Endpoints In Chemoprevention Trials Research Articles

Abstract 6333: Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue

Abstract Arachidonic acid (AA) metabolizing enzymes, including cyclooxygenases (COX), lipoxygenases, and cytochrome P450s (CYP450), are expressed in breast cancer (BC). Elevated levels of metabolites of COX-derived, pro-inflammatory prostaglandin (PGs) metabolites in urine have been linked to increased risk of developing breast cancer (BC) in two large prospective cohorts. Further, CYP450-derived EETs in BC tumors are associated with metastasis. We hypothesize that measuring levels of AA-derived oxylipins in breast tissues could enable improved BC risk prediction, provide insights into pro-carcinogenic inflammatory mechanisms, and serve as potential surrogate endpoints in chemoprevention trials. Thus, we performed a pilot study to assess the feasibility of measuring these analytes in small breast tissue samples. We analyzed two sample sets, collected under Institutional Review Board (IRB) approved protocols: 50 normal breast tissue cores donated to the Komen Tissue Bank (KTB) via vacuum-assisted biopsy and 10 tissues removed surgically at Mayo Clinic beyond resection margins of lumpectomy samples performed for atypical ductal hyperplasia (ADH). Tissues were frozen and maintained at -80°C. Normal KTB tissue cores were split in half. Tissues for analysis were weighed, extracted in cold methanol containing a cyclooxygenase inhibitor, and assayed for a panel of 42 oxylipins using validated mass spectrometric methods with a limit of detection per analyte of 0.010 ng/mL in supernatant and CVs of approximately <11%. Analyte measurements in paired cores (ng/g tissue) were compared with Spearman correlations and intra-class correlation coefficients. Measurements in surgical samples were assessed descriptively and levels were compared with normal KTB breast tissues.Median weight of the 50 split KTB cores was 0.055 grams. 17 oxylipins were detected in the tissue samples. PGD2 demonstrated the weakest correlation between split cores (r=0.12; p=0.40), whereas 15-HETE demonstrated the strongest correlation (r=0.68; p<0.0001). ICCs ranged from non-significant to nearly perfect, with highest values for 12-HETE (ICC=0.98, 95%CI: 0.97-0.99). Exploratory analyses of the KTB cores demonstrated significant associations between two BC risk factors, alcohol use and family history of breast/ovarian cancer, and several analytes. Levels of most eicosanoids tested were substantially and highly significantly elevated in tissues surrounding ADH versus KTB normal breast tissues. We conclude that eicosanoids can be readily measured in small frozen breast tissue samples obtained via percutaneous biopsy or at surgery. Future studies to assess the associations of eicosanoid levels with BC risk factors and development of BC after a benign biopsy may inform risk prediction and potentially inhibitable inflammatory mechanisms of breast carcinogenesis. Citation Format: Ginger L. Milne, Robert A. Vierkant, Amy C. Degnim, Anna Maria Storniolo, Jill E. Henry, Laura M. Pacheco-Spann, Derek C. Radisky, Mark E. Sherman. Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6333.

Short- and long-term lung cancer risk associated with noncalcified nodules observed on low-dose CT.

Chemoprevention is an important potential tool in reducing lung cancer incidence. Noncalcified nodules (NCN) observed on low-dose computed tomography (LDCT) have been proposed as intermediate endpoints in chemoprevention trials, but whether NCNs represent cancer precursors is unclear. We analyzed data from subjects in the LDCT arm of the National Lung Screening Trial (NLST) to examine short- and long-term lung cancer risks associated with NCNs and to elucidate whether some NCNs may be cancer precursors. NLST subjects received a baseline and two additional LDCT screens and were followed for a median of 6.5 years. We examined lung cancer incidence over three distinct periods from baseline-0-23 months (short-term), 24-59 months (medium-term), and 60-84 months (long-term)-in relation to baseline NCN characteristics. Spatially, lung cancer incidence was analyzed at the person, lung, and lobe levels relative to NCN location. A total of 26,272 subjects received the baseline LDCT screen, with 468, 413, and 190 lung cancers observed in the three periods. The presence of an NCN gave significantly elevated long-term lung cancer risk ratios (RR) of 1.8, 2.4, and 3.5 at the person, lung, and lobe levels; corresponding short-term RRs were 10.3, 16.8, and 38.0. Ground-glass attenuation was positively associated with long-term lung cancer risk but inversely associated with short-term risk; NCN size was positively associated with short-term risk but not significantly associated with long-term risk. That NCNs convey significantly elevated excess long-term of lung cancer lends evidence to the hypothesis that some NCNs may be cancer precursors.

