Comprehensive approach to diagnostic cytology in endometrial pathology (review of literature).

A cytological examination of uterine cavity material has proven to be the effective method of detecting and clarifying the diagnosis of cancer and non-tumor endometrial diseases. However, sometimes there are difficulties in interpreting the results in a traditional (classical) cytological examination, due to high level of inadequate samples: the presence of mucus, a large number of blood elements, structures of poorly visible cells in the wrong preparation of the smear. At present, the method of liquid cytology, based on the technology of preparation of standard thin-layer cytological preparations from liquid cell suspension, is increasingly developed and widespread. These slides, if necessary, can be used for morphometry, cytochemical, immunocytochemical studies etc. It is also possible to prepare cell blocks from this material, and to obtain information about the histological structure if small pieces of tissue are presented in cytological material, moreover, to use these blocks for immunohistochemical reactions. Material from the uterine cavity may contain tumor cells from ovarian, tubal or other non- endometrial carcinoma, and it is necessary to obtain information about their origin, to verify the morphological diagnosis and to determine the management and treatment of patients, as a lot of problems concerning ovarian and endometrial cancer remains unclear. Examination of aspirates and scrapes from the uterine cavity using advanced molecular techniques, together with existing examination methods, can help to form risk groups for uterine, tubal, ovarian and even peritoneal tumors. The review of literature contains comparative characteristics of different methods and their combinations, which allow improving diagnostics of non-tumor lesions and endometrial tumors.

[Impressionable cytology method in diagnostics of dry eye syndrome.]

Analysis of the cellular composition of the eyeball conjunctival prints and evaluation of the informative of the cytomorphological method of research in the diagnosis of dry eye syndrome (DES). Cytological examination using impression cytology was performed in 72 patients: 60 patients with DES and 12 without eye disease (control group). The main component of cytological sample in patients with DES are (a) squamous epithelium (hyperkeratocytes); (b) groups and clusters of flattened epithelial cells; (c) a small amount (up to five in the preparation) of goblet cells. Impression cytology method improves the accuracy of diagnosis of DES.

Comparative Evaluation of ß-Catenin and E-Cadherin Expression in Liquid Aspiration Biopsy Specimens of Thyroid Nodules.

We compared the results of gene molecular and immunocytochemical studies of ß-catenin and E-cadherin in different variants of nodular thyroid disease (nodular colloid goiter, follicular thyroid adenocarcinoma, papillary thyroid cancer) and revealed changes of the function of the E-cadherin/ß-catenin complex leading to switching from adhesion function of ß-catenin in nodular colloid goiter to predominantly transcriptional activity in papillary carcinoma. The results confirm the important role of disturbances in E-cadherin-ß-catenin interactions in the mechanisms of malignant transformation of follicular epithelium.

[The technique of fluid cytology in diagnostic of thyroid diseases].

The increasing of number of diseases of thyroid actualizes searching for new solutions in differential diagnostic. Nowadays, the technique of fluid cytology is started to be applied under cytological analysis of puncture sample of thyroid. However, experience of application of technique of fluid cytology in interpretation of cytological sample is limited. The article presents results of analysis of samples of fine needle aspiration biopsy of thyroid from 126 patients. In case of traditional cytological analysis aspirate was putted on glass right away. In case of technique of fluid cytology patient was re-punctured and sample was placed into vial with preservative solution. At re-puncture increasing number of analysis with non-diagnosed samples was observed (40.6% in comparison with 17.8% under traditional cytological analysis). However, in 50% of patients with non-informative samples in traditional cytological analysis implementation of technique of fluid cytology permitted to establish proper diagnosis. In diagnostic of non-tumor affections of thyroid the best results demonstrated by technique of fluid cytology (86.6% as compared with 47.2% under technique of fluid cytology). However, in diagnostic of benign tumor affections of thyroid the best accuracy was demonstrated by traditional cytological analysis (61.1% as compared with 33.3% of technique of fluid cytology). Both techniques demonstrated similar results in category of malignant affections of thyroid. The diagnosis of cancer or suspicion to cancer was established in 85.7% of cases under technique of fluid cytology and in 82.7% of cases under traditional cytological analysis). The analysis of cytological pictures of various diseases of thyroid in technique of fluid cytology demonstrated specifics and differences from traditional cytology that are to be considered. At the same time, observations convincingly testify that in number of cases traditional and fluid techniques supplement each other. Besides, technique of fluid cytology permits preserving and transport puncture samples and in case of necessity to apply additional clarifying diagnostic techniques that in turn increases effectiveness of cytological diagnostic of affections of thyroid.

