Disease Markers 20 (2004) 97–102 97 IOS Press Development tracks for cancer prevention markers Stuart G. Bakera,∗, Barnett S. Kramerb and Philip C. Proroka aBiometry Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, USA bOffice of Disease Prevention, National Institutes of Health, Bethesda, MD, USA Abstract. We provide a general framework for describing various roles for biomarkers in cancer prevention research (early detection, surrogate endpoint, and cohort identification for primary prevention) and the phases in their evaluation. 1. Introduction The evaluation of cancer prevention markers (biomarkers) depends both on the purpose to which they are used and the nature of the marker. The main purposes are early detection (with the goal of early in- tervention), surrogate endpoints (with the goal of hav- ing shorter studies) and cohort identification (of indi- viduals with the greatest benefit to harm ratio associ- ated with primary prevention). In cancer prevention, markers are also used to obtain better measures of nu- trient intake or other exposures, but these markers are beyond the scope of this article. 2. Molecular markers for early detection In the past, most markers for cancer screening in- volved changes in cell types (such as degrees of dyspla- sia) or single proteins in serum such as PSA or CA125. Recently there has been a large increase in the amount of data on molecular biomarkers to hopefully detect cancer early. One reason is simply the new types of biomarkers being developed, such as proteomic pat- terns. A second reason is the increasing number of biorepositories associated with randomized trials or co- ∗Corresponding author: Stuart G. Baker, Sc.D. National Cancer Institute, EPN 3131, 6130, Executive Blvd MSC 7354, Bethesda, MD 20892-7354, USA. Fax: +1 301 402 0816; E-mail: sb16i@nih.gov; URL: http://www.cancer.gov/prevention/bb/baker.html. hort studies, making it possible to collect biomarker data retrospectively (e.g. Baker (1998)). This large in- crease in information, which sometimes goes under the name of bioinformatics, has created a challenge for can- cer prevention. Without a clear purpose, bioinformatics may accomplish little. Pablo Picasso said, “Computers are useless. They only give us answers”. Answers to the wrong question are of little value. It is important that that computations for evaluating biomarkers an- swer the appropriate question. With early detection, the appropriate question is what biomarkers are most likely to make effective triggers of early intervention. The following phases (which are applicable to both molecu- lar markers and markers involving changes in cell type) provide a road map for answering this question. 2.1. Phase I. Molecular markers for symptomatic cancer The easiest way to collect data for investigating molecular markers is to obtain specimens from subjects without cancer and subjects with symptomatic (clini- cal) cancer, or from tumor cells and non-tumor cells in the same subject. The goal is to identify markers that discriminate well between symptomatic cancer and no cancer. As discussed in Baker [4] in more detail, the recommended outcome measures are the false positive rate (proportion of non-cancers that are positive, or one minus specificity ) and true positive rate (proportion of cancer specimens that are positive, or sensitivity). Each rule for classifying a marker as cancer or not is as- ISSN 0278-0240/04/$17.00  2004 – IOS Press and the authors. All rights reserved 98 S.G. Baker et al. / Development tracks for cancer prevention markers sociated with both a false and true positive rate. Some rules can be created by specifying different cutpoints for a particular marker. Other rules are generated by combinations of cutpoint levels among multiple mark- ers. One can plot a point for each pair of false and true positive rates, where the horizontal axis is the false positive rate and the vertical axis is the true positive rate. The points highest and farthest to left form the optimal receiver operating characteristic (ROC) curve. Certain points on the ROC curve are of primary interest. Because the eventual goal is to identify a biomarker that is useful for cancer screening, it is important that the false positive rate be very small (e.g. 1% for breast cancer screening) to avoid unnecessary biopsies. The true positive rate should be large (e.g. 80% for breast cancer screening). With many biomarkers (or combinations of biomark- ers) under investigation, chance plays a large role in making some biomarkers or their combinations appear to perform well in discriminating between cancer and no cancer in the early phases of the study. To reduce the role of chance, we recommended that the data be randomly split into training and test samples. Then, if biomarkers (or combinations of biomarkers) with a small false positive rate and large true positive rate are identified in the training sample, we recommend definitive evaluation of their performance using an ROC curve based on the test sample. One example of the use of a test sample is a proteomic study that reported FPR=0.05 and TPR =1.00 in the test sample [25]. 