BCR-ABL Translocation Research Articles

Laboratory testing in BCR-ABL1-like (Philadelphia-like) B-lymphoblastic leukemia/lymphoma.

BCR-ABL1-like B-lymphoblastic leukemia/lymphoma (BCR-ABL1-like B-ALL), also known as Philadelphia-like (Ph-like) ALL, is a neoplasm of B-lineage lymphoblasts characterized by a pattern of gene expression similar to that of B-ALL with the BCR-ABL1 translocation but lacking the BCR-ABL1 fusion protein. The diagnosis of BCR-ABL1-like B-ALL is associated with a high rate of relapse and poor clinical outcomes. In recognition of the difficulty in screening these leukemias for diagnostic workup, the 2016 update/revision to the World Health Organization (WHO) 2008 edition included BCR-ABL1-like B-ALL as a provisional entity. This review addresses the various clinical considerations and methodologies currently used in the pathologic diagnosis, subclassification, and molecular characterization of cases of BCR-ABL1-like B-ALL, with particular attention paid to emerging methods useful in identification of molecular lesions potentially amenable to targeted therapy.

Myeloproliferative neoplasms with concurrent BCR–ABL1 translocation and JAK2 V617F mutation: a multi-institutional study from the bone marrow pathology group

AbstractMyeloproliferative neoplasms arise from hematopoietic stem cells with somatically altered tyrosine kinase signaling. Classification of myeloproliferative neoplasms is based on hematologic, histopathologic and molecular characteristics including the presence of the BCR–ABL1 and JAK2 V617F. Although thought to be mutually exclusive, a number of cases with co-occurring BCR–ABL1 and JAK2 V617F have been identified. To characterize the clinicopathologic features of myeloproliferative neoplasms with concomitant BCR–ABL1 and JAK2 V617F, and define the frequency of co-occurrence, we conducted a retrospective multi-institutional study. Cases were identified using a search of electronic databases over a decade at six major institutions. Of 1570 patients who were tested for both BCR–ABL1 and JAK2 V617F, six were positive for both. An additional five patients were identified via clinical records providing a total of 11 cases for detailed evaluation. For each case, clinical variables, hematologic and genetic data, and bone marrow histomorphologic features were analyzed. The sequence of identification of the genetic abnormalities varied: five patients were initially diagnosed with a JAK2 V617F+ myeloproliferative neoplasm, one patient initially had BCR–ABL1+ chronic myeloid leukemia, while both alterations were identified simultaneously in five patients. Classification of the BCR–ABL1-negative myeloproliferative neoplasms varied, and in some cases, features only became apparent following tyrosine kinase inhibitor therapy. Seven of the 11 patients showed myelofibrosis, in some cases before identification of the second genetic alteration. Our data, reflecting the largest reported study comprehensively detailing clinicopathologic features and response to therapy, show that the co-occurrence of BCR–ABL1 and JAK2 V617F is rare, with an estimated frequency of 0.4%, and most often reflects two distinct ('composite') myeloproliferative neoplasms. Although uncommon, it is important to be aware of this potentially confounding genetic combination, lest these features be misinterpreted to reflect resistance to therapy or disease progression, considerations that could lead to inappropriate management.

Description and prognostic significance of the kinetics of minimal residual disease status in adults with acute lymphoblastic leukemia treated with HyperCVAD.

HyperCVAD is a commonly-used regimen for adults with newly-diagnosed acute lymphoblastic leukemia (ALL). However, relatively little is known about the application of minimal residual disease (MRD) detection with this treatment. To address this, we studied 142 adults with ALL treated with hyperCVAD over a 10-year period who had MRD assessed by either multi-parameter flow cytometry or (for patients with Philadelphia chromosome positive ALL) reverse transcriptase polymerase chain reaction for the BCR-ABL1 translocation. In a multivariate analysis, patients who achieved MRD negativity (MRDNeg ) at any point had significantly better overall survival (OS; hazard ratio [HR] 0.43; P = .01) and event-free survival (EFS; HR 0.27; P < .01). Of 121 patients with MRD assessed at various points within 90 days of starting hyperCVAD, 50% (n = 61) had achieved MRDNeg . Among those that became MRDNeg , the median time to MRDNeg was 68 days. Time to MRDNeg was significantly associated with EFS (P = .009), but not OS (P = .19), implying increasingly better EFS the earlier MRDNeg is achieved. These data add to our understanding of MRD assessment during treatment with hyperCVAD, aide clinicians with predicting relapse risk, and provide additional historical data on which future clinical trials can be designed.

Unique amplification of BCR-ABL1 gene fusion in a case of T-cell acute lymphoblastic leukemia

BackgroundABL1 gene translocations can be seen in precursor T-acute lymphoblastic leukemia (T-ALL). The typical translocation partner is the NUP214 gene. BCR-ABL translocations are relatively rare in this entity. Furthermore, while there have been unique patterns of amplification noted among the NUP214-ABL fusion genes, there have been few such reports among cases with BCR-ABL fusion genes.Case presentationHere we report a unique case of a 44-year old patient with T-ALL in which the blasts demonstrated a derivative chromosome 9 involving a 9;22 translocation and a dicentric Philadelphia chromosome 22 with a homogeneously staining region at the interface of the 9;22 translocation, leading to BCR-ABL1 gene amplification. Fluorescence in-situ hybridization (FISH) showed abnormal BCR/ABL1 fusions with the BCR-ABL1 gene amplification in 48% of the interphase cells analyzed. The translocation was confirmed by SNP array.ConclusionsWe present a novel derivative chromosome 9 that shows BCR-ABL gene fusion along with a dicentric Philadelphia chromosome 22 with BCR-ABL1 gene amplification. This is a unique pattern of BCR-ABL fusion which has never been described in T-ALL. It is significant that the patient responded to standard treatment with the CALGB 10403 protocol and supplementation with a tyrosine kinase inhibitor. Identification of additional patients with this pattern of BCR-ABL fusion will allow for enhanced risk assessment and prognostication.

