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In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.

A computer program has been developed to quantitatively evaluate changes in tumor growth rates of a solid tumor model (hepatoma 3924A) after a series of radiation doses from 375 R to 3750 R. The computer-derived growth curves are simulated from the volumes of the individual tumors rather than from the mean tumor volume at any specific time point after treatment. The ability to generate ata from a family of tumor growth curves permits a more precise evaluation of therapeutic effects on tumors than can beobtained with conventional methods. The quantitative determination of equivalent amounts of radiation needed to produce comparable 5-fluorouracil-induced changes in tumor growth rate has been made. The ability to determine quantitatively radiotherapeutic and chemotherapy equivalents on these solid tumor models has direct implications in regard to our effort to improve the treatment of cancer. At present no specific solid tumor or groups of solid tumors have provided al1 of the necessary information for clinical utilization in therapeutic scheduling of different forms of cancer treatment. Since solid tumors comprise the majority of human cancer, one of the primary objectives of these studies has been the establishment of a solid tumor model that could serve both as a system for devising improved therapeutic scheduling and for a better understanding of solid tumors. The rate of change of tumor volume with time is one of the basic measurements for the study of tumor growth (1-9).While Morris hepatomas have been extensively used in cancer research, no systematic study has been made of the effects of radiation on these solid tumors (10,11). This study of the effects of different x-radiation doses on tumors is part of a continuing study of the assessment of therapeutic response of different modalities of treatment in this experimental solid tumor system. Studies of the effects of radiation on cycle times, potential doubling times, cell loss factor, and the fraction of proliferating and nonproliferating cells (growth fraction) are needed along with the effects of radiation on tumor growth rates to assess treatment properly. This report is primarily concerned with the methods of evaluation of radiation-induced changes in tumor volumes and the results of different doses of radiation over a range of 375-3750 R.MATERIALS AND METHODS Female ACI rats were inoculated subcutaneously in the back with 3924A hepatoma cells by Dr. Harold Morris in Washington D.C. and shipped to this laboratory. The rats were maintained under standard laboratory conditions including commercial laboratory rat chow (Charles River Laboratories, Wilmington, Mass.) supplied ad libitum, and a 12 hr lighting schedule, the dark period beginning at 8:00 p.m.Tumor volumes, V, (mm3) were calculated from measurements of length (L), width (W), and height (H) (5). It was assumed that the tumors are approximately hemiellipsoids whose volume is lit L-W-H (1). The tumors were measured and the rats weighed daily before radiation and for 6 days afterward. The m...

Summary.-The effect of 5-fluorouracil (5-FU) on the growth and cellular proliferation of hepatoma 3924A was studied using the following parameters as indices of tumour response: (1) volume measurements, (2) cell kinetic analysis including estimates of both growth and cell loss fractions, (3) changes in tumour histology and (4) tumour DNA content and DNA synthesis. Of a series of single intraperitoneally injected doses (25-300 mg/kg body weight), 150 mg/kg interrupted tumour growth most effectively with minimal toxicity within 168 h, and after 10 days treated tumour volumes were only 42% of untreated tumour size. Doses of 25 mg/kg failed to change the rate of growth while 300 mg/kg exceeded the LD50.