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This study scans the efficacy of p16 immunostain on gynecological cytologies for the detection of high-grade lesions in ASCUS cases. This is a retrospective study that evaluates p16 immunostaining using frozen material from 109 cases diagnosed as ASCUS and followed for an average of 16 months. The diagnosis of ASCUS was made using liquid-based cytology (Thin Prep, Hologic, Marlborough, MA). High-risk HPV using Hybrid Capture II was performed. The immunohistochemical evaluation of p16 immunoreactivity was done following Wentzensen criteria. In the series p16 immunoreactivity showed a sensitivity and negative predictive value for HSIL, of 82.3 and 94.5% with specificity and positive predictive value of 100%. The sensitivity was similar to that found using the HC-II, though the specificity was much higher. The use of p16 immunostain in cytologic specimens applying the Wentzensen criteria helps to focus the attention in positive cells evaluating the cell changes, helps to detect some cells suspicious or diagnostic of HSIL, and could help to reclassify the ASCUS cases., (Copyright © 2010 Wiley-Liss, Inc.)

Cancer cells undergo significant changes in carbohydrate expression, and these alterations can be useful as biomarkers and therapeutic targets. In this study, we investigated the expression of carbohydrate antigens containing a terminal GalNAcalpha1-3Gal or GalNAcalpha1-6Gal on human cervix and cervical carcinoma. Monoclonal antibodies to each of these carbohydrates were generated by immunizing rabbits with the corresponding antigen conjugated to KLH followed by hybridoma production. Antibodies were screened and evaluated using a combination of carbohydrate microarray profiling, ELISA, dot blot and immunohistochemical staining to verify specificity. Antibody 132-3 was found to selectively recognize GalNAcalpha1-3Gal with little cross-reactivity to other structurally similar antigens such as GalNAcalpha1-6Gal, blood group A, Forssman antigen and the Tn antigen on both solution assays and human tissue. Although GalNAcalpha1-6Gal expression was not detected, GalNAcalpha1-3Gal expression was found on 55% of squamous cell carcinomas. Expression in normal tissue was observed but was restricted to the suprabasal epithelial layer. Importantly, we found expression of the antigen on cervical cancer had a statistically significant correlation with the 5-year survival rate of the patients (48 vs. 85% for antigen negative vs. positive, p = 0.017). Expression of GalNAcalpha1-3Gal did not correlate with other clinical factors including tumor stage, size and lymph node metastasis, indicating the antigen is a new, independent biomarker for the prognosis of cervical cancer., (2009 UICC.)

The objectives of the study were to evaluate 1) the diagnostic sensitivity and specificity of p16(INK4a) as a marker for high-grade cervical lesions, 2) the results of a real-time polymerase chain reaction detecting high-risk human papillomavirus, and 3) the interobserver variability of the p16(INK4a) interpretation.A total of 232 ThinPrep samples were stained for p16(INK4a), and HPV-DNA PCR was performed on 107 specimens with inclusion of both benign and abnormal cytology. Histological follow-up information was collected. The diagnostic sensitivity of ASC+ with CIN2+ in histology as endpoint was 96% for p16(INK4a) and 100% for HR-HPV DNA PCR, and the diagnostic specificity was 41% and 27%, respectively. If p16(INK4a) had been used for triage of the ASC samples, then 18 patients (42%) could have been spared unnecessary follow-up procedures compared to six patients (21%) with the HR-HPV DNA test., ((c) 2008 Wiley-Liss, Inc.)

Background and Objectives: Loss of PTEN expression has been associated with tumor progression and adverse patient outcome. The purpose of this study was to evaluate PTEN expression in the successive steps of progression in cervical neoplasia and to determine its correlation with tumor angiogenesis and clinicopathologic features in squamous cell carcinoma of the uterine cervix., Methods: Immunohistochemical staining with anti-PTEN antibody was performed in a total of 160 patients with 12 normal cervical epithelium, 63 cervical intraepithelial neoplasia (33 CIN I, 30 CIN III), and 85 cervical squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody for CD34. Computerized image analysis was used to evaluate microvessel density (MVD)., Results: Reduced PTEN expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < 0.01). There was no significant correlation between PTEN expression and MVD. On univariate analysis, stage and reduced PTEN expression were significant prognostic factors for both disease-free and overall survival. However, stage was the only independent prognostic factor by multivariate analysis., Conclusions: Our results suggest that tumor progression in the cervical epithelium is accompanied by loss of PTEN protein expression. Reduced PTEN expression is not associated with tumor angiogenesis of squamous cell carcinoma of the uterine cervix.

