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BCR-ABL1-like acute lymphoblastic leukemia (ALL) is a high-risk disease with a complex genomic background. Though extensively studied, data on the frequency and mutual associations of present mutat...

Integrating somatic mutation analysis and gene expression profiling distinguishes pediatric AML subtypes with differential prognoses and clinical risks., Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell–like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p, This study was partly funded by an unrestricted contribution from Janssen Pharmaceuticals and from GILEAD Sciences SA. A.C.L. is supported by the Peters van der Laan foundation. J.C.S. was funded by Bloodwise (11052, 12036), the Kay Kendall Leukaemia Fund (873), Cancer Research UK (C34999/A18087, ECMC C24563/A15581), Wessex Medical Research and the Bournemouth Leukaemia Fund. K.P., M.J., and S.P. are supported by the project MHCR DRO no. 65269705, the research infrastructures NCMG LM2015091, and EATRIS-CZ LM2015064, and the project CEITEC2020 LQ1601, funded by MEYS CR. R.R. is supported by Swedish Cancer Society, the Swedish Research Council, the Knut and Alice Wallenberg Foundation, Karolinska Institutet, Karolinska University Hospital, and Radiumhemmets Forskningsfonder, Stockholm.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Background: Leri-Weill syndrome (LWS) ranks among conditions with short stature homeobox gene (SHOX) haploinsufficiency. Data on possible association of SHOX aberrations with malignant diseases are scarce. Methods and Results: We report a unique case of an 8-year-old girl who was successfully treated for acute lymphoblastic leukemia (pre-B ALL, intermediate risk) and was subsequently diagnosed with LWS due to characteristic clinical appearance (short disproportionate stature, Madelung deformity of the wrist) and molecular genetic examination (complete deletion of SHOX). An identical SHOX deletion was identified also in the patient's mother. Leukemic cells of the patient were retrospectively examined by array comparative genomic hybridization (aCGH), which revealed five regions of deletions at chromosome X, including the SHOX gene locus. Conclusion: Growth retardation in children with hemato-oncologic malignancies cannot always be attributed to cytotoxic treatment and should be carefully evaluated, especially with regards to growth hormone therapy.

Background Chromosome rearrangements play an important role in cancer pathophysiology. Recently, chromothripsis has been proposed among the mechanisms leading to their formation. Chromothripsis leads to fragmentation of chromosomes and their reconstitution with tens to hundreds of rearrangements clustered in small genome regions. In contrast to the traditional concept of malignant transformation, abnormalities caused by chromothripsis are not accumulated gradually but arise during a single event. The resulting structural variants are extensive and often cause oncogene activation or tumor suppressor inactivation. Chromothripsis is associated with many tumor types, especially with brain and bone tumors. Besides that, it has been described also in congenital disorders. The exact mechanism of chromothripsis origin has not been clarified yet; however, several hypotheses have been prosed, among which DNA damage in micronucleus seems to be most likely. Similarly, an impact of chromothripsis on cellular processes has not been fully understood, yet its association with unfavorable prognosis has been observed. Purpose The purpose of this article is to summarize the current knowledge about chromothripsis and to present gathered pieces of information in a structured way. We focused on describing the basic features of chromothripsis, potential mechanisms of its origin, its impact on cellular processes and providing an overview of diseases where chromothripsis has been noted, with particular attention to cancer. Finally, we suggest a potential use of current knowledge about chromothripsis in the optimization of personalized treatment. Supported by Ministry of Health of the Czech Republic, grant no. 15-31834A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 31. 12. 2018 Accepted: 19. 3. 2019.

Cytogenetic examination and the detection of chromosomal aberrations in chronic lymphocytic leukemia (CLL) is an integral part of prognostic stratification and treatment decisions. Improvements in cytogenetic methods, notably the introduction of the cell stimulation method, have led to the detection of chromosomal aberrations in 80% of CLL cases. A comprehensive analysis of cytogenetic aberrations recently showed that complex karyotypes (CKs) defined as the detection of either three or more or five or more chromosomal changes in a karyotype have a poor prognostic impact. The current efforts at the international level are focused on the goal of including CK assessment among prognostic markers for CLL patients at diagnosis as well as after treatment. This review of the literature documents the clinical importance of CK findings in CLL and the necessity of including this factor in other poor prognostic indicators.

Recent evidence suggests that complex karyotype (CK) defined by the presence of =3 chromosomal aberrations (structural and/or numerical) identified by chromosome banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges towards routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with =5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcome, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and or TP53 mutations, TP53abs) and the expression of somatically hypermutated (M-CLL) or unmutated (U-CLL) immunoglobulin heavy variable genes (IGHV). Thus, they contrasted CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs and IGHV gene somatic hypermutation status, we propose a novel hierarchical model where patients with high-CK exhibit the worst prognosis, while M-CLL lacking CK or TP53abs as well as CK with +12,+19 show the longest overall survival. In conclusion, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with =5 chromosomal aberrations emerges as prognostically adverse, independently of other biomarkers. Prospective clinical validation is warranted before finally incorporating high-CK in risk stratification of CLL.

