Prognostic value of HIF-1α expression during fractionated irradiation.

BACKGROUND AND PURPOSE: Hypoxia and reoxygenation are important determinants of outcome after radiotherapy. HIF-1α is a key molecule involved in cellular response to hypoxia. HIF-1α expression levels have been shown to change after irradiation. The objective of the present study was to explore the prognostic value of HIF-1α expression during fractionated irradiation. MATERIALS AND METHODS: Six human squamous cell carcinoma models xenografted in nude mice were analysed. Tumours were excised after 3, 5 and 10 fractions. HIF-1α expression was quantified by western blot. For comparative analysis, previously published data on local tumour control data and pimonidazole hypoxic fraction was used. RESULTS: HIF-1α expression in untreated tumours exhibited intertumoural heterogeneity and did not correlate with pimonidazole hypoxic fraction. During fractionated irradiation the majority of tumour models exhibited a decrease in HIF-1α expression, whereas in UT-SCC-5 no change was observed. Neither kinetics nor expression levels during fractionated irradiation correlated with local tumour control. CONCLUSION: Our data do not support the use of HIF-1α determined during treatment as a biomarker to predict outcome after fractionated irradiation.

TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects.

Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. p16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. HNSCC cells and xenografts (HPV/p16-positive and -negative) were used. p16-overexpressing and small hairpin RNA-knockdown cells were generated, and the effect of p16 on radiosensitivity was determined by clonogenic cell survival and tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction; and data from The Cancer Genome Atlas HNSCC project were analyzed. p16 overexpression led to downregulation of TRIP12, which in turn led to increased RNF168 levels, repressed DNA damage repair (DDR), increased 53BP1 foci and enhanced radioresponsiveness. Inhibition of TRIP12 expression further led to radiosensitization, and overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. These findings reveal that p16 participates in radiosensitization through influencing DDR and support the rationale of blocking TRIP12 to improve radiotherapy outcomes.

Normalization of in vitro sensitivity testing of human tumor clonogenic cells.

The use of normal bone marrow (granulocyte-macrophage colony-forming units) as a point of reference to normalize the in vitro activities of anticancer agents has been investigated. The cytotoxic effects of four substituted anthraquinone derivatives, and of vinblastine on myeloid progenitors of different donors were reproducible up to a cell kill of approximately 60%. Equitoxic in vitro concentrations for normal bone marrows did not correlate with in vivo pharmacokinetic concentrations of these drugs. Breast tumor progenitor cells of 46 specimens were more sensitive than were bone marrow progenitors to the anthraquinone derivatives in 26 to 39% of instances, ratios which are similar to the clinically observed response rates of patients with breast carcinoma to these agents. Tumors were either sensitive or resistant to all four drugs in 68% (10 tumors were more sensitive, and 21 tumors were less sensitive than normal bone marrow); but in 32% of instances there were differences in tumor sensitivity for the four drugs, and the assay could select one to three drugs for which the tumor sensitivity was greater than that of bone marrow. Correlations of in vitro sensitivity and of clinical response to single agent treatments were determined in 21 patients, and the concordance was 71%. The value of the assay in predicting clinical response ranked best for sensitivity determinations within the normalized dose ranges, when testing within three different dose ranges was compared in a group of six patients. The concordance was higher in the small (1 or 2 metastatic sites) than in the large (greater than or equal to 3 metastatic sites) tumors (85 versus 50%), indicating a confounding influence of tumor load on the ability of the assay to predict efficacy of treatment. A rule of thumb is proposed for altering the in vitro sensitivity test results for large tumors that improves the overall concordance to 90%.

Protective effects of S-2-(3-aminopropylamino)ethylphosphorothioic acid against radiation damage of normal tissues and a fibrosarcoma in mice.

