Chemoprevention Trials

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Cancer prevention efforts have been a long and arduous process. As the biological basis for carcinogenesis continues to be elucidated, different strategies for prevention have emerged. The success of recent clinical trials designed to prevent cancer in patients who are at increased risk of cancer (cancer "chemoprevention" trials) suggests that chemoprevention is a rational and appealing treatment strategy. Success in the prevention of epithelial cancers suggests that chemopreventive agents can interrupt the carcinogenic process. This article focuses on current chemoprevention research both laboratory and clinical.      
Chemopreventive agents, how they might interrupt the biological effects of genetic changes to inhibit abnormal epithelial cell growth and differentiation, and the genetic events associated with epithelial carcinogenesis are discussed and the usefulness of intermediate biomarkers as markers of premalignancy is reviewed. Finally, chemoprevention trials are analyzed with emphasis on strategies of trial design and clinical outcome and future directions in chemoprevention are proposed that are based on recently acquired mechanistic insight into carcinogenesis and chemoprevention.

I. INTRODUCTION

Epithelial cancers have historically been a cause for frustration by both patients and practitioners. Since the mid-1970s, the mortality rate from epithelial malignancies has improved only slightly despite advancements in cancer therapeutics. Although some patients will present with early stage epithelial malignancies, the majority of these patients will present with locally advanced or metastatic disease. Surgical intervention with adjuvant treatment, including radiotherapy and/or chemotherapy, has offered some improvements in long-term survival rates. However, local recurrence and especially the development of second primary tumors have impacted both morbidity and mortality in this patient population. Thus, a novel approach to these epithelial cancers is needed. Epithelial carcinogenesis is a multistep process in which an accumulation of genetic events leads to a progressively dysplastic cellular appearance, deregulated cell growth, and finally carcinoma.

II. CHEMOPREVENTION

Chemoprevention can be defined as the use of specific natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. Sporn first defined the term chemoprevention in 1976. Cancer chemoprevention is a rapidly developing field that approaches carcinogenesis from a different perspective. Previously, early detection techniques were employed to reduce morbidity and mortality with respect to cancer treatment. In lung cancer, this included early chest X-ray and sputum cytology analysis in individuals at high risk. Despite these early detection techniques, overall mortality did not improve. Chemoprevention bridges basic biologic research with clinical chemical intervention and attempts to halt the process of carcinogenesis. Its principles are based on the concepts of field cancerization and multistep carcinogenesis. In field cancerization, diffuse epithelial injury results from carcinogen exposure in the aerodigestive tract, genetic changes, and premalignant and malignant lesions in one region of the field translate into an increased risk of cancer developing in the entire field. Multistep carcinogenesis describes a stepwise accumulation of alterations, both genotypic and phenotypic. Arresting one or several of the steps may impede or delay the development of cancer. This has been described particularly well in studies involving precancerous and cancerous lesions of the head and neck, which focus on oral premalignant lesions (leukoplakia and erythroplakia) and their associated increased risk of progression to cancer. As the new millennium begins, exciting new techniques to fight cancer are being devised, including biologic interventions and genetic manipulations. Intermediate markers of response are needed to assess the validity of these approaches in a timely and cost-efficient manner.

III. BIOLOGICAL BASIS OF EPITHELIAL CARCINOGENESIS

A. Field Cancerization
Field cancerization was originally described by Auerbach in 1953. In the upper aerodigestive tract (UADT), the surface epithelium, or field, is exposed in large amounts to environmental carcinogens, predominantly tobacco smoke. Pathologic evaluation of the epithelial mucosa of the UADT located adjacent to carcinomas frequently reveals hyperplastic and dysplastic changes. These premalignant changes found in areas of carcinogen-exposed epithelium adjacent to tumors are termed "field cancerization" and suggest that these multiple foci of premalignancy could progress concurrently to form multiple primary cancers.       
Second primary tumors (SPTs) have become the leading cause of mortality in head and neck cancer and this best illustrates the concept of field cancerization. Multiple genetic abnormalities have been detected in normal and premalignant epithelium of the lung and UADT in high-risk patients. In limited studies, when primary tumors and SPTs are analyzed for p53 mutations, evidence supports the independent origin of these tumors. Mutations of p53 can occur in only one of the tumors or distinct mutations occur between the primary and SPT. Continued work in analyzing molecular characteristics of primary and second primary cancers is needed.

B. Multistep Carcinogenesis
Pathological observations in field cancerization gave rise to the hypothesis of multistep carcinogenesis, which proposes that neoplastic changes evolve over a period of time progressing from normal, to hyperplastic, to dysplastic, and finally to fully malignant phenotypes. Elucidating the mechanism of multistep carcinogenesis has awaited the integration of molecular biological techniques into pathologic evaluation of epithelial lesions. This has led to the discovery of genetic abnormalities in premalignant and malignant epithelial cells. Studies have identified different carcinogenic stages, including initiation, promotion, and progression. Initiation involves direct DNA binding and damage by carcinogens and is rapid and irreversible.      
Promotion leads to premalignancy and is generally irreversible involving epigenetic mechanisms. Progression is the period between premalignancy and the cancer and is generally irreversible involving genetic mechanisms. The stages of promotion and progression are prolonged. Genetic damage appears to accumulate during neoplastic transformation, and specific genes have been discovered that, when altered, may play a role in epithelial carcinogenesis.       
These include both tumor suppressor genes and protooncogenes, which encode proteins that are involved in cell cycle control, signal transduction, and transcriptional regulation. Tumor suppressor genes inhibit clonal expansion by suppressing cell growth and genomic mutability. Some tumor suppressors that have been linked to epithelial carcinogenesis include p53, retinoblastoma (Rb), DCC (deleted in colorectal carcinoma), MCC (mutated in colorectal carcinoma), and APC (adenomatous polyposis coli) genes. Over 500 genes that play vital roles in cell signaling and growth control are altered in cancer cells and, as protooncogenes, may be involved in the process of neoplasia.       
These include ras, the myc (C-, N-, and Lmyc), erbB [erbB1 (epidermal growth factor receptor), erbB2 (her2/neu), erbB3 (her3), and erbB4/her4]. Chromosomes, also extensively damaged during epithelial carcinogenesis, are detected in the form of nuclear DNA adducts, cytoplasmic DNA fragments, or micronuclei and chromosomal abnormalities, which include aneuploidy as well as intrachromosomal deletions and amplifications. Future studies designed to discover genes disrupted by these chromosomal lesions may reveal both known and novel tumor suppressor genes and oncogenes, which play a role in epithelial carcinogenesis.

IV. BIOLOGICAL BASIS OF CHEMOPREVENTION

Chemoprevention trials are based on the hypothesis that interruption of the biological processes involved in carcinogenesis will inhibit this process and, in turn, reduce cancer incidence. This hypothesis provides a framework for the design and evaluation of chemoprevention trials, including the rationale for the selection of agents that are likely to inhibit biological processes and the development of intermediate markers associated with carcinogenesis. DNA damage associated with epithelial malignancies is thought to occur partly through oxidative processes induced by free radicals. Aberrant epithelial proliferation and differentiation are hallmarks of premalignant lesions.      
Treatment approaches include interrupting any of these processes. Development of intermediate markers for chemoprevention trials is crucial. Because treatment-induced improvements in cancer incidence require years to evaluate, monitoring intermediate markers that are both modulated by chemoprevention treatment and correlate with a reduction in cancer incidence would allow a more expeditious evaluation of potentially active chemopreventive agents.      
Premalignant lesions are a potential source of intermediate markers, and if disappearance of these lesions correlates with a reduction in cancer incidence, then markers of premalignancy will serve as intermediate end points for chemoprevention trials. Future studies in chemoprevention will continue to test this hypothesis.

