HPV DNA detection by in situ hybridization with catalyzed signal amplification on thin-layer cervical smears.

Thin layer-based technology in cervical cancer screening now allows both Papanicolaou staining and HPV testing on the same sample. Here, we show that in situ hybridization with catalyzed reporter deposition is a powerful HPV detection method when applied on thin-layer cervical smears, allowing distinction between two staining patterns suggestive of two different physical states of HPV DNA, where diffuse signals are suggestive of episomes and punctate signals are suggestive of viral DNA integration.     

Value of histiocyte detection in Pap smears for predicting endometrial pathology. An institutional experience

OBJECTIVE: To determine the clinical implications of the finding of histiocytes in Pap smears in 1 patient population. STUDY DESIGN: The medical records and Pap smears which the presence of histiocytes was mentioned in the diagnosis between August 1996 and August 2001 were reviewed in conjunction with follow-up surgical findings. The positive predictive value (PPV) for significant endometrial pathology for the isolated finding of histiocytes on Pap smear was determined. RESULTS: Of the 238,225 women screened over a 60-month period, 325 were reported to have histiocytes in their Pap smears. Of them, 238 (73.2%) had subsequent endometrial sampling, hysterectomy or both, and follow-up Pap smears. Two hundred seven smears (87%) failed to disclose endometrial pathology. Thirty-one cases (13%) resulted in significant histopathologic findings, including 12 uterine malignancies, 8 endocervical polyps, 7 endometrial polyps, 2 submucosal leiomyomata, 1 simple hyperplasia without atypia and 1 case of tamoxifen-related changes. Upon review of the clinical records, 58% (18/31) of those patients had other significant clinical and/or cytologic findings. Five of the 18 patients (27.8%) had associated postmenopausal bleeding, 11 had additional abnormal Pap smear findings (atypical glandular cells, 6/18, or 33.3%; endometrial cells, 5/18, or 27.8%), and another 2 had both postmenopausal bleeding and atypical glandular cells (2/18, or 11.1%). The PPV for significant uterine pathology for women with the isolated finding of histiocytes on a Pap smear was 5.5% and 60% with additional clinical and/or Pap smear findings. The PPV for endometrial cancer was 1.3% in women with the isolated finding of histiocytes on a Pap smear but 20% for women with histiocytes and additional clinical/or Pap smear findings. CONCLUSION: Based on the findings of this study and recently published data, we conclude that the isolated finding of increased histiocytes in the absence of postmenopausal bleeding, endometrial cells or atypical glandular cells on a Pap smear is a poor indicator of uterine disease.     

Benign cellular changes in Pap smears. Causes and significance

OBJECTIVE: We reviewed consecutive cases classified as benign cellular changes (BCC) over a four-month period. STUDY DESIGN: Cases classified as BCC were retrieved from the cytology files. A search was carried out to identify the previous Pap smears and concomitant cervical biopsies. RESULTS: One thousand one hundred three cases (23% of our gynecologic smears) were classified as BCC. Ninety-two patients (8.3%) underwent concurrent cervical biopsies. Specific infections accounted for 8% of BCC cases; reactive changes accounted for 92%. Of the biopsy specimens, 8.3% had no significant pathologic change. The most common biopsy diagnoses were cervicitis (31.5%), immature squamous metaplasia (16.3%) and reserve cell hyperplasia (10.8%). Miscellaneous benign diagnoses accounted for 21.7%. Cervical intraepithelial neoplasia (CIN) 1/human papillomavirus (HPV) was present in 14% of cases. All patients with biopsy diagnoses of CIN 1 had at least two previous abnormal Pap smears. Previous biopsy reports were available for review in 127 (12%) of the 1,103 patients. Of these 127 cases, 53.5% had a previous diagnosis of CIN/HPV; 9.4% had invasive carcinoma. A benign diagnosis was reported in 36.5%. CONCLUSION: The majority of BCC cases are due to reactive and inflammatory processes. In patients with a previous history of CIN, BCC may be of some significance. In patients with no significant prior cervical abnormalities, a Pap smear classified as BCC represents a reactive process.     

