[Computer-assisted diagnostics in cervical cytology]. / Computerassistierte Diagnostik in der Zervixzytologie.

Computer assistance has the ability to reduce screening errors. In cervical cytology only two systems are available: BD FocalPoint™ (BD) utilizes algorithms for image recognition to analyze thin-layer specimens (Surepath) and conventional slides. The ThinPrep® imaging system (Hologic) is based on the densitometric measurement of nuclei and requires a thin-layer preparation. The BD FocalPoint™ system assigns specimens according to declining probability of an abnormality to one of six groups. A further option is location-guided screening (LGS) where the 15 fields of view which are most relevant are presented on a computerized microscope. Several studies indicate that this mode is at least equivalent to manual screening with conventional and surepath (SP) slides. For the ThinPrep® imaging system which only uses the LGS mode the majority of available studies found a significantly higher sensitivity for histologically confirmed grade 2 cervical intraepithelial neoplasia (CIN 2 +). However, most were retrospective or parallel studies. Both systems allow a significantly higher productivity but require a higher technical expenditure.

Short version of the German evidence-based Guidelines for prophylactic vaccination against HPV-associated neoplasia.

Persistent infection with HPV 16 and 18 has been causally associated with the development of cervical cancer and its precursor lesions as well as with other carcinomas and their precursors, e.g. some vulvar and vaginal cancers. Furthermore HPV 6 and 11 are responsible for anogenital condylomata acuminata in more than 90% of cases. With the recently developed prophylactic bivalent (HPV 16 and 18) and quadrivalent (HPV 6, 11, 16 and 18) vaccines, it is possible to prevent infection of the cervical epithelium and other squamous epithelia, the development of premalignant lesions and, in the case of the quadrivalent vaccine, the development of condylomata acuminata. The following paper represents a summary of the full-text version of the German evidence-based Guidelines, including all evidence-based recommendations regarding the safety as well as the efficacy of the vaccines in preventing CIN, VIN/VaIN, genital warts and other HPV-associated lesions.

[Status of papillomaviruses and their diagnosis in preventive screening]. / Der Stellenwert der Papillomviren und ihrer Diagnostik bei der Vorsorge.

Cervical carcinoma and its precursors are discriminated by their epidemiology from other malignant diseases. Human papillomavirusus (HPV) have been identified as the central etiologic factor in these diseases. Certain HPV gene products are tumor specific antigens in cervical cancer which leads to new perspectives in prevention and therapy. HPV testing is a realistic option in screening, triage and follow-up of cervical cancer and its precursors which has shown significant advantages over traditional methods in numerous large studies and proven cost effectiveness in recent model simulations.

Relationship between telomerase activation and HPV 16/18 oncogene expression in squamous intraepithelial lesions and squamous cell carcinomas of the uterine cervix.

SILs (squamous intraepithelial lesions) comprise a wide spectrum of clinically and biologically heterogeneous lesions ranging from benign proliferations to precancerous lesions. Telomerase activation plays a critical role in cellular immortalization and might be important for malignant progression. The viral oncogenes E6 and E7 are the principal transforming genes of high-risk HPVs and are important in HPV-associated immortalization and neoplastic transformation. In this study we investigated the relationship between telomerase activity, telomerase RNA, and HPV 16/18 oncogene expression in low- and high-grade SILs and SCCs (squamous cell carcinomas) of the cervix uteri. Telomerase activity was examined by the TRAP-assay and expression of the telomerase RNA (hTR) and HPV 16/18 E6/E7 oncogenes by RNA/RNA-in situ hybridization (ISH). The associated HPV-type was determined by PCR. Telomerase activity was observed in 25/29 (86%) SCCs, 31/41 (76%) high-grade SILs, 6/14 (43%) low-grade SILs, and 1/28 (3.6%) normal cervical tissues. Expression of hTR and viral oncogenes increased significantly with histopathologic severity of the lesion (p < 0.0001). A correlation was found between telomerase activity and intensity of viral oncogene expression. These findings suggest that telomerase activation occurs early in cervical carcinogenesis and is predominantly found in high-grade SILs and cervical SCCs. Our findings support current experimental data that suggest that telomerase is at least partially activated by viral oncogenes of high-risk HPV types. Telomerase activity with concomitant strong viral oncogene expression might therefore characterize a subset of lesions that are at risk for malignant progression.

Evaluation of methods to detect p53 mutations in ovarian cancer.

OBJECTIVE: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. METHODS: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). RESULTS: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. CONCLUSIONS: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.

Semiquantitative analysis of human papillomavirus DNA in cervical intraepithelial neoplasia by a differential polymerase chain reaction.

