We propose to investigate minimal residual disease in all children with acute lymphoblastic and myelogenous leukemia in a referential laboratory, together with diagnostics at the onset of the disease. We will assess the practical value of each subpopulation for MRD study using polychromatic flow cytometry and cell sorting followed by molecular genetic analysis. We will study regulation of and resulting interaction through aberrantly expressed molecules correlating with genotype of leukemia.

Navrhujeme vyšetřovat v referenčních laboratořích minimální reziduální nemoc dětí s akutní lymfoblastickou i myeloidní leukémií, spolu s diagnostikou při záchytu onemocnění. Výpovědní hodnotu pro MRN u jednotlivých subpopulací ověříme pomocí mnohobarevnécytometrie a třídění buněk s následnou molekulárně genetickou analýzou. Aberantní expresi molekul korelujících s genotypem budeme studovat po stránce regulace i dopadu na interakci buňky s okolím.

• MeSH
• dítě MeSH
• imunofenotypizace MeSH
• lymfoidní leukemie diagnóza   MeSH
• myeloidní leukemie diagnóza   MeSH
• průtoková cytometrie MeSH
• reziduální nádor MeSH
• Check Tag
• dítě MeSH

• Konspekt
• Pediatrie
• NLK Obory
• pediatrie
• onkologie
• hematologie a transfuzní lékařství
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Minimal residual disease (MRD) detection using quantification of clone-specific Ig or TCR rearrangements before and after transplantation in children with high-risk ALL is an important predictor of outcome. The method and guidelines for its interpretation are very precise to avoid both false-negative and -positive results. In a group of 21 patients following transplantation, we observed detectable MRD positivities in Ig/TCR-based real-time quantitative PCR (RQ-PCR) leading to no further progression of the disease (11 of 100 (11%) total samples). We hypothesized that these positivities were mostly the result of nonspecific amplification despite the application of strict internationally agreed-upon measures. We applied two non-self-specific Ig heavy chain assays and received a similar number of positivities (20 and 15%). Nonspecific products amplified in these RQ-PCR systems differed from specific products in length and sequence. Statistical analysis proved that there was an excellent correlation of this phenomenon with B-cell regeneration in BM as measured by flow cytometry and Ig light chain-kappa excision circle quantification. We conclude that although Ig/TCR quantification is a reliable method for post transplant MRD detection, isolated positivities in Ig-based RQ-PCR systems at the time of intense B-cell regeneration must be viewed with caution to avoid the wrong indication of treatment.

• MeSH
• B-lymfocyty imunologie   MeSH
• Burkittův lymfom MeSH
• DNA nádorová genetika   MeSH
• financování organizované MeSH
• genová přestavba MeSH
• homologní transplantace imunologie   MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• polymerázová řetězová reakce MeSH
• předškolní dítě MeSH
• reziduální nádor diagnóza   genetika   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transfuze lymfocytů MeSH
• transplantace kmenových buněk MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě 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   fyziologie   genetika   MeSH
• antigeny CD44 biosyntéza   fyziologie   genetika   MeSH
• dítě MeSH
• financování organizované 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
• srovnávací studie MeSH

The ALL IC-BFM 2002 protocol was created as an alternative to the MRD-based AIEOP-BFM ALL 2000 study, to integrate early response criteria into risk-group stratification in countries not performing routine PCR-based MRD testing. ALL IC stratification comprises the response to prednisone, bone marrow (BM) morphology at days 15 and 33, age, WBC and BCR/ABL or MLL/AF4 presence. Here, we compared this stratification to the MRD-based criteria using MRD evaluation in 163 patients from four ALL IC member countries at days 8, 15 and 33 and week 12. MRD negativity at day 33 was associated with an age of 1-5 years, WBC<20,000 microl(-1), non-T immunophenotype, good prednisone response and non-M3 morphology at day 15. There were no significant associations with gender or hyperdiploidy in the study group, or with TEL/AML1 fusion within BCP-ALL. Patients with M1/2 BM at day 8 tended to be MRD negative at week 12. Patients stratified into the standard-risk group had a better response than intermediate-risk group patients. However, 34% of them were MRD positive at day 33 and/or week 12. Our findings revealed that morphology-based ALL IC risk-group stratification allows the identification of most MRD high-risk patients, but fails to discriminate the MRD low-risk group assigned to therapy reduction.