Abstract B38: Validation of a nuclear morphometric signature for prostate cancer risk in benign biopsies

Abstract Purpose: Subtle changes in nuclear shape, size and texture precede the histological recognition of prostate cancer (PCa) and thus might provide a useful biomarker for high-risk benign tissue. The objective of this study was to develop and validate a multifeature nuclear score based on direct DNA staining and test its association with field effects and the subsequent detection of PCa in benign biopsies. Methods: Whole tissue sections from 39 radical prostatectomy subjects were Feulgen-stained and scanned (400x) on a digital microscope, allowing maps of DNA content per nuclear pixel to be generated in Matlab@. Samples of PCa and benign epithelial nuclei were randomly selected for measurement of 51 morphometric features. Logistic regression models for discriminating benign from PCa nuclei were built and cross-validated by AUC analysis. Subject-level models, for discriminating benign from malignant nuclear populations taken from individual subjects, were also built and similarly validated. Both backwards elimination and all-subsets approaches were used to select covariates. Nuclear populations were randomly collected < 1mm or > 5mm from cancer foci, and from cancer-free prostates, HGPIN, and PCa Gleason grade 3-5. In addition, nuclear images were collected from negative biopsy cases and controls matched on age and date of biopsy (20 pairs); cases had PCa detected at least 2 years later, controls remained cancer-free and had at least 2 additional negative biopsies. For the case-control study, nuclei were selected using an automatic process that correlated well with results from manual selection. Frequency distributions for multifeature nuclear score from various tissue compartments were compared using the Kolmogorov D statistic. Scores for cases vs. controls were compared using a t test for paired data. Results: In prostatectomy samples, both nuclear- and populationlevel models revealed cancer-like features in benign nuclei adjacent to PCa, compared to nuclei that were more distant or were obtained from PCa-free glands. The score distribution for distant nuclei was also significantly shifted compared to nuclei from cancer-free glands. Interestingly, the best model by leave-one-out cross-validation (AUC = 0.91, 95% CI: 0.81-1.00) included only 5 variance features, reflecting the importance of nuclear pleomorphism. In negative biopsies, a validated model with 5 variance features (including features related to DNA content, size and texture) yielded significantly higher scores in cases than controls (P=0.026). This model had an AUC = 0.71 (95% CI: 0.54-0.87) for discriminating cases from controls. Conclusions: A multifeature nuclear morphometric score, obtained by automated digital analysis, was validated for discrimination of benign from cancer nuclei. This score demonstrated field effects in benign epithelial nuclei at varying distance from PCa lesions, and was associated with subsequent PCa detection in negative biopsies. This biomarker shows definite promise as an intermediate endpoint in chemoprevention trials; however, its utility as an independent predictor of PCa in the clinical setting requires much further study. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B38.

Comment re: “Sporadic Aberrant Crypt Foci Are Not a Surrogate Endpoint for Colorectal Adenoma Prevention” and “Aberrant Crypt Foci in the Adenoma Prevention with Celecoxib Trial”