Abstract 765: Prognostic impact of Philadelphia chromosome in elderly acute lymphoblastic leukemia patients ineligible for hematopoietic stem cell transplantation

Abstract Elderly acute lymphoblastic leukemia (ALL) patients are often unfit for neither dose-intensive chemotherapy nor stem cell transplantation, thus their clinical outcomes have been dismal. The increased incidence of BCR-ABL translocation with increasing age also contributed to poor prognosis but, it was our conjecture that the introduction of potent tyrosine kinase inhibitors (TKI) targeting ABL, BCR-ABL translocation might not deliver negative prognostic impact in elderly ALL. Considering that many elderly Ph- ALL patients do not tolerate cytotoxic chemotherapy required for effective tumor control, we supposed that efficacy of ABL TKIs and their combination strategy with low dose cytotoxic chemotherapy would result in better survival for elderly Ph+ ALL compared to their Ph- counterparts. This study was a multicenter longitudinal cohort study of elderly de novo ALL (≥ 60 years) recruited from 11 hospitals in Korea from January 2005 to December 2015. We considered Ph+ if it was positive in at least one of three techniques: conventional cytogenetics, fluorescence in situ hybridization, or quantitative real-time polymerase chain reaction (qRT-PCR). Among the 96 patients enrolled, there were 50 Ph- ALL patients (52.1%) and 46 Ph+ ALL patients (47.9%). There were no differences between the 2 groups regarding age, sex distribution, performance status, and induction regimens. For treatment outcomes evaluation, the 5 Ph+ ALL patients not receiving TKI were eliminated and 41 Ph+ ALL patients treated with TKI were compared with 50 Ph- ALL patients (Table). Complete remission (CR) was attained in 27 (54.0%) of patients in the Ph- group versus 35 (85.4%) in the Ph+ group (P=0.001). The median RFS was significantly longer in the Ph+ group (3.1 months for Ph- vs. 8.9 months for Ph+, P=0.017). Induction death and induction failure occurred significantly more often in the Ph- group. The median OS was 6.3 months for Ph- group versus 10.3 months for Ph+ group (P=0.033). In conclusion, using real world data, this study reports the possible favorable impact of Ph+ in TKI-era for elderly patients. Citation Format: Ja Min Byun. Prognostic impact of Philadelphia chromosome in elderly acute lymphoblastic leukemia patients ineligible for hematopoietic stem cell transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 765. doi:10.1158/1538-7445.AM2017-765

A rare e13a3 (b2a3) BCR-ABL1 fusion transcript with normal karyotype in chronic myeloid leukemia: The challenges in diagnosis and monitoring minimal residual disease (MRD)

Patients with chronic myeloid leukemia (CML) have a t (9;22)(q34;q11.2) or variant translocation that results in a BCR-ABL1 fusion gene. For many years, conventional karyotyping has been used as the standard diagnostic tool for t (9;22) (q34;q11.2). However, it has several limitations that may lead to failure for detecting BCR-ABL1 gene rearrangements in around 5% of all CML patients. Although reverse transcription polymerase chain reaction (RT-PCR) has evolved as a sensitive method for detecting BCR-ABL1 translocation, this method fail to detect certain BCR-ABL1 fusion transcript type, such as e13a3 (also known as b2a3), as a result of many commercially available and laboratory-developed primer sets. Fortunately, these two rare situations rarely appear at the same time, therefore, the combination of two methods rarely misdiagnosed the patients with CML. In this study, we report a patient with CML who tested both negative by RT-PCR and cytogenetic analysis at the time of diagnosis. She was diagnosed as atypical CML (aCML) and allogeneic hematopoietic stem cell transplantation was suggested. Further fluorescence in situ hybridization (FISH) showed cryptic insertion of ABL into BCR gene on chromosome 22, and DNA sequencing with alternative primer sets demonstrated the presence of an e13a3 BCR-ABL1 fusion. She was diagnosed as CML and received imatinib 400 mg/day. A follow-up BCR-ABL1 FISH analysis demonstrated a markedly reduced BCR-ABL1 fusion rate of 0 after 6months treatment, indicating a complete cytogenetic response.

Impact of Chromosomal Rearrangement upon DNA Methylation Patterns in Leukemia.

Genomic instability, including genetic mutations and chromosomal rearrangements, can lead to cancer development. Aberrant DNA methylation occurs commonly in cancer cells. The aim of this study is to determine the effects of a specific chromosomal lesion the BCR-ABL translocation t(9:22), in establishing DNA methylation profiles in cancer. Materials and methods We compared DNA methylation of 1,505 selected promoter CpGs in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) with and without the Philadelphia chromosome t(9:22), CD34+ hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR-ABL (acute promyelocytic leukemia (APL) and gastrointestinal stromal tumors (GIST). In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph+ ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were no influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.

