Thyroid FNA cytology: Impact of the COVID-19 pandemic and vaccination in a Brazilian series.

BACKGROUND: The coronavirus disease 2019 pandemic prompted changes in medical practice, with a reduction in cytopathology volumes and a relative increase in the malignancy rate during lockdown and the initial postlockdown period. To date, no study has evaluated the impact of these changes on the volume of rapid on-site evaluation (ROSE) or on the frequency of cases according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) categories after vaccination. METHODS: Ultrasound-guided thyroid fine-needle aspiration (FNA) and ROSE assessments performed from January 2019 to May 2022 were evaluated retrospectively according to TBSRTC categories for three periods: prepandemic (period 1), from transmission to expansion (period 2), and after vaccination (period 3). RESULTS: There were 7531 nodules from 5815 patients. FNA cases increased throughout the pandemic despite a drop during lockdown. The frequency of TBSRTC categories changed. Nondiagnostic cases had an increase of 18.1% in period 2 and 76.2% after vaccination compared with prepandemic levels. Malignant cases increased from 2.3% to 4.2% in period 2 and to 5.1% in period 3, representing increases of 83.1% and 121.2%, respectively, compared with period 1. Data corrected by time showed increases in categories IV, V, and VI and a decrease in benign nodules during the two pandemic periods. ROSE was performed in 787 cases during the prepandemic period, and there were decreases of 29.4% and 22.8% in periods 2 and 3, respectively. The ROSE-to-category I ratio was reduced significantly after vaccination. CONCLUSIONS: Increased volume with sustained lower benign rates and higher malignant rates before and after vaccination indicate better selection of patients for FNA. A worse adequacy rate was correlated with a decrease in the number of ROSE assessments.

Anaplastic lymphoma kinase 5A4 immunohistochemistry as a diagnostic assay in lung cancer: A Canadian reference testing center's results in population-based reflex testing.

BACKGROUND: The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. METHODS: Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. RESULTS: Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n = 5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9 months. CONCLUSIONS: ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.

Translation of Knowledge to Practice-Improving Awareness in NSCLC Molecular Testing.

BACKGROUND: Molecular testing in advanced lung cancer is standard in guiding treatment selection. However, population-wide implementation of testing remains a challenge. We developed a knowledge translation intervention to improve understanding among diagnostic specialists about molecular testing and appropriate diagnostic sampling in lung cancer. METHODS: Specialty-specific education programs were developed from existing literature and input from Canadian leaders in lung pathology, respirology, interventional radiology, thoracic surgery, radiation oncology, and medical oncology. The programs, including key messages, review of current data, existing guidelines, group discussion, and participant feedback, were administered at provincial and national specialty meetings. Participant knowledge was assessed before and after the intervention by using anonymous questionnaires. Molecular (EGFR) testing rates in Ontario were also evaluated before and after the intervention period. RESULTS: Ten programs were administered to diagnostic specialists, including respirologists, pathologists, thoracic surgeons, radiologists, radiation oncologists, and medical oncologists, with completion of 255 preintervention and 219 postintervention surveys. At baseline, 30% were unsure of tissue handling methods for molecular testing, 20% chose an incorrect technique, and half were unfamiliar with how to initiate testing. After intervention, specialist knowledge improved regarding tissue handling and appropriate fixation techniques and uncertainty decreased from 30% to 2% (p < 0.001). A 12% increase (relative increase 57%) in molecular (EGFR) testing requests in Ontario was observed over the intervention period (p = 0.0032). CONCLUSIONS: Significant knowledge gaps exist among diagnostic specialists regarding molecular testing and targeted therapy in lung cancer. This initiative significantly improved understanding of the importance and methods of successful molecular testing and correlated with increased testing rates.

FTA Cards for Preservation of Nucleic Acids for Molecular Assays: A Review on the Use of Cytologic/Tissue Samples.

CONTEXT: - Traditional methods for storing histologic and cytologic specimens for future use in molecular assays have consisted of either snap-freezing with cryopreservation or formalin-fixing, paraffin-embedding the samples. Although snap-freezing with cryopreservation is recommended for better preservation of nucleic acids, the infrastructure and space required for archiving impose challenges for high-volume pathology laboratories. Cost-effective, long-term storage at room temperature; relatively easy shipment; and standardized handling can be achieved with formalin-fixed, paraffin-embedded samples, but formalin fixation induces fragmentation and chemical modification of nucleic acids. Advances in next-generation sequencing platforms, coupled with an increase in diagnostic, prognostic, and predictive molecular biomarkers have created a demand for high-quality nucleic acids. To address issues of the quality of nucleic acid and logistics in sample acquisition, alternatives for specimen preservation and long-term storage have been described and include novel universal tissue fixatives, stabilizers, and technologies. OBJECTIVE: - To collect, retrieve, and review information from studies describing the use of nucleic acids recovered from cytologic/tissue specimens stored on Flinders Technology Associates (FTA, GE Whatman, Maidstone, Kent, United Kingdom) cards for downstream molecular applications. DATA SOURCES: - An electronic literature search in the PubMed (National Center for Biotechnology Information, Bethesda, Maryland) database allowed the selection of manuscripts addressing the use of FTA cards for storage of cytologic samples for molecular analysis. Only articles published in English were retrieved. CONCLUSIONS: - The use of FTA cards is a versatile method for fostering multicenter, international collaborations and clinical trials that require centralized testing, long-distance shipment, and high-quality nucleic acids for molecular techniques. Studies with controlled temperature are required to test the quality of recovered RNA after long-term storage.

Rapid On-Site Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspirations for the Diagnosis of Lung Cancer: A Perspective From Members of the Pulmonary Pathology Society.

CONTEXT: - Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as a very useful tool in the field of diagnostic respiratory cytology. Rapid on-site evaluation (ROSE) of EBUS-TBNA not only has the potential to improve diagnostic yield of the procedure but also to triage samples for predictive molecular testing to guide personalized treatments for lung cancer. OBJECTIVE: - To provide an overview of the current status of the literature regarding ROSE of EBUS-TBNA in the diagnosis of lung cancer. DATA SOURCES: - An electronic literature search in PubMed and Google databases was performed using the following key words: cytology, lung cancer, on-site evaluation, rapid on-site evaluation, and ROSE EBUS-TBNA. Only articles published in English were included in this review. CONCLUSIONS: - Rapid on-site evaluation can ensure that the targeted lesion is being sampled and can enable appropriate specimen triage. If available, it should be used with EBUS-TBNA in the diagnosis of lung cancer because it can minimize repeat procedures for additional desired testing (ie, molecular studies). Some studies have shown that ROSE does not adversely affect the number of aspirations, total procedure time of EBUS-TBNA, or the rate of postprocedure complications; it is also helpful in providing a preliminary diagnosis that can reduce the number of additional invasive procedures, such as mediastinoscopy. As EBUS technology continues to evolve, our knowledge of the role of ROSE in EBUS-TBNA for the diagnosis of lung cancer will also continue to grow and evolve.

Performance of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration for the Diagnosis of Isolated Mediastinal and Hilar Lymphadenopathy.

BACKGROUND: Although many studies have assessed the diagnostic utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the context of a specific disease, few studies have assessed the overall diagnostic yield, sensitivity, and negative predictive value in patients with isolated mediastinal and hilar lymphadenopathy (IMHL). OBJECTIVE: We evaluated the performance of EBUS-TBNA for diagnosing IMHL in a population with a high prevalence of concurrent or preexisting non-pulmonary malignancy. METHODS: A retrospective chart review of patients who underwent EBUS-TBNA from October 2008 to April 2014 was performed to identify patients with IMHL. Patients with known or suspected primary pulmonary malignancy were excluded. When available, EBUS-TBNA results were cross-referenced with further diagnostic investigation or clinical diagnosis based on follow-up. RESULTS: EBUS-TBNA was used to sample 765 lymph nodes from 350 patients. One hundred and fourteen (33.3%) patients had a concurrent or preexisting non-pulmonary malignancy. The overall yield of EBUS-TBNA for specific diagnosis was 300/350 (86%). The diagnostic yield for sarcoidosis, lymphoproliferative disease, metastatic lymphadenopathy from extrathoracic malignancy, and necrotizing granuloma was 123/149 (83%), 27/33 (82%), 20/25 (80%), and 13/19 (68%), respectively. Amongst 50 patients with non-diagnostic EBUS-TBNA, 25 yielded an insufficient sample and another 25 yielded only benign lymphoid material which was not representative of the underlying pathology. Overall, EBUS-TBNA had a sensitivity of 89%, a diagnostic yield of 86%, and a negative predictive value of 79%. CONCLUSION: For patients with isolated hilar or mediastinal lymphadenopathy and a high background prevalence of concurrent and preexisting non-pulmonary malignancy, EBUS-TBNA is a reliable first-line diagnostic investigation.

Preanalytic specimen triage: Smears, cell blocks, cytospin preparations, transport media, and cytobanking.