To the Editor: We believe that the enthusiasm of Lance and Hamilton (1) in embracing the study by Cho et al. (2) as “seminal” and their finality in dismissing aberrant crypt foci (ACF) as a surrogate end point for chemoprevention trials are misplaced. We are concerned that the Perspective of Lance and Hamilton (1) may have overlooked important evidence in arriving at these conclusions, which the authors state were based on their “diverse perspectives of gastroenterology and clinical chemoprevention and of gastrointestinal and molecular pathology.”ACF in the colon have been the focus of many mechanistic and preclinical, and a few epidemiologic, studies (3) since their initial description (4). Given the potential value of ACF as an early and readily identifiable change that might indicate colorectal cancer risk or response to preventive measures, the specifics of the Lance and Hamilton Perspective require close scrutiny.The authors' statement (regarding the first description of ACF in humans) that “at least one ACF was found in each left colon of the 13 autopsy patients without colorectal cancer” (1) is factually incorrect. Table 1 of the original article (5) clearly states that 13 patients had “left colon from autopsy,” only 1 of whom had ACF (one in this case). More important, two key references that very strongly link human ACF to colon tumorigenesis (6, 7) were omitted from the Perspective. In the first, all human ACF were monoclonal proliferations (i.e., the earliest identified neoplastic lesions in the colon; ref. 6). In the second, it seems that Wnt signaling was constitutively active in more than 90% of human ACF through promoter methylation of the genes for secreted frizzled-related protein 1 or 2 (7). In addition, a number of recent molecular and genetic analyses have shown that ACF of each histologic subtype have characteristic aberrations found in colorectal cancer (e.g., ref. 8).There is a long, impressive history of preclinical animal studies showing that ACF suppression by a wide variety of dietary factors is associated with reduced cancer incidence (9).11http://www.inra.fr/reseau-nacre/sci-memb/corpet/indexan.html In rodent studies, larger ACF and/or crypt multiplicity often correlates with tumors better than does the number of ACF (10).The clinical evidence is provocative but limited (10) and the Cho et al. study (2) is the first and only prospective randomized trial. Although innovative, it alone cannot be taken as strong evidence against human ACF as a suitable surrogate end point. The study was based on only 45 patients (of whom 10 were regular aspirin users), who were evaluated at five different sites with five different investigators. These investigators found that “the mean number of ACF at baseline was… not different between those with and without advanced adenomas,” in contrast to several other reports (10). Most important, the 45 patients in the Cho et al. study did not provide evidence that celecoxib reduced the incidence of adenomas: Adenoma detection was 37.5% in the placebo group and 45.7% in the celecoxib group at “year 1 and/or year 3.” If this study did not have the statistical power to detect a protective effect of celecoxib, it certainly did not have the power to make any definitive conclusion about ACF as a surrogate end point.We view the Perspective by Lance and Hamilton (1) as an example of “jury nullification.” They present the evidence with clarity and scholarship, but then render a verdict that is strikingly inconsistent with that evidence. Our position is not that ACF should now be accepted as a surrogate end point for human chemoprevention trials, but rather that the existing direct evidence is woefully inadequate to render a verdict at this time. In other words, more research is needed.No potential conflicts of interest were disclosed.

Randomized, Placebo-Controlled, Esophageal Squamous Cell Cancer Chemoprevention Trial of Selenomethionine and Celecoxib

Esophageal squamous cell carcinoma remains a leading cause of cancer death worldwide. Squamous dysplasia, the accepted histological precursor for esophageal squamous cell carcinoma, represents a potentially modifiable intermediate end point for chemoprevention trials in high-risk populations. We conducted a randomized, controlled trial of selenomethionine 200 microg daily and/or celecoxib 200 mg twice daily (2 x 2 factorial design) among residents of Linxian, People's Republic of China. Subjects had histologically confirmed mild or moderate esophageal squamous dysplasia at baseline. Esophagogastroduodenoscopy was performed before and after a 10-month intervention. Per-subject change (regression, stable, or progression) in the worst dysplasia grade was defined as the primary end point. Results were compared by agent group (selenomethionine vs placebo; celecoxib vs placebo). Two hundred sixty-seven subjects fulfilled all eligibility criteria, and 238 (89%) completed the trial. Overall, selenomethionine resulted in a trend toward increased dysplasia regression (43% vs 32%) and decreased dysplasia progression (14% vs 19%) compared with no selenomethionine (P = .08). In unplanned stratified analyses, selenomethionine favorably affected a change in dysplasia grade among 115 subjects with mild esophageal squamous dysplasia at baseline (P = .02), but not among 123 subjects with moderate esophageal squamous dysplasia at baseline (P = 1.00). Celecoxib status did not influence changes in dysplasia grade overall (P = .78) or by baseline histology subgroup. After a 10-month intervention, neither selenomethionine nor celecoxib inhibited esophageal squamous carcinogenesis for all high-risk subjects. However, among subjects with mild esophageal squamous dysplasia at baseline, selenomethionine did have a protective effect. Although it is based on unplanned stratified analyses, this finding is the first report of a possible beneficial effect for any candidate esophageal squamous cell carcinoma chemopreventive agent in a randomized controlled trial.

