All subjects and/or their legal guardian(s) gave their informed consent to participate. The study was approved by the Ethical Committee of the Iran University of Medical Sciences (Code: IR.IUMS.REC 1395.95-04-87-30235). Inclusion criteria were based on clinical presentations and hematological findings in the peripheral blood and bone marrow, according to the WHO 2016-guideline 14. Three patients (from 3 unrelated families) with the clinical diagnosis of blastic phase CML were recruited from the Shariati Hospital in Tehran, Iran. In the present study, we conducted integrated genomic sequencing through an in-house filtering algorithm to uncover the leukemogenic important/actionable findings and investigate the myeloid leukemogenesis model in three Iranian myeloid blast crisis CML patients. It was shown that the combination of exome, genome and RNA Sequencing data 9, called integrative sequencing 10, 11, 12 paves the way to better understand the mechanisms and pathogenesis of the cancer and can potentially improve the clinical management and finding new therapeutic targets specially in the advanced phase patients 13. Over the past decade using Massively Parallel Sequencing (MPS) or Next Generation Sequencing (NGS) the multi-dimensional genomic view with higher resolution of molecular profiling has become possible 8. In general, blast crisis in chronic myeloid leukemia is still a fatal disease 6, 7. Even in the advent of Tyrosin Kinase Inhibitor therapy, blastic phase survival is less than 12 months. Without therapeutic interventions or in the case of drug resistance, CML will progress to accelerated phase (AP) and then into an acute leukemia phase that is called blastic phase or blast crisis 5. Leukemogenic alterations mainly affect five classes of regulatory proteins: signaling pathway components, transcription factors (TFs), epigenetic regulators (ERs), tumor suppressor genes (TSGs), and components of the spliceosome 4. Further oncogenic processes cause LSCs to develop into leukemia progenitor cells (LPCs) 3. In this step, the disease may be undetectable for a decade or more. Premalignant leukemia stem cells (LSCs) are, in fact, generated in the bone marrow nich by unknown mutagenesis processes 1, 2. This strategy may be an effective method for identifying actionable findings and understanding the pathophysiological mechanisms underlying MBC-CML, as well as providing further insights into the genetic basis of MBC-CML and its management in the future.Ĭhronic Myeloid Leukemia (CML) is a unique model for the evolution of cancer and is classified as a triphasic myeloproliferative neoplasm based on clinical and pathological characteristics. Integrated genomic sequencing combined with RNA-sequencing can successfully discover and confirm a wide range of variants, from SNVs to CNVs. 2 resulted in the deletion of TP53 gene as well. The large deletion discovered on the short arm of chromosome 17 in patient No. 3, chromosome sequencing revealed multiple pathogenic deletions in the short and long arms of chromosome 7, affecting at least three critical leukemogenic genes ( IKZF1, EZH2, and CUX1). RNA-sequencing was used to confirm all of the identified variants. The GATA2-元59S in patient No.1, PTPN11-G503V and IKZF1-R208Q variants in the patient No.3 were also PAFs. Both patients No.1 and No.2 had actionable known missense variants on ABL1 (p.Y272H, p.F359V) and frameshift variants on ASXL1 (p.A627Gfs*8, p.G646Wfs*12). These variants affect genes involved in leukemia stem cell proliferation, self-renewal, and differentiation. Single nucleotide variation (SNV) and small InDel analysis by WES detected sixteen PIFs affecting all five known classes of leukemogenic genes in myeloid malignancies including signaling pathway components ( ABL1, PIK3CB, PTPN11), transcription factors ( GATA2, PHF6, IKZF1, WT1), epigenetic regulators ( ASXL1), tumor suppressor and DNA repair genes ( BRCA2, ATM, CHEK2) and components of spliceosome ( PRPF8). Standard variant interpretation guidelines were used for the interpretation of potentially important findings (PIFs) and potentially actionable findings (PAFs). An in-house filtering pipeline was applied to assess important variants in cancer-related genes. Integrated genomic sequencing via Whole Exome Sequencing (WES), Chromosome-seq and RNA-sequencing were conducted on the peripheral blood samples of three CML patients in the myeloid blast crisis. The current study identified multiple common and rare important findings in myeloid blast crisis CML (MBC-CML) using integrated genomic sequencing, covering all classes of genes implicated in the leukemogenesis model. Chronic myeloid leukemia (CML) is a model of leukemogenesis in which the exact molecular mechanisms underlying blast crisis still remained unexplored.

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