An immunotherapy effect analysis in Rasmussen encephalitis.

BACKGROUND: Immune-mediated mechanisms substantially contribute to the Rasmussen encephalitis (RE) pathology, but for unknown reasons, immunotherapy is generally ineffective in patients who have already developed intractable epilepsy; overall laboratory data regarding the effect of immunotherapy on patients with RE are limited. We analyzed multiple samples from seven differently treated children with RE and evaluated the effects of immunotherapies on neuroinflammation. Immunotherapy was introduced to all patients at the time of intractable epilepsy and they all had to undergo hemispherothomy. METHODS: Immunohistochemistry, flow cytometry, Luminex multiplex bead and enzyme-linked immunosorbent assay techniques were combined to determine: 1) inflammatory changes and lymphocyte subpopulations in 45 brain tissues; 2) lymphocyte subpopulations and the levels of 12 chemokines/cytokines in 24 cerebrospinal fluid (CSF) samples and 30 blood samples; and 3) the dynamics of these parameters in four RE patients from whom multiple samples were collected. RESULTS: Sustained T cell-targeted therapy with cyclophosphamide, natalizumab, alemtuzumab, and intrathecal methotrexate (ITMTX), but not with azathioprine, substantially reduced inflammation in brain tissues. Despite the therapy, the distributions of CD8+ T cells and the levels of C-X-C motif ligand (CXCL) 10, CXCL13, and B cell activating factor (BAFF) in patients' CSF remained increased compared to controls. A therapeutic approach combining alemtuzumab and ITMTX was the most effective in producing simultaneous reductions in histopathological inflammatory findings and in the numbers of activated CD8+ T cells in the brain tissue, as well as in the overall CD8+ T cell population and chemokine/cytokine production in the CSF. CONCLUSIONS: We provide evidence that various T cell-targeted immunotherapies reduced inflammation in the brains of RE patients. The observation that intractable epilepsy persisted in all of the patients suggests a relative independence of seizure activity on the presence of T cells in the brain later in the disease course. Thus, new therapeutic targets must be identified. CXCL10, CXCL13 and BAFF levels were substantially increased in CSF from all patients and their significance in RE pathology remains to be addressed.

Cytometric analysis of cell suspension generated by cavitron ultrasonic surgical aspirator in pediatric brain tumors.

PURPOSE: The aim of this study was to test the possibility of using specimens obtained by a cavitron ultrasonic surgical aspirator (CUSA) in flow and mass cytometry investigations of pediatric brain tumors. METHODS: CUSA specimens obtained from 19 pediatric patients with brain tumors were investigated. Flow and mass cytometry methods were applied to analyze the composition of material collected using the CUSA. Cell suspensions were prepared from CUSA aspirates. Then sample viability was assessed by conventional flow cytometry and subsequently stained with a panel of 31 metal-labeled antibodies. RESULTS: Viability assessment was performed using conventional flow cytometry. Viability of cells in the acquired samples was below 50% in 16 of 19 cases. A mass cytometry investigation and subsequent analysis enabled us to discriminate brain tumor cells from contaminating leukocytes, whose proportions varied across the specimens. The addition of the viability marker cisplatin directly into the mass cytometry panel gave the means to selecting viable cells only for subsequent analyses. The proportion of non-viable cells was higher among tumor cells compared leukocytes. CONCLUSIONS: When the analysis of the tumor cell immunophenotype is performed with markers for determining viability, the expression of the investigated markers can be evaluated. Suitable markers can be selected by high-throughput methods, such as mass cytometry, and those that are diagnostically relevant can be investigated using flow cytometry, which is more flexible in terms of time.

Genomic landscape of pediatric B-other acute lymphoblastic leukemia in a consecutive European cohort.

Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia (ALL) during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations still have to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of DUX4-rearranged, BCR-ABL1-like, ZNF384-rearranged, ETV6-RUNX1-like, iAMP21 and MEF2D-rearranged subtypes was 27%, 15%, 5%, 5%, 4%, and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in DUX4-rearranged leukemia and a strong association of ZNF384-rearranged leukemia with B-myeloid immunophenotype. Of the druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in one patient. A recently described negative prognostic factor, IKZF1plus , was found in 14% of patients and was enriched in (but not exclusive for) BCR-ABL1-like subtype. PAX5 fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and only occurred in the B-rest subset, altogether accounting for 20% of the B-other group. PAX5 P80R was associated with a specific gene expression signature, potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations. This study also strengthens and widens the current knowledge of B-other ALL and provides an objective basis for optimization of current genetic diagnostics.

