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Re: Cancer Chemoprevention: Progress and Promise The commentary by Lippman et al. (1) provides a useful review of the topic by organizing a massive amount of in- formation in an organized way. How- ever, because this review will be widely read and referenced, there are two gen- eral points made by the authors that could be challenged. The authors define two of the four criteria for identifying a “definitive” chemoprevention trial as “primary end point of cancer incidence” and “large scale (n 艌 1000) with the Journal of the National Cancer Institute, Vol. 91, No. 6, March 17, 1999 definitive sample size and duration based on anticipated event rates in the intervention arm (treatment effect) and placebo arm.” One major goal of chemoprevention research is to determine the protective effect of an agent while placing as few participants as possible at risk. One straightforward way to accomplish this goal is to understand the process of the disease (carcinogenesis) sufficiently to conduct trials with markers that predis- pose to or predict the final end point of cancer. In general, most histologically identifiable precancers (e.g., cervical in- traepithelial neoplasia, Barrett’s esopha- gus, adenoma polyps, actinic kerotoses, and dysplastic nevi) evolve to cancer with a sufficiently predictable frequency to conclude that their reversal or sup- pression can be used to predict cancer development and to assess the value of a chemoprevention agent. There are, in fact, as the authors note, several studies (2–6) that have addressed the effective- ness of chemoprevention in this manner. The medical community accepts hyper- tension and cholesterol as surrogate “preneoplasias” of cardiovascular dis- ease risk and their modulation as indica- tive of a favorable or unfavorable drug effect. Modulation of the pathobiology of precancers is as valid a marker of carcinogenesis as is the end point of can- cer. Undoubtedly, advances in our un- derstanding of carcinogenesis will allow us to identify and develop new agents by the modulation of a biochemical event earlier in the carcinogenic process and this should be a major goal of chemo- prevention research. Large numbers may provide comfort to the investigator that a definitive result has been obtained. However, there are many instances in medicine when large numbers were not required to make the point, i.e., where the underlying cause CORRESPONDENCE 563

To evaluate the effect of daily beta-carotene (30 mg) versus placebo over a 2-year period on cervical intraepithelial neoplasia (CIN) 2 and 3 lesions. Human papillomavirus (HPV) typing was done to determine whether lesion regression was related to HPV. Micronutrient levels were measured to determine whether levels were predictive of regression. Variables that influence the risk of HPV infection and CIN, such as cigarette smoking and sexual behavior, were evaluated. Women were randomized to beta-carotene or placebo, with cytology and colposcopy every 3 months. Cervical biopsies were performed before treatment and after 6 and 24 months to evaluate response. Persistence of or progression to CIN 3 resulted in removal from the study, whereas treatment continued for 2 years on all others. The presence and type of HPV was determined by PCR. Response was defined as an improvement in CIN by 2 grades. Mantel-Haenszel chi(2) test was used to analyze response to treatment. Fisher's exact test was used to determine the effect of HPV and CIN grade on response Wilcoxon's rank-sum tests were used to compare micronutrient levels between groups. Twenty-one of 124 enrolled women were not randomized because they either moved, became pregnant, voluntarily withdrew, or the pathological review of their initial cervical biopsies did not confirm CIN 2 or 3. Of the remaining 103 women, 33 experienced lesion regression, 45 had persistent or progressive disease, and 25 women did not complete the study and were considered nonresponders in the final analysis. The overall regression rate (32%) was similar between treatment arms and when stratified for CIN grade. Data on 99 women with HPV typing showed that 77% were HPV-positive and 23% HPV-negative at enrollment. HPV-positive lesions were subdivided into indeterminate-, low-, and high-risk categories; the response rate was highest for women with no HPV detected (61%), lower for indeterminate/low-risk (30%), and lowest for high-risk (18%; P =.001). CIN regression was negatively correlated with retinol levels. In conclusion, beta-carotene does not enhance the regression of high-grade CIN, especially in HPV-positive subjects.

The human melanocyte is continuously exposed to intrinsic and extrinsic sources of reactive biochemical species, but is finely tuned via the intrinsic anti-oxidant and radical properties of melanin to suppress the build-up of an altered redox phenotype. We propose that this control is lost during melanomagenesis and inappropriate redox-sensitive transcriptional factor activations occur which result in enhancement of an anti-apoptotic phenotype in the transformed cell. This conceptual framework offers testable steps to determine the role of redox alterations in the carcinogenic evolution, prevention and treatment of melanoma and other diseases of the melanocyte.

