Immunohistochemical expression of p16INK4a and bcl-2 according to HPV type and to the progression of cervical squamous intraepithelial lesions.

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.     

Diverse glandular pathologies coexist with high-grade squamous intraepithelial lesion in cyto-histological review of atypical glandular cells on ThinPrep specimens

Objective: To identify in cytology, high‐grade squamous intraepithelial lesions with endocervical glandular extension in cases previously diagnosed as atypical glandular cells (AGC), analyse possible reasons for the diagnostic pitfall and document the frequency of glandular pathology coexisting with high‐grade cervical intraepithelial lesion in histology. Methods: Thirty‐nine ThinPrep® cervical smear (Pap) tests reported as AGC of undetermined significance and showing high‐grade lesions on histology [cervical intraepithelial neoplasia (CIN) 2 or 3, endometrial or extrauterine adenocarcinoma] were reviewed retrospectively to identify the cases of high‐grade squamous intraepithelial lesion with endocervical glandular extension, using the Bethesda 2001 system. Cyto‐histological correlation was performed. Results: A high frequency of diverse glandular pathologies coexisted with high‐grade cervical intraepithelial lesions on histology. This included endocervical glandular extension in 63%, benign glandular pathology in 33% and pre‐neoplastic or malignant glandular pathology (endocervical glandular dysplasia, adenocarcinoma in situ and metastatic breast carcinoma) in 17% cases. On cytology, the sensitivity was 40%, specificity was 80% and positive predictive value was 86% for endocervical gland extension in high‐grade squamous intraepithelial lesions. Conclusions: Special efforts to recognize endocervical glandular extension in high‐grade squamous intraepithelial lesions and glandular neoplasia coexisting with squamous intraepithelial lesions from the heterogeneous category of AGC can contribute to increasing the diagnostic accuracy. The identification of endocervical glandular extension on cervical cytology would alert the gynaecologist to perform a thorough assessment of the endocervix during colposcopy. This could also help to decide on the need to perform deeper conization rather than loop electrosurgical excision procedure to ensure negative margins when colposcopic biopsy shows CIN 2 or 3.     

Observer variation in histopathological diagnosis and grading of cervical intraepithelial neoplasia.

To assess the variability among histopathologists in diagnosing and grading cervical intraepithelial neoplasia eight experienced histopathologists based at different hospitals examined the same set of 100 consecutive colposcopic cervical biopsy specimens and assigned them into one of six diagnostic categories. These were normal squamous epithelium, non-neoplastic squamous proliferations, cervical intraepithelial neoplasia grades I, II, and III, and other. The histopathologists were given currently accepted criteria for diagnosing and grading cervical intraepithelial neoplasia and asked to mark their degree of confidence about their decision on a visual linear analogue scale provided. The degree of agreement between the histopathologists was characterised by kappa statistics, which showed an overall poor agreement (unweighted kappa 0.358). Agreement between observers was excellent for invasive lesions, moderately good for cervical intraepithelial neoplasia grade III, and poor for cervical intraepithelial neoplasia grades I and II (unweighted kappa 0.832, 0.496, 0.172, and 0.175, respectively); the kappa value for all grades of cervical intraepithelial neoplasia taken together was 0.660. The most important source of disagreement lay in the distinction of reactive squamous proliferations from cervical intraepithelial neoplasia grade I. The histopathologists were confident in diagnosing cervical intraepithelial neoplasia grade III and invasive carcinoma (other) but not as confident in diagnosing cervical intraepithelial neoplasia grades I and II and glandular atypia (other). Experienced histopathologists show considerable interobserver variability in grading cervical intraepithelial neoplasia and more importantly in distinguishing between reactive squamous proliferations and cervical intraepithelial neoplasia grade I. It is suggested that the three grade division of cervical intraepithelial neoplasia should be abandoned and a borderline category introduced that entails follow up without treatment.     