Accuracy of a slide profiler for endocervical cell detection in no-further-review conventional Pap smears

OBJECTIVE: To evaluate the reliability of the Focal-Point slide profiler (TriPath Care Technologies, Burlington, North Carolina, U.S.A.) in determing the absence of endocervical cells in conventional Pap smear slides. STUDY DESIGN: A consecutive series of conventional Pap smears, designated by FocalPoint as requiring no further review (NFR) and as lacking endocervical cells, was manually screened to determine the true presence or absence of endocervical cells. These results were compared to those obtained by FocalPoint. RESULTS: From January 1, 2000, to December 31, 2001, FocalPoint indicated that 797 NFR slides did not contain endocervical cells. In contrast, manual screening revealed that 504/797 (63.2%) did contain endocervical cells. CONCLUSION: The reliability of a negative FocalPoint determination for endocervical cells is limited. Manual screening of NFR slides designated by the instrument as lacking endocervical cells appears to be necessary.     

Extrauterine malignancies. Role of Pap smears in diagnosis and management.

OBJECTIVE: To further elucidate the cytologic manifestations of extrauterine malignancies, to evaluate their possible distinction from primary cervical malignancies and to analyze their clinical significance and role in staging. STUDY DESIGN: Papanicolaou (Pap) smears in 33 cases with abnormal cells originating in histologically proven extrauterine carcinomas were evaluated. These cases came from the files of the Medical College of Pennsylvania and Lankenau Hospitals. RESULTS: Ovary, gastrointestinal tract and breast were the three most frequent primary sites, accounting for 28 of the 33 cases (85%). The histologic types encountered were adenocarcinoma, 29 cases (88%); mucoepidermoid carcinoma, 1 (3%); small cell carcinoma, 1 (3%); cloacogenic carcinoma and large cell lymphoma, 1 (3%). The following diagnoses were rendered at the time of initial evaluation: adenocarcinoma consistent with metastasis, 21 cases; carcinoma, primary versus metastatic, 2; adenocarcinoma, suspicious for endometrial primary, 2; suspicious for carcinoma, 1; and atypical glandular cells, 7. CONCLUSION: The yield for positive Pap smear diagnoses in extrauterine malignancies is best in patients with an established diagnosis of a primary neoplasm. The degree of tumor differentiation and extent of tumor involvement did not appear to correlate with diagnostic yield. There appeared to be no statistically significant association of tumor diathesis with primary versus metastatic carcinoma and presence or absence of documented local involvement of the endometrium, cervix or vagina. Therefore, while Pap smears can serve as a diagnostic tool in the evaluation of extrauterine malignancies, they are best utilized as an adjunct to tumor staging and patient management.     

Outcomes of pregnant patients with Pap smears classified as atypical glandular cells

J. Slama, P. Freitag, P. Dundr, J. Duskova, D. Fischerova, M. Zikan, I. Pinkavova and D. Cibula
Outcomes of pregnant patients with Pap smears classified as atypical glandular cells Objectives: The incidence of cervical cancers increases with age. Due to the trend of increasing age of first pregnancy, abnormal Pap smears including those classified as atypical glandular cells (AGC) are being found more often in early pregnancy. Once invasive cancer is excluded, conservative management of squamous intraepithelial lesions (SIL) in pregnancy is considered safe; however, optimal management of AGC is not well established. The aim of our study was to evaluate the outcome of patients with AGC diagnosed from smears during pregnancy. Methods: The study included 17 patients referred to us in early pregnancy with Pap smears reported as AGC: 11 not otherwise specified (AGC‐NOS), five favour neoplasia (AGC‐FN) and one adenocarcinoma in situ (AIS). Thirty‐one with high‐grade SIL (HSIL) Pap smears confirmed on punch biopsy in early pregnancy comprised a control group. Human papillomavirus (HPV) positivity was found in seven patients with persistent AGC‐NOS (including all four who had CIN3 postpartum). All the women were initially examined by expert colposcopy and those with AGC‐FN or AIS smears also by transrectal ultrasound to exclude invasive endocervical cancer. Follow‐up controls were carried out every 8–12 weeks and, if there were no signs of progression, revaluation was scheduled 6–8 weeks after delivery. Results: The mean age of the women was 31.4 years. Conization in one patient in the study group was performed in the 16th week of pregnancy due to colposcopic signs of microinvasive squamous cell cancer confirmed on histology. Progression to invasive cancer was not found in any of the other 16 patients in the study group or in the control group. Cervical intraepithelial neoplasia or AIS was confirmed postpartum by conization or punch biopsy in 47.1% (8/17) of patients in the study group and, in 77.4% (24/31) of patients in the control group. Conclusions: Conservative management of women with AGC in pregnancy is safe where invasive cancer is excluded. As histological verification of glandular pre‐cancerous lesions by punch biopsy is not reliable and the postpartum regression rate cannot be determined precisely, conization should be performed in all cases with AGC‐FN or AIS. Triage of persistent AGC‐NOS with HPV testing is useful in distinguishing significant underlying lesions.     