Potential loss of prognostic significance of minimal residual disease assessment after R-CHOP-based induction in elderly patients with mantle cell lymphoma in the era of rituximab maintenance.

Rituximab maintenance (RM) prolongs survival of elderly patients with mantle cell lymphoma (MCL). Persistent minimal residual disease (MRD) after induction repeatedly correlated with shorter progression-free survival (PFS). However, none of the published studies analyzed patients treated with RM. The main purpose was to analyze prognostic significance of MRD in the elderly patients with newly diagnosed MCL treated according to the recently published observational trial protocol (alternation of R-CHOP and R-cytarabine, 3 + 3 cycles, GovTrial number NCT03054883) at the centers that implemented RM. Minimal residual disease was evaluated by a EuroMRD standardized real-time PCR approach after 3 and 6 cycles of the induction therapy. Prognostic significance of MRD was analyzed in a subcohort of patients treated at the centers that implemented RM as a standard approach. Bone marrow proved to be a significantly more sensitive source for MRD detection than peripheral blood. In either compartment MRD (positive versus negative) after 3 or 6 cycles of the induction therapy did not correlate with PFS. The observed loss of prognostic significance of MRD after the R-CHOP-based induction appears to be a consequence of RM immune control over the residual lymphoma.

Alternating R-CHOP and R-cytarabine is a safe and effective regimen for transplant-ineligible patients with a newly diagnosed mantle cell lymphoma.

Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first-line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R-CHOP and R-cytarabine for newly diagnosed transplant-ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high-risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography-computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression-free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography-negativity independently correlated with progression-free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R-CHOP and R-cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial (clinicaltrials.gov) NCT03054883.

ETV6/RUNX1-like acute lymphoblastic leukemia: A novel B-cell precursor leukemia subtype associated with the CD27/CD44 immunophenotype.

We have shown previously that ETV6/RUNX1-positive acute lymphoblastic leukemia (ALL) is distinguishable from other ALL subtypes by CD27pos /CD44low-neg immunophenotype. During diagnostic immunophenotyping of 573 childhood B-cell precursor ALL (BCP-ALL), we identified eight cases with this immunophenotype among "B-other ALL" (BCP-ALL cases negative for routinely tested chromosomal/genetic aberrations). We aimed to elucidate whether these cases belong to the recently described ETV6/RUNX1-like ALL defined by the ETV6/RUNX1-specific gene expression profile (GEP), harboring concurrent ETV6 and IKZF1 lesions. We performed comprehensive genomic analysis using single nucleotide polymorphism arrays, whole exome and transcriptome sequencing and GEP on microarrays. In unsupervised hierarchical clustering based on GEP, five out of seven analyzed CD27pos /CD44low-neg B-other cases clustered with ETV6/RUNX1-positive ALL and were thus classified as ETV6/RUNX1-like ALL. The two cases clustering outside ETV6/RUNX1-positive ALL harbored a P2RY8/CRLF2 fusion with activating JAK2 mutations and a TCF3/ZNF384 fusion, respectively, assigning them to other ALL subtypes. All five ETV6/RUNX1-like cases harbored ETV6 deletions; uniform intragenic ARPP21 deletions and various IKZF1 lesions were each found in three ETV6/RUNX1-like cases. The frequency of ETV6 and ARPP21 deletions was significantly higher in ETV6/RUNX1-like ALL compared with a reference cohort of 42 B-other ALL. In conclusion, we show that ETV6/RUNX1-like ALL is associated with CD27pos /CD44low-neg immunophenotype and identify ARPP21 deletions to contribute to its specific genomic profile enriched for ETV6 and IKZF1 lesions. In conjunction with previously published data, our study identifies the ETV6 lesion as the only common genetic aberration and thus the most likely key driver of ETV6/RUNX1-like ALL.

Monitoring of childhood ALL using BCR-ABL1 genomic breakpoints identifies a subgroup with CML-like biology.