S-2-(3-Aminopropylamino)ethylphosphorothioic acid (WR-2721) was investigated for its protective effect against radiation-produced damage of jejunum, testis, lung, hair follicles, and a fibrosarcoma of C3Hf/Kam mice. Most of these tissues were radioprotected, and the degree of radioprotection depended on the dose of WR-2721 and the time interval between administration of WR-2721 and radiation treatment. WR-2721 increased resistance of jejunal epithelial cells and spermatogenic cells to single doses of gamma-rays by factors of 1.64 and 1.54, respectively. Protection against hair loss was less pronounced; the dose-modifying factor here was 1.24. The radiation-induced acute damage of the lung expressed by the increased formation of tumor nodules in the lung was not decreased by treatment of animals with WR-2721 before radiation. In contrast, WR-2721 augmented the radiation-induced enhancement of metastasis formation in the lung. WR-2721 protected fibrosarcoma micrometastases in the lung against therapeutic effect of radiation by a factor of 1.238. In contrast, this compound had no effect on the therapy of an 8-mm fibrosarcoma growing in the legs of mice.

Chemotherapy and hormonal therapy in combination.

The advantage of adding hormones to chemotherapy for the treatment of patients with breast carcinoma is uncertain, and benefits and disadvantages have been reported. An analysis of published randomized clinical trials reveals that the growth function (differentiation-inducing function v mitogenic function) of the hormone used may determine the ultimate benefit of combined modality treatments.

Frequency of first metastatic events in breast cancer: implications for sequencing of systemic and local-regional treatment.

PURPOSE: The sequencing of treatment for early breast cancer is controversial. The purpose of this study was to quantify the risk of delaying surgery, using estimates of the frequency of first metastases from breast primary tumors. PATIENTS AND METHODS: The probability that 560 (node-negative), 657 (with one to three positive nodes), and 505 (with more than three positive nodes) women treated without adjuvant chemotherapy would be free of distant disease at presentation was fit to a mathematical model of the seeding of distant metastases and combined with estimates of the growth rate to calculate the frequency of first distant disseminations per month. RESULTS: Frequencies of first distant metastases were approximately 1% to 2% per month, 2% to 4% per month, and 3% to 6% per month in T1 patients who were node-negative, had one to three positive nodes, or more than three positive nodes, respectively. As a result, the typical patient with T1 disease, who has a 70% to 80% chance of being free of distant disease, runs a 1% to 4% risk of distant dissemination for each month surgery is delayed. Assuming a 30% reduction in mortality caused by adjuvant chemotherapy, the model predicts that T1 patients treated with neoadjuvant chemotherapy would potentially have a higher rate of distant metastasis development than those treated with an initial surgical resection followed by adjuvant chemotherapy. CONCLUSION: We formulate the hypothesis that optimal sequencing of surgery and systemic treatment of breast cancer may be size-dependent, with a disadvantage or no benefit from neoadjuvant treatment for T1 patients but an increasing benefit with increasing size of the primary tumor.

The true predictive value of the human tumor stem cell assay: does a workable assay select for treatment responders?

In practice, the human tumor clonogenic assay is workable for less than half of the patient population to which it is applied, since the remainder of the specimens fail to produce sufficient numbers of colonies. Thereby a bias may be introduced which could result in a false predictive value positive of the test. It is therefore necessary to compare the responses to treatment of patients whose tumors could be assayed in vitro to those whose tumors failed to grow adequately, to assure that the prevalence of treatment responders has not changed within the group of patients for which the assay worked. From an analysis of the treatment response of 70 patients with stage III and IV ovarian carcinomas and 70 patients with stage IV breast cancer, no selection bias did occur and no preferential in vitro growth of tumor samples from patients with treatment response was found.

Locoregional recurrence patterns after mastectomy and doxorubicin-based chemotherapy: implications for postoperative irradiation.

