Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium.

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays.

I-SPY 2: an adaptive breast cancer trial design in the setting of neoadjuvant chemotherapy.

I-SPY 2 (investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2) is a process targeting the rapid, focused clinical development of paired oncologic therapies and biomarkers. The framework is an adaptive phase II clinical trial design in the neoadjuvant setting for women with locally advanced breast cancer. I-SPY 2 is a collaborative effort among academic investigators, the National Cancer Institute, the US Food and Drug Administration, and the pharmaceutical and biotechnology industries under the auspices of the Foundation for the National Institutes of Health Biomarkers Consortium.

Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies.

In most prostate chemoprevention studies conducted with animal models, the incidence and multiplicity of tumours have been used as endpoints for efficacy. However, the latency of tumours is usually over 1 year, making these studies costly and time consuming. The main purpose of this study was to assess the utility of prostate intraepithelial neoplasia (PIN), induced in Noble rats by continuous testosterone + oestradiol (T + E) administration, as a potential intermediate endpoint biomarker of efficacy in chemoprevention studies. Noble rats at the age of 12 weeks were treated for 36 weeks with T + E given subcutaneously via Silastic capsules. The incidence and multiplicity of PIN were assessed in various prostate glands by serial sections generated at three separate tissue levels. The efficacy of dehydroepiandrosterone (DHEA) and DHEA 8354 (1000 and 2000 mg/kg diet), difluoromethylornithine (DFMO) (1000 and 2000 mg/kg diet) and oltipraz (125 and 250 mg/kg diet) to inhibit PIN was assessed in two independent sets of experiments. T + E induced multiple PIN in the dorsolateral prostate (DLP) of 80-100% of the animals. DHEA and DHEA 8354 did not affect the incidence but decreased the multiplicity of PIN in the DLP, from 3.2 +/- 1.0 in control group to 1.5 +/- 1.0 in the low-dose and to 1.6 +/- 0.6 in the high-dose group for DHEA (P<0.05 and P<0.02, respectively), and to 1.9 +/- 0.8 in the high-dose (P<0.05) DHEA 8354. Both agents did not affect PIN in anterior prostate, seminal vesicles or ventral prostate. In a second experiment, DFMO and oltipraz were found not effective in inhibiting PIN. In this study, we provide new evidence that PIN in Noble rats, induced by continuous T + E treatment, is a useful intermediate endpoint for determining the efficacy of DHEA and other potential chemopreventive agents. The hormonal pathogenesis, high multiplicity, short latency, preferential location in the DLP, similarity in morphology and biology to PIN of human prostate, and the sensitivity to agents that suppress prostate carcinogenesis, makes PIN in Noble rats a promising intermediate endpoint for chemoprevention studies.

Identification of retinamides that are more potent than N-(4-hydroxyphenyl)retinamide in inhibiting growth and inducing apoptosis of human head and neck and lung cancer cells.

The synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4HPR), which is currently being evaluated in clinical trials for cancer prevention and therapy, inhibits the growth of a variety of malignant cells through induction of apoptosis. However, in the majority of tumor cells, this inhibitory effect of 4HPR requires high concentrations (>1 microM), which exceed the peak plasma level measured in humans. In the present study, we compared and contrasted the effects of several synthetic retinamides on the growth of human lung and head and neck cancer cells in vitro. We found that some retinamides, especially N-(2-carboxyphenyl)retinamide (2CPR), exhibited better growth inhibitory effects than 4HPR in some of the cell lines. 2CPR exerted potent growth inhibitory effects in 5 of 10 head and neck cancer cell lines and in 1 of 10 lung cancer cell lines (IC(50), <0.8 microM). 2CPR (1 microM) induced apoptosis ranging from 10 to 60% in four of five cell lines, whereas 4HPR was ineffective at the same concentration. Unlike 4HPR, 2CPR (up to 10 microM) failed to induce reactive oxygen species production in these sensitive cell lines but could activate caspases 3 and 7 as well as increase poly(ADP-ribose)polymerase cleavage. Interestingly, the effect of 2CPR on cell growth could be suppressed by the specific retinoic acid receptor pan antagonist AGN193109. Our results suggest that 2CPR acts via retinoic acid receptors and may be a good candidate for prevention and treatment of some head and neck and lung cancers.

