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.

Liquid biopsy: A promising tool for driving strategies and predicting failures in patients with classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) consists of a heterogeneous group of haematological disorders that covers undifferentiated B cell neoplasms originating from germinal centre B cells. The HL molecular characterization still represents an ongoing challenge due to the low fraction of tumour Hodgkin and Reed-Sternberg cells mixed with a plethora of non-tumour haematological cells. In this scenario, next generation sequencing of liquid biopsy samples is emerging as a useful tool in HL patients' management. In this review, we aimed to overview the clinical and methodological topics regarding the implementation of molecular analysis in cHL, focusing on the role of liquid biopsy in diagnosis, follow-up, and response prediction.

Digital Examination of LYmph node CYtopathology Using the Sydney system (DELYCYUS): An international, multi-institutional study.

BACKGROUND: After a series of standardized reporting systems in cytopathology, the Sydney system was recently introduced to address the need for reproducibility and standardization in lymph node cytopathology. Since then, the risk of malignancy for the categories of the Sydney system has been explored by several studies, but no studies have yet examined the interobserver reproducibility of the Sydney system. METHODS: The authors assessed interobserver reproducibility of the Sydney system on 85 lymph node fine-needle aspiration cytology cases reviewed by 15 cytopathologists from 12 institutions in eight different countries, resulting in 1275 diagnoses. In total, 186 slides stained with Diff-Quik, Papanicolaou, and immunocytochemistry were scanned. A subset of the cases included clinical data and results from ultrasound examinations, flow cytometry immunophenotyping, and fluorescence in situ hybridization analysis. The study participants assessed the cases digitally using whole-slide images. RESULTS: Overall, the authors observed an almost perfect agreement of cytopathologists with the ground truth (median weighted Cohen κ = 0.887; interquartile range, κ = 0.210) and moderate overall interobserver concordance (Fleiss κ = 0.476). There was substantial agreement for the inadequate and malignant categories (κ = 0.794 and κ = 0.729, respectively), moderate agreement for the benign category (κ = 0.490), and very slight agreement for the suspicious (κ = 0.104) and atypical (κ = 0.075) categories. CONCLUSIONS: The Sydney system for reporting lymph node cytopathology shows adequate interobserver concordance. Digital microscopy is an adequate means to assess lymph node cytopathology specimens.

Multiple predictive biomarker testing in melanoma: Another challenge in identifying the optimal approach on cytological samples.

BACKGROUND: The management of cutaneous melanoma has changed dramatically in recent years thanks to the development of tyrosine kinase and immune-checkpoint inhibitors (ICIs). Thus, multiple biomarker testing is becoming ever more important for the identification of patients who are potentially eligible for these treatments. One reliable approach to the molecular evaluation of metastatic melanoma is fine needle cytology (FNC). To examine the utility of this approach for assessing PD-L1 expression levels, we evaluated the cellular adequacy of residual cell block (CB) material from metastatic melanomas that were previously tested for BRAF and NRAS mutations. METHODS: We retrieved from our internal archives a series of FNC samples of metastatic melanoma that had been subjected to molecular testing on residual CB material or a dedicated needle rinse between January 2016 and July 2022. Real-time polymerase chain reaction was used to assess BRAF and NRAS status, and an SP263 assay was employed to ascertain PD-L1 expression levels. RESULTS: Overall, n = 19 cases were selected. Of these, 11 (57.9%) cases revealed a BRAF exon 15 p.V600E mutation, one case (5.3%) revealed NRAS mutation, and seven cases (36.8%) showed no mutations. Regarding PD-L1 assessment, 16/19 (84.2%) cases were deemed adequate, meaning they contained at least 100 viable cells. CONCLUSIONS: We highlighted the feasibility of assessing PD-L1 expression levels in residual CB material from metastatic melanomas previously tested for BRAF and NRAS mutations. Moreover, we pointed out that FNC needle rinses may be an alternative source of nucleic acids for molecular testing, preserving CB material for immunocytochemistry evaluation.

A roadmap for a comprehensive diagnostic approach to fine needle cytology of lymph node metastases.

