PURPOSE:Tumor cell repopulation is still considered to be a major cause of failure in radiotherapy. In this study, we investigated the influence of cell kinetic parameters on the outcome of patients treated in a randomized trial of accelerated fractionation, with or without mitomycin C, vs. conventional fractionation.METHODS AND MATERIALS:Sixty-two patients were studied using administration of bromodeoxyuridine (BrdUrd), and cell kinetic parameters were measured using flow cytometry. The patients were treated with either 70 Gy for 7 weeks or 55.3 Gy for 17 continuous days (V-CHART) with or without 20 mg/m(2) mitomycin C on day 5.RESULTS:The potential doubling time (Tpot) and labeling index (LI) failed to provide any prognostic information with regard to local control or survival. However, the duration of the S phase (Ts) revealed patients whose tumors had a long Ts had significantly worse local control (p = 0.028) and survival (p = 0.034) irrespective of treatment. A similar trend was evident within the different treatment arms particularly associated with overall survival.CONCLUSIONS:The Ts values of head-and-neck squamous cell cancers provided prognostic information that predicted clinical outcome irrespective of treatment schedule in this study. This neglected parameter of the Tpot method might provide information related to redistribution of cells during fractionated radiotherapy.

Background and purpose: Radiation therapy is often the primary treatment for advanced cases of head and neck cancers not considered suitable for radical surgery. In these cases locoregional tumour control rates are low and has warranted innovative treatment modifications, such as altered fractionation schedules and combination with chemotherapy.Patients and methods: From October 1990 to December 1997, 239 patients with squamous cell cancers originating in the head and neck region were randomized to one of three treatment options. Standard therapy consisting of conventional fractionation with 70 Gy in 7 weeks in 35 fractions (CF). The second treatment option consisted of a continuous hyperfractionated accelerated radiotherapy delivering a total dose of 55.3 Gy in 33 fractions over 17 consecutive days (V-CHART). The third study arm had identical fractionation and dose as the above accelerated treatment, with the additional administration of 20 mg/m(2) mitomycin C (MMC) on day 5 of treatment (V-CHART + MMC).Results: Main toxicity resulted from accelerated fractionation in confluent mucositis (Grade 3-4 in 95%) requiring nasogastral tube feeding, analgetics and antiphlogistics in the majority of cases. Haematological toxicity Grade 3-4 was seen after MMC administration in 18%. MMC administration did not influence mucosal reaction. Overall duration of mucositis was not different in the three treatment groups. Loco-regional tumour control was 31% after CF, 32% after V-CHART and 48% after V-CHART + MMC, respectively (P < 0.05). Overall crude survival was 24% after CF, 31% following V-CHART and 41% after V-CHART + MMC, respectively (P < 0.05). Median follow up was 48 months (assessment performed in February 1999).Conclusion: Following shortening overall treatment time from 7 weeks to 17 consecutive days and dose of radiotherapy from 70 to 55.3 Gy the results in the radiotherapy only treated patients are identical. A significant improvement regarding local tumour control and survival was seen following administration of MMC to the accelerated fractionated treatment. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

From October 1990 to March 1994, 90 patients entered a prospectively randomised trial in head and neck cancer. All patients had verified squamous cell carcinoma and were referred for primary radiation therapy. Tumours originated in the oral cavity in 25, oropharynx in 37, larynx in 15 and hypopharynx in 13 cases. Patients' stages were predominantely T3 and T4 (71/90) and had lymph node metastases (60/90). Seventy-nine male patients and 11 female patients, with a median age of 57 years (range 37-76 years) were treated. Patients were randomised to one of three treatment options: conventional fractionation (CF) consisting of 70 Gy in 35 fractions over 7 weeks or continuous hyperfractionated accelerated radiation therapy (Vienna-CHART) or Vienna-CHART with administration of a single dose of mitomycin C on day 5 of treatment (V-CHART + MMC). By the accelerated regimen a total dose of 55.3 Gy was given in 33 fractions within 17 consecutive days. Acute mucositis was the main toxicity recorded in those patients treated by accelerated fractionation, although the overall duration of mucosal reaction did not differ in the three treatment groups. There was no influence on local toxicity if MMC was added to radiation therapy or not. Those patients treated with additional MMC experienced a grade III/IV haematological toxicity in 4/28 cases. Complete remission (CR) was recorded in 48% following CF, 79% after Vienna-CHART (P < 0.05) and 71% after Vienna-CHART + MMC. The overall local failure rates were 73%, 59% and 42% (P = NS) for patients treated by CF, Vienna-CHART and Vienna-CHART + MMC respectively.

