Pitfalls in gynecological cytology - review of the common and less frequent entities in Pap test.

BACKGROUND: Pitfalls in Pap test could be defined as false positive, false negative or underdiagnosed results which can lead to unnecessary diagnostic procedures or delayed and inadequate treatment. It can be a consequence of misinterpretation of certain morphological entities which are described in this paper. SUMMARY: the paper presents the overview of the morphological features and look-alikes of the common sources of pitfalls such as atrophy, repair, IUD change, tubal metaplasia, hyperchromatic crowded groups, and radiation changes. Rare causes of pitfalls such as Arias-Stella changes, pemphigus, tumor diathesis per se, rare types of cervical cancer, including verrucous and papillary squamous cell cancer, gastric type and endometrioid adenocarcinoma are also described. KEY MESSAGES: The awareness of pitfalls in cervical cytology is important for cytopathologists and clinicians to avoid future errors. Review of Pap tests with erroneous diagnosis is important for quality control in cytology laboratory, and it must be considered as educational and experience building procedure. Cytopathologist should not pull back in significant diagnoses, especially in HPV negative cases.

Inter- and intraobserver agreement in whole-slide digital ThinPrep samples of low-grade squamous lesions of the cervix uteri with known high-risk HPV status: A multicentric international study.

BACKGROUND: High-risk human papilloma virus (HR HPV) testing and liquid-based cytology are used for primary cervical screening. Digital cytology, based on whole-slide scanned samples, is a promising technique for teaching and diagnostic purposes. The aim of our study was to evaluate the interobserver and intraobserver variation in low-grade squamous lesions, HR HPV status bias, and the use of whole-slide scanned digital cervical cytology slides. METHODS: Fifteen expert cytopathologists evaluated 71 digitalized ThinPrep slides (31 atypical squamous cells of undetermined significance [ASC-US], 21 negative for intraepithelial lesion or malignancy, and 19 low-grade squamous intraepithelial lesion cases). HR HPV data were accessible only in the second round. RESULTS: In interobserver analysis, Kendall's coefficient of concordance was 0.52 in the first round and 0.58 in the second round. Fleiss' kappa values were 0.29 in the first round and 0.31 in the second round. In the ASC-US category, Fleiss kappa increased from 0.19 to 0.22 in the second round and the increase was even higher expressed by Kendall's coefficient: from 0.42 to 0.52. In intraobserver analysis, personal scores were higher in the second round. CONCLUSIONS: The interobserver and intraobserver variability in low-grade squamous lesions was within fair agreement values in the present study, in line with previous works. The comparison of two rounds showed that expert cytopathologists are generally unbiased by the knowledge of HR HPV data, but that being informed of the HR HPV status leads to a better agreement. Stain quality and back discomfort were highlighted as factors affecting digital cytopathology use.

Organization of the 43rd European Congress of Cytology in the SARS-CoV-2 pandemic period: A report.

The 43rd European Congress of Cytology in Wroclaw, Poland, was held as a hybrid meeting in the Fall of 2021. After nearly 2 years without in-person cytology conferences, the 43rd Congress represents 1 of the first major international scientific meetings to occur during the severe acute respiratory syndrome-coronavirus 2 pandemic. Since March 2020, the pandemic situation substantially modified the organization of scientific meetings because of both domestic and international travel restrictions, new health standards, and concern among participants, resulting in new alternative forms of virtual conferencing. Cancer (Cancer Cytopathol) 2022;130:000-000.

Diagnostic Three Slides Pap Test Compared to Punch Biopsy and Endocervical Curettage in Confirmed HSIL+ Diagnosis.