E28. Chemoprevention of lung cancer: Intermediate endpoints for chemoprevention trials

E28. Chemoprevention of lung cancer: Intermediate endpoints for chemoprevention trials

Biomarkers of esophageal adenocarcinoma and Barrett's esophagus.

The rising incidence and poor prognosis of esophageal adenocarcinoma in the Western world have intensified research efforts into earlier methods of detection of this disease and its relationship to Barrett's esophagus. The progression of Barrett's esophagus to adenocarcinoma has been the focus of particular scrutiny, and a number of potential tissue and serum-based disease biomarkers have emerged. The epidemiology and pathogenesis of esophageal adenocarcinoma are outlined. Tissue biomarkers allowing risk stratification of Barrett's are reviewed as well as strategies currently being used to discover novel biomarkers that will facilitate the early detection of esophageal adenocarcinoma. Finally, the uses of biomarkers as predictive tests for targeted treatments and as surrogate endpoints in chemoprevention trials are considered.

Immunohistochemical Expression of Molecular Markers in an Avian Model: A Potential Model for Preclinical Evaluation of Agents for Ovarian Cancer Chemoprevention

Objective. A significant obstacle confronting the evaluation of potential chemopreventive compounds in ovarian carcinoma is the absence of an animal model of spontaneous ovarian carcinogenesis. A potential model of adenocarcinoma has been described in the laying hen (Gallus domesticus). The purpose of this study was to evaluate the immunohistochemical expression of available antibodies that have been utilized in chemoprevention studies in this potential model of epithelial carcinoma.Methods. Two hundred 2-year-old hens were sacrificed at Auburn University in accordance with IUACUC guidelines. Of these hens, 8 animals were thought grossly to have ovarian carcinoma and ascites. The tumors from these 8 hens were fixed in neutral-buffered formalin and processed to paraffin blocks. Hematoxylin and eosin stains were used to document the histologic presence of adenocarcinoma. Immunohistochemical evaluation for expression of antigen was performed using the following antibodies: CA125, CEA, cytokeratin, EGFR, erbB-2, Ki-67, Lewis Y, p27, PCNA, Tag 72, TGF-α, Muc 1, and Muc 2.Results. Upon microscopic examination by a pathologist eight specimens were documented as adenocarcinomas. Several antibodies to antigens that are frequently expressed in human ovarian cancer were cross-reactive in the laying hen. Of these, cytokeratin AE1/AE3, pan cytokeratin, EGFR, Lewis Y, CEA, Tag 72, and erbB-2 stained the chicken carcinomas. EGFR and p185erbB-2 stained diffusely, and cytokeratin AE1/AE3, pan cytokeratin, Lewis Y, CEA, and Tag 72 were focally positive in the tumor. The aforementioned antibodies which have been useful as surrogate endpoints in chemoprevention trials and which also stained the chicken carcinomas included PCNA, p27, and TGF-α Antibodies that were not cross-reactive include CA 125, Ki-67, Muc 1, and Muc 2.Conclusion. The data presented in this pilot study support the potential utility of an avian model of spontaneously arising adenocarcinoma in which to study chemopreventive agents. More importantly, the influence of chemoprevention protocols on the expression of relevant antigens can be determined using available antibodies that are cross-reactive in this model. Thus, changes in the phenotypic expression of surrogate endpoint biomarkers as identified by cross-reactive antibodies can aid in the development of chemoprevention trials for human ovarian cancer.

