Effects of Rapid On-Site Evaluation on Diagnostic Accuracy of Thyroid Fine-Needle Aspiration.

INTRODUCTION: Fine-needle aspiration (FNA) is well-established for the evaluation of suspicious thyroid nodules. However, a significant proportion is nondiagnostic. Rapid on-site evaluation (ROSE) has been proposed to improve the overall adequacy of FNA. METHODS: Retrospective cohort study comparing adequacy of thyroid FNA findings pre- and postimplementation of ROSE at a tertiary center in Switzerland. Patients undergoing thyroid FNA from January 2016 to December 2019 were included. The primary outcome was the rate of nondiagnostic findings (Bethesda System for Reporting Thyroid Cytopathology category I). RESULTS: In total, 410 thyroid nodule FNAs were performed. Of those, 309 with standard FNA and 101 with ROSE. The majority of patients were female (71%), with a median age of 56 years (IQR 46-68) and a nodule diameter of 1.9 cm (IQR 1.2-2.9). Implementation of ROSE led to a decrease in nondiagnostic findings from 41.1% to 23.8%, with an odds ratio of 0.42 (95% CI: 0.24-0.72; p = 0.002). Implementation of ROSE was associated with significantly higher rates of Bethesda category III (27.7% vs. 19.1%), category IV (15.8% vs. 5.5%), and Bethesda category VI (6.9% vs. 2.3%). Repeated FNA was performed in 29.1% before and 20.8% after implementation of ROSE (p = 0.18). The mean number of FNA per nodule was reduced from 1.4 (0.6) to 1.2 (0.4) with ROSE (p = 0.04). CONCLUSIONS: Implementation of ROSE of thyroid nodule specimen improved diagnostic adequacy of FNA, reducing nondiagnostic findings. However, due to increased equivocal findings (Bethesda category III), there was no significant reduction of repeat FNA.

Detection of ROS1-positive non-small cell lung cancer on cytological specimens using immunocytochemistry.

BACKGROUND: Rearrangements of the ROS1 oncogene are found in 1% to 2% of non-small cell lung cancers (NSCLC) and are regarded as mutually exclusive oncogenic driver mutations. Since the approval of targeted therapy for ROS1-positive NSCLC, ROS1 testing has become a part of the diagnostic routine. Fluorescence in situ hybridization (FISH), optionally selected for by immunohistochemistry on histological material, is a common practice for the detection of ROS1 rearrangements. However, NSCLC often is diagnosed by cytology alone, requiring predictive marker testing on cytological specimens. In the current study, the authors explored the accuracy of ROS1 immunocytochemistry (ICC) on non-cell block cytological specimens for the detection of ROS1 rearrangements. METHODS: ICC using the D4D6 antibody on an automated immunostainer was performed prospectively in the routine diagnostic setting on cytological specimens from 295 patients with NSCLC, including adenocarcinoma (241 patients), NSCLC not otherwise specified (50 patients), and other malignancies (4 patients). Any immunostaining was considered positive. RESULTS: ICC was positive in all 13 ROS1-rearranged NSCLC cases confirmed by FISH (12 cases) or next-generation sequencing (1 case). Confirmation of 282 ICC-negative cases was available for 208 patients. The sensitivity, specificity, and positive and negative predictive values for ROS1 ICC compared with the final ROS1 status all were 100%. CONCLUSIONS: ROS1 ICC is an accurate method for the detection of ROS1 rearrangements in NSCLC. Given the high costs and technical challenges of FISH and the rarity of ROS1 rearrangements, ICC is rapid and therefore well suited as a screening method. Cases with equivocal or positive findings on ICC can be confirmed by FISH or molecular tests. Cancer Cytopathol 2018;126:421-9. © 2018 American Cancer Society.

HER2 gene status in primary breast cancers and matched distant metastases.