OBJECTIVE: The aim of the study was to evaluate the accuracy of the diagnostic Pap test (DPT) on three slides and punch biopsy and endocervical curettage (PB/ECC) compared with the final biopsy material in the detection of high-grade squamous intraepithelial lesion (HSIL). MATERIALS AND METHODS: Patients treated with conization after previous DPT and PB/ECC were analyzed. The findings of the DPT and PB/ECC as well as of the endocervical brush cytology and ECC were compared with the final conus histology. RESULTS: 150 patients were analyzed, and final histology verified 145 cases of HSIL and 3 cancers. The percentage of confirmed HSIL cytology was 97%, while for PB/ECC it was 79% with 30/145 false negative results. The correlation between Pap test and PB/ECC showed that the diagnostic accuracy of DPT is significantly higher (p < 0.0001). Endocervical brush cytology confirmed HSIL+ in the endocervical canal in 83% and ECC in 35% of cases (p < 0.0001). CONCLUSION: The DPT on three slides enables better detection of HSIL compared to PB/ECC, particularly for lesions localized in the endocervical canal sampled with a cytobrush. A high quality DPT could represent a surrogate for PB/ECC and open the possibility of direct access to therapeutic procedure.

Recommendations of the European Advisory Committee of Cytotechnology and European Federation of Cytology Societies for Training and Education of Cytotechnologists in Europe.

BACKGROUND: Faced with changes in cytodiagnostics, cervical cancer screening programs, the introduction and application of new methods, the cytotechnological educational program requires the necessary changes and additions. Insufficient, uneven as well as inaccessible education of cytotechnologists in European countries was the basis for making these recommendations. SUMMARY: The results of previous research and publications related to the currently available education of cytotechnologists in Europe, the needs and suggestions were given by the European Advisory Committee of Cytotechnology (EACC) and European Federation of Cytology Societies (EFCS) for optimal education of future generations of cytotechnologists were used in the preparation of these recommendations. The EACC and EFCS propose a 1-year education and training program divided into 3 modules: gynecological, nongynecological exfoliative, and fine-needle aspiration cytology. Training programs should be organized by an accredited university, preferably a combination of internal education in a cytology laboratory and theoretical education at the university. Cytopathologists and cytotechnologists with at least 5 years of work experience in cytodiagnostics should participate in education. Upon completion of the training program, the EACC and EFCS propose an official name: EFCS certified cytotechnologist. Key Messages: The EACC and EFCS believe that it is extremely important that these recommendations are recognized and implemented by institutions that provide education for cytotechnologists so that they can meet the growing requirements of the profession with their acquired knowledge and competencies.

The Immunohistochemical Pattern of Epithelial-Mesenchymal Transition Markers In Endometrial Carcinoma.

The majority of endometrial carcinoma are diagnosed at an early stage and exhibit a favorable prognosis. However, 10% to 15% of ECs recur and the majority are type II tumors which are high-grade carcinomas. The epithelial-mesenchymal transition (EMT) has been considered as a fundamental step for the development of the invasive phenotype of cancer cells. During EMT, many of epithelial surface markers, primarily E-cadherin disappear, and mesenchymal markers including N-cadherin gain. This feature resides predominantly at the invasive front (IF) of the tumor. Therefore, we examined the immunohistochemical expression of E-cadherin and N-cadherin at the IF, in central areas of the tumor and lymphovascular space, in type I and type II endometrial carcinoma. The association of each protein with the clinicopathologic features was also evaluated. Our results confirmed a stronger E-cadherin immunostaining in type I tumors indicating that the loss of E-cadherin may be responsible for a more aggressive behavior of type II ECs. In both types, E-cadherin was strongly expressed in central areas and the reactivity decreased toward the IF. On contrary, N-cadherin was overexpressed at the IF confirming an inverse relationship between these markers. In addition, a decrease in E-cadherin expression was observed in cells within the lymphovascular space. Downregulation of E-cadherin was associated only with high-grade tumors while no correlations between both markers and other clinicopathologic features were found. Our results confirm that EMT occurs at the IF that represents a critical interface between the tumor and the host.

Adult Granulosa Cell Tumors of the Ovary: A Retrospective Study of 36 FIGO Stage I Cases with Emphasis on Prognostic Pathohistological Features.