V. CHEMOPREVENTION TRIALS

Cancer chemoprevention is still investigational, although its role in oncologic practice continues to expand. Prevention in cancer has become more prominent as frustration over the failures of current therapeutic modalities has grown. A variety of chemopreventive agents have been studied in over 40 randomized trials since 1990. Some clinical activity has been demonstrated proving the potential utility of this method of cancer prevention. Still, large randomized trials are needed before chemoprevention agents can be fully integrated into standard oncologic practice. Major trials are listed in Table I.

TABLE I Completed Randomized Chemoprevention Trials

Author Study setting Design Number Intervention Outcome
Head and neck          
Hong (1986) Oral leukoplakia Phase IIb 44 Isotretinoin (1-2 mg/kg/day) Positive
Stich (1988) Oral leukoplakia Phase IIb 65 Vitamin A (200,000 IU/week) Positive
Han (1990) Oral leukoplakia Phase IIb 61 Retinamide (40 mg/day) Positive
Lippman (1993) Oral leukoplakia Phase IIb (maintenance) 70 Isotretinoin (0.5 mg/kg/day) Positive

Chiesa (1993)

 Oral leukoplakia Phase IIb (maintenance) 80 Fenretinide Positive

Hong (1990)

Prior SCC Phase III 103 Isotretinoin (50-100 mg/m2/day) Positive
Lung          
Heimberger (1988) Metaplasia (sputum) Phase IIb 73 Vitamin B12 (500 μg/day); folic acid (10 mg/day) Positive
Arnold (1992) Metaplasia (sputum) Phase IIb 150 Etretinate (25 mg/day) Negative
Van Poppel (1992) Micronuclei (sputum) Phase IIb 114 β-Carotene (20 mg/day) Positive
Lee (1993) Metaplasia (biopsy) Phase IIb 87 Isotretinoin (1 mg/kg/day) Negative
Pastorino (1993) Prior NSCLC Phase III 307 Retinyl palmitate (300,000 IU/day) Positive (SPT)
Kurie (2000) Metaplasia/dysplasia Phase IIb 82 4-HPR Negative
Lippman (2000) Prior NSCLC Phase III 1166 Isotretinoin (30 mg/day) Negative
Colorectal          
Bussey (1982) FAP Phase IIb 36 Vitamin C (3 g/day) Positive (polyp)
McKeown-Eyssen (1988) Resected adenoma Phase IIb 137 Vitamins C (400 mg/day) and E (400 mg/day) Negative
Decosse (1989) FAP Phase IIb 58 Vitamins C (4 g/day) and E (400 mg/day); fiber (22.5 g/day) Positive (polyp)
Gregoire (1989) Prior colon cancer Phase IIb 30 Calcium (1200 mg/day) Negative (LI)
Stern (1990) Prior FAP Phase IIb 31 Calcium (1200 mg/day) Negative (LI)
Labayle (1991) FAP Phase IIb 10 Sulindac (100 mg three times/day) Positive (polyp)
Wargovich (1992) Resected adenoma Phase IIb 20 Calcium (2000 mg/day) Positive (LI)
Paganelli (1992) Resected adenoma Phase IIb 41 Vitamins A (30,000 IU/day), E (70 mg/d), and C (1 g/day) Negative (LI)
Alberts (1992) Resected adenoma Phase IIb 100 WBF (2.0 or 13.5 g/day); calcium (250 or 1500 mg/day) Negative (LI)
Nugent (1993) FAP Phase IIb 14 Sulindac (200 mg twice a day) Positive (polyp)
Giardiello (1993) FAP Phase IIb 22 Sulindac (150 mg twice a day) Positive (polyp)
MacLennan (1993) Resected adenoma Phase IIb 378 Fat (<25% of calories); WBF (11 g/day); β-carotene (20 mg/day) Negative (polyps)
Bostick (1993) Resected adenoma Phase IIb 21 Calcium (1200 mg/day) Negative (LI)
Roncucci (1993) Resected adenoma Phase IIb 209 Vitamins A (30,000 IU/day), C (1 g/day), and E (70 mg/day); lactulose (20 g/day) Positive (vitamins> lactulose)
Gann (1993) U.S. male physicians Phase III 22,071 Aspirin (325 mg qod) Negative
Steinbach (2000) FAP Phase IIb 77 Celecoxib 100 mg twice a day Negative
        Celecoxib 400 mg twice a day Positive
Schatzkin (2000) Resected adenoma Phase IIb 2079 High fiber, low fat, counseling Negative
Alberts (2000) Resected adenoma Phase IIb 1429 Wheat bran, 2.0 or 13.5 g daily Negative
Skin          
Moriarty (1982) Actinic keratoses Phase IIb 50 Etretinate (75 mg/day) Positive
Watson (1986) Actinic keratoses Phase IIb 15 Etretinate (75 mg/day) Positive
Kligman (1991) Actinic keratoses Phase IIb 527 Topical tretinoin (0.05%) Negative
Kligman (1991) Actinic keratoses Phase IIb 455 Topical tretinoin (0.10%) Positive
Moon (1993) Prior BCC/SCC Phase III 524 Isotretinoin (5-10 mg/day), retinol (25,000 IU/day) Negative
Greenberg (1990) Prior BCC/SCC Phase III 1805 β-Carotene (50 mg/day) Negative
Tangrea (1992) Prior BCC Phase III 981 Isotretinoin (10 mg/day) Negative
Moon (1993) Prior actinic keratoses Phase III 2298 Retinol (25,000 IU/day) Positive
Clark (1996) Prior BCC/SCC Phase III 1312 Selenium (200 mcg) Negative
Esophagus/stomach          
Munoz (1985) Geographic high risk (Huixan) Phase IIb 610 Retinol (50,000 IU/week); riboflavin (200 mg/week); zinc (50 mg/week) Negative (dysplasia)
          Positive (micronuclei)
Zaridze (1993) Geographic high risk (Uzbekistan) (oral leukoplakia and/or chronic esophagitis) Phase IIb 532 Riboflavin (80 mg/week); vitamins A (100,000 IU/week) and E (80 mg/week); β-carotene (40 mg/day) Negative
Blot (1993) Geographic high risk (Linxian) Phase III 29,584 Multiple vitamins/minerals Positive (stomach)
Li (1993) Geographic high risk (Linxian, dysplasia) Phase III 3318 Multiple vitamins/minerals Negative
Bladder          
Alfthan (1983) Superficial tumors (resected) Phase IIb 32 Etretinate (25-50 mg/day) Positive
Pederson (1984) Superficial tumors (resected) Phase IIb 73 Etretinate (50 mg/day) Negative
Studer (1984) Superficial tumors (resected) Phase IIb 86 Etretinate (25-50 mg/day) Positive
Cervix          
Byrne (1986) Dysplasia (CIN 2,3) Phase IIb 26 HLI (0.8 × 106 IU/week) Negative
Yliskoski (1990) Dysplasia (CIN 1,2) Phase IIb 20 HLI (9 × 106 IU/day) Negative
Frost (1990) Dysplasia (CIN 2) Phase IIb 10 IFN-α2b (4 × 106 IU/day) Negative
de Vet (1991) Dysplasia (CIN 1-3) Phase IIb 278 β-Carotene (10 mg/day) Negative
Butterworth (1992) Dysplasia (CIN 1,2) Phase IIb 235 Folic acid (10 mg/day) Negative
Chu (1993) Dysplasia (CIN 1,2) Phase IIb 298 Folic acid (5 mg/day) Negative
Meyskens (1993) Dysplasia (CIN 2,3) Phase IIb 301 Topical tretinoin (0.372%) Positive (CIN 2)
Breast          
BCPT LCIS, high-risk factors Phase III 13,388 Tamoxifen 20 mg daily Positive-early stop
Powles (1998) Family history Phase III 2494 Tamoxifen 20 mg for 8 years Negative
Veronesi (1999) Previous hysterectomy Phase III 5408 Tamoxifen 20 mg for 5 years Negative

