Endobronchial ultrasound-guided transbronchial fine needle aspiration of mediastinal lymphadenopathy: Diagnostic performance and clinical implications of the World Health Organization reporting system.

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.

Morphology and multiparameter flow cytometry combined for integrated lymphoma diagnosis on small volume samples: possibilities and limitations.

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.

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.

Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity.

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.

Unsupervised cluster analysis and subset characterization of abnormal erythropoiesis using the bioinformatic Flow-Self Organizing Maps algorithm.

BACKGROUND: The Flow-Self Organizing Maps (FlowSOM) artificial intelligence (AI) program, available within the Bioconductor open-source R-project, allows for an unsupervised visualization and interpretation of multiparameter flow cytometry (MFC) data. METHODS: Applied to a reference merged file from 11 normal bone marrows (BM) analyzed with an MFC panel targeting erythropoiesis, FlowSOM allowed to identify six subpopulations of erythropoietic precursors (EPs). In order to find out how this program would help in the characterization of abnormalities in erythropoiesis, MFC data from list-mode files of 16 patients (5 with non-clonal anemia and 11 with myelodysplastic syndrome [MDS] at diagnosis) were analyzed. RESULTS: Unsupervised FlowSOM analysis identified 18 additional subsets of EPs not present in the merged normal BM samples. Most of them involved subtle unexpected and previously unreported modifications in CD36 and/or CD71 antigen expression and in side scatter characteristics. Three patterns were observed in MDS patient samples: i) EPs with decreased proliferation and abnormal proliferating precursors, ii) EPs with a normal proliferating fraction and maturation defects in late precursors, and iii) EPs with a reduced erythropoietic fraction but mostly normal patterns suggesting that erythropoiesis was less affected. Additionally, analysis of sequential samples from an MDS patient under treatment showed a decrease of abnormal subsets after azacytidine treatment and near normalization after allogeneic hematopoietic stem-cell transplantation. CONCLUSION: Unsupervised clustering analysis of MFC data discloses subtle alterations in erythropoiesis not detectable by cytology nor FCM supervised analysis. This novel AI analytical approach sheds some new light on the pathophysiology of these conditions.

Comparison of RNA- and DNA-based methods for measurable residual disease analysis in NPM1-mutated acute myeloid leukemia.

INTRODUCTION: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. METHODS: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. RESULTS: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. CONCLUSION: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.

Is there an impact of measurable residual disease as assessed by multiparameter flow cytometry on survival of AML patients treated in clinical practice? A population-based study.

The Swedish national guidelines for treatment of acute myeloid leukemia (AML) recommend analysis of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in bone marrow in the routine clinical setting. The Swedish AML registry contains such MRD data in AML patients diagnosed 2011-2019. Of 327 patients with AML (non-APL) with MRD-results reported in complete remission after two courses of intensive chemotherapy 229 were MRD-negative (70%), as defined by <0.1% cells with leukemia-associated immunophenotype in the bone marrow. MRD-results were reported to clinicians in real time. Multivariate statistical analysis adjusted for known established risk factors did not indicate an association between MFC-MRD and overall survival (HR: 1.00 [95% CI 0.61, 1.63]) with a median follow-up of 2.7 years. Knowledge of the importance of MRD status by clinicians and individualized decisions could have ameliorated the effects of MRD as an independent prognostic factor of overall survival.

Mutational spectrum of de novo NPM1-mutated acute myeloid leukemia patients older than 75 years.

AML with mutated NPM1 occurs in all age groups. Yet, the mutational pattern is not extensively studied in the very old, which may hamper appropriate risk assessment. Herein we examined 22 cases of NPM1-mutated de novo AML in patients older than 75, with a median age of 84. All diagnostic samples were sequenced aiming for coverage of the most relevant AML-associated mutations. For comparison with younger patients, we used already published data on several cohorts. A total of 76 mutations including 50 different variants were identified in 16 recurrently mutated AML genes. Compared with younger patients, a significant enrichment of TET2 and SRSF2 was observed, together with a reduced frequency of DNMT3A mutations. Our results indicate that the mutational pattern may be different in the very old as compared to younger patients with NPM1-mutated AML.HighlightsThe mutational spectrum of NPM1-mutated AML in patients above 75 years displays distinct features.A significant enrichment of TET2 and SRSF2 mutations together with a reduced frequency of DNMT3A mutations was observed in the elderly.NPM1 mutation is a secondary event in the development of AML in the very old.

