BCR-ABL Translocation Research Articles

Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A).The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date.Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members.Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C).In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. [Display omitted] DisclosuresVoorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

Lysosomal Acid Lipase a (LIPA) Modulates Leukemia Stem Cell (LSC) Response to Venetoclax/TKI Combination Therapy in Blast Phase Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs.To establish the efficacy of Ven/TKI combination, we treated bpCML samples with Ven+Dasatinib (Das) combination for 24h, this resulted in a significant decrease in the viability of bulk and primitive populations (CD34+, CD38+). Patient-derived xenografts of bpCML samples in NSGS-mice, were treated with Ven/Das as well as single agents. The result showed a significant decrease in leukemia burden in the combination treated group, compared to either drug alone, albeit, some resistant cells survived in the combo treated group. Furthermore, using a syngeneic mouse model of bpCML, co-expressing Bcr-Abl and Nup98-HOXA9 translocations, the mouse leukemic cells treated with Ven/Dasatinib combination demonstrated a significant loss of viability of the bulk as well as phenotypically defined LSCs (Lin-/Sca1+). Treatment of leukemic mice with Ven/Das had a significant survival benefit and remained disease free at 80 days post treatment. We also showed significant survival benefits of Ven/ponatinib in NSGS-mice harboring syngeneic bpCML cells with the T315I gatekeeper mutation. Treatment of normal mice with Ven/Das combo did not affect the colony forming ability of LSK cells from the bone marrow, indicating a leukemia-specific response. Based on these results, we conclude that Ven/TKI combination effects were due to direct targeting of the LSC population.To investigate the potential mechanism of Ven/TKI activity in LSC targeting, we performed gene expression studies using RNA-seq based methods after short term treatment. Our findings indicated that the LSC population from Ven/TKI-treated mice showed enrichment of a gene signature associated with lysosome biology. Pre-treatment of mouse leukemia cells with bafilomycin, an inhibitor of lysosome function, resulted in increased sensitivity to Ven/TKI combo. Intriguingly, we also found significant induction of lysosomal acid lipase (LIPA), an enzyme involved in the generation of free fatty acids for energy needs.Metabolomic analysis of LSCs isolated after short term treatment with Ven/TKI, showed that a number of fatty acids were up-regulated in the Ven/Das treated group compared to control. Knocking down Lipa using CRISPR technology resulted in increased sensitivity to Ven/TKI combination, whereas overexpression of Lipa resulted in decreased sensitivity to the Ven/TKI combination, implicating Lipa upregulation and a resultant increase in free fatty acids as a protective response to Ven/TKI treatment. Furthermore, knocking down CPT1A, an important free fatty acid mitochondrial transporter, resulted in increased sensitivity to Ven/TKI combination both in mouse and primary human leukemic cells, leading to the hypothesis that activation of fatty acid processing through enhanced Lipa activity may represent a compensatory response to venetoclax based therapies in bpCML.In summary, we demonstrate the preclinical efficacy of Ven/TKI combination therapies for targeting of bpCML LSCs. Furthermore, our data suggest that blocking upregulation of free fatty acids through mechanisms such as inhibition of LIPA activity, might synergize with Ven/TKI combinations to eradicate LSCs, allowing for more durable response. Our findings provide a therapeutic rationale for blocking pathways involved in free fatty acids generation, as a potential strategy for increasing remission duration. DisclosuresPollyea: Amgen: Consultancy; Janssen: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Research Funding; Syndax: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Glycomimetics: Other. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding.

Genetic Landscape of Myeloproliferative Neoplasms with an Emphasis on Molecular Diagnostic Laboratory Testing.

Chronic myeloproliferative neoplasms (MPNs) are hematopoietic stem cell neoplasms with driver events including the BCR-ABL1 translocation leading to a diagnosis of chronic myeloid leukemia (CML), or somatic mutations in JAK2, CALR, or MPL resulting in Philadelphia-chromosome-negative MPNs with constitutive activation of the JAK-STAT signaling pathway. In the Philadelphia-chromosome-negative MPNs, modern sequencing panels have identified a vast molecular landscape including additional mutations in genes involved in splicing, signal transduction, DNA methylation, and chromatin modification such as ASXL1, SF3B1, SRSF2, and U2AF1. These additional mutations often influence prognosis in MPNs and therefore are increasingly important for risk stratification. This review focuses on the molecular alterations within the WHO classification of MPNs and laboratory testing used for diagnosis.

Treatment Responses in Patients with Chronic Myeloid Leukemia on Tyrosine Kinase Inhibitors (TKI) Therapy

Background: Chronic myeloid leukemia (CML) is a cancer of white blood cells results by the BCR-ABL translocation. Part of BCR gene from chromosome 22 is fused with ABL gene on chromosome 9. Aim: To observe percentage of patients achieving Cytogenetics response (CR), and Deep molecular response (DMR) in CML patients taking TKI (Imatinib and Nilotinib). Study Design: Retrospective Cohort study. Methodology: This study was conducted in 2018-2019 in about 198 CML patients to evaluate TKI therapy response and observation was based upon their Quantitative PCR test which gave percentage of BCR-ABL gene translocation in IU. Patients which were diagnosed with CML in 2016 and was regular in their treatment for about 2 years were included in the study. No intervention was given as in vivo study. Statistical analysis: Data analyzed by SPSS 25.0v. Results: Results showed that out of 198 CML patients, started on 1st line TKI (imatinib) 95 males (48%) and 103 females (52%) showed cytogenetic response at start of therapy and after 2 years of therapy 157 (79.3%) patient showed DMR. Conclusion: This study concluded that regular 2-year treatment of chronic CML patients with TKIs produced significant response in patients. Keywords: BCR-ABL Translocation, Cytogenetic Response, Major Molecular Response, Complete Molecular Response and Deep Molecular Response.