OBJECTIVE: Fine needle cytology (FNC) is widely used as a first-line procedure in the diagnostic algorithm of lymphadenopathies. In a metastatic setting, a first-line diagnostic approach identifies non-haematopoietic malignancy; however, cytopathologists could also provide a second diagnostic level, identifying the origin of the primary tumour. This paper outlines a comprehensive and practical approach to the cytological diagnosis of lymph node metastases. METHODS: Cytological diagnoses of lymph node metastases performed over a 10-year period were selected and divided into two groups. The first group, labelled "oncological," comprised patients with a previous history of malignancy; the second group, labelled "naïve," included patients with no relevant history. Pathology records were retrieved to record microscopic findings, namely, background appearance, group architecture, and specific cell features; data from cell block (CB) preparations were also collected. RESULTS: Overall, 982 cases were selected: 497 cases (50.61%) in the naïve group, and 485 (49.39%) in the oncological group. Overall, a second diagnostic level was achieved in 834/982 cases (84.92%); cases diagnosed as carcinoma not otherwise specified were more frequent in the naïve group than in the oncological group (17.51% vs. 8.04%, P < 0.01). Notably, although CB material was available in only 44.87% of the naïve cases, we were able to achieve a second diagnostic level thanks to the integration of clinical and cytomorphological findings, plus lymph node topography, in 82.49% of the cases. CONCLUSION: Our results confirmed that in a metastatic setting, FNC can reliably lead to the identification of the origin of the primary tumour.

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.

A Novel Approach to Classification and Reporting of Lymph Node Fine-Needle Cytology: Application of the Proposed Sydney System.

Fine-needle cytology (FNC) is a useful diagnostic tool in the first line evaluation of lymphadenopathy of unknown aetiology. Nevertheless, considering the large number of conditions presenting as lymphadenopathy, lymph node cytology represents a challenging scenario. Recently, an expert panel published the proposal of the Sydney system for performing classification and reporting of lymph node cytopathology; the aim of the present study was to evaluate the applicability of this system. Thus, 300 lymph node FNCs performed over 1 year were reviewed and categorized according to the Sydney system classification. Overall, n = 20 cases (6.7%) were categorized as L1-inadequate/non-diagnostic; n = 104 (34.7%) as benign (L2); n = 25 (8.3%) as atypical (L3); n = 13 (4.3%) as suspicious (L4), and n = 138 (46%) as malignant (L5). FNC diagnoses were correlated with histopathologic and clinical follow-up to assess the diagnostic accuracy and the risk of malignancy (ROM) for each diagnostic category. Statistical analysis showed the following results: sensitivity 98.47%, specificity 95.33%, positive predictive value 96.27%, negative predictive value 98.08%, and accuracy 97.06%. The ROM was 50% for the category L1, 1.92% for L2, 58.3% for L3, and 100% for L4 and L5. In conclusion, FNC coupled with ancillary techniques ensures satisfactory diagnostic accuracy and the implementation of the Sydney system may improve the practice of cytopathologists.

PD-L1 expression on routine samples of non-small cell lung cancer: results and critical issues from a 1-year experience of a centralised laboratory.

AIMS: Our laboratory is a centralised centre receiving routine non-small cell lung cancer (NSCLC) samples for programmed death ligand-1 (PD-L1) immunohistochemical (IHC) evaluation. Since literature data are not concordant here we review our clinical records to assess the rate of PD-L1 positive and negative NSCLC cases in real-world practice. METHODS: PD-L1 expression was evaluated by a validated 22C3 IHC laboratory developed test on 211 prospectively collected routine NSCLC samples, received from 10 outside institutions. PD-L1 expression was assessed by the tumour proportion score (TPS) and reported by using a three cut-point system: TPS<1, TPS 1%-49% and TPS≥50%. RESULTS: Overall, 193 out of 211 samples (91.5%) meet the criteria for adequacy (more than 100 viable neoplastic cells). In 62.7% (121/193) of samples TPS was <1%; 17.6% of samples (34/193) expressed PD-L1 with a TPS of 1%-49% and 19.7% (38/193) with a TPS of >50%. There was no significant difference in PD-L1 expression rates between different histotypes and site of sampling. Instead, a statistically significant difference was associated to the type of samples: in fact, cytological samples were more frequently negative for PD-L1 expression (TPS<1%) and less often displayed PD-L1 expression at high levels (TPS>50%) than surgical resections and biopsies. CONCLUSIONS: We present a referral laboratory experience on IHC PD-L1 expression of prospectively collected routine NSCLC samples. Data from the real-world practice can better clarify the percentage of PD-L1 positive and negative cases, to establish benchmarks for good practice standards.