Genomic and transcriptomic correlates of Richter transformation in chronic lymphocytic leukemia.

The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor-associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.

Analysis of erythroid maturation in the nonlysed bone marrow with help of radar plots facilitates detection of flow cytometric aberrations in myelodysplastic syndromes.

BACKGROUND: Accumulating data support the role of flow cytometry (FCM) in diagnostic work-up of myelodysplastic syndromes (MDS). Changes in erythropoiesis are less documented than in granulopoiesis. However, most studies were performed on bone marrow samples (BMSs) after red blood cell lysis. We have established a FCM protocol for erythropoiesis, following a no-lysis approach and live gate acquisition of nucleated cells using DNA dye DRAQ5. METHODS: The ERY tube consisted of CD36, CD71, CD105, CD117, CD13, and CD45. Comparison with cytomorphological differential counts was carried out in a learning cohort of 80 BMS. To detect aberrations, we analyzed 208 BMS from 135 patients and five normal donors, divided into three cohorts: MDS (n = 68), nonclonal cytopenia (n = 43), and normal controls (n = 29). Radar plot (RP) was created for an overview of normal and aberrant patterns. RESULTS: The proportion of erythropoiesis in the ERY tube showed better agreement with the cytomorphology, compared to FCM panels on lysed BMS. We confirmed that aberrations in coefficient of variation (CV) of CD36 fluorescence intensity (p < .001), mean fluorescence intensity of CD36 (p = .012), and CV of CD105 (p < .001) can distinguish between MDS and nonclonal cytopenia. RP facilitated evaluation of erythropoietic maturation patterns and aberrant patterns were identified in 85% of MDS patients. CONCLUSION: This study provides evidence that a no-lysis approach and RP analysis allow a more reliable evaluation of erythropoiesis and erythroid dysplasia, supporting the integration of FCM erythroid panels in the standard work-up of MDS.

Subclonal patterns in follow-up of acute myeloid leukemia combining whole exome sequencing and ultrasensitive IBSAFE digital droplet analysis.

We studied mutation kinetics in ten relapsing and four non-relapsing patients with acute myeloid leukemia by whole exome sequencing at diagnosis to identify leukemia-specific mutations and monitored selected mutations at multiple time-points using IBSAFE droplet digital PCR. Five to nine selected mutations could identify and track leukemic clones prior to clinical relapse in 10/10 patients at the time-points where measurable residual disease was negative by multicolor flow cytometry. In the non-relapsing patients, the load of mutations gradually declined in response to different therapeutic strategies. Three distinct patterns of relapse were observed: (1) one or more different clones with all monitored mutations reappearing at relapse; (2) one or more separate clones of which one prevailed at relapse; and (3) persistent clonal hematopoiesis with high variant allele frequency and most mutations present at relapse. These pilot results demonstrate that IBSAFE analyses detect leukemic clones missed by flow cytometry with possible clinical implications.HighlightsThe IBSAFE ddPCR MRD method seems applicable on virtually all newly diagnosed AML patients and was more sensitive than flow cytometry.Monitoring a few mutations captured the kinetics of the evolving recurrent leukemia.NPM1-mutation alone may not be a reliable MRD-marker.

A Proposal for the Performance, Classification, and Reporting of Lymph Node Fine-Needle Aspiration Cytopathology: The Sydney System.

BACKGROUND: The evaluation of lymph nodes (LN) by fine-needle aspiration cytology (FNAC) is routinely used in many institutions but it is not uniformly accepted mainly because of the lack of guidelines and a cytopathological diagnostic classification. A committee of cytopathologists has developed a system of performance, classification, and reporting for LN-FNAC. METHODS: The committee members prepared a document that has circulated among them five times; the final text has been approved by all the participants. It is based on a review of the international literature and on the expertise of the members. The system integrates clinical and imaging data with cytopathological features and ancillary techniques. The project has received the endorsement and patronage of the International Academy of Cytology and the European Federation of the Cytology Societies. RESULTS: Clinical, imaging, and serological data of lymphadenopathies, indications for LN-FNAC, technical procedures, and ancillary techniques are evaluated with specific recommendations. The reporting system includes two diagnostic levels. The first should provide basic diagnostic information and includes five categories: inadequate/insufficient, benign, atypical lymphoid cells of undetermined/uncertain significance, suspicious, and malignant. For each category, specific recommendations are provided. The second diagnostic level, when achievable, should produce the identification of specific benign or malignant entities and additional information by utilizing ancillary testing. CONCLUSION: The authors believe that the introduction of this system for performing and reporting LN-FNAC may improve the quality of the procedure, the report, and the communication between cytopathologists and the clinicians. This system may lead to a greater acceptance and utilization of LN-FNAC and to a better interdisciplinary understanding of the results of this procedure.