With increasing requests for the evaluation of prognostic and predictive molecular biomarkers, great attention must be paid to the preanalytical issues regarding sample quality and DNA/RNA yield from all different types of cytological preparations. The objectives of this review were: 1) to provide an update regarding the importance of specimen triage as well as specimen handling and collection; 2) to discuss the different cell preparations that can be used for molecular testing, their advantages and limitations; and 3) to highlight the strategies for biobanking cytology samples. Good-quality DNA/RNA can be harvested from fresh cells in cell suspensions, formalin-fixed paraffin-embedded cell blocks, archival stained smears, archival unstained cytospin preparations, liquid-based cytology slides, FTA cards, and cryopreserved cells. In contrast to formalin-fixed paraffin-embedded tissue specimens (small biopsies and surgical resections), the multitude of types of sample preparations as well as the diversity in sample collection and processing procedures make cytology an ideal specimen for most genomic platforms, with less DNA and RNA degradation and a purer sample, usually with a higher concentration of tumor cells. The broad incorporation of cytological specimens into clinical practice. A should increase the number of samples potentially available for molecular tests and avoid repeat invasive procedures for tissue procurement, thereby increasing patient safety. In this context, it is of utmost importance that cytopathologists become familiar with the variables that can affect test results and embrace the goal of excellence in sample quality. Cancer Cytopathol 2017;125(6 suppl):455-64. © 2017 American Cancer Society.

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

BACKGROUND: Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. METHODS: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS: All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS: Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Biomarker Testing in Lung Carcinoma Cytology Specimens: A Perspective From Members of the Pulmonary Pathology Society.

The advent of targeted therapy in lung cancer has heralded a paradigm shift in the practice of cytopathology with the need for accurately subtyping lung carcinoma, as well as providing adequate material for molecular studies, to help guide clinical and therapeutic decisions. The variety and versatility of cytologic-specimen preparations offer significant advantages to molecular testing; however, they frequently remain underused. Therefore, evaluating the utility and adequacy of cytologic specimens is critical, not only from a lung cancer diagnosis standpoint but also for the myriad ancillary studies that are necessary to provide appropriate clinical management. A large fraction of lung cancers are diagnosed by aspiration or exfoliative cytology specimens, and thus, optimizing strategies to triage and best use the tissue for diagnosis and biomarker studies forms a critical component of lung cancer management. This review focuses on the opportunities and challenges of using cytologic specimens for molecular diagnosis of lung cancer and the role of cytopathology in the molecular era.

A proposal for cellularity assessment for EGFR mutational analysis with a correlation with DNA yield and evaluation of the number of sections obtained from cell blocks for immunohistochemistry in non-small cell lung carcinoma.

AIMS: Different approaches have been described for reporting specimen adequacy for epidermal growth factor receptor (EGFR) mutation analysis. We aimed: (1) to conduct cellularity assessment and to investigate its association with DNA yield, (2) to compare the H&E slides taken before and after the thick sections (curls) obtained for EGFR testing and (3) to evaluate the number of ancillary studies performed. METHODS: Cell block (CB) slides of 110 non-small cell lung carcinoma cases submitted to EGFR analysis from 2010 to 2012 were reviewed for total cellularity (ranges 1-100, 100-250, 250-500, 500-750, 750-1000 and >1000 cells), tumour cellularity (ranges 1-50, 50-100, 100-300 and >300 cells) and the percentage of tumour cells. Precurl and postcurl H&E slides were compared using the three criteria. The number of immunohistochemistry (IHC) markers and special stains and DNA yield were recorded. RESULTS: DNA yield was significantly associated with the total cellularity, number and percentage of tumour cells. For 46 cases with precurl and postcurl slides, only three (6.5%) were classified as being different and in two of them the postcurl slide had greater cellularity than the precurl. IHC was performed in 83 cases, with a minimum of 1 and a maximum of 11 markers (median of 3) per case. CONCLUSIONS: An association between the total cellularity and the tumour cellularity with the DNA yield was demonstrated using the ranges described. Evaluation of a postcurl slide is an unnecessary practice. The majority of the CB had sufficient material for ancillary studies (up to 11 markers) and mutation testing.

Preanalytic parameters in epidermal growth factor receptor mutation testing for non-small cell lung carcinoma: A review of cytologic series.