2.2. Phase II. Molecular markers for asymptomatic cancer Biomarkers identified in Phase I that discriminate well between clinical cancer and non-cancer (as evi- denced by the ROC curve) are investigated in Phase II to determine if they discriminate well between asymp- tomatic cancer and non-cancer. Although one could measure the biomarker in all asymptomatic subjects prospectively, a retrospective study using stored speci- mens is much more practical. Typically a biorepository is created to store specimens in a prospective study that may be unrelated to the cancer of interest. As part of a nested case-control study within the prospective study, biomarkers are measured in the stored sample from all subjects with the cancer of interest and a random sample of subjects without cancer. Many of the approaches to evaluation are similar to those for Phase I. However, as discussed in Baker et al. [9], there are some special considerations and op- portunities. One special consideration is related to the detection of cancer in the prospective trial. Ideally, for purposes of analysis, the subjects in the prospective study are those who were diagnosed with symptomatic cancer, while the sample used to measure the biomarker is obtained while the subject was still asymptomatic. If cancers in the prospective study were detected by screening of asymptomatic individuals and there was substantial overdiagnosis (i.e. detection of cancers that do not cause any medical problems in a person’s life- time), the performance results of the biomarker would not be relevant. One special opportunity with stored samples in Phase II that is not available in Phase I is the ability to investigate the performance of prediction rules based on changes in marker values over time, during the asymptomatic phase of the disease. Another consideration at this phase is spectrum bias. This can occur if the spectrum of health conditions in the non-cancer control subjects differs from the spec- trum in the target population for screening. For ex- ample, the target population for lung cancer screening may be people with a heavy cigarette smoking history. Such people may have a variety of health conditions which produce a false positive test including inflam- matory lung conditions, upper respiratory infections, chronic obstructive pulmonary disease, cardiac disease, or even cancer of unrelated organ systems such as the bladder. If adequate attention is not paid to the possi- bility of spectrum bias, the false positive rate is likely to be much higher in practice than in Phase I or II studies. 2.3. Phase III. Molecular biomarkers as triggers for early intervention Even if a molecular biomarker discriminates well between cancer and no cancer, when it triggers early intervention, it may not lead to a reduction in cancer mortality or an insufficient reduction in cancer mor- tality relative to harms such as unnecessary biopsies and work-ups. Therefore it is important to evaluate the biomarker as a trigger of early intervention. The most definitive approach is via a randomized trial with a cancer mortality endpoint [9]. Because such random- ized trials are extremely large (usually with over 30,000 subjects) and costly, they should be used only for the most promising triggers for early intervention. If the costs of the aforementioned randomized trail are prohibitive, and it is possible to delay making a definitive evaluation, we recommend observational studies as an intermediate step between a Phase II study and a randomized trial of biomarker as a trigger for early S.G. Baker et al. / Development tracks for cancer prevention markers 99 intervention. One recently proposed approach, called periodic screening evaluation [9], rules out biomarker triggers for early intervention that would not likely lead to a reduction in cancer mortality in a randomized trial. Another approach is to combine before-and-after stud- ies from various geographic regions (e.g. [13,16,26], with future work involving extensions of the method- ology in Baker, Lindeman, and Kramer [10]. 3. Imaging test for early detection The results of imaging tests can be viewed as a marker for early detection. Because they involve a subjective component, the method of evaluation differs from that of molecular markers. Examples of imag- ing markers for the early detection of cancer are mam- mography and low-dose spiral CT. The evaluation of imaging markers follows the same general phases as the evaluation of molecular markers. However some details differ in Phases I and II. 3.1. Phase I. Imaging markers for symptomatic cancer As with molecular markers, the first phase is to eval- uate the ability of the markers to discriminate between subjects with clinical cancer and subjects without clin- ical cancer. However, unlike molecular markers, the classification of imaging markers involves a subjec- tive element. For example, a typical classification of mammograms (normal, probably benign, indetermi- nate, suspicious, and highly suspicious) depends both on the image and the person reading the images. For- tunately the ROC curve can also accommodate subjec- tively determined categories. It is not as useful to re- port the false and true positive rate for a particular cate- gory because the determination of the category is fuzzy due to the subjective component. Instead, the preferred outcome measure is the area under the ROC curve for a range of small false positive rates [4] which can be thought of as an "average" true positive rate over the categories with the small false positive rates of interest. 