Papillae, psammoma bodies, and/or many nuclear pseudoinclusions are helpful criteria but should not be required for a definitive cytologic diagnosis of papillary thyroid carcinoma: An institutional experience of 207 cases with surgical follow up.

BACKGROUND: Noninvasive follicular thyroid neoplasm with papillary-like features (NIFTP) was introduced in 2016 replacing noninvasive follicular variant of papillary thyroid carcinoma, with recommendations to label them "noncancer." To avoid reducing risk of malignancy (ROM) and overdiagnosing NIFTP as malignant, some authors required restricted cytologic criteria (RC) for a definitive diagnosis of papillary thyroid carcinoma (PTC), including papillae, psammoma bodies. or ≥3 nuclear pseudoinclusions. Since then, NIFTP criteria have been revised, biologic behavior better understood, and incidence reported to be much lower than initially anticipated. This study examines the impact of RC on PTC cytologic diagnoses, ROM, and detection of clinically significant carcinomas (CSC). MATERIALS AND METHODS: A total of 207 thyroid FNAs originally diagnosed as PTC and suspicious for PTC (SPTC) with surgical follow-up were evaluated. RC were retrospectively applied to cases as a requirement for diagnosing PTC, and cases that did not meet RC were reclassified as SPTC. ROMs and diagnostic accuracies of pre- and post-RC diagnoses were correlated with followup CSC. RESULTS: RC were met in 118/142 (83%) and 20/65 (31%) of cases originally diagnosed as PTC and SPTC, respectively. Post-RC, 29% (19/65) of CSC originally diagnosed as SPTC were upgraded to PTC, and 17% (24/142) of CSC originally diagnosed as PTC were downgraded to SPTC. No NIFTPs were diagnosed as malignant. CONCLUSIONS: RC should not be required for a definitive diagnosis of PTC when other nuclear features of PTC are diffuse and overt. Applying RC, however, helps the pathologist arrive at a more definitive diagnosis of PTC in suspicious cases.

Impact of education on institutional and faculty rates of atypical squamous cells.

INTRODUCTION: Papanicolaou test quality metrics include the ASC rate, ASC:SIL ratio, and ASC HPV+ rate. What a laboratory should do when metrics show a worrisome trend is not well defined. In 2015, our laboratory noted a worrisome trend in our quality metrics and decided to implement a systemic education program in 2016; we monitored the effectiveness of our program. METHODS: An educational intervention was designed for March/April 2016. Cytotechnologist education consisted of: group meeting on March 10 to discuss metrics, lecture, and written materials on ASC-US criteria, a quiz on challenging ASC-US cases, encouragement to seek consultation, and each cytotechnologist received quarterly individual metrics. The cytopathologist education consisted of: group meeting on April 16 to discuss metrics, encouragement to bring borderline cases to consensus conference, and each faculty received quarterly individual metrics. The ASC rate, ASC:SIL ratio, and ASC HPV+ rate was collected for the institution and each individual faculty in 2016 for January to March (pre-interventions, Q1), April to June (post-interventions, Q2), and July to September (post-interventions, Q3). ASC-H was included in the calculation of ASC %, ASC:SIL, and ASC HPV+ rates. RESULTS: There was a substantial decline in the lab ASC rate and ASC:SIL ratio, and the ASC HPV+ rate increased. Individual faculty changes in ASC:SIL ratio and ASC HPV+ rate also improved. CONCLUSIONS: In our institution, an educational program has been very effective in improving Papanicolaou test metrics. It is helpful to perform re-education at all levels within the department.

Role of ThinPrep liquid-based cytology in evaluation of the endocervical canal in patients with abnormal cervical screening.

