New applications for old assays and the importance of validation

INTRODUCTION The article by Bishop et al in this issue of Cancer reports on the use of the molecular hybrid capture assay for identifying human papillomavirus (HPV) in cytology samples from head and neck squamous carcinomas. In gynecologic cytology specimens, of course, the hybrid capture technique is used in 1 of the common, standard, US Food and Drug Administration (FDA)-approved commercial assays for HPV. Other HPV detection technologies have been reported both for gynecologic cytology specimens and for tissue samples, and several are commercially available, FDA-approved assays. At first blush, a reader may think that the study by Bishop et al is an obvious extension of an accepted and standard technology being used in a slightly different sample type. Of course, the same assay likely will work on cytology from another tissue site or tumor type! However, Bishop and colleagues present a lot more than just reasonably valuable research results. They importantly highlight several critical aspects of assay validation in anatomic pathology practice that often are overlooked and deserve special emphasis: 1) Clinical validity is an important consideration in new assay validation; 2) many analytes will have several different detection methodologies, and there may be considerable controversy around the ‘‘best’’ approach; and 3) quality control, assay validation, and standardization for any new laboratory-developed test (LDT) are absolutely critical. To begin, some still may be questioning whether there really is a clinical need to test head and neck squamous cell carcinomas for HPV and whether the HPV status will impact clinical decision making. These questions relate directly to what has been referred to as the ‘‘clinical validity’’ of an assay, and they are very important to ask before introducing new assays. It is my experience that most clinicians working with head and neck cancer patients have started to view HPV testing in certain squamous cell carcinomas as the standard of care. This is particularly true in tumors that involve the oropharynx, which includes tonsil, tongue base (lingual tonsil), and sometimes the soft palate. Many clinician colleagues have asked for HPV testing in oropharyngeal carcinomas to be done as a reflex testing before oncology treatment decisions are made, and they consider the HPV status in therapy selection. In contrast to concerns that have been raised in gynecologic samples, there is very little risk of false-positive results, because high-risk HPV does not appear to be present in normal tissues from the oropharynx. There is now a fairly remarkable body of literature about the importance of the association between HPV and squamous carcinomas related to both treatment responsiveness and prognosis. In fact, patients who have HPV-associated squamous cell carcinoma will have a much better prognosis than those who have squamous cell