A 7.5-year prospective study of longer than 18 months type-specific human papillomavirus persistence in a routine cytology-based cervical screening population of about 31,000 women in West Germany.

The objective of this study was to analyse the prevalence, infection pattern, duration and outcome of long-term, type-specific, persistent human papillomavirus (HPV) infections in a routine cytology-based cervical screening population of West German women followed up for 7.5 years. From a screening population of 31,000 women, a strictly selected cohort of 100 patients with > or =18-month persistent, type-specific HPV infection were prospectively followed up for a mean of 35.52 months (+/-13.0). HPV type prevalence and odds ratios for regression, progression and steady state were analysed, as well as the influence of age and HPV coinfection on outcome. Altogether, 21 different genotypes were detected. Seventy-two percent of women were infected with high-risk HPVs, 24% with low-risk and 4% with unknown risk HPV types; 44% of cases had coinfections with multiple HPV types. The risk of progression in low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions was the highest for infections with high-risk HPVs [odds ratio: 2.2 (0.79-6.11, 95% confidence interval)], whereas cases with low-risk and unknown risk HPVs tended to regress or remained unchanged during follow-up. The mean duration of infections showed considerable variation among the different HPV types and risk groups detected and ranged between 19.7 and 54.3 months. Age was not significantly associated with disease progression and infection duration, and histology had a poor sensitivity for detecting high-grade dysplasia. In conclusion, detecting long-term persistent HPV infections by genotyping may help to identify women with cervical intraepithelial lesions who are at lower and higher risk of developing high-grade precancer and cancer. This may influence future screening strategies and therapy decisions.

The prevalence and distribution of human papillomavirus genotypes in oral epithelial hyperplasia: proposal of a concept.

BACKGROUND: Evidence is accumulating for the aetiological role of human papillomavirus (HPV) in the pathogenesis of potentially malignant oral mucosal lesions and squamous cell carcinomas. METHODS: Paraffin tissue sections from 49 patients with 'white patches' of the oral mucosa were investigated histologically, by broad-spectrum PCR followed by genotyping and chromogenic in situ hybridisation (CISH). RESULTS: Histologically, 33 flat hyperplasias and 16 papillary hyperplasias were diagnosed. Twenty-two of 28 samples studied (78.6%) were positive for HPV DNA by PCR and six were negative. The following HPV types were detected in decreasing order of prevalence: HPV 35, HPV 6, HPV16, HPV 53, HPV 18, HPV 51 and HPV 55. Seventeen samples (60.7%) contained high-risk HPV DNA. Using CISH, >or= 1 HPV signals were detected at least in a few epithelial cells in 95% of cases studied. All but one case were positive with the high-risk HPV probe and all HPV infections contained low viral load. Concordant positive results both by PCR and CISH were detected in 14 of 19 cases (73.7%) analysed. CONCLUSIONS: The high prevalence of HPV infection in hyperplastic 'white patches' of the oral mucosa supports the putative role of HPV at an early stage of oral carcinogenesis. These results further indicate that the majority of white oral mucosal lesions - flat, exophytic, wart-like or papillary proliferations - could be considered as the clinical manifestations of oral HPV infection. This finding has clinical relevance regarding therapy and patient management and may help in elucidating the role of HPV infection in oral carcinogenesis.

Quantitative molecular grading of bladder tumours: a tool for objective assessment of the biological potential of urothelial neoplasias.