 

A. Trial Design
Chemoprevention trials are conducted in three phases similar to other clinical trials. Phase I trials determine the toxicity of an agent alone or in combination with other agents. However, in latter phases, the ideal end point of chemoprevention trials is a reduction in cancer incidence as patients who are enrolled in study are free of cancer as opposed to traditional phase II studies. Because cancer incidence is extremely low among the general population with no known cancer risk factors, demonstrating a treatment-induced reduction in cancer incidence among the general population requires large randomized studies, which are expensive and time-consuming.      
The time and resources required for chemoprevention trials can be significantly reduced by targeting high-risk populations and utilizing potential intermediate biomarkers. This point is best illustrated by ongoing trials of chemoprevention of upper aerodigestive tract cancer. In these studies, patients are definitively treated for a stage I or II cancer of the UADT. Although recurrence is a concern, development of second primary tumors is the leading cause of cancer-related death following treatment in early stage disease as reported by Vikram. Therefore, the end points in these trials are the occurrence of SPTs and survival. SPTs occur at a rate of 3-7% per year in these patients. To test whether a given chemopreventive agent has an effect, if a cancer incidence rate of 6% is used, it is necessary to follow 1000 patients for a period of 5 years. As of September 1, 1999, the Retinoid Head and Neck Second Primary Trial had completed accrual with 1384 registered patients and 1191 patients randomized and eligible. Interim analysis performed in May 2000 indicated significantly higher recurrence rates in active smokers vs former smokers and significantly higher smoking related SPT rates in active smokers vs never smokers, with intermediate rates for former smokers. This trial will be unblinded and definitively analyzed in September 2002, when all randomized patients have completed active therapy.     
The risk of SPTs is much lower in most other epithelial cancers. For patients who have undergone resection of a breast cancer, the risk of a SPT is 0.8% per year. When compared to UADT cancer chemoprevention trials, breast cancer chemoprevention trials require up to 5 to 10 times more patients with longer clinical follow-up. As 5000-10,000 patients are required for these trials, this is still less than the 20,000 or more required to conduct a study in the general population.      

Intermediate end points can reduce the duration of studies as far as cost and resources. By utilizing bio-markers that highly predict the development of cancer rather than assessing the actual development of cancer, chemoprevention trials can be designed to test the effects of potential agents in a smaller population in a shorter period of time.

B. Chemopreventive Agents
Approximately 2000 natural and synthetic agents have been shown in experimental systems to have chemopreventive activity. Agents that have been studied in clinical trials include retinoids, N-acetylcysteine, β-carotene, calcium, α-tocopherol, selenium, tamoxifen, finasteride, and nonsteroidal antiinflammatory drugs (NSAIDs.) Of the group, the retinoids have been studied most extensively as chemopreventive agents. Vitamin A was first noted to be an essential nutrient in 1913, and its deficiency was associated with changes in epithelial histology in 1925. Since that time, it has been shown that vitamin A deficiency is associated with bronchial metaplasia and an increased incidence of cancer. Vitamin A exists as preformed vitamin A (retinol esters, retinol, and retinal) and provitamin A carotenoids (β-carotene and metabolic precursors of retinol).      
Retinoids occur in natural (all-trans retinoic acid or ATRA, 13-cis retinoic acid or 13-cRA, 9-cis retinoic acid or 9-cRA, retinyl palmitate) and synthetic [fenretinide or N- (4-hydroxyphenyl)retinamide (4- HPR)] forms. They are important for normal cell growth and differentiation, as well as for the regulation of apoptosis. In preclinical models, they have been shown to suppress or reverse epithelial carcinogenesis in lung, oral cavity, esophagus, bladder, skin, mammary gland, prostate, and liver tissues. Clinical trials have studied naturally occurring retinoids, including tretinoin or ATRA, its stereoisomer isotretinoin or 13-cRA, and the retinoid-related molecule β-carotene. Synthetic retinoids that have demonstrated clinical activity in chemoprevention trials include retinyl palmitate, fenretinide or 4-HPR, and etretinate. A retinoid that will be studied in future clinical trials is the naturally occurring ATRA stereoisomer 9-cRA. Of all chemopreventive agents, retinoids have the best-defined mechanism of action. Retinoids function as ligands for intracellular receptors. Cytosolic receptors for retinol (CRBP) and retinoic acid (CRABP) bind retinoids and appear to regulate the transfer of retinoids into the nucleus. Nuclear retinoid receptors were discovered in 1987 and are believed to mediate the effects of retinoids and are members of a larger steroid superfamily of nuclear receptors that includes glucocorticoid, thyroid, vitamin D, progesterone, and estrogen receptors, among others. There are two families of retinoid nuclear receptors, RAR and RXR, and each family consists of three members: α, β, and γ forms.      
These receptors can exist as heterodimers or homodimers. RARs bind ATRA and 9-cRA, and RXRs bind 9-cRA and bexarotene (Targretin). Liganded nuclear receptors function as transcription factors that bind DNA and regulate the expression of genes that mediate retinoid cell functions, including growth, differentiation, and apoptosis. Retinoid nuclear receptors associate with inhibitory corepressors or stimulatory coactivators that after their transcriptional activities.      
Some retinoids, including 4-HPR, which has potent apoptosis-inducing activity, do not bind retinoid receptors. Other retinoids have not demonstrated any ability to bind either family of nuclear receptors (carotenoids), whereas their metabolites do (13-cRA, and retinol). Whether these retinoids are metabolized to another form that can bind to known nuclear receptors or bind to as yet undiscovered receptors is unknown. Studies have shown intracellular interconversion of 13-cRA to ATRA, demonstrating the importance of retinoid metabolism in determining the biological effects of retinoid treatment.      
The nuclear retinoid receptor RAR-β seems to have a major role in UADT carcinogenesis. Important observations of RAR-β made in various studies include its absence in many head and neck carcinomas and lung cancer cell lines and its ability to suppress tumorigenesis. When upregulated with isotretinoin treatment, increased RAR-β expression and clinical response correlate in 40 to 90% of cases. Thus, RAR-β is the best indicator to date of retinoid chemoprevention efficacy in human head and neck carcinogenesis and a good biomarker for continued research. Surprisingly though, strong intratumoral RAR-β expression has been associated with a poorer outcome in early stage lung cancer. Thus, the biological mechanism and clinical outcome still need further investigation.

