BACKGROUND:Fine-needle aspiration biopsy (FNAB) is the preferred first-line diagnostic tool for evaluating lymphadenopathy due to its minimally invasive nature and cost-effectiveness. However, cytopathological interpretation of lymph node FNAB remains challenging because of the wide morphological spectrum of lymphoid lesions. The World Health Organization (WHO) Reporting System for Lymph Nodes, Spleen, and Thymus Cytopathology (WHO System) incorporates the "Atypical" and "Suspicious for malignancy" ("SFM") categories to manage diagnostic uncertainty, yet their reproducibility and clinical implications remain undetermined. METHODS:This review with illustrative case-based applications of the WHO System discusses the use, diagnostic performance, and limitations of the "Atypical" and "SFM" categories. Comparative analysis of published data, including interobserver concordance and risk of malignancy (ROM) studies, has been performed, with reference to the Sydney System and the multicenter DELYCYOUS study. RESULTS:Cases categorized as "Atypical" demonstrate highly variable ROM (28.6%-76.9%, mean ≈65%), reflecting heterogeneity in application and ancillary test integration, whereas the "SFM" category consistently exhibits high ROM (82%-100%, mean ≈95%). Interobserver agreement remains poor (κ = 0.075 for "Atypical"; κ = 0.104 for "Suspicious for malignancy"), highlighting interpretive subjectivity. Diagnostic pitfalls include artifactual monomorphism mimicking high-grade lymphoma and underdiagnosis of paucicellular Hodgkin lymphoma or T-cell proliferations. Ancillary tests, while essential, may also yield misleading results-such as monoclonal B-cell populations in reactive conditions-potentially leading to overinterpretation. CONCLUSIONS:The "Atypical" and "SFM" categories are indispensable for diagnostic stratification and clinical management but suffer from limited reproducibility and intrinsic ambiguity. Their optimal application requires standardized diagnostic criteria of specific lesions, which are provided in the WHO System, institutional consistency, and integrated use of cytomorphological, immunophenotypical, and molecular data to minimize diagnostic uncertainty and improve patient outcomes.

INTRODUCTION:Accurate evaluation of lymphadenopathy requires a multidisciplinary approach integrating clinical, laboratory, and imaging data, particularly ultrasound, before and during fine-needle aspiration biopsy (FNAB). Historically, the lack of standardized reporting systems has limited the reproducibility and clinical acceptance of lymph node FNAB. This review focuses on lymph node cytopathology, which currently is supported by a stronger evidence base and more consistent integration into diagnostic algorithms, than splenic or thymic lesions. MATERIALS AND METHODS:The Sydney system (2020) and the subsequent World Health Organization (WHO) reporting system for lymph nodes, spleen, and thymus cytopathology (2024) (WHO system) provide a structured, evidence-based framework for cytopathological evaluation. The WHO system retains the 5 diagnostic categories of the Sydney system-inadequate, benign, atypical, suspicious for malignancy, and malignant-while defining cytomorphological diagnostic criteria, standardizing terminology, and defining category-specific risks of malignancy. It emphasizes clinicopathological correlation and the use of ancillary techniques, including immunocytochemistry, flow cytometry, and molecular studies, and includes specific considerations for pediatric lymphadenopathy. RESULTS:Implementation of the WHO system improves diagnostic accuracy, interobserver reproducibility, and communication between cytopathologists and clinicians. The interventional cytopathologist plays a central role through image-guided FNAB and rapid on-site evaluation, ensuring sample adequacy and appropriate triage for ancillary testing. CONCLUSIONS:By integrating cytomorphological, clinical, imaging, and ancillary data within a standardized, risk-based framework, the WHO system reinforces the pivotal role of FNAB in the diagnostic management of lymphadenopathy and establishes a globally harmonized standard for lymph node cytopathology.