Genetic analysis of leukemic cells significantly impacts prognosis and treatment stratification in childhood acute lymphoblastic leukemia (ALL). Our retrospective single center study of 86 children with ALL enrolled into three consecutive treatment protocols (ALL-BFM 90, ALL-BFM 95 and ALL IC-BFM 2002) between 1991 and 2007 demonstrates the importance of conventional cytogenetics and fluorescence in situ hybridization (FISH). Cytogenetic and FISH examinations were performed successfully in 82/86 (95.3%) patients and chromosomal changes were detected in 78 of the 82 (95.1%) patients: in 69/73 patients with B-cell precursor (BCP)-ALL and in 9/9 patients with T-lineage ALL (T-ALL). The most frequent chromosomal changes in subgroups divided according to WHO classification independent of treatment protocol and leukemia subtype were hyperdiploidy in 36 patients (with ≥50 chromosomes in 23 patients, with 47-49 chromosomes 13 patients) followed by translocation t(12;21) with ETV6/RUNX1 fusion detected by FISH in 18 (22%) patients. Additional changes were detected in 16/18 (88.8%) ETV6/RUNX1-positive ALL patients with predominant deletion or rearrangement of untranslocated ETV6 allele. Unique aberrations were detected in 4 patients and dicentric chromosomes in 8 patients, one with T-ALL. These results demonstrate that cytogenetics and FISH successfully provided important prognostic information and revealed not only recurrent but also new and rare rearrangements requiring further investigation in terms of prognostic significance.

The activity of the key DNA-damage response (DDR) kinases, ATR and ATM, is largely compromised in chronic lymphocytic leukemia (CLL) cells. While noncycling CLL cells lack ATR protein expression,[1] ATM is commonly targeted for inactivation by genetic abnormalities of the ATM gene.[2] It has been shown that checkpoint pathways, which operate in cycling somatic cells and target Cdk2 kinase activity, are to some extent activated in arrested CLL cells after DNA damage as well;[3] however, the impact of these pathways is questionable, as it could be hampered by predominantly cytoplasmic localization of Cdk2 in CLL cells.[4] Nevertheless, DDR plays a key role in sensitivity of CLL to chemotherapy. Another stress-response kinase, p38 MAP kinase (p38MAPK), was shown to promote CLL cells survival.[5] In multiple cell types, p38MAPK activates different cell cycle checkpoints (mainly through regulation of cyclindependent kinase (Cdk) inhibitors p21Cip1 and p27Kip1), and also ATM/ATR-dependent G2/M checkpoint signaling through MAPKAP kinase-2 activity.[6] While relationship between ATM/p53 and p21Cip1 expression in CLL was studied,[7] the association of ATM kinase activity with the levels of p27Kip1 after DNA damage has not been elucidated in CLL cells.

Great progress has been made in the diagnostics and treatment of childhood acute lymphoblastic leukemia (ALL) over the past decades. The vast majority of children are cured, however, there is need for further improvement, especially in specific patient subgroups. Our aim was to retrospectively evaluate disease characteristics and treatment outcomes of children with ALL enrolled in a single center into consecutive treatment protocols (ALL-BFM 90, ALL-BFM 95 and ALL IC-BFM 2002) between years 1990 and 2007 and comprehensively summarize diagnostic and therapeutic advances between protocols. In total, 97 patients aged 0 to 18 years were treated for ALL at University Hospital Olomouc in the Czech Republic and steadily high relapse-free survival (RFS), event-free survival (EFS) and overall survival (OS) were observed during the evaluated time period without significant difference between the protocols (RFS 80-86%, EFS 75-83% and OS 84-92%). In conclusion, our center has demonstrated survival rates comparable to leading international study groups for childhood ALL over a substantial period of time. This has been achieved namely due to advances in diagnostics, excellent collaboration on regional, national and international level, quality assurance and high overall standard of care. The acquired experience has been crucial for current participation in the best performing Berlin-Frankfurt-Münster (BFM)-based international trials for childhood ALL.

We describe a unique case of a woman with acute myeloid leukemia with a new, previously undescribed translocation, t(11;18)(q23;q21.2), affecting the KMT2A (MLL) gene and resulting in an KMT2A(MLL)-ME2 fusion. This disease occurred secondarily following chemotherapy for a different acute myeloid leukemia with the recurrent genetic abnormality inv(16)(p13.1;q22). The secondary leukemia was treated with intensive chemotherapy without allogeneic hematopoietic cell transplantation. Complete remission lasting more than 10 years has been achieved with concurrent and sustained remission of the primary leukemia.

BCL6 rearrangements (3q27) are the most common chromosomal abnormalities in diffuse large B-cell lymphoma (DLBCL), with numerous immunoglobulin (Ig) and non-Ig genes as partners. In DLBCL, the translocations occur predominantly in the "major breakpoint region" encompassing the first noncoding exon and a part of the first intron of BCL6; few cases with "alternative breakpoint cluster" located 245-285 kb 5' BCL6 were also described. The regulatory sequences of known Ig and non-Ig partners replace the 5' untranslated region of the BCL6 in the same transcriptional orientation. Contrary to Ig/BCL6 fusions typical by high BCL6 gene expression, in non-Ig/BCL6 translocations were observed unexpectedly low BCL6 mRNA levels. From the clinical point of view, the survival rate of DLBCL patients with non-Ig partners is inferior to those with Ig/BCL6 translocations, suggesting that non-Ig/BCL6 fusion is a poor prognostic indicator. Hereby we provide comprehensive information about known non-Ig translocation partners and clinical consequences of BCL6 rearrangements in DLBCL. Moreover, we describe a novel reciprocal translocation t(3;10) in refractory patient with DLBCL with the breaking points at 5' untranslated region of BCL6 and 5' untranslated region of the RASGEF1A gene on chromosome 10q11.21 loci; this rearrangement was associated with low BCL6 and RASGEF1A gene expressions. Our patient harbouring dual chromosomal rearrangement involving BCL2 and BCL6 genes relapsed three-times and died soon; thus, further supporting the notion that non-Ig/BCL6 fusion is a poor prognostic indicator of DLBCL. There is evidence of prognostic value of BCL6 rearrangements also in rituximab era.

