Lymphoid Blast Crisis Research Articles

Characteristics of the TCR Vbeta Repertoire and Identical Clonally Expanded T Cells in Chronic Myeloid Leukemia Patients in Advanced Phase with ABL Mutations

AbstractTumor specific or related antigen cytotoxic lymphocyte (CTL) have been identified in chronic myeloid leukemia patients, however, whether they are constituted by specific type of T cell receptor chains has not been illustrated so far. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 5 CML patients in blast crisis (BC-CML) and one in acceleration phase (AP-CML) with ABL kinase domain mutations (KDMs) including T315I, E255K, F317L+S417Y, Y-253F and L387M+T-315A. Examination of TCR Vβ expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) combined with GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in those patients, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample, which distributed in 15/24 different subfamilies (TCR Vβ4, Vβ5, Vβ6, β8, Vβ9, Vβ10, Vβ15, Vβ16, Vβ17, Vβ18, Vβ19, Vβ21, Vβ22, Vβ23, Vβ24). Intriguingly, a relatively highly expanded Vβ9 clone with the same length as CDR3 (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the other two CML patient in myeloid blast crisis (MBC-CML) or the one CML patients in accelerated phase. In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency status of these patients, and clonally expanded Vβ9 T cell clones may represent a specific immune response to leukemia-associated antigens in LBC-CML patients. DisclosuresLi:The Foundation for High-level Talents in Higher Education of Guangdong, China ([2013]246-54),and the Guangzhou Science and Technology Project Foundation (201510010211): Research Funding; National Natural Science Foundation of China (81270604, U1301226, and 81400109), the Guangdong Natural Science Foundation (S2013040016151 and S2013020012863): Research Funding.

MRD Monitoring Using Minor-BCR-ABL1 Genomic Breakpoint in Childhood ALL Identifies a Subgroup with Distinct Biology and a Very Poor Prognosis

▪Background:The exact role of minimal residual disease (MRD) testing in childhood BCR-ABL1+ ALL is still unclear and MRD levels are used rather for stem cell transplantation (SCT) indication than for frontline treatment stratification. There are two standard targets used for MRD monitoring in these ALLs: Ig/TR rearrangements and the fusion transcript quantification. However, despite similar sensitivity of the methods, we previously showed a poor correlation between them in some patients. Overall, 20% of samples negative by Ig/TR MRD were BCR-ABL1 positive (even at high levels). By demonstrating presence of BCR-ABL1 within the non-lymphoid (Ig/TR-negative) population in the non-correlating samples we showed that multilineage involvement is at least partly responsible for the discrepancy (Zaliova, Leukemia 2009). Here we established MRD monitoring based on the quantification of the patient-specific genomic (intronic) BCR-ABL1 breakpoint sequence, compared MRD levels obtained from different approaches and analyzed their prognostic significance.Methods:Our cohort totals 35 children diagnosed with minor-BCR-ABL1+ ALL. The BCR-ABL1 genomic breakpoint was found using multiplex long distance DNA PCR. The patient-specific intronic fusion sequences were used for MRD quantification by qPCR. We quantified MRD levels in 386 bone marrow (BM) samples. The results of genomic BCR-ABL1 quantification were compared with Ig/TR and BCR-ABL1 transcript levels. Samples with >1 log difference in MRD levels were considered non-correlating. For correlation analysis of methods, double-negative (DN) samples were excluded. Overall survival (OS) rates were calculated according to Kaplan-Meier and compared by log-rank test.Results:Analysis of MRD in BM samples confirmed poor correlation between Ig/TR and BCR-ABL1 mRNA quantification (Spearman correlation coefficient R=0.70; n=166 non-DN samples). Moreover, we also saw poor correlation when comparing the two DNA methods (Ig/TR vs. BCR-ABL1 DNA, R=0.69; n=254 non-DN samples) with 21% of samples positive by BCR-ABL1 (at levels up to 10e-1) while negative by Ig/TR. While in some patients the correlation of the DNA-based methods was very good, others had several subsequent follow-up samples with poor correlation (> 1 log difference) suggesting distinct biology of the disease. MRD levels determined by BCR-ABL1 DNA quantification had the best predictive value for overall survival (the best separation of survival curves was at week 12 of treatment with 10e-3 cut-off: BCR-ABL1 DNA, p=0.0003; Ig/TR, p=0.017; BCR-ABL1 mRNA, not significant).The proportion of samples with BCR-ABL1 DNA levels higher than Ig/TR by more than 1 log increased with time on treatment (day 15: 10% (2/20); day 33: 26% (7/27); week 12: 39% (7/18); week 22: 50% (7/14)). Importantly, presence of samples with this poor correlation was prognostically relevant as patients with BCR-ABL1 levels higher than Ig/TR by more than 1 log at the end of induction treatment (day 33) had significantly worse outcome (2 years OS 84+/-9% (n=21) vs. 28+/-23% (n=6) for correlating and non-correlating patients, respectively; p=0.004).Conclusion:The BCR-ABL1 quantification at genomic level probably provides the most accurate measurement of leukaemic burden. Due to multi-lineage involvement in some Ph+ ALL as well as occasional presence of subclones with different Ig/TR markers, quantification of the BCR-ABL1 gene breakpoint is more reliable than Ig/TR monitoring. Moreover, in contrast to the BCR-ABL1 transcript, measurement of the gene is not influenced by possible variability in expression between cell subtypes or during concurrent treatment. Importantly, the MRD levels determined by BCR-ABL1 DNA-based testing have higher predictive value than Ig/TR DNA or BCR-ABL1 transcript quantification. The poorer outcome observed in the subset of patients in whom BCR-ABL1 DNA-based MRD levels were higher than Ig/TR-based MRD detection possibly reflects multilineage involvement, suggesting, that some cases with minor-BCR-ABL1+ ALL are in fact CML cases identified in a lymphoid blast crisis. In view of their poor outcome, these cases might be candidates for early SCT or alternative treatments. The most reliable method for MRD detection in BCR-ABL1+ ALL needs to be further investigated within a large therapeutic protocol.Support: GAUK 554214; MH CZ-DRO UH Motol 00064203; NHMRC Australia APP1057746. DisclosuresMachova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