Magnetic resonance imaging and spectroscopic imaging: Improved patient selection and potential for metabolic intermediate endpoints in prostate cancer chemoprevention trials

In the design of prostate cancer chemoprevention trials there is a clear need for improved patient selection and risk stratification, as well as the use of biomarkers that could provide earlier assessment of therapeutic efficacy. Studies in preprostatectomy patients have indicated that the metabolic information provided by 3-dimensional magnetic resonance spectroscopic imaging (3D-MRSI) combined with the morphologic information provided by magnetic resonance imaging (MRI) can improve the assessment of cancer location and extent within the prostate, extracapsular spread, and cancer aggressiveness. Additionally, pre- and posttherapy studies have demonstrated the potential of MRI/3D-MRSI to provide a direct measure of the presence and spatial extent of prostate cancer after therapy, a measure of the time course of response, and information concerning the mechanism of therapeutic response. These studies suggest that the addition of MRI/3D-MRSI data to prostate-specific antigen and biopsy data may improve patient selection and risk stratification for chemoprevention trials, improve tissue sampling for ex vivo molecular marker analysis, and provide shorter-term endpoints in chemoprevention trials. However, future studies are necessary to establish the ability of MRI/3D-MRSI to accurately assess patients with premalignant or very early malignant changes, to validate metabolic markers as intermediate endpoints in chemoprevention trials, and to correlate metabolic endpoints with other promising intermediate biomarkers.

Biomolecular markers as intermediate end points in chemoprevention trials of upper aerodigestive tract cancer.

Head-and-neck squamous-cell cancer (HNSCC) is an important public-health problem, accounting for approximately 40,300 new cancer cases and 12,000 cancer deaths annually in the United States (Greenlee et al., [2000]). Patients with early-stage disease are often cured with surgery or radiotherapy but are at high risk for second primary tumor (SPT) development (Lippman and Hong, [1989]), and the majority of patients present with advanced disease, for which the outcomes have not markedly improved despite advances in combined-modality therapy (Vokes et al., [1993]). HNSCC arises from transformation of the genetic material of normal cells, followed by successive genetic alterations in a multistep fashion, leading to clonal evolution of progeny cells with a proliferative advantage (Vogelstein and Kinzler, [1993]), induced by tobacco carcinogens (Slaughter et al., [1953]). Chemoprevention aims at reversal of this process through re-regulation of growth and differentiation and possibly elimination of genetically and phenotypically aberrant clones. Chemoprevention studies in upper aerodigestive tract (UADT) cancers are based on these fundamental premises and the identification of molecular genetic and biologic cellular changes. These alterations represent biomarkers of the carcinogenesis process and ultimately, if validated, could serve as intermediate end points for these studies.

Tumor-Associated Antigens in Normal Mucosa of Patients With Superficial Transitional Cell Carcinoma of the Bladder

We identified premalignant lesions in tumor-surrounding histologically normal bladder mucosa from patients with superficial and muscle-invasive bladder cancer to look for "premalignant" lesions as the source of tumor recurrence. Tumor-associated antigen expression in histologically normal bladder mucosa in 23 patients with superficial bladder cancer was studied. Histologically normal bladder specimens from 21 patients with muscle-invasive bladder cancer and from 30 prostatic cancer patients served as controls. Tumor-related antigens 486p 3/12, LBS8, 19A211 and M344 were mapped quantitatively with monoclonal antibodies and immunohistological techniques. Expression of all four bladder tumor-associated antigens was significantly enhanced and more than two antigens were expressed simultaneously in histologically normal mucosa from both bladder-cancer groups, compared with histologically normal bladder mucosa of prostatic carcinoma patients (p < 0.05). The results suggest the existence of early malignantly transformed cells in the benign-looking and morphologically normal bladder mucosa of superficial bladder carcinoma patients, which possibly is the source of recurrent bladder cancer. This approach models the mapping of histopathologic premalignant lesions of the bladder to predict risk.