Karyometric image analysis for intraepithelial and invasive cervical lesions

OBJECTIVE: To derive an objective, numeric measure for the progression of intraepithelial and invasive squamous cell cervical lesions. STUDY DESIGN: Thin-layer cervical cytology preparations from colposcopically confirmed normal cervix, low grade squamous intraepithelial lesions, high grade squamous intraepithelial lesions and carcinoma were identified from a cross-sectional study. Fifty-nine cases representing 4 diagnostic categories were selected, and 2,375 nuclei from epithelial cells representative of the diagnostic category were randomly selected for imaging and measurement from these cases. Additionally, 1,378 visually normal appearing intermediate cells from low and high grade squamous intraepithelial lesions, as well as from carcinoma cases, were identified for analysis. The nuclei were quantitatively characterized, and discriminant analyses were performed to derive a progression curve from normal cytology to carcinoma. RESULTS: The lesion signatures show a clear increase in nuclear abnormality with increasing progression. A progression curve was derived based on mean discriminant function scores for each diagnostic category and on the mean nuclear abnormality values for the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. CONCLUSION: A numeric assessment of lesion progression for cervical precancerous and cancerous lesions based on karyometric measurements is possible and may provide an objective, precise characterization of each lesion as well as a basis for improved performance in automated cytology-based cervical cancer screening.     

Proliferating activity in oral dysplastic leasions and squamous cell carcinomas

The aim of the study was the quantitative analysis of AgNORs in oral squamous cell carcinomas as well as in dysplastic epithelial changes accompanied and not accompanied by oral squamous cell carcinomas. AgNOR proteins were visualized in histological slides using silver impregnation technique according to D. Ploton. In each sample 100 cell nuclei were assessed. The study used 54 cases of proliferating oral epithelial changes divided into 3 groups: group I consisting of 13 cases of dysplastic lesions not accompanied by oral squamous cell carcinomas; group II (a total of 18 cases) containing dysplastic lesions situated in the vicinity of oral carcinomas and group III (23 cases) with oral squamous cell carcinomas. Statistically significant differences were found between groups with mild dysplasia and groups with severe dysplasia as well as squamous cell carcinomas. Statistical analysis did not show any differences in the number of AgNORs between squamous cell carcinomas and epithelial lesions with severe dysplasia. Our results demonstrate that the analysis of AgNORs expression can serve only as an additional parameter to evaluate the potential of malignant transformation.     

Preclinical feasibility study of NMP179, a nuclear matrix protein marker for cervical dysplasia

OBJECTIVE: To evaluate, in a preclinical feasibility study, the efficacy of NMP179, a monoclonal antibody recognizing a cervical tumor-associated nuclear matrix antigen, for the early detection of high and low grade cervical intraepithelial neoplasia. STUDY DESIGN: In a blind study involving two clinical sites, NMP179 immunocytochemical staining data from 261 cervicovaginal Thin-Prep specimens were evaluated. Assay sensitivity and specificity were calculated based upon a positive threshold of > 10 immunostained cells per case, using cytologic diagnosis as an end point. RESULTS: Based upon the examination of squamous epithelial cells, NMP179 detected 96.7% of cases with cytologically diagnosed high grade squamous intraepithelial lesions (HSIL) and 70.5% of low grade squamous intraepithelial lesions. The antibody also reacted with 29.6% of normal (within normal limits or benign cellular changes) smears. CONCLUSION: The NMP179 assay detected HSIL with very high accuracy (96.7%). The assay was 79.3% sensitive for the detection of low and high grade cervical intraepithelial neoplasia (grades 1-3), with a specificity of 70.4%. NMP179 may be an effective marker for the early detection of preneoplastic squamous intraepithelial lesions of the cervix and may be useful as an adjunctive tool for better management of cervical intraepithelial neoplasia.     

The significance of Epstein Barr Virus (EBV) & DNA Topoisomerase II alpha (DNA-Topo II alpha) immunoreactivity in normal oral mucosa, Oral Epithelial Dysplasia (OED) and Oral Squamous Cell Carcinoma (OSCC)

Background

The glycosylation of a great number of molecules, glyco-protein or glycolipids, has been of interest for decades.

Objective

To compare the expressive patterns of the isoantigenic determinants of histo-blood groups ABH and Lewis in squamous and simple epithelium and in precursors and cancers of the cervix.

Methods

A total of 36 lesions and neoplasms (10 LG-SIL, 16 HG-SIL and 10 invasive carcinomas) have been studied with immunohistochemical techniques, using monoclonal antibodies (MoAb BG1 to BG8) for precursor chains, blood-group ABH and Lewis group Le^a, Le^b, Le^x, and Le^y, and four types of lectins. In addition, we have studied the expression of p53 protein and PCNA, establishing the rate of proliferation of each lesion. Using PCR techniques, we have also detected part of the intron of the E6 gene of HPV-16.