• MeSH
• akutní lymfatická leukemie diagnóza   terapie   MeSH
• dítě MeSH
• financování organizované MeSH
• hodnocení rizik MeSH
• imunofenotypizace MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• počet leukocytů MeSH
• předškolní dítě MeSH
• prognóza MeSH
• reziduální nádor diagnóza   MeSH
• tvar buňky MeSH
• věkové faktory MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• předškolní dítě MeSH
• Publikační typ
• klinické zkoušky MeSH
• multicentrická studie MeSH

PURPOSE: Monitoring of residual disease (RD) by flow cytometry in childhood acute myeloid leukemia (AML) may predict outcome. However, the optimal time points for investigation, the best antibody combinations, and most importantly, the clinical impact of RD analysis remain unclear. PATIENTS AND METHODS: Five hundred forty-two specimens of 150 children enrolled in the AML-Berlin-Frankfurt-Muenster (BFM) 98 study were analyzed by four-color immunophenotyping at up to four predefined time points during treatment. For each of the 12 leukemia-associated immunophenotypes and time points, a threshold level based on a previous retrospective analysis of another cohort of children with AML and on control bone marrows was determined. RESULTS: Regarding all four time points, there is a statistically significant difference in the 3-year event-free survival (EFS) in those children presenting with immunologically detectable blasts at 3 or more time points. The levels at bone marrow puncture (BMP) 1 and BMP2 turned out to have the most significant predictive value for 3-year-EFS: 71% +/- 6% versus 48% +/- 9%, P(Log-Rank) = .029 and 70% +/- 6% versus 50% +/- 7%, P(Log-Rank) = .033), resulting in a more than two-fold risk of relapse. In a multivariate analysis, using a combined risk classification based on morphologically determined blasts at BMP1 and BMP2, French-American-British classification, and cytogenetics, the influence of immunologically determined RD was no longer statistically significant. CONCLUSION: RD monitoring before second induction has the same predictive value as examining levels at four different time points during intensive chemotherapy. Compared with commonly defined risk factors in the AML-BFM studies, flow cytometry does not provide additional information for outcome prediction, but may be helpful to evaluate the remission status at day 28.

• MeSH
• akutní myeloidní leukemie * diagnóza   farmakoterapie   MeSH
• analýza přežití MeSH
• analýza rozptylu MeSH
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• mladiství MeSH
• prediktivní hodnota testů MeSH
• předškolní dítě MeSH
• přežití bez známek nemoci MeSH
• prognóza MeSH
• proporcionální rizikové modely MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   MeSH
• průtoková cytometrie * MeSH
• retrospektivní studie MeSH
• reziduální nádor * diagnóza   MeSH
• rizikové faktory MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• multicentrická studie MeSH
• práce podpořená grantem MeSH

• MeSH
• akutní lymfatická leukemie diagnóza   terapie   MeSH
• dítě MeSH
• finanční podpora výzkumu jako téma MeSH
• lidé MeSH
• mezinárodní spolupráce MeSH
• průtoková cytometrie využití   MeSH
• reziduální nádor diagnóza   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• kongresy MeSH

• MeSH
• akutní lymfatická leukemie diagnóza   genetika   MeSH
• bcr-abl fúzní proteiny diagnostické užití   MeSH
• dítě MeSH
• finanční podpora výzkumu jako téma MeSH
• lidé MeSH
• molekulární biologie metody   MeSH
• průtoková cytometrie využití   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• kongresy MeSH

• MeSH
• akutní lymfatická leukemie diagnóza   MeSH
• dítě MeSH
• finanční podpora výzkumu jako téma MeSH
• lidé MeSH
• polymerázová řetězová reakce metody   využití   MeSH
• průtoková cytometrie využití   MeSH
• reziduální nádor diagnóza   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• kongresy MeSH

TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. TCRD (TCR delta) genes are frequently incompletely rearranged in B precursor leukemias and recently were found in a significant portion of physiological B lymphocytes. Incomplete TCRD rearrangements (V-D) thus serve as natural indicators of previous V(D)J recombinase activity. Functional V(D)J recombinase has recently been found in murine NK precursors. We tested whether physiological NK cells and other leukocyte subpopulations contained TCR rearrangements in humans. This would provide evidence that V(D)J recombinase was active in the ancestry cells and suggest common pathways among the positive cell types. TCRD were rearranged in 3.2-36% of NK cells but not in nonlymphoid leukocytes. The previously known phenomenon of TCRD transcription in NK cells is a possible mechanism that maintains the chromatin open at the TCRD locus. In comparison, TCRG rearrangements were frequent in T cells, low to negative in B and NK cells, and negative in nonlymphoid cells, suggesting a tighter control of TCRG. Levels of TCRD rearrangements were similar among the B lymphocyte subsets (B1-B2, naive-memory). In conclusion, human NK cells pass through a differentiation step with active V(D)J recombinase similar to T and B lymphocytes and unlike nonlymphoid leukocytes. This contradicts recent challenges to the concept of separate lymphoid and myeloid differentiation.

• MeSH
• buněčná diferenciace genetika   imunologie   MeSH
• buňky HT-29 MeSH
• buňky NK MeSH
• finanční podpora výzkumu jako téma MeSH
• genetické markery MeSH
• genová přestavba - delta řetězec receptoru antigenů T-buněk fyziologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• podskupiny B-lymfocytů MeSH
• podskupiny lymfocytů cytologie   imunologie   metabolismus   MeSH
• VDJ-rekombinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH