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.

Hypoxia in head and neck cancer: how much, how important?

BACKGROUND: Hypoxia develops in tumors because of a less ordered, often chaotic, and leaky vascular supply compared with that in normal tissues. In preclinical models, hypoxia has been shown to be associated with treatment resistance and increased malignant potential. In the clinic, several reports show the presence and extent of tumor hypoxia as a negative prognostic indicator. This article reviews the biology and importance of hypoxia in head and neck cancer. METHODS: A review of literature was carried out and combined with our own experience on hypoxia measurements using exogenous and endogenous markers. RESULTS: Hypoxia can increase resistance to radiation and cytotoxic drugs and lead to malignant progression, affecting all treatment modalities, including surgery. Hypoxia measurements using electrodes, exogenous bioreductive markers, or endogenous markers show the presence of hypoxia in most head and neck cancers, and correlations with outcome, although limited, consistently indicate hypoxia as an important negative factor. Each hypoxia measurement method has disadvantages, and no "gold standard" yet exists. Distinctions among chronic, acute, and intermediate hypoxia need to be made, because their biology and relevance to treatment resistance differ. Reliable methods for measuring these different forms in the clinic are still lacking. Several methods to overcome hypoxia have been tested clinically, with radiosensitizers (nimorazole), hypoxic cytotoxins (tirapazamine), and carbogen showing some success. New treatments such as hypoxia-mediated gene therapy await proper clinical testing. CONCLUSIONS: The hypoxia problem in head and neck cancer needs to be addressed if improvements in current treatments are to be made. Increased knowledge of the molecular biology of intermediate, severe, and intermittent hypoxia is needed to assess their relevance and indicate strategies for overcoming their negative influence.

Rapid fluorescence ratio assay for detecting changes in radiosensitivity.

To test modifications in sensitization to radiation or drugs in preclinical studies of cancer therapy, the colony-forming assay is regarded as the gold standard. Because this assay is time consuming, somewhat laborious, and unsuitable for rapid screening, development of other assays is desirable. We describe here an assay based on the detection of enhanced green fluorescence protein (EGFP) with flow cytometry that is particularly suitable for genetic manipulation studies in which the gene of interest is introduced together with EGFP as reporter. It is easily adaptable to other reporters, however, whether naturally fluorescent or requiring immunochemical staining. Cells are irradiated as mixed populations of a known standard cell line (nonfluorescent) together with the genetically manipulated cell line expressing EGFP. Ratios of fluorescent and nonfluorescent cells are measured before treatment and several days after treatment. If the cell populations have equal radiosensitivities, the ratio remains unchanged. Changes in the ratio indicate changes in radiosensitivity. The assay was validated for two situations in which dominant negative peptides inhibiting DNA repair were expressed in A549 human lung cells and affected radiosensitivity.

Accelerated hyperfractionation (AHF) compared to conventional fractionation (CF) in the postoperative radiotherapy of locally advanced head and neck cancer: influence of proliferation.

Based on the assumption that an accelerated proliferation process prevails in tumour cell residues after surgery, the possibility that treatment acceleration would offer a therapeutic advantage in postoperative radiotherapy of locally advanced head and neck cancer was investigated. The value of T(pot) in predicting the treatment outcome and in selecting patients for accelerated fractionation was tested. Seventy patients with (T2/N1-N2) or (T3-4/any N) squamous cell carcinoma of the oral cavity, larynx and hypopharynx who underwent radical surgery, were randomized to either (a) accelerated hyperfractionation: 46.2 Gy per 12 days, 1.4 Gy per fraction, three fractions per day with 6 h interfraction interval, treating 6 days per week or (b) Conventional fractionation: 60 Gy per 6 weeks, 2 Gy per fraction, treating 5 days per week. The 3-year locoregional control rate was significantly better in the accelerated hyperfractionation (88 +/- 4%) than in the CF (57+/- 9%) group, P=0.01 (and this was confirmed by multivariate analysis), but the difference in survival (60 +/- 10% vs 46 +/- 9%) was not significant (P=0.29). The favourable influence of a short treatment time was further substantiated by demonstrating the importance of the gap between surgery and radiotherapy and the overall treatment time between surgery and end of radiotherapy. Early mucositis progressed more rapidly and was more severe in the accelerated hyperfractionation group; reflecting a faster rate of dose accumulation. Xerostomia was experienced by all patients with a tendency to be more severe after accelerated hyperfractionation. Fibrosis and oedema also tended to be more frequent after accelerated hyperfractionation and probably represent consequential reactions. T(pot) showed a correlation with disease-free survival in a univariate analysis but did not prove to be an independent factor. Moreover, the use of the minimum and corrected P-values did not identify a significant cut-off. Compared to conventional fractionation, accelerated hyperfractionation did not seem to offer a survival advantage in fast tumours though a better local control rate was noted. This limits the use of T(pot) as a guide for selecting patients for accelerated hyperfractionation. For slowly growing tumours, tumour control and survival probabilities were not significantly different in the conventional fractionation and accelerated hyperfractionation groups. A rapid tumour growth was associated with a higher risk of distant metastases (P=0.01). In conclusion, tumour cell repopulation seems to be an important determinant of postoperative radiotherapy of locally advanced head and neck cancer despite lack of a definite association between T(pot) and treatment outcome. In fast growing tumours accelerated hyperfractionation provided an improved local control but without a survival advantage. To gain a full benefit from treatment acceleration, the surgery-radiotherapy gap and the overall treatment time should not exceed 6 and 10 weeks respectively.

Hypoxia and perfusion measurements in human tumors--initial experience with pimonidazole and IUdR.

We describe our preliminary studies on the development of methods to measure hypoxia in standard paraffin sections of human tumors. Three parameters were investigated. First, image analysis of tumor vascularity yielded the parameter diffusion limited fraction (DLF), which is the amount of tumor tissue greater than a fixed distance from the nearest blood vessel. Secondly, the amount of tumor tissue stained with antibodies against bound reduced products of the bioreductive marker pimonidazole was assessed. Finally, the fraction of blood vessels showing no surrounding tumor tissue labeled with lUdR, a cell kinetic marker, was measured. DLF and pimonidazole monitor primarily chronic hypoxia, while it is hypothesized that the IUdR-negative fraction monitors acute hypoxia. Feasibility was demonstrated in a series of 10 esophageal and 10 rectal tumors (no drug administration), 10 cervix tumors (pimonidazole) and 14 head and neck tumors (pimonidazole and lUdR). Significant differences between tumors were found for all parameters. DLF correlated significantly with the pimonidazole fraction when all images of all tumors were included, although mean values per tumor showed no correlation. The IUdR-negative fraction did not correlate with either of the other two parameters. We conclude that it is feasible to measure hypoxia-related, and possibly perfusion-related, parameters on paraffin sections for predictive purposes, although each method needs further validation. Each parameter will be correlated with outcome in a larger study on head and neck tumors treated with surgery with or without postoperative radiotherapy.

Use of thymidine analogues to indicate vascular perfusion in tumours.

Temporary reduction in blood-flow within tumour blood vessels can reduce oxygen supply leading to transient perfusion-limited hypoxia. Consequent selection of cells with mutations and reduced radiosensitivity can lead to disease progression and treatment-resistance. In the present study, we investigated whether heterogeneity of labelling after thymidine analogue administration is related to perfusion variations, and if so, could it be quantified and used as a perfusion indicator. Perfusion in murine RIF1 tumours was reduced by hydralazine or increased by nicotinamide and the mice subsequently injected with IdUrd. Tumours were halved for analysis by both flow cytometry and immunohistochemistry. Tumour sections were stained for vasculature and IdUrd. Each blood vessel was scored for the density of IdUrd-labelled cells surrounding it, using a semi-quantitative scoring system. Flow cytometry showed that the IdUrd labelling index and intensity decreased by approximately 50% after hydralazine. In tumour sections of control animals, 2.9% of vessels showed no IdUrd label. In contrast, after hydralazine almost 50% of vessels had no surrounding IdUrd labelling, whereas after nicotinamide there were fewer vessels with low labelling and a higher median score. In conclusion, changes of tumour perfusion by pharmacological agents is reflected in changes in tumour-cell labelling by the thymidine analogue IdUrd, suggesting that IdUrd labelling could be used to indicate perfusion in individual vessels in human tumours.

Diffusion limited hypoxia estimated by vascular image analysis: comparison with pimonidazole staining in human tumors.

PURPOSE: To assess diffusion limited hypoxia in human tumors using image analysis of vasculature and to compare it with the bioreductive marker pimonidazole as an independent method. MATERIALS AND METHODS: To set up the method, nine rectal adenocarcinomas and ten squamous cell carcinomas were analyzed. To validate the method, ten squamous cell carcinomas of the cervix were analyzed from patients who were injected with pimonidazole and biopsied approximately 24 h later. Sections of the rectal and esophageal tumors were stained for vasculature, while cervix tumor sections were double stained for vasculature and pimonidazole. Tumor areas were delineated on digitized images, and the proportion of tumor tissue greater than a fixed distance from the nearest blood vessel (called diffusion limited fraction, DLF) was then calculated. The proportion of tumor area stained for pimonidazole was also measured. RESULTS: There was a wide variation between tumors in both the vascular-derived DLF and in the pimonidazole-stained fraction. Average DLFs varied between 1.5 and 92% for different tumors, with significant differences between them. The area stained by pimonidazole was significantly smaller than DLF for all tumors. The correlation between pimonidazole area and DLF was significant in three of seven tumors containing > or = 3 images. When images from all tumors (n=123) were analyzed together, the correlation was highly significant (r=0.47, P<0.0001). CONCLUSION: The vascular derived DLF correlates significantly with pimonidazole staining, but there was large scatter. Both methods may underestimate perfusion limited hypoxia.

In vitro differentiation characteristics of human skin fibroblasts: correlations with radiotherapy-induced breast fibrosis in patients.

PURPOSE: To determine whether there is an association between dermal fibroblast differentiation characteristics in vitro and breast fibrosis developing in patients following radiotherapy for breast cancer. MATERIALS AND METHODS: Three hundred and eighty-five patients had been characterized for the degree of breast fibrosis and the level of clinical risk factors for fibrosis as established by logistic regression. Early-passage fibroblasts from 79 patients with a high (HR) or low (LR) level of risk factors were studied in vitro. The percentage differentiated cells (%DC) 7 days after 0 and 8 Gy was scored, and unirradiated colonies were scored for the ratio of early:late fibroblast differentiation stages (E:L ratio). RESULTS: %DC: For the 0 Gy data there was a significant interpatient variation (CoV = 55%, p = 0.0001). HR patients with breast fibrosis had a higher %DC compared with patients without (p = 0.017). E:L ratio: for HR patients there was a significant interpatient variation (82%, p = 0.0030) and a lower E:L ratio for patients with fibrosis compared with those without (p = 0.086), but for LR patients this relationship was reversed (p = 0.079) CONCLUSIONS: There was a true interpatient variation in the in vitro parameters of fibroblast differentiation but insufficient correlation with observed fibrosis after radiotherapy for use as a predictive test.

Translational research offers individually tailored treatments for cancer patients.

The measurement of the effect of cisplatin on DNA has become feasible with the development of antibodies against DNA adducts. In a phase II dose escalation trial with concomitant radiotherapy and daily cisplatin in lung cancer, we found that patients with high DNA adduct levels measured in the buccal mucosa had a much higher survival rate than patients with a low or undetectable amount of cisplatin-DNA adducts. The use of this assay may therefore allow the selection of individual patients for concomitant treatment with cisplatin and radiotherapy, as has been shown to be effective in randomized trials in patients with lung, head and neck, and cervix malignancies. To predict the response to radiation treatment, assays have been developed for tumor growth potential by measuring the labeling index after intravenous injection of IdUrd or by estimating cyclin D1 expression. Intrinsic radiation sensitivity of human tumors can be estimated by conventional techniques, which are probably too slow or cumbersome for routine use, or with more rapid assays, such as those for chromosome damage with fluorescent probes. These assays should be able to guide us in the adaptation of the individual radiation doses that should be applied and to select patients for an accelerated or hyperfractionated regimen. Pretreatment levels of apoptosis may also be helpful in predicting treatment outcome, although the data so far show inconsistent results. A better understanding of the signal transduction pathways involved in radiation-induced apoptosis may help in the design of studies aimed at modulating the apoptotic response, thereby increasing cell kill. We have recently shown that alkyllysophospholipids, which inhibit mitogenic signaling, induce apoptosis in a variety of tumor cell lines. In combination with ionizing radiation, these compounds cause an enhancement of apoptotic cell kill. This type of a signaling-based intervention could form the basis for new therapeutic strategies. The role of hormonal therapy in breast cancer patients, both in an adjuvant setting and for the treatment of disseminated disease, is becoming increasingly important. The development of a functional assay for the estrogen receptor (ER-FASAY), based on a yeast growth assay, provides a better way than the classical immunohistochemistry assay of estimating abnormal function of the receptor in tumors. These assays are simply examples, illustrating how clinicians could improve the therapeutic outcome for their patients by implementing knowledge obtained in the laboratory in clinical decision making. With further optimization of these assays, this holds the promise for the future that the treatment for each patient can be tailored rationally to the biology of the individual.

DNA-adduct levels as a predictor of outcome for NSCLC patients receiving daily cisplatin and radiotherapy.

We aimed to investigate whether biological factors related to radiosensitivity and chemosensitivity have prognostic significance in non-small-cell-lung-cancer (NSCLC) patients treated with daily low doses of cisplatin and radiotherapy. We treated 27 NSCLC patients with concomitant daily low-dose cisplatin and radiotherapy between 1993 and 1995. Tumour specimens were analyzed for p53 and bcl-2 expression, and for cell proliferation using antibodies against ki-67. In addition, apoptosis was measured by an end-labeling technique (TUNEL). Finally, cisplatin-induced DNA modification in buccal cells was assessed immunocytochemically using a specific anti-serum. Univariate and multivariate analyses were performed to assess the association between the different variables and survival. The median follow-up was 41 months, and 21 patients (78%) have died. In a univariate analysis, age, tumour stage and cisplatin-DNA-adduct staining were the only factors significantly associated with survival (p < 0.05, log-rank test). p53, bcl-2, Ki-67 and apoptosis showed no relationship with outcome. Multivariate analysis revealed that cisplatin-DNA-adduct staining remained an independent prognostic factor (hazard ratio, 0.10, 95% CI, 0.02-0.49), with shorter survival times for patients with low adduct staining.

Potential of radiation-induced chromosome aberrations to predict radiosensitivity in human tumour cells.

PURPOSE: To validate whether the number of aberrations could be used as a measure of the radiosensitivity of human tumour cells. If so, this would potentially provide a more rapid method than the colony assay to predict radiocurability in human tumour biopsy material. MATERIALS AND METHODS: A panel of 13 human tumour cell lines was investigated, covering a wide range of radiosensitivities. Fluorescence in situ hybridization (FISH) employing whole chromosome probes was used to detect aberrations. RESULTS: A dose-dependent increase in radiation-induced chromosome aberrations was observed in all cell lines. A good correlation (r=0.90) was found between cell survival and total chromosome aberrations in 12 of the 13 cell lines (92%), with one exception. A poorer correlation was observed between cell survival and stable- (r=0.85) and unstable-type aberrations (r=0.81). Survival-aberration correlations for individual radiation doses were worse, although statistically significant. The exceptional cell line showed significantly more aberrations for a given level of cell kill than expected based on data for the other lines. CONCLUSION: This study indicates that radiation-induced chromosome aberrations can be used as a potential predictor of intrinsic radiosensitivity for the majority of human tumours when more than one dose level is tested. This could aid the design of radiotherapy schedules for each individual patient, or in the decision of whether to use an alternative therapy.

The value of pretreatment cell kinetic parameters as predictors for radiotherapy outcome in head and neck cancer: a multicenter analysis.