The present study aimed to assess whether patients with bladder urothelial tumours can be more objectively stratified into low- and high-risk groups for recurrence and progression using a 2-tired molecular grading scheme, than by subjective histopathological grading alone. Biopsy material from 45 consecutive patients with urothelial bladder neoplasias (2 papillary urothelial neoplasm of low malignant potentials, 18 pTa, 1 pTis, 19 pT1 and 5 pT2) was analysed for immunohistochemical Ki-67 and p53 expression. Labelling indices were assessed by automated cellular image analysis. UroVysion FISH test results were evaluated by automated signal counting, and DNA ploidy of single nuclei preparations were measured by image cytometry. Sixty-nine percent of cases showed >10% Ki-67 LI, 64% had >10% p53 LI, 53% revealed DNA aneuploidy and 56% expressed a high-risk FISH pattern. Based on a combination of single molecular markers, 75% of neoplasias were classified as high molecular grade. Tumour stage and histopathological grade were significantly associated with FISH pattern, DNA ploidy and MIB1 LI. Stage was also related with molecular grade. Clinical outcome showed a significant correlation with MIB1 LI and molecular grade. P53 had neither diagnostic nor prognostic relevance, nor was there any correlation between histological and molecular grade. Our preliminary data strongly suggest that the combination of quantitative biomarkers provides superior and objective prognostic tools in bladder urothelial neoplasias compared to classic clinicopathological features and indices.

Prevalence and genotype distribution of multiple human papillomavirus infection in the uterine cervix: a 7.5-year longitudinal study in a routine cytology-based screening population in West Germany.

The availability of vaccines against certain HPV types and the development of broad spectrum genotyping methods have increased interest in co-infections with different HPV types. In the present study, the prevalence and type-specific composition of multiple HPV infections were investigated in a routine cervical screening population in West Germany both at a cross-sectional level and longitudinally. Four hundred eighty-nine out of 8,090 women were diagnosed with multiple HPV infections once or repeatedly. During the 7.5-year study period, the cumulative prevalence of HPV co-infections was 15.3% in contrast to the cross-sectional prevalence of 3.8% at single visits. The overall cumulative prevalence within the cohort of all women screened was 6.9%. Using consensus PCR with sequencing and type-specific PCRs, two to three HPV types were detected simultaneously, whereas broad spectrum methods detected up to seven different genotypes in one sample. Nevertheless, the most common pattern of co-infection occurred with two to three HPV types irrespective of the age of the patient, cytology and histology of the lesions and the method used. The most common genotypes detected were HPV 16, 31, 53, 51, 52, and 66, and the most common pattern of co-infection was double infection with HPV 16 and 31. These results show that rates and patterns of multiple HPV infections are largely dependent on the methodology used and the time interval between tests. Given the significance of HPV vaccination and its expected influence on immunized populations, it is essential to gain additional insights into the natural course and pathogenic effect of multiple HPV infection longitudinally.

Predictive testing of early cervical pre-cancer by detecting human papillomavirus E6/E7 mRNA in cervical cytologies up to high-grade squamous intraepithelial lesions: diagnostic and prognostic implications.

The type-specific persistence of oncogenic human papillomavirus (HPV) is considered to be the true precursor of cervical cancer at which the transcription of the viral oncogenes E6 and E7 is necessary for the malignant transformation and maintenance of the neoplastic state. In the present pilot study, a cohort of 66 women was investigated from a routine office-based screening population who had an index cytological result from normal to high-grade squamous intraepithelial lesions and who were also HPV-DNA positive for at least one of the following high-risk HPV types: HPV 16, 18, 31, 33 and 45 detected by MY09/MY11 consensus and GP5+/6+ general primers, followed by sequencing. The expression of E6/E7 transcripts from the same HPV types was detected by the PreTect HPV-Proofer. Cervical status was checked 18 months after the mRNA test. The expression of E6/E7 mRNA was found in 58% of the cases showing a 97% concordance with the HPV-DNA types and a positive correlation with increasing cytological and histological grade. All HPV-mRNA positive cases were also positive for HPV DNA whereas 25 (38%) of the HPV-DNA positive cases did not express the respective mRNA. The diagnostic validity of the PreTect assay for detecting histologically-proven prevalent CIN3 lesions were: sensitivity 95%, specificity 55%, positive predictive value (PPV) 81% and negative predictive value (NPV) 86%. The prognostic power of the PreTect test for predicting cytological disease progression was as follows: 78% sensitivity, 60% specificity, 37% PPV and 90% NPV. In conclusion, our results showed that the detection of oncogenic HPV E6/E7 mRNA in cervical smears in a routine screening setting identifies prevalent CIN3 lesions with nearly 100% sensitivity and has a very high negative predictive value for disease progression during the natural course of HPV infection. Thus, testing for HPV oncogenic activity may be used as a clinically predictive marker to enhance the net effectiveness of screening and enable the prognostication of prevalent cervical lesions.