The Genomic and Epigenomic Landscapes of Blast Crisis Transformation in Chronic Myeloid Leukemia

The transition from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is characterized by reprogramming of the CML transcriptome (Radich et al. PNAS 2006), and shortened survival. Current models propose genomic instability as causal in BC transformation with enhanced DNA damage and impaired DNA repair inducing genetic mutations (ranging from large chromosomal aberrations to point mutations), altered gene function, and eventually BC transformation (Perrotti et al. JCI 2010). Consistent with this model are the phenomena of BC clonal evolution, and the increased frequency of ABL kinase domain mutations found in BC. Because different mutational processes are associated with distinct cancer-specific mutation signatures (Alexandrov et al. Nature 2013), this model also predicts the existence of a CML-specific mutation signature. In addition, recent work has highlighted the importance of epigenetic alterations in hematologic malignancies (Shih et al., Nat. Rev. Cancer, 2012). However, we lack a complete understanding of the type or frequency of genetic alterations in BC, and the relative contribution of genetic vs. epigenetic events in reprogramming the BC transcriptome.To address these knowledge gaps, we analyzed the CML progression genome, epigenome, and transcriptome in 12 CP/BC sample pairs. Whole-genome sequencing revealed the CML genome to be relatively stable with respect to structural variations, indels, and somatic single nucleotide variants. The average number of nonsynonymous coding mutations per BC genome was 5, placing the BC coding genome in the same mutation frequency range as AML and ALL genomes (Alexandrov et al. Nature 2013). In addition, we identified a novel mutation signature in all CML samples suggesting a CML-specific mutational process. 1175 genes were 'hit' by genomic, mostly copy number, alterations in >1 sample, and included TCR genes and Ikaros (IKZF1) among lymphoid BC pairs. Only 21 recurrently altered genes were affected by somatic SNVs or indels, with resistance-associated ABL1 mutations being commonest.We next used DNA methylation arrays to assess the BC epigenome, and found 20,651 CpG sites (out of 455,187) to be hyper-methylated, and 3225 to be hypo-methylated in BC compared to CP. Combined methylome and transcriptome analysis demonstrated an inverse relationship between methylation and expression changes at a subset of CpG sites enriched at promoters. Genes with increased methylation/decreased expression or decreased methylation/increased expression included those involved in cell cycle control/heme biosynthesis, and molecular mechanisms of cancer/G-protein coupled receptor signaling/MAPK signaling respectively. Unsupervised methylation-based clustering segregated samples into CP, lymphoid BC and myeloid BC groups, recapitulating expression-based clustering, and further supporting a functional role for DNA methylation in BC transcriptional reprogramming.We next performed an integrative analysis by combining the genome, methylome, and transcriptome datasets, and included data from 34 additional CML samples. Top ranking candidate genes included epigenetic modifiers, and hematopoetic differentiation- and stem cell-related genes. Functional analysis of candidate genes and epigenetic processes using genetic and epigenetic drug-based approaches are ongoing.In summary, we conclude that: 1. The genomic and epigenomic landscapes in BC are characterized by a modest number of recurring events in the former, but consistent and striking differences in the latter, 2. The BC methylome is functionally associated with the robust gene expression changes found in BC, and 3. Epigenetic modifier drugs may be of use in reversing the gene expression changes characteristic of BC. DisclosuresChuah:Children International: Honoraria; Novartis: Honoraria; Bristol Meyers Squibb: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau.

Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance.Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors.In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008).Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. DisclosuresNo relevant conflicts of interest to declare.

Dasatinib first-line: Multicentric Italian experience outside clinical trials

Dasatinib was approved for the treatment of chronic phase (CP) chronic myeloid leukemia (CML) patients in first line therapy based on the demonstration of efficacy and safety reported in patients enrolled in clinical trials. We describe a multicentric Italian “real-life” experience of dasatinib used as frontline treatment outside clinical trials. One hundred and nine patients (median age 54 years) were treated from January 2012 to December 2013. Increased incidence of high risk patients were detected according to stratification (26% according to Sokal score, 19% according to Euro score and 16% according to EUTOS) when compared to company sponsored studies. Median time from diagnosis to start of dasatinib was 18 days. Ten patients received unscheduled starting dose (6 patients 50mg and 4 patients 80mg QD), whereas 99 patients started with 100mg QD. At 3 months, 92% of patients achieved a BCR-ABL ratio less than 10%. At 6 months, the rate of CCyR was 91% and the rate of MR3 was 40%, with 8% of the patients reaching MR4.5. Ninety-three patients were evaluable at 12 months: the rate of MR3 was 62%, with MR4.5 being achieved by 19% of the patients. At a median follow-up of 12 months, 27 patients (24.7%) were receiving the drug at reduced dose. Two patients (1.8%) experienced a lymphoid blast crisis and the overall incidence of resistance was 8%. As regards safety, the major side effects recorded were thrombocytopenia, neutropenia and pleural effusions, which occurred in 22%, 10% and 8% of patients, respectively. Present results, achieved in a large cohort of patients treated outside clinical trials, further confirm the efficacy and safety of dasatinib as firstline treatment in CML.

Characteristics of the TCR Vβ repertoire in imatinib-resistant chronic myeloid leukemia patients with ABL mutations.

Diversity in the T cell receptor (TCR) repertoire provides a miniature defense ability for the T cell immune system that may be related to tumor initiation and progression. Understanding the T cell immune status of leukemia patients is critical for establishing specific immunotherapies. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 4 cases with imatinib-resistant CML in blast crisis (BC-CML) with abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase domain mutations (KDMs). Examination of TCR V expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) and GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in BC-CML patients with different KDMs, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample. Intriguingly, a relatively highly expanded Vβ9 clone with the same length as complementarity- determining region 3 (CDR3) (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the only CML patient in myeloid blast crisis (MBC-CML). In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon observed in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency of these patients. In addition, clonally expanded Vβ9 T cell clones may indicate a specific immune response to leukemia-associated antigens in LBC-CML patients.

Acute Lymphoblastic Leukemia evolving from atypical Chronic Myelogenous Leukemia: Case Report and Review of the Literature

Atypical chronic myeloid leukemia (aCML) is a rare chronic myeloproliferative disorder characterized by leukocytosis, absence of Philadelphia chromosome or BCR-ABL rearrangement, and marked myeloid dysplasia. The diagnosis of aCML is difficult and challenging, more so if the initial presentation is with blast crisis. In the absence of characteristic mutational profile, blast crisis of aCML can lead to significant diagnostic dilemma. We describe a case of refractory acute lymphoblastic leukemia (ALL), needing critical evaluation of hemopathological and cytogenetic abnormalities. It was hypothesized that patient had undiagnosed aCML and presented with lymphoid blast crisis. To our knowledge, this is the first described case of aCML presenting with lymphoid blast crisis.