INTRODUCTION: The status of the gene encoding human EGF-like receptor 2 (HER2) is an important prognostic and predictive marker in breast cancer. Only breast cancers with HER2 amplification respond to the targeted therapy with trastuzumab. It is controversial to what degree the primary tumour is representative of distant metastases in terms of HER2 status. Discrepancies in HER2 status between primary tumours and distant metastases have been described, but their reasons remain unclear. Here, we compared HER2 status on cytological specimens of distant metastases with the result from the primary carcinomas, and explored the prevalence of and the reasons for discrepant results. METHODS: HER2 status was determined by fluorescence in situ hybridisation. HER2 gene amplification was defined as a HER2/chromosome 17 signal ratio of 2 or more. HER2 results from cytological specimens of matched distant metastases were compared with the results from the corresponding primary tumours (n = 105 patients). In addition, lymph node metastases were analysed in 31 of these patients. RESULTS: HER2 amplification was found in 20% of distant metastases. HER2 status was discordant between the primary tumour and distant metastasis in 7.6% of the 105 patients. Re-evaluation revealed that in five patients (4.7%), discrepancies were due to interpretational difficulties. In two of these patients, focal amplification had initially been overlooked as a result of heterogeneity in the primary tumours or in the metastases, respectively. A further three patients had borderline amplification with a ratio close to 2. Discrepancy remained unexplained in three patients (2.9%). CONCLUSION: HER2 gene status remains highly conserved as breast cancers metastasise. However, discrepant results do occur because of interpretational difficulties and heterogeneity of HER2 amplification. Cytological specimens from distant metastases are well suited for HER2 fluorescence in situ hybridisation analysis.

An international telecytologic quiz on urinary cytology reveals educational deficits and absence of a commonly used classification system.

Urinary cytology is limited by high interobserver variability in the evaluation of cells with little atypia. We set up an online quiz on urinary cytology and tested the performance of 246 international participants. The quiz consisted of still images of 42 urinary specimens with equivocal morphologic features and 10 control cases with an unequivocal cytologic diagnosis. The nature of the cells on the 292 quiz images had been verified by multitarget fluorescence in situ hybridization in addition to the information obtained by cystoscopy, clinical follow up, and/or histologic examination. The original quiz cases and the percentage of answers given by the participants can be viewed at: http://kathrin.unibas.ch/urinzyto/. High-grade cancers were diagnosed correctly in 76.0% and low-grade cancers in only 33.9%. Remarkably, 54.5% of all participants misclassified decoy cells as malignant. This study shows that large-scale international online quizzes may be used to find educational deficits in cytopathology.

Multitarget FISH analysis in the diagnosis of lung cancer.

The aim of the present study was to explore the diagnostic usefulness of the multitarget fluorescence in situ hybridization (FISH) test, LAVysion (Vysis, Downers Grove, IL), for the detection of lung cancer cells in cytologic specimens. Specimens from bronchial washings, bronchial brushings, and transbronchial fine-needle aspirates (TBNAs) from 100 patients with suspected lung cancer and from a control group of 71 patients with nonneoplastic lung disorders were analyzed. FISH positivity was defined as more than 5 cells with gains of at least 2 chromosomes or gene loci. FISH significantly improved the sensitivity of bronchial brushings from 49% to 73%. The specificities of FISH and cytologic examination were 87% and 100%, respectively. In contrast, FISH provided no additional diagnostic information in TBNAs and bronchial washings. There was no increased prevalence of genetic changes in contralateral bronchial washings from patients with lung cancer compared with the control group. The quantity of previous smoking had no effect on the prevalence of chromosomal changes.

Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology.

BACKGROUND: Endoscopic fine-needle aspiration (FNA) and brush cytology are standard methods for the diagnosis of pancreatobiliary malignancies. Although the majority of cytological diagnoses are straightforward, there remains a difficult category of inconclusive cytology. This study explored the utility of fluorescence in situ hybridization (FISH) to improve the diagnostic stratification between reactive and malignant cells in cases of inconclusive cytology. METHODS: The multiprobe FISH assay UroVysion was used for copy number assessment of chromosomes 3, 7, 17, and the 9p21 locus on Papanicolaou-stained specimens with a diagnosis of inconclusive cytology (n = 50), adenocarcinoma (n = 31) and no evidence of malignancy (n = 9). The target cells were photographed and their coordinates saved on an automated stage prior to hybridization. A positive test was defined as increased copy number (> 2) of at least 2 chromosomes (3, 7, or 17) in at least 4 atypical cells, or loss of 9p21 in at least 12 cells. RESULTS: FISH confirmed all 31 cytological diagnoses of pancreatobiliary adenocarcinomas, and was negative in the 9 patients with negative cytology. Among the 50 cases with inconclusive cytology, FISH detected 19 of 31 cases with a final diagnosis of adenocarcinoma, and was negative in all 19 cases with no final evidence of malignancy (sensitivity of 61.3%, specificity of 100%, positive predictive value of 100%, negative predictive value of 61.3%). Loss of 9p21 was found in 43 (86%) of all 50 FISH-positive cases. CONCLUSIONS: Multiprobe FISH combined with automated relocation of atypical cells is a powerful technique to clarify inconclusive cytology of the pancreatobiliary tract, allowing for a better distinction between reactive atypia and malignancy.

Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone.

INTRODUCTION: Lung cancer is often diagnosed by cytology, necessitating predictive molecular marker analyses on cytological specimens. The gold standard for detection of predictive anaplastic lymphoma kinase (ALK)-rearrangements is fluorescence in situ hybridization (FISH), but FISH is both expensive and often challenging to interpret. The aim of our study was to investigate the accuracy of ALK immunocytochemistry (ICC) on cytological specimens of non-small-cell lung cancers (NSCLCs). METHODS: Forty-one cytological specimens with available ALK FISH results were retrospectively analyzed with the 5A4 monoclonal antibody (Novocastra; Leica Biosystems) on a fully automated slide stainer. The specimens were enriched for ALK FISH-positive NSCLCs (14 of 41; 34.1%). Evaluation of the ICC staining was performed blinded to the FISH results. The staining intensity and the percentage of stained cancer cells were recorded. Any ICC staining was regarded as a positive result. The ALK ICC results were compared with the FISH results. In case of a discrepancy the ICC-stained slide and the FISH signals were reviewed. RESULTS: ICC was evaluable on 40 of 41 specimens. Fifteen of 40 NSCLCs (37.5%) were ALK ICC-positive, with staining of the majority of cancer cells (median 100%; mean 82.3%). Twelve of the ICC-positive NSCLCs (80.0%) showed an intense staining (3+). Compared with the ALK FISH results, only one NSCLC was false-negative, and one false-positive by ICC, respectively. The sensitivity, specificity, and positive and negative predictive values for ALK ICC compared with ALK FISH were 93.3%, 96.0%, 93.3%, and 96%, respectively. CONCLUSION: ALK ICC is highly accurate for detecting ALK-rearranged NSCLCs.

Prediction of outcome in patients with low-grade squamous intraepithelial lesions by fluorescence in situ hybridization analysis of human papillomavirus, TERC, and MYC.

BACKGROUND: Cytology is an excellent method with which to diagnose preinvasive lesions of the uterine cervix, but it suffers from limited specificity for clinically significant lesions. Supplementary methods might predict the natural course of the detected lesions. The objective of the current study was to test whether a multicolor fluorescence in situ hybridization (FISH) assay might help to stratify abnormal results of Papanicolaou tests. METHODS: A total of 219 liquid-based cytology specimens of low-grade squamous intraepithelial lesions (LSIL), 49 atypical squamous cells of undetermined significance (ASCUS) specimens, 52 high-grade squamous intraepithelial lesion (HSIL) specimens, and 50 normal samples were assessed by FISH with probes for the human papillomavirus (HPV), MYC, and telomerase RNA component (TERC). Subtyping of HPV by polymerase chain reaction (PCR) was performed in a subset of cases (n=206). RESULTS: There was a significant correlation found between HPV detection by FISH and PCR (P<.0001). In patients with LSILs, the presence of HPV detected by FISH was significantly associated with disease progression (P<.0001). An increased MYC and/or TERC gene copy number (>2 signals in>10% of cells) prevailed in 43% of ASCUS specimens and was more frequent in HSIL (85%) than in LSIL (33%) (HSIL vs LSIL: P<.0001). Increased TERC gene copy number was significantly correlated with progression of LSIL (P<.01; odds ratio, 7.44; area under the receiver operating characteristic curve, 0.73; positive predictive value, 0.30; negative predictive value, 0.94) CONCLUSIONS: The detection of HPV by FISH analysis is feasible in liquid-based cytology and is significantly correlated with HPV analysis by PCR. The analysis of TERC gene copy number may be useful for risk stratification in patients with LSIL.