Cervical/endocervical cytology screening has decreased morbidity and mortality, and implementing adjunctive human papilloma virus (HPV) DNA testing for atypical squamous cells of undetermined significance has improved the specificity for detecting premalignant squamous lesions. Currently, there are no guidelines to perform HPV DNA testing on cervical/endocervical ThinPreps with atypical glandular cells (AGC). To assess the potential role of HPV DNA testing on AGC cases, Hybrid Capture 2 (Digene Corp.) testing was performed on 144 cervical/endocervical AGC specimens. One hundred three of 144 cases had follow-up; 60/103 (58.3%) were high-risk HPV negative and 43/103 (42.3%) were high-risk HPV positive. Of 43 HPV-positive patients, 37 had adenocarcinoma in situ (AIS), atypical squamous cells of undetermined significance (ASCUS), or cervical squamous intraepithelial neoplasia, while only one patient without high-risk HPV had a squamous intraepithelial neoplasia. Furthermore, most high-risk HPV positive AGC cases harbored high-grade squamous intraepithelial lesion (HSIL) rather than AIS. Our data support HPV DNA testing of all AGC specimens to detect cervical, especially squamous, neoplasia., (2006 Wiley-Liss, Inc.)

The hypothesis tested was that there is an association between the presence of proliferating (MiB-1-positive) cervical cells and clinical outcome of women infected with human immunodeficiency virus (HIV). Female partners (attending the Gynecology Outpatients Clinic of the University Hospital of Rio Grande, Brazil) of known HIV-positive (HIV+) men were used for this pilot study. Among these women, 25 were also HIV+. Papanicolaou smears of these 25 HIV+ women and of 44 HIV- women were graded as negative, CIN I, CIN II, or CIN III, using neural network screening. MiB-1 grading and HPV identification were also performed. The immune status of patients was determined using the current Centers for Disease Control classification. In agreement with the scientific literature, in these Brazilian women both CIN and HPV were associated with HIV. In the HIV+ women, the immune status tends to correlate with MiB-1 grading. Also, in the one case in whom progression from CIN I to invasive cervical carcinoma was observed, the smear contained many MiB-1-positive cells. Staining cervical smears of HIV+ women is a simple procedure to get an indication of clinical outcome of the patient., (Copyright 2001 Wiley-Liss, Inc.)

A case of granulocyte colony-stimulating factor (G-CSF)-producing small-cell carcinoma of the uterine cervix is described. In a 70-yr-old woman, clusters of small cells with hyperchromatic nuclei at a high nuclear/cytoplasmic ratio were detected cytologically in the cervix. These clusters were diagnosed as a small-cell neuroendocrine carcinoma by the concomitant use of Grimelius staining and immunohistochemical staining, in addition to electron microscopic observation. This patient showed a significant increase in peripheral leukocytes, despite the absence of infectious signs. Immunohistochemical findings, together with a high blood G-CSF level, suggested the production of G-CSF from the tumor. Consistent with the knowledge that both small-cell carcinoma and G-CSF-producing tumors have a poor prognoses, the patient had no or partial response to therapies performed, and died from the cancer 11 mo after it was diagnosed. This case strongly indicates the need for early diagnosis of this type of tumor, based on cytological features., (Copyright 2000 Wiley-Liss, Inc.)