PURPOSE: The objective of this study was to determine locoregional recurrence (LRR) patterns after mastectomy and doxorubicin-based chemotherapy to define subgroups of patients who might benefit from adjuvant irradiation. PATIENTS AND METHODS: A total of 1,031 patients were treated with mastectomy and doxorubicin-based chemotherapy without irradiation on five prospective trials. Median follow-up time was 116 months. Rates of isolated and total LRR (+/- distant metastasis) were calculated by Kaplan-Meier analysis. RESULTS: The 10-year actuarial rates of isolated LRR were 4%, 10%, 21%, and 22% for patients with zero, one to three, four to nine, or >/= 10 involved nodes, respectively (P <.0001). Chest wall (68%) and supraclavicular nodes (41%) were the most common sites of LRR. T stage (P <.001), tumor size (P <.001), and >/= 2-mm extranodal extension (P <.001) were also predictive of LRR. Separate analysis was performed for patients with T1 or T2 primary disease and one to three involved nodes (n = 404). Those with fewer than 10 nodes examined were at increased risk of LRR compared with those with >/= 10 nodes examined (24% v 11%; P =.02). Patients with tumor size greater than 4.0 cm or extranodal extension >/= 2 mm experienced rates of isolated LRR in excess of 20%. Each of these factors continued to significantly predict for LRR in multivariate analysis by Cox logistic regression. CONCLUSION: Patients with tumors >/= 4 cm or at least four involved nodes experience LRR rates in excess of 20% and should be offered adjuvant irradiation. Additionally, patients with one to three involved nodes and large tumors, extranodal extension >/= 2 mm, or inadequate axillary dissections experience high rates of LRR and may benefit from postmastectomy irradiation.

The challenge of p53 as prognostic and predictive factor in gliomas.

In recent years, increasing interest in genetic abnormalities and biologic factors such as the tumour suppressor gene p53 as possible predictive and prognostic factor in gliomas has emerged. Inactivation of p53 can result in resistance to apoptosis, one of the mechanisms thought to explain the failure to respond to DNA-damaging agents. Thus, inactivation of p53 might be associated with a worse prognosis. Considering the inconsistent results of several recent studies, it has remained controversial whether p53 actually can be related to response to treatment and patients' prognosis. Therefore, a systematic review of the literature was performed, which included 28 publications. Techniques for assessing the inactivation of p53 varied widely. Overall, approximately 50% or more of astrocytoma specimens evaluated by immunohistochemistry stained positively for p53, regardless of histologic grade. Eight studies were restricted to comparably treated patients within a single histologic group. In most instances, non-restrictive inclusion criteria and use of statistical methods, which were not sufficient to correct the possible bias, make it difficult to reach unequivocal conclusions. However, it appears that the prognostic information of p53 is at best marginal, especially when compared to established parameters such as grading, age, etc. Its predictive value, which most likely is rather limited too, can hardly be judged without prospective studies also evaluating other biological factors as well as end-points other than time to radiological progression.

Randomized phase III study of chemoradiation with or without amifostine for patients with favorable performance status inoperable stage II-III non-small cell lung cancer: preliminary results.

A prospective randomized study was conducted to determine whether amifostine (Ethyol) reduces the rate of severe esophagitis and hematologic and pulmonary toxicity associated with chemoradiation or improves control of non-small cell lung cancer (NSCLC). Sixty patients with inoperable stage II or III NSCLC were treated with concurrent chemoradiotherapy. Both groups received thoracic radiation therapy (TRT) with 1.2 Gy/fraction, 2 fraction per day, 5 days per week for a total dose 69.6 Gy. All patients received oral etoposide (VP-16), 50 mg Bid, 30 minutes before TRT beginning day 1 for 10 days, repeated on day 29, and cisplatin 50 mg/m(2) intravenously on days 1, 8, 29, and 36. Patients in the study group received amifostine, 500 mg intravenously, twice weekly before chemoradiation (arm 1); patients in the control group received chemoradiation without amifostine (arm 2). Patient and tumor characteristics were distributed equally in both groups. Of the 60 patients enrolled, 53 were evaluable (27 in arm 1, 26 in arm 2) with a median follow-up of 6 months. Median survival times were 26 months for arm 1 and 15 months for arm 2, not statistically significantly different. Morphine intake to reduce severe esophagitis was significantly lower in arm 1 (2 of 27, 7.4%) than arm 2 (8 of 26, 31%; P =.03). Acute pneumonitis was significantly lower in arm 1 (1 of 27, 3.7%) than in arm 2 (6 of 26, 23%; P =.037). Hypotension (20 mm Hg decrease from baseline blood pressure) was significantly more frequent in arm 1 (19 of 27, 70%) than arm 2 (1 of 26, 3.8%; P =.0001). Only 1 patient discontinued treatment because of hypotension. These preliminary results showed that amifostine significantly reduced acute severe esophagitis and pneumonitis. Further observation is required to assess long-term efficacy.