Leukemoid Reaction in a Case of Chronic Cholecystitis With Focal Mild Acute Inflammation

Leukemoid reaction is a rare condition of marked increase of the white blood cells (WBC) in the peripheral blood, mostly more than 50 × 109/L. Although its most common cause is infection, acute or chronic cholecystitis has been hardly mentioned. This report aimed to present the leukemoid reaction found in a Thai who had chronic cholecystitis with focal acute inflammation. A 74-year-old woman had low-graded fever and generalized vague abdominal discomfort for 2 days. The physical examination showed the body temperature 37.7 °C and unremarkable abdominal signs. Her blood tests showed: hemoglobin 9.0 g/L, WBC 52.48 × 109/L, neutrophil 91%, band form 1%, platelet 434 × 109/L, and alkaline phosphatase (ALP) 290 U/L. The blood cultures yielded no growth. The computed tomography of the abdomen revealed 2 stones in the common bile duct (CBD) causing obstruction; the gall bladder (GB) showed mild dilatation with thin wall containing multiple small gall stones; no pericholecystic fluid and normal size of spleen. She promptly underwent operative cholecystectomy, CBD choledochoscopy, stones removal and T-tube choledochostomy. Along with the surgery, intravenous ertapenem was also administered. The pathology of the gall bladder was chronic cholecystitis with focal mild acute inflammation. With these therapies, fever daily decreased and finally disappeared within 5 days meanwhile the WBC count also gradually diminished every day till 8.04 × 109/L within 7 days. The WBC count was still normal one month later. The diagnosis of leukemoid reaction was concluded and it was presumably be associated with chronic calculous cholecystitis with focal acute inflammation although the BCR-ABL translocation had never been explored.&#x0D;

INCIDENCE, RISKS AND OUTCOME OF EXTRA MEDULLARY RELAPSE IN ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN AND ADULTS: A RETROSPECTIVE COHORT STUDY

Extra medullary relapse (EMR) is the major concern of treatment failure in acute lymphoblastic leukemia (ALL). Hence, their early detection may improve prognosis. Aim of the study is to identify risk factors and outcome of EMR patients with and without concurrent medullary relapse. We retrospectively analyzed 113 patients of ALL who presented with EMR in a tertiary cancer care hospital. Patients were divided into two groups; Group 1, combined EMR (with medullary relapse) and Group 2, isolated EMR (without medullary relapse). Clinical characteristics including total leukocyte count (TLC) &amp; peripheral blood blast percentage during presentation, phenotype, initial central nervous system (CNS) involvement and BCR-ABL translocation were analyzed to identify risk factors for EMR. Incidence of EMR in our study was 15.7% (113/720 patients). Two year incidence of combined EMR and isolated EMR was 10.1% and 5.5% respectively. CNS, followed by testis was common sites of EMR. TLC &gt;50x109/L and peripheral blood blast percentage &gt;50% at presentation was common in Group 1. BCR-ABL translocation was frequent in Group 2 and in adults compared to children (p=0.027). While Group 1 patients presented with leukocytosis (TLC &gt;10X109/L), Group 2 patients presented with leukopenia (TLC &lt;4X109 p=0.001). p=0.001).&gt;50X109/L and BCR-ABL translocation were identified as risk factors for combined EMR (p=0.004) and for in adults with isolated EMR (p=0.001) respectively.

PHF6 Mutations Defines a Subgroup of Mixed Phenotype of Acute Leukemia with Aberrant T-Cell Differentiation

