Journal reading

1. Journal reading 20131021 住院總醫師 王智慧 報告 高志平醫師 指導

3. MYD88 is an adaptor protein through which most of the Toll-like receptors (TLRs) and receptors for IL-1 and IL-18 cytokines signal coordinates the assembly of a multisubunit signaling complex, including IL-1 receptor-associated kinase (IRAK) 1 and IRAK4, and also signals to JAK-STAT 3 signaling and to activation pathways and transcription factors, such as the NF-kB.

4. The genetics and pathogenesis of Waldenstrom macroglobulinemia (WM), are not fully understood A whole exome–sequencing study of 31 WM patients suggested a high frequency of MYD88 L265P mutation in WM. aim to analyze MYD88 mutation in WM and to characterize the clinical significance of this genetic alteration in a cohort of 67 WM patients.

7. whole-genome sequencing of bone marrow LPL cells in 30 patients with WM, validate by Sanger sequencing a somatic variant (T→C) in LPL cells was identified at position 38182641 at 3p22.2 predicted an amino acid change (L265P) in MYD88 triggers IRAK-mediated NF-κB signaling 27 of 30 (90%)

8. Inhibition of MYD88 signaling reduced IκBα , NF-κB p65 phosphorylation, and NF-κB nuclear staining,

9. TIRAP: TIR domain containing adaptor protien IRAK: IL-1 receptor- associated kinase TRAF6: TNF receptor-associated factor 6 TAK1: TGF ß-activated kinase 1 TAB1, TAB2: TAK1 binding protien IKK: IκB kinase

10. 67 patients (42 males, 25 females) diagnosed with WM diagnosis criteria of WM were (1) a lymphoplasmacytic bone marrow (BM) involvement; (2) any level of IgM paraprotein; (3) exclusion of other low-grade lymphomas No familial form of WM was included Patients

11. 5 MM, 9 CLL, 6 hairy cell leukemia, 23 follicular lymphoma, 10 mantle cell lymphoma, 2 IgM MGUS, 1 non-IgM lymphoplasmacytic lymphoma, 16 marginal zone lymphoma (MZL).

12. Methods DNA sequencing of exon 5 of MYD88 Single nucleotide polymorphism (SNP) array analysis

14. Results

15. High frequency of MYD88 L265P mutation in WM Fifty-three (79%) patients harbor MYD88 L265P mutation (MYDmut) MYD88 was an acquired mutation as no aberration was identified in the mutational hot spots of MYD88 in T lymphocytes isolated from WM patients (n= 4). not observed any other mutation on exon 5 of MYD88 in our cohort

16. absent in hairy cell leukemia, MM, mantle cell lymphoma, and CLL MZL (1/16; 6%) , follicular lymphoma (1/23, 4%), IgM-MGUS (1/2) , 1 case of non-IgM LPL.

17. Gain of chromosome 3 or acquired UPD, a new mechanisms of alteration of MYD88 locus in WM LOH without copy number changes  uniparental disomy (UPD). using SNP array (n =46). found UPD at the MYD88 locus in only 1 patient (2%)  homozygous for L265P mutation in tumoral cells not identified mechanisms of loss of MYD88, UPD: 單親二體症 (個體一個染色體對的二套基因僅由父或母其中一方所提供)

18. observed a gain on chromosome 3 at the 3p22 locus (including MYD88 gene) in 7 of 59 (12%) patients, including 2 (5%) patients with MYD88 mutation. gain on chromosome 3 more frequently in the MYDwild group than in the MYDmut group (P 5 .006). other cytogenetic aberrations were associated with the gain of chromosome 3 in 6 of 7 patients.

19. we identified alteration of the MYD88 locus in 91% of patients with WMgain-of-function mutation (79%) or Copy number aberration (12%)

20. MYD88 mutation is related to NF-kB abnormalities Recent studies have described genomic deletions of 2 potent regulators/inhibitors of the NF-kB pathway in WM, TNFAIP3 (located on 6q23), and deleted in lymphocytic leukemia 7 (located on 13q14) Overall, 63% of WM had at least 1 additional genetic alteration in the NF-kB pathway in our cohort, but no significant difference was observed according to the MYD88 mutation status.

21. Clinical and biological features of WM with MYDwild 21% MYDwild. MYD88 mutation was not associated with deletion 6q, gain of 4, deletion 11q, deletion 17p, or deletion 13q14 in our study not observe any difference in terms of survival according to the MYD88 mutation status

23. MYDwild expressed CD27 We found that CD27 (positive in 49% of malignant B cells from WM in our series) was expressed in a significantly lower proportion in the MYDmut group than in the MYDwild group, 30% and 92%, (P= .0001). CD27, a TNF-family member, is expressed on the cell surface of memory B cells from which WM is thought to derive a higher frequency of CD5 positive cases (positive expression in 23% of WM in our series) (P = .043) in MYDwild group

25. High expression of IL-6 was observed in WM independently of MYD mutation status no significant difference in IL-6 expression according to MYD mutation status

26. Inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth in cells expressing MYD88 L265P mutation cytotoxicity

27. inhibited cell growth

28. induced apoptosis MYD88mut cell line

31. Discussion confirmed a high frequency of MYD88 L265P mutation in this study of 67 patients diagnosed with WM, Treon and colleagues have described MYD88 L265P mutation in 90% of cases in a first series of 46 patients with WM. Few clinical and biological data distinguished MYDwild from MYDmut subgroups in our series

32. 1. Poulain S, Roumier C, Decambron A, et al. MYD88 L265P mutation in Waldenstrom’s macroglogulinemia. Blood. 2013;121(22):4504-4511. 2. Treon SP, Xu L, Yang G, et al. MYD88 L265P somatic mutation in Waldenstr¨om’s macroglobulinemia. N Engl J Med. 2012;367(9):826-833. 3. Gachard N, Parrens M, Soubeyran I, et al. IGHV gene features and MYD88 L265P mutation separate thethree marginal zone lymphoma entities and Waldenstr¨om macroglobulinemia/lymphoplasmacytic lymphomas. Leukemia. 2013;27(1):183-189. 4. Xu L, Hunter ZR, Yang G, et al. MYD88 L265P in Waldenstr¨om macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction. Blood. 2013;121(11):2051-2058.

33. 5. Landgren O, Staudt L. MYD88 L265P somatic mutation in IgM MGUS. N Engl J Med. 2012;367(23): 2255-2256, author reply 2256-2257. 6. Varettoni M, Arcaini L, Zibellini S, et al. Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom’s macroglobulinemia and related lymphoid neoplasms. Blood. 2013;121(13): 2522-2528. 7. Jim´enez C, Sebasti´an E, Del Carmen Chill´on M, et al. MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenstr¨om’s macroglobulinemia [published online ahead of print March 22, 2013]. Leukemia. 8. Ansell SM, Secreto FJ, Manske M, et al. MYD88 pathway activation in lymphoplasmacytic lymphoma drives tumor cell growth and cytokine expression [abstract]. Blood. 2012;120(21):2699. Abstract 2699.

34. The high frequency of MYD88mutation in WM highlights the TLR pathway as a new potential pathogenic pathway in WM Our preliminary observations showed that blocking of MYD88 signaling using an inhibitor of MYD88 homodimerization induced cytotoxicity and apoptosis, and reduced cell proliferation, through decreased IRAK4 and STAT3 phosphorylation

35. MYD88 gene might undergo genetic alterations through other mechanisms, such as UPD, or gain of chromosome 3. additional genetic alterations targeting NF-kB genes such as deletion of TNFAIP3, a negative regulator of NF-kB pathway, were observed.