Evidence for individual differences in the radiosensitivity of human skin.

Previously published clinical data have been re-analysed to investigate individual differences in the radiosensitivity of human skin. In the clinical studies, acute and late skin reactions were recorded for 254 breast cancer patients receiving radiotherapy to the internal mammary nodes following simple or modified radical mastectomy. Each patient was treated bilaterally with different fractionation schedules to the right and left fields. Patients were assigned prospectively to 10 different treatment groups of 11-35 patients each, with all patients in a group receiving the same pair of fractionation schedules to the right and left fields. In the present study, correlations between the skin reactions in the two treatment fields per patient were investigated. For each of three different endpoints--peak reflectance measure of erythema, peak acute skin reaction score, and a ranking measure of the progression rate of telangiectasia--significant correlations were found between the levels of skin injury to the right and left treatment fields of the patients in most treatment groups. Although there were correlations between the absorbed doses in the right and left fields, statistical analyses indicated that dose effects were not sufficient to explain fully the patient-to-patient differences in skin response. Thus, these data provide evidence for the existence of individual differences in the radiation response of human skin, both for early and late effects. Whether these differences are dominated by heterogeneity in intrinsic cell radiosensitivity or by other factors has yet to be determined. However, there was no clear evidence of a correlation between the acute and late endpoints, suggesting that the individual differences in radiosensitivity are not dominated by a common genetic component expressed equally in all cells.

Tumor radioresponsiveness versus fractionation sensitivity.

Since the introduction of mammalian cell survival curves, the parameters D0 and N have been used as quantitative measures of inherent radiation sensitivity, as was the shoulder width Dq. These parameters are more generally applicable at high doses. We propose to introduce a measure of tumor radioresponsitivity that is more applicable to the clinical treatment schedules that employ small fractional doses (1-2 Gy), the ratio alpha/E, derived from the linear quadratic model for cell inactivation as the intercept on the reciprocal-dose plot. For tumor-control experiments this ratio is the reciprocal of the TCD50 when radiation is given in very small fractions or at low dose rates (assuming negligible clonogen proliferation). The rationales for this choice are: alpha is a measure of the steepness of the initial linear segment of the dose-survival curve. Accordingly, at doses per fraction of 1-2 Gy the observed effect increases with alpha. E is by definition a positive measure of the clonogen kill required for a specified tumor response, e.g., E = -log (surviving fraction of clonogens at the 50% control level). Therefore it is also a measure of the number of clonogens present at the time of inception of treatment, which for a given dose is a prime determinant of the probability of tumor control. This measure of radioresponsitivity is to be distinguished from the ratio alpha/beta, which is a measure of fractionation sensitivity. A survey of the literature indicates that these do not correlate, except in highly hypoxic tumors (e.g., clamped); such tumors are characterized by low radioresponsitivity as well as low fractionation sensitivity (high alpha/beta ratio). There are at present only limited data for determination of this ratio, however, since reciprocal-dose analysis requires tumor control doses for several different sizes of dose per fraction.

The effect of patient-to-patient variability on the accuracy of predictive assays of tumor response to radiotherapy: a theoretical evaluation.