A major goal in the development of chemopreventive agents has been to develop markers that reflect the underlying process of carcinogenesis and which are modulatable by the agent under study. An important application of such markers will be to select cohorts that are at elevated risk for cancer development, which should allow use of smaller sample sizes in definitive phase III trials as well as shorter duration (and lower cost), without loss of statistical power. Susceptibility and surrogate end-point biomarkers are particularly important in this respect. Intermediate markers are probably best assessed in terms of proportionate rather than relative risk. The systematic development of difluoromethylornithine for use in chemoprevention against human cancer has involved pilot, phase IIa and IIb trials using participants with prior colonic polyps as the study group. A unique feature of the phase IIa study was the use of a dose de-escalation design which allowed selection of the lowest effective non-toxic dose of difluoromethylornithine. The phase IIb trial now in progress is using a combination of sulindac with difluoromethylornithine; the rationale for selection of markers for this study and for a randomized phase III registration trial is discussed. We also review the findings in phase I and IIa trials of Bowman-Birk inhibitor concentrate, in which patients with measurable oral leukoplakia are the study group.

Bowman-Birk inhibitor is a protease inhibitor derived from soybeans that has demonstrated chemopreventive activity in a number of in vitro and animal systems. We conducted a 1-month phase IIa clinical trial of Bowman-Birk inhibitor concentrate (BBIC) in patients with oral leukoplakia. BBIC was administered to 32 subjects with oral leukoplakia for 1 month. We assessed toxicity and clinical and histological response of the lesions, and oral mucosal cell protease activity (PA) and serum micronutrient levels were measured. Clinical response was determined by measurement of pre- and posttreatment individual and total lesion areas and analysis of blinded clinical judgments of photographs. On the basis of prespecified response criteria, 31% of patients achieved a clinical response (two with complete and eight with partial responses). BBIC was nontoxic in doses up to 1066 chymotrypsin inhibitory units. The mean pretreatment total lesion area decreased from 615 to 438 mm2 after BBIC treatment (P0.004). A linear fit of the dose-response relationship between dose of BBIC and decrease in total lesion area was suggested (P0.08), and analysis of blinded clinical impression from lesion photographs confirmed this relationship (P0.01). Overall, at all doses tested, a 24.2% decrease in total lesion area was observed following treatment (sign rank = -142; P0.004). High pretreatment PA was associated with greater decreases in PA after BBIC administration (P0.02). BBIC demonstrated clinical activity after oral administration to patients with oral leukoplakia. These results indicate that BBIC should be investigated for chemopreventive activity in a randomized clinical trial.

Effects of variations in agent, dose, and route of treatment administration on patient reported quality of life (QOL) were examined for 279 patients enrolled on a seven-arm randomized clinical trial (S8905) of 5-FU and its modulation for advanced colorectal cancer. Patients completed QOL questionnaires at randomization and weeks 6, 11, and 21 post-randomization with five QOL endpoints considered primary: three treatment-specific symptoms (stomatitis, diarrhea, and hand/foot sensitivity); physical functioning; and emotional functioning. Patient compliance with the QOL assessment schedule was good, supporting the feasibility of including QOL measures in cooperative group trials. However, death and deteriorating health produced substantial missing data. Cross-sectional analyses indicated that the seven therapeutic arms did not differ in their impact on QOL. Unfortunately, longitudinal analyses of the QOL data were inappropriate given non-random missing data. Graphical presentation of non-random missing data identified the seriousness of this problem and its effect on potential conclusions about QOL during treatment. This problem appears to be particularly challenging in the context of advanced-stage disease. Failure to recognize the presence of non-random missing data can lead to serious overestimates of patient QOL over time.

The molecular approach to cancer has identified specific abnormalities that contribute to malignant pathogenesis in an aetiological manner and define individuals who are at higher risk for specific malignancies. Studies of cancer distribution in families suggest that 15-20% of all malignancies may have a significant germ line hereditable mutation that directly or indirectly contributes to tumour development. Additionally, the identification of many genetically-determined polymorphisms that regulate carcinogen metabolism indicate that their assessment may contribute to selecting individuals for preventive surveillance or intervention as well. Locating individuals in the population who have moderate to high risk germ line mutations in critical oncogenic regulatory genes and assessing a panel of polymorphisms that underlie a significant attributable risk for cancer development may allow the recruitment of individuals at high risk for a particular malignancy and, therefore, represent good candidates for either directed organ surveillance and/or chemoprevention trials.

If one were to wait for the perfect set of experimental results before launching a multi-agent chemoprevention or large risk reduction study, the trial would never be launched. On the other hand, non-scientific considerations have led to the premature launching of at least three prominent studies (CARET, Carotene and Retinol Efficacy Trial; ATBC, Apha Tocopherol Beta Carotene; PCPT, Prostate Cancer Prevention Trial) and the much delayed start-up of another, BCPT, the Breast Cancer Prevention Trial. Strong epidemiologic data by itself should not be adequate to justify starting a large trial; experimental and/or clinical data should be developed. On the other hand fear of secondary adverse events that are of low incidence should not be enough to delay a trial if the overall health benefit could be high. The development of multiagent chemoprevention trials requires that each agent is active and additively or synergistically so in combination in preclinical models. Additionally, side effects of each agent should be non-overlapping and low to non-existent, preferably a feature determined in formal phase IIa and IIb trials. These principles will be discussed in the context of prior (CARET, ATBC) and ongoing (EUROSCAN, acetylcysteine/retinol), as well as proposed future trials (difluromethyl/sulindac).