Risk biomarkers and current strategies for cancer chemoprevention

Quantifiable, well-characterized cancer risk factors demonstrate the need for chemoprevention and define cohorts for chemopreventive intervention. For chemoprevention, the important cancer risk factors are those that can be measured quantitatively in the subject at risk. These factors, called risk biomarkers, can be used to identify cohorts for chemoprevention. Those modulated by chemopreventive agents may also be used as endpoints in chemoprevention studies. Generally, the risk biomarkers fit into categories based on those previously defined by Hulka: 1) carcinogen exposure, 2) carcinogen exposure/effect, 3) genetic predisposition, 4) intermediate biomarkers of cancer, and 5) previous cancers. Besides their use in characterizing cohorts for chemoprevention trials, some risk biomarkers can be modulated by chemopreventive agents. These biomarkers may be suitable surrogate endpoints for cancer incidence in chemoprevention intervention trials. The criteria for risk biomarkers defining cohorts and serving as endpoints are the same, except that those defining cohorts are not necessarily modulated by chemopreventive agents. A primary criterion is that the biomarkers fit expected biological mechanisms of early carcinogenesis-i.e., differential expression in normal and high-risk tissue, on or closely linked to the causal pathway for the cancer, and short latency compared with cancer. They must occur in sufficient number to allow their biological and statistical evaluation. Further, the biomarkers should be assayed reliably and quantitatively, measured easily, and correlated to cancer incidence. Particularly important for cancer risk screening in normal subjects is the ability to use noninvasive techniques that are highly specific, sensitive, and quantitative. Since carcinogenesis is a multipath process, single biomarkers are difficult to correlate to cancer, as they may appear on only one or a few of the many possible causal pathways. As shown in colorectal carcinogenesis, the risks associated with the presence of biomarkers may be additive or synergistic. That is, the accumulation of genetic lesions is the more important determinant of colorectal cancer compared with the presence of any single lesion. Thus, batteries of biomarker abnormalities, particularly those representing the range of carcinogenesis pathways, may prove more useful than single biomarkers both in characterizing cohorts at risk and defining modulatable risks. Risk biomarkers are already being integrated into many chemoprevention intervention trials. One example is the phase II trial of oltipraz inhibition of carcinogen-DNA adducts in a Chinese population exposed to aflatoxin B1. Also, urine samples from subjects in this trial will be screened for the effect of oltipraz on urinary mutagens. A second example is a chemoprevention protocol developed for patients at high risk for breast cancer; the cohort is defined both by hereditary risk and the presence of biomarker abnormalities. Modulation of the biomarker abnormalities is a proposed endpoint. Also, dysplastic lesions, such as prostatic intraepithelial neoplasia, oral leukoplakia and colorectal adenomas, have been used to define high-risk cohorts and as potential modulatable surrogate endpoints in chemoprevention trials.

Smokers and urinary genotoxins: Implications for selection of cohorts and modulation of endpoints in chemoprevention trials

Urinary genotoxicity assays measure the internal dose of genotoxic carcinogens, thereby providing a particularly sensitive endpoint for selecting cohorts of individuals exposed to cigarette smoke or other mutagens excreted with urines, as well as for evaluating the modulation of this parameter after administration of chemopreventive agents. Mutagenicity of urines was investigated in smoking Italian volunteers, who received oral N-acetylcysteine (NAC) at the same doses which are usually prescribed for the long-term treatment of chronic bronchitis. The daily excretion of mutagens, concentrated on XAD-2 columns and tested in Salmonella typhimurium YG1024 with S9 mix, was significantly and remarkably decreased by NAC in the majority of the subjects examined so far. Time-course experiments showed that this effect starts since the first day of drug administration and reverses when treatment is withdrawn. In addition, NAC administration almost totally prevented urinary genotoxicity in one subject whose concentrated urines induced a differential lethality in Escherichia coli strains having distinctive DNA repair capacities. The decrease of urinary genotoxicity produced by NAC in the majority of smokers correlates with the ability of this thiol to prevent tumors and to affect a variety of intermediate biomarkers in animal models. Modulation of the urinary excretion of mutagens is one of the biomarkers evaluated in two ongoing Phase II chemoprevention trials. One study involves the oral administration of NAC in Dutch smokers. The pretreatment urine samples of all the subjects so far recruited are clearly mutagenic. The other study involves the oral administration of the dithiolethione oltipraz to individuals living in the Qidong County of the People's Republic of China, an area of high endemy for HBV infection and of high exposure to aflatoxins. Additionally, a large proportion of the recruited male subjects are smokers. A total of 500 urine specimens will be assayed from 240 subjects according to a complex protocol arranged in three consecutive phases.