Mixed phenotype acute leukemia (MPAL) is a rare form of acute leukemia, and comprises 2% of all acute leukemia diagnoses. With the exception of a small subset harboring BCR/ABL1 or MLL translocations (around 15% in combination), the genetic aberrations of MPAL are largely unknown. PHF6 gene is located at Xq26-27 and encodes plant homeodomain finger 6, a zinc finger containing protein. Recurrent somatic PHF6 mutations are frequent in T-ALL (up to 30%) and rarely in AML (~3%). Here we report recurrent PHF6 mutations in approximately 20% of MPAL patients, making PHF6 the most common genetic aberrations identified in MPAL.From 2013 to 2016, a total of 1329 patients with myeloid neoplasms were sequenced at the MSKCC with a targeted next generation sequencing panel containing 30 genes that are frequently mutated in acute leukemia. We identified 38 PHF6 mutations with predicted pathologic significance in 33 patients (2.5%) spanning the whole coding sequence: 10 frameshift, 11 nonsense, 1 splicing site, and 16 missense (Figure 1). The most common co-mutant gene observed in cases with PHF6 mutations was RUNX1 (30%, 11/37). No TP53 mutations are detected within the PHF6-mutant cohort(Figure 2). The 33 patients with PHF6 mutations included patients with a diagnosis of MPAL (n=5), AML (n=19), MDS (n=5), and MPN (n=4).The disproportional distribution of PHF6 mutations in MPAL prompted us to further analyze these patients. During the same period of time, 24 MPAL patients were identified and confirmed based on WHO 2016 classification (excluding AML-MRC and t-AML). Within this cohort, 3 (12%) had BCR-ABL1 (p190) fusion, and 1 (4%) had MLL translocation, two distinct genetically defined subgroups. Importantly, 5 (21%) harbored PHF6 mutations, these mutations were mutually exclusive with BCR-ABL1 or MLL. Four of the 5 patients showed T-cell lineage differentiation, in contrast to MPAL with BCR-ABL1 or MLL translocations which were characterized by B/myeloid differentiation. The 2-year overall survival for the PHF6 mutation positive MPAL was markedly reduced compared to non-mutated cases (40% vs. 100% in PHF6 unmutated cases (p=0.005)).In the larger subset of AML cases, PHF6 mutations were nearly exclusively present in secondary AML (11 AML-MRC, 6 t-AML and only 1 de novo AML). In contrast to their rarity in de novo AML, PHF6 mutations were identified in 11% (11/88) of AML-MRC and 7.1% (6/84) of t-AML. Interestingly, T-cell markers (cytoplasmic CD3, CD2, CD5, CD7) were often expressed on the blasts of secondary AML cases regardless of PHF6 mutational status (56%, 9/16 vs. unmutated 51%, 36/70). In comparison to unmutated cases, the blasts were often negative for CD33 (69%, 11/16, vs 28%, 20/70, p=0.004 by Fisher's exact test) and CD38 (44%, 7/16, vs. 17%, 12/70, p=0.04 by Fisher's exact test) in PHF6 mutated AML, suggestive of a stemness phenotype with aberrant T-cell differentiation.In conclusion, PHF6 mutations are the most common somatic alteration identified in MPAL and define a subgroup of MPAL with altered T-cell differentiation and inferior outcome. Amongst AML, PHF6 mutations are almost exclusively seen in secondary AML and are also associated with T-cell antigen expression and a more primitive stem/progenitor immunophenotype. PHF6 mutations co-exist with RUNX1 mutations but are mutually exclusive to TP53 mutations. Notably, PHF6 mutations may provide a genetic and functional link between MPALs with poor outcome and secondary AML and provide insights into shared mechanisms of transformation. RNA sequencing studies on the sorted blast population are ongoing and will be presented at the ASH meeting. [Display omitted] DisclosuresArcila:Invivoscribe: Honoraria; Archer: Honoraria; Raindance Tecnologies: Honoraria. Levine:Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Defining the Cell Surface Proteome of MLL Rearranged Leukemia

Introduction: B-acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr ) have a particularly dismal clinical outcome. Although it is known that this oncoprotein radically alters epigenetic regulation, the precise molecular mechanisms that define the cellular phenotype of these cancers remain poorly understood and new therapeutic strategies are sorely needed. Furthermore, although flow cytometric immunophenotyping has been performed for decades on B-ALL blasts, relatively little is known about the cell surface proteome of these cancers outside of canonical CD markers. Due to its role in modulating cellular behavior as well as accessibility to immunotherapy, the cell surface represents an attractive target for unbiased proteomic profiling in order to gain insight into cellular behavior as well as enabling the development of targeted therapies. Here, we aim to resolve the cell surface proteomic landscapes of MLLr and non- MLLr B-ALL cellular models. Our goal is to generate new mechanistic insights into the molecular re-wiring that occurs in these forms of cancer and identify novel candidates for targeted immunotherapy.Methods: We examined 10 B-ALL cell lines that harbor different oncogenic fusions including the MLL-AF4, BCR-ABL, and ETV6-RUNX1 translocations, respectively, using both “classical” lines as well as low-passage patient-derived lines, in addition to non-malignant B-cells as a control. We performed unbiased labeling of cell surface proteins by biotinylation of glycosylated amino acids (3 biological replicates each) followed by affinity pull-down with streptavidin. After on-bead trypsin digestion, peptides from bound glycoproteins were analyzed on a Thermo Q-Exactive Plus Mass spectrometer using 4-hour LC runs, data-dependent acquisition, and label-free quantification to identify and quantify members of the cell surfaceome for each cell line. To confirm our proteomic findings we performed flow cytometry using a BD CytoFLEX flow cytometer on select cell lines with fluorescently conjugated monoclonal antibodies, in order to assess presence and qualitative abundance of particular cell surface targets.Results: We quantified on average >900 high-confidence membrane proteins (FDR=0.05) per cell type, comprising ~40% of all proteins identified in each experiments. Principal component analysis identified unique cell surfaceome signatures that distinguish several of the leukemia subtypes and normal B-cells from each other, implying different cell-surface phenotypes associated with specific B-ALL genetic alterations (Figure 1A). As positive controls, we found several proteins differentially expressed in MLLr that were also previously identified based on transcriptomic profiling of MLLr patient samples, such as FLT3 and PROM1. In our data, the MLLr surfaceome was surprisingly enriched for Ephrin receptors, matrix metalloproteases, and other proteins that play a role in cell motility, invasion of tissues, and modulation of the tumor microenvironment (significantly enriched membrane proteins colored in red, Figure 1B). Using flow cytometry with monoclonal antibodies specific for select cell surface markers we found excellent agreement with our proteomic data. We will next move to patient-derived xenografts and tissues for further validation. These findings suggest these novel markers may be candidates for downstream immunotherapy development, potentially specific to MLLr B-ALL, where there is particularly high clinical need.Summary and Conclusions: In this work, we profiled the cell surface proteomes of multiple B-ALL cell models, identifying cell surfaceome signatures unique to different B-ALL subtypes. Overall, our work demonstrates the power of performing un-biased interrogation of the tumor surfaceome to identify new therapeutic targets for immunotherapy, as well as provide a resource to advance our phenotypic understanding of these aggressive cancer subtypes. [Display omitted] DisclosuresWiita:TeneoBio, Inc.: Research Funding; Sutro Biopharma: Research Funding.