C. Trial Results
1. UADT Trials
The upper aerodigestive tract has served as a good model for chemoprevention and its utility. Premalignant oral lesions include erythroplakia and dysplastic leukoplakia. Retinoids, β-carotene, vitamin E, and selenium have shown activity in the reversal of oral premalignancy, but only retinoids have demonstrated positive results in randomized trials. Isotretinoin, retinal (a synthetic retinamide), and fenretinide were used in these randomized trials. Of these agents, the best characterized is isotretinoin, which has been studied in two related randomized trials. In 1986, Hong reported a placebo-controlled, double-blind study of 44 patients randomized to receive 13-cRA (1-2 mg/kg/day) or placebo for 3 months and followed for 6 months. The clinical and histologic major response rates to 3 months' treatment with high-dose isotretinoin were 67 and 54%, respectively (P = 0.0002 and 0.01), after 6 months of follow-up; clinical and histologic rates of response to placebo were 10%. Toxicity was severe with the highdose regimen, and more than 50% of treated patients relapsed within 2 to 3 months after discontinuing therapy.      
A second trial was instituted to examine the effects of low-dose maintenance isotretinoin or β-carotene for 9 months following 3 months of high-dose induction therapy with isotretinoin. Sixty-six patients completed the first phase of the study, and the premalignant disease progression rates were 8% in patients who received low-dose isotretinoin and 55% in patients who received β-carotene (P < 0.001). The percentage of patients whose lesions decreased in size was 33% in patients treated with isotretinoin and 10% in patients treated with β-carotene. Carcinoma developed in seven patients who received β-carotene and only one who received isotretinoin. This study not only confirmed the activity of isotretinoin demonstrated in the first study, but also showed that isotretinoin is superior to β-carotene in this setting and revealed that low-dose maintenance therapy with isotretinoin may yield better long-term effects than a short course of high-dose therapy. However, a 10-year update of the study revealed no differences in cancer rates between the two groups. These studies established the rationale for the treatment of premalignant disease with chemopreventive agents.       
These findings were further elaborated by studies that examined the effects of retinoid treatment in head and neck cancer patients who had been cured of their primary disease. A placebo-controlled, randomized trial of 103 patients who received high-dose isotretinoin (50-100 mg/m2 for 12 months) following surgery and/or radiotherapy for early stage disease revealed that isotretinoin reduced the rate of SPT development after 32 months (P = 0.005), but this effect decreased after a median follow-up of approximately 5 years (P = 0.04). The treatment had no effect on local, regional, or distant recurrence or overall survival rates. In addition to reversing oral premalignancy, treatment with retinoids reduced the incidence of SPTs, the major cause of mortality in head and neck cancer patients. This effect diminished with time after concluding treatment. Many patients had side effects to the retinoid, which points to the need for developing better tolerated, more efficacious therapies. To further evaluate the retinoid effect on SPTs, a randomized, double-blind, multiinstitutional trial was launched examining the effect of lowdose 13-cRA in previously definitively treated patients with early stage head and neck cancer. Patients received 30 mg of 13-cRA per day for 3 years and are followed for 4 years documenting any end points, recurrences, or SPTs. This trial is planned to be unblinded in 2002. These results, positive or negative, will help establish a new standard in head and neck cancer chemoprevention.      
Another study examined the effect of a second generation retinoid, etretinate, in patients with early stage head and neck squamous cell carcinoma treated with surgery or radiation. This randomized study followed 316 patients who were randomized to receive etretinate, 50 mg/day for the first month, followed by 25 mg/day in the following months, or placebo for 24 months. There were no significant differences regarding local, regional, and distant metastases at 5 years. Treatment was discontinued in 33% of patients taking etretinate and 23% taking placebo (P < 0.05) because of toxicity. This study failed to show any effect in prevention of SPTs with etretinate.       
Adjuvant therapy in advanced head and neck cancer has also been examined. Trials used a combination of 13cRA (50 mg/m2/day), interferon-α (3 MU/m2/t.i.w.), and α-tocopherol (1200 IU/day) given for 12 months. In a phase II study of 45 evaluable patients with definitively treated stage III/IV head and neck cancer, 39 have no evidence of disease with only 6 recurrences at 21 months median follow-up. The 2- year disease-free survival of the study is 84%. This suggests that perhaps in patients with a higher risk of developing recurrence or SPTs, more aggressive therapies for prevention are warranted.      
EUROSCAN, a large phase III study encompassing 2592 patients, reported no benefit of chemopreventive agents in patients with head and neck or lung cancer in terms of survival, event-free survival, or SPT. In the EUROSCAN study, patients were randomized to receive supplementation with retinyl palmitate, N-acetylcysteine, both drugs in combination, or placebo for 2 years. Sixty percent of patients had a history of head and neck cancer and 40% had a history of lung cancer. Patients were grouped as current/ former (93.5%) smokers and never (6.5%) smokers. However, data regarding smoking status, verification thereof (cotinine levels), and its impact relative to SPTs and recurrence were not presented. Further research and studies are needed to formulate a risk model in head and neck cancer, which can help identify that section of the general population highest at risk for development of cancer.