A differential polymerase chain reaction (PCR), which takes account of both the human papillomavirus (HPV) type and the amount of viral DNA, was applied to 100 cervical intraepithelial neoplasias (CIN) in order to improve the characterisation of the lesions. One hundred women with cytological smears suggestive of CIN I-III and a colposcopically guided punch biopsy were studied. DNA was phenol-extracted and a consensus PCR with L1 primers and a type-specific PCR for HPV-16 and-18 E6 were performed in parallel. The different sensitivity of the PCR methods allowed a semiquantitative analysis of viral DNA. HPV analysis was possible in all 100 very small cervical punch biopsies. Of the lesions 79% were HPVpositive. The percentage of HPV-16-positive lesions increased significantly (P < 0.001) with the degree of severity of CIN (CIN I: 46%, CIN II: 63%, CIN III: 85%). The percentage of CIN with a high viral load increased also with the grade of CIN (CIN I: 8%, CIN II: 33%, CIN III: 44%). Cytologically positive lesions (Pap IIID or IVa) had significantly more frequently (p < 0.02) a high viral load: 39% vs. 10% of cytologically false negative lesions. In conclusion, type-specific PCR had a very high sensitivity in the detection of HPV-DNA also in cytologically false negative CIN. The addition of the less sensitive consensusPCR allowed a semiquantitative analysis of the viral copy number. The higher amount of viral DNA in cytologically positive lesions, which may correspond to a higher rate of proliferation, reflects a possible role of the viral load in the progression of CIN. In clinical practice, differential HPVPCR could help to improve the management of CIN.

Increasing incidence of CD44v7/8 epitope expression during uterine cervical carcinogenesis.

Splice variants of the cell surface glycoprotein CD44 (CD44v) have been implicated in the progression of various human tumors. In the present study, we have examined the expression pattern of a CD44v epitope encoded by the adjacent variant exons v7 and v8 during human cervical carcinogenesis. While only l/ll normal cervical squamous epithelia was positive for this epitope by immunohistochemistry, 4/21 samples of low-grade squamous intra-epithelial lesions (LSIL), 17/35 samples of high-grade squamous intra-epithelial lesions (HSIL), 11/12 samples of the HSIL subgroup of carcinomas in situ and 17/17 cases of invasive cervical carcinoma showed CD44v7/8 epitope expression. In addition to CD44 variant expression, we have analyzed 67 lesions for the presence of HPV16/18-DNA using PCR. Most of the samples expressing the v7/8 epitope were also HPV16-positive (29/32), whereas only 17/35 of the v7/8-negative samples were HPV16-positive. HPV18 DNA was found in only one invasive carcinoma. Our data suggests that high-risk HPV infection may precede CD44v7/8 expression and that the number of samples expressing the CD44v7/8 epitope increases during carcinogenesis and reaches nearly 100% at the carcinoma in situ stage. This CD44 epitope could, therefore, serve as a diagnostic marker of cervical squamous cell carcinomas and as a possible target for CD44v7/8 epitope-directed therapies.

Human papillomavirus DNA in tumor-free regional lymph nodes: a potential prognostic marker in cervical carcinoma.

PURPOSE: The medical management of women with cervical carcinoma would benefit from a test that might predict which patients have a high risk of progression or recurrence. The detection of micrometastases in regional lymph nodes may be such a test. At least 90% of cervical carcinomas worldwide contain human papillomavirus (HPV) DNA. In a pilot study, we evaluated a polymerase chain reaction-based method for the detection of human papillomavirus DNA in archival routine sections of regional lymph nodes as a marker of micrometastasis in patients with a long-term follow-up period. PATIENTS AND METHODS: We analyzed 134 archival routine slides of histologically tumor-free lymph nodes derived from 21 patients with clinically and pathologically determined stage Ib, IIa, and IIb HPV-16 positive cervical carcinoma. The patients had been selected for good (still alive with a follow-up time of at least 5 years) or fatal outcome (dead of disease within 3 years). The amount of HPV-16 DNA was estimated by comparison with standards. RESULTS: All patients without strongly HPV-positive lymph nodes survived. In contrast, 8 of 12 women with strongly HPV-positive lymph nodes died of cervical carcinoma. CONCLUSIONS: In patients with cervical cancer, an approach based on a PCR test for HPV DNA in tumor-free regional lymph nodes may allow early identification of women at high risk for relapse who should receive adjuvant treatment.

p53 mutation and MDM2 amplification are rare even in human papillomavirus-negative cervical carcinomas.

BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single-strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg-->Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors.

