Cytology Specimens Research Articles

A novel method for making cell blocks with higher cellular yield.

Cytopathology is an important part of pathology that is used to diagnose disease on the cellular level. The application of the cell block (CB) technique plays a vital role in cytological diagnosis, as blocks and slides can be further used for special stains, immunohistochemistry (IHC), and molecular pathological analysis. Several methods for making CBs have been reported, but their procedures and cellular yield are still deemed unsatisfactory. In this article, we used gellan gum (GG) as an adjuvant for CBs, which resulted in higher cellular yield with simpler procedures. CBs were prepared by using GG, copper sulfate, plasma/thrombin, or pregelatinized starch methods. The procedures of each of these four methods were then compared. CB sections were stained with hematoxylin and eosin (H&E), and the background and morphological features seen by H&E staining were compared. A preliminary IHC and fluorescence in situ hybridization (FISH) study was performed using cytology specimens from elevenand five cases, respectively. The expression of immunocomplex by IHC and the molecular signals detected by FISH were compared in CB sections made by the four methods and a section derived from the biopsy specimen block from the same patient. Feulgen staining, Alcian blue staining, and Masson trichrome staining were performed on the CB sections from 3 cases of pleural fluid. The cellular yield of CB sections from 83 cases according to the four methods was compared using NDP analysis software. The results demonstrated that sections derived from CBs made with GG had a clear background and good morphological features by H&E staining. The expression of immunocomplex by IHC and the molecular signals of FISH detection in the sections from CBs made by GG were accurately located just as those in biopsy sections from the same patient. The DNA, acidic mucus, and fibrin could be clearly identified through special stains in the CB sections. The procedures involved in the GG method were easily controllable and the coagulated gel increased the ease by which the CB was embedded and sectioned. Specifically, sections from CBs made by the GG method contained higher cellular yield because cells could be concentrated on the bottom of the gel after centrifugation. This novel method for making CBs is a practical, simple method that can result in higher cellular yield. This method is therefore worth promoting in clinical applications.

Application of Immunohistochemistry in Cytology.

Immunohistochemistry (IHC), also referred to as immunocytochemistry in cytology literature, has revolutionized the practice of cytopathology. Because of the complexity of cytology preparation and limited diagnostic material, performing IHC remains a challenge. Formalin-fixed paraffin-embedded (FFPE) cell block (CB) is the optimal choice for IHC. In this review, the approaches for improving CB preparation will be discussed. When CB material is not available, various cytology specimens can also be used for IHC. With the utilization of Antigen Retrieval (AR) technique, these nonformalin-fixed cytology specimens can achieve successful IHC staining, comparable with the results from FFPE tissue sections. In the last part of this review, we will discuss the use of positive controls and the important role of AR in standardization of IHC in cytology.

Extended human papillomavirus genotype distribution in cervical intraepithelial neoplasia and cancer: Analysis of 40 352 cases from a large academic gynecologic center in China.

Our aim was to conduct a large epidemiologic analysis of the distribution of human papilloma virus(HPV) genotypes associated with cervical neoplasias and cancers at a major Chinese gynecologic center. The pathologic database was searched for cervical histopathologic diagnoses with prior HPV genotyping from liquid cervical cytology specimens obtained ≤6 months before biopsy. HPV testing was performed by using the Tellgenplex HPV27 or YanengBio HPV23 genotyping assays. A total of 40 352 cases meeting study criteria were identified. High risk human papillomavirus (hrHPV) was detected in 94.1% of squamous cancers compared to in only 83.3% of cervical adenocarcinomas. The prevalence of multiple HPV infections was highest in cervical intraepithelial neoplasia 1 (CIN1) (33.8%) and decreased with increasing severity of squamous lesions. The distribution of HPV genotypes was similar between CIN1 and histopathologic-negative cases. HPV16 was one of the three most common hrHPV genotypes before all histopathologic abnormalities, ranging from 72.0% for cervical cancers, 38.7% for CIN2/3/AIS, 13.1% for CIN1, and 9.1% for biopsy-negative cases. HPV16 and HPV18 accounted for over 87.2% of detected hrHPV genotypes for all glandular intraepithelial neoplastic lesions and cancers, whereas squamous lesions did not show this pattern. 80.3% of cervical cancers were associated with genotypes covered by HPV16/18 vaccines and 89.6% with genotypes covered by 9-valent vaccination.

