Aplastic Anaemia Genomics: Current data and interpretation

Transcription

1 An Academic Health Sciences Centre at the heart of a world city... Aplastic Anaemia Genomics: Current data and interpretation Austin G Kulasekararaj Consultant Haematologist King s College Hospital, London

2 Clonal Wars- clonal Force Awakens T cell clones (Immune) Escape clones- PNH, +8, 13q and 6pUPD Good Clones vs Bad clones Aged clones vs disease clones True vs Pseudo clonality Clonal Flux (Fluctuant/transient) Clonal elimination (spontaneous/therapy)- PNH and transplant (!)

3 AA associated with diseases of clonal hematopoiesis FANC AA/PNH FA VSAA SAA PIGA STAT3 PNH LGL (clonal or polyclonal) TERC, TERT,TINF2 RPS5, RPS11, RPS 19 DC NSAA RBDS5q- Low risk Hypo MDS MDS MDS/MPD High risk Hypo AML AML MDS/AML-type somatic mutations Adapted and modified from Young NS et al. Blood 26;18:

4 Del(13q) cells (%) Cytogenetic clones in AA that evade immune attack HSC WT c-myc Survivi 4 n CDK1 2 Trisomy 8 1 Changes in the proportion of del(13q) cells after IST in 8 patients Cytokin es CD8+ T cell clonal expansion Copy number neutral 6p loss of heterozygosity 2 8+ UPN 3 UPN 7 LOH del(13q) FISH (Peripheral blood) del(13q) FISH (Peripheral blood) 32.2% CTL CTL 4.% SSC-A CD11b SSC-A CD11b Del 13q 3 del(13q) FISH % Ch6, chromosome 6; CTL, cytotoxic T lymphocytes; HLA, human leukocyte antigen; Months from diagnosis HSC, GPI-AP, hematopoietic glycosylphosphatidylinositol-anchored stem cell; HSPC, hematopoietic protein; stem/progenitor FISH, fluorescent in situ hybridization; cell; UPN, LOH, unique loss patient of heterozygosity; number TCR, T cell receptor; WT1, Wilms 1. Sloand 3. Hosakawa EM et al. Blood K et al. 25;16: ; Haematologica 212;97: Katagiri T et al. tumor protein Blood 211;118: Hosakawa K et al. Haematologica 212;97: TCR 2 TCR TCR HSP C del(13q) FISH 12% CTL HLA* Recombinat 4 1 LOH 2 3 A*2:1 LOH FSC-A HLA ion of 6p A*2:6 CD55, CD59 FSC-A CD55, CD59 A*31:1 B*4:2 arms CTL CTL LOH del(13q) FISH % LOH LOH del(13q) FISH 12% 1 4