Heterogeneous BCR-ABL Signal Patterns Identified By Fluorescence in Situ Hybridization Can Predict Leukemic Clonal Evolution and Poor Prognosis in BCR-ABL-Positive Leukemia

BCR-ABL translocation occurs in approximately 90% of chronic myeloid leukemia (CML), 25% of acute lymphoblastic leukemia (ALL) and less than 5% of acute myeloid leukemia (AML) cases that produce BCR-ABL fusion proteins, which are responsible for proliferative signals and leukemogenesis. Although extensive use of tyrosine kinase inhibitors (TKIs) has resulted in high and durable response rates and prolonged survival times in BCR-ABL-positive CML and acute leukemia patients in recent years, relapse and drug resistance are still big challenges for clinicians. Monitoring the quantification of BCR-ABL fusion genes by quantitative PCR, minimal residual diseaseby flow cytometry and ABL kinase mutations by sequencing are effective means of predicting disease relapse and resistance; however, the prognostic impact of BCR-ABL signal modes by fluorescence in situ hybridization (FISH) in leukemia patients remains elusive. In the present study, we analyzed BCR-ABL signal modes using FISH in 243 CML-chronic phase (CML-CP), 17 CML-blast phase (CML-BP) and 52 ALL patients. A total of 52.9% (n=9) of the complex BCR-ABL signal modes (≥ two types of BCR-ABL signal modes) were observed in the CML-BP patients, followed by 30.8% (n=16) in the BCR-ABL-positive ALL patients, and only 2.1% (n=5) in the CML-CP patients. Conversely, typical single BCR-ABL signal modes (single 1R1G2F fusion signals) were observed in 29.4% (n=5) of the CML-BP, 53.8% (n=28) of the BCR-ABL-positive ALL and 73.7% (n=179) of the CML-CP patients. Patients with atypical single BCR-ABL signal modes (no 1R1G2F single fusion signal, such as 1R1G1F or 1R1G3F) were similar, and they accounted for 17.6% (n=3), 15.4% (n=8) and 24.3% (n=59) of the CML-BP, ALL and CML-CP patients, respectively. Additionally, multiple BCR-ABL signal modes presented very complexity and various. In total, twelve BCR-ABL signal modes were observed in this cohort: 1R1G2F, 1R1G1F, 2R1G1F, 1R2G1F, 2R2G1F, 1R2G2F, 1R1G3F, 1G3F, 2G3F, 1G4F, 1R1G4F and 1R4F. More importantly, the development of BCR-ABL signal patterns can indicate leukemic clonal evolution, disease progression and relapse by sequentially monitoring the BCR-ABL modes at different time points of disease progression using FISH. In this study, we observed five clonal evolution modes related to disease progression among our ALL patients. First, patients presented sensitive single clones at disease onset, which disappeared after treatment, whereas primary clones gradually progressed to drug-resistant clones until relapse during treatment. Second, patients presented sensitive single clones during disease onset that disappeared after treatment, whereas new single resistant clones were observed during relapse. Third, patients presented sensitive single clones during disease onset, whereas new and primary resistant subclones simultaneously occurred during relapse. Fourth, patients presented many different subclones during disease onset. Some preponderant subclones were sensitive and disappeared after treatment, whereas other minor subclones were resistant and gradually progressed to preponderant subclones until relapse. Fifth, patients simultaneously presented many different subclones during disease onset. Some minor subclones were sensitive and disappeared after treatment, whereas the preponderant subclones were resistant to TKIs or chemotherapy drugs. All of the BCR-ABL-positive ALL patients received a TKI combined with chemotherapy treatment. Furthermore, we analyzed the impact of complex BCR-ABL signal modes on survival time in the ALL patients. We observed that patients with complex BCR-ABL signal modes had very poor estimated overall survival (OS) times compared with patients with single BCR-ABL signal modes (typical and atypical single BCR-ABL signal modes) (5.0 vs. 15.0 month, P=0.006). Moreover, there were no OS differences between the BCR-ABL-positive ALL patients and the CML-BP patients (13.0 vs.10.0 month, P=0.984). Conclusion: Our data show that complex BCR-ABL signal modes usually occurred in patients with BCR-ABL-positive CML-BP, followed by ALL patients and rare in CML-CP patients. Complex BCR-ABL signal modes with various in forms predicted leukemic clonal evolution and poor prognoses. Monitoring BCR-ABL signal mode changes may be an effective means to provide prognostic guidance and treatment choices for patients with BCR-ABL-positive leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Paraneoplastic Eosinophilia (PNE): A Rare Finding in a Patient With Metastatic Medullary Thyroid Carcinoma