The diagnostic utility of trichorhinophalangeal syndrome type 1 immunohistochemistry for metastatic breast carcinoma in effusion cytology specimens.

Trichorhinophalangeal syndrome type 1 (TRPS1) is a novel immunohistochemical marker with excellent performance in distinguishing breast carcinoma from other cancers in surgical specimens. The aim of this study was to evaluate the diagnostic utility of TRPS1 compared with GATA3 for metastatic breast carcinoma in effusion cytology specimens. In total, 91 cell blocks of malignant effusion specimens, including 47 metastatic breast carcinomas (nine triple-negative breast carcinomas [TNBCs] and 38 non-TNBCs) and 44 nonmammary malignancies, were selected for TRPS1 and GATA3 immunohistochemistry. Modified H scores ≥ 200 were considered positive staining. The positive rate of TRPS1 was similar between TNBC and non-TNBC (77.8% vs 73.3%, p=.802), whereas the positive rate of GATA3 was lower in TNBC than in non-TNBC (66.7% vs 89.5%, p=.087). The positive rate of TRPS1 was significantly higher in breast carcinoma than in urothelial carcinoma (74.5% vs 0%, p<.001), whereas the positive rate of GATA3 showed no difference between these two (85.1% vs 85.7%, p=.956). Notably, diffuse and strong aberrant expression of TRPS1 was observed in one lung adenocarcinoma and one serous adenocarcinoma in this series. The overall sensitivity, specificity, positive predictive value, and negative predictive value of TRPS1 immunohistochemistry for breast carcinoma were 74.5%, 95.5%, 94.6%, and 77.8%, respectively. TRPS1 is a sensitive and specific marker for metastatic breast cancer in serous effusion cell-block specimens. It shows superior sensitivity and specificity compared with GATA3, especially in the TNBC setting and for excluding urothelial carcinoma.

Cytologic scoring of equine exercise-induced pulmonary hemorrhage: Performance of human experts and a deep learning-based algorithm.

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator's and algorithmic performance included a ground truth dataset, the mean annotators' THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

Analysis of Liquid Biopsy Performance for the Detection of Lymphoma in Dogs with a Focus on Recurrent Copy Number Variants in Matched Plasma and Lymph Node Aspirates: Implications for a Comparative Oncology Model

Introduction Liquid biopsy has shown great promise for the detection and monitoring of lymphoma in humans. Recently, similar technology has been developed and made commercially available for dogs. Because the canine and human cancer genomes share such high homology (>90%), and certain lymphoma subtypes are highly similar in terms of their biologic and molecular features between the two species, the study of lymphoma in dogs affords unique opportunities for the genomic characterization of the disease, comparative oncology studies, and the development and assessment of novel therapies. The current study describes the performance of a next-generation sequencing-based liquid biopsy assay for the detection of canine lymphoma, and in a subset of patients, analyzes concordance for prognostic copy number variants between matched plasma samples and lymph node aspirate specimens. Methods As part of a larger clinical research collection program for the CANcer Detection in Dogs (CANDiD) study (Flory, 2022), blood samples were collected following diagnosis but prior to undergoing therapy from a cohort of 116 client-owned dogs with lymphoma (63 intermediate to large B-cell, 21 intermediate to large T-cell, 6 T-zone, and 26 unphenotyped). Lymphomas with inconclusive immunophenotype or other types of indolent lymphomas were excluded. A subset of dogs analyzed for the current study also had matched cytology specimens available from lymph node fine needle aspirates (FNA). Blood samples and FNA specimens were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. Specific analysis focused on FNA-plasma concordance for recurrent copy number variants that have been previously identified in canine lymphoma patients and have prognostic potential (i.e., gains in chromosomes 13 and 31 and losses in chromosome 14) (Richards, 2015; Thomas, 2003). Results The overall detection rate for lymphoma by liquid biopsy was 92.2% (107/116); with detection rates of 57% for localized/regional disease (Stages I and II) and 95% for disseminated disease (Stages III, IV, and V). By immunophenotype, the detection rate for B-cell lymphoma was 97%, T-cell 91%, T-zone 50%, and unphenotyped 92%. In the subset of lymphoma-diagnosed dogs with matched plasma and FNA samples, the majority of cases had at least one of the prognostic copy number alterations previously reported in canine lymphoma patients (13G, 14L, 31G), demonstrating the potential of liquid biopsy to noninvasively detect clinically important biomarkers. A variety of genomic alterations were identified in patients with positive liquid biopsy results, including single nucleotide variants in common oncogenes and tumor suppressor genes (e.g., TP53 mutations), and copy number variants across the genome. Some of these variants are also important biomarkers in human lymphoma, in particular TP53 mutations (Chakravarty, 2017), highlighting the potential for canine liquid biopsy testing to support comparative oncology studies for the benefit of both species. Conclusion A novel next-generation sequencing-based liquid biopsy tool has demonstrated the ability to identify genomic alterations in blood samples of dogs with lymphoma, including recurrent CNVs previously described in canine lymphoma patients, and variants in genes previously described in human and canine lymphoma tissue. These findings, along with the high degree of homology between human and canine lymphomas, support the utility of liquid biopsy for the study of lymphoma in dogs as a comparative oncology model for accelerated biomarker discovery and therapeutic development in both species.

