1. 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

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 Hematol Educ 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 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:

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

7. Hematopoietic Growth Factor Signaling
NEJM 2006;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 Mutations Two 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 kinase nonreceptor 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 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

17. Use of tyrosine kinase inhibitors the 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 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:

21. Leukemia 2013. doi:

22. 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

23. 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

24. Thank you for your attention