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AACR Task Force Report |
Authors' Affiliations: 1 National Cancer Institute, Bethesda, Maryland; 2 University of Texas M.D. Anderson Cancer Center, Houston, Texas; 3 Weill Medical College of Cornell University, New York, New York; 4 CCS Associates, Mountain View, California; 5 Eli Lilly & Co., Indianapolis, Indiana; 6 Fred Hutchinson Cancer Research Center, Seattle, Washington; 7 Fox Chase Cancer Center; 8 The Wistar Institute, Philadelphia, Pennsylvania; 9 University of Arizona, Tucson, Arizona; 10 Emory University, Atlanta, Georgia; 11 University of Pittsburgh, Pittsburgh, Pennsylvania; 12 International Epidemiology Institute, Rockville, Maryland; 13 Mayo Clinic, Rochester, Minnesota; 14 British Columbia Cancer Agency, Vancouver, British Columbia, Canada; 15 University of California at Los Angeles, Los Angeles, California; 16 University of California at Irvine, Irvine, California; 17 Baylor Sammons Cancer Center, Texas Oncology, PA, U.S. Oncology, Dallas, Texas; and 18 Johns Hopkins University, Baltimore, Maryland
Requests for reprints: Gary J. Kelloff, National Cancer Institute, Executive Plaza North, Room 6058, 6130 Executive Boulevard, Rockville, MD 20852. Phone: 301-594-0423, Fax: 301-480-3507; E-mail: kelloffg{at}mail.nih.gov.
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Abstract |
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 Top Abstract Molecular Biomarkers in...Mechanism-Based Chemopreventive...Chemoprevention in Major Cancer...Accelerating the Development of...References |
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Knowledge of the molecular basis of carcinogenesis has increased exponentially through research elucidating signaling and metabolic pathways and defining genetic progression models. New technologies in genomics and proteomics and in functional and molecular imaging have spurred this research. As suggested in the 2002 article, this knowledge provides a basis for developing early biomarkers in IEN that will allow discovery of new chemopreventive agents, identify subjects at risk for cancer, and serve as end points for evaluating chemopreventive efficacy.
This report by the AACR Cancer Prevention Task Force provides perspectives on the current state of chemoprevention science and on the possibilities for future rapid advances. We discuss prospects for use of biomarkers in chemopreventive agent development, mechanism-based strategies for chemoprevention, and progress in the clinical development of agents to prevent cancer and prevent or treat IEN. The report concludes with recommendations for accelerating the progress of chemopreventive drug development.
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Molecular Biomarkers in Chemoprevention |
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TopAbstractMolecular Biomarkers in...Mechanism-Based Chemopreventive...Chemoprevention in Major Cancer...Accelerating the Development of...References |
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Characteristics of an ideal molecular biomarker
Generally, the more closely a biomarker resembles the carcinogenic process it is modeling, the more effective it will likely be in chemoprevention studies. For example, single genes and proteins that are overexpressed, mutated, or masked in precancers or cancers compared with normal tissue may be biomarkers of cancer risk and targets for modulation if they are indicators of a biological process associated with neoplastic progression. Cyclooxygenase-2 (COX-2) is such a target. It is overexpressed in many cancers and precancers (2), and it is a biomarker of inflammatory response to growth factors and other cellular stimuli (3–5). Although more complicated to interpret, increases in expression and activity of growth factor receptors or kinases at critical points in signaling pathways are similarly associated with early neoplastic activities, such as cellular proliferation, survival, and angiogenesis; examples are vascular endothelial growth factor (VEGF) receptors and ras and rho oncogene expression (6).
Because they are detecting overall changes in cells undergoing carcinogenesis, gene microarray, proteome, and immunogen analyses provide tools for closely modeling the process of neoplastic progression and may be the ultimate biomarkers themselves or a source of individual or coordinated clusters of molecular biomarkers. Identification of the biomarkers involves analysis of multiple coordinated molecular activities to identify those most important for cancer or alternatively to analyze pathway activation bypassing interpatient differences in activation mechanisms and feedback loops. Gene set enrichment analysis (7) provides one method for identifying critical pathway targets, in which gene occurrence is mapped to discriminate between genes that are affected or not affected in the cancer tissue. Effects on the target may be measured at one or many of the multiple possible intermediate points on the pathway(s) represented by the gene set enrichment analysis map. Gene set enrichment analysis has been used to identify tissue-specific molecular targets affected by deletion of the tumor suppressor PTEN (7, 8). As a second example, Troester and Perou have designed a strategy for applying gene expression profiling (using hierarchical cluster analysis) in breast cancer chemoprevention as risk and end-point biomarkers (9). This approach included a phase II trial design based on Fabian's model (10) with genomic analysis of fine-needle aspiration (FNA) tissue comparing high-risk women at baseline and after 6 months of treatment with either chemopreventive agent or placebo.
Changes in an ideal end-point biomarker would link to clinical benefit, directly or indirectly related to chemopreventive potential, and modulation would be associated with low toxicity (1, 11). For example, in addition to their potential effects in cancer prevention, targets of antioxidants and anti-inflammatory drugs and of cholesterol-lowering drugs are associated with clinical benefit for other diseases of aging—arthritis and cardiovascular disease, respectively. Phase II metabolic enzymes, such as the glutathione S-transferases, are targets for dietary antioxidants. Agents targeting these enzymes may provide clinical benefit by virtue of their pleiotropic chemoprotective effects, and they are likely to have low toxicity (3). Finally, ideal biomarkers can be quantified directly [e.g., epidermal growth factor receptor (EGFR) tyrosine kinase activity (12)] or via a closely related activity, such as inhibition of a specific kinase upstream or downstream from the target [e.g., S6 kinase activity or phosphorylation of 4-EBP or pS6 to measure mammalian target of rapamycin (mTOR) inhibition (13)]. In addition, measurement of these biomarkers should be reliably done on clinical specimens that are obtained as noninvasively as possible. Novel and developing molecular imaging techniques allow noninvasive assessment of the activation state of multiple signaling pathways and functional outcomes, thus offering the potential for serial analysis of effects of chemopreventive agents on the tissue of interest.
