Early warnings of unknown nonlinear shifts: a nonparametric approach.

Early warning signals (EWS) of regime shifts are challenging in cases where the true natural data-generating process is uncertain. Nonparametric drift-diffusion-jump models address this problem by fitting a general model that can approximate a wide range of data-generating processes. Drift measures the local rate of change. Diffusion measures relatively small shocks that occur at each time step. Jumps are large intermittent shocks. Total variance combines the contributions of diffusion and jumps. Nonparametric methods are well suited to emerging technology for automated, high-frequency sensors. Total variance is the most precisely measured indicator. Jump intensity appears to be a useful EWS. Estimates of the drift are highly uncertain unless long time series with many regime shifts are available. EWS computed from drift estimates (such as autocorrelation coefficients or return rates) have low precision and should be used with caution. Nonetheless, in the current state of knowledge, it is premature to disregard any potential EWS.

Leading indicators of trophic cascades.

Regime shifts are large, long-lasting changes in ecosystems. They are often hard to predict but may have leading indicators which are detectable in advance. Potential leading indicators include wider swings in dynamics of key ecosystem variables, slower return rates after perturbation and shift of variance towards lower frequencies. We evaluated these indicators using a food web model calibrated to long-term whole-lake experiments. We investigated whether impending regime shifts driven by gradual increase in exploitation of the top predator can create signals that cascade through food webs and be discerned in phytoplankton. Substantial changes in standard deviations, return rates and spectra occurred near the switch point, even two trophic levels removed from the regime shift in fishes. Signals of regime shift can be detected well in advance, if the driver of the regime shift changes much more slowly than the dynamics of key ecosystem variables which can be sampled frequently enough to measure the indicators. However, the regime shift may occur long after the driver has passed the critical point, because of very slow transient dynamics near the critical point. Thus, the ecosystem can be poised for regime shift by the time the signal is discernible. Field tests are needed to evaluate these indicators.

Rising variance: a leading indicator of ecological transition.

Regime shifts are substantial, long-lasting reorganizations of complex systems, such as ecosystems. Large ecosystem changes such as eutrophication, shifts among vegetation types, degradation of coral reefs and regional climate change often come as surprises because we lack leading indicators for regime shifts. Increases in variability of ecosystems have been suggested to foreshadow ecological regime shifts. However, it may be difficult to discern variability due to impending regime shift from that of exogenous drivers that affect the ecosystem. We addressed this problem using a model of lake eutrophication. Lakes are subject to fluctuations in recycling associated with regime shifts, as well as fluctuating nutrient inputs. Despite the complications of noisy inputs, increasing variability of lake-water phosphorus was discernible prior to the shift to eutrophic conditions. Simulations show that rising standard deviation (SD) could signal impending shifts about a decade in advance. The rising SD was detected by studying variability around predictions of a simple time-series model, and did not depend on detailed knowledge of the actual ecosystem dynamics.

Kinetic heterogeneity in density-separated murine fibrosarcoma subpopulations.

Murine fibrosarcoma cells can be separated into subpopulations by centrifugation through 10 to 35% Renografin density gradients. Previous work has shown that the heavier cell populations are rich in chronically hypoxic cells. In this study, each subpopulation was characterized for thymidine incorporation, thymidine transport, thymidine triphosphate pool sizes, and thymidine triphosphate specific activities. The heavier cell populations have less accessibility to exogenous thymidine, and they have lower endogenous pools of thymidine triphosphate and synthesize lower levels of DNA than do the lighter cell populations. However, if the cells are removed from the tumors and labeled with [3H]thymidine in vitro, all subpopulations synthesize DNA at similar rates. Two-parameter flow cytometry using acridine orange staining following partial acid denaturation of chromatin identified a small quiescent population in the most dense cell fraction, but the small number of these cells cannot account for the results of the biochemical studies. It appears that the hypoxic cells in the fibrosarcoma tumors are noncycling or slowly cycling, are in all phases of the cell cycle, and recover their ability to synthesize DNA when cultured under in vitro conditions.

Potentiation of radiation-induced regrowth delay in murine tumors by fludarabine.

Fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine-5'-monophosphate), an adenine nucleoside analogue, has previously been shown to inhibit the repair of radiation-induced chromosome damage. Thus fludarabine may have therapeutic utility in combination with photon irradiation. The purpose of this study was to determine whether fludarabine could enhance radiation-induced murine tumor regrowth delay and to determine the most effective dose and schedule of the combination. A significant (P < 0.05) absolute regrowth delay enhancement was observed in three murine tumor models (SA-NH, a sarcoma; and MCA-K and MCA-4, mammary carcinomas) when fludarabine (800 mg/kg) was given 1 h prior to 25 Gy gamma-irradiation. While fludarabine enhanced radiation-induced tumor regrowth delay when given between -36 h and +6 h of radiation (SA-NH tumor), the greatest enhancement was observed when fludarabine was given at -24 h prior to irradiation (radiation dose modification factor of 1.82 at -24 h compared to 1.57 at -3 h prior to radiation). The degree of fludarabine enhancement (at -3 or -24 h) was dose dependent at doses above 200 mg/kg. When fludarabine and radiation were administered on a fractionated schedule (fludarabine given 3 h prior to radiation each day for 4 days), the dose modification factor increased to 2.14 (1.63 if the effect of fludarabine alone is subtracted). These results suggest that fludarabine enhances radiation-induced tumor regrowth delay in a more than additive fashion after both single and fractionated treatments, and the degree of enhancement is dependent on the sequence and timing of administration, the fludarabine dose, and the tumor type. Thus, fludarabine may have clinical potential as a radiation enhancer in the treatment of solid tumors.

cis-Diamminedichloroplatinum(II)-induced sister chromatid exchange: an indicator of sensitivity and heterogeneity in primary human tumor cell cultures.

The effect of cis-diamminedichloroplatinum(II) (cPt) on sister chromatid exchange (SCE) induction was determined in 13 human primary tumor cell cultures. Primary cultures were derived from surgical specimens of solid tumors composed of a variety of histologies. Three to 16 days after biopsy, depending on the growth rate, cultures were treated with graded concentrations of cPt for 1 h and the SCE assay was performed. SCE dose-response curves (SCEs induced per chromosome versus cPt concentration) showed a wide range in cPt sensitivities that was not dependent on histology. SCE frequency histograms showed that several of the primary cultures contained both cPt-sensitive and -resistant cells. For six of the cultures, the SCEs induced per chromosome at 15 microM cPt were plotted versus the IC90 determined from a survival assay. A line fit to those points yielded a correlation coefficient of -0.74. These results show a relationship between the activity of cPt in the SCE assay and in the survival assay, which suggests that SCE analysis may be useful for predicting cPt sensitivity. In addition, characterization of cellular heterogeneity in cPt sensitivity using the SCE assay may provide additional information useful in the prediction of tumor response to treatment.

Biological effect of epidermal growth factor on the in vitro growth of human tumors.

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.

Antitumor activity against human tumor samples of cis-diamminedichloroplatinum(II) and analogues at equivalent in vitro myelotoxic concentrations.

We compared the antitumor activity of cis-diamminedichloroplatinum(II) (cisplatin; CDDP) with three CDDP analogues: cis-diammine-1,1-cyclobutanedicarboxylateplatinum(II) (CBDCA), N-methyliminodiacetato-1,2-diamino(cyclohexane)platinum(II) (MIDP), and N-(2-hydroxyethyl)-iminodiacetato-1,2-diamino(cyclohexane)platinum (II) (HIDP). Fresh human tumor samples in the adhesive tumor culture system were utilized for this comparison. The equitoxic concentrations of all four drugs were derived based on their inhibitory activity against human bone marrow samples. For these normalized concentrations, CDDP proved to have a higher cytotoxic activity than its analogues. CBDCA's in vitro activity had a significant correlation with CDDP activity (r = 0.67) in vitro. However, the structurally similar substances MIDP and HIDP demonstrated a much greater degree of association (r = 0.90). Our data suggest that CBDCA, HIDP, and MIDP have overall less activity than CDDP when tested at equitoxic in vitro concentrations. Close association between CDDP and CBDCA also reflects known clinical experience with these two drugs, suggesting the method of comparison used here is probably appropriate. These conclusions, however, must be validated by clinical trials.

Intrinsic radiosensitivity of normal human fibroblasts and lymphocytes after high- and low-dose-rate irradiation.