The results from molecular assays can be affected significantly by the preanalytic condition of cytologic samples. The authors review current knowledge on the use of cytologic samples for epidermal growth factor receptor (EGFR) mutation testing in non-small cell lung cancer with a focus on preanalytic parameters. A systematic electronic search of the MEDLINE database was performed to identify original articles that reported the use of cytologic samples for EGFR molecular analysis and included a minimum of 100 samples. The information collected included author(s), journal, and year of publication; number of patients and samples; sampling method; type of preparation; type of fixative; staining techniques; mutation analysis techniques; tumor cellularity; the percentage of tumor cells; data on DNA quantity, quality, and concentration; failed assays; and the mutation rate. EGFR mutation analysis was conducted on 4999 cytologic samples from 22 studies that fulfilled the inclusion criteria. Fine-needle aspirates and pleural effusions were the most common types of specimens used. DNA was mainly extracted from cell blocks and smears, and the most commonly reported fixatives included formalin, ethanol, and CytoLyt. Cellularity assessments and DNA yields were available from 5 studies each. The average success rate for the assays that used cytologic specimens was 95.87% (range, 85.2%-100%). The mutation rate ranged from 6% to 50.46%, and a higher mutation detection rate and lower numbers of insufficient cases were reported for pleural effusions and lymph node samples from endobronchial ultrasound-guided transbronchial needle aspiration compared with histologic specimens. Low cellularity and a low percentage of tumor cells were associated with higher test failure rates. Future guidelines should consider the current data for specific recommendations regarding cytologic samples.

Patient-derived tumor xenograft models established from samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

OBJECTIVES: There has been limited utility for laboratory tumor models to predict clinical performance of cancer drugs. Clinical drug trials usually recruit patients that have advanced disease, therefore preclinical tumor models that closely reflect this characteristic will be more reliable to test candidate drugs. We evaluated the use of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to sample metastatic lymph nodes in patients to establish patient-derived tumorxenograft (PDX) models of advanced lung cancer. MATERIALS AND METHODS: Cell suspensions from TBNA aspirates were implanted into the subcutaneous tissue of NSG (NOD scid) gamma) mice. The success rate of PDX establishment was associated with tumor histopathology and the cellularity and cytopathological diagnosis of the primary EBUS-TBNA samples. RESULTS: From December 2011 to June 2012, 19 patients were enrolled in this study. Successful engraftment was achieved in 8/19 cases (42.1%). The duration between inoculation and tumor formation averaged 62.4 days (13-144 days). The engrafted tumors included 3 adenocarcinomas (3/12: 25%), 2 squamous cell carcinomas (2/3: 67%), 1 large cell carcinoma (1/1: 100%), and 2 small cell carcinomas (2/3: 67%). CONCLUSION: EBUS-TBNA samples can be used for establishment of tumor xenograft model in immunodeficient mice.

Sample features associated with success rates in population-based EGFR mutation testing.

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing. METHODS: Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity. RESULTS: From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%). CONCLUSIONS: EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.

Subclassification of lymphoproliferative disorders in serous effusions: a 10-year experience.

BACKGROUND: Rare studies have reported the application of multiple ancillary tests to the diagnosis of lymphoproliferative disorder in serous effusions. In the current study, the authors evaluated the effectiveness of using an algorithm for the triage of serous effusions and the contribution of ancillary studies to achieve a specific subtype of lymphoproliferative disorder. METHODS: Serous effusion samples that had a final diagnosis of lymphoproliferative disorder or suspicious for lymphoma were selected from cases that were diagnosed between 2001 and 2010. Data were collected on patient and sample characteristics as well as results from immunophenotype and molecular studies. RESULTS: In total, 168 serous effusions were identified from 110 patients. The most common site of involvement was the pleural cavity (n = 133) followed by the peritoneal cavity (n = 30) and pericardial cavity (n = 5). The volume of serous effusions ranged from 2 mL to 1000 mL (mean, 238 mL). In 42 patients (38.2%), serous effusions were the primary source of diagnosis. In 129 patients who had a diagnosis of LPD, either generic (n = 82) or specific (n = 47) ancillary tests were performed as a single test in 58 samples (67.4%) or as a combination of multiple studies in 19 samples (23.2%). Immunophenotyping was successful in almost all samples that had a specific subtype with 16 B-cell and 4 T-cell lymphomas being diagnosed. More samples with a specific subtype of lymphoma underwent molecular tests compared with those who had a generic diagnosis (19.1% vs 13.4%). CONCLUSIONS: Successful, specific subtyping of lymphoproliferative disorders was achieved in approximately 33% of cases that were tested for ancillary studies following an approach for the triage and aliquoting of serous effusion samples.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples.

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377-386. © 2013 American Cancer Society.