2. Lung Trials
The rationale for prevention of lung cancer is similar to that in head and neck cancer. In both diseases, chronic exposure to tobacco is the major risk factor and dysplastic epithelial lesions are thought to represent a premalignant stage. Preclinical data indicate that retinoids reverse dysplastic bronchial epithelial lesions. Despite these data, placebo-controlled, randomized trials in smokers have revealed that retinoid treatment adds no significant benefit to the effects of smoking cessation and reversal of bronchial metaplasia. In light of results demonstrating that retinoids reduce SPTs in patients who have had a lung cancer resected (see later), bronchial metaplasia may not accurately reflect the chemopreventive effects of retinoids on bronchial epithelium. Research is underway to identify intermediate markers that predict retinoid chemopreventive effects on bronchial epithelial cells.      
In resected NSCLC patients, SPTs occur at the rate of 2-4% per year. Similar to its effects in head and neck cancer patients, retinoid treatment reduces the incidence of SPTs in lung cancer patients who have undergone resection. In the only completed trial addressing this question, 307 patients whose stage I NSCLC were completely resected were randomized to receive 12 months of treatment with retinol palmitate (300,000 IU a day) or no treatment. At a median of 46 months follow-up, patients who received retinol palmitate had a 35% lower incidence of SPTs than the control group (3.1% vs 4.8%). As in studies of head and neck cancer patients, retinoid treatment had no observed effect on survival duration or the rate of primary disease recurrence. EUROSCAN, as mentioned earlier, also showed no chemopreventive benefit in lung cancer patients. These trials in NSCLC patients point out the need to further investigate the effects of different retinoids in this setting and to extend these trials to include patients whose small cell lung cancer (SCLC) has been cured, who have SPTs rates twofold higher than in patients who have been treated for early stage head and neck cancer.      
Trials in lung cancer chemoprevention have demonstrated the importance of smoking status and use of these agents. The Alpha-Tocopherol, Beta Carotene (ATBC) Cancer Prevention study was a randomized, double-blind, placebo-controlled primaryprevention trial in which 29,133 Finnish male smokers received α-tocopherol 50 mg a day alone, β- carotene 20 mg a day alone, both α-tocopherol and β-carotene, or placebo. These men were between 50 and 69 years of age and all smoked five or more cigarettes a day. Patients were followed for 5-8 years.      
Lung cancer incidence, the primary end point, did not change with the addition of β-tocopherol alone, nor did overall mortality. However, both groups who received β-carotene supplementation (alone or with α-tocopherol) had an 18% increase in the incidence of lung cancer. There appeared to be a stronger adverse effect from β-carotene in those men who smoked more than 20 cigarettes a day. This trial raised the serious issue that pharmacologic doses of β-carotene could potentially be harmful in active smokers. The β-Carotene and Retinol Efficacy Trial (CARET) confirmed results of the Finnish trial. This randomized, double-blinded, placebo-controlled trial tested the combination of 30 mg β-carotene and 25,000 IU retinyl palmitate against placebo in 18,314 men and women age 50-69 years at high risk for lung cancer; 14,254 had at least a 20 pack-year smoking history and were either current or recent former smokers.       
Four thousand and sixty men had extensive occupational exposure to asbestos. This trial was stopped after 21 months because no benefit and even possibly harm were found. Lung cancer incidence, the primary end point, increased 28% in the active intervention group. Overall mortality also increased 17% in this group. Given these results, as well as those of the ATBC trial, high-dose β-carotene is not recommended for high-risk patients who continue to smoke. The Physicians Health Study, a randomized, doubleblind, placebo-controlled trial studied 22,071 healthy male physicians: 11,036 received 50 mg of β-carotene on alternate days and 11,035 received placebo. The use of supplemental β-carotene showed virtually no adverse or beneficial effects on cancer incidence or overall mortality during a 12-year follow-up.      
In China, a study evaluating β-carotene, α- tocopherol, and selenium in the prevention of gastric and esophageal cancer showed a nonsignificant decrease in the risk of lung cancer in a small cohort of patients. Subgroup analysis of the aforementioned studies, especially ATBC and CARET, have provided few explanations for the increase in lung cancer incidence. It seems β-carotene has a harmful effect only in high-risk heavy smokers or those with previous exposure to asbestos. Current recommendations are for these people to avoid supplemental β-carotene in large doses. Including the results of EUROSCAN mentioned previously, much work is needed before chemoprevention agents can be instituted in lung cancer. Currently, an ECOG trial is studying patients with stage I lung cancer and the effect of daily selenium supplementation.

3. Colorectal Trials
Colorectal cancer is associated with premalignant lesions, including polyps and dysplastic epithelium. Large polyps with villous elements and dysplastic epithelia are more likely to progress to carcinoma than small, tubular polyps. The premalignant stages of colon carcinogenesis present an evaluable process for chemoprevention trials.      
The agents most widely used in colorectal cancer chemoprevention trials are NSAIDs and calcium salts. In phase II trials, the NSAID sulindac achieved a statistically significant decrease in both mean polyp number and diameter compared to placebo. Aspirin also shows promise in colon cancer prevention. Epidemiologic studies suggest that aspirin inhibits colon carcinogenesis. The effects of low-dose aspirin on colon cancer incidence were examined in the large-scale U.S. Physicians Health Study. This study revealed that aspirin had no effect on polyp or cancer incidence, but the trial's premature closing because of the finding of a statistically significant effect on myocardial infarction incidence most likely limited the power of this study to detect a modest effect of aspirin in preventing colon cancer. Despite evidence of colorectal cancer prevention, NSAIDs still require more investigation.      
Agent specificity, mechanism of action, and dose and duration of treatment, in addition to adverse side effects, have made recommendations for prevention difficult. Better understanding of the role of aspirin and other NSAIDs in colon cancer chemoprevention awaits further randomized, placebo-controlled trials. Cyclooxygenase-2 (COX-2) inhibitors have shown promise. Forms of the COX enzyme demonstrated include COX-1, which is constitutively expressed, and COX-2, which is overexpressed in inflammatory cells and sites of inflammation. Most NSAIDs inhibit both enzymes. Inhibition of COX-2 specifically reduces untoward side affects such as ulcers and gastritis. In familial adenomatous polyposis (FAP), patients develop hundreds of polyps in their colon due to mutation of the adenomatous polyposis coli (APC) gene. Strong evidence for the activity of COX-2 inhibitors in the treatment and prevention of FAP has been demonstrated in mouse models and, more recently, human patients. In a recent double-blind, placebo-controlled study, 77 patients with FAP received Celebrex, a COX-2 inhibitor, 100 mg or 400 mg twice daily or placebo for 6 months. Patients who received 400 mg twice daily had a 28% reduction in the mean number of colorectal polyps and a 31% reduction in polyp burden, or the sum of polyp diameters. Based on these studies, trials to assess the prevention of adenoma development in adolescents with preclinical FAP will be needed.      
Fiber in the prevention of colon cancer has been examined in several studies. The Polyp Prevention Trial studied 2079 patients with a history of colorectal adenomas randomized to receive counseling, a low-fat, high-fiber diet rich in fruits and vegetables, or to continue their current diet without counseling. Colonoscopy after 1 and 4 years found no difference in the incidence of recurrent adenomas. Another study by the Phoenix Colon Cancer Prevention Physician's Network studied 1429 patients with a history of colorectal adenoma. These patients were randomized to receive supplemental wheat bran, 2.0 or 13.5 g a day. Again, no difference in the incidence of recurrent adenomas was found between the two groups. Currently, there is no prospective evidence that fiber supplementation is effective for colorectal cancer prevention.       
Calcium salts are available in several forms (gluconate, citrate, and carbonate). Randomized trials testing the effects of calcium as a chemopreventive agent have used cellular proliferation rates within colonic mucosal crypts as an index of response in resected colon cancer patients. Three of the four randomized trials performed to date have shown an increase in cellular proliferation rates within colonic mucosal crypts as an index of response in resected colon cancer patients.      
Three of the four randomized trials performed to date have shown an increase in cellular proliferation with calcium treatment. A randomized, doubleblind trial tested the effect of dietary supplementation with calcium carbonate in 930 patients with a recent history of colorectal adenomas. The calcium carbonatetreated group was found to have a lower risk of recurrent adenoma. The results of this study, which were modestly significant, continue to support the investigation of calcium carbonate as a possible chemopreventive agent. If calcium is found to be an effective treatment in colorectal cancer chemoprevention, then intermediate end points other than cellular proliferation will be needed for future trials.