Background Accurate evaluation of lymphadenopathy requires a multidisciplinary approach integrating clinical, laboratory, and imaging data, particularly ultrasound, before and during fine-needle aspiration biopsy (FNAB). Historically, the lack of standardized reporting systems has limited the reproducibility and clinical acceptance of lymph node FNAB. This review focuses on lymph node cytopathology, which currently is supported by a stronger evidence base and more consistent integration into diagnostic algorithms, than splenic or thymic lesions. Methods The Sydney System (2020) and the subsequent WHO Reporting System for Lymph Nodes, Spleen and Thymus Cytopathology (2024) (WHO System) provide a structured, evidence-based framework for cytopathological evaluation. The WHO System retains the five diagnostic categories of the Sydney System—Inadequate, Benign, Atypical, Suspicious for malignancy, and Malignant—while defining cytomorphological diagnostic criteria, standardizing terminology, and defining category-specific risks of malignancy (ROM). It emphasizes clinicopathological correlation and the use of ancillary techniques, including immunocytochemistry, flow cytometry, and molecular studies, and includes specific considerations for paediatric lymphadenopathy. Results Implementation of the WHO System improves diagnostic accuracy, interobserver reproducibility, and communication between cytopathologists and clinicians. The interventional cytopathologist plays a central role through image-guided FNAB and rapid on-site evaluation (ROSE), ensuring sample adequacy and appropriate triage for ancillary testing. Conclusions By integrating cytomorphological, clinical, imaging and ancillary data within a standardized, risk-based framework, the WHO System reinforces the pivotal role of FNAB in the diagnostic management of lymphadenopathy and establishes a globally harmonized standard for lymph node cytopathology.

Background: Large B-cell lymphoma (LBCL) encompasses a diverse group of aggressive lymphoid neoplasms, the most common of which is diffuse large B-cell lymphoma (DLBCL). A precise diagnosis is critical for determining prognosis and therapeutic strategies, particularly as new molecular subtypes and classification criteria have been introduced in the 5th edition of the World Health Organization (WHO) classification of hematolymphoid tumors (WHO-HAEM5, 2022). Fine needle aspiration cytology is a safe and minimally invasive diagnostic tool for assessing lymphadenopathy and extranodal masses. Summary: LBCLs are typically characterized by single cells with large nuclei, vesicular chromatin pattern, and prominent nucleoli. The diagnosis of LBCL and the distinction distinguishing between LBCL subtypes requires the integration of cytomorphologic features with ancillary techniques, including immunocytochemistry, flow cytometry, fluorescence in situ hybridization, and next-generation sequencing. Key Messages: This review synthesizes the cytologic features, immunophenotypic profiles, molecular signatures, and clinical implications of LBCL subtypes, highlighting recent advances in cytopathology and classification.

BACKGROUND:Cytologic examination of lymph nodes and serous effusions is of value in the diagnosis of small cell lymphomas. The World Health Organisation (WHO) has recently published a reporting system relating to the cytologic examination of lymph nodes, spleen, and thymus to standardise the approach to reporting such specimens. SUMMARY:An overview of the WHO reporting system is given with particular reference to the diagnosis of small B-cell lymphomas. The role of rapid on-site evaluation and ancillary testing is discussed. The cytomorphological features of specific small B-cell lymphomas and associated ancillary testing are described. KEY MESSAGES:The WHO reporting system provides a standardised approach to the reporting of cytological specimens from lymph nodes, the spleen, and the thymus. Small B-cell lymphomas pose a challenge to the cytopathologist due to their morphologic overlap with each other and reactive conditions. The importance of correlation and integration of cytomorphological appearances with clinical features and ancillary testing modalities to obtain a final diagnosis is emphasised. The present review includes a description of cytomorphological features according to pattern recognition and provides an overview of diagnostic criteria for specific entities based on cytomorphology alone and in combination with ancillary tests in different clinical settings discussing differential diagnoses and potential pitfalls.