Mathematical modeling was used to investigate the relative accuracy that might be expected from predictive assays of tumor response to radiotherapy based on one of the following four tumor characteristics: intrinsic tumor-cell radiosensitivity, doubling time of the clonogenic tumor cells, number of clonogens in the tumor at the start of treatment, and extent of hypoxia in the tumor. In particular, the influence of inter-tumor heterogeneity on predictive accuracy was investigated. Wide patient-to-patient variability in a tumor characteristic contributing to treatment response adds to the accuracy of a predictive assay based on that characteristic, but variability from patient to patient in factors influencing response, but not measured by the assay, has a confounding effect and reduces predictive accuracy. The results of this theoretical study suggest that predictive assays based on intrinsic tumor-cell radiosensitivity are much more likely to be correlated with clinical outcome than are assays based on clonogen doubling time, hypoxic fraction, or clonogen number, since individual differences in tumor radiosensitivity can seriously confound assays based on the other factors. It is concluded that it may be necessary to correct for individual differences in intrinsic radiosensitivity before predictive assays based on other tumor characteristics, such as potential doubling time, might have any detectable clinical significance.

Hyperfractionated radiotherapy of human tumors: overview of the randomized clinical trials.

PURPOSE: Hyperfractionation (HF) is the altered fractionation schedule most frequently studied in clinical Phase III trials. In this overview, surviving fractions, rates of complete responses, and estimates of the long-term locoregional tumor control probabilities after HF and conventional fractionated irradiation (CF) available from the various reports were compared. METHODS AND MATERIALS: A metaanalysis was performed of the randomized studies on hyperfractionation vs. conventional fractionation published since 1980 on different tumor types in various locations. RESULTS: Compared with CF, HF significantly reduced the odds of death for patients with head and neck tumors (three studies, odds ratio 0.48 (0.40-0.58), p < 0.0001) and bladder cancer (two studies, odds ratio 0.53 (0.36-0.78), p = 0.001), while there was a trend in nonsmall cell lung cancer (three studies, odds ratio 0.69 (0.51-0.95), p = 0.02), and malignant gliomas (three studies, odds ratio 0.67 (0.48-0.93), p = 0.02). The probability of long-term loco-regional control of head and neck tumors was significantly enhanced after HF (four studies, odds ratio for loco-regional recurrence or related events 0.35 (0.28-0.45), p < 0.0001). In trials on head and neck tumors and bladder cancer, complete responses were seen more often after HF compared with CF (odds ratio for failure of complete response: 0.43 (0.32-0.57), p < 0.0001, and 0.43 (0.27-0.70), p = 0.0007). CONCLUSIONS: This overview demonstrates that the effectiveness of radiotherapy is consistently higher for HF than for CF. The assumption that tumors have a small effective fractionation sensitivity (alpha/beta > 5 Gy) seems to be fulfilled especially for head and neck cancers.

Tumor volume and local control probability: clinical data and radiobiological interpretations.

PURPOSE: It is well established for certain human tumor histologies that increasing tumor volume leads to a decreasing probability of tumor control. The simplest explanation for these findings is that the number of tumor clonogens that must be sterilized to control a tumor increases with tumor volume. In this investigation we consider whether clinical evidence favors a further hypothesis, namely, that clonogen number increases in direct proportion to tumor volume. METHODS AND MATERIALS: Previously published data on the volume-cure relationship for breast tumors, neck nodes, malignant melanoma, and squamous cell carcinomas of the oropharynx and the uterine cervix were analyzed. RESULTS: We found in all these data sets evidence that the effect of tumor volume on tumor control probability was less than what would be expected under the assumption of proportionality between number of clonogens and volume. We describe good reasons to believe that this is the result of patient-to-patient variability in radiocurability, and possibly other factors as well. CONCLUSIONS: Clinical data do provide evidence for a highly significant reduction of tumor control probability with increasing tumor volume. However, because of heterogeneity in patient and tumor characteristics, the volume effect is less pronounced than would be expected from a simple proportionality between number of clonogens and volume. In principle this simple proportionality does hold in individual patients, so that standard approaches for treatment plan optimization in individuals may still be valid.

The clinical significance of ratios of radiobiological parameters.