BACH2 promotes Lineage-Specific Fate Decisions in BCR-ABL1-Driven Leukemia

Background and Hypothesis: CML and Ph+ ALL are both driven by the oncogenic BCR-ABL1 tyrosine kinase and CML can progress into lymphoid blast crisis (LBC), which is clinically and biologically indistinguishable from Ph+ ALL. We hypothesize that the stark differences in phenotype and clinical outcomes between CML and Ph+ ALL/LBC are due to lineage-specific transcription factors. Our group recently identified BACH2 as a B cell-specific transcription factor that mediates negative selection at the pre-B cell receptor checkpoint and functions as a tumor suppressor in Ph+ ALL and LBC (Swaminathan et al., Nature Med 2013). Here we report the surprising finding that BACH2 mediates lineage-specific fate decisions in BCR-ABL1 driven leukemia: while a potent tumor suppressor in B cell lineage in Ph+ ALL and LBC, BACH2 is required for survival and self-renewal of myeloid lineage CML cells.Results: A gene expression analysis in 99 patients with CML (Radich et al, 2006) revealed that patients with higher blast counts showed significantly higher gene expression values of BACH2 (P=8.88 x 10-9), suggesting that BACH2 mRNA levels increase with CML progression. To determine the mechanistic role of BACH2 in the progression of CML, we studied genetic deletion of BACH2 in a CML mouse model. To this end, we transformed Lin-kit+Sca-1+(LSK) cells from Bach2+/+ and Bach2-/- bone marrow with BCR-ABL1. We compared these cells alongside with Bach2+/+ and Bach2-/- pre-B cells that were transformed with BCR-ABL1 as a model for Ph+ ALL and LBC. Bach2+/+ and Bach2-/- CML-like and Ph+ ALL/LBC-like leukemia cells were then tested in a series of functional experiments to test their ability to initiate fatal leukemia in transplant recipients. While deletion of BACH2 accelerated B cell lineage leukemia (Ph+ ALL/LBC-like) in transplant recipients, we noted the opposite outcome in transplant experiments with myeloid lineage CML cells: All mice receiving Bach2+/+BCR-ABL1-transformed LSK cells developed CML-like disease within 60 days whereas recipients of Bach2-/- cells did not develop CML-like disease (Bach2+/+ vs Bach2-/-, median survival time= 33 days vs undefined; P=0.001). Additionally, we observed that deletion of Bach2 loss reduced both the colony formation ability (P=0.0059) and the S phase proliferation potential (P=0.0075) of the CML progenitors. These results were in contrast to those observed in Ph+Bach2+/+ and Bach2-/- ALLs. Reconstitution of Bach2 in Bach2-/- CML cells rescued its colony forming ability (P= 0.023). These findings were confirmed in primary human CML cells from two patients with CML in chronic phase: Inducible overexpression of Bach2 in patient-derived CML cells conferred a selective proliferative advantage of BACH2 overexpressing cells over time, in comparison to empty vector controls. Bach2 triggered a survival program in human and mouse CML cells, in contrast to B cell lineage Ph+ ALL and LBC cells. Interestingly, Bach2 did not provide a selective growth advantage to untransformed myeloid and multi-lineage progenitor cells. This demonstrates that Bach2 drives lineage-specific fate decisions and cooperates with the transforming BCR-ABL1 oncogene. Utilizing an in vitro lineage-switch assay overexpressing CEBPα we characterized the opposing roles of BACH2 in Ph+ ALL and CML. Cell viability and colony formation assays demonstrated that absence of Bach2 results in increased survival and proliferation of Ph+ ALL cells. In striking contrast, however, CEBPα-induced myeloid reprogramming induced cell cycle arrest and suppressed colony formation in Bach2-deficient cells (P=0.0001).Conclusion: While BACH2 is primarily expressed in the B cell lineage, functioning as a tumor suppressor in Ph+ ALL, we report here the surprising finding that BACH2 is required for survival and self-renewal of myeloid lineage CML cells. BACH2 mediates lineage-specific fate decisions in BCR-ABL1 driven leukemia and represents one example of how conversion of lineage identity (e.g. from CML to LBC) may impact specific requirement for malignant transformation. For instance, deletions of BACH2 at 6q15 are common in CML at the time of LBC progression but are not found in CML chronic phase or myeloid blast crisis. DisclosuresNo relevant conflicts of interest to declare.

Divergent Lineage-Specific Functions of PP2A in Chronic Phase CML and Lymphoid Blast Crisis

