Sharon L. Mount

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.

Topical Testosterone for Breast Cancer Patients with Vaginal Atrophy Related to Aromatase Inhibitors: A Phase I/II Study

PURPOSE Controversy exists about whether vaginal estrogens interfere with the efficacy of aromatase inhibitors (AIs) in breast cancer patients. With the greater incidence of vaginal atrophy in patients on AIs, a safe and effective nonestrogen therapy is necessary. We hypothesized that vaginal testosterone cream could safely treat vaginal atrophy in women on AIs. METHODS Twenty-one postmenopausal breast cancer patients on AIs with symptoms of vaginal atrophy were treated with testosterone cream applied to the vaginal epithelium daily for 28 days. Ten women received a dose of 300 μg, 10 received 150 μg, and one was not evaluable. Estradiol levels, testosterone levels, symptoms of vaginal atrophy, and gynecologic examinations with pH and vaginal cytology were compared before and after therapy. RESULTS Estradiol levels remained suppressed after treatment to <8 pg/mL. Mean total symptom scores improved from 2.0 to 0.7 after treatment (p < .001) and remained improved 1 month thereafter (p = .003). Dyspareunia (p = .0014) and vaginal dryness (p <.001) improved. The median vaginal pH decreased from 5.5 to 5.0 (p = .028). The median maturation index rose from 20% to 40% (p < .001). Although improvement in total symptom score was similar for both doses (-1.3 for 300 μg, -0.8 for 150 μg; p = .37), only the 300-μg dose was associated with improved pH and maturation values. CONCLUSIONS A 4-week course of vaginal testosterone was associated with improved signs and symptoms of vaginal atrophy related to AI therapy without increasing estradiol or testosterone levels. Longer-term trials are warranted.

Significant increase of benign endometrial cells on Papanicolaou smears in women using hormone replacement therapy.

OBJECTIVE To determine the prevalence of benign endometrial cells on Papanicolaou smears from postmenopausal women and to compare the prevalence and histologic diagnosis in women who use hormone replacement therapy (HRT) with those who do not. METHODS Papanicolaou smear diagnoses from postmenopausal women and women over age 50 between April 1995 and December 1998 were retrieved and linked with follow‐up smears and biopsies. Hormone status of women with benign endometrial cells smears was obtained from requisition forms or phone conversation with primary care providers. All surgical pathology material as well as Papanicolaou smears from women subsequently diagnosed with endometrial adenocarcinoma were reviewed. Relative prevalence with 95% confidence intervals was determined. RESULTS A total of 589 of 52,662 Papanicolaou smears from postmenopausal women were diagnosed with benign endometrial cells, a prevalence of 1.1%. Also, HRT was reported in 16,073 (31%), no HRT was reported in 33,170 (63%), and hormone status was unknown in 3379 (6%). Smears from 245 HRT users, 324 nonusers, and 20 with unknown hormone status were diagnosed with benign endometrial cells. There was a significant increased prevalence of benign endometrial cells in women on HRT compared with nonusers (relative prevalence 1.56, 95% confidence interval 1.32, 1.84, P < .001). Among 436 women with known hormonal status and follow‐up, 12 (2.7%) had endometrial carcinoma, three (1.5%) in HRT users and nine (3.7%) in nonusers (P = .175). In addition, HRT users had significantly less abnormal endometrial histology than non‐HRT users (2.6% versus 7.4%, P = .025). CONCLUSION We found that HRT is significantly associated with an increased prevalence of benign endometrial cells on Papanicolaou smears. Women on HRT who have benign endometrial cells on their Papanicolaou smears, however, have less abnormal endometrial histology compared with women not using HRT who have benign endometrial cells on their Papanicolaou smears.

Documentation of immunocytochemistry controls in the cytopathologic literature: a meta-analysis of 100 journal articles.

Although a detailed description of the procedure and tissue used as controls is considered a necessary component in surgical pathology articles in which immunohistochemistry is utilized, such documentation seems less stringent in the cytopathologic literature. A comprehensive literature search was done for articles published in English within the last 15 years on nine of the most widely used antibodies in cytopathology. Individual case reports were excluded. Of the 100 articles reviewed, 13 articles were review articles or commentaries and hence not included in the analysis. Only 11 (13%) of the remaining 87 articles described positive and negative controls run on identically prepared samples. Forty‐seven articles (54%) either did not mention controls or did not run controls as separate specimens. Sixteen articles (18%) included a vague statement about controls. Twelve (14%) commented only on the negative control, included only histology tissue controls, or included cell block controls, but the study also included other types of preparations, such as cytospins. One article (1%) did not include controls because of insufficient material. The College of American Pathologists recognizes the impracticality of maintaining separate positive control samples for every possible combination of fixation, processing, andspecimen type. However, more stringent documentation of procedure and use of controls in the cytopathologic literature will ensure that immunocytochemistry results in diagnostic cytopathology as well as in research are valid and reproducible. Diagn. Cytopathol. 2011;39:245–250.

Ultrastructural Study of a Pituitary Adenoma (Prolactinoma) Within the Clivus Bone Using Immunoelectron Microscopy

In a case of a pituitary adenoma in the clivus bone in a 71-year-old man, ultrastructural investigation using conventional aldehyde-fixed, epoxy-embedded tissue revealed the tumor to be composed of cells with euchromatic nuclei, dense nucleoli, abundant rough endoplasmic reticulum, spherical secretory granules, and granule extrusion at the lateral cell surface, all of which suggest a prolactin-producing adenoma. Using a protein A-gold immunolabeling technique on snap-frozen tissue subsequently fixed in a mild fixative and embedded in a hydrophilic resin, the presence of prolactin immunoreactivity within secretory granules at the ultrastructural level was demonstrated. This case represented the first use of protein A-gold immunolabeling at the electron microscopic level for diagnostic purposes at our institution and exemplifies the value of this technique when the need for diagnostic immunoelectron microscopy is not anticipated. Because this tumor arose in an unusual location, ultrastructural study, including immunoelectron microscopy, not only confirmed the light microscopic diagnosis of pituitary adenoma, but further allowed subclassification of the tumor.

