA specific dye Hoechst 33258. For radiobiological applications, a microtiter plate irradiation box was designed and built which allowed a variable number of wells (minimum 4, maximum 16) to be irradiated at one time. In this manner, complete dose-response curves could be obtained from one plate. The assay depends on the growth of surviving and untreated cells, and by appropriate choice of conditions (cell numbers plated, time of assay), cell survival curves for this quick fluorescence assay were in reasonable agreement with those from a clonogenic assay for cisplatin and X-ray-induced cell killing. The assay can span 1.5-2 decades of cell survival and is suitable for any cell line which grows as a monolayer. Radiobiological applications were tested using agents or conditions which modified radiation damage. Firstly, sublethal damage repair could be demonstrated in RIF1 mouse tumor cells by comparing the survival curve for a single X-ray dose with that for two fractions separated by 4 h. Secondly, incorporation of 5-iodo-2'-deoxyuridine into cellular DNA was shown to radiosensitize Chinese Hamster cells, with similar enhancement ratios obtained from the fluorescence and clonogenic assays. Thirdly, radiosensitization by cisplatin and radioprotection by cysteamine could be readily measured using the quick fluorescence assay. The ability to have multiple dose groups per plate makes it an efficient assay for both radiosensitivity and chemosensitivity testing.

Tolerance of previously irradiated mouse kidneys to cis-diamminedichloroplatinum(II).

In this study the tolerance of previously irradiated kidneys to retreatment with chemotherapy was assessed. cis-Diamminedichloroplatinum(II) (c-DDP) was given to groups of mice at 1, 3, or 6 months after bilateral renal irradiation with single doses of 8-14 Gy. Renal function was measured monthly (by clearance of 51Cr ethylenediaminetetraacetic acid) from 4-35 weeks after c-DDP injection and results were compared with function after X-rays alone or drug alone. At early testing times (during the first 11 weeks after c-DDP injection) the renal function of mice given drug at 1 or 3 months after irradiation was very similar to that seen after drug alone. c-DDP given at 6 months caused slightly more damage than either drug or X-rays alone, but these results could be explained in terms of additive toxicities. At later testing times (11-35 weeks after c-DDP injection), renal function was much worse in all animals which had received previous irradiation, with the greatest damage when c-DDP was given 6 months after X-rays. This may be partly due to additional cell killing by the drug causing the expression of subclinical radiation injury. It is also possible that c-DDP pharmacokinetics was altered in animals with previously irradiated kidneys, leading to higher drug exposures in these mice.

Reduction of systemic exposure and toxicity of cisplatin by encapsulation in poly-lactide-co-glycolide.

The tissue distribution and normal tissue toxicity of cisplatin (cDDP) administered as poly-lactide-co-glycolide (PLAGA) microspheres, developed for loco-regional administration of cDDP to the liver, were studied in Wag/Rij rats. Venoportal administration of this formulation resulted in a reduction in total systemic and renal toxicity, which correlated with a decrease in normal tissue exposure to cDDP while maintaining high liver platinum levels. Liver-to-kidney platinum level ratios were 28 times higher after 4 h and 19 times higher after 24 h with PLAGA-cDDP microspheres than with free cDDP. Liver-to-blood platinum ratios at these times were 38 times and 36 times higher using PLAGA-cDDP. In a CC531 colon carcinoma liver micrometastases model, cytotoxicity of microsphere-released cDDP was confirmed in vivo by equal inhibition of tumor growth by PLAGA-cDDP and free cDDP over a period of 26 days. Free cDDP, however, caused significantly more histological renal damage and total body weight loss. The results were supported by the finding of higher plasma creatinine and urea concentrations 26 days after administration of free cDDP. Kidney platinum levels were 7 times lower when PLAGA-cDDP was used. These findings indicate a sparing effect on normal tissues when cDDP is targeted to the liver by formulation in PLAGA. PLAGA-cDDP microspheres may, therefore, be a useful and effective addition to current techniques of loco-regional chemotherapy for disseminated hepatic tumors.