Presented are results of allogeneic hematopoietic stem cell transplantations (HSCTs) in 13 patients with high-risk acute lymphoblastic leukemia (ALL) in the first complete remission after a reduced intensity conditioning combining fludarabine (150 mg/m2) and melphalan (140 mg/m2) with thymoglobulin (4.5 mg/kg). The immunosuppressive effect of T-cell depletion reducing the risk of graft-versus-host disease (GVHD) and non-relapse mortality was compensated by early initiation of reduction and withdrawal of prophylactic immunosuppression aimed at maintaining effective immunological antileukemic control. The median post-transplant follow-up was 23 (range, 10-65) months. Stable engraftment of donor's hematopoiesis was achieved in all patients. Acute GVHD was observed in two cases (15.4%); the chronic form was not noted. Two patients (15.4%) relapsed with ALL at 3 and 16 months after transplantation. During the above post-transplant follow-up, all 13 recipients were alive, with a probability of 2-year disease-free survival of 76.9% (95% CI 51-100%). Although the results were obtained with a small pilot study group it may be assumed that, given the prognostic risk of most patients and the nearly 2-year median post-transplant follow-up, the approach may be considered as an alternative to HSCTs after traditional myeloablative or reduced conditioning regimens with standard GVHD prophylaxis.

This study aimed at mapping the frequency of IGHV3-21 and IGHV1-69 in a group of 417 patients newly diagnosed with chronic lymphocytic leukemia (CLL) and described basic characteristics, cytogenetic abnormalities and prognosis of these patient subgroups. IGHV3-21 was found in 29 patients (7%) and IGHV1-69 in 51 patients (12.4%). The median overall survival (OS) rates were 97 months and 85 months in the IGHV3-21 and IGHV1-69 groups, respectively. In this small group of patients, the study failed to show a difference in OS of IGHV3-21 patients with mutated and unmutated IGHV status (p

UNLABELLED Acute promyelocytic leukemia is a unique entity among acute leukemias. Introduction of all-trans retinoic acid and, subsequently, arsenic trioxide in its treatment has markedly improved treatment outcomes for this once frequently fatal disease. Improved outcomes have also been observed in elderly patients, including those in whom standard intensive therapy is contraindicated because of comorbidities.In our center, a total of 60 APL patients were treated in 1993-2013, of whom 9 were aged 60 or more years. Although most of them had significant comorbidities at the time of diagnosis, eight achieved complete remission. At the time of the analysis, six patients were alive and in long-term remission; two patients died of causes other than APL. The median follow-up was 59 months.Included is case report of a patient with a high comorbidity score whose treatment was markedly reduced and individualized.Our experience shows that, in APL patients a curative approach is generally tolerated and should always be attempted regardless of age and comorbidities. KEYWORDS APL - elderly patients - comorbidity.

Detection of genetic changes has improved the current risk stratification in chronic lymphocytic leukemia (CLL).1,2 Among the known recurrent chromosomal abnormalities,1 6q deletion is less frequent and controversy surrounding its prognostic significance still remains.

Presented are updated results of allogeneic hematopoietic stem cell transplantations (HSCTs) in 25 adult patients with acute lymphoblastic leukemia (ALL) in complete remission (CR) after a reduced intensity conditioning (RIC) combining fludarabine (150 mg/m2) and melphalan (140 mg/m2) with thymoglobulin (4.5 mg/kg or recently 4.0 mg/kg) followed by early initiation of reduction and withdrawal of prophylactic posttransplant immunosuppression. The median post-transplant follow-up was 32 (range, 4-87) months. Stable engraftment of donor's hematopoiesis was achieved in all patients. Acute graft versus host disease (GVHD) as well as the chronic one were equally observed in four cases (16%). Five patients (20%) relapsed with ALL in the median of 9 (range, 3-15) months after HSCT. During the above post-transplant follow-up, 4 recipients (16%) died. Disease progression and posttransplant complications were the cause of death in three (12%) and one (4%) of them, respectively. The probabilities of 2-year event-free (EFS) and overall survival (OS) were 70.3% (95% CI 51.9-88.7%) and 86.1% (95% CI 71.6-100%), respectively. Presented study confirmed our previously reported promising results and this approach may be considered as an alternative to traditional HSCTs performed in high-risk patients with ALL.

This study analyzed the prognostic significance of soluble interleukin-2 receptor α (sIL-2Rα) levels in 100 prospectively enrolled patients with previously untreated follicular lymphoma. It showed that sIL-2Rα level ≥ 115 pmol/L at the time of treatment initiation correlated with a high Follicular Lymphoma International Prognostic Index-2 (FLIPI-2), bulky disease, advanced clinical stage, number of involved lymph nodes, bone marrow involvement and elevated β2-microglobulin (B2M) level. When testing all patients, sIL-2Rα ≥ 115 pmol/L was associated with significantly shorter progression-free (PFS; p0.03, hazard ratio [HR] 2.04) but not overall (OS; p = 0.06, HR 2.36) survival rates. Subanalysis of patients receiving cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) ± rituximab showed higher predictive power for both PFS (HR 2.75, 95% confidence interval [CI] 1.24-6.11, p = 0.01) and OS (HR 3.33, 95% CI 1.15-9.63, p = 0.02). In the whole population (n = 100), only B2M proved a significant univariate predictor (p = 0.007, HR = 2.8) of PFS. When testing patients treated with CHOP ± rituximab, sIL-2Rα was found to be the best univariate predictor for PFS among all FLIPI-2 factors (HR = 2.68, p = 0.015). Serum IL-2Rα levels may help to refine risk assessment in the modern immunotherapy era complementary to FLIPI-2 factors.