Clinical characteristics and whole exome/transcriptome sequencing of coexisting chronic myeloid leukemia and myelofibrosis.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders that can be classified on the basis of genetic, clinical, phenotypic features. Genetic lesions such as JAK2 mutations and BCR-ABL translocation are often mutually exclusive in MPN patients and lead to essential thrombocythemia, polycythemia vera, or myelofibrosis or chronic myeloid leukemia, respectively. Nevertheless, coexistence of these genetic aberrations in the same patient has been reported. Whether these aberrations occur in the same stem cell or a different cell is unclear, but an unstable genome in the HSCs seems to be the common antecedent. In an effort to characterize the underlying genetic events that might contribute to the appearance of more than one MPN in a patient, we studied neoplastic cells from patients with dual MPNs by next-generation sequencing. We observed that most patients with two MPNs harbored mutations in genes known to contribute to clonal hematopoiesis through altered epigenetic regulation such as TET2, ASXL1/2, SRSF2, and IDH2 at varying frequencies (1%-47%). In addition, we found that some patients also harbored oncogenic mutations in N/KRAS, TP53, BRAF, EZH2, and GNAS at low frequencies, which probably represent clonal evolution. These findings support the hypothesis that hematopoietic cells from MPN patients harbor multiple genetic aberrations, some of which can contribute to clonal dominance. Acquiring mutations in JAK2/CALR/MPL or the BCR-ABL translocation probably drive the oncogenic phenotype towards a specific MPN. Further, we propose that the acquisition of BCR-ABL in these patients is frequently a secondary event resulting from an unstable genome.

Detection of Immunoglobulin Heavy Chain Gene Clonality by Next-Generation Sequencing for Minimal Residual Disease Monitoring in B-Lymphoblastic Leukemia.

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.

Telomere Length Shortening in the CML Leukemic Stem Cell Compartment Correlates with the Respective Clone Size: Evidence for Early Vs. Late Chronic Phase

Introduction: Chronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the bcr-abl translocation. Recent data provides evidence that CML chronic phase (CP) can be classified into early and late CP depending on the degree of expansion of the leukemic stem cell (LSC) clone. Patients in late CP have a higher LSC burden going along with an inferior response to TKI therapy. Telomeres shorten with each cell division and telomere length (TL) reflects the replicative history of a cell. We postulate that the LSC burden correlates with accelerated telomere shortening due to clonal replicative expansion of bcr-abl positive cells. Previous studies evaluating TL in peripheral blood cells of CML patients already revealed a correlation of age-adapted TL with disease stage, response to treatment and duration of CP. However, the high intra-individual, mostly genetic inter-individual variability in TL limits the predictive value of TL measurements when no patient specific bcr-abl negative cells were available for comparison. The aim of our study was to analyze TL in the LSC and non-clonal HSC compartment of patients with CP using a modified Q-FISH technique allowing the TL analysis in bcr-abl positive and negative cells.Methods and Patients: 11 patients diagnosed with CML in CP of the NCT00852566 study (Nordic CML Group) were included into our retrospective analysis. Mean age of the patients was 58.9 years. Bone marrow samples from initial diagnosis were sorted for CD34+/CD38- cells reflecting the leukemic stem cell compartment. Samples were analyzed with the FISH method using dual fusion dual color BCR-ABL1 probe following standard procedures. After capturing the bcr-abl staining using confocal microscopy, samples were re-processed for TL analysis by Q-FISH using established protocols. Optimized microscopy techniques allowed for TL to be assessed in all previously captured cells allowing the identification of bcr-abl positive and negative cells within the same sample. Analysis and quantification of bcr-abl FISH staining and TL measurement by Q-FISH were performed in single-blinded fashion.Results: Bcr-abl negative cells represent the non-clonal hematopoiesis and were used to correct TL. We observed significant shortened TL in the bcr-abl positive cells compared to bcr-abl negative cells (-2.18 ± 2.08 kilobases (kb), p=0.01) in line with previously published results. Next, we correlated the clone size (i.e. the proportion of bcr-abl positive cells) with the degree of telomere shortening in the leukemic stem cell compartment. Mean clone size of the patients was 67.4 ± 21.7 % S.D. Despite of the relatively small sample size studied, we found a significant negative correlation (R²=0.45, p=0.04) between TL and clone size strongly supporting the notion that increased expansion of the bcr-abl positive LSC pool leads to accelerated telomere shortening.Conclusions: In this study, we provide evidence for accelerated telomere shortening in bcr-abl positive LSC as compared to their normal CD34+/CD38- counterpart in CP CML samples at diagnosis. Furthermore, the degree of TL shortening correlates with the clone size in the HSC compartment, i.e. a parameter that reflects duration of CP. Thus, this allows the discrimination of early vs. late CP and might as such be used as a prognostic (and potentially predictive) biomarker in CML. DisclosuresRichter:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Fioretos:Cantargia: Equity Ownership. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Brümmendorf:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties.