Effects of hypoxia, pH, and growth stage on cell killing in Chinese hamster V79 cells in vitro by activated cyclophosphamide.

Several factors which influence the sensitivity of Chinese hamster V79 cells to cyclophosphamide (CY) have been studied in vitro in both suspension and monolayer cultures. Activated CY was obtained from the blood of mice 15 to 30 min after i.p. injection of CY (400 mg/kg). At pH 7.4, hypoxia rendered the cells more sensitive to activated CY. At lower values of pH (6.6 and 7.0), there was no difference between the sensitivities of oxic and hypoxic cells, although cells in both conditions were more sensitive to CY than at pH 7.4. Drug sensitivity was markedly affected by the stage of cell growth. Monolayer cultures were most sensitive to CY within a few hours of plating. Cultures then rapidly became less sensitive, with maximum resistance occurring between 24 and 48 h after plating, while the cells were still exhibiting rapid exponential growth. This development of resistance parallelled the formation of small colonies (2 to 4 cells), implying that intercellular contact may confer resistance to killing by activated CY.

Defective repair of DNA double-strand breaks and chromosome damage in fibroblasts from a radiosensitive leukemia patient.

A radiation-sensitive fibroblast culture (180BR) established from an acute lymphoblastic leukemia patient who died following radiotherapy is defective in the repair of radiation-induced DNA double-strand breaks. The cells also show a reduced capacity to repair interphase chromosome damage visualized by means of premature chromosome condensation and metaphase chromosome aberrations measured by fluorescence in situ hybridization on chromosome 4. This case represents the first example in humans where hypersensitivity to ionizing radiation can be ascribed directly to a defect in DNA and chromosome repair, and the defect may underlie the cancerous phenotype observed.

Characterization of resistance mechanisms to cis-diamminedichloroplatinum(II) in three sublines of the CC531 colon adenocarcinoma cell line in vitro.

Cisplatin resistance was developed in sublines of the CC531 rat colon adenocarcinoma cell line by continued low level drug exposure. Two relatively stable lines were obtained (RL2 and RL4) which were 6- and 20-fold more resistant to cisplatin. In addition, a subline more sensitive than the parent line by a factor of 2 (RLS) was obtained by subculture from a treated tumor. Mechanisms of resistance to cisplatin were investigated in these four lines, with the aim of determining the relative contributions of different resistance mechanisms at various resistance levels. Drug accumulation linearly decreased with increasing drug resistance. A 20-fold resistance was associated with only a 5-fold decrease in accumulation, suggesting that other resistance mechanisms may be involved in the total degree of resistance. Intracellular glutathione, measured fluorometrically, also increased with increasing resistance, varying by a factor of 4 between the most and least resistant lines. Reduction of glutathione levels by buthionine sulfoximine to parent line levels increased sensitivity but the cells remained considerably more resistant than parent cells. Resistant lines cultured in the absence of drug became progressively more sensitive, without accompanying changes in total glutathione levels. DNA-drug adducts, the presumed toxic lesion, were measured immunocytochemically. Initial levels decreased with increasing platinum resistance, although not proportional to resistance (factor of 5 decrease for 20-fold resistance). Drug dose ratios for equal initial adducts were similar to dose ratios for equal drug accumulation, implying that intracellular concentrations solely determine DNA adduction and that differences in glutathione level had little influence on the proportion of drug which eventually formed adducts. After 48 h, a better correlation between remaining adducts and resistance was found (factor 12 less adducts for 20-fold resistance). This implies that repair of adducts was important in determining survival. These data indicate that decreased drug accumulation played a proportionally greater role in the moderately resistant cell line and that adduct repair played a progressively greater role in the highly resistant cell line.

Monitoring of interaction products of cis-diamminedichloroplatinum(II) and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in cells from platinum-treated cancer patients.

The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.