The basic principles of lymphoma classification(s) in general have been widely evolving in a course of decades of years wiht the use of contemporary resources and recent cutting edges in hematooncology on a clinical, morphological and molecular level bring new possibilities not only in improvements of diagnostic and prognostic algorithms and also bear new opportunities in so called targeted and tailored strategies of lymphoma therapy. The pathogenesis and biologic behavior of lymphoproliferations and even lymphomas should be studied in a context of lymphocytic and (neoplastic) lymphoid stage and chronologic development. In a current more complex insight into lymphoproliferations we would like to describe huge heterogeneity of diffuse large B-cell lymphoma in relationship to mandatory WHO classification since 2008 and the next development of knowledge in this field with potential new influence on an advancement of both classification and therapy.

Introduction. MicroRNAs (miRNAs) are small non-coding single-stranded RNA molecules that regulate gene expression at the post-transcriptional level. In the pathogenesis of chronic lymphocytic leukemia (CLL), miR-15a and miR-16-1 play an important role. These miRNAs are located on chromosome 13 in the 13q14.3 region, which is deleted in more than 55% of CLL patients. This aberration affects expression of miRNAs. Objectives. The study aimed at performing a molecular genetic analysis of miR-15a and miR-16-1 expression in a group of 39 patients diagnosed with CLL and determining the association between the expression of the two miRNAs and types of deletions in the 13q14 region. Methods. We used fluorescence in situ hybridiziation (FISH) for determination of mono- or biallelic deletion 13q and quantitative polymerase chain reaction (Q-RT-PCR) to revealed expression miR-15a and miR-16-1 in 39 patients suffering from CLL. Results. The analysis comprised 19 patients with monoallelic 13q14 deletion, 3 patients with biallelic deletion, 9 patients with both monoallelic and biallelic deletions, and 8 patients without 13q14 deletion serving as controls. The results showed different levels of miRNA expression in individual patients. Significantly higher normalized levels of miR-15a expression were found in the control group and patients with monoallelic 13q14 expression compared with patients with biallelic deletion. There was a significantly decreased expression of both miRNAs in patients with biallelic deletion of the 13q14 region but only when deletions were present in 77% or more of cells, as detected by fluorescent in situ hybridization (FISH).

Aims. We assessed the long-term outcome of consecutive patients in the chronic phase of chronic myeloid leukemia (CML) treated with interferon-alpha (INF-α) in Central and Northern Moravia between 1989 and 2006. Methods. A retrospective study focused on the response, prognostic factors and side-effects of INF-α. Results. 118 patients (67 males and 51 females, median age 50 years; range 18-71) were analyzed. The median followup was 82.6 months (12.4-212.6). Thirty-six patients (30.5%) achieved major cytogenetic response (CyR) in median of 18.3 months (3.7-47.3) and maintained it for a median of 64.0 months (7.0-176.0). Sixty-one patients treated with INF-α for more than 12 months had an overall survival (OS) of 137.0 months (95% CI 117.6-156.4). Eighteen (29.5%) achieved complete CyR (CCyR). 109 patients discontinued the treatment with INF-α because of hematologic or cytogenetic resistance in 53 (48.7%), progression of CML in 31 (28.4%) and intolerance to INF-α in 17 (15.6%) patients. The percentage of peripheral blasts, leukocyte count (>50x10 9 /L), splenomegaly, anemia (Hgb≤110 g/L) and Sokal score had statistical impact on the OS in univariate assessment but only the Sokal score remained significant in multivariate analysis. Additional cytogenetic abnormalities at diagnosis were associated with poor prognosis. Conclusions. In most patients, treatment with INF-α had to be stopped because of a failure to induce response, progression of CML or side-effects but nearly one third of patients treated at least for one year had a long-term benefit from INF-α. The best prognosis was associated with achievement of CCyR and negativity of BCR-ABL in nested RT-PCR.

Using the data of 723 chronic myeloid leukemia (CML) patients in the chronic phase, we analyzed the prognostic value of the Sokal, Euro, and EUTOS scores as well as the level of BCR-ABL1 and the achievement of complete cytogenetic response (CCgR) at 3 months of imatinib therapy in relation to the so-called current survival measures: the current cumulative incidence (CCI) reflecting the probability of being alive and in CCgR after starting imatinib therapy; the current leukemia-free survival (CLFS) reflecting the probability of being alive and in CCgR after achieving the first CCgR; and the overall survival. The greatest difference between the CCI curves at 5 years after initiating imatinib therapy was observed for the BCR-ABL1 transcripts at 3 months. The 5-year CCI was 94.3% in patients with BCR-ABL1 transcripts ≤ 10% and 57.1% in patients with BCR-ABL1 transcripts 10% (P = 0.005). Therefore, the examination of BCR-ABL1 transcripts at 3 months may help in early identification of patients who are likely to perform poorly with imatinib. On the other hand, CLFS was not significantly affected by the considered stratifications. In conclusion, our results indicate that once the CCgR is achieved, the prognosis is good irrespective of the starting prognostic risks.