Background & Hypothesis: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase and functions as negative regulator of Akt- and Erk-signaling. PP2A attenuates downstream signaling of BCR-ABL1 and thereby functions as important tumor suppressor in chronic myeloid leukemia (CML). PP2A-activating drugs have been proposed for the treatment of CML and eradication of tyrosine kinase inhibitor resistant leukemia stem cells in CML (Neviani et al., 2013). Studying Cre-mediated ablation of PP2A in mouse models for BCR-ABL1-driven leukemia including myeloid CML in chronic phase (CML-CP), B lymphoid blast crisis (LBC) and B cell lineage Ph+ ALL, we were able to provide genetic evidence for this treatment concept in CML. Seemingly paradoxically, however, inducible ablation of PP2A in B cell lineage LBC and Ph+ ALL cells has the opposite effect and induced rapid cell death. These findings highlight fundamental differences in the regulation of Akt- and Erk-signaling in myeloid lineage and B cell lineage cells and have implications for specific therapy concepts that distinguish between chronic phase CML and lymphoid blast crisis/ Ph+ ALL.Results: Interestingly, PP2A protein levels are significantly higher in B cell lineage Ph+ ALL and CML lymphoid blast crisis (LBC) compared to chronic phase CML. To explore the function of PP2A during early B cell development, we deleted the α isoform of the PP2A subunit A in pro-B cells by introducing Mb1-Cre into Ppp2r1afl/fl mice. Analysis of bone marrow from Ppp2r1afl/flMb1-Cre (PP2A-cko) mice showed obvious B cell development block at the pre-B cell receptor checkpoint, although V(D) recombination and RAG1/2 activity were intact in PP2A-cko pre-B cells. Since high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL, we studied the function of PP2A in mouse models for B cell lineage ALL including Ph+ ALL and CML lymphoid blast crisis (LBC). After Cre-induced deletion of Ppp2r1a, we observed reduced protein expression of both PP2A subunit A and subunit C accompanied with decreased PP2A phosphatase. The PP2A-cKO ALL cells showed normal proliferation but significantly impaired colony formation capability and increased apoptosis, both of which could be rescued by overexpression of wildtype PP2A in PP2A-cKO ALL cells. We next transplanted luciferase expressing PP2A-KO ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although all of those mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles suggesting that these mice died from clones that had escaped Cre-mediated deletion of PP2A. Cre-mediated deletion increased phosphorylation levels of Stat5 and Erk, resulted in accumulation of p53 and increased output of the PI3K-AKT signaling pathway as measured by increased phosphorylation of FoxO factors, p70S6K and S6 ribosomal protein.Based on the involvement of PI3K-AKT in glucose metabolism, we measured the effects of inducible PP2A-deletion on glycolytic function in CML and Ph+ ALL/LBC cells. Interestingly, inducible deletion of PP2A induced profound imbalances of glucose metabolism in B cell lineage Ph+ ALL/LBC but not myeloid lineage CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-KO ALL cells indicated impaired balance of glycolytic flux and may account for increased apoptosis of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-cKO cells. In agreement with reported tumor suppressor role of PP2A in CML cells (Neviani et al., 2005), we observed that CEBPα-driven lineage conversion of B cell lineage Ph+ ALL/LBC into not myeloid lineage.Conclusion: Here we reported the requirement of PP2A in normal B cell development and lineage-specific oncogenic role of PP2A in BCR-ABL1-driven leukemia. Divergent roles of PP2A in Ph+ ALL/LBC and myeloid lineage CML may lead to future insights into mechanisms of LBC-transformation of CML and lineage-specific requirement of BCR-ABL1-transformation. DisclosuresNo relevant conflicts of interest to declare.

Modeling Chronic Myeloid Leukemia in Immunodeficient Mice Reveals an Inflammatory State with Expansion of Aberrant Mast Cells and Accumulation of Pre B Cells

Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To ultimately achieve a cure for CML, there is a need to establish improved disease models allowing increased understanding of disease pathogenesis and to evaluate novel therapeutics.Aim: In this study, we investigated if retroviral expression of BCR-ABL1 in cord blood (CB) CD34+ progenitor cells transplanted into NOD/SCID IL2–receptor gamma deficient (NSG) mice would recapitulate features observed in CP CML.Methods: CD34+ progenitor cells from human CB were transduced with a retroviral vector expressing BCR-ABL1 and subsequently transplanted into NSG mice. Mice were sacrificed at signs of illness and bone marrow (BM) and spleen cells were analyzed using flow cytometry, immunohistochemistry, FISH and RNA sequencing. Secondary transplants were also performed. BM mononuclear cells (MNC) from CP CML patients were analyzed using flow cytometry and FISH.Results: For the majority of the BCR-ABL1 transplanted (BA) mice, signs of disease were evident within 100 days post transplantation. Spleens were enlarged and both control and BA mice showed robust human engraftment in both BM and spleen. Expression of BCR-ABL1 also induced expansion of the transduced cells. In addition to the previously described features of this humanized transplantation model such as a mild expansion of human myeloid cells, BCR-ABL1 expression induced an inflammatory-like state in the BM and spleen of the BA mice as evident by an increase of human macrophages/histiocytes and T cells. In addition, an expansion of aberrant mast cells in BA mice was observed. Secondary transplantations failed to induce a similar disorder as in the primary mice, but long-term engraftment was achieved, showing only mast cells in secondary BA mice. These mast cells aberrantly expressed CD25 (IL2RA), a cell surface receptor normally only expressed on neoplastic mast cells. This was in contrast to secondary control mice where the long-term engraftment was dominated by B cells.Because CP CML can progress into lymphoid BC, we next explored if BCR-ABL1 expression in CB CD34+ cells would show a disturbance also of the B cell lineage. We observed a striking block at the pre B cell stage, resulting in an accumulation of pre B cells in the BM of BA mice. RNA sequencing of sorted BCR-ABL1-expressing pre B cells from BA and control mice revealed that BCR-ABL1 expression results in a significant de-regulation of about 700 genes. Among the up-regulated genes, many were shown to be involved in inflammation and apoptosis. Finally, by sorting BM MNCs from patients with CP CML into different populations of B cell progenitors and performing FISH for BCR-ABL1, we confirmed that a similar block in B cell differentiation is present in CP CML patients.Conclusion: This humanized mouse model of CML reveals previously unexplored features of CP CML, including expansion of aberrant mast cells and a block at the pre B cell stage, and should provide a valuable model for future studies of mechanisms involved in the disease pathogenesis of CML. DisclosuresNo relevant conflicts of interest to declare.