Inter-laboratory validation of PCR-based HPV detection in pathology specimens.

The detection and typing of human papilloma virus (HPV) in pathology specimens is gaining increasingly in importance. In the context of the initiative for quality assurance in pathology (QuIP) of the German Society of Pathology and the Professional Association of German Pathologists, four panel laboratories with experience and expertise in polymerase chain reaction (PCR)-based HPV detection were selected to establish an inter-laboratory trial. In a first step, these laboratories performed an internal testing of their own methodologies, which comprised DNA sequencing, multiplex nested PCR and hybridization techniques. Material from 39 samples including paraffin sections and DNA preparations of tissues and plasmids were evaluated by each panel institute according to their own protocols. Despite the different methodologies, a high degree of inter-laboratory reliability was achieved. In this report, we summarise the results. Pretested specimens are available for the external trail and can be ordered from the steering institute via provitro GmbH Berlin ( http://www.provitro.de ). Supplementary data are online available at http://pathologie-ccm.charite.de (rubric "Forschung"), which includes a web-based photo gallery of HPV-associated lesions and their potential association with specific virus types. The initiative is intended to foster the quality assurance of molecular HPV analysis in pathology and its correlation with morphological changes.

The spectrum of cervical diseases induced by low-risk and undefined-risk HPVS: implications for patient management.

BACKGROUND: The natural history and carcinogenicity of rare and novel HPV types is unclear. MATERIALS AND METHODS: From a total of 5,964 women tested for HPV by PCR and sequence analysis, Pap smears from 293 patients harbouring mono-infection with low-risk, undetermined-risk or novel HPV genotypes were investigated. RESULTS: Sixty-three percent of patients had ASC-US, 23% LSIL, 9% were negative and 5% had HSIL in cytology. Of 30 HPV types detected, 19 were of unknown risk (UR)-types including 3 novel genotypes. Four of the UR-HPVs (HPV 69, 30, 67 and 34) could be assigned as probable high-risk types and eight as low-risk types based on phylogenetical relationship. Morphology was not discriminative with regard to HPV type, but non-classical HPV-signs were generally present even in "normal" cytologies. CONCLUSION: HPV-typing is important for risk-adapted individual patient management. Women harbouring novel high-risk or probably high-risk HPVs require more intensive care than those bearing non high-risk infections.

Predicting treatment outcome in cervical diseases using liquid-based cytology, dynamic HPV genotyping and DNA cytometry.

BACKGROUND: In this study, our prospective experience with a multimodal follow-up protocol is summarized, with special emphasis on predicting the treatment outcome of cervical diseases. MATERIALS AND METHODS: Liquid-based cytology samples (ThinPrep) from 209 women exhibiting the whole spectrum of human papilloma virus (HPV)-related cervical diseases were investigated by cytology, PCR-based HPV genotyping and DNA cytometry pre-surgery. The first control cytology and type-specific HPV tests were performed at 3 months post-surgery. RESULTS: The success rate of surgery was 95% in eradicating high-grade cervical disease and 90% in eliminating the baseline HPV genotype. Treatment failure was significantly correlated with baseline cytology (p=0.011), resection margin status (p=0.016) and HPV positivity at 3 months post-surgery (p=0.04). Multivariate logistic regression analysis showed that type-specific persistent HPV infection (p=0.028), baseline cytology (p=0.039) and histology (p=0.065) were independent predictors of residual cervical neoplasias. CONCLUSION: Our results showed that our multimodal surveillance protocol may help to individually assess the anticipated clinical outcome of cervical diseases post-surgery.

Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer?

OBJECTIVE: To determine whether it is possible to stratify patients with superficial bladder cancer into low- and high-risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow-up testing. PATIENTS AND METHODS: Voided urine samples from 47 consecutive patients with urinary tract neoplasms (13 with no history of urothelial malignancy and 34 under follow-up after complete transurethral resection of superficial urothelial carcinoma of the bladder) were evaluated by liquid-based cytology (ThinPrep(R), CYTYC Corp., Boxborough, MA, USA) and UroVysion FISH (Vysis-Abbott, Downers Grove, IL). RESULTS: Of the 34 patients under surveillance, the UroVysion test was negative in four, 17 had loss of 9p21 sequences either alone or combined with low-frequency trisomy/ies or tetrasomy/ies of chromosomes 3, 7 and 17 in single cells (low-risk FISH), and 13 also had complex aneusomies of the remaining chromosomes (high-risk FISH). One of the four FISH-negative neoplasms, four of the 17 low-risk FISH cases and five of the 11 informative high-risk FISH-positive patients developed recurrence. Progression occurred only in patients with high-risk FISH results, showing high-frequency complex chromosomal polysomies (four of 11). CONCLUSION: The results from this pilot study indicate that the UroVysion FISH test may help to individually assess the clinical behaviour of superficial bladder cancer, based on the chromosomal pattern of exfoliated tumour cells in follow-up urinary cytology. It might be of use to identify those patients likely to progress at earlier and curable stages of disease, and lengthen the surveillance period in those with persistent or recurrent low-risk disease.

Validity of combined cytology and human papillomavirus (HPV) genotyping with adjuvant DNA-cytometry in routine cervical screening: results from 31031 women from the Bonn-region in West Germany.

Our aim was to improve the accuracy of routine cervical screening by a risk-adapted multimodal protocol with special focus on possible reduction and prognostic assessment of false positive results. A cohort of 31031 women from the Bonn-region in West Germany, median age 36 years, were screened by cytology (conventional or liquid-based), followed by PCR-based HVP detection with genotyping and adjuvant DNA image cytometry, if indicated, in a sequential manner. The true prevalence of high-grade cervical intraepithelial neoplasia and carcinoma (>/=CIN2) was 0.32% in the population as projected from cervical biopsies of 123 women (0.4%), of whom 100 showed >/=CIN2. Sensitivity of the cytology screening program at PapIIID/HSIL threshold for detecting histologically confirmed >/=CIN2 cases was 81%, with specificity, positive predictive value (PPV) and negative predictive value (NPV) of 99, 20.9 and 99.9%, respectively. Of 38 women receiving the complete screening protocol, all the 31 >/=CIN2 cases were correctly detected by cytology alone, 30 by positive high-risk HPV genotype and 30 by aneuploid DNA profile. The combination of the three methods resulted in an up to 6.9% increase in PPV for >/=CIN2 at practically unchanged detection rate with the additional benefit of being able to predict the probable outcome of CIN1 lesions detected as false positives with any single test. Multimodal cervical screening might permit identification of those women with low-grade squamous intraepithelial lesions likely to progress at an earlier and curable stage of disease and lengthen the screening interval in those with transient minor lesions caused by productive HPV infection.

Risk-adapted multimodal laboratory cervical screening -- application of new technologies to cervical cancer prevention.

The objective of screening for cervical cancer is to reduce the mortality and incidence of the disease. To date, there is extensive and strong evidence that this can be achieved by cytology-based screening programs, which continue to be the mainstay of cervical prevention worldwide despite their inherent methodological limitations. This article presents a review on the utility of conventional, ancillary, and experimental methods for cervical screening both as single tests and test-combinations, and describes possible future directions for enhanced screening accuracy using risk-adapted protocols.

Aberrant, highly hyperdiploid cells in human papillomavirus-positive, abnormal cytologic samples are associated with progressive lesions of the uterine cervix.