OBJECTIVE: Adult granulosa cell tumors (AGCTs) represent 2%-5% of all ovarian malignancies. The aim of this study was to analyze clinical and pathohistological parameters and their impact on recurrence, overall, and disease-free survival in FIGO stage I AGCT patients. METHODS: The tumor specimens analyzed in this retrospective study were obtained from a total of 36 patients with diagnosis of ovarian AGCT surgically treated at the Department of Gynecology, Rijeka University Hospital Centre, between 1994 and 2012. Clinical, pathological, and follow-up data were collected. RESULTS: The mean age at diagnosis was 54.5 years with a range of 24-84. The majority of the patients, 30 (83%), were in FIGO stage IA, 3 (8%) in stage IC1, 1 (3%) in stage IC2, and 2 (6%) in stage IC3. During follow-up period (median 117.5 months, range 26-276), recurrence occurred in 4 patients (12%) with 2 deaths of the disease recorded. In univariate analysis, the 5-year survival rates were significantly shorter in patients with FIGO substage IC (p = 0.019), with positive LVSI (p = 0.022), with presence of necrosis (p = 0.040), and with hemorrhage (p = 0.017). In univariate analysis, the 5-year disease-free survival rates were significantly shorter in patients treated with fertility surgery (p = 0.004), with diffuse growth pattern (p = 0.012), with moderate and severe nuclear atypia (p = 0.032), and with presence of hemorrhage (p = 0.022). FIGO substage IC proved to be independent predictor for recurrence (OR = 16.87, p = 0.015, and OR = 23.49, p = 0.023, resp.) and disease-free survival (p = 0.0002; HR 20.84, p = 0.02) at the uni- and multivariate analyses. CONCLUSIONS: FIGO substage IC is predictive of recurrence and disease-free survival in patients with early-stage AGCTs. LVSI, presence of necrosis and hemorrhage, diffuse growth pattern, and nuclear atypia in AGCTs seem to be associated with overall and disease-free survival, so these pathological features should be taken into consideration when managing patients with AGCT.

Expression of CD133 and CD117 in 64 Serous Ovarian Cancer Cases.

The cancer stem cells (CSCs) represent a minority of tumor cells that are able to proliferate and self-renew and might be responsible for tumor initiation and maintenance. The CD133 and CD117 are the most commonly used markers for the putative CSCs, especially for the ovarian CSCs, but its clinical significance remains uncertain. The aim of this study was to compare the immunohistochemical expression of CD133 and CD117 in 64 primary ovarian high grade serous carcinoma and peritoneal metastasis, and to examine their potential clinical role. CD133 expression was mainly seen in the apical/endoluminal cell surface of tumor cells and was found in 61% of the carcinoma samples and 41% of the metastasis. The median of CD133 positive cells in tumors was 1 (0.1-7)%, and in metastases was 0.6 (0.1-6)%. CD117 expression appeared as a cytoplasmic and/or membranous stain and was found in 81% of the carcinoma samples and 77% of the metastasis. The median of CD117 positive cells in tumors was 1 (0.1-8)%, and in metastases was 0.1 (0.1-7)%. Multivariate analysis has shown that patients with high CD133 expression in tumor cells have significantly shorter disease free survival and overall survival (p=0.025 and p=0.014, respectively). Patients with high CD117 expression in tumor cells have significantly shorter disease free survival (p=0.031). Cox's proportional hazards model identified expression of CD133 protein in tumor as an independent prognostic factor. Our study indicates that the immunohistochemical assessment of CD133 and CD117 expression may have potential clinical value in predicting disease progression and prognosis in the high grade serous ovarian cancer. CD133 proved to be an independent prognostic factor in the high grade serous ovarian cancer patients.

[Clinical recommendations for diagnosing, treatment and monitoring of patients with uterine cervical cancer -- Croatian Oncology Society and Croatian Society for Gynecology and Obstetrics as Croatian Medical Association units and Croatian Society of Gynecological Oncology]. / Klinicke upute za dijagnostiku, lijecenje i pracenje bolesnica oboljelih od raka vrata maternice hrvatskoga onkoloskog drustva i hrvatskog drustva za ginekologiju i opstetriciju hrvatskoga lije nickog zbora te hrvatskoga ginekoloskoonkoloskog drustva.