Activating Mutations Observed in De Novo Acute Myeloid Leukemia Are Also Present in a Subset of Philadelphia Chromosome (Ph)-Positive Leukemia Patients with BCR-ABL1-Independent Resistance to ABL1 Kinase Inhibitors

Activating Mutations Observed in De Novo Acute Myeloid Leukemia Are Also Present in a Subset of Philadelphia Chromosome (Ph)-Positive Leukemia Patients with BCR-ABL1-Independent Resistance to ABL1 Kinase Inhibitors

DPPIV (CD26) as a novel stem cell marker in Ph+ chronic myeloid leukaemia.

The concept of leukaemic stem cells (LSCs) has been developed to explain the complex cellular hierarchy and biology of leukaemias and to screen for pivotal targets that can be employed to improve drug therapies through LSC eradication in these patients. Some of the newly discovered LSC markers seem to be expressed in a disease-specific manner and may thus serve as major research tools and diagnostic parameters. A useful LSC marker in chronic myeloid leukaemia (CML) appears to be CD26, also known as dipeptidylpeptidase IV. Expression of CD26 is largely restricted to CD34(+) /CD38(-) LSCs in BCR/ABL1(+) CML, but is not found on LSCs in other myeloid or lymphoid neoplasms, with the exception of lymphoid blast crisis of CML, BCR/ABL1p210 + acute lymphoblastic leukaemia, and a very few cases of acute myeloid leukaemia. Moreover, CD26 usually is not expressed on normal bone marrow (BM) stem cells. Functionally, CD26 is a cytokine-targeting surface enzyme that may facilitate the mobilization of LSCs from the BM niche. In this article, we review our current knowledge about the biology and function of CD26 on CML LSCs and discuss the diagnostic potential of this new LSC marker in clinical haematology.

Early T cell precursor lymphoid blast crisis of chronic myeloid leukemia – A novel transformation

Early T cell precursor lymphoid blast crisis of chronic myeloid leukemia – A novel transformation

ABL tyrosine kinase inhibitor-induced pulmonary alveolar proteinosis in chronic myeloid leukemia.

Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by the accumulation of eosinophilic periodic acid Schiff-positive material in the intra-alveolar and bronchiolar spaces. Tyrosine kinase inhibitors, including imatinib, nilotinib, and dasatinib, have shown excellent efficacy in the treatment of chronic myeloid leukemia (CML). We report a case of acquired PAP in a patient with CML receiving tyrosine kinase inhibitors. A 67-year-old man with CML presented with progressive back pain 5 months after starting imatinib treatment. Acquired PAP was diagnosed based on physical, radiographic, and histopathological findings. The presence of granulocyte-macrophage colony-stimulating autoantibodies suggested that autoimmune mechanisms were involved in the pathogenesis. Interestingly, PAP developed in association with imatinib and dasatinib administration, but not with nilotinib treatment. The patient died of refractory leukemia in lymphoid blast crisis with a newly emerged T315I mutation. Although the incidence is very rare, imatinib and dasatinib associated with PAP development has been reported. Meanwhile, PAP in nilotinib-treated patients has not been reported. Our observation in one patient receiving multiple TKIs suggests that nilotinib may be safer than imatinib or dasatinib in avoiding the development or exacerbation of PAP.

Chronic myeloid leukemia with an e1a3 BCR-ABL fusion protein: transformation to lymphoid blast crisis

Chronic myelogenous leukemia (CML) results from the neoplastic transformation of a hematopoietic stem cell. CML is cytogenetically characterized by the presence of the Philadelphia chromosome (Ph’). Most patients with CML express e13a2 or e14a2 mRNAs that result from a rearrangement of the major breakpoint cluster regions (M-BCR) generating the 210-kDa (p210BCR-ABL) fusion proteins b2a2 or b3a2 respectively. The e1a3 CML-related atypical translocation has been reported with an indolent clinical course, low leukocyte count, long chronic phase even without treatment and good response to therapy. We report the case of a patient initially diagnosed as CML in chronic phase whose cells expressed the e1a3 variant. The patient readily responded to imatinib 400 mg with the achievement of a rapid complete cytogenetic response and the normalization of the blood count values, but after 5 months transformed into lymphoid blast crisis.

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.

Liposomal daunorubicin, fludarabine, and cytarabine (FLAD) as bridge therapy to stem cell transplant in relapsed and refractory acute leukemia.