Immunocytochemical detection of interaction products of cis-diamminedichloroplatinum(II) and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in rodent tissue sections.

Calf thymus DNA was modified in vitro by cis-diamminedichloroplatinum(II) (cisDDP), complexed with methylated bovine serum albumin and used to immunize rabbits. The anti-cisDDP-DNA antiserum obtained was applied in a double peroxidase-antiperoxidase staining procedure to localize cisDDP-DNA and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA)-DNA interaction products in cryostat tissue sections of mice and rats. Rats received cisDDP (0-10 mg/kg) and were killed after 24 h. Mice received cisDDP (0-15 mg/kg) or CBDCA (200 mg/kg), and were killed after 2 h-162 days. For each time-dose combination two mice or one rat were used; agents were given i.p. Specific nuclear staining was observed in all tissues examined from cisDDP- or CBDCA-treated animals. No significant nuclear staining could be observed in tissue sections from control rats and mice. The extent of staining after cisDDP was dose and time dependent. The lowest dose of cisDDP after which specific nuclear staining could be detected varied from tissue to tissue [e.g., 0.1 mg/kg, pancreas (mouse); 0.5 mg/kg, liver, kidney (mouse, rat)]. The longest time interval after a single dose of 6 mg/kg cisDDP in which adducts could be visualized also depended on the tissue and varied between 9 days (spleen, testis) and 162 days (kidney). The staining intensity in liver and kidney, measured microdensitometrically, decreased relatively fast in the first days after treatment, but much slower thereafter. In the kidney, cisDDP-induced DNA modification showed regional variation: inner cortex greater than outer cortex greater than medulla (rat) and cortex greater than medulla (mouse). In the mouse kidney, a small subpopulation of tubular cells in close association with the renal corpuscles showed a remarkably high staining intensity after both cisDDP and CBDCA administration. Tissues that showed clear cisDDP-induced histological alterations (kidney, pancreas, testis, and duodenum) also showed moderate to high levels of cisDDP-DNA interaction products. A correlation between cell damage (measured histologically) and cisDDP-DNA binding within one tissue type was demonstrated in the rat inner renal cortex, the murine renal cortex, and in duodenal epithelial cells of both mice and rats.

Correlation between cell killing by cis-diamminedichloroplatinum(II) in six mammalian cell lines and binding of a cis-diamminedichloroplatinum(II)-DNA antiserum.

The relationship between cell killing and the binding of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) to DNA was studied in six mammalian cell lines. Two of the human cell lines (COV413B) were of the same origin, comprising one sensitive to cis-DDP and the other with induced resistance to the drug. The four other lines, two rodent (RIF-1, Chinese hamster ovary) and two human (A2780, A1847), were unrelated. The cell lines differed in their sensitivity to cis-DDP, as tested in a clonogenic assay. cis-DDP-DNA binding was determined by quantitative immunocytochemistry using an antiserum against cis-DDP-modified DNA. The resistance factors relative to RIF-1, calculated from full survival curves for cis-DDP, were 3.8 +/- 0.4 for Chinese hamster ovary cells and 8.8 +/- 0.7 for both A2780 and A1847 lines. Using quantitative immunocytochemistry, the levels of the adduct-specific nuclear staining density compared with RIF-1 cells were 4.8 +/- 0.2 for Chinese hamster ovary cells, 9.1 +/- 0.2 for A2780, and 10.0 +/- 0.1 for A1847 cells, i.e., in good agreement with the resistance factors. In studies with the COV413B cells and their cis-DDP-resistant counterpart COV413B-PtR, immunologically detected adduct levels again correlated closely with resistance factors (correlation coefficient = 0.97). The kinetics of cis-DDP-DNA adduct formation and loss was investigated in RIF-1, A2780, and A1847 cells by the immunocytochemistry technique. Adduct levels after a 1-h incubation with approximately equitoxic doses of cis-DDP increased by 18 to 32% (average, 27%) between 0 and 6.5 h after treatment and then declined. Adduct half-lives in this latter phase did not correlate with the sensitivities of the cells for cis-DDP. These results indicate that the initial level of cis-DDP-DNA binding measured by quantitative immunocytochemistry may be a reasonable predictor of sensitivity to this chemotherapeutic drug.