Results

In the invasive cervical carcinomas, we observed a loss of expression of the Le^x antigen (p < 0.01). With regard to the progression of the different lesions studied, we found alterations in the patterns of expression of the antigens of the ABH and Lewis blood groups. There was a tendency towards a loss of expression and heterogeneous patterns in the more advanced lesions, as well as over-expression of the Le^y antigens. With PCNA, we established a proliferative rate which tended to be greater in relation to the progression of the cervix neoplasms.

Conclusion

These results indicate that there is a relation between the losses of histo-blood groups and the progression of the squamous intraepithelial lesions.     

Cervical squamous intraepithelial lesions and associated cervical infections in an HIV‐positive population in Rural Mpumalanga,South Africa

P. J. Swanepoel, P. Michelow, R. Du Plessis, I. G. Proudfoot, G. A. Tarr, S. L. Bockel, C. J. Swanepoel
Cervical squamous intraepithelial lesions and associated cervical infections in an HIV‐positive population in Rural Mpumalanga, South Africa Background: The incidences of genital human papillomavirus (HPV) infection, associated squamous intraepithelial lesions and cervical squamous cell carcinoma are significantly increased in HIV‐positive women. The role of other cervicovaginal infections in the acquisition of the HPV infection, cervical carcinogenesis and genital HIV infection remains largely speculative. Methods: A retrospective study was conducted including 1087 HIV‐positive women in rural Mpumalanga province, South Africa, for the period 1 May 2009 to 31 August 2010. For each patient, the age at first presentation, cervical cytological diagnosis, subsequent follow‐up cytology and histology, and microscopically visible infections (including endemic Bilharzia) were tabulated and statistically analysed. Results: The prevalence of low‐grade squamous intraepithelial lesion (LSIL), high‐grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma, atypical squamous cells of undetermined significance (ASC‐US) and atypical squamous cells, cannot exclude HSIL (ASC‐H) in the study population were 22.1%, 30.9%, 0.6%, 13.5% and 4.0%, respectively. LSIL, HSIL and squamous cell carcinoma were diagnosed, respectively, at the average ages of 35.7, 37.9 and 37.2 years. Four patients with cervical intraepithelial neoplasia grade 1 (CIN1), 32 with CIN2/CIN3 and two with cervical squamous cell carcinoma were also diagnosed with Bilharzia. Of the other infections only bacterial vaginosis had a positive statistical correlation with HPV‐induced cervical abnormalities (LSIL, HSIL or squamous cell carcinoma). Conclusion: This study confirms the high prevalence of progressive HPV‐associated cervical disease in a rural Southern African HIV‐positive population, which is at least equal to or worse than in other African HIV‐positive studies. The high incidence of Bilharzia infection in those cases that underwent cervical cone excision suggests a possible relationship with progressive HPV disease and cervical carcinogenesis. Bacterial vaginosis (perhaps in combination with Bilharzia) may compromise the normal barriers against HPV and HIV infection.     

Dimethylhydrazine: a reliable positive control for the short sampling time in the UDS assay in vivo

Cervical cancer represents the second most common malignant neoplasia in women world-wide. In Mexico, cervical cancer is the most common female malignancy. It has been recently seen an increased frequencies of micronuclei (MN) lymphocytes and cervical epithelial cells of cervical cancer patients. The aim of this hospital-based unmatched case-control study was to investigate the association between progressive stages in development of cervical cancer and frequency of micronucleated cells in the cervical epithelium and peripheral lymphocytes of 40 women, grouped by disease stage. Women at the Obstetrics and Gynecology Hospital of the Instituto Mexicano del Seguro Social (IMSS) in Monterrey, Mexico were diagnosed and classified on the bases of the Papanicolaou (PAP) smear and colposcopy/biopsy into control, low-grade squamous intraepithelial lesions (LGSIL), high-grade squamous intraepithelial lesions (HGSIL), and invasive groups. Analysis of the MN data in both cell types revealed (a) homogeneity among women within each of the four groups with regard to MN frequency, (b) in general, a correlation between MN frequency and grade of cervical lesion, and (c) a positive linear trend between the MN frequency and increased cervical cancer risk. In conclusion, we suggest that MN are a useful biomarker of cancer risk. Nonetheless, these results should be validated by other researchers.     

