As such, CLIA-’88 defines assay validation as “Establishment of performance specifications. Each laboratory that modifies an FDA–cleared or approved test system, or introduces a test system not subject to FDA clearance or approval (including methods developed in–house and standardized methods such as text book procedures), or uses a test system in which performance specifications are not provided by the manufacturer must, before reporting patient test results, establish for each test system the performance specifications for the following performance characteristics, as applicable: (1) Accuracy (2) Precision; (3) Analytical sensitivity; (4) Analytical specificity to include interfering substances; (5) Reportable range of test results for the test system; (6) Reference intervals (normal values); (7) Any other performance characteristic required for test performance.”

Various professional organizations have published guidelines concerning the validation of molecular assays. The CLSI has issued comprehensive guidelines for the validation and implementation of a variety of molecular diagnostic assays [3–5], as well as procedures to evaluate specific performance characteristics for qualitative and quantitative assays [6, 7]. Qualitative assays, as opposed to quantitative assays, are those capable of detecting an analyte but are not able to issue a numeric value associated with the amount of detected analyte. Historical, the field of molecular testing for infectious diseases has led the way in terms of guidelines for assay validation, primarily for quantitative assays [8, 9].

In general, molecular assays for solid tumors fall in the category of qualitative testing, and, as such, a set of performance characteristics needs to be validated before implementing such assays for clinical testing (Table 6.2).

Molecular testing for solid tumors has recently evolved to encompass the detection of an ever growing list of diagnostic, prognostic, and predictive molecular biomarkers. Therefore, when developing and/or validating a new molecular test for solid tumors, one should delineate the intended use of the assay (i.e., diagnosis, prognosis, or theragnosis—a novel concept for a combined therapeutic and diagnostic approach). This will dictate what kind of samples should be included during the validation process.

In addition, when dealing with solid tumors, one should be aware of the general issues that are unique to molecular testing of surgical pathology specimens, such as: specimen heterogeneity and sampling, sample size, and fixation. Solid tumors are often morphologically and biologically heterogeneous, with genetic markers differing among patients with the same tumor type, and even within individual tumor samples. In addition, within a specimen sample, neoplastic tissue can be admixed with benign or necrotic tissue, thereby affecting the sensitivity of molecular tests. Tumor enrichment techniques are often utilized to ensure accurate results. Such techniques, like Laser Capture Microdissection (LCM) yield very small samples for molecular testing [10]. Similarly, the adoption of less invasive diagnostic procures, such as fine-needle aspiration (FNA) specimens, contributes to the small size of samples sent for molecular testing.

In terms of tissue preservation, the ideal way of preserving the integrity of nucleic acids is to snap-freeze and indefinitely store the tissue at ultralow temperatures [11]. However, this is rarely done in routine clinical practice, where formalin-fixed, paraffin-embedded (FFPE) tissue samples are the norm. Nowadays, many molecular assays have been optimized for FFPE tissue samples. Formalin preserves tissue morphology by forming DNA–RNA–protein cross-links, which prevents ubiquitous DNases, RNases, and proteinases from digesting the cellular structure of the sample. Unfortunately, this cross-linking phenomenon affects the quality of nucleic acids isolated from such fixated tissues, in terms of amplifiability. Moreover, during fixation, nucleic acids often degrade yielding only short DNA (or RNA) molecules corresponding to fragments less than 300 base pairs in length. Therefore, when developing molecular assays intended for fixed tissue samples, one must take into account that only small amplicons should be designed. In addition, the use of fixatives containing either acids (Bouin and decalcifying solutions), which hydrolyze DNA, or heavy metals (Zenker and B5 solutions), which contain mercuric chloride that inhibits enzymes used in amplification, render these specimens unsuitable for most molecular assays [12].