The potential protective or pathogenic role of the adaptive immune response to SARS‐CoV‐2 infection has been vigorously debated. While COVID‐19 patients consistently generate a T lymphocyte response ...to SARS‐CoV‐2 antigens, evidence of significant immune dysregulation in these patients continues to accumulate. In this study, next generation sequencing of the T cell receptor beta chain (TRB) repertoire was conducted in hospitalized COVID‐19 patients to determine if immunogenetic differences of the TRB repertoire contribute to disease course severity. Clustering of highly similar TRB CDR3 amino acid sequences across COVID‐19 patients yielded 781 shared TRB sequences. The TRB sequences were then filtered for known associations with common diseases such as EBV and CMV. The remaining sequences were cross‐referenced to a publicly accessible dataset that mapped COVID‐19 specific TCRs to the SARS‐CoV‐2 genome. We identified 158 SARS‐CoV‐2 specific TRB sequences belonging to 134 clusters in our COVID‐19 patients. Next, we investigated 113 SARS‐CoV‐2 specific clusters binding only one peptide target in relation to disease course. Distinct skewing of SARS‐CoV‐2 specific TRB sequences toward the nonstructural proteins (NSPs) encoded within ORF1a/b of the SARS‐CoV‐2 genome was observed in clusters associated with critical disease course when compared to COVID‐19 clusters associated with a severe disease course. These data imply that T‐lymphocyte reactivity towards peptides from NSPs of SARS‐CoV‐2 may not constitute an effective adaptive immune response and thus may negatively affect disease severity.
Graphical
The T cell receptor beta chain (TRB) repertoire of hospitalized COVID‐19 patients revealed that TRB sequences targeting nonstructural proteins of SARS‐CoV‐2 may negatively affect disease severity.
Summary
Minimal residual disease (MRD) diagnostics are implemented in most clinical protocols for patients with acute lymphoblastic leukaemia (ALL) and are mostly performed using rearranged ...immunoglobulin (IG) and/or T‐cell receptor (TR) gene rearrangements as molecular polymerase chain reaction targets. Unfortunately, in 5–10% of patients no or no sensitive IG/TR targets are available, and patients therefore cannot be stratified appropriately. In the present study, we used fusion genes and genomic deletions as alternative MRD targets in these patients, which retrospectively revealed appropriate MDR stratification in 79% of patients with no (sensitive) IG/TR target, and a different risk group stratification in more than half of the cases.
Summary
The standardized EuroFlow protocol, including CD19 as primary B‐cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B‐cell precursor acute ...lymphoblastic leukaemia (BCP‐ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP‐ALL patients treated with CD19‐targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19‐positive, whereas this was 81% in patients that became (partially) CD19‐negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.
Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic ...stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38- fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38-LSC levels at diagnosis (n = 594), and, to identify LSC
/LSC
(n = 302) and MRD
/MRD
patients (n = 305) in bone marrow in morphological complete remission (CR). In 242 CR patients combined MRD and LSC results were available. At diagnosis the CD34
CD38
LSC frequency independently predicts overall survival (OS). After achieving CR, combining LSC and MRD showed reduced survival in MRD
/LSC
patients (hazard ratio HR 3.62 for OS and 5.89 for cumulative incidence of relapse CIR) compared to MRD
/LSC
, MRD
/LSC
, and especially MRD
/LSC
patients. Moreover, in the NPM1mutant positive sub-group, prognostic value of golden standard NPM1-MRD by qPCR can be improved by addition of flow cytometric approaches. This is the first prospective study demonstrating that LSC strongly improves prognostic impact of MRD detection, identifying a patient subgroup with an almost 100% treatment failure probability, warranting consideration of LSC measurement incorporation in future AML risk schemes.
Summary
Given its myeloid‐restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a ...robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte‐based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.
The World Health Organization (WHO) classification contributes to refined classification and prognostication of myelodysplastic syndromes (MDSs). Flow cytometry might add significantly to diagnostic ...and prognostic criteria. Our analysis of bone marrow samples from 50 patients with MDS showed aberrant expression of differentiation antigens in the myelomonocytic lineage. This also accounted for refractory anemia (RA) with or without ringed sideroblasts (RS), indicating multilineage dysplasia. In 38% of patients, CD34+ myeloid blasts expressed CD5, CD7, or CD56. Flow cytometry data were translated into a numerical MDS flow-score. Flow-scores increased significantly from RA with or without RS, refractory cytopenia with multilineage dysplasia (RCMD) with or without RS up to refractory anemia with excess of blasts-1 (RAEB-1) and RAEB-2. No significant differences were observed between WHO cytogenetic subgroups. Flow-scores were highly heterogeneous within International Prognostic Scoring System (IPSS) subgroups. Patients in progression to advanced MDS or acute myeloid leukemia had a significantly higher flow-score compared with non–transfusion-dependent patients. In 60% of patients with transfusion dependency or progressive disease, myeloid blasts expressed CD7 or CD56, in contrast to only 9% of non–transfusion-dependent patients. Moreover, all patients with pure RA with or without RS with aberrant myeloid blasts showed an adverse clinical course. In conclusion, flow cytometry in MDS identified aberrancies in the myelomonocytic lineage not otherwise determined by cytomorphology. In addition, flow cytometry identified patients at risk for transfusion dependency and/or progressive disease independent of known risk groups, which might have impact on treatment decisions and the prognostic scoring system in the near future.
Background
Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress ...has been made in the FCM field concerning technical issues (including software and hardware) and pre‐analytical procedures.
Methods
Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group.
Results
We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies.
Conclusions
These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.
This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes ...(MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow‐up of MDS patients.
In acute myeloid leukemia (AML), aberrant expression and mutations of transcription factors have been correlated with disease outcome. In the present study, we performed expression and mutation ...screening of GATA2, which is an essential transcription factor for regulation of myeloid lineage determination, in de novo pediatric AML patients. GATA2 mutations were detected in 5 of 230 patients, representing a frequency of 2.2% overall and 9.8% in cytogenetically normal AML. GATA2 expression analysis demonstrated that in 155 of 237 diagnostic samples (65%), GATA2 expression was higher than in normal BM. In complete remission, normalization of GATA2 expression was observed, whereas GATA2 expression levels stayed high in patients with resistant disease. High GATA2 expression at diagnosis was an independent poor prognostic factor for overall survival (hazard ratio HR = 1.7, P = .045), event-free survival (HR = 2.1, P = .002), and disease-free survival (HR = 2.3, P = .004). The prognostic impact of GATA2 was particularly evident in specific AML subgroups. In patients with French-American-British M5 morphology, inv(16), or high WT1 expression, significant differences in survival were observed between patients with high versus normal GATA2 expression. We conclude that high GATA2 expression is a novel poor prognostic marker in pediatric AML, which may contribute to better risk-group stratification and risk-adapted therapy in the future.
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