Evaluation of chromosomal aberrations in patients with benign conditions and reactive changes in urinary cytology.

BACKGROUND: Fluorescence in situ hybridization (FISH) is routinely used to help clarify atypical urinary cytology. However, to the authors' knowledge, little is known regarding the frequency of chromosomal aberrations in non-neoplastic conditions that could potentially lead to false-positive FISH results. The objective of the current study was to evaluate the frequency of chromosomal aberrations in benign cells of the urinary tract using the UroVysion FISH test. METHODS: The authors analyzed 77 Papanicolaou-stained benign urine cytology specimens with reactive epithelial atypia using a FISH assay detecting the chromosomes 3, 7, and 17 and the gene locus 9p21. A positive test result was defined as an increased copy number of at least 2 chromosomes in ≥ 4 of 25 cells, or > 10 cells with a tetraploid or octaploid pattern, or homozygous or heterozygous deletion of 9p21 (≥ 12cells). RESULTS: FISH was positive in 27 of 77 bladder washings (35.1%) including 25 of 65 bladder washings (40.5%) and 2 of 15 voided urines (13.5%) from patients with irritative bladder (15 of 36 patients), a history of radiotherapy (7 of 12 patients), nonspecific cystitis (3 of 11 patients), hematuria (3 of 8 patients), and lithiasis (1 of 4 patients) . In 7 of 27 FISH-positive urothelial specimens, the positivity was solely due a polyploid pattern (tetraploid/octaploid pattern) in > 10 of the cells. CONCLUSIONS: Chromosomal aberrations can occur in reactive urothelial cells, with a tetraploid pattern being the most common. Even an aneuploid pattern of FISH signals does not always prove malignancy because it rarely occurs in reactive urothelial cells. Correlation of FISH results with cytomorphology and patient history is crucial to avoid false-positive diagnoses.

Assessment of mean EGFR gene copy number is a highly reproducible method for evaluating FISH in histological and cytological cancer specimens.

The aims of this study were first, to systematically assess the inter-observer reproducibility of mean Epidermal Growth Factor Receptor (EGFR) gene copy number (MCN) in histological and cytological specimens from lung and non-lung cancers, second to compare the performance of this quantitative approach to the current Colorado criteria for the assessment of fluorescence in situ hybridization (FISH) positivity and third to develop a model to convert cytology into histology MCN. EGFR FISH analysis was performed on 170 histological and 153 cytological specimens. The MCN and Colorado criteria were assessed by two independent observers and agreement evaluated using Bland-Altman plots and kappa values. Conversion of cytology into histology MCN was tested on two randomized subgroups of specimens. Applying the Colorado criteria, agreement between observers was moderate with histology (k=0.5) and excellent with cytology (k=0.94). Positivity in histology versus cytology led to fair agreement (k=0.33). MCN was significantly greater in cytology compared to histology (p<0.001) with excellent inter-observer agreement in both sample types (r=0.99 and 0.89, respectively). The average difference in MCN between observers was -0.003 (95%CI: -0.05 to 0.05) and 0.008 (95%CI: -0.09 to 0.11) for cytology and histology, respectively. A reliable conversion model with an R(2)-value of 0.91 in the validation subgroup (p<0.001) was obtained. The MCN is a reproducible scoring method for evaluating EGFR FISH in samples from lung and non-lung cancers. This quantitative scoring system may constitute an alternative to current methods of gene copy number assessment and will further help to optimize patient selection for targeted therapies.

Fluorescence in situ hybridization in the definitive diagnosis of malignant mesothelioma in effusion cytology.