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor and a potential cancer chemopreventive agent for humans. In this Phase I clinical trial, BBI concentrate was administered as a single oral dose to 24 subjects with oral leukoplakia. Pharmacokinetics of BBI was analyzed, and subjects were monitored clinically for toxic effects. Subjects received between 25 and 800 chymotrypsin inhibitor units (CIU) of the compound in a dose escalation trial. BBI was taken up rapidly, and a metabolic product of BBI was excreted in the urine within 24-48 h. No clinical or laboratory evidence of toxicity was observed in the study. Protease activity was also measured in buccal cells to evaluate usefulness as a biomarker. Single-dose BBI concentrate administered up to 800 CIU was well tolerated and appeared to be nontoxic. Further investigation in Phase II clinical trials is being done.

The protease catalyzing the hydrolysis of the tripeptide fluorescence substrate, butoxycarbonyl-valine-proline-arginine-(7-amino-4-methylcoumarin) (Boc-Val-Pro-Arg-MCA) and the neu oncogenic protein are potentially useful biomarkers for human cancer prevention studies. In the present study, we standardized a specific substrate hydrolysis method for measuring this protease activity in human oral mucosal cells and characterized the relationship between neu oncogene expression and protease activity in patients enrolled in an oral cancer prevention trial using Bowman Birk Inhibitor Concentrate (BBIC) as the cancer preventive agent. The results demonstrate that changes in the protease activity in oral mucosal cells after BBIC treatment correlated with the changes in the neu protein levels in oral mucosal cells (r = 0.726, P0.001) and serum (r = 0.675, P0.001), suggesting that the Boc-Val-Pro-Arg-MCA hydrolyzing activity can be as useful as neu oncogene expression as a cancer biomarker. In the 25 patients enrolled in the study, the level of neu protein in oral mucosal cells correlated with the serum neu protein concentration in the patients before BBIC treatment (r = 0.645, P0.001). However, such a correlation was not observed after the BBIC treatment, suggesting that BBI may inhibit serine protease(s) involved in the cleavage of neu protein on the cell surface, thereby preventing the release of the extracellular domain of neu protein into the circulation. By inhibiting the cleavage of neu protein on the cell surface, BBI could prevent malignant and premalignant cells expressing high levels of neu protein antigen from escaping host immunological surveillance control.

The biological basis for the general pharmacological resistance of human melanoma is unknown. A unique biochemical feature of the melanocyte is the synthesis of melanin, which leads to the generation of hydrogen peroxide and the consumption of reduced glutathione. This activity produces a state of chronic oxidative stress in these cells. We demonstrated previously that the expression of the c-jun family was dysregulated in metastatic melanoma cells compared with normal human melanocytes (D. T. Yamanishi et al., J. Invest. Dermatol., 97: 349-353, 1991). In the current investigation, we measured the levels of two major redox response transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1, in metastatic melanoma cells and normal melanocytes and their response to oxidative stress. The basal DNA-binding activity of NF-kappaB as measured by the electrophoretic mobility shift assay in metastatic melanoma cells was increased 4-fold compared with that of normal melanocytes. This level of binding was paralleled by a 1.5- to 4-fold increase in the expression of p50 (NF-kappaB1), p65 (Rel-A), and IkappaB-alpha as measured by Northern blot analysis. In contrast, the expression of p75 (c-rel) was markedly decreased (60%) in melanoma cells compared with normal melanocytes. Following oxidative stress produced by enzyme-generated H2O2, free H2O2, or incubation with buthionine sulfoximine, NF-kappaB binding activity increased 1.5- to 2.5-fold in melanoma cells (buthionine sulfoximineH2O2), but only slightly in normal melanocytes. In contrast, activator protein-1 binding activity was unaffected or increased in normal melanocytes in response to oxidative stress, but was either unaffected or decreased in melanoma cells. These results suggest that the redox regulation of melanoma cells at the molecular level is fundamentally different from normal melanocytes and may offer a unique avenue for preventive or therapeutic intervention as well as new insights into the pathogenesis of melanocyte transformation.