Genetic progression models/intrinsic properties of neoplasia
A key concept supporting use of molecular biomarkers for developing chemopreventive agents is that cancer is a disease of genetic progression. Progression has been mapped in tissues by the appearance of specific molecular and more general genotypic damage associated with increasingly severe histologic phenotypes (14–17). Early, critical steps include inactivation of tumor suppressor genes, such as APC (15, 16, 18), BRCA1 or BRCA2 (19), and PTEN (13, 20, 21), and activation of oncogenes, such as ras and PI3KCA (22). In some cases, progression has been correlated to the appearance of a cluster of genetic defects, such as point mutations and loss of heterozygosity (LOH) in p16 and p53 genes and p16 promoter methylation in esophagus (23, 24). Progression may also be influenced by factors specific to the host tissue's environment, such as production and action of hormones and growth factors in stroma in the microenvironment of the developing epithelial tumor; activation of VEGF receptor leading to angiogenesis and macrophage-mediated inflammatory response are examples (25–27).
These events, manifested in cells and tissue as intrinsic properties of neoplasia, were described by Hanahan and Weinberg as six acquired characteristics of cancer in their landmark review (26). Because of the imputed association with neoplastic progression, cellular signaling pathways with genetic lesions producing these effects are a rich source of potential molecular targets. Table 1 lists the six properties of neoplasia along with candidate molecular targets for intervention.
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Mechanism-Based Chemopreventive Agent Development |
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TopAbstractMolecular Biomarkers in...Mechanism-Based Chemopreventive...Chemoprevention in Major Cancer...Accelerating the Development of...References |
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There is increasing evidence from epidemiologic, experimental, and clinical data suggesting that inhibition of insulin-like growth factor (IGF) signaling, particularly via phosphatidylinositol 3-kinase (PI3K)/AKT–activated pathways, may be a target for chemoprevention. IGF levels are increased in many cancers, and levels of IGF-binding protein-3 (which, when bound to IGF, inhibits IGF signaling) are decreased (30–37). For example, clinical studies have shown that retinoids and selective estrogen receptor (ER) modulators (SERM) lower the IGF-I/IGF-binding protein-3 ratio in breast and that this activity is associated with antiproliferative chemopreventive activity (38–42). mTOR signaling is also a potential target for chemoprevention, because mTOR integrates signals from a host of environmental factors, including amino acids, energy, hormones, and growth factors, to regulate cell cycle. For example, AKT is upstream of mTOR, and activation of AKT1 in transgenic mice leads to the rapid development of high-grade prostatic IEN (HGPIN). The mTOR inhibitor RAD-001 (a rapamycin analogue) reversed the PIN phenotype, and this effect was associated with increased apoptosis (8).
Oncogene pathway addiction and tumor suppressor hypersensitivity
As described by Weinstein and others (43–46), oncogene addiction is physiologic dependence of cancer cells on the continued activation or overexpression of single oncogenes for maintaining the malignant phenotype. This dependence occurs in the milieu of the other changes that mark neoplastic progression. For example, addiction has been observed in mice transgenic for ras in melanoma; BCR-ABL in leukemia; HER-2/neu in breast; and c-MYC in pancreas, skin, and leukemia. Addiction has also been observed in many human cancer cells [e.g., ras in pancreas; EGFR in lung; the PI3K pathway in multiple tumor types; and cyclin D1 in esophageal, colon, and pancreatic adenocarcinomas (44, 46)]. Because of the selective sensitivity of addicted cells to inhibition of the oncogene or its pathway, these are good candidates for chemoprevention. Particularly relevant to chemoprevention, damping rather than eliminating activity may be effective as was seen for cyclin D1 in esophageal cancer cells (43, 44). Absence of tumor suppressors may confer a similar procancer addiction. For example, APC, p53, and Rb have shown selective antiproliferative and growth inhibition when inserted into cells in which they have been inactivated (43). Clinical studies characterizing precancerous lesions by microarrays and other new technologies are confirming the value of inhibiting these sensitive targets (43).
Infection/inflammation and vaccines
Abundant epidemiologic (47–50) and experimental (51–54) data implicate infection and inflammation as factors in neoplastic progression via production of oxygen and nitrogen radical oxidants, production of growth-promoting cytokines, tumor suppressor inhibition, and stimulation of signal transduction pathways. Some of the prominent infective carcinogens are human papillomaviruses (HPV; ref. 55), EBV (56), human herpesvirus-8 (57), human hepatitis viruses [hepatitis B virus (HBV) and hepatitis C virus (HCV; ref. 58)], schistosomes (59), and Helicobacter pylori (60, 61). Because of the frequency of these infections, their prevention or treatment is a potentially fruitful cancer prevention strategy (Fig. 1
). Progress in the development of anti-infectives and vaccines that target the carcinogenic mechanisms of these agents has been significant (60). For example, HBV and HCV infections are prominent causes of chronic liver disease, including hepatocellular carcinoma, one of the most common cancers worldwide (58). HCV infection may also be a risk factor for other cancers, including non-Hodgkin's lymphoma and multiple myeloma (62). EBV is a ubiquitous human herpesvirus that is associated with a spectrum of malignant diseases, including Burkitt's lymphoma and nasopharyngeal carcinoma (63).
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Prevention of HPV infection can be achieved by induction of capsid-specific neutralizing antibodies. One of the most advanced vaccines of this type is Gardasil, an experimental vaccine targeting the four most common strains of sexually transmitted HPV that cause cervical cancer or genital warts (68, 69). This vaccine was 89% effective in preventing infection with the viral strains and 100% effective in preventing precancerous lesions [cervical IEN (CIN)] or genital warts in a phase II trial. In a phase III trial, Gardasil raised antibodies in >99% of 1,529 people who received a three-dose regimen over a 6-month period. The long-term effects on prevention of cervix cancer and the applicability to third world countries where cervix cancer is particularly prevalent are under evaluation.