Background. The Recommendations of the European Leukemia Net (ELN) have become an essential tool in the management and prognosis of patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs). However, the definition of suboptimal response remains under discussion. Methods. We used conventional cytogenetics for the detection of clonal changes in Ph-positive and negative clones. RT-PCR and sequencing were carried out on peripheral blood leukocytes to detect the type of BCR-ABL1 transcript. The BCR-ABL1 mutational status was assessed using sequencing of RT-PCR products. High performance capillary electrophoresis for determination of imatinib (IMA) plasma concentration was used. Results. A retrospective study of 110 patients diagnosed with chronic-phase (CP) CML treated with IMA or 2 nd generation TKIs in the years 2000-2009 focused on analysis of patients with suboptimal response according to ELN criteria. 40 patients were administered IMA as first-line therapy and 70 had been pretreated with interferon-alpha (IFN-α) with or without Ara-C and/or hydroxyurea (HU) for a median 12 months (range, 1-92 months). After adjusting for the ELN criteria, major molecular response (MMR) was achieved after median 34 and 39 months in 66.7% and 41.7% of patients after the first and second-line IMA therapy with suboptimal response defined as lack of achievement of MMR at the 18 th month of treatment, respectively. In comparison to patients with optimal response, patients with suboptimal response did not show significant differences in overall survival (OS) or progression-free survival (PFS). Cytogenetic assays demonstrated additional chromosome abnormalities (ACAs): chromosome 8 trisomy in a Ph-negative clone during the IMA treatment (in 1 case) and der(9q) in Ph-positive clone (in 2 cases); in patients receiving first-line IMA only chromosome 8 trisomy was observed which was associated with myelodysplastic syndrome – this was the only case where hematopoietic stem cell transplantation (HSCT) was performed. During the treatment with IMA in both subgroups no regulatory mutations in the ABL kinase domain were confirmed. Conclusion. We believe that the category of suboptimal response should be redefined or withdrawn from the ELN 2009 recommendations for management of CML patients treated with TKIs. Patients with suboptimal response who have no additional risks (additional cytogenetic abnormalities or BCR-ABL1 regulatory mutations) may remain on IMA treatment while patients with these risks should be switched to the 2 nd generation TKIs.

The significance of positron emission tomography/computed tomography (PET/CT) in chronic lymphocytic leukemia (CLL) has not yet been systematically studied. This prospective study was aimed at assessing the benefit of PET/CT in patients with newly diagnosed or relapsed CLL and Richter transformation (RT). PET/CT examination was performed in 23 patients with newly diagnosed disease, 13 with relapsed disease and eight with suspected or histopathologically confirmed RT. In all patients, the maximum standardized uptake value (SUV(max)) was calculated. The median SUV(max) was 3.4 (range: 1.5-6.3) and 3.1 (range: 1.2-5.9) in newly diagnosed and relapsed patients, respectively. The median SUV(max) of patients with suspected or confirmed RT reached 16.5 (range: 7.2-25.3), a value different from that of the previous groups (p0.001). 2-[18F]fluoro- 2-deoxy-D-glucose ((18)F-FDG) PET/CT revealed inflammatory lesions in seven patients (16%) and synchronous tumors in two newly diagnosed patients. (18)F-FDG PET/CT may be a beneficial imaging method when used in individuals with CLL and suspected RT.

We evaluated responses to the treatment and long-term outcomes of chronic myeloid leukemia patients treated with imatinib as first-line treatment in routine clinical setting from two countries with centralized tyrosine kinase inhibitors (TKIs) treatment. We assessed prognostic significance of European LeukemiaNet (ELN) 2006- and 2009-defined responses and the prognostic value of molecular responses at defined time points on 5-year survivals. Among the cumulative rates of incidence of hematologic, cytogenetic, and molecular responses and all important survival parameters, we evaluated the prognostic significance of different BCR-ABL transcript-level ratios (≤1%; >1%-≤10%; >10%) at 3, 6, 12, and 18 months (n = 199). The ELN optimal response criteria and their predictive role were significantly beneficial for event-free survival at all given time points. We found significant improvement in survivals of patients with BCR-ABL lower than 10% in the 6th and 12th months. Significantly better outcome was found in patients who achieved major molecular response (MMR) in the 12th month. The cumulative incidences of complete cytogenetic response (CCyR) and MMR were significantly associated with the molecular response in the 3rd month. The ELN response criteria and their predictive role were helpful at given time points; however, the 2009 definition did not significantly alter the prognostic accuracy compared with that of the 2006 definition. The significant value was observed for cytogenetic responses at the 6th and 12th month. Moreover, progression-free and event-free survivals were improved with MMR at the 12th month.

Assessment of serum levels of free light chains (FLC-κ and FLC-λ) and recently heavy/light chain pairs of immunoglobulin (HLC-κ and HLC-λ) and their ratio (FLC-r and HLC-r) has significantly enriched traditional algorithm of multiple myeloma (MM) evaluation. The aim of the presented study was to assess the relationship of classical prognostic parameters of MM, standard FLC-κ/λ and HLC-κ/λ ratio ((s)FLC-r and (s)HLC-r), modified ratio of "involved/uninvolved" FLC and HLC ((m)FLC-r and (m)HLC-r ), the difference between "involved - uninvolved" FLC and HLC (FLC-dif. and HLC-dif.) to current stratification models of MM based on the result of cytogenetic analysis.In a group of 97 patients with MM we assessed serum levels of FLC by FreeliteTM method, and we calculated (s)FLC-r, (m)FLC-r and FLC-dif. indices by HevyliteTM method. For cytogenetic analysis we used FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms). For MM stratification we used standard staging systems according to Durie-Salmon (D-S) and International Staging System (ISS) as well as novel stratification systems based on the results of cytogenetic analysis, ie. "Mayo Stratification of Myeloma and Risk-Adapted Therapy" (mSMART) and "Revised International Staging System" (R-ISS).Stratification mSMART and R-ISS has significantly different representation of "standard" or "low-risk" (71, 15.5, 11.3 a 29.9 %), "intermediate risk" (15.5, 53.6, 34 a 33 %) and "high risk" patients (13.4, 30.9, 54.7 a 37.1 %) compared to standard staging systems. mSMART stratification was compared to prognostic factors of MM (Hb, albumin, β(2)-M, creatinine and LDH), and the only significant relationship was found in the case of β(2)-M, R-ISS had relationship only to Hb and creatinine. In the case of D-S staging we found significant relationship of stages 1-3 and substages A and B to the levels of (m)FLC-r, FLC-dif. and (m)HLC-r, ISS had moreover relationship to k HLC-dif. and MIg concentration. Analysis of mSMART stratification showed primarily significant relationship of risk categories 1-3 to (m)FLC-r and (s)HLC-r indices, and R-ISS to (m)HLC-r index and MIg concentration. In both cytogenetics-based stratifications there was a lack of relationship to (s)FLC-r, FLC-dif. and HLC-dif. indices.Comparison of the results of standard staging systems according to D-S and ISS with cytogenetics based models mSMART and R-ISS showed different representation of risk groups, and significantly different relationship to classical prognostic factors together with original relationship of sMART stratification to (m)FLC-r and (s)HLC-r, and R-ISS to (m)HLC-r and MIg concentration.