Micronuclei as biomarkers for evaluating the risk of malignant transformation in the uterine cervix

We evaluated micronucleus and apoptosis occurrence among women with normal smears and women with different kinds of cervical abnormalities, i.e., inflammatory processes and low- and high-grade squamous intraepithelial lesions (N = 12, N = 10 and N = 27, respectively). The sample included 59 women who were seen at a public medical service for cervical cancer prevention in Feira de Santana, Bahia, Brazil. The diagnosis was established by means of cytological, colposcopic, and histopathological examination. Cytogenetic analysis was performed on 2000 cells from each woman and included assessment of micronuclei and nuclear degenerative abnormalities indicative of apoptosis (karyorrhexis, pyknosis and condensed chromatin). Micronucleus frequency was significantly higher in the women with high-grade squamous intraepithelial lesions than in the women without cervical abnormalities or inflammatory processes (P< 0.001) or in the women with low-grade squamous intraepithelial lesions (P < 0.005). The frequency of apoptosis was similar in women without cervical abnormalities and women showing high-grade squamous intraepithelial lesions (P > 0.50), and significantly lower in women without cervical abnormalities and in women showing high-grade squamous intraepithelial lesions than in women showing inflammatory processes or low-grade squamous intraepithelial lesions (P < 0.0001). These results indicate that, in addition to Papanicolaou cervical cytological analysis, it would be useful to use micronucleus analysis to screen women who are at risk of developing cervical cancer. The assessment of nuclear degenerative abnormalities indicative of apoptosis increased the sensitivity of this test.     

Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US,ASC‐H,LSIL and HSIL)

G. Bigras, J. Wilson, L. Russell, G. Johnson, D. Morel and M. Saddik
Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US, ASC‐H, LSIL and HSIL) Objectives: Given the well‐known poor reproducibility of cervical cytology diagnosis, especially for atypical squamous cells of undetermined significance (ASC‐US) and low‐grade squamous intraepithelial lesion (LSIL), this study surveyed reproducibility in the assessment of individual cytomorphological features. Methods: One hundred and fifty cells or groups of cells, with a variety of morphological appearances, including normal cells, high‐grade squamous intraepithelial lesion (HSIL), LSIL, ASC‐US and ASC cannot exclude HSIL (ASC‐H), were precisely marked on 150 different liquid‐based cytological preparations. They were analysed by 17 observers who assessed 17 cytological features including nuclear features (chromatin texture, nuclear outline, nuclear shape, etc.), cytoplasmic features (cell shape, cytoplasmic staining, cytoplasmic clearing, etc.) and group characteristics (nuclear polarity, cellular density, etc.). A total of 43 350 data scores were collected in a database using a web‐based survey. Kendall’s W and relative entropy indexes were utilized to compute concordance indexes of respectively ordinal and nominal variables. Results: Nuclear features have significantly lower reproducibility (0.46) compared with other cytological features (0.59). The feature with least agreement is assessment of chromatin texture. A small but significant difference in concordance was found between two subsets of observers with different levels of experience. Conclusion: Most previous studies assessing reproducibility of cytological diagnoses show, at best, moderate reproducibility among observers. This study focused on agreement regarding the presence of constituent morphological features used to recognize dyskaryosis and various grades of squamous intraepithelial lesions. A map of reproducibility indexes is presented that highlights, for daily practice or teaching, the robustness of features used for cytological assessment, recognizing that diagnosis is always based on a combination of features.     