High-risk human papillomaviruses (HPV), particularly HPV 16, are associated with invasive cervical cancer (ICC), and persistent high-risk HPV infection is considered to be a marker for progressive cervical intra-epithelial neoplasia (CIN). However, most high-risk, HPV-infected, pre-cancerous lesions will not progress to invasion. Several reports suggest that specific HPV 16 E6 and/or E7 sequence variations may be associated with a high risk for progression. No data from German patients have so far been reported. Therefore, we analyzed intra-type variations of these oncogenes in women with normal histology or CIN 1 (< or = CIN 1), CIN 2/3 or ICC. Cervical scrapes from 75 patients with normal histology or CIN and biopsies from 37 ICC patients all positive for HPV 16 were analyzed. The open reading frames of oncogenes HPV 16 E6 and E7 were amplified by nested PCR followed by primer cycle sequencing. From each cervical scrape, 2 independent PCR amplicons were generated and sequenced from both orientations. The prototype sequence of HPV 16 E6 and E7 was identified in 33% and 87% of < or = CIN 1, in 62% and 69% of CIN 2/3 and in 43% and 86% of ICC, respectively (not significant). Of all variants identified, the E6 variant 350G (L83V) and the E7 variant 822G were most frequently detected irrespective of histology and showed prevalence rates of 27% to 43% and 7% to 20%, respectively. No statistically significant differences in the prevalence of the E6 or E7 prototype sequences, any variants or multivariants in German women with < or = CIN 1, CIN 2/3 or ICC were found.

Background and Objective: Fluorescence spectroscopy has been shown to provide information useful in the detection of cervical dysplasia. The goal of this study was to determine if substances found on the cervix such as acetic acid, mucus, and vaginal medications can influence the fluorescence in the spectral region useful for discriminating normal cervical tissue from abnormal tissue., Study Design/materials and Methods: Fluorescence spectra were collected at 337 nm excitation from the cervix in vivo both before and after application of acetic acid; the data were analyzed to identify the effects of the acetic acid on the spectra. Cervical mucus was acquired from patients referred for colposcopy and frozen until measurements were taken. Fluorescence excitation-emission matrices (EEMs) were measured for the mucus samples. Additionally, the transmission spectra of mucus were measured to determine if its absorption could influence the fluorescence signal measured from the tissue. EEMs were measured for samples of commonly prescribed vaginal medications. All EEMs were compared to those of cervical biopsies., Results: Acetic acid introduces changes in both the lineshape and intensity of the spectra. On average, the changes are more significant in spectra of abnormal tissue. Cervical mucus was found to have no significant absorption bands, but the measured fluorescence was approximately the same order of magnitude as that measured from the cervix in vitro. Most medications exhibited significant fluorescence in the spectral region of diagnostic interest for the cervix., Conclusions: Acetic acid appears to increase the differences in fluorescence emission spectra of normal and pre-cancerous cervical tissues; thus, its use is beneficial. The presence of cervical mucus can possibly interfere with the collection of fluorescence spectra for tissue classification. Patients should not use vaginal preparations during the 48 hours prior to tissue fluorescence measurements., (Copyright 1999 Wiley-Liss, Inc.)

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears.

The factors responsible for the initial interaction between maternal and fetal epithelium leading to the establishment of pregnancy remain poorly understood. Temporal and spatial expression of specific endometrial peptides in response to ovarian steroids is thought to contribute to the development of a period of uterine receptivity, whereby the endometrium becomes hospitable to the implanting blastocyst. The failure to establish receptivity may account for a significant percentage of the cases of infertility in the female, especially affecting women with luteal phase deficiency, leiomyomata uteri, endometriosis, habitual abortion, and unexplained infertility. In addition, despite increasing global experience with advanced reproductive technologies, the majority of In Vitro Fertilization (IVF) attempts remain unsuccessful, most likely on the basis of implantation failure. In this article, we review the concepts involved in the study of uterine receptivity in the human, highlight potential immunohistochemical (IHC) markers that have recently been discovered, and discuss how IHC assessment of the endometrium is a potentially valuable method for the evaluation of the receptive endometrial state. Using this approach we have examined several new potential markers of uterine receptivity. Endometrial progesterone receptors and one of the integrin cell adhesion molecules appear to undergo changes in expression around the time of implantation, and may be sensitive indicators of the receptive state. Further, these markers are delayed in women with infertility and luteal phase deficiency. These studies illustrate the utility of IHC diagnosis for the evaluation of endometrial function.