D,L-alpha-difluoromethylornithine (DFMO) was synthesized over 20 years ago. It was hoped that this enzyme-activated, irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis, would be effective as a chemotherapy for hyperproliferative diseases, including cancer and/or infectious processes. DFMO was generally found to exert cytostatic effects on mammalian cells and tissues, and its effectiveness as a therapeutic agent has been modest. DFMO was also found to cause treatment-limiting (but reversible) ototoxicity at high doses. This side effect, along with its minimal therapeutic activity, contributed to the loss of interest by many clinicians in further developing DFMO as a cancer therapeutic agent. However, DFMO was subsequently shown to inhibit carcinogen-induced cancer development in a number of rodent models, and interest in developing this compound as a preventive agent has increased. The rationale for the inhibition of ornithine decarboxylase as a cancer chemopreventive agent has been strengthened in recent years because this enzyme has been shown to be transactivated by the c-myc oncogene in certain cell/tissue types and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. Recent clinical cancer chemoprevention trials, using dose de-escalation designs, indicate that DFMO can be given over long periods of time at low doses that suppress polyamine contents in gastrointestinal and other epithelial tissues but cause no detectable hearing loss or other side effects. Current clinical chemoprevention trials are investigating the efficacy of DFMO to suppress surrogate end point biomarkers (e.g., colon polyp recurrence) of carcinogenesis in patient populations at elevated risk for the development of specific epithelial cancers, including colon, esophageal, breast, cutaneous, and prostate malignancies.

The field of chemoprevention of cancer in humans is at a teenage level of maturity. There is anticipation and energy, and some promising results have come in, but it's unclear whether the entire enterprise is worth the effort. Reflecting on the status of the organism and where we are in its developmental history is therefore an important exercise at this time. Empirical and philosophical perspectives are offered for several key questions: Why prevent Cancer? What is the preclinical evidence that chemoprevention of cancer in humans should work? What is the clinical evidence that chemoprevention agents work? What is the clinical evidence that chemoprevention agent don't work? What is the status of ongoing randomized phase III/IV chemoprevention trials? The answers to each of these questions provide a part of the scaffold for a logical platform for the launching of the chemoprevention imperative as an integral part of our approach to the overall management of human cancer.

Interferon alfa has modest but definite activity in the treatment of metastatic melanoma and is the only agent currently available for adjuvant therapy of high-risk resected disease. A variety of retinoic acid derivatives have been shown to be synergistic with interferon alfa in vitro and in vivo, with nonoverlapping toxicities. If promising combinations of interferon alfa and retinoids could be developed for melanoma patients, they would have clinical relevance for the treatment of advanced as well as localized disease.To determine the efficacy and toxicity of a combination of interferon alfa-2a and all-trans-retinoic acid in patients with measurable metastatic melanoma, the South-west Oncology Group conducted a phase II clinical trial.Fifty-seven patients with measurable metastatic melanoma (American Joint Committee on Cancer stage IV) were entered; five patients were unevaluable. Treatment consisted of oral all-trans-retinoic acid (37.5 to 75 mg/m2 orally twice daily for 21 days followed by 7 days' rest) plus subcutaneously administered interferon alfa-2a (6 MU/m2 three times a week).Two complete and three partial responses were observed among 52 evaluable patients, for an objective response rate of 10% (95% confidence interval 3% to 21%). Responses were seen only in patients with pulmonary, nodal, or subcutaneous metastases, and lasted from 4 to 23+ months. Median survival for the 52 patients was 8 months. Side effects were tolerable but significant, with one case of grade IV anemia and 92% of patients experiencing at least grade II toxicity. Flu-like symptoms were the most commonly reported side effects. There was one case of grade III hyperlipidemia.The combination of recombinant human interferon alfa-2a with all-trans-retinoic acid did not result in a greater percentage of objective responses or a longer overall survival than that associated with interferon alfa alone. This combination cannot be recommended for further evaluation in melanoma in either the advanced disease or the adjuvant settings.

The Beta-Carotene and Retinol Efficacy Trial tested the effect of the combination of beta-carotene (30 mg) and retinyl palmitate (25,000 units) daily on the incidence of lung cancer in high-risk individuals. In study centers located in Seattle, WA; Portland, OR; and Irvine, CA, we recruited current and recent ex-cigarette smokers, aged 50-69 years. Our primary method of recruitment was by mailing study information and eligibility questionnaires to age-selected health insurance subscribers. A total of 1,216,549 subscriber households were contacted, which resulted in 16,449 enrollments and 12,184 randomizations. Other methods of recruitment yielded 1421 enrollments and 1002 randomizations. Seventy-four % of those participants who enrolled in the 3-month placebo run-in were randomized. The major reasons for nonrandomization once subjects were enrolled were: becoming ineligible (13%), concern about or development of side effects attributed to the study vitamins (18%), loss of interest or being too busy (23%), and not showing up at the appointed time or not willing to come to the study center (23%). Here, we discuss the reasons for nonparticipation and for subjects leaving the trial prior to randomization and possible modifications of trial design and procedures to address these problems. This recruitment approach provided a constant flow of potentially eligible participants, screened out many ineligible and uninterested persons prior to the scheduling of a study center visit, and ensured randomization of committed participants. A major limitation of this study was that the pool of minorities that was reached was small.

