Interpretation

In general, the interpretation of occult disease detection assays consists of either a positive or negative result, given that the assay controls are appropriate. For IHC markers, nonspecific or aberrant expression of protein markers requires that careful evaluation of the cytologic characteristics of the positive cells be performed. The ability to visualize cell morphology with IHC can reduce false-positive results because the interpretation of a positive result can be limited to IHC positive cells with tumor cell morphology. In the case of genomic or mRNA targets, cytologic evaluation is not possible, making target choice, and assay design and validation even more critical. Carefully designed and validated molecular assays will include positive and negative controls, as well as internal controls to demonstrate the presence of intact and amplifiable nucleic acid isolated from the patient sample. In particular, the internal control should be present at a constant level, and its expression should be low enough to accurately reflect RNA degradation in the sample.

Issues with sensitivity and specificity exist for all occult disease detection assays. The analytical sensitivity of each assay should be determined using serial dilutions of a positive cell line or cell type into a negative cell population. However, actual tumor cells may vary widely in the expression of the target mRNA or protein, and thus actual sensitivity may be significantly less than the sensitivity using the control. Additionally, some markers may fail to be expressed by tumor cells, or may be expressed inappropriately by other cell types.21,22 Some groups have utilized multiplex assays in an effort to detect an increased range of tumor cells.23-25 Others have used quantitative assays to define a threshold value above which a positive signal indicates the presence of tumor cells, and below which nonspecific or inappropriate expression of the marker is assumed.26,27 However,because tumor cells may vary widely in their expression of a particular marker, it is difficult to define a clinically meaningful positive threshold above background. These and many issues must be addressed for assays to become more standardized, and thus for meaningful for clinical use.

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