The objective of the recently published World Health Organization Reporting System for Lymph Node, Spleen, and Thymus Cytopathology (WHO system) is to standardize the diagnostic approach to fine-needle aspiration biopsies of hematolymphoid tissues. By categorizing specimens into five diagnostic groups-inadequate/insufficient/nondiagnostic, benign, atypical, suspicious for malignancy, and malignant-the system provides a structured framework that enhances diagnostic clarity and facilitates communication between cytopathologists and clinicians. Each category is associated with a defined risk of malignancy, supporting informed clinical decision making regarding further diagnostic workup. Accurate categorization requires the integration of cytomorphologic features and clinical context, and final and specific diagnoses often require ancillary techniques such as flow cytometry, immunocytochemistry, in situ hybridization, and molecular diagnostics. To assist cytopathologists, especially those less familiar with hematolymphoid neoplasms, the WHO system incorporates a pattern-based diagnostic approach. Four cytopathologic patterns-mixed lymphoid cell; predominantly small/intermediate cell; predominantly large/pleomorphic/blastic cell; and single, very large, atypical cell-serve as guides to narrow down differential diagnoses. However, interpretation can be challenging because of overlapping features, variable inflammatory backgrounds, and limited sample material. This review provides a brief overview of the WHO system and its application to hematolymphoid proliferations, emphasizing the importance of clinical correlation and the use of relevant ancillary techniques. It then provides in-depth discussion of the pattern-based approach to diagnosing hematolymphoid neoplasms on cytopathology. It highlights the strengths and limitations of cytopathologic evaluation in hematolymphoid neoplasms and provides practical insights for applying the WHO system in routine practice.

INTRODUCTION:A World Health Organization (WHO) system has recently been introduced to standardize reporting of lymph node fine-needle aspiration biopsy (LN-FNAB) cytopathology. Herein, we analyze its diagnostic performance in a routine setting. MATERIALS AND METHODS:We reviewed 2274 consecutive lymph node fine-needle aspiration biopsies categorized according to the WHO system. Corresponding histological material, radiological images, or uneventful clinical follow-up over 12 months were used as ground truths for classifying LNs as malignant or benign. RESULTS:The diagnostic accuracy of the WHO system was 91.8% without and 95.9% with ancillary techniques. The risk of malignancy (ROM) was 21.8% for the "Insufficient/Inadequate/Non-diagnostic", 6.6% for the "Benign", 29.7% for the "Atypical", 91.2% for the "Suspicious for malignancy", and 99.6% for the "Malignant" category. ROM increased with lymph node size, with a significant rise to 33.1% in the 10-14 mm size interval. The ratio of risk of lymphoma (ROL) to risk of metastasis (ROMet) was significantly lower in lymph nodes <15 mm and depended on the anatomical site with the highest ROL in abdominal/pelvic and the highest ROMet in axillary/mammary lymph nodes. The ROL-to-ROMet ratio was higher if the question was lymphoma (36.7%-8.0%) or unspecified (8.3%-8.3%) compared to any other question. CONCLUSIONS:The WHO system demonstrates high diagnostic accuracy, increasing with the use of ancillary techniques, particularly flow cytometry. The ROM, ROL, and ROMet depend on lymph node size, the anatomical site, and the clinical question. A lymph node size of 10 mm may be an appropriate threshold for further investigation, depending on the clinical context.

The clinical course of Mantle Cell Lymphoma (MCL) varies between individual patients. Early detection of risk is crucial to assign MCL patients to novel treatment strategies. Most of the established biomarkers of outcome require specifically trained pathologists or molecular analysis. Here we introduce MAIPI (MCL Artificial Intelligence Prognostic Index), a deep learning algorithm trained only on Hematoxylin and Eosin (H&E) images of diagnostic biopsies of n = 428 MCL patients from clinical trials to assess prognosis. The capability of MAIPI to predict disease outcome was validated in an independent cohort of n = 140 patients treated with immunochemotherapy with and without ibrutinib. MAIPI selects areas of interest by itself and provides prognostic information independent of the MCL International Prognostic Index (MIPI) and Ki67 and without the need of molecular testing or expert pathologists evaluation.

