No abstract

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

Our patient is a 36-year-old man referred by his general physician to the Department of Hematology because of mild neutropenia in a routine analysis at work. There was no history of previous diseases, and examination was normal. Blood investigations confirmed the neutropenia and showed elongation of prothrombin time. A bone marrow examination was performed revealing about 10% of myeloblasts on the aspirate smears. A cytogenetic study showed chromosome 16 inversion in all of these cells and tetraploidy only in some of them, which were extremely large in size. According to the revised WHO classification of tumours (2008), the patient was diagnosed as a case of acute myeloid leukaemia with chromosome 16 inversion.

Background Acute promyelocytic leukemia (APL) is characterized by pathologic promyelocytes, chromosomal translocations t(15:17) (q22; q21) and clinical and cytological response to all-transretinoic acid (ATRA). A significant minority of patients with APL have variant translocations involving RARα gene with a different partner, not involving PML. These APL variant translocations may or not respond to ATRA treatment. Case report A routine laboratory evaluation, before minor surgery, revealed leukocytosis, thrombocytopenia and blasts presence in peripheral blood (PB), in an otherwise asymptomatic 70-year-old man. He had a previous history of hypertension, dyslipidemia, ischemic heart disease, double coronary bypass and prostatectomy for non-malignant prostatic hypertrophy. Peripheral blood (PB) count: hemoglobin concentration 12g/dL, platelet count: 21x109/L and white blood cell count (WBC): 35x109/L. Peripheral blood smear: myelemia exhibiting 74% blasts (including myeloid blast and promyelocytes) with fine chromatin pattern, heterogeneous and frequently convoluted nuclei and one to three prominent nucleoli. The cytoplasm of these cells was basophilic with scanty purple granules. No Auer rods were found. The basic coagulation tests were normal(INR, APTT and fibrinogen) but D Dimer was 33.87 µg/ml. Normal biochemistry was shown, except for GGT 85 U/L and lactate dehydrogenase 2244 U/L. Bone marrow (BM) aspirate was unsuccessful (”dry tap”) but the bone trephine revealed an infiltration of 80% myeloid blasts. Cytochemistry: myeloperoxidase stain was deeply positive and chloroacetate and non-specific esterase stains were negative. Flow cytometry immunophenotype on PB was consistent with AML, likely M3 leukemia, with an atypical phenotype (MPO+, CD117+low, CD34+, CD13++, CD45+, CD15-,CD133-, CD33+, HLA-DR+low het , CD5-, CD7-, CD11b- , CD56+het, CD10-, CD8- ,CD4-, CD45RA+, CD19-, CD2-, Glya-, CD71+low, CD38-, CD1a-, CD41-, CD14-). The analysis of the PML/RARα fusion gene, according to standard protocols, was negative. FISH studies showed 2 PML signals and 3 RARα copies, suggesting a possible variant rearrangement of this gene. FISH and molecular biology did not detect transcript for t(15:17), (5:17) or (11:17); so RARα was translocated with a strange partner. Suspecting of a variant APL, the patient was treated with ATRA plus Idarrubicin. After 4 weeks treatment, deep neutropenia was observed but no granulocytic maturation. That suggested us resistance to ATRA. Thus, we changed the treatment to a classical induction scheme for AML. The patient attained cytologic remission, which he mantains so far with conventional chemotherapy. Karyotype study was subsequently performed, identified an unbalanced rearrangement between 7q and 17q. Currently, we are identifying and sequencing the fusion partner of RARα. Conclusion We report a novel case of variant acute promyelocytic leukemia with the karyotype der (7) t(7;17) (q11;q21). The morphology of PB and BM was critical for initial diagnosis and treatment decisions. Disclosures: No relevant conflicts of interest to declare.

Background: Kinase domain (KD) mutations is a common resistance mechanism, secondary to the tyrosine-kinase inhibitors (ITKs) treatment in the case of chronic myeloid leukemia (CML) and Philadelphia (Ph)-positive acute lymphoblastic leukemia (ALL) patients. Sanger sequencing is the gold standard technique and already the currently recommended method for BCR-ABL1 KD mutation detection. However, Sanger sequencing has limited sensitivity and cannot firmly identify populations with variant allele frequencies (VAF) < 15-20%. Next-generation sequencing (NGS) allow us the screening of mutations in the whole KD with variants with a VAF greater than 1%. The aim of this study is to evaluate the clinical and prognostic implications of CML and Ph-positive ALL patients who have been studied for mutations in BCR-ABL1 by NGS. Methods: Seventy CML and Ph-pos ALL patients have been studied for BCR-ABL1 mutations between years 2015-2017. The study reason was warning or failure according to European Leukemia Net recommendations in the case of CML patients, and diagnostic or relapse in the case of ALL patients. Clinical characteristics of the patients are depicted on Table 1. Categorical variables are described as frequency, and quantitative variables as medians. Contingency tables were used to analyze associations between categorical variables (χ2). Median test was used to compare medians of continuous variables between groups. Overall survival (OS) was estimated using the Kaplan-Meier method and compared between patients using the log-rank test. Results: We have found 37 patients with mutations (51%), the most frequent being p.T315I, p.L248V and p.L387M. 28 out of 59 were found in CML (47%) vs 9 out of 13 (69%) in ALL. Of the 37 patients with mutations, double mutations have been found 10 times (27%). In the 72 analyses performed, 62 mutations were found in total, 41 of them were variants of uncertain significance (VUS) and 21 were well-known mutation. The median levels of BCR-ABL1 (IS) at the time of analysis were 3.00 (0.01-196.18) %. Regarding CML patients, we have found 12 and 16 cases with pathogenic mutations and VUS, respectively. The mean survival for CML and ALL were 75.2 months (CI 95%, 65.7-84.6) and 24.7 months (13.3-36.2), respectively. There are significant differences between the overall survival curves for patients with CML who have mutations in BCR-ABL1 compared to those who have VUS or do not (p-value = 0.024, n=59), suggesting a second role of the VUS variants in the resistance of the patients to the TKI. These two groups have no significant differences in ALL patients (p-value= 0.32, n=13). Overall survival at 10 years from the date of diagnosis is 74% for CML patients with mutations and 90% for CML patients without mutations. Data dropped significantly for ALL patients, but the number of cases is too low. Conclusions: - Mutations have been identified in 47% of CML patients studied in the case of failure or warning and 69% of the patients of ALL at diagnosis or relapse moments. - The identification of pathogenic variants has poor prognosis in patients with CML (p = 0.024), however no differences were observed in ALL. - The identification of VUS is not associated to poor prognosis and these variants could not confer resistance to ITK. Disclosures Sevilla: Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Steegmann:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. García Gutiérrez:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.