Detection of false negative Pap smears by rapid reviewing. A metaanalysis

OBJECTIVE: To explore the diagnostic validity of rapid reviewing (RR) as a quality control method in cytologic laboratories. STUDY DESIGN: Fourteen studies dealing with the detection of false negative Pap smears by RR were included in a metaanalysis. RESULTS: The overall additional yield of positive slides, expressed as the percentage of all reviewed slides, is: 0.18% (95% confidence interval [CI]: .14-.21) for all cytologic abnormalities; 0.07% (CI: .05-.09) for squamous intraepithelial lesions (SIL) and 0.02% (CI: .01-.03) for high grade SIL. The false negative rate of primary screening, evaluated by RR, was 2.0% (CI: 1.5-2.6) for all cytologic abnormalities and 1.4% (CI: .8-2.1) for high grade SIL. The specificity of rapid rescreening was estimated as 97.2% (CI: 96.4-98.1). The positive predictive value of suspicion at RR is about 8.8%. Seven references contained historical data on full rescreening of a random sample of slides reported originally as negative. The results were also pooled and compared with RR. Complete rescreening is more sensitive, but if applied on only 10% of the negative workload, it would yield, on average, 4.7 times fewer extra positives, 5.6 times fewer SIL and 7.9 times fewer high grade SIL in comparison with RR of all sides. CONCLUSION: RR of all smears initially reported as nonpositive is a more effective and a fortiori a more cost effective quality control method in comparison with full rescreening of a 10% random sample.     

Significance of AGUS Pap smears in pregnant and postpartum women

OBJECTIVE: To study the clinical significance of atypical glandular cells of undertermined significance (AGUS) in pregnant and postpartum women. STUDY DESIGN: We evaluated 35 women who were pregnant (30) or within three months postpartum (5) and had a cytologic diagnosis of AGUS. Twenty-seven (77%) patients had follow-up: 17 (63%) patients underwent colposcopic examination and biopsy, and 10 (37%) had repeat Pap smears. Eight patients were lost to follow-up. RESULTS: Five (29.4%) patients had a squamous intraepithelial lesion (SIL), including three high grade and two low grade, on subsequent biopsy. The remaining (70.6%) patients had benign pathology, which included 5 chronic cervicitis, 4 endocervical and/or endometrial polyps, 2 Arias-Stella reaction and 1 microglandular hyperplasia. Among the patients with repeat Pap smears, two had persistent AGUS/atypical squamous cells of undetermined significance, the remaining cases were within normal limits. CONCLUSION: Pregnancy-related changes may present with glandular atypia. In addition, about one-third of pregnant and postpartum women with a diagnosis of AGUS had SIL on subsequent biopsy; that rate is similar to that in nonpregnant women. Therefore, pregnant women with a cytologic diagnosis of AGUS should be followed closely.     

Combined Pap smear, cervicography and HPV DNA testing in the detection of cervical intraepithelial neoplasia and cancer

OBJECTIVE: The sensitivity of the Pap smear (PAP) continues to be the subject of debate. During the past several years, cervicography (CER) and HPV DNA testing have been suggested as optional tools in the screening of cervical cancer precursors. STUDY DESIGN: The performance characteristics of PAP, CER and HPV DNA testing (hybrid capture test [HCT]) in all potential combinations were evaluated in a series of 1,030 women (aged 16-70, median, 33), subjected to colposcopy (COLPO) as the reference tool. RESULTS: Of the 992 evaluable cases, 402/992 (41%) had positive COLPO (i.e., an abnormal transformation zone). Of them, 298 women underwent directed punch biopsy, while of the COLPO negative patients, 18/93 positive by at least one of the three tests had endocervical curettage. Of the 402 COLPO positive women, 146 (36%) remained negative on all tests, whereas 256 (64%) had at least one positive test. There were 84 cervical intraepithelial neoplasia (CIN) 2 and 3 lesions and 6 invasive carcinomas. Of the former, 10 were detected by PAP alone, 4 by CER alone and 3 by HCT alone. Three of the 6 carcinomas were HCT negative. The predictive value (PPV) of a positive test was 45% for PAP, 51% for CER and 48% for HCT. The combinations of PAP with CER (for PAP negative cases) and PAP with HCT were more sensitive for CIN 2 and 3 (95% and 94%, respectively) as compared with PAP alone but were associated with a significant decrease in specificity (44% and 46% vs. 57%, respectively). However, both combinations retained a PPV (43%) similar to that of PAP alone (45%). CONCLUSION: The potential combinations of PAP with CER and with HCT were more sensitive in detecting CIN 2 and 3 as compared with PAP alone and retained a PPV similar to that of PAP.     