Validation of a Cytological Classification System for the Rapid On-Site Evaluation (Rose) of Pulmonary and Mediastinal Needle Aspirates.

Rapid on-site evaluation (ROSE) is a procedure that allows immediate assessment of adequacy of cytological specimens obtained by fine needle aspiration (FNA). The application of ROSE diagnostic categories has been applied in various organs, but not in thoracic pathology. We aimed to retrospectively assess the concordance with the final diagnosis of a categorization from C1 (inadequate) to C5 (neoplastic) during ROSE performed with bronchoscopic or percutaneous sampling procedures of thoracic lesions in a large series of consecutive cases. This retrospective single-center study evaluated 2282 consecutive ROSEs performed on 1827 patients from January 2016 to December 2020 in 994 cases of transbronchial needle aspiration (TBNA) in peripheral pulmonary lesions, in 898 transthoracic FNAs, in 318 ultrasound-guided TBNAs, in 50 conventional TBNAs and in 22 endobronchial TBNAs. False positive and false negative cases of ROSE were 43 (1.88%) and 73 (3.2%), respectively, when compared with the definitive diagnosis. The sensitivity, specificity and the positive and negative prognostic values of ROSE were 94.84%, 95.05%, 96.89% and 91.87%, respectively. Overall concordance between ROSE and the final diagnosis was 0.8960 (Cohen's kappa). No significant differences were observed in terms of sampling procedures and type and location of the lesions. A tiered classification scheme of ROSE from C1 to C5 during bronchoscopic and percutaneous sampling procedures is helpful in effectively guiding clinical management of patients with thoracic lesions.

The utility of WhatsApp-based off-site evaluation for rapid cytology of EBUS-TBNA samples.

Whether off-site evaluation of slides by a cytologist viewing the images shared by WhatsApp improves the on-site evaluation by a pulmonologist (P-ROSE) remains unknown. This study's objective was to compare the sensitivity of P-ROSE and WHOSE for adequacy and diagnosis of cytology specimens obtained by endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA). We retrospectively reviewed our bronchoscopy database to identify subjects who underwent EBUS-TBNA for lymph node sampling and had reports of P-ROSE and WHOSE. We collected data on the adequacy of samples as reported by the pulmonologist (P-ROSE), remotely by the cytologist (WHOSE), and finally after detailed cytologic evaluation. The study's primary outcome was to assess the increment in sensitivity for adequacy and diagnostic category (using the final cytology report as reference) by incorporating WHOSE. We included 264 (P-ROSE, n=184; WHOSE, n=80) subjects. The sensitivity (95% CI) for sample adequacy by P-ROSE and WHOSE was 65.3% (57.9%-72%) and 92% (83.6%-96.2%), respectively. There was a 26.6% (95% CI, 16%-35.2%) increment in the sensitivity for adequacy. The sensitivity (95% CI) for diagnosis by P-ROSE and WHOSE was 53.9% (46%-61.1%) and 89.8% (79.5%-95.3%), respectively. There was a 35.9% (95% CI, 23.4%-45%) increment in the sensitivity for diagnosis with WHOSE. The agreement between P-ROSE and final cytology in adequacy was poor (κ=-0.023, p=0.616). The agreement between WHOSE and final cytology was moderate for adequacy (κ=0.491, p=<0.001). We found WHOSE significantly improves the performance of P-ROSE for rapid assessment of cytology specimens obtained by EBUS-TBNA.

