Is the Magnitude of Difference Between the Two Groups Reliable

The hallmark of any scientific observation is, of course, repro-ducibility. With few exceptions, tumor markers seem to pass through a "life cycle" in which the original report is extraordinarily positive with great acclaim, but subsequent studies fail to live up to the promise. There are several elements regarding both technical variability of the assay and clinical trial design that account for this phenomenon, and these may hinder acceptance of the assay for routine clinical use. There are fundamentally three reasons for this conundrum: (1) technical variability of the assay; (2) variations in the manner in which different assays for the same marker are performed; and (3) inadequate and variable study designs.

The assay must be technically reliable and reproducible. Assay reproducibility is critical for any clinical test. Repro-ducibility hinges on several factors, all of which must be standardized and validated. Too often, an assay is developed in an individual investigator's laboratory based on personal preferences and subjective techniques that are not easily transported to other investigators and laboratories. For an assay to be useful clinically, it must be shown to be accurate throughout a broad dynamic range of values and reproducible at each of these levels as well. Concern must be taken regarding fixatives and other processing of samples, because these can have an enormous impact on the results of an assay from one laboratory to the next, resulting in false positives or negatives.

Analysis and Quantitation of Results

How the assay is "scored" or "read" is also critical for repro-ducibility. For example, when immunohistochemistry is performed, does the reader report the results as percent cells that stained, the intensity of staining, or a combination of both? Are the results reported as such, or in an index, such as 0-3+? Furthermore, selection of the cutoff that distinguishes positive from negative populations can give incredibly different results for the same assay. Several means of establishing a cutoff are employed, and there is no consensus regarding the optimal method.25 One method is to arbitrarily select a cutoff, based on some preconceived reason, such as the mean level of the assay in an affected population or the mean plus two standard deviations (2 SD) of the level in an unaffected population. A second method is to test several potential cutoffs in one population, selecting the one that appears most robust relative to separation of the outcomes of the two groups or to apparent statistical significance. Regardless of the method used, it is essential to validate the results in a separate group of patients.26 Thus, even if the same assay using the same reagents is applied in two different studies, use of different cutoffs will substantially affect the results.

Do Two Studies Use the Same Assay?

Because of competition among scientists and commercial interests, different assays are often developed to evaluate the same marker. Thus, when reading what appears to be a confirmatory study of a given marker, one must be certain that the same assay was used in both studies. For example, HER2 status can be determined by examination of cancer tissue amplification of the erbB2 gene using a variety of techniques including Southern blotting, slot-blot quantification, or fluorescence in situ hybridization, and by evaluation of the protein using Western blotting, immunohistochemistry, immunofluorescence, or enzyme-linked immunosorbent assays (ELISA). Moreover, the circulating extracellular domain of HER2 can be quantified in human serum using ELISA.27 Although they are all correlated, each of these assays, which in one way or another provides an indication of overproduction of HER2, appears to differ from the other and to provide different results in regard to prediction of outcome. Furthermore, even if the assay format is the same, use of different reagents or conditions may affect results. For example, it has been clearly shown that different antibodies against HER-2 can provide very different results in immuno-histochemical (IHC) assays.28 Thus, it is not surprising that results from study to study are not validated, if the assays that are being compared are not identical.

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