Effects of novel phenylretinamides on cell growth and apoptosis in bladder cancer.

Superficial bladder cancer is a major target for chemoprevention. Retinoids are important modulators of epithelial differentiation and proliferation and are effective in the treatment and prevention of several epithelial cancers. One class of compounds, the retinamides, is structurally similar to other retinoids but have the added feature of being potent apoptosis inducers. Among these, fenretinide (N-[4-hydroxyphenyl]retinamide), or 4HPR, has promise for bladder cancer chemoprevention and is currently under Phase III study in this setting. In addition to 4HPR, there are several new structurally related phenylretinamides bearing hydroxyl, carboxyl, or methoxyl residues on carbons 2, 3, and 4 of the terminal phenylamine ring [designated N-(2-hydroxyphenyl)retinamide, N-(3-hydroxyphenyl)retin amide, N-(2-carboxyphenyl)retin- amide, N-(3-carboxyphenyl)retin amide, N-(4-carboxy- phenyl)retinamide, and N-(4-methoxyphenyl)retinamide, respectively]. The objective of this study was to compare the growth inhibitory and apoptotic effects of these phenylretinamides with 4HPR in human bladder transitional cell cancer-derived cell lines of varying histological grade (RT4, grade 1; UM-UC9 and UM-UC10, grade 3; and UM-UC14, grade 4) by cell counting, cell cycle fluorescence-activated cell sorter analysis and a dual stain apoptosis assay. All of the seven phenylretinamides reduced cell number, altered the cell cycle distribution, and induced apoptosis when administered at a concentration of 10 microM, which is within the pharmacologically achievable range. Although the relative potencies of the phenylretinamides varied depending on the cell line, N-(3-hydroxy phenyl)retin- amide was the most active with significantly greater growth inhibition than 4HPR in all of the four cell lines. These in vitro findings warrant further study of these novel phenylretinamides, which may have potential as preventive or therapeutic agents in transitional cell cancer.

Chromosome 3p tumor-suppressor gene alterations in cervical carcinomas.

Loss of heterozygosity (LOH) on chromosome 3p is a common event in cervical cancer and typically occurs in a dispersed pattern involving several loci. This implies that more than one resident tumor-suppressor gene is involved in the genesis of these tumors; however, specific targets remain to be identified. The region of 3p14.2-pter encompasses a region of frequent loss and contains at least three tumor-suppressor genes: fragile histidine triad (FHIT), transforming growth factor-beta receptor II (T beta R-II), and Von Hippel-Lindau. To identify those loci within 3p14.2-pter that are important in cervical cancer, invasive tumors were first subjected to high-density LOH analysis. With 25 microsatellite markers, LOH was detected in seven of 15 cervical carcinomas (47%). Losses always included markers mapping to 3p22, and markers at this location were exclusively lost in two tumors, implicating this as a site of a cervical tumor-suppressor gene. Because it is a known tumor-suppressor gene located at 3p22 and thus a potential target for inactivation in these tumors, the T beta R-II gene was subsequently screened for mutation and altered expression levels. Whereas no tumor-derived mutations were detected in any of the tumors, six of ten tumors showed T beta R-II transcript levels reduced by > or = 50% when compared with normal cervical epithelium. Nine of 15 (60%) tumors exhibited LOH at 3p22 or reduced expression of T beta R-II, suggesting that reduced T beta R-II levels contribute to cervical tumorigenesis. Two cases exhibited silent germline polymorphisms of T beta R-II: one corresponding to a C1167T transversion and the other to an A1266G transition. The FHIT gene, which is located at 3p14.2, also frequently incurred LOH and abnormal transcription in these tumors. LOH of FHIT was observed in five of the 15 tumors analyzed. Neither mutations nor homozygous deletions of FHIT were detected in the tumors. However, aberrantly short transcripts of the FHIT gene were evident in six of nine (67%) tumors. Only one of these also displayed LOH, indicating that this gene was altered in at least 10 of 15 (67%) tumors. These results provide evidence that the inactivation of two known tumor-suppressor genes, TbetaR-II and FHIT, on chromosome 3p is involved in cervical carcinogenesis. Mol. Carcinog. 30:159--168, 2001.