Human papillomavirus DNA detection in Papanicolaou-stained cervical smears with a nonradioactive, in situ hybridization assay.

Archived Papanicolaou-stained cervical smears from women with different cervical pathologies were processed for human papillomavirus (HPV) DNA detection and typing with an in situ hybridization (ISH) assay that employed commercial biotinylated HPV DNA probes. Two HPV DNA probes were utilized: one included HPV genotypes 6/11 and the other, 16/18. The method yielded positive results for HPV DNA 6/11 in 5 cases with condylomata acuminata (100%) and in 2 of 47 with flat warty lesions (4.2%), whereas HPV DNA 16/18 was detected in 29/47 of the latter group (61.7%). In cases with cervical intraepithelial III or invasive squamous cell carcinoma the yield was lower: positive results for HPV DNA 16/18 were obtained in only one of the five cases with one or the other cervical pathology (20%). An analysis of the results showed that the sensitivity of the assay correlated with evidence in the Papanicolaou specimens of pathognomonic cell injury from HPV infection. In the presence of such cytologic features, HPV DNA typing was possible in 37/52 cases (65.4%). In view of the modest difficulty and relatively quick execution of the nonradioactive ISH assay, the authors believe that Papanicolaou cervical smears with cytologic changes of HPV infection could be processed by this method in order to acquire information on the HPV type or types involved in the cervical infection.     

Parathyroid adenoma diagnosed as papillary carcinoma of thyroid on needle aspiration smears

Needle aspiration of a neck mass was performed on a 55-year-old white female with hypercalcemia. The cytologic material showed cohesive clusters of monomorphic hyperchromatic cells resembling follicular cells. Papillary formation and follicle-like arrangements suggested papillary carcinoma of the thyroid. The histopathologic section of the excised mass, however, revealed a parathyroid adenoma. The diagnostic difficulties in differentiating parathyroid adenoma from well-differentiated thyroid carcinoma are discussed. Data published in the literature emphasize similar difficulties, and it is concluded that a final diagnosis can be established only on histopathologic sections.     

PCR diagnosis of HPV in cervical biopsies of CIN and invasive neoplasia formerly diagnosed as HPV negative

OBJECTIVE: To evaluate whether the polymerase chain reaction (PCR) enhances the diagnosis of human papillomavirus (HPV) in biopsies of the uterine cervix with cervical intraepithelial neoplasia (CIN) or invasive neoplasia. STUDY DESIGN: Samples of 71 paraffin-embedded cervical tissue blocks from patients seen in the period 1997-1998 were analyzed. Samples were selected according to age (18-60 years old) and an active sexual life and divided in to 3 groups: test (samples with CIN or invasive neoplasia and a negative HPV diagnosis), positive controls (samples with CIN or invasive neoplasia and a positive HPV diagnosis) and negative controls (samples without CIN or invasive neoplasia and a negative HPV diagnosis). Samples were subjected to DNA extraction and PCR for HPV detection. RESULTS: PCR analysis matched the colposcopic and cytopathologic diagnoses in the positive and negative controls. However, 77% of samples in test group were HPV positive. CONCLUSION: CIN, an invasive neoplasm, is associated with the presence of HPV. Colposcopy and cytopathology are efficient but not sufficient to identify HPV. Thus, despite the high cost, PCR can be used as an additional examination, in women with cervical lesions.     

Utility of HPV DNA detection in thin-layer,liquid-based tests with atypical squamous metaplasia

OBJECTIVE: To determine if testing for HPV is useful in the management of patients with atypical squamous metaplasia (ASM) and to evaluate a small group of patients with atypical squamous cells in the setting of an atrophic cellular profile (estrogen test [ESTT]). STUDY DESIGN: Presence of HPV DNA was determined on 104 ASM and ESTT cases on residual ThinPrep specimens using Hybrid Capture II. Results of the HPV DNA test were correlated with subsequent biopsy or repeat Pap test results. RESULTS: Of 63 patients with ASM on ThinPrep Pap tests, 52% were associated with histologically proven SIL when HPV DNA was detected by Hybrid Capture II. None of the 17 patients who were negative for HPV DNA had subsequent evidence of HPV infection by biopsy or repeat Pap test. Among 14 patients with ESTT, none of the 12 who were negative for HPV DNA had subsequent evidence of HPV infection. CONCLUSION: ASM and ESTT with a negative HPV DNA test can be followed routinely, and a colposcopic examination is not warranted.     