[ß-Catenin: Structure, Function and Role in Malignant Transformation of Epithelial Cells].

The article presents the data on the structure and mechanisms of ß-catenin functioning. The basic aspects of the role of ß-catenin in malignant transformation have been studied at various tumors. Primary structure of ß-catenin allows it to interact with many factors and ligands, including transcription factors, α-catenin, cadherin, Axin, Rho family GTPases, Bcl9 et al. This interaction is the base for ß-catenin's intracellular multi-functioning. The review presents data on the participation of ß-catenin in the mechanisms of adhesion, regulation of RNA metabolism, formation contacts with the cytoskeleton and its role in the canonical Wnt signaling pathway, marked examples pro-inflammatory and anti-inflammatory effects of ß-catenin. The ß-catenin involvement in malignant transformation and progression of certain tumors is not in doubt. The data on the changes in ß-catenin expression in the given examples of colon cancer, prostate cancer, different forms of thyroid cancer and hepatocellular carcinoma are presented with the prospects of its use as a marker and a predictor of malignant transformation. Continued research in this area will not only make use of ß-catenin as a potential predictor of malignant tumors, but also to develop approaches to targeted therapy.

[THE VIRTUAL CYTOLOGIC SLIDES FOR EXTERNAL EVALUATION OF QUALITY OF IMPLEMENTATION OF CYTOLOGIC ANALYSES IN CLINICAL DIAGNOSTIC LABORATORIES: POSSIBILITIES AND PERSPECTIVES].

The article considers application of technology of analysis of cytological slides in external quality control of clinical diagnostic laboratories. The advantages of virtual slides are demonstrated against other applied technologies of external evaluation of quality i.e. slide plate and digital micro-photography. The conditions of formation of virtual slides for external evaluation of quality of clinical diagnostic laboratories. The technology of their application is described. The success of practical application of considered technology in the Federal system of external evaluation of quality is emphasized.

[THE MORPHOMETRY IN CYTOLOGICAL ANALYSIS OF EXUDATIVE FLUIDS].

The cytological technique takes a leading position in diagnostic of tumor processes according exudative fluids. However, its results depend on large number of subjective factors. The morphometry is one of techniques by virtue of which objectification of data of cytological analysis is possible. The study was carried out to establish differences of morphometric parameters of benign and malignant cells of pleural effusion. The morphometric analysis of cells of mesothelium, breast cancer, adenocarcinoma of lung and adenocarcinoma of stomach was implemented. The parameters characterizing size (area, perimeter) and form (form factor) of nucleus and cell, nucleus-cytoplasm ratio. The results demonstrated that in pleural effusion between cells of proliferating mesothelium and malignant neoplasms exist significant differences in morphometric parameters (p<0.001). The differences between area of nuclei and cells are especially significant. The comparison of data of morphometry of cells of breast cancer; adenocarcinoma of lung and adenocarcinoma of stomach demonstrated that despite of some morphological similarities, analysis of morphometric parameters can provide important data for proper establishment of cytological diagnosis.

Training and practice of cytotechnologists: a discussion forum focused on Europe.

OBJECTIVES: To discuss the role and training of cytotechnologists (CTs) in Europe, to identify areas of good practice and to provide an informed opinion to those providing guidelines for training and practice in Europe. METHODS: All members of the Editorial Advisory Board of Cytopathology were invited to take part in a 'discussion forum' for which six topics were circulated in advance concerning the roles of CTs with regard to: (1) pre-screening slides; (2) 'signing out' reports; (3) carrying out ancillary techniques; (4) supervising laboratory staff; (5) taking part in rapid on-site evaluation (ROSE) of fine needle aspirates (FNAs); and (6) whether CTs were trained specifically in cytopathology or in general histopathology. Notes of the meeting were circulated by email and a final report was agreed by 22 participants from 17 predominantly European countries. RESULTS: Training for CTs throughout Europe was variable, especially for non-gynaecological cytology, which was inconsistent with the range of activities required. The participants recommended graduate entry, preliminary training in general laboratory technology, and subsequent training to take account of the probability and, in some centres, the reality of primary cervical cancer screening changing from cytology to human papillomavirus (HPV) testing. They further recommended that CTs should perform HPV tests and take part in ROSE for FNAs, and they supported the European Federation of Cytology Societies developing guidelines for training and practice. CONCLUSION: With CT training added to a university-based education in laboratory or biomedical science, a career in cytotechnology should be an attractive option involving a diverse range of laboratory and clinically based activities.