Modulation of biomarkers by chemopreventive agents in smoke-exposed rats.

Chemoprevention opens new perspectives in the prevention of cancer and other chronic degenerative diseases associated with tobacco smoking, exploitable in current smokers and, even more, in exsmokers and passive smokers. Evaluation of biomarkers in animal models is an essential step for the preclinical assessment of efficacy and safety of potential chemopreventive agents. Groups of Sprague Dawley rats were exposed whole body to a mixture of mainstream and sidestream cigarette smoke for 28 consecutive days. Five chemopreventive agents were given either with drinking water (N-acetyl-L-cysteine, 1 g/kg body weight/day) or with the diet (1,2-dithiole-3-thione, 400 mg; Oltipraz, 400 mg; phenethyl isothiocyanate, 500 mg; and 5,6-benzoflavone, 500 mg/kg diet). The monitored biomarkers included: DNA adducts in bronchoalveolar lavage cells, tracheal epithelium, lung and heart; oxidative damage to pulmonary DNA; hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol-9,10-epoxide; micronucleated and polynucleated alveolar macrophages and micronucleated polychromatic erythrocytes in bone marrow. Exposure of rats to smoke resulted in dramatic alterations of all investigated parameters. N-Acetyl-L-cysteine, phenylethyl isothiocyanate, and 5,6-benzoflavone exerted a significant protective effect on all alterations. 1,2-Dithiole-3-thione was a less effective inhibitor and exhibited both a systemic toxicity and genotoxicity in alveolar macrophages, whereas its substituted analogue Oltipraz showed limited protective effects in this model. Interestingly, combination of N-acetyl-L-cysteine with Oltipraz was the most potent treatment, resulting in an additive or more than additive inhibition of smoke-related DNA adducts in the lung and hemoglobin adducts. These results provide evidence for the differential ability of test agents to modulate smoke-related biomarkers in the respiratory tract and other body compartments and highlight the potential advantages in combining chemopreventive agents working with distinctive mechanisms.

Quantitative nuclear morphometry by image analysis for prediction of recurrence of ductal carcinoma in situ of the breast.

Clinical management of ductal carcinoma in situ (DCIS) remains a challenge because significant proportions of patients experience recurrence after conservative surgical treatment. Unfortunately, it is difficult to prospectively identify, using objective criteria, patients who are at high risk of recurrence and might benefit from additional treatment. We conducted a multi-institutional, collaborative case-control study to identify nuclear morphometric features that would be useful for identifying women with DCIS at the highest risk of recurrence. Tissue sections of archival breast tissue of 29 women with recurrent and 73 matched women with nonrecurrent DCIS were stained for DNA, and nuclei in the DCIS lesions were evaluated by image analysis. A clear correlation between mean fractal2_area (FA2) and nuclear grade was observed (P < 0.001), allowing an objective determination of nuclear grade. Several nuclear morphometric features, including mean and variance of variation of radius, mean area, mean and variance of frequency of high boundary harmonics (FQH), and variance in sphericity, were found to be useful in discriminating recurrent from nonrecurrent DCIS subjects. However, the nuclear features associated with recurrence differed between high- and low-grade lesions. For lesions with high FA2 (nuclear grade 3), mean variation of radius, mean FQH, and mean area alone yielded recurrence odds ratios of 4.55 [95% confidence interval (CI) 0.45-45.96], 3.86 (95% CI, 0.88-16.98), 2.90 (95% CI, 0.31-27.2), respectively. Using a summed feature model, high-FA2 lesions showing three poor prognostic features had an odds ratio of 15.63 (95% CI, 1.22-200), compared with those with zero or one poor prognostic feature. Lesions with low mean FA2 (nuclear grade 1 or 2) showing high variances in sphericity and FQH had an odds ratio of 7.71 (95% CI, 1.77-33.60). Addition of other features did not enhance the odds ratio or its significance. These results suggest that nuclear image analysis of DCIS lesions may provide an adjunctive tool to conventional pathological analysis, both for the objective assessment of nuclear grade and for the identification of features that predict patient outcome.

Detection of differentially expressed genes in mouse lung adenocarcinomas.