Therapeutic options for patients with relapsed or refractory acute leukemia are still undefined and often unsatisfactory. We report the outcome of 79 patients with relapsed-refractory acute leukemia treated with fludarabine, cytarabine, and liposomal daunorubicin (FLAD regimen) followed by hematopoietic stem cell transplantation (HSCT), when clinically indicated, between May 2000 and January 2013. Forty-one patients had acute myeloid leukemia (AML), and 38 had acute lymphoblastic leukemia (ALL). Two patients with myeloid blast crises of CML and three with lymphoid blast crises were included in the AML and ALL subgroups, respectively. Median age was 48 years (range 13-77). FLAD was well tolerated with negligible, nonhematological toxicity. Six patients (7.5 %) died before response evaluation. Forty-seven patients achieved hematologic complete response (CR). Complete remission rate was 53 and 65 % among AML and ALL patients, respectively. No CR was recorded among 11 refractory AML patients. Twenty-four patients (30 %) underwent HSCT. Nine patients received stem cells from an HLA identical sibling, and 15 from an alternative donor (3 unrelated matched, 12 haploidentical sibling). Median overall survival in AML and ALL patients receiving FLAD therapy was 9 and 8 months, respectively. A 5-year projected OS for patients receiving the whole program (FLAD + HSCT) was 24 % for AML patients (median survival 43 months), 28 % for ALL patients treated in relapse (median survival 15 months), and 0 % for ALL patients treated for refractory disease. In this paper, we show that FLAD seems to be an effective bridge therapy to HSCT for a part of poor prognosis acute leukemia patients. However, prospective studies are needed to confirm our results.

Differential analysis of BM cell morphology, immunophenotypic, cytogenetic characters and prognosis between myeloblastic and lymphoblastic crisis of CML

This study was purposed to investigate the difference of morphology, immunophenotype, cytogenetic features and prognosis between myeloid blast crisis and lymphoid blast crisis of chronic myelogenous leukemia (CML). A total of 31 patients with CML in blastic crisis in Department of Hematology, the First Affiliated Hospital of Xi'an Jiaotong University school of Medicine from 2009 January to 2014 January were enrolled in this study. Out of 31 CML patients, 24 cases were patients with myeloid blast crisis and other 7 cases were patients with lymphoblastic crisis. The clinical data, blast cell percentage in peripheral blood and bone marrow, eosinophil and basophil percentage, immunophenotype, cytogenetic characteristics and prognosis were analyzed. The results indicated that there was no significant difference of blastic cell percentage in peripheral blood and bone marrow of CML with myeloid blast crisis, and the eosinophil and basophil cells could be easily detected. The ratio of blastic cells in BM was higher than that in PB in lymphoid blastic crisis of CML, eosinophil and basophil cells were rare. 7 cases of CML with lymphoid blastic crisis were B ALL with CD10, CD19, CD34, HLA-DR expression, and 2 cases with CD13 and CD33 expression. The lymphoid score was in all CML patients with lymphoid blastic crisis was greater than or equal to 1.5;and 2 patients with CD13 and CD33 expression, and with 1 myeloid score.24 cases of myeloid blastic crisis of CML patients mainly expressed CD33, CD13, CD38, CD34, CD11b and HLA-DR, and their myeloid score greater than or equal to 2, among them the lymphoid scores of 2 patients were 0.5 and 1 score, respectively. All the 31 patients showed 100% Ph(+) chromosome, among them 3 cases also showed other new chromosome aberrations. There was no significant difference of overall survival rate between lymphoid and myeloid blastic crisis of CML, but the overall survival rate of patients treated with tyrosine kinase inhibitor (TKI ) was higher than that in the patients without TKI treatment. It is concluded that eosinophil and basophil cells in peripheral blood of lymphoid blastic crisis were less than that of CML patients with myeloid blastic crisis. Lymphoid blastic crisis of CML patients occurred mostly in B ALL cases with expression of CD10 and CD19. Patients with myeloid blastic crisis of CML mainly expressed CD33, CD13, CD38, CD34, CD11b and HLA-DR, and could be accompanied by other lineage antigen expression, but the score was less than 2. New chromosome aberration is easily observed in myeloid blastic crisis of CML. There is no significant difference of overall survival rate of between CML patients with lymphoid and myeloid blastic crisis, but the overall survival rate of patients treated with TKI is higher than the patients without TKI treatment.