Guest Lecture 8.45–9.30 Monday 15 September 2003 Endocervical Adenocarcinoma and its Precursors

The incidence of malignant and pre‐malignant endocervical glandular lesions is increasing. Part of this is an apparent increase due to a reduction in the number of invasive cervical squamous carcinomas but there is evidence that there is a real increase in malignant and pre‐malignant endocervical glandular lesions. Different terminologies are in use in the UK where the term cervical glandular intraepithelial neoplasia (CGIN) is commonly used and the rest of the world where pre‐malignant lesions are classified as glandular dysplasia and adenocarcinoma in situ (AIS) (WHO classification). It is well established that high‐grade CGIN (AIS in WHO terminology) is a precursor lesion of cervical adenocarcinoma but it is controversial whether a recognizable precursor to high grade CGIN (namely low‐grade CGIN) exists and criteria for diagnosing this are poorly established and poorly reproducible. Most cases of CGIN are of usual or endocervical type but other morphological subtypes described include endometrioid, intestinal, tubal and stratified mucinous intraepithelial lesion (SMILE). The presence of skip lesions and lesions high up the endocervical canal has been overemphasised in CGIN with most cases occurring close to the transformation zone. Treatment is on an individualized basis but local excision with negative margins and close cytological follow‐up may be employed. There is evidence in the literature that early invasive adenocarcinomas behave in a similar fashion to early invasive squamous carcinomas and that, on selected occasions, conservative therapy can be safely undertaken. However, further studies are needed to ascertain the behaviour and natural history of early invasive cervical adenocarcinoma. In 10%–15% of cases it may be impossible to ascertain whether a malignant endocervical glandular lesion is invasive or in situ. There are many benign mimics of CGIN and adenocarcinoma, including tuboendometrial metaplasia (TEM), endometriosis and microglandular hyperplasia (MGH). Although careful morphological examination usually allows confident distinction of these lesions, a panel of immunohistochemical stains including MIB1, bcl2 and p16 may assist.     

Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions

D. Demirel, N. Akyürek and I. Ramzy
Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions Objective: To study the DNA ploidy pattern of uterine cervical squamous intraepithelial lesions (SILs) and its diagnostic and prognostic significance. Methods: The study included 31 cases of SIL: 11 low‐grade (LSIL) and 20 high‐grade (HSIL). Feulgen–pararosaniline staining was performed on previously Papanicolaou‐stained smears and a DNA image cytometric study was performed. An internal reference was used to calibrate the samples. Results: All 31 cases of SIL, either LSIL or HSIL, were non‐diploid. Of the 11 cases of LSIL, four were tetraploid and seven were aneuploid, whereas, of the 20 cases of HSIL, four were tetraploid and 16 were aneuploid. Stemline aneuploidy was not a significant discriminator between LSIL and HSIL (P = 0.32). Based on single‐cell analysis, HSIL cases had significantly higher DNA content than LSIL cases (P < 0.01). When a mean of 30% or more was used for the 6c‐exceeding event (6cEE) value, the sensitivity and specificity to indicate HSIL were 83% and 64%, respectively, with a positive predictive value (PPV) of 81% and negative predictive value (NPV) of 65%. All HSIL cases were cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on biopsy. In addition, cases which showed recurrence had more DNA content by single‐cell analysis than those with an indolent clinical behaviour: P = 0.04 and P = 0.03 for LSIL and HSIL, respectively. Conclusions: Image cytometric DNA analysis is a useful technique for diagnostic and prognostic purposes in uterine cervical SIL when appropriate ‘c’ values are used in single‐cell analysis. We propose that a >6c DNA content of 30% is useful as a cut‐off level for predicting cases with CIN2+ in DNA image cytometry of cervical smears.     

Natural history of cervical intraepithelial neoplasia: a meta-analysis

OBJECTIVE: To determine the probabilities of transition of stages in the cervical cancer by conducting a meta-studies on the topic. STUDY DESIGN: We identified health states of interest in the natural history of cervical precancer, identified all possible papers that could meet selection criteria, developed relevance and acceptability criteria for inclusion, then thoroughly reviewed the selected studies. To determine the transition probability data we used a random effects model. We determined probabilities for 4 health state transitions. The 6-month mean predictive transition probability (95% confidence intervals with "prediction interval" in parentheses) for high grade squamous intraepithelial lesions (HSIL) to cancer was 0.0037 (0.00004, 0.03386), for low grade squamous intraepithelial lesions (LSIL) to HSIL was 0.0362 (0.00055, 0.23220), for HSIL to LSIL was 0.0282 (0.00027, 0.35782), and for LSIL to normal was 0.0740 (0.00119, 0.42672). CONCLUSION: The transition probabilities between cervical cancer health states for 6-month intervals are small; however, the cumulative risk of cervical cancer is significant. Markers to identify the cervical precursors that will lead to the transition to cervical cancer are needed.     