Clonal Evolution Underlying the Progression from Myelodysplastic Syndromes to Ph-Positive Acute Lymphoblastic Leukemia

Introduction:Myelodysplastic syndromes (MDS) are considered as a "stem cell disorders", in which hematopoietic stem cells and lineage-committed progenitor cells acquire genetic and epigenetic alterations and provide aberrant, clonal hematopoiesis, sometimes resulted in the progression to acute myeloid leukemia (Elias HK et al, Oncogene 2014). We previously reported a rare case of which the patient developed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) 2.5 years after being diagnosed with MDS (Kohno T et al, Br J Haematol 1996). p190 BCR-ABL1 mRNA was detected in the Ph+ALL cells. Metaphase cytogenetics showed the karyotypes: 46, XY, 20q- in MDS phase and 46, XY, t(9;22)(q34;q11), 20q- in ALL phase, indicating that MDS and Ph+ALL in this patient were of the same clonal origin.To uncover the detail of the clonal evolution, we analyzed bone marrow samples of MDS and Ph+ALL in this patient by targeted massively parallel sequencing with a panel of 154 genes including known driver genes of hematologic malignancies.Methods:Genomic DNAs (gDNAs) were extracted from the bone marrow mononuclear cells of MDS and Ph+ALL in this patient. Targeted sequencing was performed on the Illumina HiSeq2500 platform. Single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using HaplotypeCaller of Genome Analysis Toolkit (GATK) version 3.4-46. We also attempted to detect the breakpoint of BCR-ABL1 translocation from the targeted sequencing data using the computational method, BreaKmer (Abo RP et al, Nucleic Acids Research 2015). The candidates of the mutations and structural variations were validated by amplicon-based deep sequencing and Sanger sequencing. Copy number variations were analyzed using Affymetrix CytoScan HD Array.Results:The mutations in ASXL1 and U2AF1 genes were identified in the MDS sample with variant allele frequencies (VAFs) of about 45%. At the progression of Ph+ALL, the mutations in SETBP1, SMC1A, and SLC5A8 genes were newly acquired while the ASXL1 and U2AF1 mutations were also identified with the same level of VAFs (about 50%) as the other mutations. VAFs of all of the mutations were decreased to about 20% after the chemotherapy for Ph+ALL, and then increased to about 40% at the recurrence of the disease. Furthermore, we identified the breakpoint of BCR-ABL1 translocation at intron 1 of ABL1 genes and intron 1 of BCR genes, that is the well-known cluster region, m-bcr, only among the samples of Ph+ALL. Copy number analysis confirmed that both MDS and Ph+ALL samples harbored the deletion of chromosome 20q. And the deletion of IKZF1 gene, which is frequently identified in Ph+ALL cases (Mullighan CG et al, Nature 2008), was not identified during the progression from MDS to Ph+ALL. These results demonstrated that the MDS clone harboring 20q- and ASXL1 and U2AF1 mutations acquired the mutations in SETBP1, SMC1A, and SLC5A8 genes and the p190 BCR-ABL1, resulted in the development of Ph+ALL in this patient.Conclusion:The alterations of SETBP1, SMC1A, and SLC5A8 genes are usually reported in myeloid malignancies (Makishima H et al, Nat Genet 2013, Kon A et al, Nat Genet 2013, Whitman SP et al, Blood 2008). Previous study in transgenic mouse demonstrated the distinct role of p190 BCR-ABL1 in the development of an ALL (Voncken JW et al, Blood 1995). Recapitulating this scenario, p190 BCR-ABL1 may play a critical role in the development of Ph+ALL from the MDS stem cells in this patient. This study may provide a new insight into the stem cell origin of MDS and the role of p190 BCR-ABL1 in the development of Ph+ALL. DisclosuresNo relevant conflicts of interest to declare.

Epidemiology Data of Patients with Chronic Myeloid Leukemia in West Central Mexico

Background:Chronic myeloid leukemia (CML) treatment in Mexican mestizos since the use of TKIs has been well described. Most of the studies only refers to the response rather than epidemiology. There are two large cohorts reported this years (Gad Med Mex 2016;152:208-212 and Clin Lymphoma Myeloma Leuk 2016;16:57-62), but all national information is not available.Methods:Epidemiological data from samples referred to Laboratorios Fatima de Michoacan for identification of BCR-ABL translocation by chromosome or molecular analysis in patients with suspected chronic myeloid leukemia.Results:77 positive samples for identification of BCR-ABL were analyzed. By physician indication 46 karyotype and 31 PCR. Male 40 and females 37, ratio 1.08/1. Age range from 15 to 87 years, mean 44.8. Three less than 19 years, thirteen from 20 to 29, twenty from 30 to 39 years, twelve from 40 to 49, twelve from 50 to 59, seven from 60 to 69, nine from 70 to 79 and one more than 80 years.Conclusions: Compared with data from other Mexican CML studies (cited above) the age at diagnosis was similar: 44.8, 45.8 and 47.0 years; important to mention that in the three studies there are patients less than 15 years old; in the ratio M/F there was a difference: 1.08, 1.37 and 1.32. Compared to CLL epidemiology data that we presented last year in the 57thASH Annual Meeting (Blood 2015;126:5269), CML presented more than 20 years earlier, 66.0 to 44.8 years; M/F ratio is elevated in CLL 1.6 a 1; and CML is more frequent, 77 to 52 cases in CLL. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Genetic Mutations in Plasma Exosomes As a Biomarker Tool for Detecting the Clonal Evolution of Leukemic Cells in Pediatric AML