PURPOSE: Interindividual heterogeneity of the radiobiological characteristics of malignant and normal tissues hampers the derivation of radiobiological parameters from clinical data. Focusing on the ratio Dprolif, i.e., the dose to compensate 1 day of treatment interruption, this article investigates the hypothesis that ratios of parameters might be less sensitive to interpatient heterogeneity and may constitute a more reliable description of the radiobiological properties of tissues than the parameters themselves. METHODS AND MATERIALS: Analytic calculations were performed in an idealized example in which the only source of heterogeneity was the number of clonogenic cells. Computer simulations were used to assess the effects of heterogeneity in radiosensitivity and in proliferative capacity. Treatment outcome was simulated in pseudopatients with increasing dose-time correlation. RESULTS: Interindividual heterogeneity in clonogenic cell number, radiosensitivity, or proliferative ability results in a marked underestimation of the response parameters describing these processes. In contrast, the estimates of the ratio Dprolif were more stable. The coefficients of variation increased with increasing heterogeneity. However, this only became unacceptable when heterogeneity in radiosensitivity was marked, or when total dose and treatment time were closely correlated. CONCLUSION: Parameter ratios may provide more robust radiobiological information than single parameters estimated from clinical data except when interindividual heterogeneity is very large or when the treatment modalities are too highly correlated. As usual, caution is advised in the presence of patient selection, a correlation between treatment prescription and expected outcome, or limited ranges of dose-time treatment patterns.

Flexure dose: the low-dose limit of effective fractionation.

Total radiation dose often can be increased without subsequent increases in the severity of tissue injury by using reduced doses per fraction. The flexure dose, df, is defined as the largest fractional dose for which further fractionation produces no significant change in the total dose required to reach a specified effect level. Thus, df is clinically relevant in that it represents the limit of effective dose fractionation. For those tissues in which injury reflects depletion of a critical proportion of target cells, the flexure dose is a measure of the extent of the initial, nearly linear portion of the dose-survival curve. More generally, the flexure dose is a measure of the extent of the initial, nearly linear portion of a dose-response curve in organized tissue, whatever its relationship to clonogenic target cells might be. Several quantitative expressions for df are derived. The characteristic common to these is that each defines the flexure dose as a multiple of the ratio alpha/beta of the parameters of the linear-quadratic model of cell survival or dose response, where the multiple is a measure of experimental or statistical resolution. These multiples tend to fall within a limited range, thereby defining the "region of flexure" via the inequality 0.05 (alpha/beta) less than or equal to df less than or equal to 0.15 (alpha/beta). Estimates of the region of flexure are presented for a variety of normal and neoplastic tissues.

Fractionation sensitivity of a functional endpoint: impaired shoulder movement after post-mastectomy radiotherapy.

A clinical radiobiological study of the fractionation sensitivity of impaired shoulder movement after postmastectomy radiotherapy is presented. From 1978 to 1980, 163 breast cancer patients received postmastectomy irradiation delivered in 12 fractions, 2 fractions per week, over a period of 37 to 46 days. The total dose was specified either as a maximum absorbed dose of 51.36 Gy, or as a minimum target dose of 36.6 Gy specified at the level of midaxilla. From 1981 to 1982, 66 patients received a minimum target dose of 40.92 Gy in 22 fractions with 5 fractions per week over a period of 29 to 35 days. The treatment technique remained essentially unchanged during the whole period of 1978 to 1982. Maximal flexion and abduction of the arm were measured with unfixed scapula. A relative score, indicating the grade of impaired shoulder movement on a nominal scale from 0-3, was established by comparison with the mobility of the contralateral arm. In addition, working ability and pain at movement and at rest were evaluated. These performance parameters significantly correlated with the grade of impaired shoulder movement. The occurrence of moderately or severely impaired shoulder movement was analyzed using a mixture model incorporating dose-fractionation, latency, and predisposing clinical factors. The risk of shoulder problems was significantly higher among patients above 60 years of age. The analysis pointed to subcutaneous fibrosis as a disposing factor and indicates that postoperative physical exercise programs are beneficial. Arm edema had no statistically significant influence on shoulder performance. The alpha/beta ratio was estimated at 3.5 Gy with 95% confidence limits (c.l.) [0.7, 6.2] Gy. The length of time to expression of 90% of the ultimate frequency of moderate and severe shoulder impairment was estimated at 3.9 years with 95% confidence limits [3.1, 4.6] years. The nominal standard dose (NSD) formalism failed to produce isoeffective total doses with respect to impaired shoulder movement in the two fractionation schedules. Although the deleterious effects of larger-than-conventional dose fractions have been documented in a number of clinical series, this study represents the first quantitation of the fractionation sensitivity of a functional clinical endpoint.