The detection of endocervical gland involvement by high grade squamous intraepithelial lesions in smears prepared from endocervical brush specimens

The cytologic features of squamous cell carcinoma in situ with endocervical gland involvement have been described in cervical smears. We evaluated the presence of two types of cellular fragments in 43 cervical smears of high grade squamous intraepithelial lesions (HGSIL) to assess their ability to predict glandular involvement by HGSIL in subsequent cone biopsies. an endocervical brush was used to obtain all endocervical specimens. of 16 cases without glandular involvement, fragments were present in 13 smears. of 27 cases with glandular involvement, fragments were absent in 11 smears. No statistical association was identified between the presence of abnormal cellular fragments on cervical smears of HGSIL and endocervical gland involvement on cone biopsies.     

Opportunistic testing of medically underserved women for cervical cancer in South Africa

OBJECTIVE: To determine the yield of opportunistic Pap smears taken in an unscreened and medically underserved population in the Transkei Region of South Africa. STUDY DESIGN: Cross-sectional study of 22,160 cervical cytology specimens from an unscreened population attending gynecologic outpatient clinics between January 1990 and December 1996. RESULTS: The overall prevalence of atypical squamous cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL) was, respectively, 34.7%, 8.3% and 2.4%. The ASCUS: SIL ratio was 3:1. The prevalence of invasive squamous cell carcinoma was 1.6%. The yield of opportunistic Pap smears was 10.7% including only LSIL and HSIL. CONCLUSION: The pathologic process of precursor lesions of cervical cancer appears to start at an early age since > 20% of cases are diagnosed before the age of 30 years. In the absence of a national screening program, opportunistic testing of medically underserved women needs to be maintained and encouraged.     

Polymorphism of Ag(+)-NORs in cervical smears from women with cervical cancer

OBJECTIVE: To evaluate Ag(+)-stained (Ag(+)-NOR) polymorphism in four groups of patients with various grades of cervical lesions and in a control group. STUDY DESIGN: Forty-five women were selected, diagnosed and classified on the bases of the Pap smear and colposcopy/biopsy at Hospital de Ginecologia y Obstetricia del Instituto Mexicano del Seguro Social in Monterrey, Mexico. Five categories were considered: (1) inflammatory, (2) low grade squamous intraepithelial lesions (LSILs), (3) high grade squamous intraepithelial lesions (HSILs), (4) invasive cervical cancer, and (5) normal. The cervical smears were stained by the Ag(+)-NOR method. One hundred cells per slide were counted and classified according to the polymorphism of Ag(+)-NOR dots: typical (spherical) and atypical (large, kidney shaped and clustered). The four shapes of Ag(+)-NORs were quantified by percentage and transformed using the arcsine root procedure. RESULTS: Statistical analysis showed a significant decrease in spherical shape according to neoplastic development. The three atypical shapes showed a significant increase in patients with HSIL and invasive carcinoma in respect to LSIL. Principal components analysis grouped the data at five locations in the plane formed by the first two principal components according to the diagnosis. CONCLUSION: These findings suggest the potential diagnostic and prognostic value of the determination of Ag(+)-NOR polymorphism in cervical cytology studies.     

Clinical significance of atypical glandular cells of undetermined significance. A follow-up study from an academic medical center

OBJECTIVE: To determine the rate of atypical glandular cells of undetermined significance (AGUS) and the incidence of subsequent clinically significant lesions. STUDY DESIGN: A computer-based search of our cytology laboratory files was performed for cervicovaginal smears diagnosed as AGUS from January 1996 to December 1996. RESULTS: In 43,456 cervicovaginal smears examined during the 12-month period, AGUS was reported in 222 (0.5%) cases, with follow-up in 191 (86.0%) (133 [59.9%] biopsies and 58 [26.1%] repeat cervicovaginal smears). Among the patients with repeat cervicovaginal smears, 1 (1.7%) had a high grade squamous intraepithelial lesion, and 10 (17.2%) had persistent AGUS/atypical squamous cells of undetermined significance; the remainder were within normal limits. Thirty-three (24.8%) patients had preneoplastic or neoplastic, squamous or glandular lesions on biopsy (8 [6.0%] cervical intraepithelial neoplasia [CIN] 1, 18 [13.5%] CIN 2/3 and 7 [5.3%] endometrial adenocarcinomas). Half the patients with CIN 2/3 also had evidence of endocervical gland involvement. Squamous lesions were seen more commonly in premenopausal women, while glandular lesions were noted predominantly in postmenopausal women. Patients with a prior abnormal gynecologic history or a concomitant diagnosis of squamous intraepithelial lesion (SIL) had a higher incidence of significant lesions on subsequent biopsy. CONCLUSION: Our incidence of AGUS was 0.5%, similar to that in other published reports. AGUS is associated with a significant number of squamous or glandular, premalignant or malignant lesions. A majority of these lesions are high grade SIL, often with endocervical gland involvement. A small but significant number of patients had a glandular malignancy. Our results justify close and persistent follow-up for patients with a diagnosis of AGUS on cervicovaginal smears.     

