Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML. N Engl J Med. 2013 May 9; 368:1781 Speaker: CR 呂學儒醫師 Moderator: VS 蕭樑材醫師

Definition of CNL and atypical CML Leukocytosis and hypercellularity of BM, predominantly of granulocytic cell Absence Ph chromosome , absence of rearrangements of PDGFR A/B and FGFR1 CNL: neutrophils in both periphreal and BM blood (segmented neutrophils and band forms > 80% of WBC) Atypical CML : granulocytic dysplasia, increased neutrophil precursors in both peripheral and BM blood (typically, ≥10% of white cells) Hematology Am Soc Hematol Educ Program. 2011;2011:250-6

Clinical, hematological and cytogenetic characteristics of aCML Background: an infrequent chronic MPN leukocytosis, absence of Ph/BCR-ABL, marked myeloid dysplasia immature granulocytes > 15%, some with absolute monocytosis, D/D CMML Result: median age: 65 years 55% had moderate anemia and 36% had thrombocytopenia Most with marked dysplasia, particularly in the granulocytic lineage Cytogenetic data: trisomy 8, 5q-, 13q-, 17p-, 12q-, and 11q-, t(6;8)(p23;q22) median survival: 14 months Ann Oncol. 2000;11:441

Histopathology of peripheral blood and BM of CNL N Engl J Med. 2013;368:1781

G-CSF and its receptor in myeloid malignancy Granulocyte colony-stimulating factor(G-CSF/CSF3): major regulator of neutrophil production granulopoiesis during infections and enhances multiple neutrophil functions inducing proliferation and survival of myeloid progenitor cells The receptor for CSF3 (CSF3R) 17 exons and its protein 813 amino acids Activation with Jak/STAT, Ras/Raf/MAP kinase, and PKB/Akt pathways Colony-stimulating factor 3 receptor gene (CSF3R) mapping to chromosome 1p provides the proliferative and survival signal for granulocytes contributes to their differentiation and function CSF3R mutations: severe congenital neutropenia, secondary developed at AML (1%) Blood. 2010;115:5131 Leukemia 2013. doi: 10.1038

Use of Hematopoietic Growth Factors in the Survival and Differentiation of Hematopoietic Cells NEJM 2013;368:1131

Hematopoietic Growth Factor Signaling NEJM 2006;354:2034

Sequential gain of mutations in severe congenital neutropenia (SCN) progressing to acute myeloid leukemia (AML) Blood. 2012;119:5071

Model for Activation and Signaling of CSF3R Mutations Two different classes of CSF3R mutations N Engl J Med. 2013;368:1781

The mutations in CSF3R are a defining molecular abnormality of CNL/atypical CML Testing for CSF3R mutations could aid in the diagnosis of these diseases 5 patients: both membrane proximal and truncation mutations AML(SCN progression): Q741X mutation ETP-T-ALL: one of T618I

CSF3R deep sequencing CNL, CSF3R (S783fs mutation)

Dependence on SRC Family-TNK2 or JAK Kinases (Truncation mutations) CNL, CSF3R (S783fs mutation) for 66 kinase inhibitor Hypersensitive to dasatinib Insensitive to JAK kinase inhibitors IC50: 50% inhibitory concentration; SFK: SRC family kinase; TNK2: tyrosine kinase nonreceptor 2 small interfering RNAs (siRNAs) silencing of TNK2 and FGR (an SFK) inhibited by dasatinib

Dependence on Src Family-TNK2 or JAK Kinases (membrane proximal mutations) CLL, CSF3R (T618I mutation) for 66 kinase inhibitor Insensitive to dasatinib Sensitive to JAK kinase inhibitors

Two different classes of CSF3R mutations Truncation mutations(S783fs) Dysregulation of SRC family–TNK2 kinases sensitivity to dasatinib but not to JAK kinase inhibitors Membrane proximal mutations(T618I) Dysregulation of JAK family kinases sensitivity to JAK kinase inhibitors but not to dasatinib

Distinct signaling-pathway dysregulation To test the relative transforming capacity Expressing wild-type CSF3R, membrane proximal mutations, or truncation mutations Infected with murine retrovirus Over a 10-day period CSF3R mutations were capable to induce transformation Membrane proximal mutations faster

The potential signaling differences between the two classes of CSF3R Immunoblot analysis for JAK–STAT phosphorylation T618I mutant induced high levels of STAT3 -JAK2 phosphorylation; S783fs mutant was low Two classes of CSF3R mutations have different transformation potential and downstream signaling consequences

Use of tyrosine kinase inhibitors the sensitivities of CSF3R, mice

Clinical efficacy of Ruxolitinib in a patient with CSF3R T618I

CNL vs.atypical CML CNL and atypical CML separate neoplasms by the WHO But challenging for clinicians and hematopathologists The categorization relies on arbitrary thresholds Total WBC ( ≥25,000 for CNL; ≥13,000 for atypical CML) immature granulocytes (<10% for CNL; ≥10% for atypical CML) the presence or absence of dysgranulopoiesis (absent in CNL and characteristic of atypical CML) CSF3R mutations : a biologically unifying feature of CNL and atypical CML the molecular classification of MPD and MDS/MPD

Sequenced CSF3R in 54 cases CNL (n=35) or atypical CML(n=19) WHO defined: 12 patients diagnosed CNL; 5 monoclonal gammopathy (MG)-associated CNL; 9 aCML 14 CSF3R mutations detected in 13 patients CSF3R T618I is the most mutation: 10/13 CSF3R T618I frequency: 83% (10/12) in WHO-defined CNL  CSF3R mutations not in aCML or MG-associated CNL CSF3RT618I also absent in PMF(n=76) and CMML(n=94) CSF3RT618I highly sensitive and specific molecular marker for CNL should be incorporated into current diagnostic criteria Leukemia 2013. doi: 10.1038

Leukemia 2013. doi: 10.1038

Genetically Informed Therapy in Leukemia Jerald Radich, M. D Genetically Informed Therapy in Leukemia Jerald Radich, M.D. Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance Julia E. Maxson A TK mutation which play a major role in myeloid cancer Identified a novel mutations in CSF3R Tested in vitro with kinase inhibitors Different types sensitivity to different therapeutic agents Treated a patient with dramatic improved in WBC, Neutrophil, and PLT Therapeutic benefit in these rare disorders power of genetic screening to uncover new potential drug targets This is how we will beat cancer, one gene, one disease at a time. NEJM 2013;368:1838

Take home message CSF3R mutations: identified in >50% CNL/aCML Consider as a diagnostic criteria The oncogenic CSF3R mutations Truncation mutations or membrane proximal mutations sensitivity to inhibitors of SRC family–TNK2 and JAK kinases

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