Longitudinal outcomes of high-risk human papillomavirus (HPV) infections as competing-risks events following cervical HPV test at baseline visit in the NIS-LAMS cohort.

BACKGROUND: The complex natural history of human papillomavirus (HPV) infections following a single HPV test can be modeled as competing-risks events (i.e., no-, transient- or persistent infection) in a longitudinal setting. The covariates associated with these competing events have not been previously assessed using competing-risks regression models. OBJECTIVES: To gain further insights in the outcomes of cervical HPV infections, we used univariate- and multivariate competing-risks regression models to assess the covariates associated with these competing events. STUDY DESIGN AND METHODS: Covariates associated with three competing outcomes (no-, transient- or persistent HR-HPV infection) were analysed in a sub-cohort of 1,865 women prospectively followed-up in the NIS (n = 3,187) and LAMS Study (n = 12,114). RESULTS: In multivariate competing-risks models (with two other outcomes as competing events), permanently HR-HPV negative outcome was significantly predicted only by the clearance ofASCUS+ Pap during FU, while three independent covariates predicted transient HR-HPV infections: i) number of recent (< 12 months) sexual partners (risk increased), ii) previous Pap screening history (protective), and history of previous CIN (increased risk). The two most powerful predictors of persistent HR-HPV infections were persistent ASCUS+ Pap (risk increased), and previous Pap screening history (protective). In pair-wise comparisons, number of recent sexual partners and previous CIN history increase the probability of transient HR-HPV infection against the HR-HPV negative competing event, while previous Pap screening history is protective. Persistent ASCUS+ Pap during FU and no previous Pap screening history are significantly associated with the persistent HR-HPV outcome (compared both with i) always negative, and ii) transient events), whereas multiparity is protective. CONCLUSIONS: Different covariates are associated with the three main outcomes of cervical HPV infections. The most significant covariates of each competing events are probably distinct enough to enable constructing of a risk-profile for each main outcome.

Covariates of high-risk human papillomavirus (HPV) infections are distinct for incident CIN1, CIN2 and CIN3 as disclosed by competing-risks regression models.

BACKGROUND: In addition to the oncogenic human papillomavirus (HPV), several cofactors are needed in cervical carcinogenesis, but whether the HPV covariates associated with incident (i) CIN1 are different from those of incident (ii) CIN2 and (iii) CIN3 needs further assessment. OBJECTIVES: To gain further insights into the true biological differences between CIN1, CIN2 and CIN3, we assessed HPV covariates associated with incident CIN1, CIN2, and CIN3. STUDY DESIGN AND METHODS: HPV covariates associated with progression to CIN1, CIN2 and CIN3 were analysed in the combined cohort of the NIS (n = 3187) and LAMS study (n = 12,114), using competing-risks regression models (in panel data) for baseline HR-HPV-positive women (n = 1105), who represent a sub-cohort of all 1865 women prospectively followed-up in these two studies. RESULTS: Altogether, 90 (4.8%), 39 (2.1%) and 14 (1.4%) cases progressed to CIN1, CIN2, and CIN3, respectively. Among these baseline HR-HPV-positive women, the risk profiles of incident CIN1, CIN2 and CIN3 were unique in that completely different HPV covariates were associated with progression to CIN1, CIN2 and CIN3, irrespective which categories (non-progression, CIN1, CIN2, CIN3 or all) were used as competing-risks events in univariate and multivariate models. CONCLUSIONS: These data confirm our previous analysis based on multinomial regression models implicating that distinct covariates of HR-HPV are associated with progression to CIN1, CIN2 and CIN3. This emphasises true biological differences between the three grades of CIN, which revisits the concept of combining CIN2 with CIN3 or with CIN1 in histological classification or used as a common endpoint, e.g., in HPV vaccine trials.