The results of Southwest Oncology Group Study 8711 (Group 2B) are presented. The objective was to evaluate the natural history of sperm concentration and selected hormonal parameters in patients with testicular cancer treated with orchiectomy and radiotherapy.Of a total of 207 patients enrolled on SWOG 8711, 53 pure seminoma patients were identified who were treated with orchiectomy and radiotherapy only. Sperm concentration, follicle-stimulating hormone (FSH) levels, and sexual satisfaction scores were the main parameters followed.A fraction of the patients were infertile prior to receiving radiotherapy. Our analysis indicates that incidental radiation dose to the remaining testicle affects time to recovery of fertility, and at an aggregate level, changes in FSH mirror changes in sperm concentration over time. This phenomenon is the same as that described in patients free from testicular cancer. These men evaluated their sexual activity as good after orchidectomy.Our data support the use of clamshell-type testicular shields as a means of providing maximum protection to the remaining testicle.

It is essential to identify intermediate marker endpoints of carcinogenesis for the evaluation of the effectiveness of cancer-chemopreventive agents. We have observed that levels of proteolytic activities (as detected by 4 different substrates) are increased 2-3-fold (P0.003) in oral buccal mucosa cells of smokers and patients with oral leukoplakia or erythroplakia as compared to a nonsmoking comparison group. In addition, proteolytic activity levels in the buccal cells were increased nearly 3-fold in patients with oral trauma (P0.01) or diabetes (P0.02), as well as pregnant women (P0.04). Excluding these subgroups of patients in epidemiological studies increase the differences in levels of proteolytic activities between both the nonsmoking comparison group and smokers and between the comparison group and patients with oral leukoplakia or erythroplakia. Evaluation of prerandomization levels of proteolytic activities of patients in cancer chemoprevention trials will increase the statistical power by allowing stratified randomization based on levels of proteolytic activities. The observed increases in levels of proteolytic activities in tissues at higher than normal risk of cancer development suggest that levels of proteolytic activities should be used as immediate marker endpoints in human cancer prevention trials using protease inhibitors as potential anticarcinogenic agents.

Several epidemiological reports and experimental investigations have suggested a preventive role for folic acid in the etiology of cervical cancer. The effect of p.o. folic acid supplementation on the natural history of cervical intraepithelial neoplasia (CIN) was evaluated in a multiinstitutional prospective, randomized, double-blind, placebo-controlled trial. Three hundred thirty-one women with biopsy-proven koilocytic atypia, mild CIN, or moderate CIN were randomized to receive oral folic acid (5 mg) or a similar-appearing placebo daily for 6 months following a 1-month run-in placebo period. Colposcopy, Papanicolaou smear, and serum vitamin levels (folate, retinol, alpha-tocopherol, beta-carotene, and retinyl palmitate) were monitored every 3 months. Demographic, medical, dietary, and sexual history data were obtained from personal interviews. The primary end point of the study was improvement in both Papanicolaou smear and colposcopic picture after 3 and 6 months of treatment as compared to the start of treatment. After 6 months of treatment there was no significant difference between the two study groups in the percentage of patients improved. Median serum folate levels in the treatment arm at 3 and 6 months (29.0 and 20.0 micrograms/dl) were significantly higher than those in the placebo arm (7.8 and 7.1 micrograms/dl, respectively). Mean serum levels of retinol, retinyl palmitate, alpha-tocopherol, and beta-carotene did not differ significantly between the two treatment arms. Our data support the conclusion that supplementation with folic acid (5 mg/day) does not enhance the regression of early epithelial abnormalities of the cervix.(ABSTRACT TRUNCATED AT 250 WORDS)

In summary, prospective studies within the last 10 years have made a significant impact on our understanding of the natural history of early melanoma. Surgeons can now excise the melanoma with narrower and more selective margins that preserve function and cosmetic appearance. Early melanomas are now, and will continue to be the most common presentation of this cutaneous malignancy. This reflects an enhanced awareness of the public and the medical community about the value of increased detection, and serious attempts at protection from intense ultra-violet light exposure. Current research is now focusing on the inherited forms of melanoma, the effects of ultra-violet light on melanocytes and the immune cells of the skin, and the molecular measurement of the abnormal cytogenetics seen in malignant melanocytes. These studies will allow physicians to detect a melanoma at an earlier and earlier time point in its natural history, and perhaps prevent or reverse the formation of melanoma early in life.