Abstract Introduction: AML is the second most of pediatric leukemia with relapse in &gt;30% of the patients.Clonal evolution of rare primary leukemic cells that survived the initial therapy or gained additional mutations independent of therapy stress, could be the potential cause of relapse in pediatric AML.Therefore, sensitive and reliable methods to measure accurately the mutational shifts between diagnoses, during therapy and relapse could provide useful information on the disease progression.Exosomes are extracellular vesicles of 30-150nm in diameter that are released by both healthy and malignant cells. Exosomes derived from tumor cells or leukemia blasts have emerged as potential valuable biomarkers as they have been illustrated to feature disease specific protein, lipid and nucleic acid signatures that represent the pathological state of the respective cells. While leukemia cells release relatively higher amounts of exosomes compared to healthy cells, mutational profiling of exosomes could be more sensitive than analyzing rare leukemia sub-clone in thehematopoietic compartment. Methods: Secreted exosomes from conditioned media of K-562leukemia celllines and plasma of pediatric AML patients were isolated using differential ultracentrifugation at 100000g. Absolute DNA amount was quantified usingQuantiFluordsDNA System fromPromega. DNA deep sequencing was performed to analyze theexosomal DNA from K-562 cell line. The mutational profiling of the DNA from primary material, plasma and exosomes was performed using next generation sequencing platform, Illumina. Results: To establish the diagnostic and prognostic potential of exosomes in measuring leukemia associated mutations we isolated exosomes from the conditioned media of the cultured leukemia cell lineK-562. We used this cell line as model because it harbors the classical BCR-ABL translocation that is relevant in deciding treatment options in chronic myeloid leukemia. Analysis of DNA isolated from these exosomes revealed the presence of genomic double-stranded DNA (dsDNA) fragments. Using deep sequencing approach we could further detect the classical BCR-ABL translocation in exosomes. The presence of leukemia specific mutation in exosomes that is potentially derived from parental cells suggests the utility for exosomes in leukemia diagnosis and to forecast treatment response and relapse. In the next step, we analyzedexosomal dsDNA from plasma exosomes of primary pediatric AML patients. We compared the sensitivity of DNA associated from exosomes isolated from blood plasma of pediatric AML patients and compared it to free floating cell free DNA (cfDNA) in the plasma supernatant. In our study we used the plasma volume ranging from 350 microliters to 1ml from peripheral blood of AML patients at diagnosis and relapse (n=9). We performed DNA isolation from the starting plasma material and then compared it with the exosomes isolated. As control, DNA from the exosomes depleted fraction was also analyzed. Comparison of these three fractions (input plasma, pelleted exosomes and exosome depleted plasma) revealed a significant enrichment of dsDNA in the exosome fraction after ultracentrifugation in 7 out of 9 patients (Figure A). This suggests that isolation of exosomes from supernatant can enrich the amount of DNA and thereby the sensitivity of downstream mutational analysis in diagnostic. By next generation sequencing (NGS) (myeloid panel, Illumina) using the DNA materials from plasma supernatants and the respective exosomes we screened for AML associated mutations (n=7). We were able to detect the known primary mutation detected at diagnosis or relapse in both plasma and exosomes of 6 out of 7 patient material. In one patient the detection of mutations were not possible in both the plasma andexosomal fraction. Interestingly we could detect additional mutations like FLT-ITD (patient 2) or CUX1 (patient 4) only in the exosome fraction (Table B). Conclusion: These results suggest that the combined evaluation of DNA from plasma and exosome fraction could provide novel and additional information about the clonal hierarchy or evolution of AML, and if used for monitoring, might be of prognostic relevance. Disclosures Reinhardt: Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Abstract 2039: Exploring the longitudinal transcriptomic landscape of tyrosine kinase inhibitor treatment response in chronic myeloid leukemia patients

Abstract The interpretation and implementation of large-scale genetic profiles into clinical practice remains a challenge despite substantial growth in our understanding of genetic contributors to drug response. Most current omic studies focus on identifying genetic features that are distinct between normal and tumor samples, but fail to capture the dynamics of association between omic profiles, treatment response and disease progression over time. The focus of this research is to analyze the longitudinal transcriptomic profile of chronic myeloid leukemia patients (CML) in context of tyrosine kinase inhibitor (TKI) treatment and clinical status. The main objectives were to compare a series of post-TKI treatment transcriptome profiles to their baseline levels, and characterize the impact of TKI treatment and CML disease status on the individual's transcriptome over time. Our ultimate goal is to develop TKI response predictors using the longitudinal expression data collected over the treatment course. Peripheral blood samples, buccal swabs and detailed clinical data were collected from each study participant (screened for BCR-ABL1 translocation) for a period of 6 months, in addition to pre-therapy baseline. RNA was extracted from granulocytes isolated from peripheral blood samples, and profiled using RNA sequencing. RNAseq profiles over TKI treatment course were compared to baseline, as well as against hematologic response (complete blood count), cytogenetic response (FISH), and clinical disease progression. We investigated dynamic trends in RNAseq profiles associated TKI response, as well as with the clinical status of the patient over time. We identified genetic features that were either 1) Differentially expressed between baseline and post-TKI time points; 2) Showed non-random spikes in expression levels at specific time points; 3) Associated with hematological and clinical phenotypes, including white blood cell count, percentage granulocytes and percentage cells with BCR-ABL1 translocation; 4) Demonstrated highly correlated patterns of expression over time. Through clustering and enrichment analysis of the selected transcripts, we identified several pathways and molecular features associated with TKI-response, and altered disease state. Of note, we found mTOR signaling, and pro-apoptotic pathways to be significantly altered between baseline and TKI-responding individuals. In addition, we observed significant changes in transcription regulatory network of several transcription factors, notably AP-1, over the treatment time course. To our knowledge, this is the first study to establish the utility of comprehensive longitudinal multiple transcriptome profile analysis of TKI-response in CML. We believe this study will pave way for future large-scale longitudinal omic profiling of CML and other cancer-types. Citation Format: Aritro Nath, Fan Wang, Divya Lenkala, Bonnie LaCroix, Nancy Glavin, Kristen Kipping-Johnson, Paul Geeleher, Michael Thirman, Lucy Godley, Gordana Raca, Richard Larson, R. Stephanie Huang. Exploring the longitudinal transcriptomic landscape of tyrosine kinase inhibitor treatment response in chronic myeloid leukemia patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2039.