Increasing evidence suggests that altered gene expression is associated with the induction and maintenance of malignancy in various organs including mouse lung adenocarcinomas. A competitive cDNA library screening (CCLS) was used to examine gene expression in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung adenocarcinomas from (C3H/HeJ x A/J])F1 mice. Comparisons of RNA expression in lung adenocarcinomas to those of normal surrounding lung tissue revealed altered expression in 220 clones from more than 50,000 clones screened. Fifty clones were selected for quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis to verify altered expression. PCR primers were designed based on partial sequence analysis of the clones. Twenty-two clones were found to be differentially expressed in lung adenocarcinomas compared with normal lungs. GenBank database analysis showed that 14 of the 22 clones were homologous with known genes, whereas 8 clones contained novel sequences. Thirteen clones were down regulated in tumors compared to normal lung tissues, and 9 were overexpressed. The clones underexpressed or absent include adipocyte p27, carbonic anhydrase III, carbonyl reductase, cytochrome CYP2E1, skelemin, myosin, major urinary protein, and contrapsin. Overexpressed clones include Bruton's tyrosine kinase, cyclin D3, poly(A)-binding protein, alpha-fetoprotein, transferrin, and mouse B2 family repetitive sequence. Further examination of biologic implications of the differentially expressed genes in lung adenocarcinomas is necessary to understand their role(s) in mouse lung carcinogenesis.

Agents, biomarkers, and cohorts for chemopreventive agent development in prostate cancer.

Chemoprevention is the use of agents to slow progression of, reverse, or inhibit carcinogenesis thereby lowering the risk of developing invasive or clinically significant disease. With its long latency, high incidence and significant morbidity and mortality, prostate cancer is a relevant target for chemoprevention. Developing rational chemopreventive strategies for prostate cancer requires well-characterized agents, suitable cohorts, and reliable intermediate biomarkers of cancer. Chemopreventive agent requirements are experimental or epidemiologic data showing efficacy, safety on chronic administration, and a mechanistic rationale for activity. Current promising agents include antiandrogens and antiestrogens; steroid aromatase inhibitors; retinoids and their modulators; 5alpha-reductase inhibitors; vitamins D, E, and analogs; selenium compounds; carotenoids; soy isoflavones; dehydroepiandrostenedione and analogs; 2-difluoromethylornithine; lipoxygenase inhibitors; apoptosis inducers; and nonsteroidal anti-inflammatory drugs. Identifying biomarkers and validating them as surrogate endpoints for cancer incidence are critical for prostate chemoprevention trials. Potentially useful biomarkers for prostate chemoprevention are associated with histologic, proliferative, differentiation-related, biochemical, and genetic/regulatory features of prostatic disease. In that the prostate is not easily visualized, critical issues also include adequacy and consistency of tissue sampling. Various drugs for the chemoprevention of prostate cancer are now under evaluation in phase 1, 2, and 3 clinical trials. Cohort selection should be based on various patient characteristics (stage of the disease, previous cancers or premalignant lesions, or high risk factors) and should be conducted within the context of standard treatment.

Bayesian monitoring of a phase 2 chemoprevention trial in high-risk cohorts for prostate cancer.

The objective of phase 2 cancer chemoprevention trials is to evaluate whether a chemopreventive agent will cause significant modulation of intermediate endpoint biomarkers (IEB) in patients at high risk for the disease. A phase 2 chemoprevention trial of 4-hydroxyphenyl retinamide (4-HPR) versus placebo was conducted in men with a histologic diagnosis of early prostate cancer and scheduled to have radical prostatectomy. A Bayesian monitoring method was used to sequentially monitor this trial for evidence of biological activity or ineffectiveness based on a single IEB variable. Different prior distributions were used and posterior distributions were obtained to calculate the probability that treatment differences are greater than or less than a predetermined clinically significant effect. The interim analysis of transforming growth factor-alpha expression indicated a high probability of insufficient biological activity of 4-HPR on this IEB. This study demonstrates the potential utility of Bayesian methods in the decision-making process in the conduct of phase 2 chemoprevention trials.

Modulation of apoptosis by cigarette smoke and cancer chemopreventive agents in the respiratory tract of rats.