The existence of heritable radiosensitivity syndromes and clinical observations in radiotherapy patients suggests that human cellular radiosensitivity differs among individuals. We report here an in vitro study of radiosensitivity in 30 fibroblast and 29 lymphocyte cultures obtained from cancer patients and controls. In 25 cases, both fibroblasts and lymphocytes were obtained from the same donors. Fibroblasts were cultured from skin biopsy samples, and peripheral T-cell lymphocytes were cultured from blood. Clonogenic survival assays were performed by using high- and low-dose-rate irradiation; lymphocytes were in G0 phase and fibroblasts in confluent plateau phase. Various end points were calculated and compared (i.e., surviving fraction at 2 Gy, initial slope of the survival curve, and doses resulting in 10 and 1% survival, respectively). Depending on the end point, the coefficient of variation of the survival parameters ranged from 31 to 68% for lymphocytes and 21 to 41% for fibroblasts following high-dose-rate irradiation. Similar ranges were obtained after low-dose-rate irradiation. Variance analysis performed on replicate assays in cultures derived from the same patient showed that variation due to technical or sampling errors was significantly lower than variation between individuals (P = 0.00034 and 0.014 for fibroblasts and lymphocytes, respectively). No correlation was observed between the radiosensitivity of lymphocyte and fibroblast cultures derived from the same donors. We conclude that there is significant variation in normal cell radiosensitivity among individuals. On the other hand, comparisons of lymphocyte and fibroblast radiosensitivities suggest that tissue-specific characteristics, such as differentiation status, may variably modulate radiosensitivity.

Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium.

The limitations of the agar suspension culture method for primary culturing of human tumor cells prompted development of a monolayer system optimized for cell adhesion and growth. This method grew 83% of fresh human tumor cell biopsy specimens, cultured and not contaminated, from a heterogeneous group of 396 tumors including lung cancer (93 of 114, 82%); melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a miscellaneous group consisting of gastrointestinal, genitourinary, mesothelioma, and unknown primaries (78 of 91, 86%). Cell growth was characterized morphologically with Papanicolaoustained coverslip cultures and cytogenetically with Giemsastained metaphase spreads. Morphological features such as nuclear pleomorphism, chromatin condensation, basophilic cytoplasm, and melanin pigmentation were routinely seen. Aneuploid metaphases were seen in 90% of evaluable cultures, with 15 of 28 showing 70% or more aneuploid metaphases. Colony-forming efficiency ranged between 0.01 and 1% of viable tumor cells, with a median efficiency of 0.2%. This culture system uses a low inoculum of 25,000 viable cells per well which permitted chemosensitivity testing of nine drugs at four doses in duplicate from 2.2 X 10(6) viable tumor cells and radiation sensitivity testing at five doses in quadruplicate from 0.6 X 10(6) cells. Cultures were analyzed for survival by computerized image analysis of crystal violet-stained cells. Drug sensitivity studies showed variability in sensitivity and in survival curve shape with exponential cell killing for cisplatin, Adriamycin, and etoposide, and shouldered survival curves for 5-fluorouracil frequently seen. Radiation sensitivity studies also showed variability in both sensitivity and survival curve shape. Many cultures showed exponential cell killing, although others had shouldered survival curves. This method for growing cells from primary human biopsy specimens is more efficient than the agar culture method, enables easier and better biological analysis of the actual cells grown, and permits improved characterization of drug and radiation survival curves.

The role of fludarabine-induced apoptosis and cell cycle synchronization in enhanced murine tumor radiation response in vivo.

