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The variability in radiosensitivity across the human population is governed in part by genetic factors. The ability to predict therapeutic response, identify individuals at greatest risk for adverse clinical responses after therapeutic radiation doses, or identify individuals at high risk for carcinogenesis from environmental or medical radiation exposures has a medical and economic impact on both the individual and society at large. As radiotherapy incorporates particles, particularly particles larger than protons, into therapy, the need for such discriminators, (i.e., biomarkers) will become ever more important. Cellular assays for survival, DNA repair, or chromatid/chromosomal analysis have been used to identify at-risk individuals, but they are not clinically applicable. Newer approaches, such as genome-wide analysis of gene expression or single nucleotide polymorphisms and small copy number variations within chromosomes, are examples of technologies being applied to the discovery process. Gene expression analysis of primary or immortalized human cells suggests that there are distinct gene expression patterns associated with radiation exposure to both low and high linear energy transfer radiations and that those most radiosensitive are discernible by their basal gene expression patterns. However, because the genetic alterations that drive radio response may be subtle and cumulative, the need for large sample sizes of specific cell or tissue types is required. A systems biology approach will ultimately be necessary. Potential biomarkers from cell lines or animal models will require validation in a human setting where possible and before being considered as a credible biomarker some understanding of the molecular mechanism is necessary.

Previous reports have suggested that measuring radiosensitivity of normal and tumor cells would have significant clinical relevance for the practice of radiation oncology. We hypothesized that radiosensitivity might be predicted by analyzing DNA end-binding complexes (DNA-EBCs), which form at DNA double-strand breaks, the most important cytotoxic lesion caused by radiation. To test this hypothesis, the DNA-EBC pattern of 21 primary human fibroblast cultures and 15 tumor cell lines were studied. DNA-EBC patterns were determined using a modified electrophoretic mobility shift assay and were correlated with radiosensitivity, as measured by SF2. DNA-EBC analysis identified a rapidly migrating ATM-containing band (identified as "band-A") of which the density correlated with SF2 (0.02

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.

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.

Clinically defined groups of tumors are usually characterized by shallow dose-response curves, and this results from heterogeneity among individual dose-response curves, each of which is very likely quite steep. A review of published results for human tumors indicates that a 10% escalation of dose to tumors controlled at the 50% level, where changes in outcome are most likely to be detected, will be detectable in a population of unselected patients only in sizable clinical trials (130-300 patients per dose level). With a few exceptions, a dose escalation of 20% will be detectable in much smaller trials (50-130 patients per dose level). Therefore, clinical trials of improved treatment modalities will be confounded by patient heterogeneity, and modest improvements may go undetected in all but the largest trials. Mathematical modeling was used to study the effect on the steepness of the dose-response curve of selecting patients on the basis of the radiosensitivity measure SF2 (surviving fraction at 2 Gy). If SF2 is a faithful predictor of response in a group of tumors, then heterogeneity could be reduced by excluding the patients with the most sensitive (controlled with near certainty) and most resistant (recurring with near certainty) tumors. The resulting "stochastic fraction" (tumors for which treatment outcome is probabilistic) would be characterized by a steep dose response, and the number of patients required to demonstrate the effect of dose escalation would be substantially reduced (by about 50%).

Several predictors of tumor radiocurability are already integrated into clinical practice, for example, tumor size, gross morphology (that is, infiltrative or exophytic), histologic type and grade. These are nonspecific and relatively imprecise. The aim of research into predictive assays is not only to refine the discrimination of existing predictors, but also to suggest specific experimental approaches for overcoming tumor radioresistance in individual patients. Two broad categories of predictive assays can be defined: direct and indirect measurement of tumor cell survival and/or repair capability following irradiation, and measurement of cellular and extracellular parameters affecting radiosensitivity. The ongoing research at The University of Texas M. D. Anderson Hospital is overviewed to illustrate potential methods for predicting radiocurability.

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.

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 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.

The present method for predicting the radiocurability of individual human tumors is based upon considerations of tumor size, site, histological type and grade, and host factors such as sex and age. Small tumors located such that normal tissues do not seriously limit total dose and those with "favorable" histology are more radiocurable than large tumors located over a critical normal tissue. However, the precision of prognosis based upon those features is relatively low. The need for other parameters for more accurate predictability is greater than ever because of the existence of different radiation modalities, including neutrons, and the development of a broad range of chemotherapeutic drugs that can be used alone or in combination with radiation and surgery. In this laboratory we are testing the micronucleus (MN) assay for measuring the relative biological effectiveness of high- and low-LET irradiations in mouse tumors and a new primary human tumor cell culture system for making direct measurements of tumor cell radiosensitivity. The potential usefulness of these two systems in predicting human tumor response to radiotherapy is discussed.

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.

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.