We used the genomic breakpoint between BCR and ABL1 genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of IKZF1 deletion, we observed very good correlation for the methods in a majority of patients; however, >20% of children (25% [8/32] with minor and 12.5% [1/8] with major-BCR-ABL1 variants in the consecutive cohorts) had significantly (>1 log) higher levels of BCR-ABL1 fusion than Ig/TCR rearrangements and/or IKZF1 deletion. We performed cell sorting of the diagnostic material and assessed the frequency of BCR-ABL1-positive cells in various hematopoietic subpopulations; 12% to 83% of non-ALL B lymphocytes, T cells, and/or myeloid cells harbored the BCR-ABL1 fusion in patients with discrepant MRD results. The multilineage involvement of the BCR-ABL1-positive clone demonstrates that in some patients diagnosed with BCR-ABL1-positive ALL, a multipotent hematopoietic progenitor is affected by the BCR-ABL1 fusion. These patients have BCR-ABL1-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)-like disease manifesting in "lymphoid blast crisis." The biological heterogeneity of BCR-ABL1-positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like BCR-ABL1-positive ALL.

Lymphocyte enrichment using CD81-targeted immunoaffinity matrix.

In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.

Slower early response to treatment and distinct expression profile of childhood high hyperdiploid acute lymphoblastic leukaemia with DNA index < 1.16.

Acute lymphoblastic leukaemias (ALL) with 51-67 chromosomes are defined as high hyperdiploid (HHD) and are generally associated with good prognosis. However, several studies show heterogeneity in HHD ALL and suggest that the favourable prognosis is associated rather with higher ploidy defined by DNA index (DNAi) ≥ 1.16 or with a presence of specific single or combined trisomies. HHD ALL with DNAi < 1.16 are only rarely studied separately. Using single nucleotide polymorphism array, we analysed 89 childhood HHD ALL patients divided into groups with lower (<1.16; n = 34) and higher (≥1.16; n = 55) DNAi. We assessed treatment response, presence of secondary aberrations, mutations in RAS pathway genes and CREBBP and also gene expression profile (GEP) to reveal differences between the two subgroups. Cases with 51-54 chromosomes had DNAi 1.1-1.16 and cases with 55-67 chromosomes had DNAi ≥ 1.16. The groups with lower and higher DNAi had distinct response to early treatment and distinct GEP. The better response of the group with higher DNAi was associated with specific trisomies (trisomy of chromosome 10 or combined with trisomies 4 and/or 17). Our results suggest that cytogenetically defined HHD ALL can in fact be divided into two biologically distinguishable subgroups and that DNAi 1.16 is a relevant value to separate between the two. © 2016 Wiley Periodicals, Inc.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells.

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients.To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p< 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation.In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study.

Cytokines, growth, and environment factors in bone marrow plasma of acute lymphoblastic leukemia pediatric patients.

Acute lymphoblastic leukemia (ALL) cells depend on the microenvironment of the host in vivo and do not survive in in vitro culture. Conversely, the suppression of non-malignant tissues is one of the leading characteristics of the course of ALL. Both the non-malignant suppression and malignant cell survival may be partly affected by soluble factors within the bone marrow (BM) environment. Here, we aimed to identify proteins in BM plasma of children with ALL that may contribute to ALL aggressiveness and/or the microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the time of ALL diagnosis was compared to control plasma of bone marrow (CBMp) or peripheral blood (CPBp) using a cytokine antibody array. The cytokine antibody array enabled simultaneous detection of 79 proteins per sample. Candidate proteins exhibiting significantly different profiles were further analyzed and confirmed by ELISA. mRNA expression of one of the candidate proteins (TIMP1) was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The cytokine antibody array experiments identified 23 proteins that differed significantly (p<0.05); of these, two proteins (TIMP1 and LIF) withstood the Bonferroni correction. In contrast, little difference was observed between CBMp and CPBp. At the diagnosis of ALL, changes in the soluble microenvironment are detectable in BM plasma. These changes probably participate in the pathogenesis and/or result from the changes in the cell composition.

High expression of cytoskeletal protein drebrin in TEL/AML1pos B-cell precursor acute lymphoblastic leukemia identified by a novel monoclonal antibody.

