Multinucleated giant cells' incidence, immune markers, and significance: a study of 172 cases of papillary thyroid carcinoma.

Multinucleated giant cells (MGCs) are often detected in cases of papillary thyroid carcinoma (PTC). Their origin and significance, however, has not been established. One possibility is that they form in response to injury induced by fine needle aspiration biopsy (FNAB). Other hypotheses are that the chemically-altered colloid produced by PTC induces MGCs to act as colloidophages, or else MGCs are a non-specific immune response ingesting neoplastic follicle cells. We assigned 172 cases of PTC a semi-quantitative score for MGCs. Cases with "many" MGCs were immunohistochemically stained for AEI/AEIII, CD68, and CD163 to assess for epithelial vs histiocytic differentiation, and for thyroglobulin and TTF-1 to assess for MGC ingestion of colloid or thyroid follicle cells respectively. Overall, we identified MGCs in 100/172 (58.1%) PTC specimens; in 45 (26.2%), "many" MGCs were found, while in 55 (31.9%) MGCs were "few." The mean sizes of PTC in cases with many as opposed to rare/no MGCs was 2.50 cm vs 1.8 [P = 0.003]. The cases of PTC with many MGCs had higher multifocality (26/45 vs 51/127 [P = 0.06]), extrathyroidal extension (21/45 vs 36/127 [P = 0.03]), and recurrence (8/45 vs 9/127 [P = 0.08]), than did cases with rare or no MGCs. The majority of patients both with and without numerous MGCs had previous histories of FNA or hemilobectomy: 40/45 and 99/127 respectively (P = 0.062). The majority of MGCs were positive for CD68 (45/45), CD163 (44/45), thyroglobulin (34/45) and negative for AEI/AEIII (44/45) and TTF-1 (44/45). These results indicate that MGCs in PTC are of histiocytic origin. Cases of PTC with many MGCs have a significantly greater likelihood of extrathyroidal extension and greater tumor size than cases with few/no MGCs. MGCs appear to be functioning largely as colloidophages.

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.

Clinical and molecular differences between clear cell and papillary serous ovarian carcinoma.

PURPOSE: The aim of the current study is to compare clear cell ovarian carcinoma (CCOC) and papillary serous ovarian carcinoma (PSOC) with respect to their clinical features and expression of different regulators of cell cycle, apoptosis, and chemoresistance. EXPERIMENTAL DESIGN: Women with stage III CCOC (n = 9) and those with stage III, poorly differentiated PSOC (n = 21) seen between 1996 and 2000 and treated with cytoreductive surgery followed by paclitaxel and platinum chemotherapy were compared in their demographic features, tumor marker profile, surgical substage, results of cytoreductive surgery, thromboembolic complications, response to chemotherapy, and tumor recurrence. Tumor samples were compared in their expression of p53, Bcl(2), Bcl(x), Bax, p21, p-glycoprotein (PGP), multi-drug resistance-associated protein (MRP), lung resistance protein (LRP), and glutathione S-transferase (GST) using immunohistochemistry. RESULTS: Women with CCOC had significantly lower mean preoperative CA-125 values, lower surgical substage, less expression of p53, and more expression of p21 than women with PSOC (P = 0.037, 0.012, 0.008, and 0.009, respectively). Women with CCOC had less ascites, smaller amount of residual tumor, higher incidence of thromboembolism, chemoresistance, more expression of Bcl(2), and less expression of PGP than women with PSOC (P = 0.067, 0.078, 0.108, 0.114, 0.091, and 0.118, respectively). CONCLUSIONS: Women with CCOC exhibit certain clinical and molecular differences compared to stage- and grade-matched women with PSOC. Women with CCOC have a smaller tumor volume and manifest different expressions of p53, p21, and Bcl(2) than women with PSOC. Although further studies with larger number of patients are needed, our findings indicate that chemoresistance in CCOC is probably not p53-dependent.

Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus.

BACKGROUND: The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. METHODS: Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng-100 ng) diluted in a background of C-33A DNA (100 ng-2 microg). Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. RESULTS: HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 microg or 1 microg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (approximately 28 copies HPV-16) in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 microg or 1 microg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Multiple infections were also detectable using a touchdown approach. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. CONCLUSION: Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity.

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. p16, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16 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, p16 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). p16 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 p16 staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16 staining showed no evidence of viral integration. Both increased p16 immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16 immunostaining in CIN 1 appears independent of HPV punctate signal type.

Factors associated with cytoreducibility among women with ovarian carcinoma.

OBJECTIVES: The aim of the current study is to investigate the clinical and molecular factors associated with cytoreduction among women with advanced stage epithelial ovarian carcinoma EOC. METHODS: Seventy-two women with FIGO stage III and IV EOC or primary peritoneal carcinoma (PPC) underwent similar attempt at surgical cytoreduction, mostly by the same surgeon. The histologic material of these patients was reviewed and the histologic subtype and grade were assigned. Immunohistochemical tests were performed for expression of molecular regulators of apoptosis (p53, p21, Bcl(2), Bcl(x), Bax) and chemoresistance (PGP, MRP, LRP, GST). The following factors were assessed for their association with complete (no residual tumor) and optimal (residual tumor < 1 cm) cytoreduction: type of carcinoma (EOC versus PPC), stage, CA-125 values, ascites, histology, tumor grade, and p53, p21, Bcl(2), Bcl(x), Bax, PGP, MRP, LRP, GST expression using the odds ratio and associated 95% confidence intervals. Significant univariate odds ratios were assessed jointly in a multivariate logistic regression model. Receiver operating characteristic curve analysis was performed to determine the CA-125 level with the maximal cytoreduction prognostic power. RESULTS: Twenty-three (31.9%) women had no residual tumor, 35 (48.6%) had 1 cm. Factors with significant univariate associations with complete cytoreduction included stage, CA-125 level, ascites, histology, and p53. p53 expression was the only factor which remained significant in the multivariate analysis (odds ratio 7.2, 95% CI 1.5, 34.9). A preoperative CA-125 value of

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.