Ellen E. Sheets

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)

During the development of neoplasia, epithelial tissues undergo biochemical and structural changes that can manifest in tissue fluorescence. There have been several reports on different in vivo fluorescence characteristics between normal and precancerous (dysplastic) tissues. However, it has been difficult to identify and quantify the origins of these changes, mainly because of distortions introduced in measured tissue fluorescence spectra by tissue scattering and absorption. Such distortions can be removed by combining information in simultaneously measured fluorescence and reflectance spectra. Thus, we can recover the intrinsic (undistorted) tissue fluorescence. In this report, we show that extraction of the intrinsic fluorescence allows us: (a) to determine the fluorescence spectra of NAD(P)H and collagen in an in vivo environment, and (b) to use these NAD(P)H and collagen spectra to describe, quantitatively, diagnostically significant biochemical changes between normal and dysplastic tissues. Specifically, by analyzing intrinsic fluorescence of human epithelial tissue as it becomes deoxygenated in vivo, we can resolve the fluorescence spectra of NAD(P)H and collagen, two of the major tissue fluorophores. This is important because fluorescence depends on the local environment of the chromophore. Then, we extract the intrinsic fluorescence spectra of sites from 35 patients with suspected cervical lesions and 7 patients with Barrett's esophagus and describe them accurately as a linear combination of NAD(P)H and collagen contributions. In both tissue cases, we find that low collagen and high NAD(P)H fluorescence characterizes the high-grade dysplastic lesions when compared with nondysplastic tissues. These data present evidence for the presence of detectable levels of NAD(P)H fluorescence in human epithelial tissues in an in vivo setting and demonstrate that NAD(P)H and collagen may be used as quantitative fluorescence biomarkers for in vivo detection of dysplasia in the cervix and the esophagus.

Garrett, Audrey P. MD, MPH; Wenham, Robert M. MD, MSc; Sheets, Ellen E. MD Author Information

BACKGROUND: Squamous (CIN) and glandular (ACIS) intraepithelial lesions often coexist in the same cervical specimen. However, a less common and little studied variant consists of a stratified epithelium resembling CIN in which conspicuous mucin production is present (Stratified Mucin-producing Intraepithelial LEsions (SMILE). This report describes the phenotypic characteristics of the SMILE, its associated lesions, and its immunophenotype. METHODS: Eighteen SMILEs were identified by the presence of conspicuous cytoplasmic clearing or vacuoles in lesions otherwise resembling CIN. The morphologic spectrum of SMILEs was detailed; including associated intraepithelial and invasive cervical neoplasms. In addition, selected cases were stained for mucicarmine, markers of squamous cell/reserve cell differentiation (keratin-14 and p63), and proliferative activity (Mib-1). RESULTS: Stratified neoplastic epithelial cells with a high Mib-1 index and a rounded or lobular contour at the epithelialstromal interface characterized SMILEs. In contrast to CIN, in which mucin droplets are confined to surface cells, mucin was present throughout the epithelium, varying from indistinct cytoplasmic clearing to discrete vacuoles. SMILEs were distinguished from benign metaplasia by nuclear hyperchromasia and a high Mib-1 index. All but three coexisted with either a squamous (CIN) or glandular (ACIS) precursor lesion. Nine of nine coexisting invasive carcinomas contained glandular, adenosquamous differentiation, or both. SMILEs stained negative for keratin-14 and variably for p63. When present, staining with p63 was confined to basal areas of SMILEs and was absent in areas of columnar differentiation. CONCLUSIONS: SMILEs are unusual cervical intraepithelial lesions best classified as variants of endocervical columnar cell neoplasia based on immunophenotype. The distribution and immunophenotype of SMILEs are consistent with a neoplasm arising in reserve cells in the transformation zone. The coexistence of a wide spectrum of intraepithelial and invasive cell phenotypes suggests that SMILEs are a marker for phenotypic instability, emphasizing the importance of identifying SMILEs and ensuring a complete examination of specimens containing this unusual precursor lesion.