Abstract Introduction: Medullary thyroid cancer (MTC) is a rare tumor of neuroendocrine origin that co-secretes various peptides leading to diarrhea and vasodilation. Ectopic Cushing syndrome due to production of adrenocorticotropic or corticotrophin-releasing hormone is a well-recognized but uncommon paraneoplastic manifestation of advanced MTC. We report an extremely rare presentation of eosinophilia in a patient with MTC that correlated with disease progression. Case: A 58-year-old woman with sporadic metastatic MTC harboring somatic RET M918T mutation developed metastatic disease to the liver, lung and bones 2 years after surgery for locally advanced disease. Calcitonin (Ctn) was 123 pg/ml and CEA was 2575 ng/ml. At that time, leukocytosis [WBC 30.8 K/ul (4-11k/ul)] and eosinophilia [eosinophil count 16.01 k/ul (0.04-0.40 k/ul)] were noted. She was asymptomatic. Extensive evaluation of hypereosinophilia ruled out hematological or infectious causes. The patient initiated vandetanib with a partial response (PR) with decrease in lung and liver metastases and a significant improvement of the paraneoplastic eosinophilia [eosinophil count 2.39 k/ul] after 10 weeks of treatment. After a year on treatment, there was progressive disease (PD) with increasing hilar and abdominal lymphadenopathy associated with increased eosinophilia of 4.34 K/ul. Vandetanib was discontinued. She was enrolled on a clinical trial with a highly potent and selective RET inhibitor. The patient achieved PR to study drug by the 2nd month on treatment and durable response for 30 months. The eosinophil count normalized [0.32 k/ul] 4 weeks after starting the new treatment. She developed PD in liver metastases associated with recurrent leukocytosis (WBC 58.6 K/ul) and eosinophilia of 28.13 K/ul. Ctn was 1646 pg/ml. CEA was 8722 ng/ml. Her bone marrow biopsy showed marked eosinophilia, focal MTC metastatic infiltrate, no increased blasts, and was negative for the BCR-ABL1 translocation and the FIP1L1-PDGFRA fusion. She was switched to a different RET Inhibitor but passed away 1 month after starting the new protocol. Discussion: Paraneoplastic eosinophilia should be considered after excluding other causes (e.g. infections, allergy, collagen, vascular or malignant hematopoietic diseases). Thyroid tumors producing colony-stimulating factors, associated with neutrophilia and/or eosinophilia have been described almost exclusively in patients with anaplastic thyroid cancer. This patient had a poorly differentiated MTC as evidenced by the disproportionally high CEA relative to Ctn. The course of the eosinophilia paralleled the clinical behavior of her disease. To our knowledge, this is only the 2nd report of eosinophil trends corresponding with MTC disease course, consistent with a paraneoplastic process. Conclusion: PNE is very rare in MTC and its presence suggests a poor prognosis.

Genetics and Diagnostic Approach to Lymphoblastic Leukemia/Lymphoma.

Our understanding of the genetics and biology of lymphoblastic leukemia/lymphoma (acute lymphoblastic leukemia, ALL) has advanced rapidly in the past decade with advances in sequencing and other molecular techniques. Besides recurrent chromosomal abnormalities detected by karyotyping or fluorescence in situ hybridization, these leukemias/lymphomas are characterized by a variety of mutations, gene rearrangements as well as copy number alterations. This is particularly true in the case of Philadelphia-like (Ph-like) ALL, a major subset which has the same gene expression signature as Philadelphia chromosome-positive ALL but lacks BCR-ABL1 translocation. Ph-like ALL is associated with a worse prognosis and hence its detection is critical. However, techniques to detect this entity are complex and are not widely available. This chapter discusses various subsets of ALL and describes our approach to the accurate classification and prognostication of these cases.

Arterial Thrombotic Complications of Tyrosine Kinase Inhibitors.

Abnormal expression or function of several classes of kinases contribute to the development of many types of solid and hematologic malignancies. TKs (tyrosine kinases) in particular play a role in tumor growth, metastasis, neovascularization, suppression of immune surveillance, and drug resistance. TKIs (tyrosine kinase inhibitors) targeted to TKs such as BCR-ABL1, VEGF receptors, PDGF receptors, have transformed therapy of certain forms of cancer by providing excellent efficacy with relatively low adverse event rates. Yet some of these agents have been associated with high rates of vascular events, presumably from prothrombotic complications that result in myocardial infarction, stroke, and critical limb ischemia. This review describes the scope of the problem evidenced by clinical experience with some of the most commonly used TKIs, with a focus on TKIs targeted to the BCR-ABL1 (breakpoint cluster region-Abelson 1) translocation. We also discuss the potential mechanisms responsible for arterial thrombotic complications that could lead to mitigation strategies or unique TK targeting strategies to reduce adverse event rates without compromising efficacy.

The efficacy and safety of a homoharringtonine-based protocol for children with acute myeloid leukemia: A retrospective study in China

The standard chemotherapy for acute myeloid leukemia (AML) is usually composed of anthracyclines and cytarabine. We previously reported that homoharringtonine (HHT) was incorporated into regimens for pediatric AML with acceptable efficacy and tolerable toxicity. We treated newly diagnosed AML patients aged 0–18 years on the AML-SCMC-2009 protocol. A total of 102 de novo newly diagnosed AML patients aged 0–18 years were enrolled. All patients were treated with ten courses of chemotherapy including double induction, high dose cytarabine consolidation, and maintenance. The cumulative dose of HHT was 165 mg/m2 and the daunorubicin dose was 120 mg/m2. Complete remission (CR), overall survival (OS) rate, event free survival (EFS) rate, adverse effect response and prognosis factors were retrospectively evaluated to investigate the long-term outcome and safety of this protocol. Eighty-two patients (80.4%) achieved complete remission with the first induction. The 5-year overall survival and event-free survival rates were 65.0% (SE, 4.9%) and 53.3% (SE, 5.2%), respectively. A first induction failure, age older than 2 years, and BCR-ABL translocation were associated with a significantly worse outcome (p < 0.05). No significant drug-related cardiac toxicity was observed. AML-SCMC-2009 protocol which use HHT as backbone drug is efficacious and tolerated. HHT could partially replace anthracycline to be an effective option for childhood AML.