The effectiveness of accelerated fractionation and hyperfractionation in cancer of the head and neck has been confirmed by randomized studies. These new fractionation strategies are almost invariably accompanied by an increase of early normal tissue reactions, in particular mucosal reactions. This paper presents a survey of the available experimental and clinical mucositis data and aims to assess to what extent the upper aerodigestive tract mucosa is limiting to treatment intensification by altered fractionation.The rate of dose delivery is the most important determinant for early radiation reactions. With accelerated radiotherapy, relative to a conventional treatment of 7 weeks, the achievable gain in treatment time is 2 weeks at most with the mucosa being the limiting tissue. Any further acceleration requires a reduction of dose. Manipulations with the temporal distribution of dose, fraction dose, and optimization of interfraction intervals can improve tolerance but probably do not allow significant further intensification of the existing accelerated schedules. Dose escalation by hyperfractionation does not seem to be directly limited by early mucosal reactions. Late reacting tissues are more likely to limit intensification of these schedules.Suggestions for further improvement of treatment outcome include: the generation of a potent agent which can ameliorate radiation mucositis and so permit further intensification of radiotherapy schedules; combination of altered fractionation schedules with hypoxic modifiers; and tailoring of the treatment strategy based on patient and tumour characteristics.

The mean extracellular pH (pHe) within solid tumours has been found to be lower than in normal tissues. Agents which cause intracellular acidification at low pHe might have selective toxicity towards cells in tumours. Weak acids (or their anions) with pKa values in the range of 4-6 have a higher proportion of molecules in the uncharged form at low pHe and can diffuse more rapidly into cells. The effects of organic acids including succinate, monomethyl succinate and malonate to acidify cells have been evaluated under conditions of different pHe in the acidic range. These weak acids caused intracellular acidification of murine EMT-6 and human MGH-U1 cells in a concentration and pHe dependent fashion. At concentrations of 10 mM and above, these acids also caused in vitro cytotoxicity to these cells at low pHe (< 6.5). The rate and extent of cellular acidification caused by these weak acids, and their cytotoxicity at low pHe, were enhanced by exposure to amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA), agents which inhibit Na+/H+ exchange, and hence the regulation of intracellular pH. Acid dependent cytotoxicity was also investigated in a murine solid tumour using the endpoints of growth delay and colony formation in vitro following treatment in vivo. Agents were tested alone or with 15 Gy X-rays to select a population of hypoxic (and presumably acidic) cells. Achievable serum concentrations of succinate were about 1 mM and no antitumour activity of succinate was detected when used in this way. It is concluded that weak acids are selectively taken up into cells, and can cause selective cellular acidification and toxicity, at low pHe in culture. Weak acids that are normal cellular metabolites are not toxic in vivo, but weak acids carrying cytotoxic groups offer the potential for selective uptake and toxicity under the conditions of low pHe that exist in many solid tumours.

From May 1990 to May 1991, 23 patients with advanced, inoperable squamous cell cancers, clinically staged as III or IV, were treated by unconventional fractionation radiotherapy. Treatment consisted of a continuous hyperfractionated accelerated radiotherapy, delivering a total dose of 55.3 Gy within 17 consecutive days. In ten patients radiation therapy was combined with chemotherapy; 20 mg mitomycin C/m2, administered by intravenous bolus injection on day 5 of treatment. Apart from a confluent mucositis, treatment tolerance was good. Haematological toxicity from mitomycin C was minor and did not require any specific therapy. The mucosal reaction lasted six weeks (median duration) and was not thought to be increased by additional chemotherapy. In twelve of 23 patients a complete remission of the primary tumour was seen, in patients with lymph node metastases there was a complete response in 14 out of 20 patients. After a median follow-up of 18 months, ten of 23 patients have survived (8/23 without evidence of disease). Eleven patients have died due to local tumour progression and one patient died with distant metastases, being without evidence of local tumour. The advantage of this unconventional fractionation, which takes the described short potential tumour doubling time for head and neck cancers into account, is discussed.

An examination of the effects of radiation combined with either 5-fluorouracil, Mitomycin C, or both drugs in vitro has been made using a mouse squamous tumor cell line SCC VIITo and cell viability as an endpoint. Depending on how the survival endpoint was calculated, the interaction of 5-fluorouracil, Mitomycin C, or 5-fluorouracil plus Mitomycin C with radiation was greater than additive (plating efficiency) or only additive (viable cells per flask). These results suggest that the cytostatic effect of these drugs may be an important aspect of their action clinically.