Carcinogen‐DNA and protein adducts: Biomarkers for cohort selection and modifiable endpoints in chemoprevention trials

Carcinogen‐DNA and protein adducts: Biomarkers for cohort selection and modifiable endpoints in chemoprevention trials

Smokers and urinary genotoxins: Implications for selection of cohorts and modulation of endpoints in chemoprevention trials

Smokers and urinary genotoxins: Implications for selection of cohorts and modulation of endpoints in chemoprevention trials

Carcinogen-DNA and protein adducts: Biomarkers for cohort selection and modifiable endpoints in chemoprevention trials

Chemical-specific markers have been developed for a number of environmental carcinogens for use as molecular dosimeters of individual exposure. In addition to contributing substantially to the specificity and sensitivity of epidemiological studies aimed at determining the role of environmental agents in the etiology of human cancers, some of these biomarkers may prove to be useful endpoints for assessing the efficacy of preventive interventions including exposure avoidance or remediation and chemoprevention. Biomarkers of the biologically effective dose may be particularly useful in this context in that they provide a mechanistic linkage between exposure and disease outcome. The biologically effective dose reflects the amount of toxicant that has interacted with its critical molecular target and can be measured through a variety of analytical techniques as either carcinogen-DNA or -protein adducts. Approaches for the development and validation of aflatoxin adduct biomarkers are presented as a paradigm for the application of carcinogen-specific markers for cohort selection and as modifiable endpoints for assessing efficacy in chemoprevention trials.

Micronuclei and carcinogen DNA adducts as intermediate end points in nutrient intervention trial of precancerous lesions in the oral cavity

In cancer chemoprevention trials, biomarkers as intermediate end points have gained importance. A variety of biomarkers have been proposed as intermediate end points for upper aerodigestive tract cancers. This study was aimed at studying the frequency of micronucleated cells and carcinogen DNA adducts as indicators of DNA damage and intervention end points in chemoprevention trials. Reverse smokers of chutta (rolled tobacco) from four villages numbering 298 in total were selected. Out of these, 150 were supplemented with four nutrients (vitamin A, riboflavin, zinc and selenium) and 148 controls received placebo, one capsule twice a week for 1 year. Slides of buccal smears were prepared and stained with Fuelgen reaction and counterstained with Fast Green and examined microscopically for the presence of micronucleated cells. Oral cell washings were collected and centrifuged. The DNA adducts were evaluated by the 32P post-labelling assay method. Protein and RNA free DNA (adducted) isolated from the cells was digested with MN/SPD and the DNA adducts isolated by the butanol enrichment procedure. The DNA adducts were identified and quantitated by multidimensional chromatography on PEI-TLC sheets by screen enhanced autoradiography and presented as RAL (relative adduct labelling) values. Both the micronuclei and DNA adducts were significantly elevated in subjects with lesions. At the end of 1 year the frequency of micronuclei decreased significantly (P < 0.001) in the supplemented subjects with or without lesions. The DNA adducts in the supplement group at the end of 1 year also reduced significantly. The adducts decreased by 95% in subjects with all categories of lesions and by 72% in subjects without lesions. No such effects were noted in the placebo group. The two biomarkers investigated in the case study appear to be modifiable by the administration of micronutrient supplements.(ABSTRACT TRUNCATED AT 250 WORDS)

Micronuclei, a biomarker for chemoprevention trials: Results of a randomized study in oral pre‐malignancy

