Barbara G. Beatty

p16INK4A immunoexpression and HPV in situ hybridization signal patterns: potential markers of high-grade cervical intraepithelial neoplasia.

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16INK4a, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16INK4a immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16INK4a immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P < 0.001). p16INK4a immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P < 0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16INK4a staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16INK4a staining showed no evidence of viral integration. Both increased p16INK4a immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16INK4a immunostaining in CIN 1 appears independent of HPV punctate signal type.

Biotinyl-Tyramide-Based In Situ Hybridization Signal Patterns Distinguish Human Papillomavirus Type and Grade of Cervical Intraepithelial Neoplasia

In this study, the prevalence of human papillomavirus integration in cervical intraepithelial neoplasia Grades I, II, and III has been investigated using a highly sensitive biotinyl-tyramide–based in situ hybridization methodology. This method is able to demonstrate integrated viral DNA by punctate signals within the nucleus and episomal viral DNA by a diffuse signal throughout the nucleus. Fifteen viral types were identified by General Primer 5+/6+ polymerase chain reaction assay among 26 Grade I and 22 Grade II/III lesions. High-risk human papillomavirus (Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) was found in 20 (77%) Grade I and in 22 (100%) Grade II/III lesions (P =.025). Human papillomavirus Type 16 was identified in 2 (7%) Grade I and in 15 (68%) Grade II/III samples (P <.0001) and was distinguished from other high-risk types by its demonstration in both Grade I and Grade II/III lesions as frequent punctate signals, detectable at all levels of the epithelium including the basal layer. In contrast, punctate signals, when detected among Grade I lesions that were positive for other high-risk types, did not involve the basal layer and were restricted to occasional cells in the superficial layers. However, Grade II/III lesions positive for high-risk types other than human papillomavirus Type 16 demonstrated frequent punctate signals throughout the epithelium. Overall, punctate signals were detected in 22 (100%) high-risk human papillomavirus–positive Grade II/III lesions and in 5 (25%) high-risk positive Grade I lesions (P <.0001). These data are consistent with human papillomavirus Type 16 possessing a high potential for integration, which may explain its frequent association with cervical intraepithelial neoplasia Grade III and carcinomas. Acquisition of the punctate correlate, especially in the basal layer, is also indicated as important in the development of Grade II/III lesions. The data illustrate the unique potential of biotinyl-tyramide–based in situ hybridization to address key issues concerning the biology of cervical intraepithelial neoplasia.

HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in situ hybridization and immunocytochemical analysis.

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers. Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise kappa association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, kappa = 0.848; ThinPrep, kappa = 0.918) and moderate (ThinPrep, kappa = 0.692; formalin fixation, kappa = 0.667) to poor (ethanol, kappa = 0.300) pairwise kappa agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated. We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.

HER-2/neu Detection in Fine-Needle Aspirates of Breast Cancer

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers. Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise κ association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, κ = 0.848; ThinPrep, κ = 0.918) and moderate (ThinPrep, κ = 0.692; formalin fixation, κ = 0.667) to poor (ethanol, κ = 0.300) pairwise κ agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated. We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.

FISH detection of t(14;18) in follicular lymphoma on Papanicolaou‐stained archival cytology slides

The t(14;18)(q32;q21) translocation is present in about 85% of follicular lymphomas (FL) and can be identified using fluorescence in situ hybridization (FISH). In the diagnostic laboratory setting, the cytologic archival material consists of stained slides, and only rarely is material saved for molecular testing. The authors proposed FISH for FL using Papanicolaou‐stained archival cytology material as a practical ancillary technique for diagnosing FL.

The protein C system in placental massive perivillous fibrin deposition.

Massive perivillous fibrin deposition (MPFD) is associated with intrauterine growth retardation and first-trimester and second-trimester spontaneous abortion. Histologically, villi near the maternal interface are completely surrounded by fibrinoid material. This work compared the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in early miscarriage specimens with and without MPFD. Ten specimens with a gestational age of 7–12 weeks (mean 10 weeks) and 10 age-matched miscarriage specimens lacking MPFD were sampled. Formalin-fixed paraffin-embedded sections were stained with monoclonal antibodies against TM and EPCR using an immunoperoxidase method. The slides were independently reviewed by two pathologists using a semiquantitative grading system. Among unaffected villi, there was no difference in staining for TM or EPCR in cases of massive perivillous fibrin deposition compared with the control group. In the MPFD cases, loss of membrane positivity was noted for both TM and EPCR at the junction between normal villous epithelium and villous epithelium with deposition of fibrin. This could imply an underlying defect of trophoblastic protein C activation. Alternatively, it may represent a degenerative change secondary to impedence of oxygen and nutrient supply to the trophoblastic epithelium.