Cervical cancer, in comparison with other gynecological malignancies, mainly affects younger women. It can be prevented trough educational programs, screening and early detection. It also can be efficiently treated when it appears. Treatment modalities include surgery, chemotherapy and radiotherapy, according to the stage of the disease and patient condition. Treatment decisions should be made after multidisciplinary team discussion. Due to the significance of this disease it is important to define and implement standardized approach for diagnostic, treatment and monitoring algorithm as well. The following text presents the clinical guidelines in order to standardize the procedures and criteria for the diagnosis, management, treatment and monitoring of patients with uterine cervical cancer in the Republic of Croatia.

Evaluation of the HPV L1 capsid protein in prognosis of mild and moderate dysplasia of the cervix uteri.

Cervical intraepithelial neoplasia (CIN) can be detected in the cytologic smears years before invasive squamous cancer arises, but no reproducible morphologic criteria exist to predict behavior of cervical lesions. The possibility of predicting the clinical course of cervical lesions could be of high value in clinical practice and some women will spare of unnecessary treatment. HPV L1 capsid protein represents about 90% of the total protein on the surface of the virus and can be detected in mild to moderate dysplasia and rarely in severe dysplasia. The purpose of the study was to evaluate the use of immunodetection of HPV L1 protein on archival Pap smears with findings of mild and moderate dysplasia in predicting its clinical course. Immunochemical analyses with L1 antibody revealed positively stained nuclei of squamous epithelial cells in 56 of 114 smears (49.1%). The staining results were correlated with follow-up smears or with histologic verification. Regression (negativisation of the Pap smear for 24 months or longer) was noticed in 31 of 56 (55.4%) L1-positive cases and in 20 of 58 (34.5%) L1-negative cases. Persistent disease occured in 13 (23.2%) L1-positive cases and in 14 (24.1%) L1-negative cases. Progressive disease occured in 12 (21.4%) L1-positive cases and in 24 (41.4%) L1-negative cases. The difference in the clinical course between the L1-positive and L1-negative patients was statistically significant (p = 0.025). Also, the difference in the clinical course of the L1-negative staining in the under-30 and over-30 years age group was statistically significant (p = 0.04). For conclusion, our data confirm that immunostaining for HPV L1 capsid protein could offer prognostic information about mild and moderate intraepithelial cervical squamous lesions.

Cervical cancer screening programme in Primorsko-Goranska County, Croatia--the results of the pilot study.

The opportunistic cervical cancer screening has been conducted in Croatia since its introduction in the 1960s, in the context of a high quality gynaecological cytology with a long tradition and a wide network of primary care gynaecologists. In 2006, a pilot screening programme under the title "Early detection of cervical cancer was conducted in Primorsko-Goranska County (PGC)", as the first organised cervical cancer screening ever conducted in the Republic of Croatia. The pilot screening programme targeted women aged 20-64 years. The pilot group consisted of 6,000 randomly sampled primary care patients of six gynaecologists. The women were invited via a personal letter and were given a questionnaire. The results of the first and the second year of screening, as well as of both years together were analysed. The response rate to the anamnestic questionnaire was 49.1%. The participation rates to the screening were 35.2% in 2007, and 46.5% in 2008, total of 42.7%. The increase in participation between years 2007 and 2008 was statistically significant (p = 0.01). According to the age, the lowest participation rate of 33.3% was observed in the youngest group of women (20-29) and the highest of 60.7% in the oldest group (60-64). The detection rate of cytological abnormalities was 4.6% with 2.6% of borderline (ASCUS) cytology and referral rate of 1.2%. The highest abnormal Pap test frequencies of 6.8% and 7.1% were observed in the youngest age groups (20-29 and 30-39), and the lowest (2%) in the age group of 60-64. Specimen adequacy was generally of high quality with unsatisfactory rate of 0.8%, with statistically significant improvement in 2008, compared to the previous year (p = 0.001). Although to a limited extension, during two-year pilot cervical cancer screening programme in PGC the participation rates and Pap smear adequacy have improved. We expect that the continuation of the programme will result in further increase of participation and higher overall quality of the programme.

Villoglandular papillary adenocarcinoma of the uterine cervix with aggressive clinical course--a case report.