Cultivation of human melanocytes requires several growth factors for cell proliferation. For example, basic fibroblast growth factor (bFGF) is an essential growth agent for melanocyte proliferation in vitro and has been proposed to be an autocrine growth factor in human melanoma cells. Studies using either anti-bFGF antibodies or antisense oligonucleotides partially inhibited the proliferation of human melanoma cells. However, one group was unable to detect bFGF RNA transcripts in human melanoma cells using a human complementary DNA probe. These contradictory results prompted us to investigate the bFGF gene expression in human primary melanocytes and metastatic melanoma cells using Southern, Northern, and Western blot analyses. No gross rearrangements in the bFGF gene were detected in the genomic DNA. Although high levels of bFGF RNA transcripts were detected in melanocytes, no bFGF protein was detected using Western blot analysis. In contrast, melanoma cells expressed much lower levels of bFGF RNA transcripts, and cells from three of four cell strains synthesized the multiple isoforms of bFGF protein. In one of the melanoma cell strains, no bFGF protein was detected using Western blot analysis. Although three of four melanoma cell strains expressed bFGF protein, this molecule does not appear to function as an autocrine growth factor, and expression of the bFGF protein was not a consistent alteration in all melanoma cell strains.

A substantial amount of epidemiologic and laboratory data indicates that the majority of human cancers should be preventable. In this article, the biologic basis of cancer prevention is presented and the major factors contributing to the initiation, promotion, and progression of cancer in humans detailed. These include tobacco, ultraviolet and ionizing (radon) radiation, certain chemicals (asbestos, aniline dyes), and viruses--both sexually transmitted and endemic. Dietary factors both enhance and inhibit carcinogenesis in humans. In this article, the roles of fat, fiber, and beta-carotene are reviewed with respect to the genesis of breast, colon, prostate, lung, and oral cancers. A large number of prevention trials involving most common malignancies are being conducted in the United States and abroad. This article considers the major strategies and methodology of prevention trials and outlines the major trials now in progress.

Difluoromethylornithine (DFMO) is an investigational chemopreventive agent that inhibits ornithine decarboxylase (ODC) activity, lowers cellular polyamine concentrations, and decreases cell proliferation in vivo and in vitro. In five subjects we have compared the polyamine concentrations in rectal mucosal biopsies and in exfoliated buccal mucosal cells (EBM) before and after DFMO treatment to assess the suitability of EBM as an easily accessible marker tissue for DFMO suppression of polyamine synthesis in the rectal mucosa. One month of 3 g/m2/day of DFMO treatment caused a statistically significant decrease in putrescine and spermidine concentrations in rectal mucosa biopsy specimens but not in EBM samples. ODC activity in EBM was high (approximately 1 mumol/min/mg protein), resistant to DFMO inhibition (Ki = 4200 microM), dependent on GTP concentration (maximal at 0.1 mM), and was reduced concomitantly with bacterial concentration by antiseptic mouthwashing. Bacteria adherent to EBM were visible by electron microscopy. Forty bacterial colonies/ng protein were culturable from washed EBM samples. Oral bacteria preclude the use of EBM samples as a marker tissue of DFMO effect in the rectal mucosa, but oral DFMO therapy is effective in depleting polyamines in rectal mucosa.

Detection of cancer is the definitive endpoint in the conduct of chemoprevention trials. There are, however, several reasons why cancer as the endpoint may not be feasible or ethical: 1) the usage of patients with easily followable preneoplasias may preclude the development of cancer, and 2) the time to a cancer event may be long or the incidence uncommon, even in individuals at high risk. Two major types of biomarker intermediate endpoints should be considered: 1) those that identify individuals at high risk and 2) those that serve as a surrogate for cancer. Various epidemiologic features, including family history, have been used to estimate relative risk. This approach, however, only slightly decreases the size of populations needed for chemoprevention trials and only little addresses the question of individual risk. Advances in understanding the genetic basis for cancer will lead to the development of probes that will help assess risk for many cancers. Innumerable biomarker intermediate endpoints can be identified as associated with cancer formation, including genetic, epigenetic, and histologic features. The challenge is not in identifying potential biomarker intermediate endpoints but in showing that they are relevant. Carcinogenesis has been shown to be carcinogen, inhibitor, dose, tissue, and species specific; it is likely that relevant biomarker intermediate endpoints will need to be identified, studied, and verified in human models. The upper aerodigestive system should be a rich source for biomarker intermediate endpoint studies, as tissue is readily available, the carcinogenic process can be monitored, and there are currently available reasonable compounds to use in biomarker intermediate endpoint modulation and chemoprevention trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemoprevention is a relatively new area of clinical cancer research. In this article we have presented a general overview of the concepts as well as the status of ongoing clinical trials using the most promising agents in selected populations. The reader is referred to recent excellent reviews presenting more detailed discussions of the various topics discussed, such as the selection process for new agents, in vitro and animal model screening procedures, epidemiologic studies, and other agents now being screened and tested for potential clinical study. Increased understanding of the biology of carcinogenesis will undoubtedly lead to new ideas and approaches. Pathways for conversion of proto-oncogenes to tumor-supporting oncogenes, the mechanisms of action of tumor suppressor genes, and the involvement of abnormal growth factor responses in producing "field cancerization" are some examples of future targets for chemopreventive intervention. Long-term goals of this effort will include application of the results of early promising prevention studies, such as those in oral premalignant lesions, to the design of carefully controlled trials to demonstrate actual inhibition of cancer occurrence. In the laboratory, basic studies need to be conducted to increase our understanding of the molecular events underlying carcinogenesis, in order to identify additional targets for new agents to enhance the clinical efforts. Some of the most exciting developments in the control of cancer over the next decade will very likely result from this chemopreventive approach, with application not only to prevention of the first or primary malignancy, especially in high risk populations, but also to clinical situations traditionally considered to be in the domain of chemotherapeutic strategies, such as adjuvant treatment after definitive therapy of a primary cancer.