We have previously reported that fludarabine, an adenine nucleoside analogue, significantly enhances radiation-induced tumor regrowth delay and local cure in several mouse tumors. Although fludarabine potentiated tumor regrowth delay at various times from -36 h to +6 h in a SA-NH mouse sarcoma model, the greatest enhancement was observed when fludarabine was administered 24 h before irradiation. The purpose of this study was to understand the basis for in vivo enhancement of radiation efficacy by fludarabine. To examine the effect of fludarabine on DNA synthesis and cell cycle progression, tumor-bearing mice were given fludarabine by an i.p. route and then bromodeoxyuridine at various times up to 36 h, followed 0.5 h later by tumor harvest. Two-parameter flow cytometry analysis of the tumor cells using an anti-bromodeoxyuridine antibody demonstrated that an 800-mg/kg fludarabine dose stops DNA synthesis within 3 h with recovery starting at 12 h. By 24 h after fludarabine treatment, a synchronized wave of cycling tumor cells appeared in G2-M phase. The degree of DNA synthesis shutdown and the timing of the reinitiation of DNA synthesis and cell cycle progression were all fludarabine dose dependent. Interestingly, DNA synthesis reinitiated only at the G1-S boundary; cells in the S phase at the time of fludarabine administration appeared to disappear from the tumor population. To confirm these observations more directly, we pretreated tumor-bearing mice i.p. with chlorodeoxyuridine to mark the cells in the S phase, gave them fludarabine 0.5 h later, and then gave them iododeoxyuridine 0.5 h before tumor harvest. Flow cytometry analysis using antibodies specific for chlorodeoxyuridine- and iododeoxyuridined-labeled cells confirmed that cells in the S phase at the time of fludarabine administration never reinitiated DNA synthesis and disappeared from the tumor population. Immunohistological analysis of tumor sections obtained after fludarabine administration demonstrated that prelabeled S-phase cells took on an apoptotic appearance and gradually disappeared from the tumors. An in situ DNA end labeling assay demonstrated DNA fragmentation in these morphologically apoptotic cells. These results suggest that the mechanism of fludarabine enhancement of radiation response involves induced S-phase cell loss through an apoptotic pathway and subsequent synchronization of the remaining cells to a more radiosensitive cell cycle phase at the time of irradiation.

Radioprotectors in tumor radiotherapy: factors and settings determining therapeutic ratio.

WR-2721 and DDC have been used most frequently in our studies on radioprotective agents. WR-2721 was a much more potent radioprotector of murine normal tissues, both against early and late injuries of several organs and tissues, than was DDC. Protection factors for WR-2721 usually ranged between 1.5 and 2.5. Both agents protected solid murine tumors only minimally. While WR-2721 increased therapeutic ratios commonly, DDC did so only rarely. Micrometastatic foci were amenable to radioprotection more than established solitary tumors. Additional factors that influenced the degree of therapeutic benefit included dose of WR-2721, dose of irradiation (single versus fractionated), and time of WR-2721 administration in relation to radiation delivery. The ability of WR-2721 to prevent radiation-induced immunosuppression, metastatic spread, and carcinogenesis are additional benefits in the therapeutic use of this agent. Our current research on the improvement of radioprotectors for therapeutic use is focused on (a) a search for new radioprotective agents that are equal to or better than WR-2721 but less toxic and/or more specific for normal tissue, (b) understanding the basic mechanisms of action of these radioprotective agents at the molecular level, both in cells and tissues, and thus understanding the mechanisms leading to selective or preferential radioprotection of normal tissues, and (c) in vitro testing of primary human tumor cultures for their (non)susceptibility to radioprotection.

Hormone levels during prostaglandin F 2 infusions for therapeutic abortion.

PIP: This study determines the effect of prostaglandins (PGs) on fetal-placental hormone production or luteal steroidogenesis early in pregnancy by measuring plasma levels of unconjugated estrone, 17-beta estradiol, estriol, progesterone, 17-hydroxyprogesterone, human chorionic gonadotropin (HCG), and human chorionic somatomammotropin (HCS) in 7 healthy women aged 15-30 years receiving PGF2alpha for therapeutic abortion. The patients were 7-20 weeks pregnant and were all from the Clinical Research Unit of the Yale-New Haven Hospital. 5 patients participated in a dose-response tolerance study in which the drug was given over a 12-hour period at predetermined dose levels from 25-200 mcg/minute. The remaining 2 patients received 50 mcg for 12 consecutive hours, and 2 6-hour periods respectively. Heparinized blood samples were collected prior to the beginning of the infusion, at least hourly during the infusion, and also 24 hours after the beginning of the study. Transabdominal and transcervical catheters were used to monitor intrauterine pressures. A definite decline in estradiol levels (from 50-70% of initial levels) was observed during the PGF2alpha infusions. Plasma levels of unconjugated estriol were found to decline earlier and more markedly than the estradiol levels. 17-hydroxyprogesterone was undetectable in all but 1 patient who was 7 weeks pregnant. There were no significant changes in HCG levels in 4 patients until abortion and or curettage was performed. HCS levels gradually declined in 3 patients during the infusion process. This study shows that PGF2alpha does not exert a luteolytic effect in terminating pregnancy from 7-20 weeks gestation, confirming the study of Wiqvist et.al. Further study of the 1st few weeks of gestation should be done before ruling out the possibility of luteolysis in humans.^ieng

Increase in radiosensitivity of lung micrometastases by hyperbaric oxygen.

