Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML .

1. OncogenicCSF3R mutations in chronic neutrophilic leukemia and atypical CML.OncogenicCSF3R 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

2. 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 HematolEduc Program. 2011;2011:250-6

3. 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

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

5. 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 neutrophilfunctions • 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/Aktpathways • 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

6. Use of Hematopoietic Growth Factors in the Survival and Differentiation of Hematopoietic Cells NEJM2013;368:1131

7. Hematopoietic Growth Factor Signaling NEJM2006;354:2034

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

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

10. 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

11. CSF3R deep sequencing CNL, CSF3R(S783fs mutation)

12. 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 kinasenonreceptor 2 small interfering RNAs (siRNAs) silencing of TNK2 and FGR (an SFK) inhibited by dasatinib

13. 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

14. 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

15. 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

16. The potential signalingdifferences 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

17. Use of tyrosine kinase inhibitorsthe sensitivities of CSF3R, mice

18. Clinical efficacy of Ruxolitinib in a patient with CSF3R T618I

19. 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

20. Sequenced CSF3Rin 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 Leukemia2013. doi: 10.1038

21. Leukemia 2013. doi: 10.1038

22. Genetically Informed Therapy in LeukemiaJerald 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

23. Take home message • CSF3R mutations: identified in >50% CNL/aCML • Consider as a diagnostic criteria • The oncogenicCSF3R mutations • Truncation mutations or membrane proximal mutations • sensitivity to inhibitors of SRC family–TNK2 and JAK kinases

24. Thank you for your attention