BACKGROUND: Infection with oncogenic-type human papillomavirus (HPV) and consecutive cytologic abnormalities of the uterine cervix precede the evolution of carcinoma. However, the specificity of both changes is too low to predict the true malignant potential of the change in a given time point, because the majority of the HPV infections revert to normal with time. In preliminary studies, the authors demonstrated that, among many dysregulatory phenomena at the cytologic level, the occurrence of significant DNA content aberrations were in good correlation with progressive cervical changes; and, as a marker for this, the significance of cells with nuclear DNA content > 9c (9c cells) was investigated using slide-based cytometry. The objective of the current study was to determine whether 9c cells in cytologic samples that presented with dysplasia and with high-risk HPV types were associated with the development of higher grade cervical intraepithelial neoplasia (CIN II+). METHODS: Samples with positive cytologic diagnoses (atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion [LSIL], and high-grade squamous intraepithelial lesion [HSIL]) were evaluated for both parameters and were related to outcomes and, if available, to histology results over a follow-up of up to 4 years. RESULTS: Although the presence of high-risk HPV was demonstrable in almost all samples with CIN II+ (96.7%), the virus genome alone proved to have a poor positive predictive value (56.1%) for higher grade CIN. Cytology and the demonstration of 9c cells resulted in less sensitivity compared with the occurrence of high-risk HPV (70.4% and 73.7%, respectively) but in significantly greater specificity (91.3% and 89.2%, respectively). Moreover, a positive predictive value of 81.8% for CIN II+ could be calculated for 9c cells. HSIL morphology, unfavorable HPV type, and DNA cytometry together excluded the highest rate of CIN > or = II with 100% sensitivity and 91.3% specificity. CONCLUSIONS: Biologic parameters, in addition to cytology, help to define the nature of cervical dysplasias more accurately. Oncogenic HPV types and hyperdiploid cells with DNA content > 9c disclosed cytologic changes with the greatest malignant potential. Cancer

DNA ploidy and chromosome (FISH) pattern analysis of peripheral nerve sheath tumors.

BACKGROUND AND METHODS: 44 peripheral nerve sheath tumors (PNST) (27 schwannomas, 9 neurofibromas and 8 malignant peripheral nerve sheath tumors (MPNST)) were analyzed to determine DNA ploidy pattern and to clarify the conflicting data in the literature concerning this topic (whether benign PNSTs are aneuploid or not). For further insight we analyzed 6 schwannomas, one atypical neurofibroma and five MPNSTs by fluorescence in situ hybridization (FISH) technique using centromeric chromosome probes (7, 17 and 18) and automatic image analysis station, Metafer 4. RESULTS: Benign schwannomas (including the problematic variants as ancient, cellular, neuroblastoma like and multiplex schwannomas) could be characterized by euploid-polyploidisation and by their 4c peak height value which was usually more than 10% of total cell number measured. These characters were not found among neurofibromas and MPNST-s. FISH analysis revealed and confirmed that the 'normal' euploid-polyploid cells are mainly eusomic-polysomic containing two, four, eight or sixteen signals for each chromosomes examined, but in a small proportion aneusomy was found among tumor cells of benign schwannomas (average: 2.58; range 1.33-3.44). In contrast, the atypical neurofibroma displayed marked aneusomy (18.44%) but it contained normal eusomic and polysomic cells too. Two diploid MPNSTs proved to be clearly aneusomic with trisomy of chromosome 17 and monosomy of chromosome 18. CONCLUSIONS: All these data suggest that ploidy pattern determination combined with FISH analysis may be a very useful supplementary tool for making a right diagnosis (to differentiate benign versus malignant schwannomas in problematic variants) and to understand better the malignant transformation in PNSTs.

Chromosomal aberrations accumulate in polyploid cells of high-grade squamous intraepithelial lesions (HSIL).