Preclinical studies may elucidate the meaning of biomarkers applicable to epidemiologic studies and to clinical trials for cancer prevention. No study has explored so far the effect of cigarette smoke on apoptosis in vivo. We evaluated modulation of apoptosis in cells of the respiratory tract of smoke-exposed Sprague-Dawley rats both by morphological analysis and TUNEL method. In a first study, exposure of rats to mainstream cigarette smoke for either 18 or 100 consecutive days produced a significant and time-dependent increase in the proportion of apoptotic cells in the bronchial and bronchiolar epithelium. Oral N:-acetylcysteine did not affect the background frequency of apoptosis but significantly and sharply decreased smoke-induced apoptosis. In a second study, exposure of rats to a mixture of sidestream and mainstream smoke for 28 consecutive days resulted in a >10-fold increase in the frequency of pulmonary alveolar macrophages undergoing apoptosis. Dietary administration of either 5,6-benzoflavone, 1,2-dithiole-3-thione or oltipraz did not affect the frequency of smoke-induced apoptosis, whereas phenethyl isothiocyanate produced a further significant enhancement. Again, N-acetylcysteine and its combination with oltipraz significantly decreased smoke-induced apoptosis. In both studies exposure to smoke resulted in a sharp increase of cells positive for proliferating cell nuclear antigen (PCNA), which was unaffected by the examined chemopreventive agents. These findings highlight the concept that modulation of apoptosis has diversified meanings. Different meanings (as explained in the following lines). First, the apoptotic process is triggered as a defense system against genotoxic agents, such as the components of cigarette smoke. The further induction produced by phenethyl isothiocyanate, favoring removal of damaged cells, represents an example of a detoxification mechanism. Inhibition of smoke-induced apoptosis by N:-acetylcysteine should be interpreted as an epiphenomenon of antigenotoxic mechanisms, as shown in parallel studies evaluating modulation of DNA alterations in the respiratory tract of the same animals. Thus, it is important to discriminate between whether the opposite modulation of apoptosis is per se a protective mechanism or the beneficial outcome of other mechanisms inhibiting genotoxicity.

Effects of acute and chronic body weight gain reductions in the evaluation of agents for efficacy in mammary cancer prevention.

Studies were performed to determine the effects of moderate decreases in body weight gain on mammary carcinogenesis. The levels of depressions in weight gain were those often observed in the evaluation of chemopreventive agents. In the first experiment, the effects of acute and chronic reductions of body weight gain when started after carcinogen treatment were examined in young rats (MNU at 50 days of age). Significant decreases (36%) in mammary cancers occurred in groups of rats that underwent a 12% acute reduction in body weight gain as compared with ad libitum controls. In contrast, chronic weight reductions of up to 12% had minimal effects on cancer multiplicities, while a 15% chronic reduction significantly decreased cancer numbers (26%). A second experiment evaluated the efficacy of toremifene (7.0 mg/kg diet), an estrogen/anti-estrogen, and the effect of toremifene-matched body weight gain reduction that occurred during the study. Toremifene caused a chronic reduction in body weight that resulted in a 10% decrease in final body weight at the end of the study. While toremifene-treated rats exhibited a 67% decrease in the number of mammary cancers, the rats which similarly exhibited a 10% decrease in final body weight showed only a 14% decrease in cancer number. Thus, the weight effects observed with toremifene, similar estrogens/anti-estrogens, and other classes of chemopreventive compounds (where chronic body weight reductions are 10% or less) imply that the body weight reduction has a limited effect on overall chemopreventive activity. A third study examined the effect of chronic body weight gain reduction on mammary cancers induced in older rats (MNU given at 100 days of age). This model more closely resembles the status of the breast tissue of mature women currently enrolled in clinical trials of chemopreventive agents. Under these conditions chronic reductions in body weight up to 15% had minimal effects on mammary carcinogenesis. These data further demonstrated that acute body weight reductions in young rats at the time of carcinogen treatment can be a concern in interpretation of the chemopreventive activity of an agent, but that moderate chronic depressions of body weight gain probably do not play a significant role.

Pilot studies evaluating the lung tumor yield in cigarette smoke-exposed mice.