BACKGROUND:Fine-needle aspiration biopsy (FNAB) of lymph nodes is a widely used method for evaluating lymphadenopathy. FNAB offers general advantages of rapid turnaround time, low cost and minimal morbidity, and more specific advantages in various clinical situations, such as deeply located lymph nodes or patients with significant comorbidities. The FNAB sample can be utilized for a wide range of ancillary tests, including microbiological studies, immunocytochemistry for primary and metastatic neoplasms and flow cytometry immunophenotyping in cases of lymphoid-rich samples, where there is a suspicion for lymphomas. SUMMARY:The increasing application of FNAB in lymph node pathology has led to the development of a standardized reporting system, formalized in the World Health Organization (WHO) Reporting System for Lymph Node, Spleen and Thymus Cytopathology (WHO System). This system is equally applicable to lymph node, spleen and thymus; however, this article focuses on lymph nodes. The WHO System was established through a joint project of the WHO, the International Agency for Research on Cancer (IARC) and the International Academy of Cytology (IAC) and is structured into five diagnostic categories: inadequate/insufficient/non-diagnostic, benign, atypical, suspicious for malignancy, and malignant. The WHO System provides a standardized and reliable means of categorizing various lymph node lesions based on cytopathology findings and enables pathologists to make more accurate and reproducible diagnoses, thereby improving clinical management and treatment decisions. Integrating cellular morphology and clinical-imaging data help distinguish benign from malignant lesions, significantly reducing diagnostic variability. The primary goal was to reduce diagnostic uncertainty and improve patient outcomes through greater consistency and clarity in lymph node cytopathology reports. The WHO System emphasizes the use of rapid on-site assessment (ROSE) to improve diagnostic accuracy and reduce the need for additional diagnostic procedures. The risk of malignancy (ROM) varies by diagnostic category, with higher risks of malignancy in the "Suspicious for malignancy" and "Malignant" categories. The system also includes recommendations for ancillary tests and performance of additional biopsies when further clarification is needed. The WHO System represents a significant advancement in the standardization of lymph node, spleen, and thymus cytopathology, facilitating interdisciplinary communication and improving risk stratification. However, diagnostic challenges remain, particularly in managing inadequate samples and interpreting atypical lesions, necessitating a multidisciplinary approach that integrates clinical, imaging, ancillary testing and, in some cases, core needle, or excision biopsy material. KEY MESSAGES:The WHO System serves as a crucial tool for refining the diagnosis of the broad range of inflammatory, infectious, metastatic, and lymphomatous processes in lymph node pathologies. In addition, it is suitable for high income as well as most obviously low- and middle-income countries leading to optimizing therapeutic decision-making.

OBJECTIVES:To determine the diagnostic value of disclosing monotypic T-cell populations by expression analysis of the constant region 1 of T-cell receptor β (TRBC1) by flow cytometry, for the detection of T-cell neoplasms, in a routine hematopathology practice setting. METHODS:A panel of antibodies ECD-CD16/PECy5.5-CD4/PECy7-CD2/APCAF700-CD7/KO-CD8/FITC-TCRγδ/PE-TRBC1/APC-CD26/APC-CD57/APC-CD30/APCH7-CD45/BV421-CD3 was applied. One thousand and twenty-nine cases investigated during 26 months were reviewed and categorized as monotypic or polytypic depending on the expression of TRBC1 in all identifiable CD3+ T-cell subsets. RESULTS:Although TRBC1 restriction proved to be sensitive (90.7%) with a high negative predictive value (99.1%), in some T-cell neoplasms, particularly those with significant inflammation, including angioimmunoblastic T-cell lymphoma, monotypic T-cells were not detected. The specificity was high (95.6%) albeit with a modest positive predictive value (67.1%), reflecting T-cell clones of uncertain significance (T-CUS) without T-cell neoplasm. Of these clones, 53.6% exhibited a T-cell large granular lymphocytic leukemia-like phenotype, most commonly found in the peripheral blood or bone marrow, while others were identified in association with unrelated primary tumors or other comorbidities. Seventy-nine percent of the T-CUS cases remained stable over time during 1-77 months of follow-up. CONCLUSION:Including TRBC1 antibodies in a routine flow cytometry panel facilitates the identification of T-cell neoplasms. The analysis must be interpreted within its clinical context since T-cell lymphomas with a small and sometimes surface CD3-negative neoplastic T cell population, may display normal patterns of TRBC1-expression with a background of reactive T cells. Conversely, monotypic T-cells can be found in the absence of T-cell neoplasm.