CML-143: BCR-ABL1 Translocation Combined with JAK2V617F or CALRtype-1,2 Mutations in Russian CML Patients Undergoing TKI Therapy: Transcript Level and Mutation Allele Burden

Background Frequency of BCR-ABL1 translocation and JAK2V617F or CALRtype-1,2 mutation co-occurrence in chronic myeloid leukemia (CML) patients varies in different populations. According to Soderquist et al., it amounts to 0.4% of cases in USA; Martin-Cabrera et al. – 0.2% in Germany; Lewandowski et al. – 0.7% in Poland. Combination of BCR-ABL1 translocation with CALRtype-1,2 mutations in CML patients during tyrosine kinase inhibitor (TKI) therapy has not been reported yet. Prognostic value of the BCR-ABL1 transcript level kinetics and of JAK2V617F or CALRtype-1,2 mutations allele burden (AB) in Russian CML patients undergoing TKI therapy has not been assessed so far. Aims To evaluate frequency of coexistence and kinetics of the BCR-ABL1 transcript level and AB of JAK2V617Fand CALRtype-1,2 mutations in CML patients undergoing TKI therapy. Materials and Methods mRNA and DNA samples isolated from blood and bone marrow cells of 683 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2020 period were included in the study. The BCR-ABL1 transcript level and AB of JAK2V617F mutation were determined using a highly sensitive (Log-5) quantitative real-time polymerase chain reaction. CALRtype-1,2 mutations were investigated using fragment analysis followed by Sanger sequencing. Results The combination of the BCR-ABL1, JAK2V617F, and CALRtype-1,2 mutations among CML patients receiving TKIs occurred in 8 of 683 cases (1.17%). Out of these, the combination of BCR-ABL1 with JAK2V617F, and the combination of BCR-ABL1 with CALRtype-1,2 mutations were detected in 0.88% (6/683) and 0.29% (2/683) of cases, respectively. During TKI therapy, in 6 out of 8 cases, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these cases, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second generation TKIs. Conclusions The combination of the BCR-ABL1 translocation with JAK2V617F or CALRtype-1,2 mutations is a rare event in our cohort of patients and occurred in 0.88 and 0.29% of cases, respectively. The combination of the BCR-ABL1, JAK2V617F, and CALRtype-1,2 mutations may not interfere with the achievement of major MR under TKI treatment.

Abstract 3529: Detection of genomic structural variations associated with drug sensitivities and resistance in AML using novel whole genome imaging

Abstract Acute myeloid leukemia (AML) is a particularly devastating collection of hematological cancers and whilst somewhat rare, the patient survival rate is abysmal without bone marrow transplantation. Traditional chemotherapies administered to AML patients cause significant and devastating side effects. Additionally, more than 30% of patients fail to respond to these initial treatments and most patients that do respond will eventually relapse within 5 years. As such, understanding the evolution of AML to identify novel targets, and therefore drug treatment regimens, is a significant medical need. Genomic rearrangements and other Structural Variations (SVs) have long been known to be causative and pathogenic in multiple cancers, including leukemias. Indeed the discovery of the “Philadelphia chromosome” (eventually identified as a BCR-ABL translocation) as causative in Chronic Myeloid Leukemia has prompted much research into SVs in cancers, including the development of targeted therapeutics against oncogenic proteins resulting from genomic rearrangements. These SVs may be involved in cancer initiation, progression, clonal evolution, and drug resistance, and a better understanding of SVs from individual AML patients may help guide therapeutic options. Here we show utilization of an innovative whole genome imaging technology to detect known, and novel, SVs in AML patients' samples. Importantly, this new technology provides an unprecedented level of granularity and quantitation unavailable to other current techniques and it allows an unbiased detection of novel SVs, which may be relevant for disease pathogenesis and/ or drug resistance. Coupled with standard gene expression analyses we have also assessed the chemosensitivities of these samples to 120 FDA approved oncology drugs and 335 epigenetic modulating agents. Here we show how integrative analysis of these diverse datasets is used to associate the detected genomic rearrangements with drug sensitivity profiles, potentially identifying novel therapeutic targets for individual AML patients. Citation Format: Darren Finlay, Rabi Murad, Karl Hong, Joyce Lee, Andy Pang, Chi-Yu Lai, Carol Burian, James Mason, Alex Hastie, Jun Yin, Kristiina Vuori. Detection of genomic structural variations associated with drug sensitivities and resistance in AML using novel whole genome imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3529.