Treatment of bacterial infection with antibiotics, thus reducing associated chronic inflammation, is another promising chemopreventive strategy exemplified by treatment of H. pylori. The 2005 Nobel Prize in medicine was awarded to Barry J. Marshal and J. Robin Warren for their discovery of the role of H. pylori in gastritis (inflammation of the stomach), gastric ulcers, and more severe lesions resulting from chronic inflammation. Individuals with gastric atrophy and intestinal metaplasia are at risk for developing cancer of the stomach, and chronic H. pylori infection is one the most important factors in the development of these precancerous gastric lesions (61). In addition to bacterial factors, polymorphisms in the host cytokine genes that modulate inflammatory responses have a synergistic effect on the development of gastric cancer and precancerous lesions. Individuals with a positive family history of gastric cancer and/or proinflammatory polymorphisms of the interleukin-1 and tumor necrosis factor-
genes and who are infected by H. pylori virulent strains (cagA-positive, vacA s1-positive, vacA m1-positive, and babA2-positive) have been found to have a high risk of gastric cancer development.
Inflammation and oxidation
Inflammation not associated with infection may be associated with cancer risk (11, 51, 70–74) in gastrointestinal tract (53, 75), bladder (76, 77), skin (78), lung (78–80), head and neck (81), breast (82, 83) and prostate (84, 85). Moreover, agents modulating molecular targets of inflammation, such as COX (53, 86), inducible nitric oxide synthase (87, 88), and lipoxygenase (LOX; refs. 89, 90), have shown promising chemopreventive activity. Particularly, COX inhibitors, including aspirin, traditional nonsteroidal anti-inflammatory drugs (NSAID), and COX-2 selective inhibitors, have shown chemopreventive efficacy in epidemiologic analyses as well as in clinical studies and are being evaluated in numerous cancer targets where COX-2 overexpression or inflammation is observed. Nuclear factor-
B (NF-B), a protein induced during inflammation that serves as a transcription factor regulating genes for other inflammatory and tumor-promoting proteins [such as COX-2, BCL-2, interleukin-1, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-, LOX, inducible nitric oxide synthase, cyclin D1, cell adhesion molecules, c-MYC, matrix metalloproteinase-9, VEGF, survivin, and telomerase (hTERT)] could prove to be a key molecular target for chemoprevention (25, 27, 91).
Many natural antioxidants (e.g., green tea polyphenols, lycopene, resveratrol, curcumin, and sulforaphane) have broad-spectrum anti-inflammatory and free radical trapping activities. These agents have shown chemopreventive activity in animal models (92, 93), are associated with lower cancer risk in human studies, and seem to be good candidates for development. The green tea polyphenols are particularly noteworthy. These catechins have shown potential chemopreventive activity in numerous animal models (92). Moreover, a recent clinical study showed that tea could potentially prevent prostate cancer in men with HGPIN (94). The characterization of molecular targets of antioxidant activity has been difficult because of the pleiotropic activities of these agents. Attention is now being directed to Nrf2, a transcription factor that activates genes with products involved in deactivating electrophilic toxic compounds. Nrf2 is sequestered by Keap1 until antioxidant inducers cause a conformational change in the Nrf2-Keap1 complex and release Nrf2, which then interacts with an antioxidant response element to induce antioxidant gene expression (95–98). Nrf2 can also be activated by phosphorylation [e.g., via signal transduction involving mitogen-activated protein kinase (MAPK), protein kinase C, or PI3K]. Synthetic antioxidants (e.g., oltipraz, CDDO, and its derivatives) also have potential chemopreventive activity via these pathways (97, 98).
Epidemiologic and experimental evidence
Epidemiologic data associating cancer preventive activity or cancer incidence with the use of certain drugs, foods, lifestyles, or the presence of germ-line mutations or gene variations historically have been a primary means for identifying possible molecular targets for chemoprevention. Combining epidemiologic leads with experimental data provides a rationale for use of these targets in chemoprevention. For example, numerous studies associate lower incidence of colon cancers, colorectal adenomas, and colorectal cancer mortality with use of aspirin and NSAIDs (86, 99). These data led to the exploration of COX inhibition, the primary mechanistic activity of these drugs, for chemoprevention. The compelling body of epidemiologic, experimental, and mechanistic evidence implicating estrogen in promotion of breast and other cancers (100, 101) led to the development of anticancer drugs, such as tamoxifen and exemestane, targeting the ER and steroid aromatase, respectively. Hormonal contraceptives induce at least a 50% decrease in development of life-threatening ovarian cancers. Epidemiologic data associating vitamin A and carotenoids with reduced cancer incidence (102) led to the identification of retinoid receptors as molecular targets for prevention, and epidemiologic data on the association of IGF with cancers of the breast, prostate, and lung raised interest in the IGF signaling pathway as a target for chemoprevention (34, 35, 103). Data associating statin usage with reduced risk of colon, prostate, and breast cancers and melanoma (104, 105) provide valuable leads that are being followed up with experimental investigations, both clinical and preclinical to elucidate the target mechanism(s) for this observed effect. Recent reports of large studies and meta-analyses that show no association between statin usage and cancer risk illustrate the complexity of interpreting epidemiologic data (106–109).
Collateral targets of mechanistically targeted drugs
Collateral targets for chemopreventive agents are molecules in signal transduction and metabolic pathways or networks that are upstream or downstream of the direct target of the agent. These indirect targets are also associated with neoplastic progression, possibly more directly than the mechanistic target. Mechanism-based chemoprevention strategies may involve collateral targets in several ways—that is, in combinations of agents to increase efficacy or reduce toxicity of the individual agents, as new direct mechanistic targets for identifying potential chemopreventive agents, and as chemopreventive targets for which the mechanistic targets of agents are surrogates (where it is difficult to design agents that modulate the chemopreventive target). For example, aromatase can be considered a collateral target of COX-2 in that inhibition of prostaglandin synthesis also inhibits prostaglandin-mediated induction of aromatase, so use of NSAIDs in combination with aromatase inhibitors may allow lower doses of aromatase inhibitors and reduced toxicity in prevention of breast cancers (5, 51). Interaction of COX-2 and EGFR is described in Combination strategies. Chan et al. have suggested arachidonic acid as a collateral and alternative target to COX-2, because inhibition of COX-2 raises the level of cellular arachidonic acid, thereby potentially activating ceramide-mediated apoptosis (110). In addition, evidence that telomerase, which is activated early in prostate carcinogenesis, is regulated by ER and is thus a collateral target of ER (111), provides a rationale for exploring ER as a molecular target for chemoprevention in prostate.