4. Skin Trials
Chemoprevention of skin cancer has been reported in two large trials using selenium and retinols. The objective of the selenium study was to determine whether supplemental selenium would decrease the incidence of cancer, specifically basal cell and squamous cell carcinomas of the skin. This multicenter, double-blind, randomized trial evaluated 1312 patients ages 18-80 years having a history of skin cancer who were given 200 μg of selenium (0.5 g high selenium brewer's yeast tablet) or placebo daily. Selenium had no effect on skin cancer incidence. However, secondary end point analyses revealed that selenium supplementation was associated with significantly lower incidences of total nonskin cancer and total nonskin and overall cancer mortality rates. In addition, lung cancer, prostate cancer, and colon cancer incidences were significantly reduced. Future trials will test the efficacy of selenium in lung and with α-tocopherol in prostate cancer.      
Retinoids have demonstrated activity in the reversal of actinic keratoses, a premalignant skin disorder. Trials have tested either topical tretinoin or various retinoids given systemically. Small-scale trials in patients at high risk for skin cancers such as xeroderma pigmentosa or in renal transplant patients have shown that prolonged treatment with high-dose isotretinoin or etretinate reduced the incidence of invasive skin cancers. In a phase III trial, 2297 patients who were at a lower risk for skin cancer, including patients with actinic keratoses, received oral retinol (25,000 IU) or placebo daily for 5 years. Retinol treatment was effective in reducing the incidence of squamous cell skin cancer, but not basal cell carcinoma. However, in patients with a prior skin cancer, three phase III trials that tested the effects of retinol, β-carotene, and low-dose isotretinoin demonstrated that these retinoids had no effect on the incidence of SPTs. Further studies are needed to validate the effects of these agents in skin and other cancers.

5. Breast Trials
Like UADT and lung cancers, breast cancer provides a model to study the effects of chemopreventive agents. Chemoprevention strategies in breast cancer target the development of SPTs, which occur at a rate of 0.8% per year, as well as preventing breast cancer in high risk patients. Pooled data from over 40 randomized adjuvant trials revealed that tamoxifen, given over prolonged periods, reduces the incidence of SPTs by 39% in postmenopausal women who have undergone resection. Observing that tamoxifen reduced the incidence of contralateral breast cancer when used as adjuvant therapy, investigators hypothesized that a possible benefit could exist in the prevention of breast cancer in high-risk patients. This initiated several large studies, including the U.S. Breast Cancer Prevention Trial (BCPT) or NSABP P-1 (National Surgical Adjuvant Breast and Bowel Project) trial that was launched in 1992. They studied 13,388 patients who were at increased risk for breast cancer: greater than age 60 years, elevated Gail assessment in those aged 35-59 years, and patients with a history of lobular carcinoma in situ.      
Patients were randomized to take tamoxifen or placebo for 5 years. This trial was stopped early and unblinded after interim analysis results demonstrated a 50% reduction in new tumors. Two other studies, the Royal Marsden Hospital (RMH) Tamoxifen Chemoprevention Trial with 2494 patients and the Italian Tamoxifen Prevention Trial with 5408 patients, are still blinded because preliminary results did not indicate a reduction in breast cancer incidence. The Italian study, a multicenter, double-blind, placebo-controlled trial, evaluated the effect of taking tamoxifen for 5 years in healthy women. The RMH study included women age 30 to 70 years with a family history of breast cancer.      
Another trial, MORE, is studying 3 years of treatment with raloxifene to prevent osteoporosis. It too has reported a reduction in breast cancer incidence in women taking raloxifene. Currently, the NSABP-P2 or STAR (Study of Tamoxifen and Raloxifene) is ongoing in the United States. It will enroll tens of thousands of patients to help determine whether a difference exists in treatment between the two drugs. Raloxifene is another type of SERM, which binds with high affinity to estrogen receptors and preliminarily has shown a marked effect on estrogen receptor-positive tumors. The final results of these trials are still pending.      
Fenretinide, a synthetic vitamin A derivative, has been studied in breast cancer since 1979. A phase III trial initiated in 1987 randomized 2972 women with a history of stage I breast cancer to receive 200 mg of fenretinide daily or no intervention for 5 years in an attempt to reduce contralateral breast cancer. Although no significant difference was found between the two groups after a median of 8 years, there was a trend for benefit in premenopausal women. This has led to a current trial studying the effect of tamoxifen and fenretinide in premenopausal women at increased risk for breast cancer.      
Other strategies for chemoprevention trials in breast cancer include studying the effects of lutenizing hormone-releasing hormone (LHRH) agonists in high-risk premenopausal women as well as aromatase inhibitors in postmenopausal women.

6. Esophageal/Gastric Trials
Esophageal carcinoma has been associated with tobacco and alcohol exposure in the United States and with nutrient deficiencies and exposure to N-nitrous compounds in China. Randomized chemoprevention studies in China, which tested the effects of combinations of agents such as retinol, riboflavin, zinc, and vitamin E, have revealed a reduction in micronuclei frequency but not in the incidence of premalignant lesions in the esophagus. A cooperative study with the United States in Linxian, China, studied the effects of one of four combinations of vitamins/minerals for 5 years (retinol and zinc, riboflavin and niacin, vitamin C, molybdenum and β-carotene, vitamin E and selenium). No statistically significant relationships were correlated with the intervention. However, in secondary analysis, selenium, β-carotene, and vitamin E use was associated with a statistically significant lower mortality rate in all cancers, predominantly gastric cancer. No effect was seen with esophageal cancer. The interpretation of these studies is made difficult by the use of readily available vitamin supplements in the control arm, blurring potential differences from the treatment arm. This represents an inherent flaw in trials utilizing vitamin supplements and dietary intervention. Other trials include examining the effects of vitamin/ mineral supplementation in patients with esophageal dysplasia; 3,318 patients with defined histologic evidence received treatment for 6 years and demonstrated lowered mortality as well as a reduced risk of esophageal or gastric dysplasia. Other trials currently ongoing are comparing gastric dysplasia and treatment of Helicobacter pylori and chronic atrophic gastritis with oltipraz treatment.

7. Bladder Trials
Previous studies using retinoids to prevent recurrence and SPTs in bladder cancer patients have been small and limited by high toxicity. Two trials with prolonged low-dose etretinate appeared to have some effect. One double-blind, placebo-controlled trial studied 30 patients with superficial bladder tumors and the preventive effect of etretinate. A reduction in recurrence was seen in the treated group. Another trial, which was multicenter and randomized, evaluated 79 patients with superficial papillary bladder tumors with 25 mg of etretinate or placebo daily. Time to first recurrence was similar in the two groups; however, the interval to subsequent tumor recurrence was significantly longer in the treated group. Some mild toxicity was experienced in these trials and, although small, indicate beneficial effects of retinoids in prevention. Better tolerated retinoids such as fenretinide are being used in ongoing bladder cancer trials, and combinations of these with other treatment modalities, such as intravesical chemotherapy, NSAIDs, and oltipraz, are under investigation.

8. Cervical Trials
Cervical cancer has well-recognized premalignant dysplastic stages that are accessible for study. Interferon, folic acid, β-carotene, and retinoids have been used in the treatment of cervical dysplasia, and only retinoids have yielded positive results. Nutritional studies have helped define micronutrients of interest (folate, carotenoids, vitamin C, vitamin E). Other interesting medications under evaluation include retinoids [4-hydroxyphenylretinamide (4-HPR), retinyl acetate gel, topical all-trans retinoic acid], polyamine synthesis inhibitors [α-difluoromethylornithine (DFMO)], and nonsteroidal anti-inflammatory drugs (ibuprofen). Phase I chemoprevention studies of the cervix have tested retinyl acetate gel and all-trans retinoic acid. Topical all-trans retinoic acid has been shown to increase the histological regression rate in women with moderate cervical intraepithelial hyperplasia (CIN 2). Phase II trials of all-trans retinoic acid, β-carotene, and folic acid have been and are being carried out, whereas phase III trials of all-trans retinoic acid have been completed and have shown significant regression of CIN 2 but not CIN 3. Clearly, further studies are warranted to formulate chemoprevention strategies in cervical cancer.