Human papillomavirus DNA in cervical carcinoma--correlation with clinical data and influence on prognosis.

Human papillomavirus (HPV) is a main factor in cervical carcinogenesis. However, data on the correlation of HPV with clinical features and the prognosis of cervical carcinoma remain controversial. The HPV status (positivity, type, copy number) in unfixed tissue specimens of 205 primary invasive cervical carcinomas was determined by Southern blot hybridization. A correlation with comprehensive clinical and histopathologic data and long-time survival was evaluated. HPV DNA was detected in 73% of the cases; 83% of the HPV-positive tumors contained HPV 16. HPV 16 was predominant among squamous-cell carcinomas (SCC) (p = 0.05). HPV 16 copy number was higher in keratinizing tumors (p < 0.05), and elevated levels of the SCC antigen were more common in patients positive for HPV 16 (p < 0.03). No association was found between the HPV status and 8 other clinical and histopathologic variables. Multivariate analysis after a median follow-up of 73 months demonstrated longer survival for patients with lower clinical stage (p = 0.001) and keratinizing SCC (p = 0.005). Women with HPV-negative tumors had a higher risk of death (RR 1.51; p = 0.07). HPV analysis does not clearly define biologically distinct sub-sets of cervical carcinoma. This underlines the importance of additional factors in cervical carcinogenesis.

HPV DNA in autopsy-derived material from multiple metastases of a cervical carcinoma.

Fresh tissue from primary tumor and a metastasis of a cervical carcinoma and 13 autopsy-derived tissue specimens of the same patient were analyzed for HPV DNA by Southern blot hybridization and PCR. Primary tumor and 7 of 10 histologically proven distant metastases contained HPV 16 DNA by Southern blot. PCR detected HPV 16 in all 10 metastases and in 2 of 3 additional tumor-free autopsy-derived tissues. The restriction pattern was identical in all HPV-positive lesions and only slight variations in copy number occurred. Two-dimensional gel electrophoresis showed the viral DNA fully integrated in the cellular genome without any difference between primary tumor and metastasis. The relevance of HPV also for the metastatic spread of the malignant disease is indicated by its conserved presence in multiple distant metastases of cervical carcinoma.

Human papillomavirus DNA in locally recurrent cervical cancer.

Ten vaginal and central recurrences of cervical carcinoma were analyzed by Southern blot hybridization under nonstringent and stringent conditions for the presence of human papillomavirus (HPV) type 11, 16, 18, 31, and 35 DNA. Cases judged HPV-negative by this assay were tested with the polymerase chain reaction (PCR) for HPV 16 and 18. All 10 recurrences were HPV-positive. Seven tumors contained HPV 16, one contained HPV 18, one contained HPV 16 and 18, and one contained HPV-related sequences. In three cases, where the corresponding primary tumor could be studied, the identical HPV type was contained. The consistent presence of HPV DNA in local recurrences of invasive cervical carcinoma argues in favor of a role of these viruses also in the maintenance and reappearance of malignancy.

Human papillomavirus, type 16, DNA in multicentric anogenital neoplasia associated with idiopathic panmyelopathy. A case report.

A 27-year-old woman suffering from panmyelopathy for six years presented with a cervical low grade squamous intraepithelial lesion (SIL), vulvar high grade SIL and perianal squamous cell carcinoma with an inguinal metastasis. Southern blot hybridization with 32P-labeled human papillomavirus (HPV) DNA revealed HPV 16 DNA in varying copy numbers in material from the four locations. HPV 16 genomes persisting after surgery on the perianal tumor area were no longer detectable after betatron radiotherapy.

Human papillomavirus DNA in distant metastases of cervical cancer.

Eighteen distant metastases from cervical cancer to the extrapelvic abdomen, extraabdominal lymph nodes, vulva, suburethral region, skin, and breast in 17 patients were analyzed by Southern blot hybridization under nonstringent and stringent conditions for the prevalence of human papillomavirus (HPV) type 11, 16, 18, 31, 33, and 35 DNA. Fourteen metastases in thirteen patients were HPV-positive. Thirteen tumors contained HPV-16 and one HPV-related sequences with varying copy number. In 9 of 11 cases, where the corresponding primary tumor could be studied, HPV positivity and type were identical. Two HPV-negative primary lesions had HPV-positive metastases; in three cases differences in restriction pattern or copy number were revealed. The HPV status showed no clear association with age of the patient, latency period between primary tumor and metastasis, histological findings, therapy, and clinical course of the disease after metastasis. The rather conserved presence of HPV DNA in distant metastases of cervical carcinoma underlines the importance of these viruses also for the maintenance of the malignant state.