Cyclin D-1, interleukin-6, HER-2/neu, transforming growth factor receptor-II and prediction of relapse in women with early stage, hormone receptor-positive breast cancer treated with tamoxifen

Abstract:  We hypothesized that amplification or overexpression of HER‐2 (c‐erbB‐2), the Ki‐67 antigen (Mib1), cyclin D‐1 (CD1), interleukin‐6 (IL‐6), or the transforming growth factor beta II receptor, (TGFβRII), would predict relapse in women with early stage, estrogen (ER) and/or progesterone receptor (PR) positive breast cancer treated with tamoxifen. Conditional logistic regression models and a new novel analytic method––support vector machines (SVM) were used to assess the effect of multiple variables on treatment outcome. All patients had stage I–IIIa breast cancer (AJCC version 5). We paired 63 patients who were disease‐free on or after tamoxifen with 63 patients who had relapsed (total 126); both disease‐free and relapsed patients were matched by duration of tamoxifen therapy and time to recurrence. These 126 patients also served as the training set for SVM analysis and 18 other patients used as a validation set for SVM. In a multivariate analysis, larger tumor size, increasing extent of lymph node involvement, and poorer tumor grade were significant predictors of relapse. When HER‐2 or CD1 were added to the model both were borderline significant predictors of relapse. The SVM model, after including all of the clinical and marker variables in the 126 patients as a training set, correctly predicted relapse in 78% of the 18 patient validation samples. In this trial, HER‐2 and CD1 proved of borderline significance as predictive factors for recurrence on tamoxifen. An SVM model that included all clinical and biologic variables correctly predicted relapse in >75% of patients.

Laser scanning cytometry for the detection of neoplasia in urologic cytology specimens

The objective of the current pilot project was to assess the efficacy of laser scanning cytometry (LSC) for DNA ploidy analysis of atypical urologic cytology specimens to enhance the distinction between benign and malignant changes.

FUNCTION OF THE TRANSCRIPTIONAL REGULATING PROTEIN OF 132 kDa (TReP-132) ON HUMAN P450scc GENE EXPRESSION

Cytochrome P450scc catalyzes the important first step in the steroid synthesis pathway; however, it is clear that additional factors regulating the temporal and spacial specific expression of the CYP11A1 gene remain to be identified. To isolate novel transcription factors that regulate this gene, a cis-acting element of the 5′-flanking region from nucleotides −155 to −131 (−155/−131) was used to screen a human placental λgt11 cDNA expression library, and an interacting clone was isolated. The open reading frame of the cDNA encodes several domains that are characteristic of transcription factors including an acidic region, a region rich in prolines and three zinc-finger motifs. Expression of the cDNA by in vitro transcription/translation and by transient transfection in HeLa cells yielded a protein of 132 kDa, which concurs with the predicted size. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells, stimulate expression of a reporter construct controlled by the P450scc gene 5′-flanking region from nucleotides −1676 to +49. This transcriptional regulating protein of 132 kDa (TReP-132) when expressed in HeLa cells was demonstrated to interact with the −155/−131 region in bandshift analysis, and tandem copies of this region was shown to confer activation of the heterologous HSV thymidine kinase minimal promoter. Coexpression of CBP/p300 with TReP-132 further increased promoter activity, and the proteins were demonstrated to interact physically. RNA analysis demonstrated the highest levels of expression in the adrenal cortex and testis; and transcript expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines, but not in non-steroidogenic HepG2 and HK293 cells. Subsequently it has been shown that TReP-132 interacts with steroidogenic factor-1 (SF-1) through specific domains; and along with the interaction with CBP/p300 these factors are postulated to form a complex to regulate expression of the P450scc gene.

GLUT1 antibody staining in thin-layer specimens of benign and malignant body cavity effusions.

OBJECTIVE To determine whether GLUT1 antibody could replace one or more of the currently used antiepithelial antibodies and to assess whether ThinPrep methodology is suited to immunocytochemical (ICC) evaluation. STUDY DESIGN In a prospective study of 10 fluids containing malignant cells from cases of proven adenocarcinoma and 10 cytologically benign effusions, multiple slides were prepared by ThinPrep technology for staining with four commercially available antibodies and appropriate isotype-matched negative controls. The antibodies used were GLUT1, CEA, B72.3 and Leu-M1 (CD 15). Tissue sections and ThinPrep slides were used as positive controls. Specimens were batched to ensure similar conditions for all antibody reactions. RESULTS Of the 11 cases ultimately proven to be carcinoma, GLUT1 and B72.3 stained 7 each (63.6%), and CEA and Leu-M1 6 each (54.5%). No false positive staining was encountered, but one case chosen as a benign control was shown to contain immunopositive cells by three of the four epithelial markers used; this case was therefore an occult true positive rather than a false positive. CONCLUSION In this small but controlled prospective analysis, GLUT1 demonstrated strong positive staining, with sensitivity similar to that of currently used epithelial markers. Using GLUT1 in conjunction with B72.3, no cases of carcinoma were missed. GLUT1 could be used in a panel of antibodies designed to confirm the presence of adenocarcinoma. ThinPrep methodology, which enables multiple slides to be prepared after routine microscopy determines the need for ICC, appears suited to this adjuvant investigation.