9. Prostate Trials
Chemoprevention in prostate cancer is a relatively new area of research. The Prostate Cancer Prevention Trial (PCPT) was launched in 1993 with its principle end point being a reduction of biopsy-proven prostate cancer incidence. Patients included 18,882 males age 55 years and older who are received finasteride, 5-α- reductase inhibitor, 5 mg or placebo daily for 7 years. The trial is still blinded and will achieve its primary end point in 2004. Selenium and vitamin E have been observed to decrease prostate cancer incidence in several trials. Therefore, the National Cancer Institute will launch a trial entitled "SELECT." This phase III trial will enroll more than 32,000 men and assess prostate cancer prevention with selenium, vitamin E, selenium and vitamin E, and placebo. Results of this trial, which will last over 10 years, will help elucidate new strategies in prostate cancer chemoprevention.

10. Other Cancers
Studies of retinoids in other cancers such as hepatocellular carcinoma have been described as well. A prospective randomized study reported in 1996 studied 89 patients who were definitively treated for primary hepatoma. Patients received polyprenoic acid (600 mg daily) or placebo for 12 months. There was a significant decrease in recurrence as well as second primary hepatomas in the retinoid-treated group. At 38 months, 27% of treated patients had recurrence or SPT versus 49% in the placebo group (P = 0.04). Further studies are needed to verify these encouraging results.

VI. FUTURE DIRECTIONS

A. Risk Models
Treatment of epithelial malignancies is moving toward prevention. Since the early 1980s, many patients at high risk for epithelial cancers, mostly those with premalignant lesions or a history of epithelial malignancy, have been enrolled in chemoprevention trials. While this accounts for a substantial number of patients, the vast majority of epithelial malignancies arise in patients with no history of either of these risk factors. This population is, at present, unrecognizable and is therefore not entering chemoprevention trials. While this accounts for a substantial number of patients, the vast majority of epithelial malignancies arise in patients with no history of either of these risk factors. This population is, at present, unrecognizable and is therefore not entering chemoprevention trials. This points out the need to develop a risk model to use in identifying high-risk people from the general population.      
Developing a risk model will require the identification of markers that will predict the likelihood of cancer development. Ongoing research will determine the cancer risks associated with the presence of premalignant epithelial lesions and the genetic abnormalities they contain. These studies will be advanced by improved techniques in the diagnosis of premalignant epithelial foci. Spectroscopic analysis of epithelial tissues performed at endoscopy can differentiate normal, dysplastic, and malignant areas. This technique is based on fluorescent emission spectra of epithelial cells following laser excitation and this reflects differences between normal and malignant cells in their endogenous flavins, riboflavins, and other fluorophors.      
These endoscopic techniques will aid in the identification and acquisition of premalignant tissue for histologic and genotypic analyses. By using this technique, genetic events that are associated with malignant progression can be analyzed, including changes in chromosomal ploidy, micronuclei, proliferation antigens, point mutations in ras and p53, amplification of the myc or erbB-2 genes, and deletions incorporating these markers into ongoing chemoprevention trials in patients with prior malignancies, clinical correlation will reveal the power of these markers to predict the likelihood of an SPT. To determine whether these variables can be used in a model to predict cancer risk among the general population with no history of cancer, prospective studies must be performed. Because cancer risk among the general population is low, population sample size for these studies will be extremely large and will require years of clinical follow-up. Analysis of biopsy material and patient examinations will be extremely expensive to carry out. However, these studies are necessary to the construction of a framework on which chemoprevention trials can be built. Not until high-risk patients are identified can potential chemopreventive agents be tested.

B. Intermediate End Points
Work is underway to identify cellular and molecular markers that change during chemopreventive treatment and correlate with a treatment-induced reduction in cancer incidence. Intracellular pathways activated or inhibited by chemopreventive agents offer potential intermediate markers of response. Of the pathways known to be important in mediating the effects of chemopreventive agents, the retinoid pathway is the best characterized. Cytoplasmic and nuclear retinoid receptors are key elements of retinoid signal transduction. In vitro studies have shown that the expression of RARs is activated within hours of retinoid treatment. A growing body of evidence reveals that the activated expression of specific RARs is crucial to the process of retinoid-induced tumor differentiation. For example, in P19 murine teratocarcinoma cells, retinoid refractoriness correlates with aberrant RAR-α expression. The mechanism by which retinoids induce tumor differentiation may be different from that of their chemopreventive effects.       
Growth inhibition, apoptosis, reversal of dysplasia, and stabilization of the DNA damage process are potential mechanisms of retinoid actions. In acute promyelocytic leukemia patients, tretinoin induces differentiation of promyelocytic cells within weeks. The minimum duration of retinoid treatment necessary to prevent epithelial cancers is not known. If months of treatment are required, then events other than receptor activation may be necessary for retinoidinduced chemoprevention. In addition to retinoid receptors, specific growth factors, their receptors, and carbohydrate antigens have been shown to be modulated by retinoid treatment in vitro and may be important in mediating retinoid effects in epithelial cells. Previous studies have shown that retinoid treatment downregulates the expression of transforming growth factor α, epidermal growth factor receptor, and fibroblast growth factor-4. To examine the roles of these and other biological events as potential intermediate markers for chemoprevention trials, prospective clinical studies that correlate treatmentinduced changes in these cellular components with clinical outcome are needed.      
Premalignant lesions are another source of intermediate end points. Phenotypic analysis of hyperplastic and dysplastic changes can be performed by light microscopy and immunostaining for proliferation antigens such as proliferating cell nuclear antigen and nuclear protein that binds the Ki-67 antibody. Analysis of genetic changes associated with premalignancy offers a potentially more objective means of diagnosis. For example, mutations in p53 have been found in preneoplastic lesions associated with most epithelial cancers. Because mutations can occur over a large region of the p53 gene, a mutation at a specific site, involving an alteration to a specific nucleotide, may be considered a clonal marker. Thus, disappearance of a clonal marker during treatment with a chemopreventive agent could represent regression of a premalignant lesion and may have prognostic value. Work is underway to develop the ability to detect the presence of such clonal markers in sputum, urine, and stool. This will pave the way to testing their usefulness as intermediate markers in chemoprevention trials.