Association of Myeloid Aberrant Antigens and the Presence of BCR-ABL Gene Re-arrangements in ALL Patients

Background: BCR-ABL translocation is the most common genetic abnormality associated with adult Acute Lymphoblastic Leukaemia (ALL) with poor outcome. Objective: The aim of the study was to determine the association of myeloid aberrant antigens and the presence of BCR-ABL gene rearrangements in acute lymphoblastic leukaemia (ALL) patients in our context. Method: A total of 38 ALL patients were included in this cross-sectional study from August 2018 to July 2019 according to selection criteria. BCR-ABL was detected by Real Time-Polymerase Chain Reaction (RT-PCR). Results: The median age at diagnosis was 27.5 years with male (76.3%) predominance. Aberrant myeloid markers, e.g.CD13 was present in 9(64.3%) patients who were BCR-ABL positive which was statistically significant (p:&lt;0.05). Conclusion: Early suspicion about BCR-ABL positivity can be made in ALL patients who show aberrant myeloid expression.

The Current Genomic and Molecular Landscape of Philadelphia-like Acute Lymphoblastic Leukemia.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk B-cell Acute Lymphoblastic Leukemia (B-ALL) characterized by a gene expression profile similar to Ph-positive B-ALL but lacking the BCR-ABL1 translocation. The molecular pathogenesis of Ph-like B-ALL is heterogenous and involves aberrant genomics, receptor overexpression, kinase fusions, and mutations leading to kinase signaling activation, leukemogenic cellular proliferation, and differentiation blockade. Testing for the Ph-like signature, once only a research technique, is now available to the clinical oncologist. The plethora of data pointing to poor outcomes for this ALL subset has triggered investigations into the role of targeted therapies, predominantly involving tyrosine kinase inhibitors that are showing promising results.

A Rare Entity in Chronic Myelocytic Leukemia: Coexistence of BCR ABL1 Translocation and JAK2 V617F Mutation. Case Report

Myeloproliferative neoplasms (MPNs) are diseases of myeloid series usually with an increase in peripheral blood cells. Among this disease group, only chronic myeloid leukemia (CML) has t(9; 22) (Philadelphia chromosome) leading to the production of BCR-ABL1 fusion gene with tyrosine kinase activity. The main distinction between the diagnosis and treatment of this heterogeneous group is the presence of BCR-ABL1 or JAK-2 V617F mutation. The coexistence of these two mutations may be observed, although not frequently, and can lead to delay in the diagnosis. The case here presents the detection and clinical management of JAK-2 V617F mutation after the suspicion of other MPNs due to fluctuations in clinical and hematological responses of this rare entity in a 78-year-old female patient with CML. To the best of our knowledge, this is the first CML case with coexistence of BCR-ABL1 translocation and JAK2 V617F mutation from Turkey.

Role of IGF2BP1 and Target Genes in ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia is the most prevalent hematological neoplasm in Indian children and around 85% are of B-cell origin (B-Acute Lymphoblastic Leukemia (B-ALL)). ETV6-RUNX1 (E/R) is one of the common translocations seen in B-ALL. We have identified Insulin like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1), an oncofetal protein, to be specifically overexpressed in bone marrow samples of E/R positive B-ALL patients. We have tried to characterize the role of this protein in B-ALL. We collected 193 new paediatric pre B-ALL bone marrow samples. Most of the patients were under 5 years of age (73%) and only 3% of patients were more than 10 years. 66% of patients had no known translocation. Among the ones with translocation, ETV6-RUNX1 and BCR-ABL translocation were the commonest (11% each). 10% were E2A-PBX positive and only 3% had an MLL translocation. The translocation profile was very different from that of previously published reports. E/R translocation generally portends a good prognosis. There was a significantly lower initial WBC count in E/R positive group when compared to the other groups. However, there was no significant difference in other blood parameters including initial Hemoglobin levels and platelet counts. Interestingly, the proportion of patients with a high number of blasts in peripheral blood on day 8 post chemotherapy was actually significantly higher in the E/R +ve group indicating that there might be a proportion of patients in the E/R +ve group with a worse prognosis. There was no difference in minimal residual disease between the E/R translocation positive and negative groups. Real time RT-qPCR analysis of IGF2BP1 and ETV6 mRNA expression in bone marrow samples revealed significantly higher expression of IGF2BP1 in E/R positive B-ALL compared to E/R negative B-ALL samples. ETV6 mRNA relative expression on the other hand was found to be significantly lower in the E/R positive group. This corresponds to existing data that there is usually loss of the other wild type ETV6 allele in these patients. We used data from multiple studies to narrow down on true targets of IGF2BP1 in B-ALL. We used a published eCLIP dataset of IGF2BP1 to identify cognate RNA targets. Over expression of these targets in E/R +ve samples was confirmed from the other previously published high throughput gene expression studies on B-ALL (MILE (microarray innovations in leukemia) study (n = 3242) and Palanichamy et al, 2016, J Clin Invest). We identified 13 such genes which were bound by IGF2BP1 and regulated by it in E/R +ve B-ALL. One of these targets, EGFL7, has previously been shown to play an important role in haematopoiesis and involved in Acute Myeloid Leukemia pathogenesis. c-MYC is a known target of IGF2BP1 and. being a proto oncogene has an important role in B-cell proliferation. However, the biological and clinical role of cMYC and EGFL7 genes had not been evaluated in ETV6-RUNX1 positive Pre B-ALL. Real time analysis of EGFL7 and cMYC expression in the patient bone marrow samples showed significantly altered expression in both groups. As expected, EGFL7 showed a significantly higher expression in E/R +ve samples. Unexpectedly, cMYC showed lower expression in E/R positive group. The expression of EGFL7 also showed a significant positive correlation with initial hemoglobin levels and a negative correlation with initial WBC counts. To study the effect of IGF2BP1 binding to the EGFL7 or c-MYC UTRs, they were cloned downstream of firefly luciferase and coexpressed with IGF2BP1 in HeLa cells. Interestingly, the dual luciferase assay revealed no stabilization of either of these UTRs by IGF2BP1. This reveals a much more complex regulation by IGF2BP1 of its targets which might involve RNA methylation. To utilize the expression of IGF2BP1 and its target genes as a diagnostic marker for the presence of the E/R translocation, ROC curves using FISH/PCR as the gold standard test were prepared. The AUC (Area under curve) for ROC of the expression of IGF2BP1, c-MYC and EGFL7 were 0.917, 0.7 and 0.78 respectively. We also analyzed ROC curves using various combinations of these genes. Gene expression of IGF2BP1 alone was the best indicator of the presence of the ETV6-RUNX1 translocation with the highest sensitivity and specificity. In conclusion, relative expression of IGF2BP1 can serve as an excellent diagnostic marker for the E/R translocation with more than 90% sensitivity and 85% specificity. Disclosures No relevant conflicts of interest to declare.