In this editorial, we discuss the added value of the novel TCRBC1 antibody for immunohistochemistry in the differential diagnostics in haematopathology and promote its implementation in current diagnostic algorithms.

BACKGROUND:The International Academy of Cytology in collaboration with the International Agency for Research on Cancer has developed a standardized World Health Organization Reporting System (WHO System) for the cytopathology of the lymph nodes, spleen, and thymus. Fine-needle aspiration biopsy (FNAB) of the spleen and thymus, performed under ultrasound or computed tomography guidance, constitutes a minimally invasive, well-tolerated, and cost-effective diagnostic technique. Despite a limited number of published studies on the diagnostic accuracy of FNAB in these organs, it remains a valuable tool particularly when combined with rapid on-site evaluation and ancillary techniques, such as immunophenotypic analysis by flow cytometry or immunocytochemistry and molecular testing. SUMMARY:The WHO System categorizes thymic and splenic cytopathology into five diagnostic categories: "Inadequate/Insufficient/Nondiagnostic," "Benign," "Atypical," "Suspicious for malignancy," and "Malignant." This standardized approach aims to enhance diagnostic accuracy, provide risk assessment, and facilitate clinical decision-making. However, applicability of FNAB in the thymus and spleen is still debated, particularly concerning sample adequacy, diagnostic accuracy, and potential complications such as hemorrhage, especially for splenic FNAB. Thymic FNAB is primarily used for diagnosing thymic hyperplasia, thymoma, thymic carcinomas, and lymphomas. However, distinguishing these entities and their subtypes is challenging. Splenic FNAB is valuable in evaluating splenomegaly, infections, lymphomas, and other neoplasms, particularly in staging and monitoring disease progression. While using FNAB to evaluate these organs is not yet a widely adopted standard practice, its role in minimizing unnecessary surgical interventions and guiding therapeutic strategies is being increasingly recognized. KEY MESSAGES:The WHO System for the thymus and spleen seeks to establish a harmonized, evidence-based framework for cytopathological diagnosis, incorporating key diagnostic criteria, malignancy risk assessment, and standardized reporting protocols. Future research is needed to refine and develop diagnostic role of FNAB, enhance its integration with advanced molecular techniques, and optimize its use in personalized medicine.

INTRODUCTION:Lymph node fine-needle aspiration cytology (LN-FNAC) is a common, rapid, minimally invasive and cost-effective diagnostic method. For mediastinal lymph nodes, endobronchial ultrasound (EBUS) guided LN-FNAC is a first-line investigation and has an indispensable role in the diagnosis and staging of patients with suspected lung cancer. Recently, a new WHO system has been proposed for classification of LN-FNAC heralding five different diagnostic categories; insufficient, benign, atypical, suspicious for malignancy and malignant. The aim of this study was to evaluate the diagnostic accuracy and risk of malignancy (ROM) of these categories in EBUS-guided LN-FNAC from mediastinal lymph nodes. METHOD:We evaluated 2110 consecutive mediastinal lymph nodes during this one-year retrospective study. Corresponding radiological images and histologic material were used as ground truth to calculate accuracy, sensitivity, specificity and ROM. RESULTS:The WHO system showed an overall accuracy of 93.7% with a sensitivity of 83.0% and a specificity of 97.5%. The positive predictive value was 92.3% and the negative predictive value 94.2%. The overall ROM for each category in the WHO classification system was 12.8% for the inadequate, 2.4% for the benign, 47.4% for the atypical, 81.0% for the suspicious for malignancy and 93.6% for the malignant category. CONCLUSION:The results of the present study indicate that the new WHO system entails a high diagnostic accuracy regarding EBUS-guided LN-FNAC assessment of mediastinal lymph nodes and supports its integration into clinical practice. Application of the WHO system standardizes risk assessment thus facilitating communication between cytopathologists and clinicians and minimizes the need for histopathological analysis.