In spite of the major role played by cigarette smoking in the epidemiology of lung cancer, it is very difficult to reproduce the carcinogenicity of this complex mixture in animal models. We implemented a series of pilot experiments in three mouse strains, exposed either to environmental cigarette smoke (ECS) or mainstream cigarette smoke (MCS) or its condensate (MCSC). The whole-body exposure of Aroclor-treated A/J mice to ECS resulted in a rapid and potent induction of micronuclei in peripheral blood erythrocytes. After 6 months of exposure, 6 h a day, followed by 4 months of recovery in filtered air, both lung tumor incidence and multiplicity were significantly increased as compared to sham-exposed mice (77.8% vs. 22.2%, and 1.11+/-0.26 vs. 0.22+/-0.15, means +/- SE). Multiple i.p. injections of butylated hydroxytoluene did not significantly enhance the tumor yield. Another experiment confirmed the responsiveness of A/J mice exposed to ECS for 5 months, followed by 4 months of recovery in air (75.0% vs. 25.0%, and 1.05+/-0.17 vs. 0.25+/-0.10). In contrast, the increase in lung tumor yield after exposure to ECS for 2 months, followed by recovery in air for 7 months, was not significant, and the continuous exposure to ECS for 9 months was totally ineffective. These data, in agreement with previous results of others, show that exposure of A/J mice to ECS for 5-6 months, followed by recovery in air for 4 months, is successful in inducing a weak but significant and reproducible increase in lung tumor yield. Furthermore, the simultaneous exposure to the light emitted by halogen quartz bulbs for 9 months and to ECS for 5 months, followed by 4 months in air, was again weakly tumorigenic (incidence of 55.0% and multiplicity of 0.75+/-0.19), whereas exposure to both ECS and light for 9 months was devoid of effect. The whole-body exposure of A/J mice to MCS, 1 h a day for 5 months, or weekly i.p. injections of MCSC for 5 months, followed in both cases by 4 months of recovery in air, failed to enhance the lung tumor yield. The whole-body exposure of SKH-1 hairless mice to ECS for 6 months, followed by exposure to halogen light for 8 months, resulted in the formation of multiple skin tumors but failed to produce lung tumors. The whole-body exposure of C57BL/6 mice to ECS for 6 months failed to induce any lung tumor but caused alopecia, gray hair, and hair bulb cell apoptosis, which were prevented by the oral administration of N-acetylcysteine.

Surrogate end-point biomarkers in chemopreventive drug development.

Relevant and feasible surrogate end-points are needed for the evaluation of intervention strategies against cancer and other chronic, life-threatening diseases. Carcinogenesis can be viewed as a process of progressive disorganization. This process is characterized by the accumulation of genotypic lesions and corresponding tissue and cellular abnormalities, including loss of proliferation and apoptosis controls. Potential surrogate end-points for cancer incidence include both phenotypic and genotypic biomarkers of this progression. In the US National Cancer Institute chemoprevention programme, histological modulation of a precancer (intraepithelial neoplasia) has so far been the primary phenotypic surrogate end-point in chemoprevention trials. Additionally, high priority has been given to biomarkers measuring specific and general genotypic changes correlated with the carcinogenesis progression model for the targeted cancer (e.g., progressive genomic instability as measured by loss of heterozygosity or amplification at specific microsatellite loci). Other potential surrogate end-points include proliferation and differentiation indices, specific gene and general chromosome damage, cell growth regulatory molecules, and biochemical activities (e.g., enzyme inhibition). Serum biomarkers thought to be associated with cancer progression (e.g., prostate-specific antigen) are particularly appealing surrogate end-points because of accessibility. Potentially chemopreventive effects of the test agent may also be measured (e.g., tissue and serum estrogen levels in studies of steroid aromatase inhibitors). To establish chemopreventive efficacy, prevention of virtually all biomarker lesions, or of those lesions with particular propensity for progression, may be required. Ideally, the phenotype and genotype of any new or remaining precancers in the target tissue of chemopreventive agent-treated subjects would show less, and certainly no greater, potential for progression than those of placebo-treated subjects.

Modulation of Ki67, p53 and RARbeta expression in normal, premalignant and malignant human oral epithelial cells by chemopreventive agents.

