BACKGROUND: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. METHODS: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. RESULTS: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. CONCLUSIONS: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.

• Klíčová slova
• Acute lymphoblastic leukemia, CD304, CD73, CD86, Flow cytometry, Minimal residual disease,
• MeSH
• 5'-nukleotidasa analýza   biosyntéza   MeSH
• antigeny CD86 analýza   biosyntéza   MeSH
• dítě MeSH
• GPI-vázané proteiny analýza   biosyntéza   MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• neuropilin-1 analýza   biosyntéza   MeSH
• pre-B-buněčná leukemie patologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky patologie   MeSH
• reziduální nádor MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5'-nukleotidasa MeSH
• antigeny CD86 MeSH
• CD86 protein, human MeSH Prohlížeč
• GPI-vázané proteiny MeSH
• nádorové biomarkery MeSH
• neuropilin-1 MeSH
• NRP1 protein, human MeSH Prohlížeč
• NT5E protein, human MeSH Prohlížeč

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34high pre-B-I cell immunophenotype. These CD34-dim pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34-dim pre-B-I cells. Our results indicate that newly identified CD34-dim pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34-dim pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements.

• Klíčová slova
• B cells, acute leukaemia, flow cytometry, minimal residual disease,
• MeSH
• antigeny CD34 metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• genová přestavba B-lymfocytů imunologie   MeSH
• imunofenotypizace metody   MeSH
• kostní dřeň fyziologie   MeSH
• lidé MeSH
• mladiství MeSH
• pre-B-buněčná leukemie diagnóza   imunologie   patofyziologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky imunologie   patologie   fyziologie   MeSH
• průtoková cytometrie MeSH
• regenerace MeSH
• reziduální nádor MeSH
• těžké řetězce imunoglobulinů imunologie   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD34 MeSH
• těžké řetězce imunoglobulinů MeSH

• MeSH
• Asijci MeSH
• běloši MeSH
• buněčná diferenciace MeSH
• buňky kostní dřeně imunologie   patologie   MeSH
• chronická lymfatická leukemie etnologie   imunologie   patologie   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• počet lymfocytů MeSH
• prekurzorové B-lymfoidní buňky imunologie   patologie   MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

Minimal residual disease (MRD) monitoring is an essential tool for risk group stratification in current treatment protocols for childhood acute lymphoblastic leukaemia (ALL). Although quantitative detection of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is currently considered to be the standard method, leukaemia fusion genes provide other possible targets for MRD follow-up, as already demonstrated in TEL/AML1-positive ALLs. We analysed and compared MRD levels quantified by BCR/ABL transcript detection and by the standard Ig/TCR-based method in 218 bone marrow specimens from 17 children with BCR/ABL-positive ALL. We found only a limited overall correlation of MRD levels as assessed by the two methods (correlation coefficient R(2)=0.64). The correlation varied among patients from excellent (R(2)=0.99) to very poor (R(2)=0.17). Despite identical sensitivity of the approaches, 20% of the samples were negative by the Ig/TCR approach whereas positive by the BCR/ABL method. We show that multilineage involvement is at least partly responsible for the discrepancy. Moreover, our data demonstrate that BCR/ABL monitoring enables better and earlier prediction of relapse compared to the standard Ig/TCR methodology. We conclude that BCR/ABL-based MRD monitoring of childhood ALL is a clinically relevant tool and should be performed in parallel with the standard Ig/TCR follow-up.

• MeSH
• akutní lymfatická leukemie klasifikace   genetika   MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• dítě MeSH
• genová přestavba T-lymfocytů genetika   MeSH
• geny pro imunoglobuliny genetika   MeSH
• indukce remise MeSH
• kultivované buňky MeSH
• lidé MeSH
• lokální recidiva nádoru genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mladiství MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky metabolismus   patologie   MeSH
• prognóza MeSH
• retrospektivní studie MeSH
• reziduální nádor genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• messenger RNA MeSH

The expression of CD27 and CD44 correlate with the genotype of B-precursor acute lymphoblastic leukemia (ALL). Based on the expression of these antigens, we identified counterparts of TEL/AML1(pos) and TEL/AML1(neg) leukemic cells in nonmalignant bone marrow. Although CD27 is known as a marker of mature memory B cells, we recently showed that CD27 is also expressed by malignant and nonmalignant B precursors. Here, we show that CD27 and CD44 delineate stages of B-precursor development. Well-established differentiation markers showed that the developmental sequence starts from undetermined progenitors, expressing CD44. Upon B-lineage commitment, cells gain CD27 and lose CD44. The CD27(pos)CD44(neg) (CD27 single positive, 27SP) cells are the earliest stage within CD10(pos)CD19(pos) B precursors and express RAG-1 and TDT. These cells correspond to TEL/AML1(pos) ALL (1/4 pediatric B-precursor ALL). The development follows to CD27/CD44 double-positive (27/44DP) stage, 44SP stage and CD27/CD44 double-negative (27/44DN) stage. Before exit to periphery, CD44 is reexpressed. The 27/44DP cells are mostly large and profoundly suppress RAG-1. Despite their presumably high proliferation potential, 27/44DP cells rarely dominate in leukemia. At 44SP stage, which corresponds to TEL/AML1(neg) leukemias, RAG-1 is reexpressed and Ig light chain gene starts to be rearranged.

• MeSH
• antigeny CD27 biosyntéza   genetika   fyziologie   MeSH
• antigeny CD44 biosyntéza   genetika   fyziologie   MeSH
• dítě MeSH
• genová přestavba B-lymfocytů imunologie   MeSH
• imunofenotypizace MeSH
• leukemie B-buněčná diagnóza   genetika   imunologie   MeSH
• lidé MeSH
• lymfopoéza genetika   imunologie   MeSH
• prekurzorové B-lymfoidní buňky cytologie   imunologie   patologie   MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny CD27 MeSH
• antigeny CD44 MeSH