The diagnosis of lymphoma relies mainly on clinical examination and laboratory explorations. Among the latter, morphological and immunohistochemical analysis of a tissue biopsy are the cornerstones for proper identification and classification of the disease. In lymphoma with blood and/or bone marrow involvement, multiparameter flow cytometry is useful. This technique can also be applied to fresh cells released from a biopsy sample. For full comprehension of lymphomas, surgical biopsies are best and indeed recommended by the hematopathological community. Currently, however, there is a global trend towards less invasive procedures, resulting in smaller samples such as core needle biopsies or fine needle aspirations which can make the diagnosis quite challenging. In this review, the possibilities and limitations to make an accurate lymphoma diagnosis on such small volume material are presented. After recalling the major steps of lymphoma diagnosis, the respective value of histology, cytology, and flow cytometry is discussed, including handling of small specimens. The benefits of an integrated approach are then evoked, followed by discussion about which attitude to adopt in different contexts. Perhaps contrary to the prevailing view among many pathologists, a full diagnosis on small volume material, combined with relevant ancillary techniques, is often possible and indeed supported by recent literature. A glimpse at future evolutions, notably the merit of artificial intelligence tools, is finally provided. All in all, this document aims at providing pathologists with an overview of diagnostic possibilities in lymphoma patients when confronted with small volume material such as core needle biopsies or fine needle aspirations.