Loss of heterozygosity at chromosome 6 as a marker of early genetic alterations in cervical intraepithelial neoplasias and microinvasive carcinomas

Oncogenic human papillomaviruses (mostly HPV types 16 and 18) are the major cause of cervical intraepithelial neoplasia (CIN), which progresses into cervical cancer (CC). To reveal early genetic alterations of chromosome 6 that are important for CC progression, we analyzed the loss of heterozygosity (LOH) in DNAs from 45 CIN cases, 47 microcarcinomas, and 19 invasive squamous cell carcinomas stage IB. LOH analysis of DNA samples prepared with microdissection from all CIN foci, as well as from CC lesions and synchronous CIN, permitted investigation of CIN and CC heterogeneity. Out of all CC stage I cases, 79% showed LOH with six microsatellite markers at chromosome 6. LOH with the microsatellite markers D6S276 (6p22) and TNFa (6p21.3) was found in 50% of the CC cases. LOH frequency in CIN lesions synchronous with CC was higher then in CIN cases without cancer; the statistical significance (P = 0.004) was shown for D6S291 (6p21.2). The finding suggests that the high frequency of LOH in CIN lesions is a marker of unfavorable prognosis for CIN. Progression from microcarcinoma to invasive CC of stage IB was associated with a higher LOH frequency at D6S344 (6p25) and TNFa (6p21.3). Early genetic alterations were found in CIN with microsatellites D6S273 and TNFa located at 6p21.3. Moreover, LOH frequency at D6S273 remained the same in both CIN and CC cases. Based on HPV typing, LOH analysis, and X-chromosome inactivation, the polyclonality of CC lesions, as well as CIN, was observed in a few patients.     

Comparison of the conventional cervical smear and liquid-based cytology: results of a controlled, prospective study in the Abruzzo Region of Italy

OBJECTIVE: To compare conventional cervical testing (CCT) and liquid-based cytology (LBC) within a randomized trial performed during 2001-2002 in the Abruzzo Region of Italy, including a cost-outcome comparative analysis. STUDY DESIGN: Study subjects were recruited in the framework of a controlled, randomized study organized in the Abruzzo Region. Women aged 2 6-64 years were randomized to an active arm (LBC) or control arm (CC1). The particip ating laboratories had no previous ex perience with LBC. RESULTS: The inadequacy rate was 4.3% in CCT and 1.3% in the LBC arm (D < 0.001). Atypical squamous cells of undetermined sign ifi cance and atypical glands of undetermined significance reports were more frequent at CCT vs. LBC. A small, insignificant excess of low grade squamous intraepithelial lesions or high grade squamous epithelial lesions+ reports was observed in the LBC arm. The cervical intraepithelial neoplasia 2+ (CIN2+) detection rate was not statistically different in the 2 arms (CCT=0.54%, LBC= 0.66%, p = 0.28). In the overall series positive predictive value was slightly but not significantly higher in the LBC arm. LBC increased costs by 4.2% per both screened women and CIN2+ detected. CONCLUSION: The study reflects the introductory phase of LBC in laboratories without prior LBC experience. In this setting LBC reduced the inadequacy rate and decreased reading and was at least as sensitive as and more specific than CCT. Utilization of LBC in organized screening programs will be based on local feasibility, considering that the high cost of LBC is only partially compensated for by other benefits, such as residual cellular material, available for molecular testing, including human papillomavirus testing.