The expression of drebrin, a cytoskeletal protein newly estimated by expression profiling to correlate with the genotype and prognosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), was examined by independent methods. After demonstrating its higher expression in TEL/AML1(pos) BCP-ALL by quantitative reverse transcriptase polymerase chain reaction, we developed an anti-drebrin monoclonal antibody (mAb). In a cohort of 86 children with BCP-ALL, we found increased expression of drebrin in TEL/AML1(pos) ALL. In conclusion, relationship of drebrin expression and prognosis or genotype can now be assessed using flow cytometry.

TEL/AML1-positive patients lacking TEL exon 5 resemble canonical TEL/AML1 cases.

BACKGROUND: The TEL/AML1 fusion gene which represents the most frequent genetic abnormality in childhood ALL, usually results from genomic breakpoints in TEL intron 5 and AML1 intron 1 or 2. At the protein level, the helix-loop-helix domain and exon 5-coded central region of TEL are typically fused to almost entire AML1 including DNA-binding domain. PROCEDURE: We identified two ALL patients with genomic breakpoints within TEL intron 4 resulting in variant TEL/AML1 fusion lacking the TEL exon 5-coded central region. This region was supposed to play an important role in TEL/AML1 function, particularly in TEL/AML1-mediated transcriptional repression of AML1 targets. We aimed at investigating the impact of the loss of this region on disease behavior and TEL/AML1 function. We compared clinical and biological characteristics, treatment response, and outcome of the variant versus classical TEL/AML1 cases, analyzed genome wide gene expression profiles and performed reporter gene assay. RESULTS: No distinct differences between variant and classical TEL/AML1 cases were observed including gene expression profiling and detailed immunophenotyping. By using reporter gene assay, we showed that the loss of the central region does not influence the TEL/AML1-mediated transcriptional repression. CONCLUSIONS: The deletion of the central region did not affect the TEL/AML1-specific phenotype; we did not find any relevant differences in clinical and biological features when variant versus classical TEL/AML1-positive cases were compared. Thus, our data does not support hypothesis that the central region of TEL is indispensable for TEL/AML1 driven leukemogenesis.

Detection of residual B precursor lymphoblastic leukemia by uniform gating flow cytometry.

BACKGROUND: Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)-based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients. PROCEDURE: We focused at the design of standardized and reproducible RD monitoring in ALL. RD was investigated by a uniform gating strategy, which was designed internationally and tested in one center by Ig/TCR rearrangements. RESULTS: For each gate, positivity cutoff value was assigned using quantification of non-leukemic background. Comparing to Ig/TCR at 0.1% level, 80 of 103 specimens were correctly diagnosed by FC. The predictive value of FC RD at day 15 was then analyzed. In B lineage ALL, day 15 FC significantly correlated with Ig/TCR results at day 33 and/or week 12 (P < 0.01). No significant correlation was found in T lineage ALL. CONCLUSIONS: Thus, FC with preset uniform gating at day 15 predicts PCR-detectable MRD in B precursor ALL. Presented data may be used to define new polychromatic cytometric diagnostics of MRD including semiautomatic assessment. Pediatr Blood Cancer 2010; 54:62-70. (c) 2009 Wiley-Liss, Inc.

Adaptor molecules expression in normal lymphopoiesis and in childhood leukemia.

Transmembrane adaptor proteins are key mediators of antigen receptor signaling in lymphocytes. By influencing proliferation and differentiation, these molecules might play a role in ethiopathogenesis of acute lymphoblastic leukemia (ALL). The aim of this study was to characterize expression of PAG, LAT, NTAL and LIME adaptors at the mRNA and protein levels in normal B- and T-precursors. Moreover, diagnostic samples of childhood ALL cases were analyzed. During normal lymphocyte development, some adaptors show significant dynamics (gradual decrease of NTAL and increase of LAT and LIME during the T-cell maturation, decrease of PAG in B-precursors, high levels of LIME in peripheral B-lymphocytes). Analysis of childhood ALL samples revealed that in B-cell precursor ALL, the TEL/AML1 subgroup have unique adaptor profile compared to other leukemias. Moreover, NTAL expression separates T lineage leukemias into two subgroups with good and poor response to initial prednisone therapy showing prognostic impact of this molecule in T-ALL.

Structure-function analysis of delta trafficking, receptor binding and signaling in Drosophila.