Nationwide differences in cytology fixation and processing methods and their impact on interlaboratory variation in PD-L1 positivity

Programmed death ligand-1 (PD-L1) immunostaining, which aids clinicians in decision-making on immunotherapy for non-small cell lung cancer (NSCLC) patients, is sometimes performed on cytological specimens. In this study, differences in cytology fixation and cell block (CB) processing between pathology laboratories were assessed, and the influence of these differences on interlaboratory variation in PD-L1 positivity was investigated. Questionnaires on cytology processing were sent to all Dutch laboratories. Information gathered from the responses was added to data on all Dutch NSCLC patients with a mention of PD-L1 testing in their cytopathology report from July 2017 to December 2018, retrieved from PALGA (the nationwide network and registry of histo- and cytopathology in the Netherlands). Case mix-adjusted PD-L1 positivity rates were determined for laboratories with known fixation and CB method. The influence of differences in cytology processing on interlaboratory variation in PD-L1 positivity was assessed by comparing positivity rates adjusted for differences in the variables fixative and CB method with positivity rates not adjusted for differences in these variables. Twenty-eight laboratories responded to the survey and reported 19 different combinations of fixation and CB method. Interlaboratory variation in PD-L1 positivity was assessed in 19 laboratories. Correcting for differences in the fixative and CB method resulted in a reduction (from eight (42.1%) to five (26.3%)) in the number of laboratories that differed significantly from the mean in PD-L1 positivity. Substantial variation in cytology fixation and CB processing methods was observed between Dutch pathology laboratories, which partially explains the existing considerable interlaboratory variation in PD-L1 positivity.

Biomarker testing of cytology specimens in personalized medicine for lung cancer patients.

Every patient with advanced non-small cell lung cancer (NSCLC) should be tested for targetable driver mutations and gene arrangements that may open avenues for targeted therapy. As most patients with NSCLC in the advanced stage of the disease are not candidates for surgery, these tests have to be performed on small biopsies or cytology samples. A growing number of other genetic changes with targetable mutations may be treatable in the near future. To identify patients who might benefit from novel targeted therapy, relevant markers should be tested in an appropriate context. In addition, immunotherapy of lung cancer is guided by the status of programmed death-ligand 1 expression in tumor cells. The variety and versatility of cytological specimen preparations offer significant advantages for molecular testing; however, they frequently remain underused. Therefore, evaluating the utility and adequacy of cytologic specimens is important, not only from a lung cancer diagnosis, but also for the large number of ancillary studies that are necessary to provide appropriate clinical management. A large proportion of lung cancers is diagnosed by aspiration or exfoliative cytology specimens; thus, optimizing strategies to triage and best use the tissue for diagnosis and biomarker studies forms a critical component of lung cancer management. In this review, we discuss the opportunities and challenges of using cytologic specimens for biomarker testing of lung cancer and the role of cytopathology in the molecular era.