Abstract 395: Patient stratification and drug combination strategy based on drug response and genomic information from PDX clinical trials (PCTs)

Abstract Cancer is a heterogeneous disease with various molecular lesions and drug response profiles within the same tumor type. Patient stratification in clinical trials based on molecular features has contributed to recent success of several targeted cancer drugs on molecular aberrations such as BCR-ABL translocation in CML, Her2 amplification in breast and gastric cancers, EGFR mutations and ALK fusions in lung cancer and BRAF mutation in melanoma. However, in most cancers, the molecular features which can be used for patient stratification are not as simple as a single genetic aberration. Multiple drug resistance mechanisms caused by various mutations in cancer signaling pathways can also increase the uncertainty of clinical outcomes. To increase the chance of success in human clinical studies, patient-derived xenograft (PDX) clinical trials (PCTs) have increasingly been used for predictive biomarker validation, resistance mechanism investigation and combination therapy selection. PDX tumor models have been demonstrated to have high correlations with human patients in tumor pathology, molecular characteristics and drug responses. Large scale PCTs have also shown consistency in results when compared to related human clinical trials. At GenenDesign, we have established over 1000 PDX tumor models and more than 100 resistance models against various cancer drugs. Many of these PDX models have been characterized at RNA/Exome sequence, gene expression, gene copy number and hot-spot mutation levels. We carried out our PDX clinical trials by testing multiple approved drugs and clinical drug candidates such as targeted inhibitors against FGFRs, c-Met/ALK, HER2, EGFR, cell cycle regulators, Ras/Raf pathway, PI3K/Akt pathway, as well as chemotherapy drugs in biomarker-driven multi-drug multi-arm expanded PDX clinical trials. So far, we have accumulated more than 3000 efficacy data sets and associated PD samples. Analysis of drug response and associated genomic information from PDX clinical trials yielded rich information for predictive biomarker identification and validation. At the same time, many potential resistance mechanisms were also revealed. These information can make human clinical trial better prepared, more efficient and focused. More importantly, testing of a targeted drug with multiple chemotherapies in the same models can also provide guidance on future combination selection strategy. Citation Format: Jingjing Jiang, Tengfei Yu, Ying Yan, Wei Du, Tingting Tan, Xuqin Yang, Jiali Gu, Xin K. Ye, Zhenyu Gu. Patient stratification and drug combination strategy based on drug response and genomic information from PDX clinical trials (PCTs). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 395.

Deciphering “B-others”: Novel fusion genes driving B-cell acute lymphoblastic leukemia

Deciphering “B-others”: Novel fusion genes driving B-cell acute lymphoblastic leukemia

Concomitant BCR-ABL1 positive chronic myelogenous leukemia emerging in a patient with MPL W515L associated primary myelofibrosis

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more cell lineages in the bone marrow. At present, the main criterion in the 2008 World Health Organization classification of MPNs is the presence of an underlying genetic abnormality. These mutations are generally mutually exclusive except for rare reports in the literature. We report for the first time a detailed analysis of the clinical, histologic and cytogenetic/molecular features of a patient who initially presented with MPL W515L positive primary myelofibrosis and over the course of five years developed an MPN associated with both BCR-ABL1 and MPL W515L mutation. We discuss the diagnostic challenges and therapeutic implications of concomitant BCR-ABL1 translocation with MPL W515L mutation. Multiple genetic alterations may simultaneously coexist in patients exhibiting features of myeloproliferative disorders.

Genetic Characterisation of Immunoglobulin Heavy Chain Locus CCAAT Enhancer-Binding Protein Translocated Acute Lymphoblastic Leukaemia