INTRODUCTION: Endocervical sampling is frequently used as an adjunct to colposcopy. Few studies address the role of endocervical brushing (ECB) with liquid-based cytology (LBC) for evaluation of the endocervical canal. We assessed the roles of ThinPrep (TP) LBC of ECB specimens and cell blocks (CBs). MATERIALS AND METHODS: Pathology archives were searched for ECB specimens from 2010-2015. Preceding Papanicolaou test interpretation, human papillomavirus status, concurrent and follow-up surgical specimens, and ECB diagnoses were recorded. CB cellularity, when available, was scored on a scale of 0 to 4. The cellularity of the TP and CBs was compared. RESULTS: Of 365 ECB cases, 6 (1.6%) were insufficient for diagnosis, compared with a 5% rate for endocervical curettings. Eleven ECB cases (3%) showed low cellularity. Of the 241 (66%) cases with concurrent biopsies, the ECB diagnosis agreed with the biopsy diagnosis (within 1 grade) in 198 (82%) cases. In 9 (2.5%) cases, ECB was the only means of diagnosis of a high-grade squamous intraepithelial lesion / adenocarcinoma in situ confirmed on follow-up. Compared with TP LBC, the CBs (performed in 84 [23%] of cases) were of greater cellularity in 30 (42%) and of equal cellularity in 17 (24%). None of the CBs showed an additional epithelial abnormality missed in TP LBC. CONCLUSIONS: TP LBC is capable of detecting endocervical epithelial abnormalities and may be used as a substitute for endocervical curettings. Performing a CB did not lead to detection of additional abnormalities, although it complemented TP findings and facilitates the performance of ancillary studies.

Utility of Sry-Related HMG-Box Gene 10 (SOX10) as a marker of melanoma in effusion cytology.

BACKGROUND: The cytodiagnosis of melanoma in effusions can be challenging. Although immunohistochemical (IHC) stains such as HMB45, Melan A, and S-100 are often utilized; the role of Sry-related HMG-box gene 10 (SOX10) in the diagnosis of melanoma in effusions has not been previously reported. METHODS: A total of 14 confirmed melanoma cases diagnosed on effusion cytology (nine pleural and five peritoneal) were collected from 2000 to 2016. IHC stain for SOX10 was performed and compared with HMB45, Melan A, and S-100. To evaluate the specificity of SOX10, we stained 47 previously diagnosed nonmelanocytic malignant effusions. A cut-off of >1 positively staining cells was considered positive. The intensity of staining was graded as weak, moderate, and strong. RESULTS: All 14 melanoma cases were positive for SOX10 and HMB45 (100%), compared to 12 cases (86%) using Melan A and S-100. Among 47 previously diagnosed nonmelanocytic malignant effusions (4 malignant mesotheliomas, 12 müllerian tumors, 9 breast carcinomas, 9 lung adenocarcinomas, and 13 hematologic tumors), SOX10 and HMB45 showed a specificity of 98%, whereas Melan A and S100 had a specificity of 100%. CONCLUSIONS: The sensitivity of SOX10 for melanoma in effusions is comparable with HMB45 with a 100% sensitivity. In terms of staining intensity, HMB45 appeared to be superior as it showed 100% moderate to strong intensity compared to 72% for SOX10. All four markers showed near-100% specificity in differentiating melanoma from nonmelanocytic malignant effusions. A combination of HMB45 and SOX10 might be useful as the initial stains of choice in diagnosing melanoma in effusion cytology.

Unsatisfactory exfoliative anal cytology samples, 15-year experience with histologic, cytologic, and molecular follow-up.

BACKGROUND: The incidence of anal carcinoma has risen in recent decades. Exfoliative cytology screening of selected high risk patients is performed in many centers. Unsatisfactory cytology results are frustrating to patients, clinicians, and laboratorians. The aim of this study is to ascertain outcomes of patients with non-diagnostic anal cytology. METHODS: A retrospective review of anal cytology testing performed at the Cleveland Clinic between 01/01/2001 and 12/31/2015 was performed. All cases were received as liquid-based samples and processed as ThinPreps (Hologic, Marlborough, MA). Co-testing for HR-HPV DNA was performed using Hybrid Capture 2® (Qiagen, Germantown, MD) in the majority of patients. RESULTS: Of 1,276 ThinPrep anal cytology samples, 130 (10%) were deemed unsatisfactory. 77% of patients were HIV positive. 85% were males. Of the unsatisfactory cases, 116 (89%) were co-tested for HR-HPV DNA. Of those, 40 patients (34%) had a simultaneous positive HR-HPV DNA. Adequate follow up cytology within a one year and a two year period revealed that 18/130 (14%) and 26/130 (20%) of patients had ASC or SIL respectively. Histologic follow-up within one and two years showed 3 patients (2%) and 8 patients (6%) with HSIL or worse. CONCLUSIONS: High risk patients with unsatisfactory anal cytology are not "negative". At least one-third proved to be concomitantly HR-HPV DNA positive with one-fifth showing subsequent cytologic squamous abnormalities and with more than 5% being diagnosed with a high grade intraepithelial lesion within two years. Prompt repeat cytology and/or HR-HPV DNA is recommended for high risk patients with non-diagnostic cytology.