PD-L1 Expression in Cytological and Histological Lung Cancer Specimens

Abstract Introduction/Objective Several studies have explored the feasibility of measuring PD-L1 in cell block cytology and indicated cytological materials could be a reliable source for PD-L1 evaluation in non-small cell lung carcinoma patients. A few studies have investigated the compatibility and performance of PD-L1 clone SP263 testing between cytology and histology specimens. The study was pursued to evaluate PD-L1 expression in cell blocks from EBUS- TBNA compared to that in biopsied tissues from patients with lung carcinoma in our institution to evaluate a feasibility of PD-L1 clone SP263 in cell blocks and histology samples. Methods/Case Report A total of 57 specimens cytologically diagnosed lung carcinoma using endobronchial ultrasound guided transbronchial needle aspiraton (EBUS-TBNA) from Jan 1st, 2020, to Dec 31st, 2021 were screened for enrollment. Among them, 24 patients diagnosed with lung carcinoma using EBUS-TBNA and matched transbronchial biopsy (TBB) specimens were reviewed for study. After careful selection, 13 paired formalin-fixed tissues from lung carcinoma patients, including cell blocks and matched histology samples, were included. PD-L1 expression was assessed using the SP263 assay, and the tumor proportion score (TPS) was evaluated. PD-L1 expression was finally divided into three categories according to the TPS: &amp;lt; 1% (negative), 1–49% (low expression) and ≥ 50% (high expression). Results (if a Case Study enter NA) Of the 13 matched pairs, 12 (92.3%) showed concordant PD-L1 expression. On cytology, 3 cases were positive (2 high expressors and 1 low- expressors) of which 2 were concordant and 1 discordant with matched histology specimens. Ten cytology samples were negative for PD-L1 expression, and they were concordant to histology samples. The correlation coefficient for TPS was 0.75 considered as having good value. Conclusion: With an overall concordance rate of 92.3% between cytology and histology specimen, this study demonstrates the feasibility of PD-L1 IHC with SP263 clone on limited quality and quantity of cytology samples from lung carcinoma in our institute. It is required for further evaluation with additional specimens to conclude that the usefulness of cytology cell blocks for PD-L1 expression analysis.

Morphological Characteristics of Malignancy in Circulating Tumor Cells (CTCs): A New Approach to Liquid Biopsies Utilizing Affordable Standard Cytology Techniques and the FDA Cleared Parsortix® System

Abstract Introduction/Objective The process of metastasis is responsible for most cancer deaths. Identification of tumor cells circulating in blood (CTCs) has the potential to offer important information to patients and clinicians regarding disease status, longitudinal monitoring, remission status and assessment of patient prognosis, among others. However, currently CTC tests in the market are expensive and are not covered by medical insurance, making them unaffordable for most cancer patients. Legacy CTC technologies also require the use of expensive immunofluorescence microscopes and reagents, and are not easily implemented in standard clinical laboratory settings, thus preventing their use as standard of care. Onc-ADaPT™ Clinical Laboratories have explored a method to identify CTCs using the FDA cleared Parsortix® System (ANGLE plc, Guildford, UK) followed by standard cytology processing, staining and analysis, significantly reducing the cost of operations and increasing the likelihood of affordability to patients everywhere. Methods/Case Report The basic features of malignancy were listed by a Senior Cytologist (CT) and reviewed by another CT. Cancer cell lines were spiked into normal donor blood collected in Streck tubes. Non-spiked healthy volunteer blood was used to establish the background. The samples were then processed through the Parsortix® system producing 200µL suspension of separated cells. The harvest was then processed using Cytospin, stained with standard Pap Stain and reviewed with bright field microscopy by qualified CTs. Results (if a Case Study enter NA) Spiked tumor cells were identified and correctly categorized by the Cytologists. Non-spiked normal donor samples were negative for malignancy. Cells of interest were identified in the non-spiked HNV samples and classified as non-malignant (NOS). Using the training set, CTs later analyzed the available patient sample. The positive cancer patient sample showed cells with obvious characteristics of malignancy. Conclusion The basic characteristics of malignancy used for analysis of standard cytology specimens are adequate to identify CTCs using the established background to eliminate the NOS cells. CTC testing can be performed using the combination of Parsortix® System, standard cytology staining and analysis by qualified Cytologists.

