Gilda da Cunha Santos

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.

Background: Accurate classification of lung cancer subtypes has become critical in tailoring lung cancer treatment. Our study aimed to evaluate changes in diagnostic testing and pathologic subtyping of advanced non-small-cell lung cancer (NSCLC) over time at a major cancer centre. Methods: In a review of patients diagnosed with advanced NSCLC at Princess Margaret Cancer Centre between 2007–2009 and 2013–2015, diagnostic method, sample type and site, pathologic subtype, and use of immunohistochemistry (IHC) staining and molecular testing were abstracted. Results: The review identified 238 patients in 2007–2009 and 283 patients in 2013–2015. Over time, the proportion of patients diagnosed with adenocarcinoma increased to 73.1% from 60.9%, and diagnoses of NSCLC not otherwise specified (NOS) decreased to 6.4% from 18.9%, p < 0.0001. Use of diagnostic bronchoscopy decreased (26.9% vs. 18.4%), and mediastinal sampling procedures, including endobronchial ultrasonography, increased (9.2% vs. 20.5%, p = 0.0001). Use of IHC increased over time to 76.3% from 41.6% (p < 0.0001). Larger surgical or core biopsy samples and those for which IHC was performed were more likely to undergo biomarker testing (both p < 0.01). Conclusions: Customizing treatment based on pathologic subtype and molecular genotype has become key in treating patients with advanced lung cancer. Greater accuracy of pathology diagnosis is being achieved, including through the routine use of IHC.

Cytology is a viable and underused medium for obtaining molecular testing in lung cancer. There are various obstacles to the widespread adoption of molecular diagnostics using cytologic specimens, and these should be addressed to ensure optimal patient care.

Minimally invasive procedures such as endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) must yield not only good quality and quantity of material for morphological assessment, but also an adequate sample for analysis of molecular markers to guide patients to appropriate targeted therapies. In this context, cytopathologists worldwide should be familiar with minimum requirements for refereeing cytological samples for testing. The present manuscript is a review with comprehensive description of the content of the workshop entitled Cytological preparations for molecular analysis: pre‐analytical issues for EBUS TBNA, presented at the 40th European Congress of Cytopathology in Liverpool, UK. The present review emphasises the advantages and limitations of different types of cytology substrates used for molecular analysis such as archival smears, liquid‐based preparations, archival cytospin preparations and FTA (Flinders Technology Associates) cards, as well as their technical requirements/features. These various types of cytological specimens can be successfully used for an extensive array of molecular studies, but the quality and quantity of extracted nucleic acids rely directly on adequate pre‐analytical assessment of those samples. In this setting, cytopathologists must not only be familiar with the different types of specimens and associated technical procedures, but also correctly handle the material provided by minimally invasive procedures, ensuring that there is sufficient amount of material for a precise diagnosis and correct management of the patient through personalised care.

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.

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.

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.

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.

Background: Although molecular testing has become standard in managing advanced nonsquamous non-small-cell lung cancer (nsclc), most patients undergo minimally invasive procedures, and the diagnostic tumour specimens available for testing are usually limited. A knowledge translation initiative to educate diagnostic specialists about sampling techniques and laboratory processes was undertaken to improve the uptake and application of molecular testing in advanced lung cancer. Methods: A multidisciplinary panel of physician experts including pathologists, respirologists, interventional thoracic radiologists, thoracic surgeons, medical oncologists, and radiation oncologists developed a specialty-specific education program, adapting international clinical guidelines to the local Ontario context. Expert recommendations from the program are reported here. Results: Panel experts agreed that specialists procuring samples for lung cancer diagnosis should choose biopsy techniques that maximize tumour cellularity, and that conservation strategies to maximize tissue for molecular testing should be used in tissue processing. The timeliness of molecular reporting can be improved by pathologist-initiated reflex testing upon confirmation of nonsquamous nsclc and by prompt transportation of specimens to designated molecular diagnostic centres. To coordinate timely molecular testing and optimal treatment, collaboration and communication between all clinicians involved in diagnosing patients with advanced lung cancer are mandatory. Conclusions: Knowledge transfer to diagnostic lung cancer specialists could potentially improve molecular testing and treatment for advanced lung cancer patients.

Objective: Fine needle aspiration (FNA) is widely used in the diagnosis of metastatic melanoma, both at initial presentation and in the setting of recurrent disease. The purpose of this study was to evaluate the performance of confirmatory immunohistochemistry (IHC) and molecular analysis of the BRAF mutation in cytological preparations of metastatic melanoma.Methods: A 2‐year retrospective review of pathology reports was performed on cytological samples of metastatic melanoma at the University Health Network (Toronto, Canada) and the Santa Casa Medical School (Sao Paulo, Brazil). IHC was performed on cell block sections prepared from formalin‐fixed, fresh samples or residuum of CytoLyt/PreservCyt post‐fixed in formalin. BRAF V600E/K mutations were assessed by amplification refractory mutation system (ARMS) analysis.Results: A total of 104 samples (94 FNAs and 10 fluids) from 83 patients (20 women, 63 men) were included. IHC was attempted in 43 cases (41.3%) and successful in 41 (95.3%). The panel number of antibodies ranged from 1 to 15 (median 3). The most frequently used melanoma markers included HMB‐45, melanoma cocktail and S100 protein, used in 25 (58.1%), 23 (53.5%) and 18 samples (41.9%). Thirty cases (69.8%) used three or fewer markers. The BRAF V600E/K mutation was tested in eight samples, being successful in seven (87.5%) and positive in three (37.5%).Conclusions: Cytological samples are a reliable and sufficient source for IHC and subsequent molecular analysis, allowing a reduced diagnostic time and rapid, appropriate treatment options in patients with advanced melanoma.