Knowledge of the HPV status biases cytotechnologists' interpretation of Pap tests originally diagnosed as negative for intraepithelial lesion or malignancy.

BACKGROUND: Because of cervical cancer screening recommendations and forthcoming first-line human papillomavirus (HPV) screening, many Papanicolaou (Pap) tests will be performed in patients with known concurrent HPV results. This study was designed to evaluate whether knowledge of the HPV status affects cytotechnologists' interpretation of Pap tests. METHODS: A retrospective search of cervical screening Pap tests with known concurrent HPV results provided 250 ThinPrep Pap tests, which were chosen to reflect an atypical rate similar to the rate of the Cleveland Clinic's normal practice. Fifty percent of negative for intraepithelial lesion or malignancy (NILM) and atypical squamous cells of undetermined significance (ASCUS) cases were from patients with positive HPV results. Slides were re-evaluated twice by 8 cytotechnologists blinded to the diagnosis and study purpose. The HPV status was provided for 50% of the cases in the first phase; after a washout period, knowledge of the HPV status for each case was reversed in the second phase. Follow-up information was collected from the medical record. RESULTS: In both phases, there was a significant bias for HPV-positive NILM cases to be upgraded to ASCUS or worse when the HPV-positive status was provided (P < .001). When the HPV status was withheld, there was no difference in upgrading NILM cases (phase 1, P = .69; phase 2, P = .066). A combined analysis showed a significant bias in referral to the pathologist when the HPV-positive status was provided rather than withheld (P < .001). Follow-up data revealed no significant effect of bias when the HPV-positive status was provided between patient groups with benign, low-grade, or high-grade follow-up. CONCLUSIONS: A known HPV-positive status biases cytotechnologists' interpretation of Pap tests, and this results in a higher rate of upgrading to ASCUS or worse; however, it does not improve sensitivity for disease detection. Cancer Cytopathol 2017;125:60-69. © 2016 American Cancer Society.

Time consumed by microscopic and nonmicroscopic tasks in image-assisted gynecologic screening: Implications for workload assessment.

BACKGROUND: Gynecologic screening cytology is a complex task that includes microscopic activities and nonmicroscopic activities. The authors sought to determine the amount and percentage of time that cytotechnologists spend on those activities using the ThinPrep imaging system. METHODS: In arm 1, a total of 550 consecutive unselected slides were reviewed by 11 cytotechnologists, and the time used for individual subtasks of the screening process was recorded. In arm 2, a total of 20 unselected slides were each screened by 10 different cytotechnologists (200 slides in total) and total screening times and full manual review (FMR) times were recorded. RESULTS: In arm 1, cases with and without FMR required an average of 5.6 minutes and 3.0 minutes, respectively, to screen. Overall, review of fields of view (FOVs) took 95 seconds. FMR took an average of 2.6 minutes. The average screening times for FOV-only cases was significantly longer than the US Food and Drug Administration/Centers for Medicare and Medicaid Services (FDA/CMS) workload limit of 2.4 minutes (P = .005). However, in arm 2, the time needed to screen a case increased by an average of 1 minute compared with arm 1, including 1.1 minute for FOV-only cases and >2 minutes for FMR plus FOV cases. Approximately 100% of cases screened as FOV only exceeded the FDA/CMS workload limit of 2.4 minutes. CONCLUSIONS: The FDA/CMS workload limits for FOV-only cases appears to significantly underestimate the time needed to screen those cases, but seems to be appropriate for the majority of FMR plus FOV cases. Approximately 60% and 30% of the time designated to screening slides was spent on nonmicroscopic activities for FOV-only cases and FMR cases, respectively. Cancer Cytopathol 2016;124:501-7. © 2016 American Cancer Society.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

BACKGROUND: BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. METHODS: This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. RESULTS: Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. CONCLUSIONS: This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society.