The experimental platform in hematooncology is still searching for more valid prognostic and predictive factors on clinical, morphological and molecular levels. The bridge closer to daily practice is so-called translation medicine and from this point of view we have tried to sort diffuse large B-cell lymphoma not otherwise specified. The applied methodological approaches are morphology, indirect immunohistochemistry on formaline-fixed, parrafin-embeded tissue, Hans classifier sorting, expression of Bcl-2, CD5, CD20, CD30 and NfκB proteins in comparison with the clinical (Ann Arbor stage, IPI, aa-IPI, PFS, OS), laboratory and cytogenetic results (complex and simplex karyotypes). Statistical analysis included Cox regressive analysis, Mann-Whitney and Kruska-Wallis test. The interval of PFS and OS has been assessed according to Kaplan-Meier analysis. According to Hans classifier 11 cases (18.7 %) could not be sorted exactly into GCB/nonGCB- like subgroups. All relapsing cases bear negative expression of CD10 and 28 cases of non-relapsing cases showed positive expression of CD10. The "third" - GCB-like/nonGCB-like unsortable subgroups shared a very similar course of PFS with the nonGCB-like subgroup and a worse clinical course of OS. Statistically nonsignificantly better response to chemotherapy was shown by cases with positive Bcl-2 expression of more than 30 %. Statistically nonsignificantly better OS and PFS was shown by cases with a proliferation index Ki67 more than 70 %. The study detected 17 cases (28.8 %) with a nuclear expression of p50 and one case with nuclear expression of p65 (1.7 %) which may imply the possibility of NfκB signaling pathway activation. A statistically nonsignificant realationship of p50 expression and OS/PFS was indicated.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype of non-Hodgkin lymphomas in Western countries. Implementation of immunotherapy using monoclonal antibodies to therapeutic protocols has led to dramatic improvements in overall survival. DLBCL became a model of a successful immunochemotherapy concept. Despite this fact, there is still a proportion of patients who do not respond to or relapse early after treatment. Growing evidence suggests that host antitumor immunity is suppressed by lymphoma cells in many ways. First, host cytotoxic T cells are directly suppressed by interaction with programmed cell death (PD) ligand on lymphoma cell surface and a similar mechanism enhances the activity of suppressive regulatory T cells (Tregs). Second, tumor cells escape host cytotoxic cells due to lower immunogenicity caused by reduced expression of HLA antigens. Both mechanisms have an origin in primary genetic events in lymphomagenesis. Rearrangement of MHC class II transcriptional activator (CIITA) gene and amplification of Janus kinase (JAK2) gene lead to enhanced expression of PD ligands 1 and 2, higher proliferation activity, and lower expression of HLA. This paper summarizes current knowledge about clinically relevant immune escape mechanisms in DLBCL.

Genetic background of mature B cell neoplasms with villous lymphocytes is poorly understood. We identified a novel breakpoint region at 14q32.13 that was rearranged together with IGH/14q32.33 in 4 BRAF/V600E-negative leukemia/lymphoma cases with villous lymphocytes carrying either t(14;14)(q32.13;q32.33) (3 patients) or del(14)(q32.13q32.33) (1 patient). The 14q32.13 breakpoints were mapped by FISH in the region harboring the TCL1A/TCL1B/TCL6 genes, known to be affected by TCRA/D-mediated t(14;14)(q11;q32)/inv(14)(q11q32) occurring in T-cell leukemia/lymphoma. To identify the target of t(14;14)(q32.13;q32.33) and del(14)(q32.13q32.33), qRT-PCR analysis of 25 candidate genes located centromerically and telomerically to the 14q3213 breakpoint was performed. Any of the analyzed genes was commonly overexpressed in the presented cases. Of note, upregulated transcription of TCL1A was observed in 2 cases. In summary, we provide evidence that IGH-mediated chromosomal aberrations affecting the 14q32.13/TCL1A-TCL6 region are recurrent in mature B-cell neoplasms with villous lymphocytes. Despite extensive qRT-PCR studies, molecular consequences of these novel aberrations remain elusive. ispartof: Leukemia & Lymphoma vol:53 issue:12 pages:2449-2455 ispartof: location:United States status: published

We assessed long-term outcome of 118 consecutive patients in chronic phase of chronic myeloid leukemia (CML) treated with interferon-alpha (IFN-α) in the Central and Northern Moravia region between 1989 and 2006 with focus on operational cure. The median follow-up was 82.6 months (range 12.4–212.6). Eighteen (15.3%) patients achieved complete cytogenetic response (CCyR) after median 16.7 (3.7–40.8) months. Nine of these patients (7.6%) achieved BCR-ABL negativity in nested reverse transcriptase–polymerase chain reaction [“complete” molecular response (CMR)] and 6 of them have been operationally cured without any treatment for median 6 (4–10) years, while 2 continue with IFN-α and 1 died from CML-unrelated cause. Operationally cured patients had a significantly lower percentage of initial peripheral promyelocytes, blasts, and erythroblasts than the rest of patients treated for more than 12 months (P=0.01–0.03). Unlike patients with sole CCyR, the majority of whom lost CCyR despite continuing IFN-α therapy and required imatinib, patients who achieved CMR had excellent long-term outcome.