BACKGROUND: Aberrant expression of Ki67, p53 and RARbeta are characteristic of many tumor types including those of the oral cavity. Chemopreventive agents may act by modulating their expression to more normal levels. MATERIALS AND METHODS: The effects of 21 chemopreventive agents on the expression of Ki67, p53 and RARbeta were determined using a human in vitro model of normal, premalignant and malignant oral epithelial cell lines. RESULTS: Ki67 and mutant p53 (mtp53) were overexpressed in both the premalignant and malignant cell lines, whereas expression of RARbeta was high in the normal, low in the premalignant and not detectable in the malignant cell lines. Most of the agents selectively inhibited the expression of Ki67 in the premalignant and malignant cell lines. Eight of the 21 agents increased, while four agents decreased, the levels of mtp53 protein in the premalignant cell line. In the malignant cell line, five of the agents increased, while ten agents decreased mtp53 protein levels. The agents increased RARbeta expression to near normal levels in the premalignant cell line. CONCLUSION: The data suggest that the suppression of Ki67 and mtp53 are good indicators of the effectiveness of agents in premalignant and malignant oral cells, whereas the enhancement of RARbeta is a measure of effectiveness in premalignant oral cells.

A quantitative angiogenesis model for efficacy testing of chemopreventive agents.

One of the approaches in chemoprevention to prevent or delay the progression of precancerous lesions, is to apply chemopreventive agents that can potentially block angiogenesis. A quantitative in vivo angiogenesis inhibition assay was developed to test the efficacy of twelve chemopreventive agents that represent different chemical classes and multiple biological activities, using the chick chorioallantoic membrane (CAM) model and an oncogene-transfected angiogenic cell line (6 Ti ras/SV myc # 4). These tumorigenic cells held by a primary agarose pellet, were placed alone or with a secondary pellet incorporating five concentrations of the test agent, on an exposed CAM of 7-day-old chick embryo for 72 hours in a humidified chamber at 35 degrees C. The cell-induced angiogenic blood vessels, including the microvessels radiating from the cell pellet focal area, were scored using a computerized custom image analysis system. The results show that nonsteroidal antiinflammatory drugs (NSAIDS); aspirin, sulindac, sulindac sulfide and sulindac sulfone, were effective inhibitors of cell-induced angiogenesis (23-66%). Aspirin displayed a dose-dependent response with the highest inhibition at 300 microM and an EC50 (the effective molar concentration that inhibits angiogenesis by 50%) of 26 microM. Sulindac sulfone was more effective than sulindac with an EC50 of 5 microM versus 85 microM. However, sulindac sulfide showed an intermediate response with an EC50 of 41 microM. The retinoids; all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9-cis-RA), and 13-cis-retinoic acid (13-cis-RA) were also highly effective inhibitors of cell-mediated CAM-angiogenesis. 13-cis-RA with an EC50 of 3.6 nM, has been the most efficacious test agent. > 400-fold more effective than 9-cis-RA (1.5 microM). ATRA exhibited an intermediate response between 9-cis-RA and 13-cis-RA with an EC50 of 0.3 microM, and was 100-fold more efficacious than 9-cis-RA. However, the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR), was not an effective inhibitor of CAM angiogenesis. Thalidomide, a compound with multiple biological activities, exhibited dose-dependent inhibition ranging from 10-1000 microM with an EC50 of 19 microM. Other agents that exhibited dose-dependent inhibition included Bowman-Birk inhibitor (BBI), EC50: 10 microg/ml, tamoxifen, EC50, 0.05 microM and difluoromethyl omithine (DFMO), with an EC50 of 13 microM. These results suggest that tumor-associated angiogenesis can be modulated by non-toxic concentrations of chemopreventive agents representing multiple biological activities and multiple targets.

Chemoprevention with theaflavins of rat esophageal intraepithelial neoplasia quantitatively monitored by image tile analysis.

