Interobserver variability in cytopathology: How much do we agree?

Interobserver variability remains a major challenge for cytopathologists despite the development of standardized reporting and classification systems. Indeed, whereas moderate-to-good interobserver agreement is generally achievable when the differential diagnosis between benign and malignant entities is straightforward, high levels of variability make the diagnostic interpretation of atypical and suspicious samples not consistent. This review explores the landscape of interobserver agreement in cytopathology across different anatomical sites.

Delphi expert consensus for whole slide imaging in thyroid cytopathology.

OBJECTIVE: Despite an increase in thyroid fine needle aspiration (FNA) and advances in whole slide imaging (WSI) adoption, digital pathology is still considered inadequate for primary diagnosis of these cases. Herein, we aim to validate the utility of WSI in thyroid FNAs employing the Delphi method strategy. METHODS: A panel of experts from seven reference cytology centres was recruited. The study consisted of two consecutive rounds: (1) an open-ended, free-response questionnaire generating a list of survey items; and (2) a consensus analysis of 80 selected shared WSIs from 80 cases by six investigators answering six morphological questions utilising a 1 to 5 Likert scale. RESULTS: High consensus was achieved for all parameters, with an overall average score of 4.27. The broad majority of items (84%) were ranked either 4 or 5 by each physician. Two badly scanned cases were responsible for more than half of the low-ranked (≤2) values (57%). Good to excellent (≥3) diagnostic confidence was reached in more than 95.2% of cases. For most cases (78%) WSI assessment was not limited by technical issues linked to the image acquisition process. CONCLUSION: This systematic Delphi study indicates broad consensus among participating physicians on the application of DP to thyroid cytopathology, supporting expert opinion that WSI is reliable and safe for primary diagnostic purposes.

Evaluation of the Molecular Landscape in PD-L1 Positive Metastatic NSCLC: Data from Campania, Italy.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) have increased and improved the treatment options for patients with non-oncogene-addicted advanced stage non-small cell lung cancer (NSCLC). However, the role of ICIs in oncogene-addicted advanced stage NSCLC patients is still debated. In this study, in an attempt to fill in the informational gap on the effect of ICIs on other driver mutations, we set out to provide a molecular landscape of clinically relevant oncogenic drivers in programmed death-ligand 1 (PD-L1) positive NSCLC patients. METHODS: We retrospectively reviewed data on 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our clinic for molecular evaluation of five driver oncogenes, namely, EGFR, KRAS, BRAF, ALK and ROS1. RESULTS: Interestingly, n = 93 (55.7%) patients showed at least one genomic alteration within the tested genes. Furthermore, analyzing a subset of patients with PD-L1 tumor proportion score (TPS) ≥ 50% and concomitant gene alterations (n = 8), we found that n = 3 (37.5%) of these patients feature clinical benefit with ICIs administration, despite the presence of a concomitant KRAS gene alteration. CONCLUSIONS: In this study, we provide a molecular landscape of clinically relevant biomarkers in NSCLC PD-L1 positive patients, along with data evidencing the clinical benefit of ICIs in patient NSCLC PD-L1 positive alterations.

PD-L1 and beyond: Immuno-oncology in cytopathology.

Over the past decade, immunotherapy has emerged as one of the most promising cancer treatments. Several monoclonal antibodies targeting the programmed death 1 (PD-1)/ programmed death ligand-1 (PD-L1) pathway have been integrated into standard-of-care treatments for a wide range of cancer types. Although all the available PD-L1 immunohistochemistry (IHC) assays have been developed on formalin-fixed histological specimens, a growing body of research has recently suggested the feasibility of PD-L1 testing on cytological samples. Although promising results have been reported, several important issues still need to be addressed. Among these are pre-analytical issues, cyto-hystological correlation, and inter-observer agreement. This review will briefly summarise the knowledge gaps and future directions of cytopathology in the immuno-oncology scenario.

A MiR181/Sirtuin1 regulatory circuit modulates drug response in biliary cancers.