Association of trough imatinib plasma levels (IPL) with cytogenetic or molecular response to treatment in patients with chronic myeloid leukemia (CML) was repeatedly reported. We analyzed their value in the routine clinical setting in 131 patients with chronic phase CML in whom imatinib was applied as first- or second-line treatment. A total of 1,118 measurements were obtained by ultra-performance liquid chromatography-tandem mass spectrometry assay in patients treated with daily dose of imatinib ranging from 100 to 800 mg. Samples were obtained from 1 to 96 h after drug ingestion. High inter (36%) and intraindividual variability (9-33%) of IPL was observed. For analysis of correlation of IPL with treatment response, two sets of samples were selected according to the European LeukemiaNet (ELN) criteria. The first set consisted of 241 samples taken 24 ± 2 h after dosing in 54 patients, and the second one consisted of 329 samples taken 24 ± 4 h after imatinib ingestion in 84 patients. In both sets, only patients treated with 400 mg imatinib once daily for at least 18 months were included. From multiple measurements in individual patients, mean IPL were used. In both sets, we were not able to demonstrate a statistically significant correlation between IPL and response to treatment according to the ELN. We believe that this was due to the differences in patients' compliance, leukemia biology, and other variables that are difficult to eliminate in the routine clinical practice. The use of IPL for prognostic estimation in CML treatment outside the clinical trials is probably limited.

Epigenetic therapy with hypomethylating agent (5-azacytidine; AZA) is common in the management of specific subtypes of myelodysplastic syndrome (MDS), but there are only few studies in chronic myelomonocytic leukemia (CMML) patients. In this paper our experience with 3 CMML patients treated with AZA is described. In one patient transfusion independency was observed after 4 treatment cycles; in one case a partial response was recorded, but a progression to acute myeloid leukemia (AML) after 13 AZA cycles has appeared. In one patient, AZA in reduced dosage was administered as a bridging treatment before allogeneic stem cell transplantation (ASCT), but in the control bone marrow aspirate (before ASCT) a progression to AML was recorded. Future studies are mandatory for evaluation of new molecular and clinical features which could predict the efficiency of hypomethylating agents in CMML therapy with respect to overall survival, event-free survival, quality-adjusted life year, and pharmacoeconomy.

Down-regulation of miR-150 and up-regulation of its target FOXP1 is associated with transformation of follicular lymphoma.Down-regulation of miR-150 and up-regulation of its target FOXP1 is associated with transformation of follicular lymphoma.

PURPOSE: Treatment of childhood acute lymphoblastic leukemia (ALL) was unified in the year 1986 in the Czech Republic using BFM protocols. PATIENTS AND METHODS: Children were treated in 10 and later on in 8 centers localized at the pediatric departments of the biggest hospitals. More than 1.000 children and adolescents up to the age of 18 years were treated between 1986 and 2002 on three consecutive studies ALL-BFM 83, ALL-BFM 90 and ALL-BFM 95. RESULTS: Event-free survival and overall survival improved gradually from 58/62% on ALL-BFM 83 through 70.5/76.6% on ALL-BFM 90 until 72.1/80.2% on ALL-BFM 95. At the same time the incidence of toxic deaths was decreasing and the success in the achievement of complete remission was increasing. Diagnostics in the Czech Republic improved remarkably in the 1990 with gradual introduction of centralized flow cytometry and molecular genetic analysis in one reference laboratory (CLIP = Childhood Leukemia Investigation Prague), which since then has become an internationally respected research center. CONCLUSIONS: Building up a network of closely collaborating leukemia centers covering the whole country, together with the establishment of reference and research laboratories has paved the way for the implementation of the Czech Pediatric Hematology Working Group (CPH) into the international studies Interfant 99, EsPhALL and for the active role in ALL IC-BFM 2002, the I-BFM-SG international randomized trial for treatment of children and adolescents with non-B ALL starting in 2002.

Most results on the treatment of chronic myeloid leukaemia (CML) with imatinib were obtained from clinical trials that may differ from the routine practice. We report the results of treatment of consecutive patients with CML at ten major centres during 2000–2008. Patients and methods: Data reporting was retrospective in 2000–2004 and prospective from 2005 on. A total of 661 patients [301 women and 360 men; median age 51 (range, 15–83)] with Ph + CML were registered. The median follow-up was 46.1 months (0–122.2). Results: Most patients were treated with first- (379; 57.3%) or second-line (193; 29.2%) imatinib; some of the patients underwent allogeneic hematopoietic stem cell transplantation (AHSCT) (83; 12.6%), but 6.1% were treated with other modalities [40 patients; median age 66 (range, 32–83)]. The probability of overall survival (OS) at 5 years, according to Kaplan and Meier, was 88.9%, 77.5% and 68.7% for chronic-phase patients treated with first-line imatinib, second-line imatinib and first-line AHSCT, respectively, but only 25.2% for patients receiving other modalities. The OS was dependent on the disease phase and Sokal, Hasford and European group for blood and marrow transplantation (EBMT) risk scores (P < 0.001; each). Only 46.2% of deaths in patients treated with other modalities were attributable to CML. Elderly patients over 65 years achieved similar response rates and progression-free survival to the younger ones. There was a trend for inferior results of AHSCT performed after the failure of imatinib (P = 0.075), probably as a result of differences in EBMT risk scores (P < 0.001). Conclusions: The ability to achieve results comparable to those of previous clinical studies in our CML cohort was influenced by centralised care. Decisions not to initiate imatinib or to delay AHSCT may have a negative impact on OS, but comorbidities may limit the treatment potential of imatinib in the elderly.