A system was designed to allow imaging of control and drug treated multicellular spheroids with a high frequency backscatter ultrasound microscope. It allowed imaging of individual spheroids under good growth conditions. Since little data were available on cellular toxicity of ultrasound at these high frequencies (80 MHz), studies were undertaken to evaluate effects on cell survival, using a colony forming assay. No toxicity was observed on cell monolayers subjected to pulsed ultrasound at the intensities used for imaging experiments. Spheroids were also subjected to pulsed ultrasound and no growth delay was observed when exposed spheroids were compared with mock-exposed spheroids. Imaging studies were performed and pictures of untreated spheroids were obtained in which the necrotic and viable regions are clearly distinguishable. When the hypoxic cell cytotoxin 1-methyl-2-nitroimidazole (INO2) was added to the spheroid, dramatic changes were observed in the backscatter signal. The interior viable cells of the spheroid were selectively affected. Changes in the backscatter signal were also observed when the reduction product 1-methyl-2-nitrosoimidazole (INO) was added to spheroids. With INO however, the changes were located at the periphery of the spheroid, presumably due to the high reactivity of INO which limits diffusion of the drug into the spheroid. The present work demonstrates the potential usefulness of ultrasound backscatter microscopy in following the action of selected drugs in this in vitro tumour model.

We have investigated the ability of the weak acids, lactate, succinate, and the monomethylester of succinate, to cause intracellular acidification of EMT-6 and MGH-U1 cells. Each of the three substances caused a decrease of intracellular pH (pHi) when the cell lines were exposed at low extracellular pH (pHe) in the range 6.0-6.5. Only monomethylsuccinate caused intracellular acidification at neutral pHe. The fall in pHi increased with increasing dose of each agent and with decreasing pHe. The pHi recovered to almost normal values after exposure of 30 minutes to 50 mM lactate, but there was little or no recovery of pHi in the presence of succinate or monomethylsuccinate. Succinate and its methylester were toxic to cells at low pHe (less than 6.5), and cell killing increased with exposure time and with dose of the agents used. Lactate did not cause cell death at low pHe, and none of the three substances exhibited any cytotoxicity at neutral pHe. Solid tumors are known to have an acidic microenvironment, and pHe may be particularly low in regions of hypoxia. Succinate and its monomethylester may have the potential to kill cells in acidic regions of tumors and might therefore enhance the effect of radiation.

From May 1985 to June 1988, 70 evaluable patients with advanced squamous cell cancers of the oral cavity and the oropharynx were treated with preoperative combined radio-chemotherapy. Treatment consisted of 50 Gy/25 fractions/5 weeks, combined with concomitant administration of mitomycin C on day 1 (15 mg/m2, i.v. bolus) and 5-fluorouracil during the first 5 days of irradiation (750 mg/m2/24 hours, continuous infusion). Surgery was performed 3 to 5 weeks following irradiation. Treatment tolerance was good and local mucosal reaction was increased, but no major systemic side effects were recorded. At surgery, 3-5 weeks following irradiation, 48.6% of the operation specimens did not contain any histologically detectable residual tumor. Overall survival is 61%, being 69% in T2 and T3, while none of the patients with bone invasion has survived. Median survival is 28, 26, and 9 months in T2, T3 and T4 stages, respectively. Loco-regional relapses have been recorded in 33% of the patients, occurring in 27% of T2, 25% of T3, and 88% of T4 stages. Patients have been spared mutilating radical neck dissection because of combined presurgical treatment without impaired survival or loco-regional relapse rate.

Advanced head and neck tumours have a poor prognosis due to the high frequency of local recurrence. Multimodality treatment has been shown to be effective in decreasing local recurrence. In this study, 51 patients with advanced oral and oropharyngeal carcinoma were entered in a trial of preoperative radio-chemotherapy. After exclusion of 10 protocol violations (no surgery or no chemotherapy), 41 patients remained evaluable.

Forty-one patients with Stage III and IV squamous cell carcinomas (SCC) of the head and neck were treated preoperatively with mitomycin C and 5-fluorouracil and concomitant radiotherapy. Operation specimen were examined histologically, the percentage of vital tumor cells and devitalized tumor cells were graduated. The grade of regression was classified according to a four-stage scale. Tumor regression was elevated as good (Grades 1, 2) and bad (3, 4) response to combined preoperative therapy. After a follow-up of 18 to 30 months, 14 of 41 patients have experienced a locoregional recurrence; all these patients were bad responders (Grades 3, 4) to preoperative radiochemotherapy. There was a statistically significant correlation between tumor regression grade and probability of survival (P less than 0.001). The authors conclude that the prognosis of patients with pretreated SCC of head and neck depends on the histologic grade of tumor regression.