CXCR4 Signaling Has a CXCL12-Independent Essential Role in Murine MLL-AF9-Driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is defined by an accumulation of immature myeloid blasts in the bone marrow. To identify key dependencies of AML stem cells in vivo, here we use a CRISPR-Cas9 screen targeting cell surface genes in a syngeneic MLL-AF9 AML mouse model and show that CXCR4 is a top cell surface regulator of AML cell growth and survival. Deletion of Cxcr4 in AML cells eradicates leukemia cells in vivo without impairing their homing to the bone marrow. In contrast, the CXCR4 ligand CXCL12 is dispensable for leukemia development in recipient mice. Moreover, expression of mutated Cxcr4 variants reveals that CXCR4 signaling is essential for leukemia cells. Notably, loss of CXCR4 signaling in leukemia cells leads to oxidative stress and differentiation in vivo. Taken together, our results identify CXCR4 signaling as essential for AML stem cells by protecting them from differentiation independent of CXCL12 stimulation.

Measurable residual disease testing for personalized treatment of acute myeloid leukemia.

This review summarizes - with the practicing hematologist in mind - the methods used to determine measurable residual disease (MRD) in everyday practice with some future perspectives, and the current knowledge about the prognostic impact of MRD on outcome in acute myeloid leukemia (AML), excluding acute promyelocytic leukemia. Possible implications for choice of MRD method, timing of MRD monitoring, and guidance of therapy are discussed in general and in some detail for certain types of leukemia with specific molecular markers to monitor, including core binding factor (CBF)-leukemias and NPM1-mutated leukemias.

Minimal residual disease assessed with deep sequencing of NPM1 mutations predicts relapse after allogeneic stem cell transplant in AML.

Mutations in NPM1 can be used for minimal residual disease (MRD) analysis in acute myeloid leukemia (AML). We here applied a newly introduced method, deep sequencing, allowing for simultaneous analysis of all recurrent NPM1 insertions and thus constituting an attractive alternative to multiple PCRs for the clinical laboratory. We retrospectively used deep sequencing for measurement of MRD pre- and post-allogeneic hematopoietic stem cell transplantation (alloHCT). For 29 patients in morphological remission at the time of alloHCT, the effect of deep sequencing MRD on outcome was assessed. MRD positivity was defined as variant allele frequency ≥0.02%. Post-transplant MRD status was significantly and independently associated with clinical outcome; 3-year relapse-free survival 20% vs 85% (p < .001), HR 45 (95% CI 2-1260), and overall survival 20% vs 89% (p < .001), HR 49 (95% CI 2-1253). Thus, the new methodology deep sequencing is an applicable and predictive tool for MRD assessment in AML.

Signaling via Smad2 and Smad3 is dispensable for adult murine hematopoietic stem cell function in vivo.

Transforming growth factor-ß (TGFß) is a member of a large family of polypeptide growth factors. TGFß signals mainly through the intracellular proteins Smad2 and Smad3, which are highly similar in amino acid sequence identity. A number of studies have shown that these proteins, dependent on context, have distinct roles in the TGFß signaling pathway. TGFß is one of the most potent inhibitors of hematopoietic stem and progenitor cell proliferation in vitro, but its role in hematopoiesis in vivo is still being determined. To circumvent possible redundancies at the receptor level and to address specifically the role of the Smad circuitry downstream of TGFß and activin in hematopoiesis, we studied the effect of genetically deleting both Smad2 and Smad3 in adult murine hematopoietic cells. Indeed, TGFß signaling is impaired in vitro in primitive bone marrow (BM) cells of Smad2 and Smad3 single knockout models. However, blood parameters appear normal under steady state and in the transplantation setting. Interestingly, upon deletion of both Smad2 and Smad3 in vivo, mice quickly develop a lethal inflammatory disease, suggesting that activin/TGFß signaling is crucial for immune cell homeostasis in the adult context. Furthermore, concurrent deletion of Smad2 and Smad3 in BM cells in immune-deficient nude mice did not result in any significant alterations of the hematopoietic system. Our findings suggest that Smad2 and Smad3 function to mediate crucial aspects of the immunoregulatory properties of TGFß, but are dispensable for any effect that TGFß has on primitive hematopoietic cells in vivo.