Persistant infection with human papillomavirus (HPV) of the uterine cervix is related with cytological atypia (SIL), the oncogenic potential of which is unclear in a given time point of monitoring. HPV-induced genetic instability result in polyploidization as well as in low frequency random chromosome aberrations in squamous cells. In the present work we analyzed whether highly polyploid/aneuploid cells reflect genomic changes at the chromosomal level. 13 samples with the cytological diagnosis of HSIL were analyzed for HPV type and nuclear DNA content measured by laser scanning cytometry (LSC). Hyperdiploid cells with >5c and with >9c DNA content were further analyzed for numerical aberrations of the chromosomes 3 and 17 by fluorescence in situ hybridization (FISH) following repositioning. Cells with >5c DNA content were found more frequently than cells with >9c DNA content (5-98 and 1-44 cells, respectively). The FISH analysis demonstrated frequent polysomies, however, the rate of aneusomy (other than 2, 4, 8 or 16 chromosome copies) was significantly higher in cells with >9c DNA content than in cells with >5c DNA content or the normal diploid cells. The imbalance of chromosome 3 and 17 copy number was also increased in cells with >9c DNA content. Moreover, in three out of the 13 analyzed HSIL samples, recurrent abnormal chromosome 3/17 ratio was demonstrated in a significant part of the cells, indicating a common origin of these cells. Highly polyploid/aneuploid cells in HSIL accumulate cytogenetic aberrations detectable by FISH analysis. These cells may reflect early changes with tumorigenic potential in a very concentrated fashion.

Outcome and prognostic factors in ocular adnexal lymphoma.

AIM: To classify ocular lymphomas in patients treated at the Zagreb University Hospital Center according to the new classification of the World Health Organization (WHO) and to determine factors with prognostic significance. METHODS: From 1986 to 2003, histological diagnosis of ocular lymphoma was made in 24 patients. The median age of patients was 62 years, with 2:1 female predominance. The patients underwent staging procedures and clinical evaluations prior to the date of the initial therapy. Histopathologic slides were reviewed and tumors were classified according to the new WHO classification. Additional immunohistochemical studies were performed on 35 available specimens. The antibodies used were CD3, CD5, CD10, CD20, CD43, and bcl-6; and in a few cases cyclin D1, bcl-2, CD23, CD79a, and CD138. The main outcome measures were development of distant recurrence after new presentation with solely ocular adnexal disease, and death attributable to widespread lymphoma. RESULTS: Ocular adnexal lymphomas were found in orbit in 20 patients, in eyelid in two, and conjunctiva in two patients. Twenty patients had lymphoma stage IE, one had IIE, and three had stage IV. Three patients had prior or concurrent systemic disease and 21 patients had primary lymphoma. The main subtypes of non-Hodgkin lymphoma according to the WHO classification were extranodal marginal zone B-cell lymphoma (n=20), diffuse large cell B-cell lymphoma (n=2), mantle cell lymphoma (n=1), and plasmacytoma (n=1). Six lymphomas were CD43 positive and five of them were extranodal marginal zone B-cell lymphomas. Radiotherapy was given to 11 patients, chemotherapy in 8 patients, whereas radiotherapy and chemotherapy were implemented in three patients. Two patients underwent only surgical excision of the tumor. Local relapse was found in three and distant recurrence in four patients. Distant recurrence was found in four patients with stage IE (two of them also had a local relapse). In the group of patients with extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (B-EMZL), the estimated 5-year overall survival was 92.9+/-6.6% (mean+/-standard deviation) and the 5-year failure-free survival was 80.1+/-10.3%. Age, sex, side of involvement, anatomic localization of the lesion, clinical stage of disease, and mode of therapy did not have any prognostic significance during the follow-up period (median, 53; range, 9-131 months). Immunohistochemical marker CD43 was the only parameter of prognostic significance (p=0.035). Patients with B-EMZL had almost 14 times higher chance for an unfavorable outcome if the tumor cells expressed CD43 on their surface, than the CD43-negative cases. CONCLUSION: Most ocular adnexal lymphomas usually have a B-cell immunophenotype, the morphologic and immunohistochemical features of extranodal marginal zone B-cell lymphoma, and a favorable prognosis. Our data suggest that CD43 could be useful to separate the group of patients with extranodal marginal zone B-cell lymphomas with unfavorable prognosis from those that have a good prognosis. CD43 positive ocular lymphomas are associated with a higher rate of subsequent distant recurrence and the rate of lymphoma-related death.

Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area.

Human papillomaviruses (HPVs) are aetiological agents for cervical cancer. More than 70 different HPV types that infect genital mucosa have been found. In order to develop a sensitive and specific detection and typing assay, a PCR/direct sequencing approach was used. Two pairs of consensus primers were used for amplification of HPV DNA and the PCR products obtained were analysed by automated sequencing. Sequences were compared with those in GenBank by using the BLAST program. In this study, 2916 cytological samples were screened for HPV, as well as for triage. Nine hundred and forty-eight (32.5%) samples were positive for HPV, of which 134 harboured more than one HPV type. Of the 948 PCR-positive samples, 648 were typed. Thirty-nine different HPV types were identified by sequencing. The two most frequently found HPV types, 16 and 31, together accounted for 36.3% of the sequences (26.2 and 10.1%, respectively). This group was followed by HPV types 6 (5.7%), 18 (5.3%), 58 (4.5%), 61 (4.5%), 53 (4.4%), 42 (4.3%) and 51 (4.0%). All other types were detected at frequencies <4% and eight types were detected only once. PCR/direct sequencing is a reliable method for routine detection of HPV in cytological samples. The data presented here suggest a complex distribution of HPV types in the population tested. The results accentuate the importance of PCR-based techniques in HPV diagnosis, as hybridization-based methods can only detect a limited number of infections. This method can also be applied easily to the analysis of tissue samples and it therefore also allows type-specific follow-up of women who have been treated for cervical intraepithelial neoplasia.

Measuring telomerase activity in various human tumors in routine histology and cytology.

The aim of this study was to investigate telomerase reactivation, to quantitatively measure the human telomerase reverse transcriptase (hTERT) content and telomerase activity level (TA) in routine histological and cytological samples, and to examine the relationship between these values and morphological factors. We analyzed 86 (35 cytological and 51 histological) lesions which were divided into four main groups: renal tumors, soft tissue tumors, bladder-urine and thyroid gland lesions. The relative expression of mRNA of hTERT was examined by real-time polymerase chain reaction (RT-PCR). Almost all of the renal cell carcinomas showed TA. In soft tissue tumors no correlation was seen between TA and histogenesis, aggressiveness and prognosis. Concerning cytological material a very good correlation was seen between TA and the benign or malignant nature of these tumors (92.3% specificity and 60% sensitivity). Our results indicated that TA can be used beside histology also in cytologic samples, for example in the preoperative differential diagnosis of the thyroid gland lesions and urine samples. Telomerase reactivation may not play an important role in tumorigenesis in STT. Useful observations can be made by concentrating not only on one group of diseases or localization but the unselected analysis of routine diagnostic cases can also be of help both in diagnosis and therapy.

Determination of features indicating progression in atypical squamous cells with undetermined significance: human papillomavirus typing and DNA ploidy analysis from liquid-based cytologic samples.