PURPOSE: The aim of this study was to assess the potential of pre-treatment cell kinetic parameters to predict outcome in head and neck cancer patients treated by conventional radiotherapy. MATERIALS AND METHODS: Data from 11 different centers were pooled. Inclusion criteria were such that the patients received radiotherapy alone, and that the radiotherapy was given in an overall time of at least 6 weeks with a dose of at least 60 Gy. All patients received a tracer dose of either iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) intravenously prior to treatment and a tumor biopsy was taken several hours later. The cell kinetic parameters labeling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were subsequently calculated from flow cytometry data, obtained on the biopsies using antibodies against I/BrdUrd incorporated into DNA. Each center carried out their own flow cytometry analysis. RESULTS: From the 11 centers, a total of 476 patients conforming to the inclusion criteria were analyzed. Median values for overall time and total dose were 49 days and 69 Gy, respectively. Fifty one percent of patients had local recurrences and 53% patients had died, the majority from their disease. Median follow-up was 20 months; being 30 months for surviving patients. Multivariate analysis revealed that T-stage, maximum tumor diameter, differentiation grade, N-stage, tumor localization and overall time correlated with locoregional control, in decreasing order of significance. For the cell kinetic parameters, univariate analysis showed that only LI was significantly associated with local control (P=0.02), with higher values correlating with a worse outcome. Ts showed some evidence that patients with longer values did worse, but this was not significant (P=0.06). Tpot showed no trend (P=0.8). When assessing survival in a univariate analysis, neither LI nor Tpot associated with outcome (P=0.4, 0.4, respectively). Surprisingly, Ts did correlate with survival, with longer values being worse (P=0.02). In the multivariate analysis of local control, LI lost its significance (P=0.16). CONCLUSIONS: The only pretreatment kinetic parameter for which some evidence was found for an association with local control (the best end-point for testing the present hypothesis) was LI, not Tpot, and this evidence disappeared in a multivariate analysis. It therefore appears that pretreatment cell kinetic measurements carried out using flow cytometry, only provide a relatively weak predictor of outcome after radiotherapy in head and neck cancer.

The presence of wild-type TP53 is necessary for the radioprotective effect of the Bowman-Birk proteinase inhibitor in normal fibroblasts.

In the present study we have demonstrated that the Bowman-Birk proteinase inhibitor (BBI) protected normal fibroblasts from a radiation-induced reduction in cell survival, whereas in transformed fibroblasts no radioprotective effect was observed. It was shown that BBI reduced the radiation-induced protein stabilization and DNA-binding activity of TP53 (formerly known as p53) in normal fibroblasts. In transformed fibroblasts, BBI failed to induce these effects. The analysis of the TP53 gene in transformed fibroblasts revealed a mutation in exon 5. As a consequence of this mutation, the expression of the TP53 downstream gene CDKN1A (p21/WAF1/Cip1) is blocked. Based on experiments using TP53 antisense oligonucleotides, the radioprotective effect of BBI could be correlated with the function of wild-type TP53. Thus BBI can be considered as a selective radioprotective agent for normal human fibroblasts.

Automatic detection of stable and unstable chromosome aberrations visualized by three-color imaging after fluorescence in situ hybridization with a painting and a pancentromeric DNA probe.

Two-color fluorescence in situ hybridization (FISH) in combination with digital image analysis was used to develop an automatic system for the detection and classification of chromosome aberrations. Algorithms were developed for the automatic thresholding of the three digitized images: an FITC image representing specific painted chromosomes, a TRITC image representing the centromeres of all chromosomes, and a DAPI image representing all the counterstained chromosomes. A further algorithm was developed for the automatic classification of the different types of chromosome aberrations, such as translocations, dicentrics, and fragments. For this study, a dataset of 252 metaphases were digitized and analyzed automatically as well as manually. Of these metaphases, 81.3% could be correctly classified by the algorithm. The error rate was reduced to 9.3% by automatically excluding the detected clusters and artifacts. The average analysis time per metaphase was 34.5 s without any user intervention.

Low predictive value of intrinsic fibroblast radiosensitivity for fibrosis development following radiotherapy for breast cancer.