The transmembrane proteins Delta and Notch act as ligand and receptor in a conserved signaling pathway required for a variety of cell fate specification events in many organisms. Binding of Delta to Notch results in a proteolytic cascade that releases the Notch intracellular domain, allowing it to participate in transcriptional activation in the nucleus. Recent research has implicated the endocytic and ubiquitylation machinery as essential components of Delta-Notch signaling. Our analysis of chimeric and missense Delta variants has delineated a number of structural requirements for Delta trafficking, receptor binding, and signaling. We find that while the Delta N-terminal domain is necessary and sufficient for binding to Notch, the integrity of the epidermal-growth-factor-like repeat (ELR) 2 is also required for Notch binding. Screening of 117 Delta mutant lines for proteins that exhibit aberrant subcellular trafficking has led to the identification of 18 Delta alleles (DlTD alleles) that encode "trafficking-defective" Delta proteins. We find, unexpectedly, that many DlTD alleles contain missense mutations in ELRs within the Delta extracellular domain. Finally, we find that two DlTD alleles contain lysine missense mutations within the Delta intracellular domain (DeltaICD) that may identify residues important for DeltaICD mono-ubiquitylation and subsequent Delta endocytosis and signaling.

TEL/AML1 and immunoreceptor gene rearrangements-which comes first?

TEL/AML1 fusion gene is present in 20-25% of childhood acute lymphoblastic leukaemias. In order to unravel at which stage of B-cell precursor development the fusion is originated, we analysed frequency and pattern of immunoreceptor (immunoglobulin and T-cell receptor) gene rearrangements in 47 TEL/AML1-positive and 43 TEL/AML1-negative cases of the same CD10+ immunophenotype. Moreover, we compared corresponding immunoreceptor gene rearrangements in 11 cases of TEL/AML1-positive leukaemia at diagnosis and relapse. More mature immunogenotype of TEL/AML1-positive cases and changes in 37% of rearrangements between diagnosis and relapse suggest that in most cases the TEL/AML1 fusion is formed during immunoreceptor gene rearrangement process.

Myeloid antigens in childhood lymphoblastic leukemia: clinical data point to regulation of CD66c distinct from other myeloid antigens.

BACKGROUND: Aberrant expression of myeloid antigens (MyAgs) on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon, although its regulating mechanisms are unclear. MyAgs in ALL are interpreted e.g. as hallmarks of early differentiation stage and/or lineage indecisiveness. Granulocytic marker CD66c -- Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is aberrantly expressed on ALL with strong correlation to genotype (negative in TEL/AML1 and MLL/AF4, positive in BCR/ABL and hyperdiploid cases). METHODS: In a cohort of 365 consecutively diagnosed Czech B-precursor ALL patients, we analyze distribution of MyAg+ cases and mutual relationship among CD13, CD15, CD33, CD65 and CD66c. The most frequent MyAg (CD66c) is studied further regarding its stability from diagnosis to relapse, prognostic significance and regulation of surface expression. For the latter, flow cytometry, Western blot and quantitative RT-PCR on sorted cells is used. RESULTS: We show CD66c is expressed in 43% patients, which is more frequent than other MyAgs studied. In addition, CD66c expression negatively correlates with CD13 (p < 0.0001), CD33 (p = 0.002) and/or CD65 (p = 0.029). Our data show that different myeloid antigens often differ in biological importance, which may be obscured by combining them into "MyAg positive ALL". We show that unlike other MyAgs, CD66c expression is not shifted from the onset of ALL to relapse (n = 39, time to relapse 0.3-5.3 years). Although opposite has previously been suggested, we show that CEACAM6 transcription is invariably followed by surface expression (by quantitative RT-PCR on sorted cells) and that malignant cells containing CD66c in cytoplasm without surface expression are not found by flow cytometry nor by Western blot in vivo. We report no prognostic significance of CD66c, globally or separately in genotype subsets of B-precursor ALL, nor an association with known risk factors (n = 254). CONCLUSION: In contrast to general notion we show that different MyAgs in lymphoblastic leukemia represent different biological circumstances. We chose the most frequent and tightly genotype-associated MyAg CD66c to show its stabile expression in patients from diagnosis to relapse, which differs from what is known on the other MyAgs. Surface expression of CD66c is regulated at the gene transcription level, in contrast to previous reports.