Linda Simmons-Arnold

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.

Detection of Sarcocystis Parasites in Retail Beef : A Regional Survey Combining Histological and Genetic Detection Methods

Sarcocystis spp. are parasitic protists acquired when undercooked, cyst-laden meat is consumed. While both Sarcocystis hominis and S. cruzi encyst in beef, only S. hominis is pathogenic to humans. In this study, we used histological methods and novel molecular techniques to determine the regional prevalence and identity of Sarcocystis spp. in retail beef. Of 110 samples, 60 supported amplification of parasite rRNA by PCR. All 41 sequenced representatives were identified as S. cruzi. To compare detection methods, 48 samples were then examined in parallel by histology and PCR, and 16 and 26 samples, respectively, were positive. Five samples positive by initial histologic sections were not amplified by PCR. Fifteen PCR-positive samples did not contain sarcocysts on initial histologic section, but additional sections from these samples revealed sarcocysts in an additional 12 samples. When combined, histology with additional sections and PCR detected 31 positive specimens of the 48 total specimens. We found no evidence of human pathogen S. hominis and confirm that cattle pathogen S. cruzi is highly prevalent in this regional sample. PCR assays may increase the detection sensitivity of Sarcocystis spp. and contribute diagnostic precision.

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.

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.

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.