Cytopathology Practice in the Covid-19 Pandemic, During the Lockdown and Post-lockdown Period: A Tertiary-Care Center Experience

Background: Covid-19 pandemic has changed the healthcare delivery and cytopathology practice worldwide. We evaluated our cytopathology laboratory workload during the lockdown and post-lockdown period and compared it with the same period in 2019, to see the impact of the Covid-19 pandemic. Methods: The cytological reports issued during the lockdown (10 March - 31 May 2020) and the post-lockdown period (1 June -31 August 2020); and the corresponding periods in 2019 were retrospectively reviewed from the database. Sample type, sampling site, and diagnostic categories were recorded. Results: During the Covid-19 lockdown period, the total number of cytological specimens, was reduced from n=3197 to n=745, with a rate of 76,7%. The most reduction was in thyroid fine-needle aspiration (FNA) and cervicovaginal smears. Relative increases were observed for soft tissue, lung, and liver FNA samples (p&lt;0.05) and cerebrospinal fluid, peritoneal and pleural fluid samples (p&lt;0.05). In the post-lockdown period, the total number reduced from n=2461 to n=2032 with a rate of 17.4%. Significant reduction continued for thyroid FNAs, but other samples have nearly reached the pre-covid levels. During the total six months period, the rate of the malignant category increased while the negative for malignancy category decreased compared to 2019. Conclusion: During the Covid-19 lockdown period, the reduction was primarily observed in the samples taken for screening purposes, and high-risk oncological patients continued to receive healthcare services. In the initial phase of the post-lockdown period, health services and cytopathology practice have rapidly reached almost the levels of the pre-pandemic period.

Comprehensive Review of Metastatic Breast Carcinoma in Cytology Specimens

Breast carcinomas are known to metastasize to various organs of the human body. Fine needle aspiration cytology or exfoliative cytology often are the standard method for diagnosis at these metastatic sites due to ease of procurement of diagnostic material, accessibility, less complications, high sensitivity, and specificity of diagnosis and evaluation of biomarker status needed to guide future management. This comprehensive review article discusses in detail metastatic patterns, cytomorphology of metastatic breast cancer at different body sites, immunohistochemistry needed for diagnosis of breast carcinoma, sensitivity and specificity of diagnosis and breast biomarker assays in the cytology material.

A multivalent activatable aptamer probe with ultralow background signal and high sensitivity for diagnosis of lung adenocarcinoma

Lung adenocarcinoma is the most common subtype of lung cancer. Developing a simple, rapid, and sensitive method for the detection of lung adenocarcinoma is helpful to reduce the clinical workload and facilitate clinical treatment. To detect lung adenocarcinoma cells, this study designed a multivalent activatable aptamer probe (multi-AP) using S1 aptamer that can specifically recognize A549 cells (a lung adenocarcinoma cell line). Firstly, the monovalent activatable aptamer probe (mono-AP) was designed by strand-displacement strategy and fluorescent resonance energy transfer (FRET). The multi-AP was constructed by mono-AP assembled on the nucleic acid scaffold obtained by hybridization chain reaction (HCR). The presence of the target cell activated the multi-AP, the complementary strand was released, and the fluorescence detection signal was reported based on the FRET. The multi-AP has good specificity and sensitivity for detecting A549 in different matrices and greatly reduces the background signal with the lowest detection limit of 6 cells/200 μL. Furthermore, the multi-AP could directly detect the lung adenocarcinoma cells in cytology specimens, indicating a good potential of the multi-AP in clinical cytology specimens for rapid diagnosis, such as rapid on-site cytological evaluation. Besides, the multi-AP was further modified with biotin (bio-multi-AP) and used to detect clinical cytology cell blocks, implying the value of bio-multi-AP in immunocytochemistry for accurate diagnosis of lung adenocarcinoma.