Tumour cell repopulation during fractionated radiotherapy: correlation between flow cytometric and radiobiological data in three murine tumours.

This study tested whether the potential doubling time of tumour cells measured before or during treatment could predict the repopulation rate of surviving clonogens during fractionated radiotherapy. Tumours used for the study were a fibrosarcoma (SSK 2), an adenocarcinoma (AT 7) and a squamous cell carcinoma (AT 478), all grown subcutaneously in the C3H mouse. Potential doubling times (Tpot) were measured using the thymidine nanalogue iododexyuridine (IUdR) and flow cytometry. Results were compared with previous radiobiological studies on these tumours in which repopulation rates during radiotherapy were estimated using the tumour growth delay and tumour cure assays. Fractionated treatments consisted of daily doses of 4 or 8 Gy to clamped (hypoxic) tumours, 6 days per week for 1-3 weeks. Tpot values increased markedly during therapy for two of the tumours (SSK 2 and AT 478), by a factor of more than 10 for AT 478 in the third treatment week. Tpot remained approximately constant for the third tumour (AT 7). In no case was there evidence from the labelling studies of a shortening of Tpot which would suggest accelerated repopulation. From the radiobiological data, effective clonogen doubling times during radiotherapy were calculated from the doses required to produce a given effect in short and long treatment schedules. In the second week of treatment, effective clonogen doubling times in two tumours were approximately equal to the pretreatment Tpot, and shorter than the pretreatment Tpot in the third tumour. At some time during treatment, the surviving clonogens in these tumours therefore proliferated at the same rate or faster than before treatment. The difference between the labelling and radiobiological measurements was ascribed to the fact that, shortly after the start of a fractionated treatment, the IUdR labelling technique measures primarily doomed cells. These results show that kinetic measurements using DNA labelling techniques made during fractionated radiotherapy in most cases do not reflect the proliferation status of the surviving cells which are responsible for treatment outcome. Pretreatment Tpot measurements give a much better indication of the proliferation rate of surviving cells but in some cases may underestimate repopulation during radiotherapy.

Intraperitoneal cisplatin with regional hyperthermia in advanced ovarian cancer: pharmacokinetics and cisplatin-DNA adduct formation in patients and ovarian cancer cell lines.

The purpose of this study was to investigate the influence of hyperthermia on cisplatin pharmacokinetics and DNA adduct formation. The latter was investigated both in tumour cell lines in vitro and in tumour cells and buccal cells from cancer patients. The patients had advanced ovarian carcinoma and were entered into a phase I study for cytoreductive surgery followed by hyperthermia in combination with intraperitoneal cisplatin administration. The cisplatin-DNA modifications in vivo and in vitro were studied by an immunocytochemical method with the polyclonal antiserum NKI-A59. The patient samples for pharmacokinetic determinations were analysed by flameless atomic absorption spectrometry. In vitro, the combination of hyperthermia and cisplatin enhanced cell killing compared with either treatment alone, such that the cisplatin-resistant ovarian cell line A2780/DDP became almost as sensitive as the parent A2780 cell line (resistance factor reduced from 30 to 2 at the IC50). In addition, increased cisplatin-DNA adducts were observed in the resistant cell line after the combined treatment compared with cisplatin alone. A good correlation was found between nuclear staining density and surviving fraction for all groups, indicating that the DNA adducts generated are an important determinant of toxicity and that the mechanism by which hyperthermia enhances kill is by increasing adduct levels. In the patients, the ratio of drug concentration in the peritoneal perfusate compared with that in plasma was found to be approximately 15, indicating a favourable pharmacokinetic ratio. Cisplatin-DNA adduct formation in tumour cells from patients was higher than in buccal cells, reflecting this higher drug exposure, i.e. local plus systemic versus systemic only. In addition, the tumour cells but not buccal cells were exposed to hyperthermia. The higher number of tumour adducts also suggests that a favourable therapeutic ratio could be achieved. Platinum-DNA adduct formation was found to decrease with distance from the surface of the tumour nodules. However, at a distance of 3-5 mm, the nuclear staining density levels were still measurable and higher than in buccal cells. In conclusion, the combined pharmacokinetic and adduct data in patients support the advantages of the intraperitoneal route for drug administration, and the addition of heat.