BACKGROUND: The Bethesda System of cervical cytologic findings introduced the term ASCUS (atypical squamous cells of undetermined significance) to cover the broad zone separating normal cytomorphology from definitive squamous intraepithelial lesions (SILs). The management of patients with ASCUS is particularly problematic as approximately 10% of ASCUS patients develop SIL and 1 per 1000 develop cervical carcinoma. METHODS: Our aim was to demonstrate the combined use of polymerase chain reaction for human papillomavirus (HPV) typing and laser scanning cytometry for DNA content measurements in the subcategorization of ASCUS cases according to the risk for progression toward cancer. Liquid- based monolayer preparation (ThinPrep, Cytyc, Boston, MA) of the cytologic material was used for cytomorphologic analysis. DNA content measurements using laser scanning cytometry and direct sequencing of HPV using the consensus primers GP5+/GP6+ and MY09/MY11 were performed from the same material. RESULTS: Twelve of the 44 cases (27.2%) with ASCUS carried a high-risk HPV genome whereas only 3 of the 195 normal control cases (1.5%) showed positivity for a high-risk HPV genome. Six of 12 (50%) of the high-risk HPV-positive ASCUS cases presented isolated cells with a DNA content above 5c, whereas cells with a DNA content above 9c were found in 3 of 12 cases (25%) and were exclusively found in combination with high-risk HPV infection. In these three cases, the histologic follow-up resulted in cervical intraepithelial neoplasia (CIN) I (one case) and CIN III (two cases). None of the other ASCUS or normal cases displayed DNA aneuploidy above 9c. They returned to normal cytology (within normal limits/benign cellular changes) in the follow-up. CONCLUSIONS: Human papillomavirus typing and DNA content measurements may delineate a distinct group of ASCUS. Our preliminary data suggest that ASCUS cases with high-risk HPV positivity and with rare cells with abnormally high DNA content represent similar biologic features as high-grade SIL and are at elevated risk to develop cancer.

Human papillomavirus typing and DNA ploidy determination of squamous intraepithelial lesions in liquid-based cytologic samples.

BACKGROUND: Infection by high-risk human papillomavirus (HPV) plays a role in the evolution of cervical carcinoma. Cellular atypia and consecutive DNA content alterations in cytologic samples are consequences of a preexisting viral infection. METHODS: We analyzed the frequency and association of HPV types and the presence of rare cells with abnormally high DNA content. We also evaluated whether these findings support the cytologic diagnosis in 112 routine cases with low and high-grade squamous intraepithelial lesions (LSIL/HSIL) when performed from liquid-based cytologic samples (ThinPrep). For DNA content measurements, laser scanning cytometry was applied and at least 10,000 cells were analyzed. HPV typing was performed by a direct sequencing approach using the consensus primers GP5+/GP6+ and MY09/MY11. RESULTS: Of 112 SIL cases, 110 (98.2%) were HPV positive and 95 (84.8%) had a high-risk type HPV infection. Almost one-half of the cases (46 of 95, 48.4%) with a high-risk HPV infection presented aneuploid squamous cells with greater than 9c DNA content, whereas none of the low-risk HPV-positive or HPV-negative SIL cases showed any aneuploid cells in this range. Although 91.8% of the HSIL cases displayed greater than 9c aneuploid cells, only 7.9% of the LSIL cases were positive for such cells with abnormally high DNA content. CONCLUSIONS: HPV typing and DNA measurements help in the objectivation of cytologic atypia and both can be performed efficiently from the same liquid-based cytologic samples.

Determination of ploidy and steroid receptor status in breast cancer by laser scanning cytometry.

BACKGROUND: Measurements on DNA content and steroid receptor status in breast cancer are of great clinical interest. Objective determination of estrogen and progesterone receptor expression should help to define the lowest levels of positivity still responding to adjuvant antihormonal therapy. For this purpose, a simple protocol for laser scanning cytometry is presented. METHODS: Analysis of 54 routine breast cancer samples was performed by laser scanning cytometry (LSC). To obtain single cell preparations from fresh tumor tissue, slides were prepared using the Cervisoft cytological device. Exact determination of tumor cell DNA content was done by referring to the CD45-positive tissue leukocyte fraction as the internal diploid reference cell population. Steroid receptor-expressing cells were detected by indirect immunolabeling. RESULTS: Indirect immunofluorescence allowed the best quantification of both the estrogen and progesterone receptor-expressing cell fractions by LSC. The number of receptor-expressing cells could be given in percentage. For comparison, the 10% cutoff value was used to determine receptor positivity. CONCLUSION: LSC enabled a simple, reliable, and inexpensive determination of DNA index and steroid receptor expression in breast cancer specimens by objective criteria.