Combination strategies
A major aspect of molecular carcinogenesis research is the identification of multiple targets for combinations of drugs that may have greater efficacy than would single agents. Agent combinations targeting the EGFR and COX-2 signaling pathways exemplify combined-agent development for cancer prevention. The independent and interactive signaling of EGFR and COX-2 has been shown in lung, head and neck, and colon carcinogenesis. Several processes linked to carcinogenesis (cell proliferation, apoptosis, angiogenesis, and invasiveness) can be influenced by the stimulation of EGFR signaling or enhanced synthesis of prostaglandin E2 (PGE2).
Data supporting the cross-talk and potential feedback loops between EGFR and COX-2 strengthens the rationale for combination regimens aimed at both targets (refs. 4, 53; Fig. 2 ). Mutually independent EGFR and COX-2 effects also are important to the potential efficacy of combined inhibitors of these targets. EGFR and its downstream effectors can be activated independently of COX-2/PGE2, and COX-2/PGE2 and its downstream effectors can be regulated independently of EGFR signaling (53). For example, PGE2 can stimulate cell proliferation by an EGFR-independent mechanism (112). Illustrating the potential benefit of independent plus interactive effects, combined inhibitors of COX and EGFR tyrosine kinase almost completely prevented adenoma development in APC(Min) mice (113) and subsequently were shown to be active in a head and neck cancer xenograft model (114).
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ligands enhance histone deacetylase inhibitor activity (117); combined inhibition of IGF-I and mTOR inactivates a potent feedback loop (118), and the combination of a histone deacetylase inhibitor with a DNA methyltransferase inhibitor is highly active in vitro and in vivo (119). Single agents targeting each of these classes of molecules are at various stages of clinical development and show promise for combination approaches. |
Chemoprevention in Major Cancer Target Organs—Update |
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TopAbstractMolecular Biomarkers in...Mechanism-Based Chemopreventive...Chemoprevention in Major Cancer...Accelerating the Development of...References |
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2% to 3%, but the prevalence of latent prostate cancer detected in autopsy series is 30% (1, 121, 122). The introduction and rapid dissemination of prostate-specific antigen (PSA) screening between 1986 and 1992 caused an increase in the overall age-adjusted incidence of prostate cancer likely due to lead time bias, but thereafter this apparent increase in incidence declined. There is no clear evidence that early detection has reduced prostate cancer mortality because this rate has not declined in association with the use of PSA screening (123).
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Implicated in the multiple pathways that lead to carcinogenesis, proliferative inflammatory atrophy (PIA) is a result of inflammation and dietary influences (85, 133). PIA effects include increased cell death and regenerated cells with DNA damage. It is believed that prostate carcinogenesis progresses from normal epithelium to PIA to PIN to HGPIN and finally to cancer. PIA also is associated with glutathione S-transferase P1 and COX-2 expression. A PIA pathology consensus conference has completed a standardized classification system for PIA lesions, and the future publication of this system should enhance comparisons of PIA research results from different groups and advance the design of preventive interventions.
Spectral imaging and other newer techniques should be investigated and implemented for identifying and characterizing alterations during prostate carcinogenesis. The development of quantitative tools will overcome subjective interpretations and accelerate the understanding of gene regulation within prostate carcinogenesis. New tools for this work include genomics, transcriptional profiling, and proteomics along with novel bioinformatics approaches (such as gene set enrichment analysis cited above). When combined with well-characterized, diverse, and ample clinical samples with long-term clinical follow-up, these tools could lead to a clearer understanding of the molecular biology of prostate cancer.
Completed in 2003, the National Cancer Institute (NCI)–funded Prostate Cancer Prevention Trial was the first large-scale phase III prostate cancer prevention study (134), randomizing 18,882 subjects to receive finasteride (a 5-reductase inhibitor) or placebo. The prevalence of prostate cancer over a period of 7 years was 24.8% lower in the finasteride than placebo arm (95% confidence interval, 18.6-30.6; P < 0.001). Tumors of a higher grade (Gleason 7-10) were detected 1.25 times more often in the finasteride arm (6.4% of graded tumors) than in the placebo arm (5.1%; P < 0.001). Speculation on the reason for the higher grades in the finasteride arm include the possibility that finasteride increases the risk of high-grade cancer through changes in intraprostatic androgen and/or estrogen signaling. Higher Gleason grades have been detected in tumors of men with lower testosterone levels (versus normal levels; refs. 135–138), possibly reflecting effects of lower dihydrotestosterone levels, which also occur with finasteride. It also is possible that the increased high-grade disease with finasteride was more apparent than real, either because finasteride caused tumor cell morphologic changes that mimic higher Gleason scores or because finasteride significantly shrank prostate volume, raised the tumor-to-gland ratio (135–137, 139), and thus improved the detection of high-grade tumors. Efforts to explain the high-grade Prostate Cancer Prevention Trial results include extensive analyses of tumor specimens and a NCI-supported follow-up study of long-term outcomes of men diagnosed with high-grade tumors (140).
The Selenium and Vitamin E Cancer Prevention Trial is a large-scale NCI-supported Prostate Cancer Prevention Trial that met its accrual goal with the randomization of 35,534 men between July 2001 and June 2004 (141). Selenium and Vitamin E Cancer Prevention Trial has a prospectively collected biorepository designed for future research, such as the development of comprehensive models for identifying men at the greatest risk of prostate cancer (especially high-grade prostate cancer) and most likely to benefit from chemoprevention with selenium and vitamin E.