Four-day-old artificial pulmonary micrometastases of two murine fibrosarcomas, designated FSA and NFSA, showed increased sensitivity to ionizing radiation by a factor of 1.13 when animals were exposed to hyperbaric oxygen breathing before and during irradiation, implying the presence of hypoxia in the micrometastases. At the time of irradiation the diameter of FSA and NFSA metastases was smaller than 200 and 100 microns, respectively, which, on the basis of oxygen diffusion, could not be responsible for hypoxia. It is assumed that hypoxia of micrometastases is passive, reflecting the radiobiological hypoxia of lung tissue that could exist under normal breathing conditions.

The hybrid spheroid clonogenic assay for the intrinsic radio- and chemo-sensitivities of human tumors.

The Hybrid Spheroid assay is based on packaging tumor cells into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates provide an in vivo-like environment for entrapped test cells. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of noncolony forming spheroids and the Poisson distribution, the average number of clonogens per spheroid can be calculated. The clonogenicity and radiation survival curves of cells derived from human tumors (of the maxilla, tongue, larynx, mouth floor, lung, breast, ovary, and colon) were so determined. Plating efficiency was increased in these normally poorly plating tumor cells, thus enabling survival measurements which are not practical using conventional methods. The Hybrid Spheroid assay has also been applied to determine the chemosensitivity of colon cancer cells.

Radiosensitization of primary human tumor cell cultures by N-methylformamide.

The effects of the differentiation-inducing agent N-methylformamide (NMF) on the radiation response of ten primary human tumor cell cultures were investigated. Cell survival was determined using an adhesive tumor cell culture system. NMF (1%) was added to cultures on Day 1 and was left for 6 days; cultures were irradiated with graded doses of X rays (1.0-6.0 Gy) on Day 4. Using survival at 2.0 Gy as a comparative endpoint, eight of ten cultures tested exhibited enhanced radiosensitivity upon exposure to NMF. In sensitized cultures, the dose-enhancement factors ranged from 1.3 to 2.5. The NMF-mediated radiosensitization did not appear to be dependent on the histologic cell type. The results presented support previous data obtained from established cell lines and suggest that NMF may offer clinical benefits against a variety of tumor types when used in combination with radiotherapy.

Modification by dexamethasone of radiation response of in vitro cultured cells.

Because of the potential clinical significance of the report that dexamethasone is a radioprotector of Chinese hamster V-79 cells, the effect of dexamethasone treatment on the radiosensitivity of five other cultured mammalian cell lines (including two human cell lines) was tested and preliminary investigations into the mechanism of protection of V-79 cells were undertaken. In agreement with the published results of others, we found that treatment of V-79 cells with dexamethasone results in a 1.3-fold increase in D0. Conversely, dexamethasone had no effect on the radiosensitivity of Chinese hamster ovary cells, murine fibrosarcoma, rat glioma cells, human diploid fibroblasts, or human mammary carcinoma cells. To study the mechanism of the radioprotective effect of dexamethasone on V-79 cells, the cell cycle was examined. Dexamethasone treatment causes a change in cell cycle distribution in V-79 cells, resulting in a dose-dependent reduced fraction of S-phase and an increased fraction of G1- and G2 + M-phase cells. However, these kinetic changes cannot explain the observed radioprotection of asynchronous populations, since purified G1 cells are more radiosensitive. Furthermore, cells synchronized in G1 by centrifugal elutriation were shown to be protected by dexamethasone to the same extent as was the unsorted population, thereby ruling out the mechanism of protection being a redistribution in the cell cycle.

Radiosensitivity measurement of keratinocytes and fibroblasts from radiotherapy patients.