3.2. Phase II. Imaging markers for asymptomatic cancer If images are taken periodically in a prospective study and stored digitally for later evaluation, one can retrospectively read the images from subjects who later developed cancer and a random sample of subjects who did not develop cancer. The retrospective analysis fol- lows the same recommendations as with the molecular markers. However, as mentioned above, due to the sub- jective nature of the readings, the preferred outcome measure is an area under part of the ROC curve. Sometimes a prospective study is implemented for evaluating the performance of imaging in discriminat- ing between asymptomatic cancer and no cancer. In the prospective study images are read, classified, and a rec- ommendation for biopsy or no biopsy is made. Because cancer is rare in asymptomatic subjects, the prospective study needs to be large (e.g. 20,000 subjects.) However the prospective study has the advantage of a relatively short duration. The difficulty for evaluating the prospective study is that a biopsy is performed only if the image is classified as positive, leading to a severe bias known as “con- founding by indication.” Therefore it is not possible to directly estimate false and true positive rates, which require knowing the number of subjects with and with- out cancer at the time of screening. However, because cancer is rare among screened subjects, one can obtain an estimate of the false positive rate by assuming that all subjects not biopsied are free of cancer. For estimat- ing true positive rates, some investigators assume that all subjects negative on imaging who developed cancer within, say, one year, in fact, had undetected cancer at the time of screening. The validity of this assump- tion depends on the distribution of the length of time at which asymptomatic cancer is detectable on screen- ing. See Day [14]. If classification data are collected, then, due to the subjective nature of the readings, the preferred outcome measure is an area under part of the ROC curve corresponding to small false positive rates, say 0.5% to 1.5%. This area can be used to compute an average TPR in the range of small false positive of interest. Ideally one would want an average TPR of 80%. Alternatively as this is a more definitive study than Phase I, one might base the criteria for going to Phase III on the performance of a decision to biopsy or not. In that case the numerical values of false positive rate (e.g. 1% for breast cancer screening) and high true positive rate (e.g. 80% for breast cancer screening) are analogous to the values for molecular markers. 3.3. Phase III. Imaging biomarkers as triggers for early intervention The same considerations for evaluating molecular markers as triggers for early intervention apply to eval- uating imaging markers as triggers for early interven- tion. 100 S.G. Baker et al. / Development tracks for cancer prevention markers 4. Surrogate endpoint markers A surrogate endpoint is an endpoint that is obtained sooner, less invasively, or at less cost than a true end- point for a health outcome. Surrogate endpoints have either been used to replace true endpoints or to pre- dict true endpoints. Evaluation methodology is more controversial than with markers for early detection. Some endpoints that were thought to be good surro- gate endpoints later turned out to give incorrect infer- ence when compared to true endpoints [17]. Despite this strong caveat, we can outline various development stages and identify the gaps that need addressing. Most of the evaluation techniques can rule out poor surrogate endpoints. However, more methodological research is needed to develop criteria for deciding if a surrogate endpoint is useful in practice and thus avoid unintended harm based on incorrect assumptions. Given all the difficulties we discuss, the main use of surrogate end- points may be in a preliminary study of an intervention to decide on whether or not to evaluate the intervention in a definitive randomized trial with a true endpoint. 4.1. Phase I. Surrogate and true endpoint in an observational study A surrogate endpoint should be strongly associated with a true endpoint. To estimate association, data are needed on both a surrogate and true endpoints in indi- viduals. In preliminay studies, the data will be obser- vational. For example the strong association between human papilloma virus (HPV) infection and cervical intraepithelial neoplasia (CIN) [27] indicates the po- tential for using HPV as a surrogate endpoint for CIN. The challenge in this phase is deciding what level of as- sociation is sufficiently large to move to the next phase, and here the published methodology is lacking. Lev- els of association adequate or even high for an etio- logic risk factor may not be sufficient to warrant further investigation as a surrogate endpoint since they may still explain a low proportion of variability in the true endpoint. More importantly, a high or even perfect correlation between surrogate and true endpoints does guarantee that the surrogate endpoint will provide reli- able inference about the true endpoint in a randomized trial [4]. 