Promising new agents for prostate cancer prevention include antioxidants (e.g., tea polyphenols), SERMs (e.g., toremifene), NSAIDs, soy isoflavones, and statins. For example, recent observational studies suggest that statins are associated with reduced prostate cancer risk. A study involving 34,438 men within the ongoing prospective Health Professionals Follow-up Study (142) indicated that statins and other cholesterol-lowering drugs were associated with a significant 46% reduced risk of advanced prostate cancer, and this association got stronger with longer drug use (P = 0.008). Risks of metastatic and fatal disease were reduced 66%. This study did not find an association with reduced overall prostate cancer incidence. Statins were the most likely active agents because they constituted 90% of all cholesterol-lowering treatment at the end of study, when the strongest risk reductions were observed. In a case-control study conducted within the Veterans Affairs system, there were significant inverse associations between statin use and prostate cancer, and the inverse associations strengthened with high-grade prostate cancer and longer statin use (143). A significant, duration-dependent inverse association between statin use and prostate cancer risk also was found in another Veterans Affairs case-control study (144). These results are supported by the limited available preclinical data on the effects of statins in prostate carcinogenesis (145). In contrast, as noted above, two recent epidemiologic studies have shown no effect of statins on cancer incidences at multiple sites, including prostate (106, 107). Tea polyphenols (94) and toremifene (146–148) have both prevented progression of HGPIN to prostate cancer in phase II clinical trials; NSAIDs and soy isoflavones are under evaluation in phase II models.
There is a need for more efficient clinical evaluation of chemopreventive agents that lead to promising models involving serial biopsy, including high-risk men, men with HGPIN, watchful waiting in low-grade cancer patients, and preprostatectomy models. The international, multicenter, double-blind, placebo-controlled Reduction by Dutasteride of Prostate Cancer Events trial is obtaining serial biopsy specimens from at-risk patients being treated with dutasteride or placebo (149). These patients are established to be free of cancer by a negative 6- or 12-core biopsy taken within 6 months of enrollment and are being monitored for type 1 and 2 5-reductase, which are increased in association with prostate carcinogenesis (150) and are inhibited by dutasteride. The serial biopsy model is expected to help improve the understanding of the natural history of prostate cancer and to facilitate detecting correlations between clinical and pathologic data. This model also is expected to cut the expenses and accelerate the advances of preventive drug development.
Available data support the conclusion that the presence of PIN on prostate biopsy predicts for an increased risk for prostate cancer and that some PIN lesions give rise to prostate cancers. Thus, PIN lesions detected on prostate biopsy identify men at high risk for developing prostate cancer. However, the limitations of prostate biopsy sampling preclude repeated monitoring of PIN lesions to assess their natural history (138). When a diagnosis of HGPIN is combined with other risk factors, such as serum PSA, age, race, and/or family history, cohorts of men at very high risk for developing prostate cancer are identified who have prostate cancer incidence rates of 40% over 3 years and 80% over 10 years (151, 152). Because extent of PIN cannot be reliably measured by serial sampling, a decrease in the extent of PIN with treatment is not a conclusive efficacy end point. Thus, clinical trials targeted at eliminating or reducing the extent of PIN are not likely to show net clinical benefit without additional data indicating that prostate cancer incidence (risk) has been reduced. Because prostate cancer incidence can be estimated in cohorts of patients with HGPIN, PSA abnormalities, and other risk factors, phase III placebo-controlled trials that have prostate cancer incidence as the primary end point can be conducted with 300 to 500 patients per arm, with the control group having an expected 40% prostate cancer incidence over 3 years. This trial design will more definitively evaluate prostate cancer risk-reduction candidates and will validate extent of HGPIN as a suitable efficacy end point (1, 133, 151, 152). A 30% reduction in prostate cancer incidence in the HGPIN patients who are safely treated with the new agent compared with control patients should likely constitute clinical benefit.
The watchful waiting model involves an arm of no surgery in the setting of low-risk, localized prostate cancer, which is known for its lengthy indolent phase. Consenting patients are put under surveillance with no treatment and evaluated against patients undergoing immediate treatment for organ-confined disease. Surveillance is conducive to embedding phase II chemopreventive (or therapeutic) end points designed to identify potential preventive biomarkers and correlate them with clinical outcomes. The primary objective of surveillance trials is to determine if men with organ-confined disease can avoid or postpone the financial, emotional, and morbidity (e.g., sexual dysfunction) costs of treatment without developing a worse outcome (e.g., progression to advanced disease; efs. 137, 153–156). Furthermore, a 10-year follow-up of a Swedish study of 695 men showed significant differences favoring surgery over watchful waiting in overall mortality (27.0% versus 32.0%; P = 0.04), disease-specific mortality (9.6% versus 14.9%; P = 0.01), local progression (19.2% versus 44.3%; P < 0.001), and distant metastasis (15.2% versus 25.4%; P = 0.004; refs. 153, 156). Additional research is needed to determine if these outcomes would parallel outcomes in the United States, where men typically are diagnosed at an earlier stage than were the men in the Swedish study (157) and where different ethnic and behavioral issues may be involved. The ongoing Prostate Cancer Intervention versus Observation Trial is expected to determine whether the Swedish results will be replicated in United States patients (158).
The preprostatectomy model examines effects of chemopreventive agents on biomarker end points in the short interlude between histologic diagnosis and prostatectomy. After prostatectomy, the whole gland can be examined to help define zonal patterns of prostate carcinogenesis and delineate cell-type characteristics and prostate cancer precursor lesions (159). The short intervention duration (typically from a few days to a month) causes problems for detecting and interpreting biomarkers and meeting accrual goals and creates statistical demands on the biomarker end points that recently have been the subject of systematic investigation, including the development of proteomic end points based on comparison of study patients with subjects without prostate cancer or intervention (160). Efforts at standardization will help meet the accrual goals and statistical demands of evaluating biomarker end points, lessening the risk of false-negative findings in these brief intervention trials. Development and validation of effective molecular imaging approaches that allow assessment of changes during treatment will also facilitate this trial design.
Colorectal cancer
Colorectal cancer is the third most common cancer in both men and women, constituting 10% of new cancer cases in men and 11% in women, and it is the second most common cause of death from cancer in the United States (Table 4). In 2005, there were an estimated 145,000 new cases in the United States and 56,000 related deaths (a rate second only to that of lung cancer; ref. 120). Although the incidence of colorectal cancer decreased between 1998 and 2001 (annual percent change –2.4%) in the United States, 6% of Americans will eventually develop invasive colon or rectal cancer, and >6 million Americans who are alive today will die of the disease (an individual's lifetime risk of dying from colorectal cancer in the United States has been estimated to be 2.5%). Globally, it is the fourth most common cancer in men and the third most common in women, with mortality paralleling incidence. In the year 2002, there were >1 million new cases of colorectal cancer worldwide (161). Despite evidence that 5-year survival is 90% when colorectal cancer is diagnosed at an early stage, <40% of cases are diagnosed when the cancer is still localized.