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is a heterogeneous disease in which patient outcome is influenced by genetic lesions. Cytogenetic classification has improved survival through risk stratification for treatment. Translocations involving the Immunoglobulin Heavy Chain Locus (IGH) comprise 5% of BCP-ALL and lead to overexpression of juxtaposed genes, due to the powerful IGH enhancer elements. Multiple IGH partner genes have been described in BCP-ALL, including five members of the Ccaat Enhancer-Binding Protein (CEBP) transcription factor family: CEBPA, CEBPB, CEBPD, CEBPE and CEBPG, which comprise 12% of the IGH-translocation subgroup.Patients with potential IGH-CEBP translocations were identified by mining cytogenetic data from UK ALL trials. The translocations were confirmed by fluorescence in situ hybridisation (FISH), followed by screening for commonly occurring BCP-ALL lesions using Multiplex Ligation-dependent Probe Amplification (MLPA) (SALSA P335 kit, MRC Holland), and detection of novel genetic lesions using SNP6.0 arrays. A total of 33 IGH-CEBP patients were identified; CEBPD (n=11, 34%); CEBPA (n=10, 30%); CEBPB (n=8, 24%); CEBPE (n=3, 9%); CEBPG (n=1, 3%). Cohort median age was 15 years (range, 2-65 years). Interestingly the majority of IGH-CEBPD patients were under the age of 10 years, while CEBPA and CEBPB patients were older, all over the age of 10 years (p=0.005). The median white blood cell (WBC) count of the cohort was low at 8.24x109/l (range, 0.9-430 109/l), the highest WBCs were observed in the CEBPB subgroup (p=0.04). Associated abnormalities were mixed: 5 Down syndrome (DS) patients were identified in the cohort all exclusive to the IGH-CEBPD subgroup, an observation which has been previously reported, BCR-ABL1 translocation (n=1), high hyperdiploidy (n=1) and hypodiploidy (n=1). Although numbers were small, some trends were observed: 27% (n=9) of patients were deceased: CEBPB (n=4, 44%), CEBPA (n=2, 22%), CEBPD (n=2, 22%) and CEBPE (n=1, 13%). Overall 15% (n=5) relapsed: CEBPD (n=3, 60%), CEBPA and CEBPB (each n=1, 20%). Minimum residual disease data (MRD) at day 28 were available for 8 (24%) patients, CEBPD (n=3, 38%), CEBPA (n=2, 25%), CEBPB, CEBPE and CEBPG (n=1, 13% each). Although numbers were low, all 3 MRD negative patients were IGH-CEBPD while the remaining patients, all MRD positive, comprised the remaining CEBP subgroups.MLPA screening of 28 patients identified that 25% had whole gene deletions of CDKN2A/CDKN2B (n=7): the most common copy number abnormality (CNA) observed in IGH-CEBP. IKZF1 deletions of exons 4-7, a deletion resulting in the formation of the dominant negative isoform IK6, were present in 21% (n=6) patients: CEBPB (n=4, 67%) and CEBPD (n=2, 33%) (P=0.04). Four CNAs of the PAX5 gene were observed: individual gains of exon 5 and exon 7 (n=1 each, 50%); single exon gains of PAX5 have been predicted to lead to altered protein activity, whole gene deletion (n=1), deletion of exon 1 along with gain of exons 7 and 8 (n=1).Using SNP6.0 arrays (n=13) a recurrent focal deletion of the ABL2 gene was discovered (n=4, 31%). These patients had a common region of deletion covering exon 2 of the gene. This deletion covers the Src Homology 2 domain (SH2), which allows for the recognition of phosphorylated tyrosine residues, vital for cell signalling. Patients spanned three subgroups; CEBPB (n=2, 50%), CEBPA (n=1, 25%) and CEBPD (n=1, 25%).In this study, we show that in the IGH-CEBP subgroup, the CEBP partner gene influences disease phenotype. CEBPB patients showed more aggressive BCP-ALL characteristics, with the highest WBC, death rate (50%), older age and highest levels of associated abnormalities, with all patients showing one or more genetic lesions. The CEBPD subgroup comprises the youngest patients, a statistically significant finding both with and without inclusion of DS status. This subgroup also had the highest number of relapses in the cohort, and was the only CEBP subgroup to be MRD negative at day 28, albeit based on low patient numbers. A focal deletion within ABL2, a paralog of the ABL1 gene, is an interesting new abnormality in this subgroup, which may contribute to disease development. DisclosuresNo relevant conflicts of interest to declare.

An Accurate and Sensitive RT-qPCR Method That Reliably Detects BCR-ABL1 Beyond MR4.5

▪Introduction: The BCR-ABL1 translocation is a hallmark of chronic myeloid leukemia (CML). As levels of BCR-ABL1 reflect leukemia burden, quantitation of BCR-ABL1 transcripts with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an essential tool that defines molecular responses in CML monitoring. The prognostic significance of molecular responses has been established by large-scale clinical trials. Achieving designated levels of BCR-ABL1 on the international scale (IS) within specific time frames is an important measure for assessing patient response, probability for relapse and progression, and overall survival. Recent evidence suggests that achievement of MR4.5 at 4 years may portend a better survival than those who achieve 0.1-1% IS (MR3-MR2). With deeper responses generated by second-generation tyrosine kinase inhibitors at a higher frequency, need for detecting a very low level of minimal disease has been increasing. Herein, we evaluate the performance of Asuragen's Quantidex™ (QDX) BCR-ABL IS CMR Kit to determine if the method would meet this need.Materials and Methods: Forty-nine samples with BCR-ABL1 IS values previously determined by a clinically validated BCR-ABL1 RT-qPCR assay (Ipsogen BCR-ABL1 Mbcr Kit) were reevaluated with the Asuragen QDX BCR-ABL assay. Samples were tested in singlet by two operators over four independent runs using an ABI 7500 FAST instrument, software version V2.0.6. The auto threshold setting was used for BCR-ABL1 analysis; ABL1 analysis was performed using a manual threshold. The analytical specificity of the assay was assessed with six non-CML leukemic RNA samples from cell lines. Assay linearity was evaluated using a set of BCR-ABL1 positive cell line dilutions ranging from MR1.0-MR4.0, while precision was established testing this set of cell line dilutions an additional three times each in quadruplicate between two operators. The limit of detection of the assay was determined using four contrived, pre-diluted human-derived specimens with BCR-ABL1 values ranging from MR4.0 to MR 4.9 at 0.1 intervals.Results: There was 96% concordance between the Ipsogen and the QDX BCR-ABL assay results. Two clinical samples that were historically positive for BCR-ABL1 transcripts, and later tested negative using the Ipsogen assay were detected as weak positives by the QDX BCR-ABL assay. Repeat testing of one of the samples with the QDX BCR-ABL assay confirmed the low positive result. All non-CML samples tested negative for BCR-ABL1. One sample yielded a low positive result. Repeat testing yielded the expected negative result. Results were linear between 0.1 and 10% IS. A few outliers were detected above 10% IS, at which point, greater variation is expected. BCR-ABL1 transcripts were detectable down to MR4.9 (0.0013% IS); however, the results were not reproducible for all tested replicates. BCR-ABL1 transcripts were reproducibly detected down to MR4.5. Assay precision was high at 0.1-10%IS values (CV% of 7.5-8.7%), and decreased at %IS values greater than 10% IS and less than 0.1% IS.Conclusions: The QDX BCR-ABL IS CMR Kit is a robust and sensitive assay that generates results sufficient for studies in low-level molecular responses. DisclosuresNo relevant conflicts of interest to declare.