The objective of the study was to compare three methods of monitoring the inhibition by dietary theaflavins of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal intraepithelial neoplasia: the mean tile grade, measured by computer-assisted quantitative image tile analysis; tumor multiplicity; and mean tumor size. A "tile" is defined as a small portion of a microscopic image at x 40, 87 x 292 microm in size. The computer divided the image of esophageal intraepithelial neoplasia into a grid of contiguous tiles and measured four tissue features within each tile based on cytonuclear and tissue architectural changes used by pathologists to diagnose intraepithelial neoplasia. The tile grade is defined as the weighted sum of the four feature measurements within a tile, the weights being determined by Fisher linear discriminant analysis. The mean tile grade of 300 tiles is used to grade rat esophageal intraepithelial neoplasia. NMBA was given s.c., 0.5 mg/kg, three times a week for 5 weeks. Theaflavins were given in the drinking water at 360 ppm (low dose) and 1200 ppm (high dose) throughout the experiment. In a given set of four groups of rats, one group received theaflavins alone, one NMBA alone, one NMBA plus low-dose theaflavins, and one NMBA plus high-dose theaflavins. One set of four groups, four rats/group, was sacrificed at the 15th week and another at the 20th week after starting NMBA; a final set with 15 rats/group was sacrificed at 25 weeks. At the 15th and 20th weeks, the mean tumor grade was the only variable that responded significantly (P < 0.01) to the low dose of dietary theaflavins. In fact, tumor multiplicity and mean tumor size sometimes showed enhancement at these doses. At the 25th week, when there were 15 instead of 4 rats/group, the mean tile grade, tumor multiplicity, and mean tumor size were all significantly (P < 0.01) decreased by both low and high doses of theaflavins. The mean tile grade is a more sensitive and reproducible variable than tumor multiplicity and mean tumor size in detecting the chemopreventive effects of theaflavins on intraepithelial neoplasia in the rat esophagus. This suggests that the mean tile grade may be a useful intermediate end point for use in human chemoprevention trials.

Celecoxib inhibits N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder cancers in male B6D2F1 mice and female Fischer-344 rats.

Epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) may have a role in the prevention of human cancers. A number of preclinical studies have also suggested that inhibition of cyclooxygenase (COX) with NSAIDs has an anticancer effect in animal models of colon, urinary bladder, skin, and breast. In these studies, we evaluated the COX-2 inhibitor celecoxib in two rodent models of urinary bladder cancer. Male B6D2F1 mice treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) developed transitional and squamous cell urinary bladder cancers, many of which grew rapidly and caused substantial morbidity that required sacrifice of the mice. Groups of mice received various daily doses of celecoxib in the diet (1250, 500, or 200 mg/kg of diet) beginning 7 days before the initiation of 12 weekly doses of OH-BBN. Mice were checked weekly for the presence of palpable urinary bladder masses. The study was terminated at 8 months following the initial treatment with OH-BBN. The percentage of mice with large palpable bladder lesions, which necessitated sacrifice of the mice, was 40% in the OH-BBN control group. In contrast, only 10% of all celecoxib-treated mice required sacrifice before the scheduled termination of the experiment, implying that all three doses of celecoxib inhibited the formation of large palpable lesions. Celecoxib did not significantly alter the incidence of preneoplastic bladder lesions, but did dose-dependently decrease the total number of urinary bladder cancers/mouse, palpable plus microscopic, by 77, 57, and 43% at dosages of 1250, 500, and 200 mg of celecoxib/kg of diet, respectively. In the second model, female Fischer-344 rats were administered OH-BBN twice/week for a period of 8 weeks. After 8 months, all rats developed preneoplastic lesions, whereas roughly 60% of the rats developed relatively small urinary bladder cancers. Rats were treated continually with celecoxib in the diet (500 or 1000 mg/kg of diet) beginning either 1 week prior to the initial OH-BBN treatment or beginning 1 week following the last OH-BBN treatment. Neither celecoxib treatment regimen significantly altered the number of preneoplastic lesions. Whereas celecoxib treatment initiated prior to OH-BBN administration decreased cancer incidence roughly 65%, celecoxib treatment initiated beginning 1 week after the last dose of OH-BBN profoundly decreased cancer incidence (>95%). Celecoxib did not alter the body weights of the mice or rats, or cause other signs of toxicity at any of the doses studied. Taken together these results demonstrate that: (a) celecoxib effectively inhibits tumor growth and enhances survival in the mouse model of urinary bladder cancer; and (b) celecoxib profoundly inhibits development of urinary bladder cancers in the rat model even when administered following the last dose of OH-BBN. Clinical trials will be necessary to determine whether COX-2 inhibitors will provide a clinical benefit in human bladder cancer.