Introduction: Although most patients with mantle cell lymphoma (MCL) are elderly, there is no universal established standard therapy for this population. Accumulating evidence show that BTK inhibitors may have an important role in front line regimens. Ibrutinib+ rituximab was recently compared to chemoimmunotherapy in the phase 3 ENRICH trial, but in vitro suggest that ibrutinib partially antagonizes the activity of rituximab through NK-cell inhibition. Acalabrutinib is not associated with NK-cell inhibition potentially and may thus be a more attractive combination partner with rituximab (AR). In this multicenter open-label phase II trial, we evaluated safety and efficacy of AR in elderly patients with MCL, including an MRD guided treatment duration strategy, as previously explored in the NLG-MCL7 (VALERIA) trial. Methods: Main eligibility criteria were age ≥60 years, untreated MCL, non-eligible for transplant, WHO PS 0-3, and measurable disease by imaging. The primary endpoint was progression-free survival, and to provide a context for the single arm trial, an external comparator cohort was created using data were compared to a matched dataset from the population-based Swedish MCLcompleteproject enrolling 1367 patients treated in the period 2006-2018. Matching was done by matching on sex and using nearest neighbor on MIPI score, in a 1:5 ratio for MCLcomplete. The most common regimens in the matched dataset were R-Bendamustine (33%) and NLG-MCL2 (18%). Minimal residual disease (MRD) monitoring by RQ-PCR was performed every 3 months during follow-up, according to EuroMRD criteria. Sequencing of the most frequently mutated genes in MCL was performed on formalin-fixed tumor tissue. Treatment schedule: Acalabrutinib 100 mg BID in combination with rituximab 375 mg/m2 IV on day 1 every 28 days for 6 cycles, followed by treatment every 8 weeks. The minimum duration of treatment was 13 months. Patients in molecular remission in blood and bone marrow and in complete remission, would stop acalabrutinib, but continue with rituximab for up to 36 months. Patients with TP53 aberrations and/or blastoid histology continued with treatment until progression or unacceptable toxicity regardless of MRD results. Results: In total, 81 patients were enrolled, from 18 sites in Sweden, Norway, Denmark, Finland and South Korea. The median age was 75 years, 61 (75%) were male and 56 (69%) were MIPI high risk. A TP53 mutation was detected in 19 (24%) of diagnostic biopsies, high Ki67 expression (>30%) in 18 (22%), and blastoid histology in 5 (6%). At 6 months, the ORR (n=61) was 95% (37 CR, 21 PR). The median follow-up is currently 15.5 months. PFS at 1 year is 86.5% (95% CI 78.9-94.8) , compared to 71.8% (95% CI 69.5-74.2) in MCLcomplete (p=0.016). OS at 1 year is 92.9% (95% CI 87.1-99.2) compared to 79.6% (95% CI 77.5-81.7) in MCLcomplete (p=0.012). Patients with TP53-mutated disease showed inferior outcome with a PFS of 69% at 1 year. At 12 months, 27 (59%) of 46 evaluable patients were MRD-negative, 14 were MRD+, 2 were not evaluable by MRD, and 3 are awaiting results. 19 out of 27 MRD-neg (70.4%) have stopped acalabrutinib at this point, and 5 continued treatment due to high risk biology. Another 2 patients stopped acala at 18 months, and one pt at 24 months, due to MRD-negativity. The most common grade 3-4 AE were neutropenia in 11 pts, infection in 10 pts, and cardiovascular toxicity in 7 pts. There have been 9 deaths, 3 due to MCL, 2 due to treatment-related toxicity (aplastic anemia and sepsis), and 4 due to other causes. Conclusions: A response adapted treatment strategy, by stopping treatment in molecular remission appears to be feasible in MCL, even in an international multicenter trial setting. By comparison to matched population-based data, single arm clinical trial data may be set into a meaningful context, and frontline AR appear to be favorable compared to current standard therapy. Updated results will be presented at the meeting, including prolonged follow-up of MRD-negative patients after stopping acalabrutinib.

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.

Acute myeloid leukemia (AML) is initiated and propagated by leukemia stem cells (LSCs), a self-renewing population of leukemia cells responsible for therapy resistance. Hence, there is an urgent need to identify new therapeutic opportunities targeting LSCs. Here, we performed an in vivo CRISPR knockout screen to identify potential therapeutic targets by interrogating cell surface dependencies of LSCs. The facilitated glucose transporter type 1 (GLUT1) emerged as a critical in vivo metabolic dependency for LSCs in a murine MLL::AF9-driven model of AML. GLUT1 disruption by genetic ablation or pharmacological inhibition led to suppression of leukemia progression and improved survival of mice that received transplantation with LSCs. Metabolic profiling revealed that Glut1 inhibition suppressed glycolysis, decreased levels of tricarboxylic acid cycle intermediates and increased the levels of amino acids. This metabolic reprogramming was accompanied by an increase in autophagic activity and apoptosis. Moreover, Glut1 disruption caused transcriptional, morphological, and immunophenotypic changes, consistent with differentiation of AML cells. Notably, dual inhibition of GLUT1 and oxidative phosphorylation (OXPHOS) exhibited synergistic antileukemic effects in the majority of tested primary AML patient samples through restraining of their metabolic plasticity. In particular, RUNX1-mutated primary leukemia cells displayed striking sensitivity to the combination treatment compared with normal CD34+ bone marrow and cord blood cells. Collectively, our study reveals a GLUT1 dependency of murine LSCs in the bone marrow microenvironment and demonstrates that dual inhibition of GLUT1 and OXPHOS is a promising therapeutic approach for AML.