Detection of minimal/measurable residual disease (MRD) is a critical prognostic marker in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The EuroFlow Consortium previously developed an 8-color flow cytometric MRD protocol, effective for >98% of BCP-ALL patients treated with chemotherapy. This study aimed to enhance MRD detection, particularly for patients treated with CD19-targeted therapies, by expanding the EuroFlow protocol to a 12-color panel. This new panel incorporates additional B-cell markers and exclusion T/NK-cell markers (CD3 and CD7). Through an evaluation of 237 diagnostic BCP-ALL samples, CD22, CD24, and HLA-DR were selected as additional B-cell gating markers. Two 12-color tubes were technically optimized and clinically validated across 101 patient follow-up samples, demonstrating excellent concordance with molecular MRD levels (R 2 = 0.88). The 12-color BCP-ALL MRD tubes were compatible with the previously developed 8-color automated gating and identification (AGI) tool and demonstrated good reproducibility. Our findings indicate that the 12-color panel performs comparably to the 8-color BCP-ALL MRD panel, including both CD19-positive and CD19-negative cases. However, it offers an improved definition of the B-cell lineage, particularly for expert-guided manual data analysis, and provides additional information on the expression of the targetable marker CD22.

• Publikační typ
• časopisecké články MeSH

• MeSH
• akutní myeloidní leukemie * farmakoterapie   MeSH
• antigeny CD28 MeSH
• antigeny CD3 imunologie   MeSH
• antigeny CD38 * antagonisté a inhibitory   metabolismus   MeSH
• humanizované monoklonální protilátky * terapeutické užití   MeSH
• lidé MeSH
• membránové glykoproteiny MeSH
• protilátky bispecifické terapeutické užití   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD28 MeSH
• antigeny CD3 MeSH
• antigeny CD38 * MeSH
• CD38 protein, human MeSH Prohlížeč
• humanizované monoklonální protilátky * MeSH
• isatuximab MeSH Prohlížeč
• membránové glykoproteiny MeSH
• protilátky bispecifické MeSH

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

• MeSH
• antigeny CD34 genetika   MeSH
• dítě MeSH
• juvenilní myelomonocytární leukemie * diagnóza   genetika   MeSH
• lidé MeSH
• monocyty patologie   MeSH
• průtoková cytometrie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD34 MeSH

Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML-not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL-other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile.

• Klíčová slova
• AMKL, Down syndrome, EuroFlow, immunophenotyping, transient abnormal myelopoiesis,
• Publikační typ
• časopisecké články MeSH

MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

• MeSH
• akutní myeloidní leukemie genetika   MeSH
• epigeneze genetická MeSH
• fúzní onkogenní proteiny genetika   MeSH
• karcinogeneze genetika   MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protoonkogenní protein MLL genetika   MeSH
• regulace genové exprese u leukemie MeSH
• RNA-polymerasa II genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fúzní onkogenní proteiny MeSH
• mikro RNA MeSH
• MIRN139 microRNA, human MeSH Prohlížeč
• MIRN139 microRNA, mouse MeSH Prohlížeč
• MLL-AF9 fusion protein, human MeSH Prohlížeč
• POLR2M protein, human MeSH Prohlížeč
• Polr2m protein, mouse MeSH Prohlížeč
• protoonkogenní protein MLL MeSH
• RNA-polymerasa II MeSH

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD10+ diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD10- diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms.

• MeSH
• difúzní velkobuněčný B-lymfom * MeSH
• dospělí MeSH
• folikulární lymfom * diagnóza   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH

• MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• mnohočetný myelom * genetika   MeSH
• mutace MeSH
• reziduální nádor MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Plasma cell leukaemia (PCL) is a rare and very aggressive plasma cell disorder. Preventing a dismal outcome of PCL requires early diagnosis with appropriate analytical tools. Therefore, the investigation of 33 patients with primary and secondary PCL was done when the quantity of circulating plasma cells (PCs) using flow cytometry (FC) and morphology assessment was evaluated. The phenotypic profile of the PCs was also analysed to determine if there is an association with clinical outcomes and to evaluate the prognostic value of analysed markers. Our results revealed that FC is an excellent method for identifying circulating PCs as a significantly higher number was identified by FC than by morphology (26·7% vs. 13·5%, P = 0·02). None of secondary PCL cases expressed CD19 or CD20. A low level of expression with similar positivity of CD27, CD28, CD81 and CD117 was found in both PCL groups. A decrease of CD44 expression was detected only in secondary PCL. Expression of CD56 was present in more than half of PCL cases as well as cytoplasmic nestin. A decreased level of platelets, Eastern Cooperative Oncology Group score of 2-3 and lack of CD20+ PC were associated with a higher risk of death. FC could be incorporated in PCL diagnostics not only to determine the number of circulating PCs, but also to assess their phenotype profile and this information should be useful in patients' diagnosis and possible prognosis.