The polymerase chain reaction (PCR) was used to investigate the prevalence and distribution of human papillomavirus (HPV)-16 DNA in paraffin sections of all pelvic lymph nodes removed from 14 patients with Stage Ib-cervical cancer at the time of resection of their primary tumours. The results were compared with those obtained from 8 women with no known history of cervical abnormality. In all, 22 cervical biopsies and 40 I lymph nodes (296 paraffin blocks) were examined. Nine of the 14 cervical cancer patients had primary tumours that were positive for HPV-16 DNA: only 3 of these had lymph nodes with histological evidence of metastasis, and HPV 16 DNA was detected in each of the corresponding paraffin blocks. HPV 16 DNA was also detected in varying proportions (8%-92%) of the histologically-negative lymph nodes from these women. There was no correlation between the HPV DNA-positive lymph nodes and their proximity to the primary tumour. HPV-16 DNA was not identified in any of the lymph nodes from the 5 women whose cancers were not HPV-16-related, or in those of women with no evidence of cervical abnormality. This preliminary survey suggests that HPV DNA is frequently transported from HPV-16-related cervical tumours to regional lymph nodes. However, its practical significance will not be clear until sufficient time has elapsed for correlation of the results with the clinical outcome.

We investigated the immunohistochemical localization of parathyroid hormone-related protein (PTHrP), a major factor responsible for the humoral hypercalcemia of malignancy, in uterine cervical lesions. Formalin-fixed paraffin-embedded specimens from 16 cases of normal and reactive conditions, 45 cases of cervical intra-epithelial neoplasm (CIN) and 63 cases of invasive cancer were studied immunohistochemically by the avidine-biotin-peroxidase method, using an anti-PTHrP monoclonal antibody (MAb), 4B3. In normal and reactive conditions, PTHrP was positive in parabasal cells, squamous metaplasia, and hyperplastic reserve cells. In neoplastic conditions, 96% (43/45) of invasive squamous-cell carcinomas were positive for PTHrP, regardless of the patients' serum calcium levels. Two cases with hypercalcemia were strongly positive for PTHrP and showed prominent stromal interaction of the scirrhous type. In CIN, including koilocytic atypia, 76% (32/42) of cases were positive for PTHrP. In contrast, 91% (10/11) of adenocarcinomas were negative for PTHrP. In conclusion, we found, first, that in non-neoplastic conditions, the presence of PTHrP was correlated with the transformation of progenitor cells into squamous epithelia and with the maturation of keratinocytes and, second, that in squamous-cell carcinoma, the degree of keratinization and stromal interaction was higher in direct proportion to the apparent incidence of detectable PTHrP.

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.

Cytomorphology, mucin stains, and immunocytochemistry were used to determine the characteristics of intracytoplasmic vacuoles in cervical smears and their relationship to chlamydial infections. Three study groups were examined: A) cervical smears containing intracytoplasmic vacuoles suggesting chlamydial infection: B) cervical smears from patients known to be either culture positive or culture negative for chlamydia; and C) control smears of chlamydia-infected McCoy cell cultures. Forty-nine smears containing vacuoles suspected of being chlamydial inclusions were studied. In 21 of 31 smears the vacuoles were shown by histochemistry to contain mucin. Immunoperoxidase staining performed on the remaining 18 smears revealed only two vacuoles to be chlamydial inclusions. Of 38 smears from chlamydia-culture-positive patients, only 4 showed chlamydial inclusions by immunoperoxidase methods. Of 30 smears from chlamydia-culture-negative patients, none were positive by immunoperoxidase staining. The "nebular body" of Shiina appears to be the most specific cytomorphologic evidence of chlamydial infection in cervical smears, but distinction from mucin vacuoles and other nonspecific vacuoles is difficult. The immunoperoxidase technique is unlikely to be useful in diagnosis in cervical smears unless samples are specifically obtained from the endocervical canal by endocervical brushing.