DNA copy number alterations mark disease progression in paediatric chronic myeloid leukaemia

Early recognition of children with chronic phase chronic myeloid leukaemia (CML-CP) at risk for developing a lymphoid blast crisis (LyBC) is desirable, because therapy options in CML-LyBC are limited. We used Multiplex Ligation-dependent Probe Amplification to determine whether B-cell lymphoid leukaemia-specific copy number alterations (CNAs) (e.g. IKZF1, PAX5, CDKN2A deletions) could be detected in CML-CP and may be used to predict disease progression to LyBC. CNAs were detected in all patients with CML-LyBC, but in none of the 77 patients with CML-CP. Based on this study we conclude that CNAs remain a hallmark of disease progression.

The Cytogenetic Profile In Lymphoid and Myeloid CML Blast Crisis

IntroductionIn acute leukemias, specific cytogenetic aberrations frequently correlate with myeloid or lymphoid phenotype of blasts and influence risk stratification. In chronic myeloid leukemia (CML) blast crisis (BC) it is not clear whether myeloid or lymphoid phenotype of blasts could be distinguished by specific chromosomal aberrations and have prognostic value. At diagnosis of CML, major route additional cytogenetic aberrations (ACA) like +8, i(17)(q10), +19, +der(22)t(9;22)(q34;q11) and minor route ACA like -X, del(1)(q21), del(5)(q11q14), +10,-21, resulting in an unbalanced karyotype have been described to adversely affect outcome. Patients with minor route ACA (for example reciprocal translocations other than the t(9;22)(q34;q11) (e.g. t(1;21), t(2;16), t(3;12), t(4;6), t(5;8), t(15;20)) resulting in a balanced karyotype did not show differences in overall survival and progression free survival compared to patients with the standard translocation, a variant translocation or the loss of the Y chromosome. Aim of this study was to analyze the impact of the phenotype (myeloid or lymphoid) on time to BC and on cytogenetic pattern. Methods73 out of 1524 evaluable patients (4.8%) randomized until March 2012 to the German CML-Study IV (a 5-arm trial to optimize imatinib therapy) progressed to BC. Cytogenetic data of 23 out of 32 patients with myeloid BC and 14 out of 21 patients with lymphoid BC were available. In 15 patients, cytogenetic analysis were missing whereas 2 and 3 patients had megakaryoblastic and mixed phenotype, respectively and were not considered in this analysis. Karyotypes of lymphoid and myeloid BC were divided in major route and minor route ACA and balanced and unbalanced karyotypes. Categorical covariates were compared with Fisher's exact test, while continuous covariates were compared with the Mann-Whitney-Wilcoxon test. Survival probabilities after BC were compared using the log-rank test. ResultsOut of 23 patients with myeloid BC, 14 (61%) had major route unbalanced ACA (n=10) or minor route unbalanced ACA (n=4), 4 had minor route balanced ACA and 5 patients had the translocation t(9;22)(q34;q11) or a variant translocation t(v;22) without ACA.13 out of 14 (93%) patients with lymphoid BC had major route unbalanced (n=10) or minor route unbalanced ACA (n=3) and 1 had the standard translocation t(9;22)(q34;q11) only. Between myeloid and lymphoid BC, the difference in the distribution of unbalanced ACA was apparent, but not statistically significant (p=0.06). The most frequently observed major route ACA was trisomy 8 in both groups (7 vs. 6), +der (22)t(9;22)(q34;q11) was more frequently found in myeloid than lymphoid BC (6 vs. 2), +19 was found in both phenotypes (3 vs. 3) whereas an isochromosome i(17)(q10) and an isoderivative chromosome ider(22)t(9;22)(q34;q11) were less frequent and found only in myeloid BC (1 for each vs 0 for each aberration). In lymphoid BC, 5 of 14 patients (36%) had ACA which involved chromosome 7 (del(7)(q22) and -7) whereas in myeloid BC only 2 patients (9%) had -7 (p=0.08). The balanced karyotype with a translocation t(3;21)(q26;q22) and the translocation t(9;11)(p22;q23) described in acute myeloid leukemia was observed in 3 patients with myeloid CML (2 and 1, respectively) and in none with lymphoid phenotype. No differences were observed in time to BC for patients with lymphoid vs. myeloid BC (p=0.31, median time: 409 vs. 453 days) and survival after onset of BC (p=0.9, median time: 544 vs. 284 days). ConclusionsThe proportion of unbalanced karyotypes was higher in lymphoid than in myeloid BC. In lymphoid BC alterations of chromosome 7 were more often present whereas +der(22)t(9;22)(q34;q11) was observed more frequently in myeloid BC. The reciprocal translocations t(3;21)(q26;q22) and t(9;11)(p22;q23) described in acute myeloid leukemias were only observed in myeloid BC. However these cytogenetic differences do not seem to alter the course of BC. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hehlmann:Novartis: Research Funding; BMS: Consultancy, Research Funding. Hochhaus:Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Travel Other. Müller:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding; Ariad: Honoraria. Saussele:Pfizer: Honoraria; BMS: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Research Funding, Travel Other.