Aim. This is a case report of a 51 year old male with marked splenomegaly, basophilia, severe thrombocytopenia, anemia and high SFKL phosphorylation downstream of Bcr-Abl, investigated for association of the e6a2 BCR-ABL fusion gene and marked basophilia. The treatment strategy implications in patients with Philadelphia positive CML are described. Methods. RT-PCR and sequencing were carried out on the peripheral blood leukocytes to detect the type of BCRABL transcript. The BCR-ABL mutational status was assessed using sequencing of the RT-PCR products. The in vitro test of sensitivity to TKIs was based on detecting inhibited phosphorylation of the Crkl and Phospho-Src family kinases (SFK, Tyr416) using immunodetection. Results. The cytogenetics revealed 90% of Ph+ (Philadelphia) cells in the bone marrow aspirate with no additional clonal chromosomal abnormalities at diagnosis. This correlated with an accelerated phase of the CML. Sequencing analysis of reverse transcribed and PCR amplified BCR-ABL transcript revealed a rare e6a2 fusion, with no evidence for Bcr-Abl kinase domain mutation. Western blot analysis showed high phosphorylation (activation) of Crkl and the Src family of kinases (P-SFK). In vitro test of sensitivity of the patients’ leukemic cells to imatinib demonstrated sensitivity of Bcr-Abl tyrosine kinase to imatinib, as assessed by a decrease in phosphorylated Crkl and the disappearance of P-SFK, suggesting that P-Src reflects only the Bcr-Abl–dependent Src activity. The initial treatment strategy was reduced imatinib and search for an unrelated hematopoietic stem cell donor (according to the ELN recommendations). The patient was allografted with peripheral stem cells from an HLA- identical male donor but on day +70 graft failure occurred. He was allografted again with the peripheral stem cells from an HLA-identical female donor, engrafted on day +15 and showed 100% donor chimerism with no evidence of the e6a2 BCR-ABL fusion transcript on day +30. Conclusion. The clinical disease course in patients with the rare e6a2 BCR-ABL transcript variant is aggressive. This may be the result of increased kinase activity due to partial loss of the guanine exchange factor/dbl-like domain which mediates the interaction with several Ras-like G-proteins involved in cell proliferation, signal transduction, and cytoskeletal organization. For the above reasons, these patients should receive stem cell transplant immediately after a short course of treatment with imatinib/ dual Src/Abl kinase inhibitor or they should be registered in clinical trials with experimental agents.

Pheochromocytomas (PCCs) are rare tumors among children and adolescents and therefore are not genetically well characterized. The most frequently observed chromosomal changes in PCC are losses of 1p, 3q and/or 3p, 6q, 17p, 11q, 22q, and gains of 9q and 17q. Aberrations involving chromosome 11 are more common in malignant tumors. Unfortunately information about gene aberrations in childhood PCC's is limited. We used comparative genomic hybridization (CGH) and array comparative genomic hybridization (aCGH) to screen for copy number changes in four children suffering from pheochromocytoma or paraganglioma. Patients were diagnosed at the age 13 or 14 years. Bilateral pheochromocytoma was associated with von Hippel-Lindau syndrome (VHL). Multiple paraganglioma was associated with a germline mutation in SDHB. We found very good concordance between the results of CGH and aCGH techniques. Losses were observed more frequently than gains. All cases had a loss of chromosome 11 or 11p. Other aberrations were loss of chromosome 3 and 11 in sporadic pheochromocytoma, and loss of 3p and 11p in pheochromocytoma, which carried the VHL mutation. The deletion of chromosome 1p and other changes were observed in paragangliomas. We conclude that both array CGH and CGH analysis identified similar chromosomal regions involved in tumorigenesis of pheochromocytoma and paragangliomas, but we found 3 discrepancies between the methods. We didn't find any, of the proposed, molecular markers of malignancy in our benign cases and therefore we speculate that molecular cytogenetic examination may be helpful in separating benign and malignant forms in the future.

Monosomy 7 and/or deletion of the long arm of chromosome 7 is a common cytogenetic aberration in children with myelodysplastic syndrome (MDS) and is associated with poor outcome. In this report, we present an unusual cytogenetic abnormality leading to loss of both the whole short and whole long arms of chromosome 7, which was found in the bone marrow cells of three pediatric patients with MDS. Using a combination of conventional and molecular cytogenetic methods, a tiny "dot-like" marker chromosome was found and described as der(7)del(7)(p11)del(7)(q11). Together with one previously published case, this chromosomal aberration represents a new rare recurrent karyotypic abnormality involving chromosome 7 in children with MDS.

Despite the prognostic value of trough imatinib plasma levels (IPL) identified in some studies, no recommendations for the use of IPL results in routine management of CML patients have been issued. We report two patients in whom daily imatinib dose was increased from 400 to 600 or 800 mg because of low IPL found at various intervals from the beginning of treatment (7 measurements; mean IPL values = 616.33 and 764.5 ng/mL, respectively). Both patients achieved suboptimal response according to the European LeukemiaNet criteria (complete cytogenetic response was not achieved after 1 year of treatment in patient 1 and major molecular response after 47 months of standard-dose imatinib therapy in patient 2). In addition, we have demonstrated low hOCT-1 expression at diagnosis in both patients, retrospectively. Escalation of imatinib daily dose resulted in a significant increase of IPL (6 measurements; mean = 1790 and 1416.66 ng/mL, respectively) and in the achievement of complete cytogenetic response in patient 1 after 3 months and major molecular response within 15 and 6 months in both patients. Our cases demonstrate that low IPL identified at various non-predefined intervals from the beginning of therapy may be used for deciding on dose escalation in selected CML patients in the routine clinical setting, especially in cases with suboptimal treatment response.

Background Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines. Design and Methods Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed. Results The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53±10% and 76±2% at 5 years, respectively, P =0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts. Conclusions Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.