Screening has become a compelling strategy for prevention of colorectal disease. Current evidence indicates that screening for colorectal cancer reduces mortality. This has prompted the U.S. Preventative Services Task Force, the American Cancer Society, the Agency for Health Care Policy Research, and other agencies to recommend that average-risk individuals (those without a family history of colorectal neoplasia or other predisposing conditions, such as inflammatory bowel disease) be screened for colorectal cancer beginning at age 50 years (162, 163). Current evidence-based guidelines provide a menu of options for screening, which includes fecal occult blood testing yearly, flexible sigmoidoscopy every 5 years, the combination of fecal occult blood testing and flexible sigmoidoscopy, colonoscopy every 10 years, or air-contrast barium enema every 5 years. The concept of a menu of choices does not imply equal performance characteristics but rather reflects the need to increase screening compliance. Because millions of individuals yearly in the United States reach screening age, there is a growing need for cost-effective, compliance-driven strategies for screening. Computed tomography (CT) colonography and stool-based genetic testing have recently been explored as options.
CT colonography or "virtual" colonoscopy involves the use of helical CT to generate high-resolution, two-dimensional images of the abdomen and pelvis. The accuracy and potential of virtual colonoscopy as a screening tool for colorectal neoplasia has been hotly debated because initial studies yielded a wide range of sensitivity. Large recently published multicenter trials continued to fuel this controversy (164, 165). Several key issues will need to be addressed as the use of virtual colonoscopy becomes more widespread. Principal among these is determination of the acceptable size cutoff of a lesion detected by virtual colonoscopy that will necessitate a follow-up colonoscopy.
Specific genetic tests are not currently available for the majority of patients at risk for developing colorectal cancer. A molecular approach to colorectal cancer screening is attractive because it targets genetic changes that are fundamental to the neoplastic process. The feasibility of detecting altered DNA in stool has been shown using a multitarget assay panel of molecular markers (166). A recent multicenter study compared fecal DNA testing with fecal occult blood testing and colonoscopy (167). Although the majority of lesions identified by colonoscopy were not detected by either noninvasive test, multitargeted fecal DNA testing detected a higher proportion of important lesions compared with Hemoccult. Risk models based on these factors are under development (168).
Most sporadic colorectal cancers are believed to develop from a precursor IEN, sporadic adenomas (86, 99). The adenoma-to-carcinoma sequence describes the common pathway taken by neoplasms that arise as the result of the progressive accumulation of genetic changes, which may include alterations in proto-oncogenes, loss of tumor suppressor gene activity, and abnormalities in genes involved in DNA repair. These changes commonly involve chromosomal instability with widespread chromosomal deletions, duplications, and rearrangements that produce aneuploidy (169, 170). Alternatively, increased rates of mutation, often in tandemly repeated DNA sequences known as microsatellites (microsatellite instability) or a form of epigenetic instability called the CpG island methylator phenotype, in which genes are inappropriately silenced by promoter methylation (171), are mechanisms that can lead to progressive multistep carcinogenesis (Fig. 3). Subtle alterations in the regular pattern of the intestinal crypts known as aberrant crypt foci (ACF) are one of the first histologically detectable changes that may be associated with development of colorectal cancers. ACF seem to arise as the result of premalignant genetic alterations; they often show APC loss and K-ras mutations. The number, size, and dysplastic features of ACF correlate with the number of adenomatous polyps (adenomas). The stool-based genomic panel cited above targets 19 alterations associated with colorectal neoplasia (including mutational hotspots on K-ras, APC, and p53 as well as long-fragment DNA).
Over the past several years, several nutrition and drug prevention trials have been completed, including studies of fiber, calcium, and NSAIDs. The potential benefit of low-fat, high-fiber diets based on descriptive epidemiology and case-control studies has generally been accepted, but current data from prospective human trials are thus far equivocal or negative. Two large randomized trials that examined the effects of fiber supplementation on adenoma recurrence failed to show a chemopreventive effect (172). Recently, a prospective double-blind, placebo-controlled trial showed that supplemental calcium (3 g/d calcium carbonate equivalent to 1,200 mg elemental calcium) reduced the incidence and number of recurrent adenomas in subjects with a recent history of these lesions (173). The effect of calcium was modest (19% reduction in adenoma recurrence and 24% reduction in the number of adenomas over 3 years).
The most promising results come from trials using aspirin and NSAIDs for colorectal cancer prevention. Case-control and cohort studies suggested that the risk for developing of adenoma and carcinoma may be substantially reduced (40-50%) among aspirin and NSAID users compared with controls. A double-blind, placebo-controlled trial studied the effects of celecoxib, a selective COX-2 inhibitor, on colorectal polyps in patients with familial adenomatous polyposis (FAP). Treatment with high doses of this agent for 6 months was associated with a significant reduction from baseline in the number of colorectal polyps compared with placebo (28.0% versus 4.5%; P = 0.003; ref. 174). The reduction in polyps was mirrored by the polyp burden representing the sum of polyp diameters. This led Food and Drug Administration (FDA) to approve this drug as an adjunct to standard therapy in patients with FAP. In the sporadic setting, three recently published trials showed that aspirin reduced adenoma recurrence. The magnitude of the effect varied depending on the magnitude of risk in the group studied. Data from a large randomized prospective trial using aspirin in patients with sporadic adenomas showed a modest but significant effect of low-dose (81 mg) aspirin on adenoma recurrence of 19% (175). A 45% risk reduction in adenoma recurrence was shown using 325 mg aspirin in a higher-risk group of patients with previous colorectal cancer. Data from three large randomized trial, which employed COX-2 inhibitors for chemoprevention of sporadic adenomas, were presented recently. The APPROVe trial showed a highly significant 25% reduction in adenoma occurrence to be associated with intake of 25 mg rofecoxib compared with placebo during 3 years of follow-up in patients with a previous history of colorectal adenomas. Celecoxib showed even more striking efficacy in preventing sporadic adenomas in two studies in similar cohorts. In the Adenoma Prevention with Celecoxib study, in which 2,035 patients were randomized to placebo or 200 or 400 mg celecoxib b.i.d., adenoma incidence at 3 years was reduced by 45% in patients taking celecoxib compared with placebo (P < 0.0001; ref. 176). The Prevention of Colorectal Sporadic Adenomatous Polyps observed a similarly highly significant (P < 0.0001) reduction in adenoma incidence at 3 years in patients taking 400 mg celecoxib q.d. among 1,561 patients randomized in a 3:2 ratio to treatment or placebo (177). These trials and others found that the use of this class of drugs is associated with an increased cardiovascular risk (178, 179). Future trials, which use this class of agents, will need to assess the potential for risk versus benefit.