PURPOSE: To test whether the intrinsic radiosensitivity of skin fibroblasts from breast cancer patients correlates with the degree of breast fibrosis after breast conserving therapy. METHODS: In a systematic study design, 79 patients were selected from an earlier study group of 385 patients based on observed fibrosis and seven identified clinical risk factors for fibrosis development. In vitro radiosensitivity of patients' dermal fibroblasts was determined by clonogenic assay of early passage cultures. Survival was determined after irradiation at 0, 2 and 4 Gy, given in two fractions of 2 Gy with a 6 h interval. RESULTS: There was a significant inter-patient variation for SF2 values (coefficient of variation = 40%). The ratio of SF2 values for fibroblasts from patients with breast fibrosis versus those without was 0.80 (95% CI: 0.60-1.07). This was a statistically non-significant trend (p = 0.13). The same ratio for a derived value for SF2 ((SF2 + square root of SF4)/2) was 0.88 (p = 0.19). CONCLUSIONS: A significant variation in intrinsic radiosensitivity of breast cancer patients' dermal fibroblasts was observed. However, the degree of radiosensitivity did not show a significant correlation with fibrosis development. This indicates that the use of fibroblast radiosensitivity will have a limited usefulness for predicting fibrosis following breast irradiation.

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.

Accelerated fractionation (AF) compared to conventional fractionation (CF) improves loco-regional control in the radiotherapy of advanced head and neck cancers: results of the EORTC 22851 randomized trial.

BACKGROUND AND PURPOSE: A 5 week-hyperfractionated and accelerated radiotherapy regimen without reduction of the total dose was developed to fight tumour repopulation during treatment and tumour hypoxia. The purpose of the study was to try to improve loco-regional control in high risk head and neck carcinoma treated with curative radiotherapy. METHODS AND MATERIALS: From 1985 to 1995, a randomised controlled trial of the EORTC Cooperative Group of Radiotherapy (EORTC 22851) compared the experimental regimen (72 Gy/45 fractions/5 weeks) to standard fractionation and overall treatment time (70 Gy/35 fractions/7 weeks) in T2, T3 and T4 head and neck cancers (hypopharynx excluded). The end-point criteria were local and loco-regional control, overall and disease-free survival, and acute and late toxicities. Five hundred twelve patients were accrued. RESULTS: Patients in the AF (accelerated fractionation) arm did significantly better with regard to loco-regional control (P = 0.02) resulting at 5 years in a 13% gain (95% CI 3-23% gain) in loco-regional control over the CF (conventional fractionation) arm. This improvement is of larger magnitude in patients with poorer prognosis (N2-3 any T, T4 any N) than in patients with more favourable stage. Multivariate analysis confirmed AF as an independent prognostic factor of good prognosis for loco-regional control (P = 0.03). Specific survival shows a trend (P = 0.06) in favour of the AF arm. ACUTE AND LATE TOXICITIES: Acute and late toxicity were increased in the AF arm. Late severe functional irradiation damage occurred in 14% of patients of the AF arm versus 4% in the CF arm. Two cases of radiation-induced myelitis occurred after doses of 42 and 48 Gy to the spinal cord. CONCLUSIONS: This trial shows that accelerated radiotherapy improves loco-regional control in head and neck squamous cell carcinomas. A less toxic scheme should, however, be investigated and documented before using accelerated radiotherapy as a standard regimen of curative radiotherapy for head and neck cancers.

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.

Detection of radiation-induced chromosome aberrations using fluorescence in situ hybridization in drug-induced premature chromosome condensations of tumour cell lines with different radiosensitivities.

A potential assay for radiosensitivity of human tumours is that of radiation-induced chromosome damage determined on metaphase spreads of human solid tumours. It is often difficult, however, to obtain enough metaphases for cytogenetic analysis after radiation. A possible solution would be to use the technique of premature chromosome condensation (PCC), enabling the study of interphase cells. The induction of PCCs using mitotic inducer cells is technically difficult, however, and the frequency of induction relatively low. We have attempted to use another approach, to induce PCCs using the phosphatase inhibitors okadaic acid and calyculin A. Both inhibitors were found to induce PCCs in several human tumour cell lines, with calyculin A producing the higher incidence. Determination of radiation-induced chromosome aberrations using fluorescence in situ hybridization on these chemically induced PCCs showed a clear difference between a radiosensitive (SCC61) and a radioresistant (A549) tumour cell line, with more aberrations in the sensitive line. Owing to incomplete condensation compared with that in standard metaphases, accurate classification of aberration types was not possible. Despite this limitation, the present data indicate that this relatively quick and simple method may be useful for determining chromosome aberrations in interphase cells and potentially in human solid tumours for predictive assay purposes.