Additivity versus repair inhibition in fractionated treatments combining drugs and X rays: a theoretical analysis.

Drugs which inhibit the repair of radiation damage could potentially be useful for enhancing the effects of radiotherapy. In pre-clinical combined modality studies, however, it is often difficult to state with certainty whether or not a drug has inhibited radiation damage repair. This paper shows that several commonly used parameters for assessing repair can give the wrong answer regarding the presence of drug-induced repair inhibition. These parameters are; the difference in radiation dose between 1 and n fractions to give the same effect, the fractional recovered dose per fraction interval, FR, and the related parameter FREC. A further parameter used for treatment comparisons is the enhancement ratio for the drug (D.E.R.; ratio of radiation doses, with and without drug, to cause a given effect). An increasing D.E.R. with increasing number of radiation fractions has been taken as an indication that the drug inhibited repair. The present report demonstrates that this, too, can be misleading. From an analysis based on a linear-quadratic survival curve for X rays, it is suggested that deriving and comparing alpha/beta ratios (ratio of the linea to quadratic coefficients) gives the best indication of drug-induced changes in survival curve shape which may reflect underlying changes in repair capacity.

Cisplatin and radiation: interaction probabilities and therapeutic possibilities.

This paper examines the probability of interactions occurring between drug lesions and radiation lesions in DNA for the cytotoxic and radiosensitizing agent cisplatin. The number of cisplatin-induced DNA adducts and radiation-induced strand breaks after a given dose of each agent are known for given cell systems, from which the probability that these lesions will interact can be estimated. Results of these calculations indicate that the probability of interaction could be high, depending on the distance over which two lesions can interact and the probability of repair of the interaction lesion. Calculated lesion numbers have been compared with known data on radiation modification, including illustrations of inconsistencies. In the second part of the paper, ways in which combined therapy with cisplatin and radiation can be improved are described. Development of methods to predict which types of tumor and which individual tumors within a given type are sensitive to the cytotoxic and radiosensitizing effects of the drug would aid rational selection of patients for combination treatments. Immunocytochemical methods sensitive enough to monitor cisplatin-DNA interactions in patients are available and may be useful in this context. The delivery and maintenance of higher tumour concentrations of radiosensitizer offers a further possibility for improvement. Studies of intratumoral injection of cisplatin have shown promise for achieving this goal while limiting normal tissue toxicity.

Repair of sublethal radiation injury after multiple small doses in mouse kidney: an estimate of flexure dose.

Functional kidney damage in mice was measured after a series of fractionated X-irradiations. Doses per fraction of 0.75-12.5 Gy were given as 2, 5, 10, 30, 40, 60, or 80 equal doses in a total treatment time of 4 weeks. Renal function (measured by clearance of 51CrEDTA or hematocrit levels) deteriorated progressively, in a dose related manner, from 20 to 46 weeks after the start of treatment. The changes in renal function versus time were fitted by a polynomial regression through all data and interpolated values for 51CrEDTA clearance were then calculated at 30 and 40 weeks after treatment. Steep dose response curves were obtained and these were used to calculate isoeffective doses for the different fractionation schedules. There was a marked increase in total isoeffective doses from 2-30 fractions and these data were well described by a linear quadratic (L.Q.) expression for damage with an alpha/beta ratio of 2.3 +/- 0.2 Gy. There was only a slight increase in the total isoeffect dose as the size of the dose per fraction was decreased below 2 Gy and the measured isoeffect doses after 40 to 80 fractions were lower than predicted on the basis of an L.Q. model assuming complete repair between successive irradiations. The flexure dose for mouse kidneys irradiated 3 times per day was, effectively, 1 to 2 Gy and hyperfractionation using lower doses per fraction did not lead to significant, additional repair.