Despite recent advances, biliary tract cancer (BTC) remains one of the most lethal tumor worldwide due to late diagnosis, limited therapeutic strategies and resistance to conventional therapies. In recent years, high-throughput technologies have enabled extensive genome, and transcriptome sequencing unveiling, among others, the regulatory potential of microRNAs (miRNAs). Compelling evidence shown that miRNA are attractive therapeutic targets and promising candidates as biomarkers for various therapy-resistant tumors. The analysis of miRNA profile successfully identified miR-181c and -181d as significantly downregulated in BTC patients. Low miR-181c and -181d expression levels were correlated with worse prognosis and poor treatment efficacy. In fact, progression-free survival analysis indicated poor survival rates in miR-181c and -181d low expressing patients. The expression profile of miR-181c and -181d in BTC cell lines revealed that both miRNAs were dysregulated. Functional in vitro experiments in BTC cell lines showed that overexpression of miR-181c and -181d affected cell viability and increased sensitivity to chemotherapy compared to controls. In addition, by using bioinformatic tools we showed that the miR-181c/d functional role is determined by binding to their target SIRT1 (Sirtuin 1). Moreover, BTC patients expressing high levels of miR-181 and low SIRT1 shown an improved survival and treatment response. An integrative network analysis demonstrated that, miR-181/SIRT1 circuit had a regulatory effect on several important metabolic tumor-related processes. Our study demonstrated that miR-181c and -181d act as tumor suppressor miRNA in BTC, suggesting the potential use as therapeutic strategy in resistant cancers and as predictive biomarker in the precision medicine of BTC.

Evaluating local thyroid cytopathology practices by molecular quality metrics: A multi-institutional study on 4651 FNAs with a focus on the role of the interventional cytopathologist.

BACKGROUND: The diagnostic accuracy of thyroid fine-needle aspiration (FNA) can be highly influenced by the technical skills of the operator performing the procedure and by interobserver variability in microscopic interpretation. This is particularly true for the indeterminate categories. Recently, molecular testing has been proposed as an ancillary tool for monitoring the performance of different thyroid cytopathology practices. The objective of this multicenter study was to evaluate the quality of different local cytopathology practices by assessing the impact of interventional cytopathologists on FNA adequacy for molecular testing and the variations in mutation rates across different health care centers operating in the Campania region. METHODS: The study included 4651 thyroid FNA samples diagnosed in different Southern Italian clinical laboratories belonging to the TIRNET (the Tiroide Network). FNA samples were collected by different proceduralists and were classified by local cytopathologists according to The Bethesda System for Reporting Thyroid Cytopathology. FNAs classified as atypia of undetermined significance, follicular neoplasm, suspicious for malignancy, and malignant were centralized for a real-time polymerase chain reaction-based, seven-gene test at the authors' institution. RESULTS: Centers that employed interventional cytopathologists obtained fewer unsatisfactory FNA samples for molecular testing (11.3%) than centers that employed noncytopathologists (16.7%; p < .05). Furthermore, a significant variation in the mutation rate was observed in FNAs diagnosed by different local cytopathologists; indeterminate categories had the highest percentage of mutation rate variability among centers. CONCLUSIONS: Interventional cytopathologists obtained higher yields of diagnostic material for molecular testing. Finally, the current results suggest that the variability in mutation rates among different centers may highlight the low reproducibility of microscopic criteria among cytopathologists, particularly for indeterminate cases.

Methods for actionable gene fusion detection in lung cancer: now and in the future.

Although gene fusions occur rarely in non-small-cell lung cancer (NSCLC) patients, they represent a relevant target in treatment decision algorithms. To date, immunohistochemistry and fluorescence in situ hybridization are the two principal methods used in clinical trials. However, using these methods in routine clinical practice is often impractical and time consuming because they can only analyze single genes and the quantity of tissue material is often insufficient. Thus, novel technologies, able to test multiple genes in a single run with minimal sample input, are being under investigation. Here, we discuss the utility of next-generation sequencing and nCounter technologies in detecting simultaneous gene fusions in NSCLC patients.

Next Generation Sequencing in Cytopathology: Focus on Non-Small Cell Lung Cancer.

Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations (cell blocks, direct smears, and liquid-based cytology) and supernatants.

A narrative review of lung cancer cytology in the times of coronavirus: what physicians should know.

In the modern era of personalized and precision medicine, lung cancer management needs to be carried out in a multidisciplinary manner. Among other disciplines, also cytopathology is key in diagnosis and treatment management of these patients. Indeed, cytopathology specimens are often the only source of available tissue material for morphological diagnosis and molecular purposes in order to guarantee an adequate treatment decision making, since surgical resection specimens are not available when lung cancer is diagnosed at advanced disease stages. Today, as an effect of the current severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) pandemic, cytopathology is reorganizing and reshaping many of its procedures and workflows, in order to ensure the safety of cytopathologists and laboratory personnel. In particular, careful attention should be paid on biosafety procedures when pulmonary cytological specimens are handled. In addition, also molecular cytopathology, that provides relevant information on the molecular status and on the potential sensitivity to target treatments, is undergoing major changes. In this setting, fully automated technologies, requiring minimal hands-on work, may be a valid option. The aim of this narrative review is to keep updated all the different professional figures involved in lung cancer management and treatment on how SARS-CoV-2 is modifying lung cancer cytopathology.

The continuing role of breast fine-needle aspiration biopsy after the introduction of the IAC Yokohama System For Reporting Breast Fine Needle Aspiration Biopsy Cytopathology.

BACKGROUND: Ultrasound-guided fine-needle aspiration biopsy (FNAB) (US-guided FNAB) is a rapid and cost-effective procedure for the diagnosis of breast lesions. Our Institution has a long tradition in breast FNAB performed by cytopathologists; recently we adopted both US guidance and a five-tiered classification system similar to that proposed by the International Academy of Cytology (IAC). The aim of this study was to demonstrate the continuing role of US-guided FNAB in the diagnosis of breast lesions, despite the growing adoption of core-needle biopsy (CNB). METHODS: The laboratory information database system was searched to obtain the breast FNAB diagnostic reports recorded from 2010 to 2017 and classified using a five-tiered Classification System; each entry was matched with the available histology. RESULTS: A total of 4624 breast FNAB samples were retrieved. Of these, 1745/4624 cases (37.7%) had histological follow-ups. The risk of malignancy (ROM) was 4.9% for benign, 20.7% for atypical, 78.7% for suspicious of malignancy, and 98.8% for malignant. When the atypical category was evaluated as a negative index, the positive predictive value was 93.73%, and the negative predictive value was 90.78%, reaching an overall diagnostic accuracy of 92.82%. CONCLUSIONS: The IAC Yokohama System for Reporting Breast FNAB Cytopathology clearly identifies different diagnostic categories with increasing ROM. Most of the FNAB samples were classified as benign or malignant (65.3%), warranting prompt management for these patients. Moreover, the inclusion of the atypical category as a low-risk indeterminate category avoided overtreatment of benign lesions. Thus, despite the well-established merits of CNB, US-guided FNAB still represents a cost-effective and rapid nonoperative diagnostic approach.

Is the Idylla EGFR Mutation Assay feasible on archival stained cytological smears? A pilot study.

AIM: The rapid and fully automated Idylla EGFR Mutation Assay has been specifically designed to process formalin-fixed, paraffin-embedded sections without requiring preliminary DNA extraction. This study evaluates whether this approach can also process archival smears from patients with non-small cell lung cancer (NSCLC) by scraping the stained cellular material directly into the cartridge. METHODS: The study was divided into two parts. In the first part, we carried out Idylla EGFR Mutation Assay on archival stained smears from 39 patients with NSCLC. Among these, 14 cases harboured a mutation in either exon 19 (n=11) or exon 21 (n=3), previously detected on DNA extracts by fragment length and TaqMan assays. In the second part, we evaluated whether de-staining of the smears could reduce background fluorescence. RESULTS: The Idylla EGFR Mutation Assay confirmed the presence of EGFR mutation in 11 instances (78.6%). However, concordance was higher for exon 19 deletions (10/11) than for exon 21 p.L858R assessments. Raw data showed a high background fluorescence in channel 2, where the EGFR exon 21 p.L858R mutation was detected. This interference, due to dye residues from the original staining, was partially reduced by de-staining the cytological material. CONCLUSIONS: Our data, although preliminary, show that the Idylla EGFR Mutation Assay can reliably process most archival smears without requiring preliminary DNA extraction. Results may be further improved by de-staining the cellular material before insertion into the cartridge.

EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non-small cell lung carcinoma patients.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are reliably detected by referral laboratories, even if most lung cancer cytology specimens sent to such laboratories contain very few cells. However, EGFR mutations may be distributed heterogeneously within tumors, thereby raising concerns that mutations detected on cytology are not representative of the entire tumor and, thus, are less reliable in predicting response to tyrosine kinase inhibitor (TKI) treatment than mutations detected on histology. To address this issue, the authors reviewed their clinical practice archives and compared the outcome of TKI treatment among patients who were selected by cytology versus patients who were selected by histology. METHODS: From July 2010 to July 2012, 364 cytology samples and 318 histology samples were received. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing; discrepancies were resolved by cloning polymerase chain reaction products. The response rate (RR) and progression-free survival (PFS) at 12 months (range, 3-34 months) were evaluable in 13 EGFR-mutated patients who were selected for treatment by cytology and 13 patients who were selected by histology. RESULTS: The mutation rate was similar in histology samples (8.5%) and cytology samples (8.8%). The RR (54%) and PFS (9.2 months) were similar in histologically selected patients and cytologically selected patients (RR, 62%; PFS, 8.6 months; P = .88). The disease control rate (responsive plus stable disease) was 92% in histologically selected patients and 100% in cytologically selected patients. CONCLUSIONS: EGFR mutations detected on cytology specimens by a centralized laboratory can predict TKI treatment response equally well as mutations identified on histology samples.

Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

BACKGROUND: The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples. METHODS: Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. RESULTS: The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR. CONCLUSIONS: The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society.

Sanger sequencing in routine KRAS testing: a review of 1720 cases from a pathologist's perspective.

BACKGROUND: Sanger sequencing (SS) of PCR products is still the most frequent method to test colorectal cancer for KRAS mutations in routine practice. METHODS: An audit of SS on 1720 routine cases was carried out, taking into account age, gender, specimen type (resection vs biopsies), tumour site (primary vs metastasis), tumour stage, neoplastic cells abundance (>30% vs <30%) and fixation type (buffered formalin vs simple formalin). In a subset of 50 wild-type (WT) patients correlations between SS findings and response rate (RR), progression-free survival (PFS) and overall survival (OS) were also evaluated. RESULTS: The tests were informative in 1691 cases (98.3%). Mutations were detected in 671 cases (39.6%). No significant differences in mutation rates were observed with respect to age (p=0.2), gender (p=0.2), specimen type (p=0.3) and formalin fixation (p=0.08). Conversely, KRAS mutant rate was higher in metastatic tissue (50% vs 39%, p=0.02), in samples with over 30% of neoplastic cells (43.4% vs 26.6%, p=0.02) and in tumours tested in stage IV (p=0.05). The RR of SS KRAS WT patients was 26% (one complete and 12 partial responses). The disease control rate (objective responses plus stable disease) was 56%. Median PFS was 4.4 months and median OS was 10.4 months. CONCLUSIONS: Pathological criteria that make SS a more robust method for KRAS testing and treatment response prediction are neoplastic cell abundance, metastatic tissue sample and stage IV primary tumour.

KRAS testing on colo-rectal carcinoma cytological imprints.

Anti-EGFR monoclonal antibodies, cetuximab, and panitumumab, are administrated under the condition that advanced colo-rectal cancer (CRC) carries a wild-type KRAS gene. Thus, clinicians request pathologists to genotype KRAS before treatment. In the near future routine mutation testing at the same time of the surgery may be implemented. The reliability of a rapid KRAS testing on ex vivo cytological samples obtained by direct scraping of the colon tumour tissue is here evaluated. A consecutive series of 20 surgically resected, primary CRC specimens was analysed. Fresh tissue from CRC was scraped with a scalpel blade, smeared on uncoated glass slides, air-dried and Diff-Quik stained to ensure malignant cell presence. The same tissue area was also histologically processed. Exon 2 KRAS gene mutations were evaluated on both cytological and histological specimens by dideoxy sequencing and by the DxS KRAS Mutation Test Kit (DxS, Manchester, England). Data obtained on on imprint cytology and matched histological samples showed full concordance; however, the mutation frequency was slightly higher (35%) by the DxS KRAS Mutation Test Kit than by the dideoxy sequencing (30%). Thus, colon cancer imprint cytology sample is a reliable biospecimen for both dideoxy-sequencing and DxS KRAS Mutation Test Kit analysis and it may be useful to abbreviate the KRAS assay turnaround time.