Risk estimates for persistent high-risk human papillomavirus infections as surrogate endpoints of progressive cervical disease critically depend on reference category: analysis of the combined prospective cohort of the New Independent States of the Former Soviet Union and Latin American Screening studies.

To make feasible future clinical trials with new-generation human papillomavirus (HPV) vaccines, novel virological surrogate endpoints of progressive disease have been proposed, including high-risk HPV (HR-HPV) persistence for six months (6M+) or 12 months (12M+). The risk estimates (relative risks [RRs]) of these 'virological endpoints' are influenced by several variables, not yet validated adequately. We compared the impact of three referent groups: (i) HPV-negative, (ii) HPV-transient, (iii) HPV-mixed outcome on the risk estimates for 6M+ or 12M+ HR-HPV persistence as predictors of progressive disease. Generalized estimating equation models were used to estimate the strength of 6M+ and 12M+ HR-HPV persistence with disease progression to squamous intraepithelial lesions (SILs), cervical intraepithelial neoplasia (CIN) grade 1+, CIN2+, CIN/SIL endpoints, comparing three optional reference categories (i)-(iii) in a prospective sub-cohort of 1865 women from the combined New Independent States of the Former Soviet Union (NIS) and Latin American Screening (LAMS) studies cohort (n = 15,301). The RRs of these viral endpoints as predictors of progressive disease are affected by the length of viral persistence (6M+ or 12M+) and the surrogate endpoint (SIL, CIN1, CIN2, CIN/SIL). Most dramatic is the effect of the referent group used in risk estimates, with the HPV-negative referent group giving the highest and most consistent RRs for both 6M+ and 12M+ viral persistence, irrespective of which surrogate is used. In addition to deciding on whether to use 6M+ or 12M+ persistence criteria, and cytological, histological or combined surrogate endpoints, one should adopt the HPV-negative referent group as the gold standard in all future studies using viral persistence as the surrogate endpoint of progressive disease.

Co-factors of high-risk human papillomavirus infections display unique profiles in incident CIN1, CIN2 and CIN3.

In addition to oncogenic 'high-risk' human papillomaviruses (HR-HPV), several co-factors are needed in cervical carcinogenesis, but it is poorly understood whether these HPV co-factors associated with incident cervical intraepithelial neoplasia (CIN) grade 1 are different from those required for progression to CIN2 and CIN3. To gain further insights into the true biological differences between CIN1, CIN2 and CIN3, we assessed HPV co-factors increasing the risk of incident CIN1, CIN2 and CIN3. Data from the New Independent States of the Former Soviet Union (NIS) Cohort (n = 3187) and the Latin American Screening (LAMS) Study (n = 12,114) were combined, and co-factors associated with progression to CIN1, CIN2 and CIN3 were analysed using multinomial logistic regression models with all covariates recorded at baseline. HR-HPV-positive women (n = 1105) represented a subcohort of all 1865 women prospectively followed up in both studies. Altogether, 90 (4.8%), 39 (2.1%) and 14 (1.4%) cases progressed to CIN1, CIN2 and CIN3, respectively. Baseline HR-HPV was the single most powerful predictor of incident CIN1, CIN2 and CIN3. When controlled for residual HPV confounding by analysing HR-HPV-positive women only, the risk profiles of incident CIN1, CIN2 and CIN3 were unique. Completely different HPV co-factors were associated with progression to CIN1, CIN2 and CIN3 in univariate and multivariate analyses, irrespective of whether non-progression, CIN1 or CIN2 was used as the reference outcome. HPV co-factors associated with progression to CIN1, CIN2 and CIN3 display unique profiles, implicating genuine biological differences between the three CIN grades, which prompts us to re-visit the concept of combining CIN2 with CIN3 or CIN1.

[The liquid cytology in clinical practice: a lecture].

The lecture covers one of the most perspective directions in clinical cytology. The emergence of liquid cytology is related to preparation of thin-layer specimens of organism liquids. Nowadays, the liquid cytology spreads more and more due to eephi application in different areas of cytological diagnostics, including screening of cervix uteri diseases, automatic screening, punctate tests, endoscopic and other materials. The lecture presents the basics of method, the characteristics of pre-analytic stage, the approaches to preparation of cytological specimens and cell blocks. The main possibilities, characteristics and perspectives of liquid cytology on the stage of rapid development of molecular genetic studies are discussed.