4.2. Phase II. Randomized trial of intervention known to affect true outcome As a preliminary requirement for a surrogate end- point to be effective, an intervention that is known to affect a true endpoint should also affect a surrogate endpoint. The best way to check is to randomize sub- jects to either an intervention with a known effect on a true endpoint or a control group, where the outcome is the surrogate endpoint. If the surrogate endpoint is promising for this phase, the intervention would affect the surogate endpoint in the same direction as the inter- vention is known to affect true endpoint. With a reason- able sample size, if there is no statistically significant difference in surrogate outcomes between randomiza- tion groups, one could likely rule out the biomarker as a good surrogate endpoint. The dilemma is that it is often unlikely to have an intervention that is known to affect the true endpoint and be acceptable for a randomized trial. Nevertheless in some situations it is possible. For example, suppose that adenoma recurrence is a true endpoint. (In reality it is only a surrogate endpoint for colorectal cancer, but let us ignore that for now). Based on a randomized trial, calcium supplement was found to reduce adenoma recurrence [20]. Because calcium supplement did not affect rectal mucosal proliferation (surrogate endpoint) in another randomized trial [1], one can rule out rec- tal mucosal proliferation as a promising surrogate end- point for adenoma recurrence. If the outcome of this type of randomized trial is a panel of surrogate endpoint markers, it is important to adjust for multiple comparisons. The simplest, albeit conservative, approach is a Bonferroni correction: if there are k markers then statistical significance is not 5% (two-sided) but 5/k%. Although the magnitude of the effect is also important, it is not clear what size effect is needed for proceeding to the next phase. 4.3. Phase III. Randomized trials of multiple interventions with surrogate and true endpoints The very nature and complexity of cancer pathogen- esis makes the results of Phase I and II very tenta- tive. Most cancers have multiple causal pathways and each pathway has multiple steps that can take years to evolve. If a surrogate marker occurs early in the path- way but the intervention blocks a “downstream” event, measurement of a surrogate endpoint can give a mis- leading answer even if the marker is in the causal chain. Likewise, if the marker is in an entirely different causal S.G. Baker et al. / Development tracks for cancer prevention markers 101 pathway, its measurement can be misleading. This is why extrapolation of results from one particular set of surrogate and true outcomes can be hazardous. For this reason the most informative evaluation of surro- gate endpoints comes from data on surrogate and true endpoints either in multiple randomized trials involv- ing different interventions or a single randomized trial with multiple arms, each with a different intervention. Ideally the set of interventions should affect all major pathways so results are applicable to new interventions. The methodological research in this area has focused on evaluating multiple trials with surrogate and true endpoints [19,23]. The data may be divided into a training set involving all but one trial and a test set involving the remaining trial. Based on data in the training set, a model is developed to predict the true endpoint from the surrogate endpoint. This prediction could be at a “meta-analytic” level [19,23] or based on an individual-level model (extending the methodology in Morrison 1989 and Day and Duffy, 1996 to multiple previous trials). Confidence intervals should incorpo- rate sampling variability as well as variability among parameters in previous trials. For validation, the model is used to predict true endpoint in the test trial based on surrogate data in the test trial. The predicted true endpoint in the test trial is compared with the observed true endpoint in the test trial. However there is no guid- ance on how to decide when the overlap in confidence intervals warrants acceptance of the surrogate endpoint as a replacement for the true endpoint. An alternative design, which we have not seen ap- plied or discussed, is a single trial involving random- ization to multiple interventions with surrogate and true outcomes. This design has several advantages over the multiple trial design. First it would likely take less time to implement. Second, it does not involve duplication of control groups. Third, it might be easier to set up a biorepository for implementing a nested case-control study in which the surrogate endpoint is measured in stored samples from a random sample of subjects [21]. Even if this more rigorous approach finds that a sur- rogate endpoint adequately predicts a true endpoint un- der a variety of