Villoglandular papillary adenocarcinoma (VGA) of cervix is an uncommon but well recognized histologic subtype of cervical adenocarcinoma which usually affects young women. Based on the favorable outcomes reported in most previous cases the tumor is generally considered to have an indolent clinical course with excellent prognosis. We present a case of a 22-year-old woman admitted at our Department for glandular abnormality on cervical smear and episodes of vaginal discharge. In the Pap smear, the cytological features were suspicious but not diagnostic of adenocarcinoma, therefore reported as atypical glandular cells (AGC). Histological examination confirmed VGA associated with lymphovascular space invasion. The patient underwent radical operative procedure. Intraoperative cytologic examination detected pelvic lymph nodes metastasis. The patient was confirmed to be in an advanced stage - III B (FIGO). During a two years follow-up period a rapid dissemination of the tumor occurred and resulted with a fatal outcome. Although VGA has been reported to have a favorable prognosis, several cases with lymph node involvement have already been described. Cervical smears examination would be helpful for an early diagnosis of VGA, however the cytologic recognition is often difficult. Further investigation of the pathogenesis, diagnosis and therapy of the tumor is needed.

Cervical cytology and HPV test in follow-up after conisation or LLETZ.

The patients treated with conservative surgical therapy for cervical intraepithelial neoplasia (CIN) have an increased risk to develop invasive cervical carcinoma compared to the general population. Cervical cytology and HPV test are included in the protocols for the detection of treatment failure. The purpose of the study was to analyse cytology-histology correlation after conisation or Large Loop Excision of the Transformation Zone (LLETZ), resection margin status, compliance to the follow-up protocol and evaluation of cervical cytology and HPV testing in two year period after surgical treatment. We retrospectively reviewed 251 cases of conisation or LLETZ performed between January and December, 2006. Conventional cervical smears were analysed and abnormal cytology was defined as atypical squamous cells of undetermined significance or worse (ASCUS+). The digene Hybrid capture 2 test was used for detection of high-risk HPV types. Histology analysis demonstrated CIN1 + lesion in 234 cases (93.2%) with cytology-histology correlation in 97.9% of cases. A preoperative HPV test was made in 142 histologically confirmed CIN1 + lesions and 137 (96.5%) tested positive. The resection margins were involved in 48 (20.8%) cases. In 24 (10.3%) cases the margins were difficult to determine. Abnormal cytology was found in 33 (15.2%) cases of the 217 (86.5%) patients that attended the post-treatment visits. The post-treatment HPV test was performed on 159 women and it was positive in 25 (15.7%) cases. The complete follow-up control cytology, with at least three Pap smears in the subsequent two years or with second treatment, was registered in only 146 (58.2%) patients. 14/217 (6.5%) patients underwent second treatment with histologically confirmed treatment failure. In all patients with control smear, repeated cytology found HSIL. On six women, the control HPV test was performed. In five cases, it was positive and in one case with histological diagnoses of VAIN2, it was negative. Our study confirms the important role of cervical cytology in the diagnosis of cervical intraepithelial lesions and monitoring after treatment. In the future we will have to improve compliance to the follow-up protocols and use of the HPV test in the selection of women at risk of treatment failure.

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints.

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.

HPV testing for cervical cancer screening in Croatia.

Opportunistic screening based on the Pap smear has been undertaken in Croatia since 1953. However, cervical cancer remains an important health problem in Croatia when compared to European countries with organised screening programmes. In Croatia, in addition to screening based on well established cytology, Human papillomavirus (HPV) testing is widely used as secondary test as a triage to borderline cytology and as a follow-up after treatment of severe cervical lesions. Many different approaches for HPV testing arose in Croatia over the last decade depending on the needs of each medical institution involved. Presently, there is an urgent need for better networking between the laboratories, the implementation of quality assessment and the adaptation of a uniform system of referring to and reporting of HPV testing. In conclusion, the best possible organisation for HPV testing would be essential for implementation of HPV testing as primary screening test in Croatia, an thus ultimately and hopefully, the more successful cervical cancer control.