Influence of cis-diamminedichloroplatinum (II) on mouse duodenal crypt stem cell survival after multifraction X ray treatment.

The mechanism of interaction of cis-platinum and X rays was investigated in mouse duodenal crypts using the microcolony assay. Mice were exposed to 1, 2, 5, 10, or 15 fractions of X rays, either alone or preceded by a single i.p. injection of cis-platinum, 8 mg/kg, one-half hour before the first fraction. In all fractionation regimens, cisplatinum caused a shift of the X ray survival curve for crypt cells towards lower doses. The vertical distances between the survival curves after X rays and those in combination with cis-platinum were about the same, giving a mean value of 0.89 +/- 0.12 log10 cells. After cis-platinum treatment alone, a crypt cell survival curve was established in the high dose range. The estimated cell kill by 8 mg/kg of cis-platinum, obtained by extrapolation of this curve, was 1 log10 cell number. These data imply independent cell killing mechanisms for cis-platinum and X rays. However, even after correction for cell kill by the drug, cis-platinum tended to inhibit slightly sublethal damage repair after X rays. This was supported by linear quadratic analyses, in which the alpha/beta value after combined treatment was found to be slightly higher than after X rays alone (20 +/- 4 Gy versus 13.0 +/- 1.7 Gy).

Cell kinetic measurements in prostate cancer.

PURPOSE: Two approaches have been suggested for escalating the total dose in radiotherapy treatment of prostate cancer. One is conformal radiotherapy; the other is hyperfractionation using many small fractions. Both imply some possible prolongation in overall treatment time. To judge whether prolonged treatment schedules would be detrimental, it is necessary to know the proliferation rates in human prostate tumors, specifically, the potential doubling time (Tpot). There is a lack of data on this parameter in the literature. METHODS AND MATERIALS: Seven patients with adenocarcinoma of the prostate were studied. A tracer dose of 100 mg/m2 of IUdR was infused intravenously 4-12 h before biopies were taken. Biopsies were fixed in 70% ethanol, stored at 4 degrees C, and later prepared and stained by standard methods for flow cytometry, using the red fluorescence signal for DNA and the green fluorescence signal (fluorescein isothiocyanate) for 5-iodo-2'-deoxyuridine. The duration of DNA synthesis (Ts) was determined by the relative movement (RM) method, knowing the interval between tracer administration and biopsy. Tpot was calculated as the quotient of Ts by labeling index (LI). RESULTS: In two of the seven tumors the LI was too low (<0.6%) for a reliable estimate of RM to be made, so no determination of Tpot was possible for these tumors. The mean LI values in the other five tumors were 2.4%, 1.4%, 1.0%, 3.0%, and 0.9%. The durations of Ts were 13.2, 9.5, 10.0, 11.7, and 12.7 h, respectively. The resulting values of Tpot were 23, 28, 42, 16, and 61 days, respectively. CONCLUSION: The low labeling indices in prostate tumors, also reported by others, made estimation of Ts by RM impossible in about a third of these tumors. However, five tumors yielded long estimates for Tpot, implying that prolongation from 6 to about 8 weeks should not be detrimental.

The predictive value of cell kinetic measurements in a European trial of accelerated fractionation in advanced head and neck tumors: an interim report.