Other anti-inflammatory agents have shown promise in preclinical studies and are being evaluated in clinical studies. NO-NSAIDs are particularly interesting in this regard. They are potent chemopreventive agents in mouse models and show low toxicity in clinical settings (88, 180). An alternative downstream pathway from arachidonic acid ends with the leukotrienes. A metabolic product of an enzyme (15-LOX-1) in this pathway down-regulates peroxisome proliferator-activated receptor-
, thereby possibly inducing apoptosis (181).
In addition to having mostly promising epidemiologic data, statins are efficacious in animal models of colorectal cancer. For example, atorvastatin alone and in combinations with other chemopreventive agents was active in the colon ACF assay (182). Besides providing a rationale for evaluating the chemopreventive efficacy of atorvastatin in colon, these results also suggest that combinations of atorvastatin with NSAIDs may be an effective chemopreventive strategy, allowing the individual agents to be administered at subtoxic doses (182).
Because the natural history of colorectal cancer is protracted, clinical randomized trials have often concentrated on prevention of colorectal adenomas, the precursors to carcinoma. There has been recent interest in identifying earlier intermediate end points that can be used in chemoprevention trials. Magnifying endoscopy is being used to study and characterize ACF as dysplastic ACFs are thought to be precursors of adenomas in the colon (183). Standardization of techniques to identify and quantify these lesions will be crucial to the successful interpretation of intermediate end-point data. These ACF trials would be followed by the definitive adenoma prevention trials.
Breast cancer
In 2005, breast cancer was expected to be diagnosed in 212,930 women and to cause 40,870 deaths, making this the most common and second deadliest cancer in U.S. women (Table 4). Early detection from widespread mammographic screening has led to earlier diagnosis and a trend to reduced breast cancer mortality. Currently, the presence of atypia (i.e., the abnormal cytologic features primarily of increased nuclear size, abnormal shape, and variation in size or shape in cytologic or histologic specimens) in breast tissue is a known marker associated with breast cancer risk.
Because of their increased risk of developing breast cancer, individuals with abnormal breast histology, including atypical ductal hyperplasia, lobular carcinoma in situ, ductal carcinoma in situ (DCIS), and BRCA1 or BRCA2 mutations, are candidates for chemopreventive interventions. As cited above, Troester and Perou have designed a strategy for applying gene expression profiling to identifying and associating risk with breast cancer subtypes (9). Significant features of genetic progression in breast IEN (Fig. 3) were discussed in the 2002 review. Briefly, in the early stages of IEN, the expression of tumor suppressor genes is reduced partially due to methylation of gene promoter regions. The predominant phenotype in breast IEN is a progressive increase in the proportion of cells expressing ER- along with increasing growth factors, receptor tyrosine kinase activity and expression, and diminished apoptosis. Oxidative stress and DNA damage may result in increased expression of wild-type p53 in early IEN, but mutated p53 may not appear until later stages of IEN (DCIS) and invasive cancer. Aneuploidy and LOH have been observed in early-stage IEN and seem to be progressive through late-stage IEN and invasive cancer. Twenty percent to 40% of simple and atypical hyperplasias and 80% of DCIS are found to have LOH regions that are also present in synchronous invasive breast cancers (1).
Biomarkers in breast cancer that have particular relevance to chemoprevention include the following: markers associated with neoplastic phenotypes, such as alterations of nuclear morphology and angiogenesis; expression of mRNAs or proteins likely to be required for response to putative chemopreventive agents (e.g., ERs and retinoid receptors); markers indicative of intact downstream response pathways (e.g., progesterone receptors); oncogenes and tumor suppressor genes regulated by chemopreventive agents (e.g., HER-2/neu, transforming growth factor-ß, IGF-I, and IGF-II); and markers of genetic instability, such as microsatellite alterations and DNA methylation in high-risk breast epithelium.
Several recent clinical trials focused on treatment of breast IEN. Fabian et al. reported results of a randomized phase II trial of oral -difluoromethylornithine (DFMO) using imaging, serum, and urine biomarkers in high-risk women (10, 184). DFMO is an irreversible inhibitor of ornithine decarboxylase, which is a limiting enzyme of polyamine synthesis that is often up-regulated in breast cancer. Eligible women in this trial had random periareolar FNA (RPFNA) cytology that revealed hyperplasia or hyperplasia with atypia but no evidence of breast cancer on clinical examination or mammogram. The women were at high risk for the development of breast cancer based on family history and also had FNA evidence of breast IEN. One hundred nineteen high-risk women were randomized to receive 6 months of oral DFMO (0.5 g/m2/d) versus placebo and underwent repeat FNA of both breasts in a periareolar location at the completion of 6 months of treatment. There was no difference in cytology results at 6 months comparing DFMO to placebo compared with the baseline FNA results. There was also no difference between the DFMO and placebo groups for the secondary end points, including expression of proliferating cell nuclear antigen, p53, and EGFR expression. In addition, there was no difference between the DFMO and placebo groups regarding changes in mammographic breast density or serum IGF-I/IGF-binding protein-3 ratio. A modest reduction in average total urine polyamines was obtained in the DFMO group and there was no reduction in the spermidine-to-spermine ratio, suggesting that the dose of DFMO was too low to definitively affect polyamine synthesis. Although DFMO was not effective, this study design set a precedent for identification of breast IEN in high-risk women using RPFNA and for short-term intervention trials. This clinical trial design has been used to complete a randomized phase II trial of the SERM, arzoxifene versus placebo. Preliminary results from this treatment intervention trial are expected.