Characterization of BCR-ABL Specific CD4+ T Cells and Their Role in Controlling Ph+ Acute Lymphoblastic Leukemia

Background: Patients with BCR-ABL+ acute lymphoblastic leukemia (Ph+ ALL) can experience significant disease reduction after induction chemotherapy, but subsequently incur adverse long-term outcomes, due to the presence of minimal residual disease (MRD). This suggests a need for novel therapeutic strategies to eliminate MRD. Checkpoint blockade has been considered of little benefit in ALL due to the overall low frequency of somatic mutations. However, the protein product of the BCR-ABL translocation region forms a tumor associated antigen (TAA). Prior studies have demonstrated that patients with Ph+ ALL harbor CD8+ and CD4+ T-cells specific for peptides derived from this junctional region, presented in MHCI or MHCII contexts. This suggests that relapse in Ph+ ALL may be countered by immunotherapeutic strategies that expand sufficient numbers of BCR-ABL-specific effector T-cells. The current study uses a murine model of Ph+ ALL to characterize effector cell types and mechanisms responsible for leukemia control and eradication. We also investigated the impact of commonly used chemotherapy agents on effector cell function, to determine the viability of a combination immunotherapy and vaccination approach at eliminating MRD after induction. Methods: Bone marrow cells from p19ARF-null C57BL/6 mice were transduced with a BCR-ABL expressing retrovirus. Transformed cells were injected into immunocompetent B/6 mice, resulting in a uniformly fatal leukemia in 20-25 days. We used a peptide: MHC-II tetramer to label endogenous CD4+ T-cells specific for a BCR-ABL-derived peptide ("BAp") presented in an MHC II I-Ab context (hereafter referred to as "BAp"-specific T-cells). Results: Previously, we found that BAp-specific CD4+ T-cells were elicited during leukemia development but were limited by regulatory-T-cells (Tregs) that were cross-reactive with endogenous ABL protein. Nonetheless, a heterologous vaccination strategy using BAp peptide, combined with dual CTLA-4 and PD1 checkpoint blockade, was able to extend survival and eradicate leukemia in a subset of mice. Prolonged survival correlated with the robust expression of an ensemble of antigen-presentation molecules by host leukemia cells, as well as the presence of polyfunctional, cytotoxic CD4+ BAp-specific T-cells. To formally evaluate the relative contributions of CD4+ vs. CD8+ T-cells, we next established leukemia and allowed it to progress after the selective depletion of CD4+, CD8+, or both CD4+ and CD8+ T-cells. All mice were also treated with nilotinib and PD-L1 checkpoint blockade. Depletion of CD4+ T-cells led to markedly shortened survival. In contrast, depletion of CD8+ T-cells had no effect, and the majority of mice survived long-term. As this confirmed that CD4+ T-cells were critical for leukemia control, we next characterized the impact of common induction chemotherapy regimens on this subset. Combinations of cytotoxic chemotherapies mimicking either a standard intensity Hyper-CVAD-like regimen or a reduced-intensity regimen were administered to naïve mice, followed by vaccination with BAp. Mice treated with a Hyper-CVAD-like regimen generated fewer numbers of BAp-specific CD4+ T-cells in response to subsequent vaccination. Moreover, scRNAseq analysis of activated CD4+ T-cells demonstrated that standard intensity regimens induced dysfunctional gene expression signatures. These changes were not observed after treatment with a reduced intensity regimen. Dysfunctional signatures were characterized by the upregulation of senescence-associated genes including p21, as well as decreased expression of T-stem-central-memory (Tscm) genes including TCF7 and Tox2. Altogether these findings suggest that Hyper-CVAD-like chemotherapy inhibits proliferation as well as the self-renewal of CD4+ T-cells responding to leukemia antigens. Conclusions: CD4+ T-cells specific for peptides derived from the BCR-ABL protein are responsive to PD-L1 checkpoint blockade, and are critical for Ph+ ALL clearance. Standard intensity induction chemotherapy leads to significant dysfunction of these cells. Ongoing experiments are exploring the mechanisms underlying this dysfunction, and methods to overcome it via BCR-ABL-based peptide vaccination combined with checkpoint blockade. Validation experiments utilizing human samples are also underway and will be reported. Disclosures No relevant conflicts of interest to declare.