Genetic diversity is believed to influence cellular radiosensitivity and individual variability in normal tissue reactions to radiotherapy. To measure normal cell radiosensitivity in vitro, we investigated a culture technique that yields keratinocyte and fibroblast cell cultures from small skin biopsy samples (average weight 32 mg). This technique uses 3T3 NIH cells as feeder cells, culture medium containing dialyzed fetal calf serum, low calcium, and various growth factors for keratinocyte growth. A calcium concentration of 4 x 10(-3) M and the use of lethally irradiated NIH 3T3 feeder cells were critical to the success of this method. Primary keratinocyte cultures were successfully obtained from nine biopsy specimens, and radiosensitivity measurements were obtained in six of the resulting strains. Keratinocytes were, in general, more radioresistant than fibroblasts derived from the same specimen. We conclude that radiosensitivity assessment of keratinocyte and fibroblast cultures derived from small punch biopsy specimens is feasible. Further studies can now be carried out to determine the degree of variability between individuals and the relationship between in vitro keratinocyte and fibroblast radiosensitivity and their value in predicting normal tissue responses to radiotherapy.

Prospective comparison of in vitro normal cell radiosensitivity and normal tissue reactions in radiotherapy patients.

PURPOSE: This pilot study was undertaken to assess the relationship between in vitro radiosensitivity of different normal cell types and the type and severity of normal tissue reactions in individual patients after radiotherapy. METHODS AND MATERIALS: Twenty-one patients with head and neck cancer were studied prospectively; four with head and neck and two with breast cancer were studied retrospectively. The retrospective cases were chosen because they exhibited unusual (severe or minimal) normal tissue reactions after radiotherapy. Small skin biopsies and blood samples were obtained and used to generate in vitro fibroblast and lymphocyte cultures, respectively. Clonogenic assays were used to measure in vitro fibroblast and lymphocyte radiosensitivity after high- and low-dose rate irradiation. Head and neck patients were treated by conventional, hyperfractionated, or concomitant boost regimens, which have been found to yield an equal probability of late normal tissue reactions. The highest dose received by each normal tissue in the target volume was estimated using computed tomography treatment plans. The median patient follow-up time was 19 months (range: 13-25). RESULTS: The distributions of in vitro radiosensitivity parameters and the grade of tissue reaction scores in the patients showed a broad range between individuals. When in vitro parameters were compared to the acute and late tissue reactions, the radiosensitivity of fibroblasts, measured as surviving fraction at 2 Gy after high-dose rate irradiation, showed a highly significant correlation with the maximum grade of late effects (p < 0.0001 for the whole group and p = 0.0013 for the group of patients studied prospectively). No significant correlation was found between fibroblast radiosensitivity and maximum grade of acute effects or between lymphocyte radiosensitivity and either acute or late effects. CONCLUSION: We conclude that individuals vary in normal cell radiosensitivity, and that in vitro measurements of fibroblast radiosensitivity may predict the magnitude of late normal tissue reactions after radiotherapy. These preliminary results, however, need to be validated in a larger group of patients.

Cellular radiosensitivity of primary head and neck squamous cell carcinomas and local tumor control.

The low dose survival parameters of human tumor cell lines have been shown to correlate with the perceived clinical radiosensitivity of different tumor histologic types. This conclusion has been generated from the analysis of a large number of cell lines and has, therefore, served as the basis for attempts to develop predictive assays of tumor radiocurability. In this study, the tumors from 72 patients with head and neck squamous cell carcinoma have been grown in an adhesive tumor cell assay system and their sensitivity to radiation has been measured. All patients in this study were treated with post-operative radiotherapy, the surgical margins were negative, and any patient that had received chemotherapy was excluded. The average S2 (survival at 2.0 Gy) value of the 72 cultures was 0.33, with the values ranging from 0.11 to 0.91. All patients were evaluated for local tumor control. They have been followed for about 1 year and continued follow-up is still in progress. The average survival at 2.0 Gy of cultures derived from the 12 patients that have had recurrences so far is slightly higher (0.40) than that from those who appear so far to have local tumor control (0.30). Although the general trend is that recurrent tumors yield primary cultures that are slightly more resistant, the difference is not statistically significant.