Fabian et al. reported the results of a phase I evaluation of biomarker modulation with arzoxifene in breast IEN and early breast cancer patients (185). Arzoxifene has potent antiestrogen activity against breast cancer cell lines and has proven antitumor activity in women with metastatic breast cancer and does not have estrogen activity in the uterus (186). In the phase I multicenter trial, women with newly diagnosed DCIS or T1/T2 invasive breast cancer were randomized to receive 10 versus 20 or 50 mg arzoxifene daily in the interval between diagnostic biopsy and definitive surgery. An additional group of patients was enrolled as the no-treatment control group. The phase I experience defined 20 mg arzoxifene as the optimal dose for biomarker modulation. Subsequently, 76 postmenopausal women with DCIS or early-stage breast cancer were randomized to receive 20 mg arzoxifene versus placebo daily during the interval between diagnostic biopsy and definitive surgery. In both trials, increases in serum sex hormone-binding globulin and decreases in IGF-I and the IGF-I/IGF-binding protein-3 ratio were noted. In the dose-finding portion of this study, in 45 evaluable women, decreases in proliferation indices were more prevalent in the arzoxifene-treated group than in the control group. In the 58 evaluable women in the randomized control portion of the study, a decrease in ER expression was observed with arzoxifene compared with placebo. However, no statistically significant difference in the reductions in proliferation was observed with arzoxifene compared with placebo, a finding felt to be due to the confounding effects of the women stopping hormone replacement therapy at the time of diagnosis. This IEN and early breast cancer treatment "window of opportunity" trial showed the feasibility of conducting such trials in the United States in multiple centers with central pathology assessment and biomarker determination.
Bundred et al. reported the results of their randomized, double-blind, placebo-controlled trial of short-term treatment with gefitinib (Iressa, ZD1839) in patients with DCIS (187). They have shown previously that blockade of EGFR with gefitinib led to reduced epithelial proliferation and increased apoptosis in immunosuppressed mice who were implanted with human DCIS obtained from 16 women (188). Subsequently, 65 patients with intermediate or high-grade DCIS were treated with gefitinib, 250 or 500 mg/d, during the interval from their diagnostic biopsy to definitive surgery. Of the 49 patients assessed, reduction in Ki-67 proliferation was seen in 47% of patients and a >10% decrease in activated nuclear MAPK was seen in 54% of patients (187). The reduction in proliferation correlated with reduced expression of activated MAPK. These results suggest that gefitinib may hold promise in the treatment of intermediate or high-grade DCIS.
Guix and Arteaga et al. showed similar findings with a short course of the EGFR tyrosine kinase inhibitor erlotinib (Tarceva) in women with DCIS or early-stage breast cancer treated between the time of diagnostic biopsy and definitive surgery (189). In these studies, significant reductions in Ki-67 (
75% inhibition) and substantial reductions in activated MAPK (
85% inhibition) were shown after 14 days of treatment in 50% of patients. Interestingly, erlotinib did not affect the expression of total or nuclear phosphorylated AKT, suggesting that AKT, unlike MAPK, may not be under regulation by the EGFR pathway in DCIS and early-stage breast cancer.
Fabian et al. recently reported decreased breast epithelial cell proliferation after 6 months of the aromatase inhibitor letrozole in high-risk women on hormone replacement therapy who had RPFNA evidence of hyperplasia with atypia (190). Twenty-six postmenopausal women, who were on a stable dose of estrogen with or without progestin for at least 6 months before baseline RPFNA and who had evidence of hyperplasia with atypia, were treated with letrozole 2.5 mg/d in addition to continuing their hormone replacement therapy. There were no significant increases in hot flashes or arthralgias with letrozole, although there was an increase in fatigue. At the initial report of this trial, 17 patients had completed the 6 months of letrozole and had undergone a repeat RPFNA. Of the 10 subjects who had a baseline Ki-67 value of 1.5%, all 10 had a reduced Ki-67 expression in their breast epithelial cells at 6 months, with 9 of the 10 showing a reduction in Ki-67 of >50%. The preliminary results from this ongoing trial show that letrozole decreased proliferation in atypical breast epithelial cells in high-risk women who had an elevated Ki-67 even while on hormone replacement therapy.
Although some IEN treatments may induce apoptosis and eradicate IEN in a subpopulation of women, the available evidence suggests that modulation of proliferation, as evidenced by a decrease in Ki-67 in DCIS or hyperplasia with atypia, without evidence of apoptosis, is feasible and is a promising end point for future IEN treatment trials. Although it is possible that short-term treatment of IEN may eradicate high-risk subclones of IEN in some patients, as carcinogenesis is a multidecade process, prolonged treatment of breast IEN with safe and well-tolerated agents that can chronically suppress proliferation of breast IEN may be a realistic treatment strategy for the future.
The most important advances in the practical application of targeted chemoprevention have focused on the hormonal modulation of breast cancer development. ER has proven to be a primary target for the treatment of breast cancer and the most practical target for chemoprevention of breast cancer (191). Since the publication of the National Surgical Adjuvant Breast and Bowel Project Breast Cancer Prevention Trial P1 in 1998 (192, 193), tamoxifen received supplemental approval from the FDA for risk reduction in high-risk premenopausal and postmenopausal women. Extensive analysis of symptoms data from the National Surgical Adjuvant Breast and Bowel Project study has provided additional practical information for the clinical use of tamoxifen (192, 194–197). Because of concerns about the side effects of tamoxifen, only a small proportion of women eligible for risk reduction consider tamoxifen treatment (198). This led to the Study of Tamoxifen and Raloxifene as a second-generation chemoprevention study with SERMs.
Raloxifene is a SERM that was tested for the treatment and prevention of osteoporosis in the Multiple Outcomes of Raloxifene Trial. Multiple Outcomes of Raloxifene is a multicenter randomized blinded placebo-controlled study that recruited 7,705 women, ages 31 to 80 years in 25 countries, who had been postmenopausal for at least 2 years a