Rapid on-site evaluation of endobronchial ultrasound-guided fine-needle aspirates: correlation of adequacy assessment and final diagnosis in patients with bronchogenic carcinoma.

INTRODUCTION: We review endobronchial ultrasound-guided fine-needle aspirate samples and investigate cases with discrepancies between rapid on-site evaluation (ROSE) and final diagnosis in patients with bronchogenic carcinoma. MATERIALS AND METHODS: Endobronchial ultrasound-guided fine-needle aspirates from 2009 to 2010 were studied. On-site adequacy assessments were compared with final diagnoses. Concordant diagnoses showed agreement between ROSE interpretation and final diagnosis. If the initial interpretation differed from the final diagnosis, the case was discordant. Slides from discordant aspirates were reviewed. Discordant results were categorized as sampling error or interpretive/screening error at ROSE. RESULTS: A total of 340 endobronchial ultrasound-guided procedures were performed in 335 patients (168 men, 167 women, median age 65 years). Diagnostic discrepancies between ROSE and final diagnoses occurred in 65 aspirates (11%) from 51 patients with carcinoma. Of the 65 discrepant cases, 52 (83%) were subsequently called positive for carcinoma. Rescreening of slides in 47 available cases with a final positive diagnosis showed insufficient tumor for diagnosis in 28 of 47 cases (60%). The remaining 19 of 47 cases (40%) were classified as interpretive/screening errors at ROSE. Most errors occurred in aspirates called atypical or atypical suspicious, which upon rescreening were considered diagnostic (16 aspirates, 84%). CONCLUSIONS: Initial and final diagnoses were concordant in 89% of aspirates from patients with carcinoma. All aspirates that were positive at ROSE were concordant. In discordant cases, all aspirates deemed "atypical suspicious for malignancy" and 86% of aspirates deemed "atypical cells" on ROSE had a final diagnosis of carcinoma. The majority of discordant cases with a positive final diagnosis were due to sampling (60%).

Chromosomal biomarkers for detection of human papillomavirus associated genomic instability in epithelial cells of cervical cytology specimens.

The goal of this study was to compare how accumulation of chromosomal aberrations in human papillomavirus (HPV)-infected cells correlates with the severity of cervical dysplastic lesions. We assessed the frequency of genomic alterations for 35 different loci in a pilot biopsy study and selected two loci (3q26 and 8q24) with the highest frequency of copy number gains found in high-grade dysplasia and cancer. These probes were labeled with gold and red fluorophores and combined with HPV biotin-labeled probes for subsequent detection using a tyramide signal amplification system with a green fluorophore. Cells that were both HPV positive and chromosomally abnormal were designated as "double-positive cells." Cervical cytology specimens from 235 patients were used for this blinded study. The average number of double-positive cells increased from two cells in patients with a cytological interpretation of atypical squamous cells of undetermined significance to 22 cells in low-grade squamous intraepithelial lesion and 99 cells in high-grade squamous intraepithelial lesion, reflecting an accumulation of chromosomal abnormality with disease progression. Using a cutoff of four or more double-positive cells as the criterion for the presence of a cervical intraepithelial neoplasia 2 or 3 lesion, we demonstrated that low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion cytology specimens with underlying cervical intraepithelial neoplasia 2/3 histology showed positive test results in more than 80% of cases. Correlation of 3q26 and 8q24 aneusomy with concurrent HPV infection may thus serve as a biomarker of genetic instability in HPV-infected cells.