• Klíčová slova
• flow cytometry, multiple myeloma, phenotype, plasma cell, plasma cell leukaemia, prognosis,
• MeSH
• antigeny nádorové analýza   MeSH
• buňky kostní dřeně chemie   MeSH
• časná detekce nádoru MeSH
• CD antigeny analýza   MeSH
• doba přežití bez progrese choroby MeSH
• dospělí MeSH
• falešně negativní reakce MeSH
• imunofenotypizace MeSH
• Kaplanův-Meierův odhad MeSH
• kostní dřeň patologie   MeSH
• krevní obraz * metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové cirkulující buňky * MeSH
• plazmatické buňky * chemie   ultrastruktura   MeSH
• plazmocelulární leukemie krev   mortalita   MeSH
• průtoková cytometrie metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny nádorové MeSH
• CD antigeny MeSH

Minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is the most effective tool to define a deep response in multiple myeloma (MM). We conducted an MRD correlative study of the EMN02/HO95 MM phase III trial in newly diagnosed MM patients achieving a suspected complete response before maintenance and every 6 months during maintenance. Patients received high-dose melphalan (HDM) versus bortezomib-melphalan-prednisone (VMP) intensification, followed by bortezomib-lenalidomide-dexamethasone (VRd) versus no consolidation, and lenalidomide maintenance. Bone marrow (BM) samples were processed in three European laboratories, applying EuroFlow-based MFC protocols (eight colors, two tubes) with 10-4-10-5 sensitivity. At enrollment in the MRD correlative study, 76% (244/321) of patients were MRD-negative. In the intention-to-treat analysis, after a median follow-up of 75 months, 5-year progression-free survival was 66% in MRD-negative versus 31% in MRD-positive patients (HR 0.39; p < 0.001), 5-year overall survival was 86% versus 69%, respectively (HR 0.41; p < 0.001). MRD negativity was associated with reduced risk of progression or death in all subgroups, including ISS-III (HR 0.37) and high-risk fluorescence in situ hybridization (FISH) patients (HR 0.38;). In the 1-year maintenance MRD population, 42% of MRD-positive patients at pre-maintenance became MRD-negative after lenalidomide exposure. In conclusion, MRD by MFC is a strong prognostic factor. Lenalidomide maintenance further improved MRD-negativity rate.

• MeSH
• autologní štěp MeSH
• bortezomib aplikace a dávkování   MeSH
• buňky kostní dřeně metabolismus   MeSH
• dexamethason aplikace a dávkování   MeSH
• lenalidomid aplikace a dávkování   MeSH
• lidé středního věku MeSH
• lidé MeSH
• melfalan aplikace a dávkování   MeSH
• míra přežití MeSH
• mnohočetný myelom * metabolismus   mortalita   terapie   MeSH
• přežití bez známek nemoci MeSH
• protokoly protinádorové kombinované chemoterapie aplikace a dávkování   MeSH
• průtoková cytometrie * MeSH
• reziduální nádor MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze III MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• bortezomib MeSH
• dexamethason MeSH
• lenalidomid MeSH
• melfalan MeSH

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.

• Klíčová slova
• ALL, AML, acute leukaemia, flow cytometry, immunophenotyping, myeloperoxidase,
• MeSH
• akutní nemoc MeSH
• imunofenotypizace normy   MeSH
• leukemie * diagnóza   enzymologie   imunologie   MeSH
• lidé MeSH
• nádorové proteiny * krev   imunologie   MeSH
• peroxidasa * krev   imunologie   MeSH
• průtoková cytometrie normy   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MPO protein, human MeSH Prohlížeč
• nádorové proteiny * MeSH
• peroxidasa * MeSH