Biology and evolution of cervical squamous intraepithelial lesions: a hypothesis with diagnostic prognostic implications.

Recent advances in the understanding of HPV-associated cervical squamous intraepithelial lesions, specifically with respect to HPV DNA integration into basal cervical epithelial cells, need to be incorporated into strategies for diagnosing and classifying these lesions. The biology and evolution of HPV-associated cervical squamous intraepithelial lesions is reviewed, along with recent developments using tyramide-based in situ hybridization and MIB-1 immunoreactivity. It is proposed that HPV DNA integration into the basal cells of cervical squamous epithelium precedes the transformation of low-into high-grade lesions. The HPV DNA tyramide-based in situ hybridization system may prove to be a powerful diagnostic/prognostic tool in this regard. It is also proposed that the presence of mitoses (especially atypical forms) in the upper layers may be a discriminatory hallmark in the morphologic distinction between low- and high-grade lesions. Further, since the biologic changes manifest between these two lesions are reflected in their respective phenotype, it appears plausible to adopt the Bethesda System two-tiered/binary classification of LGSIL and HGSIL for histopathologic diagnoses.

Smoking increases the risk of high-grade vaginal intraepithelial neoplasia in women with oncogenic human papillomavirus

OBJECTIVES In a large retrospective study, the association of smoking with human papillomavirus (HPV) genotype and vaginal intraepithelial neoplasia (VAIN) grade was analyzed. METHODS A SNOMED search was performed for vaginal biopsy or resection specimens diagnosed as VAIN over an 11-year period. The diagnosis of VAIN grade was confirmed by histological review. HPV genotype was determined by GP5+/6+ PCR and dot blot hybridization with type-specific oligonucleotide probes. Smoking history was obtained by chart review. Statistical analysis was performed using the chi-square test. RESULTS We identified specimens from 111 patients (age range 15-84); 64% (n=71) were diagnosed with high-grade VAIN (HGVAIN) and 36% (n=40) with low-grade VAIN (LGVAIN). High-risk (HR) HPV genotypes were identified in 83% of specimens (n=92), other types in 17% (n=19). Twenty-one different HPV genotypes were detected in total. Smoking history was available for 81% (n=90). Forty-one percent (n=37) had a positive smoking history. There was no significant difference in infection with HR vs. other types (p=0.92) among smokers when compared to non-smokers. In patients with HR HPV genotypes, smokers were at an increased risk for HGVAIN lesions when compared to patients who had never smoked (83% vs. 59%, p=0.02). CONCLUSIONS These data indicate an increased risk for HGVAIN in HR HPV positive women who smoke compared to HR HPV positive non-smokers.

False positive diagnosis in conventional and liquid-based cervical specimens.

OBJECTIVE To examine conventional and liquid-based cervical smears falsely diagnosed as malignant at our institution and to investigate, through cytologic-histologic correlation, factors influencing false positive diagnoses. STUDY DESIGN Cervical cytologic diagnoses of malignancy from May 1, 1995, to April 30, 2001, were retrieved through a computer search. A retrospective review of hospital records and pathology reports was performed. Cases identified as false positives were reviewed and correlated with histologic follow-up specimens. RESULTS A group of 68 patients with malignancy reported on cervical smears and with histologic follow-up was identified. Conventional smears numbered 32 (47%); the remaining 36 (53%) were liquid-based samples. Of the total, 7 false positive cases (10.3%) were identified in 4 conventional and 3 liquid-based preparations. Cytologic diagnosis in these cases was squamous cell carcinoma in 5 and adenocarcinoma in 2. On histologic follow-up, all 7 patients were ultimately found to have high grade squamous intraepithelial lesions (HSILs) without invasion. Review of the original slides confirmed most, or all, of the following features in all cases: major cellular pleomorphism, extensive cytoplasmic keratinization, intense nuclear pyknosis, background necrosis and severe atrophy. CONCLUSION There was no significant difference in rates of false positive diagnoses between conventional (12.5%) and liquid-based (8.3%) samples. The chief reason for overdiagnosis in this series was the capacity of HSIL to exfoliate cells mimicking invasive malignancy, particularly when keratinized and especially in an atrophic milieu. The other cause of false positivity was superimposition of inflammation and atypical reparative change on a background of HSIL, which then suggested invasion.

Chlamydia trachomatis in the endometrium: can surgical pathologists identify plasma cells?

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease. The authors examined the relation between chronic endometritis, which they term plasma cell endometritis (PCE), and chlamydial infection using plasmid-based polymerase chain reaction (PCR) and immunohistochemical staining (IHC) of paraffin-embedded endometrial sections. C. trachomatis infection was detected in 5 (24%) of 21 cases of PCE and in 1 (4%) of histologically normal endometrium. The diagnosis of chronic endometritis (with plasma cells confirmed by methyl green pyronin staining) was correctly made in 74% of the cases originally diagnosed as PCE and in 23% of control cases originally diagnosed as normal. The authors conclude that the histopathologic finding of plasma cells in endometrial samples should encourage further examination for chlamydial infection.

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer

BackgroundOvarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression.MethodsProtein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α).ResultsThere was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062).ConclusionsTARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.