interventions, there still could be a se- rious problem with using a surrogate endpoint. Most, if not all, cancer prevention trials involve both benefits and harms.A surrogate endpoint that is very accurate in predicting a true endpoint of benefit may not be very accurate in predicting harms. This could occur if the harms arise after the time of the surrogate endpoint. If trials are stopped early because of an apparently favor- able change in a surrogate endpoint, a net harm asso- ciated with the intervention can be missed. For exam- ple if a chemoprevention agent were to delay or pre- vent polyps but cause strokes that occur between the detection of polyps and the occurrence of colon cancer, the harm from strokes would be missed if studies are stopped due to a “valid” surrogate endpoint based on polyps. 5. Cohort identification for primary prevention Another use of biomarkers in cancer prevention is to target preventions to individuals with biomarkers that indicate a high ratio of benefits to harms, either by virtue of having greater benefits, fewer harms, or both. For simplicity of this discussion, we focus on benefits. 5.1. Phase I. Preliminary observational studies One type of preliminary observational study is a small study in which all subjects receive the primary prevention, data are collected on biomarkers, and the outcome is a surrogate endpoint indicating response or non-response. The goal is to identify what biomarkers, if any, differ statistically significantly between respon- ders and non-responders. Usually a very large number of markers are considered, so there is a need to adjust for multiple comparisons. A second type of preliminary observational study is a case-control study to identify genes that substantially elevate the risk of cancer. The underlying premise is that subjects with genes indicative of a higher can- cer risk have the greatest potential to benefit in abso- lute terms from a primary prevention. Again, if many biomarkers are under consideration an adjustment for multiple comparisons is necessary. 5.2. Phase II. Randomized trials If a primary prevention was shown to be beneficial in a randomized trial, but the benefits did not outweigh the harms, it would be of great interest to investigate whether the benefits (i.e. reduction in cancer) would be greater for subjects with a certain gene. The problem is that testing for the gene in all subjects is not possible. Although only cases need to be tested [4,22] to estimate relative risk among subjects with the gene, relative risk is not the most clinically relevant outcome. A more relevant outcome is the age-specific absolute risk dif- ference due to the intervention. As discussed in Baker and Kramer [26], this outcome can be estimated using 102 S.G. Baker et al. / Development tracks for cancer prevention markers a nested case control design involving data from all subjects with cancer and a random sample of subjects without cancer in a randomized study. If the randomized trial is in the planning stage and the focus is on a single gene, one could test for the gene in all subjects or subjects with a family history of cancer and randomize the subjects with the gene to intervention or not [5]. If many genes are involved, it would be preferable to randomize all subjects, collect specimens for a biorepository, and then perform the aforementioned nested-case control study. 6. Discussion The purpose of this article is to outline development tracks for some markers that play a role in cancer pre- vention. Not all markers fit neatly into these devel- opment phases, as for example prospective studies of sputum cytology markers for early detection of lung cancer [8], but some of the same principles apply, such as using the appropriate portion of the ROC curves for evaluation. This article should help investigators understand how marker research fits into the general strategy of cancer prevention and identify the gaps in methodological work that need to be addressed in or- der to avoid potential unintended harms that can occur when biomarkers or surrogate endpoints are accepted prematurely. References [1] J.A. Baron, et al., Calcium supplements for the prevention of colorectal adenomas, New England Journal of Medicine 340 (1999), 101–107. [2] S.G. Baker, Identifying combinations of cancer biomarkers for further study as triggers of early intervention, Biometrics 56 (2000), 1082–1087. [3] S.G. Baker, The Central Role of Receiver Operating Charac- teristic (ROC) Curves in Evaluating Tests for the Early De- tection of Cancer, J. Natl Cancer Inst. 95(7) (Apr 2 2003), 511–515. [4] S.G. Baker, D. Erwin, B.S Kramer and P.C. 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Petricoin, et al., Use of proteomic patterns in serum to identify ovarian cancer, The Lancet 359 (2002), 572–577. [26] L. Peron, L. Moore, I. Bairati, P. Bernard and F. Meyer, PSA screening and prostate cancer mortlaity, Canadian Medical Association Journal 166 (2002), 586–591. [27] M.H. Schiffman et al., Human popillomavirus and cervical in- traepithelial neoplasia response, Journal of the National Can- cer Institute 85 (1993), 1868–1870. 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