BACKGROUND: Distinction of malignant mesothelioma (MM) from reactive mesothelial cells (RM) in effusions is notoriously difficult. The aim of our study was to test chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in the diagnosis of MM in effusion cytology and to explore the potential role of p16, p14, and p15 gene methylation as an alternative mechanism of tumor suppressor gene inactivation. METHODS: Fifty-two effusions of biopsy-proven MM and 28 benign effusions were retrospectively analyzed by multitarget FISH assay for aberrations of chromosomes 3, 7, 17, and 9p21. In case of a negative result, the corresponding MM biopsy specimen was analyzed. Methylation-specific polymerase chain reaction (MSP) for p16, p14, and p15 was performed on FISH-negative MM biopsy specimens. RESULTS: Seventy-nine percent of effusions with biopsy-proven MM had chromosomal aberrations, with loss of 9p21 as the most common finding. All benign effusions were FISH negative. Sensitivity, specificity, and positive and negative predictive values for detection of MM by FISH were 79%, 100%, 100%, and 72%, respectively. Six of nine FISH-negative effusions with biopsy-proven MM were also FISH negative in the MM biopsy specimens. Four of five FISH-negative biopsy specimens showed promoter methylation in p16 and p14 as compared with one of 12 benign controls. CONCLUSIONS: FISH is a sensitive and highly specific method for the definitive diagnosis of MM in effusion cytology. In the subset of FISH-negative MM, tumor suppressor genes on the chromosomal region 9p21 are often inactivated by promoter methylation.

An online quiz uncovers limitations of morphology in equivocal lung cytology.

BACKGROUND: Equivocal atypia in respiratory cytology can be a diagnostic challenge. In such cases fluorescence in situ hybridization (FISH) may be used for the analysis of chromosomal aberrations and often allows a reliable distinction of benign and malignant cells. METHODS: An online picture gallery of 30 respiratory cytologic preparations comprising 23 specimens with equivocal cytology as well as 5 positive and 2 negative controls was prepared (www.unibas.ch/patho/lungenzyto/loesung). The final diagnoses were confirmed by clinical follow-up or biopsy or both. Each of the illustrated cell groups was analyzed by multitarget FISH after PAP image capturing and automatic relocalization. RESULTS: The online questionnaire was completed by 137 cytomorphologists from all continents. The control cases were assessed accurately to a significantly higher percentage than the equivocal cases. In equivocal cases participants more often made false-positive than false-negative diagnoses. In 2 patients with benign conditions (tuberculosis and pulmonary capillaritis) the rate of false-positive answers was remarkably high (31.4% and 62.8% respectively). The result of the 20 best-performing participants for the 5 cases with the highest percentage of inaccurate answers was not better than if they had chosen their answer by chance. CONCLUSIONS: These data illustrate that single cells or cell clusters of a subgroup of equivocal lung cytology are a diagnostic challenge even for highly experienced morphologists. Internet-based tests are able to reveal limitations of cytomorphology.

DNA image cytometry and fluorescence in situ hybridization for noninvasive detection of urothelial tumors in voided urine.

BACKGROUND: Cystoscopy and histologic examination remain the standard methods for initial tumor diagnosis and monitoring for early detection of recurrences, since the sensitivity of conventional urinary cytology for the detection of urothelial tumors in urinary specimens is low. DNA image cytometry (ICM) and fluorescence in situ hybridization (FISH) have been suggested as ancillary tools. The goal of the current study was to compare the diagnostic value of DNA image cytometry and FISH for the noninvasive detection of urothelial tumors in voided urine. METHODS: Cytospin preparations were prepared from voided urine collected prior to the resection of 26 noninvasive (pTa) and 11 invasive (pT1-2) tumors. Specimens from 14 patients with benign prostatic hyperplasia were used as negative controls. DNA ICM was performed using the AUTOCYTE trade mark cell analytical system on Feulgen-stained cytospin specimens. The commercially available UroVysion trade mark FISH multiprobe was used to analyze chromosomes 3, 7, and 17, and 9p21. RESULTS: The overall sensitivity of cytology improved from 24% to 54% and to 78% if supplemented by ICM or FISH, respectively. Image cytometry detected all invasive tumors (pT1-2), while FISH missed one; FISH identified 19 of 26 (73%) pTa tumors, while only 9 (35%) of these tumors were aneuploid by ICM. The results of ICM and FISH were concordant in 37 of 51 (72%) cases. CONCLUSIONS: The current study shows that both FISH and ICM can successfully be used as supplementary methods to detect the clinically most relevant group of invasive bladder carcinomas. However, UroVysion FISH is more sensitive in the detection of pTa tumors than ICM, as it recognizes individual chromosomal alterations that frequently prevail in urothelial tumors.