An important variable in determining in vitro cell survival is the time interval between treatment and assay. One manifestation of radiation damage is the introduction of a division delay in which the post-radiation division rate is less than the pre-radiation division rate. We have examined the influence of length of incubation on radiosensitivity of murine and human melanoma cells in a soft-agar assay. In general, the longer the incubation period the larger was the median colony diameter within control or experimental groups. Hence apparent radiosensitivity decreased with increased length of incubation.

Development of contingency recruitment plans in cancer chemoprevention research is as important as formulation of the initial plan. We found that requesting recruitment information from our initial CTCD subjects provided a framework for our contingency plan. The revised recruitment plan consisted of: 1) calling and sending letters to community gynecologists in private practice or affiliated with HMO's to explain the study and ask for referrals; 2) continued personal contact by the principal investigator with referring physicians; 3) sending thank you and follow-up letters to every physician who referred patients to the study; 4) soliciting Papanicolaou smear reports from HMO's if physicians of women with abnormal Papanicolaou smears gave permission to pathologists to release this information; 5) utilizing free media such as feature articles on the CTCD in local papers, public service announcements, and television "spots;" 6) continued use of brochures and posters printed for the initial recruitment effort; and 7) continued presentations to local professional physician and nurse groups about the study. Our contingency plan to date has provided us with 100% of our projected accrual. Thus, our recruitment methods have proved to be effective in accruing subjects for this cancer chemoprevention trial.

Human thymus was disrupted by mechanical and enzymatic techniques yielding tissue fragments and single-cell suspensions. Cell types and their distribution were determined before and after density separation. Single-cell suspensions produced by both separation procedures yielded comparable percentages of lymphoid and monocytic cells, whereas only the enzyme-treated tissue yielded epithelial cells. Less than 1% of all monocytic cells were mature. Monolayer cultures were established from fragments as well as unfractionated cells and gradient fractions. Analysis of these cultures showed that: (1) gradient separation of mechanically-prepared cell suspensions produced cultures enriched for macrophages, while epithelial cell cultures could be produced from fractionated enzyme treated tissue; (2) spindle-shaped cells may be fibroblasts or epithelial cells; (3) explant cultures have a growth pattern characteristic of other epithelial cells in vitro; (4) macrophages constitute a definite population of explanted cells and their percentages increase with time in culture; (5) macromolecular synthesis by epithelial cells in culture may be donor age dependent. Nonlymphoid cells of the human thymus can be identified by a variety of separation techniques and after monolayer culture. Characteristics of these cells are defined which may affect cell-cell interaction studies of the thymus.

A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.

Retinoids, including isotretinoin, have demonstrated antiproliferative and antineoplastic activity in laboratory and clinical trials. In a phase II trial, 25 patients with extensive mycosis fungoides were evaluated for response to isotretinoin. There was a 44% (11 patients) objective clinical response rate with three clinical complete responses without concomitant evidence of pathologic clearing of the disease. An additional 24% (six patients) showed a minor degree of clinical improvement. The median time to response was two months (range, 0.5 to eight months) and the median response duration was eight months or longer (range, one to 25 months). Chronic toxic reactions consisted primarily of drying of the skin and mucous membranes and resulted in dose reduction in the majority of patients. It is concluded that isotretinoin produces significant clinical benefit to some patients with mycosis fungoides.

Pimozide, a potent neuroleptic which inhibits the release of pituitary releasing factors and is an effective dopamine antagonist, was administered to 30 patients with previously treated metastatic melanoma. Six patients were inevaluable because of poor drug tolerance (two), disease progression within 1 week and death within 2 weeks (three), and death from other causes (one). Among the 24 evaluable patients, two had complete response, two had partial response, and two had disease stabilization. Responses were observed in soft tissue, lymph nodes, liver, and lung. Toxic effects consisted of extrapyramidal manifestations in nine patients and malaise in seven. Pimozide has activity in patients with previously treated metastatic melanoma (17% response rate in evaluable patients) and merits consideration of further study in combination regimens.