Combination of Anti-CD123 and Anti-CD19 Chimeric Antigen Receptor T Cells for the Treatment and Prevention of Antigen-Loss Relapses Occurring after CD19-Targeted Immunotherapies

Introduction: Anti-CD19 chimeric antigen receptor T cells (CART19) and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generate unprecedented complete response rates of 45-90% in relapsing/refractory acute lymphoblastic leukemia (r/r B-ALL). However, a subset of patients treated with these targeted approaches will relapse and a significant portion of these relapses are characterized by the loss of detectable CD19 (about 30% of relapses after blinatumomab and up to 50% after CART19), a clear manifestation of immunoediting. Hence, novel effective strategies are needed in order to be able to treat those patients and ideally prevent antigen-loss. CD123, the interleukin-3 receptor alpha, is involved in hematopoiesis and has been shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia (AML) and more recently also B-ALL. Targeting CD123 with chimeric antigen receptor T cells (CART123) was shown to lead to deep and long-term responses in human primary AML xenografts. The goal of this study was to pre-clinically evaluate the impact of targeting CD123 and CD19 with chimeric antigen receptor T cells for the treatment and prevention of CD19-negative leukemia relapses occurring after CD19-directed therapies.Results: We analyzed the expression of CD123 in 42 r/r B-ALL samples and found that CD123 is highly and homogeneously expressed on the surface of most B-ALL blasts (81.75%, range: 5.1-99.6), making it a promising candidate for targeted therapy in B-ALL. Moreover, CD123 was also found to be expressed in the putative leukemia stem cells (LSC), identified as CD34-pos CD38-neg. Notably, in some Ph+ B-ALL samples we found CD19-ve CD123+ve cells with a BCR-ABL translocation by FISH, suggesting that these cells too are malignant. The expression of CD123 was detected in all (n=6) CD19-negative B-ALL blasts analyzed after relapse from CART19 treatment (Figure 1). These findings indicate that CD123 represents an ideal marker to target CD19-neg ALL blasts occurring after CART19 or blinatumomab. Therefore, we generated anti-CD123 chimeric antigen receptor T cells costimulated with 4-1-BB using a lentiviral vector (CART123). We then evaluated the CART123 anti-B-ALL efficacy both in vitro and in vivo against primary B-ALL and cell line (NALM-6). CART123 showed intense anti-B-ALL ex vivo activity, as defined by specific CD107a degranulation, cytokine production, cytotoxicity and proliferation, not statistically different from that of CART19. In order to test the role of CART123 to target CD19-negative relapses we developed a novel in vivo model, engrafting immunodeficient NSG mice with blasts obtained from a patient relapsing with CD19-ve disease after CART19 treatment. At day 14 mice were randomized to receive CART19, CART123 or control T cells (untransduced, UTD). CART19 and control T cell treated mice had no anti-tumor activity, while CART123 led to complete eradication of the disease and long-term survival. We next developed a murine model to test the hypothesis that a combined approach simultaneously targeting CD123 and CD19 could treat and prevent CD19-negative relapses. NSG mice were injected with a mixture of primary CD19-neg and CD19-pos blasts from the same patient that were labeled with different click beetle luciferases (red or green) in order to be able to track the respective clones in vivo. Mice were then randomized to receive UTD, CART19 or the combination of CART19 and CART123 (same total dose of T cells). As shown in Figure 2, mice treated with UTD had progression of both leukemic clones (CD19-pos and CD19-neg) while CART19 showed rapid progression mostly of the CD19-neg disease (red luciferase); on the contrary only mice treated with the combination of CART123 and CART19 showed rapid clearance of the disease, with improved overall survival (64 days for CART19, not reached for CART19+CART123). As a final strategy, we expressed both CAR19 and CAR123 in the same T cells and showed potent anti-leukemia activity (CD107a degranulation 81.7%).Conclusions: Here we demonstrate that CD123 is expressed in CD19-negative B-ALL relapses occurring after CD19-directed therapies, and that combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in B-ALL murine xenografts. [Display omitted] [Display omitted] DisclosuresRuella:Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Scholler:Novartis: Patents & Royalties. Lacey:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Hunter:Surface Oncology - SAB: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.