Biomarkers are being sought that could serve as surrogate end points for chemoprevention trials. Micronuclei, cytoplasmic fragments of DNA, have been proposed as a biomarker and studied in oral pre-malignancy. This study evaluated micronuclei frequency in a randomized chemoprevention trial of oral pre-malignancy. A recent clinical trial evaluated the responses of pre-malignant oral lesions to 3 months of therapy with isotretinoin followed by 9 months of either low-dose isotretinoin or beta-carotene. For 57 study participants, micronuclei were counted in mucosal scrapings of the lesion and in normal-appearing mucosa at baseline and following 3 months and 12 months of therapy. Micronuclei counts were higher in scrapings from the lesion than in the normal-appearing mucosa. Following 3 months of isotretinoin, the micronuclei counts in scrapings of the lesion were significantly reduced. With treatment, the mean micronuclei count declined at 3 months. In a randomized comparison, both isotretinoin and beta-carotene maintained the suppression of micronuclei. The change in micronuclei count was not associated with the clinical or histological response to treatment. Chemoprevention treatment with isotretinoin led to a reduction in frequency of micronuclei, a marker of recent DNA injury, which was then maintained by both isotretinoin and beta-carotene.

Biomarkers in chemoprevention for upper aerodigestive tract tumors.

A chemopreventive approach to cancers of the upper aerodigestive tract (including those of head and neck and lung) to reduce the incidence and mortality rates for these cancers has become an important strategy because therapies such as surgery, radiation, and chemotherapy have only marginally improved the five-year survival rate over the last two decades. However, chemopreventive trials have been hampered by serious feasibility problems, including high cost, the requirement of large numbers of patients, and long-term follow-up necessary to determine cancer incidence, which served as the study end point. Thus, the use of biomarkers, the identification of which would serve as an intermediate end point of the study has recently emerged as a subject of great interest. To try to understand the process of tumorigenesis from normal tissues through the premalignant tissue stage to malignant lesions, there has recently been a search for genetic and/or phenotypic changes that qualify as candidates for biomarkers. These candidates include genomic markers, certain specific genetic markers (such as oncogenes, growth factors and their receptors, and tumor suppressor genes), cell proliferation markers, and cell differentiation markers. This review covers genomic markers (including micronuclei and specific chromosomal alterations) and specific genetic markers (such as the ras gene family, the myc family, erb B1, int-2/hst-1, and the p53 tumor suppressor gene). As a consequence of genetic alteration, we also reviewed cell proliferation markers such as proliferating cell nuclei antigen (PCNA) and the squamous cell differentiations markers, including keratins, involucrin, and transglutaminase 1. These biomarker candidates are important adjuncts to the development of the new chemopreventive agents and to the rational design of future intervention trials. However, it should be emphasized that these biomarkers must first be validated in clinical trials; only then can they replace cancer incidence as the sole end point in chemoprevention trials.

Surrogate endpoints in chemoprevention of breast cancer: guidelines for evaluation of new biomarkers.

Markers of early events in the development of breast cancer are potential candidates for surrogate endpoints in chemoprevention trials. There are many such markers and the challenge is to identify truly relevant markers. If successful, surrogate endpoints offer several potential benefits in the conduct of prevention trials, including: shorter latency and hence shorter trials; reductions in size and cost of trials; and the opportunity to study prevention measures where use of primary outcomes would be excessively invasive or unethical. Although there are currently no validated surrogate endpoints for breast cancer, criteria for the discovery and validation of surrogates have been proposed. Putative surrogate endpoints should be biologically plausible, represent an early event in the causal pathway, be measurable by a standardized and reliable assay, and exhibit a dose-response. Perhaps most importantly, surrogates should statistically capture the effect of the intervention on outcome. The identification and establishment of a biomarker as a valid surrogate for cancer is a stepwise process that involves both smaller "transitional" studies and larger second-generation chemoprevention trials in which both primary outcome and putative surrogates are measured. Transitional studies are used to move new markers from the laboratory into use in human populations, and are designed to address specific questions of assay validity, treatment/marker associations, marker/disease associations, and inter- and intra-individual variability. Promising markers should be added to current and planned, large, traditionally designed chemoprevention trials in order to definitively address the issues of optimal representation, and to test the adequacy with which the marker(s) captures the effect of treatment on outcome.(ABSTRACT TRUNCATED AT 250 WORDS)