[Information technologies in clinical cytology (a lecture)].

The lecture is devoted to the urgent problem that is to increase the quality of cytological diagnosis, by diminishing the subjectivism factor via introduction of up-to-date computer information technologies into a cytologist's practice. Its main lines from the standardization of cytological specimen preparation to the registration of a cytologist's opinion and the assessment of the specialist's work quality at the laboratories that successfully use the capacities of the current information systems are described. Information technology capabilities to improve the interpretation of the cellular composition of cytological specimens are detailed.

Survey of medical training in cytopathology carried out by the journal Cytopathology.

This report of the Editorial Advisory Board of Cytopathology gives the results of a survey of medical practitioners in cytopathology, which aimed to find out their views on the current situation in undergraduate and postgraduate training in their institutions and countries. The results show that training in cytopathology and histopathology are largely carried out at postgraduate level and tend to be organized nationally rather than locally. Histopathology was regarded as essential for training in cytopathology by 89.5% of respondents and was mandatory according to 83.1%. Mandatory cytopathology sections of histopathology were reported by 67.3% and specific examinations in cytopathology by 55.4%. The main deficiencies in training were due to its variability; there were insufficient numbers of pathologists interested in cytology and a consequent lack of training to a high level of competence. Pathologists without specific training in cytopathology signed out cytology reports according to 54.7% of responses, more often in centres where training was 3-6 months or less duration. Although 92.2% of respondents thought that specialist cytology should not be reported by pathologists without experience in general cytopathology, that practice was reported by 30.9%, more often in centres with small workloads. The survey report recommends that 6-12 months should be dedicated to cytopathology during histopathology training, with optional additional training for those wanting to carry out independent practice in cytopathology. Formal accreditation should be mandatory for independent practice in cytopathology. When necessary, temporary placements to centres of good practice should be available for trainees intending to practise independently in cytopathology. There should be adequate numbers of pathologists trained in cytopathology to a high level of competence; some of their time could be released by training cytotechnologists and trainee pathologists to prescreen cytology slides and assess adequacy of fine-needle aspiration samples when immediate diagnosis was not required. The survey demonstrated a clear need for European and international guidelines for training in cytopathology.

[Proposals for standardization of clinical laboratory studies: technology "cytological study of lymph node specimens"].

This proposal contains unique requirements for a cytological study of lymph node specimens at the cytological and clinical diagnostic laboratories of health care facilities certified for this kind of work. The paper gives requirements for the performance of technologies, for specialists and auxiliary personnel, specialists' knowledge and skills, medical staff safeguarding. The conditions for rendering a service, necessary equipment, ware, and reagents are described in details. The specific features of a pre-analytical stage, methods for the fixation and staining of smears, the interpretation and recording of findings, and the control of the quality of a performed study, as well as the cost characteristics in conventional units of man-hours. The developed technology may be of great importance to the health care facilities certified for rendering this service.

The role of breast FNAC in diagnosis and clinical management: a survey of current practice.

Most participating countries have now adopted a triple assessment approach, i.e. clinical,imaging and pathology, to breast diagnosis, with FNAC as the first-line pathological investigation in both screening and symptomatic populations, with the exception of microcalcifications. Pathologists specialized in cytopathology are best qualified to collect and interpret FNAC samples, but this is not always possible or practical. Radiologists involved in breast imaging should ensure that they have the necessary skills to carry out FNAC under all forms of image guidance. Best results are achieved by a combination of both techniques, as shown in the image-guided FNAC in the presence of the cytopathologist. The majority of European countries use similar reporting systems for breast FNAC (C1-C5), in keeping with European Guidelines for Quality Assurance in Breast Cancer Screening and Diagnosis, although some still prefer descriptive reporting only. When triple assessment is concordant, final treatment may proceed on the basis of FNAC, without a tissue biopsy. ER and PR assessment can be done safely on FNAC material. However, not all institutions may have expertise in doing this. HER-2 protein expression on direct cytological preparations is insufficiently reliable for clinical use, although its use for FISH is possible, if expertise is available. The majority of participants practise a degree of one-stop diagnosis with a cytopathologist present in the out-patient clinic. Formal recognition of the importance of the time spent outside the laboratory, both for cytopathologist and cytotechnologist, is necessary in order to ensure appropriate resourcing. The use of core biopsy (CB) has increased, although not always for evidence-based reasons. CB and FNAC are not mutually exclusive. FNAC should be used in diagnosis of benign, symptomatic lesions and CB in microcalcifications, suspicious FNAC findings and malignancies where radiology cannot guarantee stromal invasion.