The value of cell kinetic measurements in head and neck tumors in predicting which patients will benefit from accelerated fractionation radiotherapy regimens is being tested in a multicenter European trial (EORTC trial 22851). This paper reports on the first analysis of the correlation of kinetics with outcome in this trial. A proportion of patients in both the accelerated arm (72 Gy in 5 weeks, 1.6Gy per fraction, 45 fractions) and the conventional arm (70-72 Gy in 7-8 weeks, 1.8-2.0 Gy per fraction, 35-40 fractions) were given an i.v. injection of 100 mg/m2 IUdR (iododeoxyuridine) before treatment, and a tumor biopsy was taken several hours later. The potential doubling time of the tumor (Tpot) was obtained from a flow cytometric analysis of tumor cell nuclei using an anti-IUdR antibody. From a total of 260 patients entered in the trial, 53 have undergone kinetic analysis. Adequate IUdR labeling was seen in 47 patients (88.7%), from which the mean value for Tpot was found to be 4.5 +/- 2.5 days (+/- S.D.). Of the IUdR labeled patients, 30 have now been followed up for at least 1 year, 17 with conventional and 13 with accelerated radiotherapy. These patients were split into those with fast and those with slowly growing tumors, the dividing line being the median Tpot value of 4.6 days. After conventional 7-week radiotherapy, 2 of 6 patients with "fast" growing tumors obtained local control compared with 8 of 11 with "slow" growing tumors. A small difference in local control was seen been fast and slow tumors in the accelerated arm (5/9 vs. 3/4). These preliminary data support the hypothesis that patients with fast growing tumors do poorly with conventional radiotherapy and that pretreatment kinetic measurements can select patients at risk. The predictive power of the method must await the final analysis of trial results.

The sensitivity of normal stroma to fractionated radiotherapy measured by a tumour growth rate assay.

The growth rate of implanted tumours has been used as an assay for radiation injury in normal stroma. The subcutaneous tissue was irradiated in an unstimulated state and was then stimulated to produce new blood vessels by the inoculation of a syngeneic tumour 3 days after the last irradiation. Steep dose-response curves were obtained with a dose resolution of approximately 1 Gy. The time between irradiation and stimulation of the stroma by the tumour implant was shown to have no effect on tumour growth rate for times ranging from 1 h to 14 days after single doses. This functional assay of stromal damage has been used after irradiation with 1, 2, 5, 10 or 20 fractions of X-rays. Isoeffect data were well fitted by a linear-quadratic equation, for which the ratio of linear to quadratic coefficients (alpha/beta) was 6.2 +/- 0.6 Gy. This is on the high end of the range of published alpha/beta values for late reacting tissues and other stromal/vascular assays, but lower than those for all early reacting tissues. Overall treatment times ranging from 1 to 11 days were tested with the 2 and 5 fraction schedules. No effect attributable to slow repair or repopulation could be demonstrated over this period.

The effect of cisplatin on the repair of radiation damage in RIF1 mouse tumours in vivo.

The effect of the antitumour agent cisplatin on repair of X-ray-induced damage was studied in RIF1 mouse tumours treated in situ. The response of tumours, assessed by growth delay, to 4 fractions of X-rays given at 5-h intervals was compared with that after single doses. The displacement between the curves was taken as a measure of repair. A single dose of 6 mg.kg-1 cisplatin given 0.5 h before the first fraction resulted in no detectable inhibition of repair despite a significant growth delay caused by drug alone. A dose of 2 mg/kg cisplatin given 0.5 h before each of the X-ray fractions did, however, cause some repair inhibition; a result confirmed by tumour control experiments. The schedule dependence for repair inhibition was the same whether the irradiations were carried out on clamped (fully hypoxic) tumours or under ambient conditions. Significant enhancement of radiation damage was seen after correcting for the effects of drug alone, whether or not repair inhibition occurred. The effects of cisplatin on normal stroma within the tumour (vascular damage) was also investigated by monitoring the regrowth rates of recurrent tumours. In contrast to the effects on tumour cells, no enhancement of damage or inhibition of repair was seen for this assay in the combined treatment schedules.