C. Chemopreventive Agents
There is a long list of potentially effective chemopreventive agents that await clinical trials. As shown in Table I, the list includes a broad range of compounds, including vitamins, minerals, antioxidants, antiinflammatory agents, and steroid hormone antagonists. As single agents alone, these compounds will provide material for important clinical trials over at least the next decade.     
Retinoids have undergone extensive development as chemopreventive agents. In addition to 13-cRA, another retinoic acid stereoisomer, 9-cRA, has been developed for clinical trials. 9-cRA is able to bind and activate both RAR and RXR receptor families. Because of its novel nuclear receptor affinities, 9-cis RA may have biochemical effects that other retinoic acid stereoisomers do not have. In addition, synthetic retinoids have been developed that have receptorspecific activity. These agents may have greater efficacy than natural retinoids if particular receptors are implicated in epithelial carcinogenesis. One site in which this may prove true is UADT. RAR-β expression is lower in cancers in this region than in adjacent histologically normal mucosa. In UADT cancer patients, treatment with retinoic acid leads to a chemopreventive response that occurs in UADT cancer patients who have undergone resection. The mechanisms by which retinoids induce a chemopreventive effect in the UADT and lung are unknown, but insight was provided by in vitro experiments that showed that RAR-β appears to suppress cell growth.      
In a lung cancer cell line, overexpression of RAR-β through stable transfection led to a suppression of growth and tumorigenicity in nude mice. Thus, novel retinoids that target RAR-β may be more efficacious than other retinoids in UADT and lung cancer chemoprevention trials. Recently though, several studies have suggested an adverse effect of retinoids in current smokers producing a higher incidence of lung cancer. If RAR-β regulates growth in epithelial cells of the lung and UADT, then the RAR-β gene might be considered as a therapeutic agent in gene therapy trials in the future. The settings in which RAR-β might be most efficacious are unknown, but trials in lung cancer prevention should be considered. The rationale for its use in prevention trials rests on the observation that benign bronchial epithelial cells are more responsive to the growth suppressive effects of retinoids than lung cancer cells, suggesting that retinoid receptors may have greater biological activity in benign bronchial epithelium than in lung cancer cells. Trials such as these await the development of vectors that can safely and effectively deliver genes of interest to the target tissues.       
Specific gene mutations found in premalignant lesions, such as ras and p53 point mutations, may offer additional opportunities for intervention at the molecular level. For example, posttranslational farnesylation of mutant ras is necessary for activation of its transforming properties. FTIs block the enzyme farnesyltransferase or its substrate, farnesyl. Although the technology to deliver efficiently peptides or proteins in clinical trials has not yet been developed, this is the goal of several ongoing studies. Similarly, in vitro studies suggest that changes in mutant p53 phosphorylation can induce wild-type p53 properties. Such an approach might be considered to restore p53 tumor suppressor activities within tumor cells.      
Another direction in chemoprevention trials that may prove fruitful is combination therapy. Combining agents that mediate their effects through different pathways might enhance the ultimate chemopreventive effect. For example, agents that decrease the accumulation of intracellular-free radicals, such as oltipraz, might be combined with retinoids that activate a separate pathway, i.e., the modulation of cell growth and differentiation. Other agents have enhanced the effects of retinoids. In tumor differentiation models, for example, the effects of retinoids are augmented by agents such as phorbol esters or cyclic AMP, which activate protein kinase C (PKC) and protein kinase A, respectively. In a human teratocarcinoma cell line, activation of these kinases enhanced the effects of retinoic acid on RAR-β activation, demonstrating coupling of these kinase pathways with retinoid receptors. In addition to retinoid receptors, another mechanism through which retinoids and kinases might couple is transforming growth factor-β (TGF-β). Intracellular TGF-β production is increased by either retinoic acid treatment or PKC activation.      
Thus, retinoid and protein kinase pathways converge on TGF-β. Transforming growth factor-β has been shown to induce profound growth suppression in many cell types. Studies that illuminate the convergence of different intracellular pathways by molecules that mediate growth suppression, such as retinoid receptors and TGF-β, provide a basis for combination therapy. Combination therapy might be used in the design of future chemoprevention trials. These trials might await the development of agents that interact with specific kinase pathways. One drug that should be considered in future chemoprevention trials is bryostatin, a PKC activator that is now entering phase I trials. Other agents that could potentially be effective preventive agents in high-risk patients because of their favorable side effect profiles include farnesyltransferase inhibitors, which inhibit ras, monoclonal antibodies and small molecules such as tyrosine kinase inhibitors to epidermal growth factor or vascular endothelial growth factor. Cyclooxygenase of lipoxygenase inhibitors and other NSAIDs need to be further studied as chemopreventive agents. Immunotherapy with interferon-α combinations is also under investigation. These agents all have promise in high risk patients as primary prevention agents or in the adjuvant setting.

VII. SUMMARY

Since the mid-1980s, the goal of treatment for epithelial cancers has begun to shift from the eradication of metastatic disease to the prevention of cancer. Along these lines, advancements have been made in the prevention of UADT cancer by treatment with retinoids as well as the use of tamoxifen in breast cancer and targeting COX-2 in FAP colon cancer. Further advances in epithelial cancer prevention await the development of cancer risk models and intermediate markers that can be incorporated into the design of chemoprevention trials. These needs may be met through advancements in our understanding of genetic events that occur during epithelial carcinogenesis, such as point mutations of ras and p53. Pathways activated or inhibited by chemopreventive agents may offer additional intermediate markers of response, including retinoid receptor expression, which increases following retinoid treatment, epidermal growth factor, and cyclooxygenase. Prospective phase III clinical trials are necessary to examine these possibilities and to establish eventually chemoprevention strategies as standard of care in health policy.

Edward S. Kim
Fadlo R. Khuri
Waun Ki Hong
University of Texas M. D. Anderson Cancer Center, Houston

See Also
CHEMOPREVENTION, PRINCIPLES OF ; COLORECTAL CANCER: MOLECULAR AND CELLULAR ABNORMALITIES ; HEAD AND NECK CANCER ; LUNG CANCER: MOLECULAR AND CELLULAR ABNORMALITIES ; MOLECULAR EPIDEMIOLOGY AND CANCER RISK ; MULTISTAGE CARCINOGENESIS

GLOSSARY
chemoprevention Treatment with natural or synthetic agents that prevent the development of cancer in cancernaive patients (primary chemoprevention) or in patients who have been cured of a prior cancer (secondary chemoprevention).

DNA damage Genomic damage resulting from carcinogen exposure that leads to abnormalities in coding sequence (point mutations, amplifications, deletions, additions) or in chromosomal content (aneuploidy).

epithelial cancer Cancer that originates in the epithelial lining of any organ.

field cancerization Hypothesis in which diffuse epithelial injury results from chronic carcinogen exposure; genetic changes and the presence of premalignant and malignant lesions in one region of the field are associated with an increased risk of cancer developing throughout the entire field.

intermediate end point Any biological or genetic variable that changes during chemopreventive treatment and that can be used as a marker to predict a reduction in cancer incidence in chemoprevention trials.

multistep carcinogenesis Hypothesis stating that a cancer cell evolves from a normal cell as a result of a sequence of genomic injuries, which leads to a progressively dysplastic appearance and abnormal biological behavior.

oncogene A gene that, when expressed at abnormal levels or in a mutated state, contributes to the carcinogenic process through dysregulation of cell growth and differentiation.

premalignancy An epithelial region that is histologically abnormal but not fully malignant in appearance or behavior and that is at risk for neoplastic progression in the future.

retinoid receptor A cytoplasmic or nuclear protein that binds retinoids and functions in retinoid signal transduction.

retinoids A chemically diverse group of natural and synthetic compounds that are related to vitamin A and bind a specific receptor or set of receptors.

second primary tumor A tumor that occurs in a patient who has been cured of a prior cancer and that is of a different histology and/or occurs in a separate location from the original cancer.

tumor suppressor gene A gene that normally functions as a physiologic inhibitor of abnormal clonal expansion, preventing the outgrowth of cells that have undergone oncogenic mutations.

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