Response of fibrosarcoma cell subpopulations to small doses of radiation delivered in situ.

The survival of cells from 2 tumor subpopulations after gamma-ray doses ranging from 1 to 19 Gy was determined using a lung colony assay. Methylcholanthrene-induced fibrosarcomas grown in the hind legs of C3H/Kam pathogen-free mice were irradiated in situ when the tumors were 8-10 mm in diameter. Single cell suspensions prepared from excised tumors were separated on a linear density gradient, and the clonogenicity of predominantly oxic Band 2 (density 1.08 g/cm3) and predominantly hypoxic Band 4 (density 1.14 gm/cm3) cells was measured. The surviving fraction of cells after doses of 1, 2, and 3 Gy was estimated from that measured after total doses of 5 Gy = 5 X 1 Gy, 10 Gy = 5 X 2 Gy, and 15 Gy = 5 X 3 Gy, under the assumption of equal effect per fraction (checked by estimating survival at 3 Gy after different numbers of fractions). Very little curvature was evident in the survival curves of Band 2 and Band 4 cells (beta/alpha = .013-.034 Gy-1). The initial segment of the survival curve of the predominantly oxic Band 2 cells was steeper (1Do = 3.6 Gy) than that of the predominantly hypoxic Band 4 cells (1Do = 5.2 Gy); both remained linear over a large range, to doses in excess of 3 Gy. These results imply that these tumor subpopulations will be insensitive, in their response to multifractionated regimens, to changes in size of dose per fraction in the range 0 to 3 Gy, a trait shared by two acutely responding normal tissues (murine testis and jejunum).

Tumor sensitizing effect by misonidazole in a clinically relevant radiation dose range.

The survival of cells from two murine fibrosarcoma (FSa) subpopulations after exposure to radiation only or radiation in combination with misonidazole (0.2 mg/g/fraction) was determined using a lung colony assay. FSa tumors grown in the hind legs of C3Hf/Kam pathogen-free mice were irradiated in situ when the tumors were 8 to 10 mm in diameter. Single cell suspensions prepared from excised tumors were separated on linear density gradients of Renografin, and the clonogenicity of predominantly oxic Band 2 (density 1.08 g/cm3) and predominantly hypoxic Band 4 (density 1.14 g/cm3) cells were measured. The surviving fraction of cells after doses of 1, 2, and 3 Gy, alone or preceded 30 minutes earlier with an i.p. injection of misonidazole (0.2 mg/g) was estimated from that measured after total radiation doses of 5 Gy = 5 X 1 Gy, 10 Gy = 5 X 2 Gy, and 15 Gy = 5 X 3 Gy, with the misonidazole-treated groups receiving a total drug dose of 1 mg/g, under the assumption of an equal effect per fraction. Under these conditions the initial slopes of Band 2 cells following irradiation only or irradiation plus misonidazole were 1D0 = 3.6 Gy and 2.74 Gy, respectively, giving rise to a sensitizer enhancement ratio of 1.3. Band 4 cells exhibited a 1D0 of 5.15 Gy to radiation only and 2.75 Gy to radiation plus misonidazole (SER of 1.9). In addition, misonidazole when administered alone in a single dose or up to 5 fractions of 0.2 mg/g each separated by 4-hour intervals, was effective in killing 50% of the Band 4 cells. The target population at risk appeared to remain constant regardless of the number of dose fractions administered. In contrast, Band 2 cells were not affected by the cytotoxic action of misonidazole. These data suggest that misonidazole is effective in sensitizing hypoxic cells in the clinical dose range, and that it is directly cytotoxic to hypoxic tumor cells.