EGFR testing in paraffin-embedded cell block cytology material is reliable with increased detection for effusion fluid

IntroductionCytology is integral to lung cancer diagnosis. Aspiration and exfoliative fluid specimens represent valuable tumor material for molecular testing. In this study, a large retrospective cohort of EGFR tests was reviewed to address the adequacy, detection and discrepancy rate in tests performed with cytology material. MethodsEGFR tests performed from 2013 to 2022 were reviewed and classified by the modality of obtaining tissue and by tissue type. EGFR tests for tissue specimens were performed on unstained sections of paraffin-embedded material on glass slides. Adequacy and types of mutation(s) detected were analysed. Cases where multiple EGFR testing was performed on the same patient were reviewed for discordance. ResultsThere were 5,504 tests retrieved, with 1,855, 3,607 and 42 performed on cytology, surgical and blood specimens. Lung and excision specimens were more often adequate (p < 0.001). Cytology material showed lower adequacy rates (p < 0.01). EGFR detection (positive) rate was higher in pleural fluid compared to biopsy (59.8 % vs 50.7 %, p = 0.022), but similar between lung and lymph node cytology and non-cytology specimens. Effusion fluid specimens had the highest adequacy (81.5 %) and detection rate (59.3 %) among cytology specimens (p < 0.001). Four (4.4 %) cases showed discordant results in cytology specimens. Two were false negatives in the non-cytology material. Only in one case was cytology material genuinely discrepant. The remaining discordance was attributed to the interval treatment effect. ConclusionThe findings support that EGFR testing in cell block is reliable and complements tissue material. In addition, pleural fluid appears to be superior to pleural biopsies for molecular testing.

Molecular testing of cytology specimens: overview of assay selection with focus on lung, salivary gland, and thyroid testing.

Ancillary and molecular testing of cytopathology specimens has emerged as a reliable and useful tool to provide diagnostic information and treatment-related biomarker status for the management of cancer patients. The cytology specimens obtained through minimally invasive means have proven suitable testing substrates for a variety of ancillary tests, including immunohistochemistry, fluorescence in situ hybridization, as well as polymerase chain reaction and next generation sequencing molecular techniques. By focusing specifically on the cytology specimen, this review provides an overview of basic testing considerations and assay selection in addition to updates on the ancillary testing of cytologic tumor specimens from the lung, salivary gland, and thyroid.

Utility of TRPS-1 immunohistochemistry in diagnosis of metastatic breast carcinoma in cytology specimens

At present, GATA binding protein 3 (GATA-3) is the most frequently used diagnostic immunohistochemical (IHC) marker for breast carcinoma (BC). However, it is not specific and has very low sensitivity for triple-negative BC (TNBC). SRY-box transcription factor 10 (SOX-10) and trichorhinophalangeal syndrome type 1 (TRPS-1) have been suggested for inclusion in the diagnostic workup of TNBC. TRPS-1 has not been established in cytology specimens as a diagnostic IHC marker for metastatic BC (MBC). Hence, in the present study we evaluated the utility of TRPS-1 in diagnosing MBC in cytology specimens. MBC cases diagnosed on cytology specimens from January to October 2020 were included in the present study. Only cases with hormonal status available and ≥20 tumor cells on cell blocks were included in the study. The cell blocks were assessed for TRPS-1, GATA-3, and SOX-10 IHC marker positivity (intensity and percentage of tumor cells). The results were correlated with the specimen type (fine needle aspiration [FNA] versus body fluid) and various BC prognostic subgroups. We analyzed 61 cases, including 33 body fluid and 28 FNA (13 lymph node, 10 bone, 2 liver, 2 soft tissue, and 1 lung) specimens. TRPS-1 had 97.2% positivity in ER/PR+ (estrogen receptor/progesterone receptor-positive) MBC compared with GATA-3, which had 100% positivity in the same group. TRPS-1 showed high positivity in 35 of 37 cases (94.6%) and intermediate positivity in 1 (2.6%) and was negative/low positive in 1 case (2.7%). In contrast, GATA-3 showed high positivity for all 37 cases (100%). SOX-10 showed positivity in only 1 of 37 cases (2.7%), with intermediate positivity. In the HER2+ (human epidermal growth factor receptor 2-positive) group, TRPS-1 showed high positivity in 5 of 7 cases (71.4%), intermediate positivity in 1 case (14.3%), and negativity in 1 case (14.3%). However, GATA-3 showed high positivity in 6 of 7 cases (85.7%) and negative/low positivity in 1 case (14.3%). SOX-10 was negative in all 7 cases. In TNBC, TRPS-1 showed high positivity in 16 of 17 cases (94%) and intermediate positivity in 1(5.9%), and GATA-3 showed high positivity in 9 (53%), intermediate positivity in 2 (11.8%), and low positive/negative in 6 of the 17 cases (35.3%). TRPS-1 expression was significantly higher than GATA-3 expression for the number of positive cases (P = 0.07), mean percentage of positive tumor cells (P=0.005), and intensity of reactivity (P = 0.005). SOX-10 expression was present in only 5 of 17 cases (29%), with a mean percentage of positivity in the tumor cells of 26.5% and intensity of 0.8. No differences were found in the IHC results between the different specimen types (FNA versus fluid) in any group. TRPS-1 is a highly sensitive new diagnostic IHC marker for breast carcinoma, with a similar positivity rate in ER/PR+ and HER2+ BC compared with GATA-3 and a higher positivity rate than GATA-3 and SOX-10 in TNBC in cytology specimens. In particular, when only a few clusters of tumor cells are present on the cell block, TRPS-1 can be highly useful, because its mean percentage of positive tumor cells and intensity are higher than those of other IHC markers.