13-cis-Retinoic acid (13-CRA) is a synthetic analog of vitamin A effective reversing preneoplastic lesions in both humans and animals. To study its physiochemical properties and disposition kinetics, we developed a rapid, sensitive, and precise high-performance liquid chromatography assay for 13-CRA in biological samples. This assay system resulted in a clear separation of 13-CRA from all-trans-retinoic acid and retinol and had a detection limit of 20 ng/ml plasma. Recovery was 89 +/- 6% (S.D.) at equivalent physiological concentrations with a precision of 8%. To study the disposition kinetics in humans, 13 patients received a p.o. bolus of 13-CRA and had blood samples collected at timed intervals. For the 10 patients studied on the first day of 13-CRA administration, the mean time to peak plasma concentration was 222 +/- 102 min. Interpatient peak 13-CRA plasma concentrations were found to be variable, suggesting irregular gastrointestinal absorption. Beta-Phase t 1/2 was approximately 25 hr. The prolonged terminal-phase plasma half-life may represent biliary excretion and enterohepatic circulation.

The recognition of recurring sites of chromosome change in human cancers has pinpointed the location in the genome of several important growth-regulatory sequences (e.g., cellular oncogenes). This report details the finding of a recurring translocation site involving the long arm of chromosome 6 (6q) in malignant melanoma. We have observed a translocation (t) between chromosomes 1 and 6 in five different cases of malignant metastatic melanoma. All five melanomas evidencing t(1;6) involved band regions 6q11-13, while two different regions of chromosome 1 (p22, q12-q21) were shown to be translocated to 6q. In reviewing previously published cases of melanoma, an additional two cases of t(1;6) and 13 cases of other translocations to 6q11-13 have been identified. Chromosome 6q contains several biologically important gene sequences including the proto-oncogenes ros, myb, and mas1. However, based on current mapping studies, the breakpoint of this translocation (6q11-13) is not within the region encoding these sequences. By analogy to other systems, molecular analysis of the translocation breakpoints may identify a gene(s) which plays a role in melanoma tumorigenesis.

The authors have studied the natural history of 377 patients with Stage I cutaneous malignant melanoma followed at the Arizona Cancer Center, Tucson. Two hundred eight patients, or 55%, remained free of metastatic disease after a median follow-up of 30 months. The survival at 5, 8, and 10 years was 69, 65, and 63%, respectively. Natural breakpoints in Breslow thickness for survival occurred at 0.85, 1.95, and 4.00 mm. These are not significantly different from those found by other investigators. A step-down multivariate analysis using the Cox regression model yielded four factors as highly significant in predicting survival: Breslow thickness (P less than 0.001), an age/sex interaction (P = 0.0012), clinical ulceration (P = 0.0039), and a prophylactic node dissection (P = 0.019). No predictive value for a BANS or non-BANS location was detected. These results are discussed in reference to other large series which describe the natural history of cutaneous melanoma.

Three short-term human melanoma cell lines were tested for sensitivity to human recombinant alpha-tumor necrosis factor (TNF) in a semisolid agar colony formation assay. Cells from three pigmented and one amelanotic strain displayed low sensitivity to TNF. The ID50 for the inhibition of melanoma colony formation ranged from 2,500 to 20,000 U/ml. We then tested the ability of human recombinant alpha-interferon (IFN-alpha) and gamma-interferon (IFN-gamma) to interact with TNF to inhibit melanoma colony formation. Analysis of the TNF-IFN mixtures using the median effect method demonstrated that both IFNs interacted synergistically with TNF to inhibit melanoma colony formation. On a unit basis, IFN-gamma was more active with TNF than IFN-alpha. The addition of the second interferon to the mixture enhanced the ability of TNF to promote the cytolysis of human melanoma cells. The enhanced killing effect seen with the combination of IFN-alpha, IFN-gamma, and TNF suggests an interesting strategy for the treatment of human melanoma.

A human platelet sonicate was evaluated for its effects on the growth of human metastatic melanoma colony-forming cells in soft agar from cells in culture and from biopsies. The addition of platelet sonicate increased both cloning efficiency and proliferative capacity in that more and larger colonies were formed. In more detailed studies under growth-limiting conditions, melanoma cellular responses to known growth factors were compared to the activity found in the platelet sonicate. None of the growth factors tested either alone or in combination, including platelet-derived growth factor, epidermal growth factor, alpha-type transforming growth factor, and beta-type transforming growth factor, were capable of inducing melanoma colony formation to the 12-fold stimulation observed with the platelet sonicate. Treatment of platelet sonicate with dithiothreitol, trypsin, or acid resulted in loss of activity for human melanoma. Our results suggest that human platelets contain an acid-sensitive protein which can support the expression of the transformed phenotype of human melanoma, and this factor is distinct from acid-stable activities previously characterized from human platelets.

A substantial amount of data suggests that both RNA and DNA tumor viruses are associated with human cancer. Correction of malignant transformation at the genotypic level may be possible in the future but will require significant advances in our understanding of genomic material. The use of simple defined models such as tumor viruses seems like a reasonable place to start. The use of antipromotors (e.g. retinoids) and inhibitors of promotors will prove as important to the successful treatment of cancer in the next decade as chemotherapy has been in the last two decades.