Epidemiological, clinical and viral determinants of the increased prevalence of high-risk human papillomavirus (HPV) infections in elderly women.

BACKGROUND: Population-based studies have reported a second peak of human papillomavirus (HPV) prevalence among women > 55 years, but reasons for this U-shaped HPV prevalence curve are poorly understood. OBJECTIVES: To analyse determinants of high-risk HPV (HR-HPV) infections among postmenopausal women. STUDY DESIGN AND METHODS: A cohort of 3,187 women was stratified into three age categories: i) youngest age group < 25 years (n = 1.103); ii) women between 26-55 years (n = 2.004), and iii) women > 55 years (n = 80), analysed for epidemiological, clinical and virological determinants of their HR-HPV infections. Real-time PCR was used for HPV genotyping, analysis of viral loads for HPV16, 18/45, 31, 33/52/58, 35 and 39, and load of integrated HPV16. RESULTS: Age-standardised prevalence of HR-HPV infections showed a second peak among women > 55 years, with a perfect U-shaped curve (R2 = 0.966). The factors explaining this increased HR-HPV prevalence among older women include: i) cohort effect, ii) higher viral loads for HR-HPV types with cubic model curve (R2 = 0.714) for HPV 16, iii) distinct shift (p = 0.0001) from multiple-type infections to single HR-HPV types, iv) transition from episomal to integrated HPV16 (p = 0.009), v) higher load of integrated HPV16 (p = 0.009), and, vi) higher proportion of incident infections, higher rate of viral persistence, and lower rate of HR-HPV clearance. CONCLUSIONS: These data suggest that in women who fail to eradicate their HR-HPV infection until menopause, selection of integrated viral clone has taken place, driving the process towards progressing disease. Consequent to this, most of the HR-HPV infections in women > 55 years were associated with high-grade CIN or invasive carcinoma.

The future of cytopathology in Europe. Will the wider use of HPV testing have an impact on the provision of cervical screening?

The emphasis of the EFCS Congress held in Venice in October 2006 was on the future of Cytopathology in relation to events in Europe. Much of the discussion centred on the role of human papilloma virus testing and its impact on the provision of cervical screening. The following is a transcript of the discussion that took place at the Advisory Board Meeting for the journal Cytopathology, with some additional written comments received prior to the meeting. A brief summary has been provided as a conclusion by Dr A. Herbert.

Early integration of high copy HPV16 detectable in women with normal and low grade cervical cytology and histology.

BACKGROUND: Integration of human papillomavirus (HPV) DNA has been considered a late event in cervical carcinogenesis. However, integrated forms of HPV were recently detected in cancer precursor lesions using a new real time polymerase chain reaction (PCR) to detect the deletions at the 3362-3443 region of HPV16 E2 OBJECTIVE: To study the frequency of HPV16 DNA integration in cervical lesions and compare the sensitivity of an additional upstream region of the E2 ORF (2962-3138) in detecting HPV integration. METHODS: Using the TaqMan based PCR, HPV16 positive DNA samples were analysed in 164 cervical scrapings from women participating in a multicentre screening trial. Biopsy confirmation was available in 62 cases. RESULTS: Primers targeting the 3362-3443 region detected the majority of E2 deletions. In only 23% of the samples was the E2 upstream region equal or better target than the 3362-3443 region. Mixed (episomal/integrated) pattern was the most prevalent physical state of HPV16, also present in PAP smears with normal morphology. Pure integrated form was most prevalent in HSIL and cancer lesions, but also detectable in low grade abnormalities (NSIL, ASC-US, LSIL). Women with only integrated HPV16 were almost 10 years older than those with episomal HPV16. Viral load of integrated HPV16 was related to cytological abnormality (p = 0.003) but not to histology. CONCLUSIONS: Integrated HPV16 is present in low grade cervical lesions, mostly mixed with the episomal form. Women with the pure integrated form of HPV16 are older than those with the other forms.