Sensitivity of HPV tests on stained vs. unstained cervical smears

OBJECTIVE: To examine the effect of Pspanicolaou staining of cervical smears on the sensitivity of molecular biologic HPV tests. STUDY DESIGN: Two sensitive HPV tests were used, HPV DNA sequence analysis after polymerase chain reaction (PCR) amplification and the Hybrid Capture II method (HC II) (Digene Diagnostics Inc., Silver Spring, Maryland, USA). Papanicolaou-stained and unstained smears taken simultaneously were examined from 265 women readmitted for examination due to an atypical squamous cells of undetermined significance diagnosis. RESULTS: After an HPV test with the PCR method on unstained slides, 66% of the women were HPV positive, whereas the same women were HPVpositive in 54% when Papanicolaou-stained slides were analyzed. However, this difference was not statistically significant (p > 0.1). With the HC II method, 55% of unstained smears were HPV positive whereas 29% were HPV positive, when Papanicolaou-stained slides were examined. This difference was significant (p < 0.001). The same strong differences in sensitivity were observed when both the PCR and HC II methods were studied on the same Papanicolaou stained glass slides, whereas on unstained slides no significant difference was found. CONCLUSION: The results demonstrate that Papanicolaou staining of a cervical smear significantly decreases the sensitivity of an HPV test performed with the HC II method, whereas the PCR method is less affected. With the Papanicolaou method, the hematoxylin bath is followed by HCl treatment, and strong acid treatment destroys DNA.     

Cost analysis of PAPNET-assisted vs. conventional Pap smear evaluation in primary screening of cervical smears

OBJECTIVE: To assess the difference in costs between PAPNET-assisted and conventional microscopy of cervical smears when used as a primary screening tool. STUDY DESIGN: We performed time measurements of the initial screening of smears by four cytotechnologists in one laboratory. Time was measured in 816 conventionally screened smears and in 614 smears with PAPNET-assisted screening. Data were collected on the components of initial screening, clerical activities and other activities in the total work time of cytotechnologists in the routine situation and on resource requirements for both techniques. RESULTS: PAPNET saved an average of 22% on initial screening time per smear. Due to costs of processing and additional equipment, the costs of PAPNET-assisted screening were estimated to be $2.85 (and at least $1.79) higher per smear than conventional microscopy. The difference in costs is sensitive to the rate of time saving, the possibility of saving on quality control procedures and the component of the initial screening time in the total work time of cytotechnologists. CONCLUSION: Although PAPNET is time saving as compared with conventional microscopy, the associated reduction in personnel costs is outweighed by the costs of scanning the slides and additional equipment. This conclusion holds under a variety of assumptions. Using PAPNET instead of conventional microscopy as a primary screening tool will make cervical cancer screening less cost-effective unless the costs of PAPNET are considerably reduced and its sensitivity and/or specificity are considerably improved.     

Characteristics of false-negative smears tested in the normal screening situation.

It is common practice to rescreen false-negative (FN) smears. However, it is inevitable that this is done with some foreknowledge; at least it is known that the test smears contain one or more special cases. Therefore, we decided to test smears in the normal screening situation, when cytotechnologists are completely unaware of being tested. This experiment was done with five FNs and five true-positive (TP) smears. In a third experiment the FNs were tested with the cytotechnologists aware of their presence. Finally, 10 qualities of FNs and TPs were analyzed. In the normal screening situation, only in 1 of 25 tests was the FN recognized as malignant, while all the TPs were detected at the first testing. However, when the cytotechnologists were aware of being tested, the FN was detected in seven of eight tests. The FNs differed from the TPs in 5 of the 10 analyzed characteristics. FNs contained few (if any) large neoplastic epithelial fragments. Detached malignant cells were nearly lacking. The cancer cells had small nuclei that presented little anisokaryosis. The FNs possessed generally hypochromatic nuclei. These features explain why the malignant cells were almost never identified in the normal screening situation. We conclude that FNs and TPs differ and that it seems virtually impossible to avoid all false-negative diagnoses.