PSAT379 Performance of a Raman Fingerprint in Thyroid Nodules with Indeterminate Cytology: A Prospective Blinded Monocentric Study

Abstract Background Molecular analysis of thyroid fine-needle aspiration biopsy (FNA) was proposed to improve indeterminate nodules management. However, sensibility and specificity as well as the cost of molecular diagnostics require to be improved to increase their cost-effectiveness for medical practice setting. Raman spectroscopy (RS) demonstrated ability in separating benign from malignant thyroid lesions in surgically removed tissues, based on specific RS profile. This study aimed to investigate the diagnostic performance of RS on cytological samples obtained by thyroid FNA. Methods In this prospective, blinded monocentric study, we enrolled 123 patients with indeterminate or worse cytological diagnosis, candidate to surgery according to international guidelines, and submitted to RS analysis of FNA samples. Cytology specimens were evaluated in agreement to Italian Reporting System for Thyroid Cytology1 as follows: TIR1 (non-diagnostic), TIR1C (non-diagnostic-cystic), TIR 2 non-malignant/benign, TIR3A (low-risk indeterminate lesion), TIR3B (high-risk indeterminate lesion), TIR 4 (suspicious of malignancy), or TIR 5 (malignant). As previously published2, the two diagnostic subcategories referred to indeterminate nodules with low (TIR3A) and high risk (TIR3B) of malignancy, may be respectively compared to the class III and Class IV of The Bethesda System for Reporting Thyroid Cytopathology. We compared RS, cytology and final histology, as reference standard, using various statistical approaches. Findings: Our study population included 37 TIR3A, 32 TIR3B, 16 TIR4 and 38 TIR5; the 30.9% of patients had benign histological diagnosis after surgery. In particular, 72.9% of patients classified TIR3A and 31.3% TIR3B had benign histological diagnosis. RS analysis of FNA samples had overall specificity of 86.8% in predicting thyroid malignancy. In indeterminate cytological categories, RS specificity was 86.5%. In patients with TI-RADS score four or five, the specificity of RS increased to 87.5% for TIR3A and reached 100% in TIR3B.; if considering RS positive test, unnecessary surgery was reduced to 7.4% in the whole sample, 33.3% in TIR3A, and 6.7% in TIR 3B. Interpretation: We demonstrated for the first time that RS represents a valuable tool for thyroid cytology and a valid alternative to molecular analyses, able to improve management and reduce unnecessary surgery in indeterminate nodules. Funding: This study was supported by Ministero della Salute, through TIRAMA project (RF-2018-12366568). 1 J Endocrinol Invest. 2014;37(6): 593-599. doi: 10.1007/s40618-014-0062-0 2Cytopathology. 2021;32(6): 714-717. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Diagnosis of Large Cell Neuroendocrine Carcinomas on Cytology Specimens: How Reliable Are We?

Diagnosis of Large Cell Neuroendocrine Carcinomas on Cytology Specimens: How Reliable Are We?