5 Frequency and incidence of mutations ASXL1 (n=12) DNMT3A (n=8) BCOR (n=6) One each of: SRSF2 U2AF1 TET2 MPL IKZF1 ERBB2 The median allele burden was 2% (range, %) All heterozygous except one hemizygous BCOR 41% of mutations had <1% clone Mutations detected were missense (n=12), nonsense (n=12), frameshift (n=6), non-frameshift deletion (n=1) and splice-site changes (n=1) UPN Gene Mutant allele burden (%) 142 DNMT3A 1.5 Non-synonymous SNV c.c5644t:p.r882c C>T p.w1356 X Variant class Nucleotide and protein change 7 ASXL1 2 Stopgain SNV c.g226t:p.e676x 81 ASXL1 3 Stopgain SNV c.t2324g:p.l775x 88 ASXL1 7 Frameshift deletion c.2126delc:p.a79fs 2 ASXL1 3 Frameshift insertion c.1927_1928insg:p.g643fs 16 ASXL1 3 Frameshift insertion c.2469_247insag:p.l823fs 17 ASXL1 3 Stopgain SNV c.t2468g:p.l823x 4 ASXL1 31 Non-frameshift deletion c.2894_2896del:p.965_966del 69 ASXL1 34 Stopgain SNV c.277t:p.r693x 79 ASXL1 36 Stopgain SNV c.g226t:p.e676x 66 ASXL1 37 Frameshift deletion c.2433delt:p.n811fs 6 ASXL1 38 Stopgain SNV c.c2242t:p.q748x 29 ASXL1 41 Stopgain SNV c.g468a:p.w1356x 129 DNMT3A 5 Non-synonymous SNV c.g227a:p.r736h 13 DNMT3A 5 Non-synonymous SNV c.g2645a:p.r882h Mutant allele burden = 41% 97 DNMT3A 7 Non-synonymous SNV c.c5644t:p.r882c 93 DNMT3A 8 Stopgain SNV C2311T:p.R771X 18 DNMT3A 31 Non-synonymous SNV c.c2644t:p.r882c 2 DNMT3A 42 Non-synonymous SNV c.c154g:p.l514v 64 DNMT3A 47 Non-synonymous SNV c.c2644t:p.r882c 21 BCOR 5 Stopgain SNV c.c526t:p.q176x 14 BCOR 5 Frameshift deletion c.476delc:p.p1587fs 73 BCOR 6 Frameshift insertion c.4834_4835insc:p.l1612fs 7 BCOR 14 Stopgain SNV c.t912g:p.y34x 94 BCOR 3 Splice site Splice site c.352-2a>g 33 BCOR 68 Stopgain SNV c.g4832a:p.w1611x 2 ERBB2 44 Non-synonymous SNV c.g922a:p.v38m 19 IKZF1 14 Non-synonymous SNV c.c64g:p.h214d 46 MPL 1 Non-synonymous SNV c.g1544t:p.w515l 1 SRSF2 2 Non-synonymous SNV c.c284t:p.p95l 5 TET2 5 Stopgain SNV c.c31t:p.q134x 67 U2AF1 19 Non-synonymous SNV c.c11a:p.s34y Bone marrow Skin Kulasekararaj AG et al. Blood 214;124:

6 AA (excluding PIGA mutations)? Do they predict risk of evolution to MDS? Disease duration Mutations (n=29) Wild type (n=121) <6 months (n=63) 9 54 P-value Transformation into MDS 3 3 <.3 Median mutant allele burden <1% 7 NA >6 months (n=87) 2 67 Transformation into MDS 8 3 <.2 Median mutant allele burden <1% Median follow-up 46 months (range, 1 36) Somatic mutation found in 29/15 (19%) In the presence of a somatic mutation: 4 NA 1. Risk of MDS = 11/29 (38%) compared with 6/121 (6%); P<.1 2. If disease duration >6 months, risk of MDS is 4% compared with 4% Kulasekararaj AG et al. Blood 214;124:

7 Monosomy 7 (% by FISH) Monosomy 7: Diverse origins 1 8 Control GCSF 4 ng/ml Postauto HSCT Donor leukemia Postcord Myeloid neoplasia Road to monosomy 7 Growth factors AA GCSF Kostmanns TPO mimetic Eltrombopag Transient -7 (disappears post-atg) Persistent -7 (MDS/AML) Patient UPN Stressed hematopoiesis Proliferative pressure Depleted stem cell pool Telomere loss Genomic instability Age/ sex Diagnosi s Initial therapy Disease duration (months) Respons e MC 73/M NSAA ATG + CSA 44 CR Normal Mutation(s) ASXL1 DNMT3A Clonal evolution RCMD/-7 35/F NSAA ATG + CSA 45 NR 45,XX,-7 ASXL1 RCMD/ /M VSAA ATG + CSA 2 NR Normal ASXL1 hmds/-7 31 RCMD- 83/M NSAA CSA 62 CR 45,XY,-7 DNMT3A 47 CR, complete response; CSA, cyclosporine A; GCSF, granulocyte-colony stimulating RAEB2-AML factor; hmds, hypoplastic MDS; HSCT, hematopoietic stem cell transplant; NR, non response; RAEB, refractory anemia with excess blasts; RCMD, refractory cytopenia with multilineage dysplasia; TPO, thrombopoietin; VAF, variable allele frequency Kulasekararaj AG et al. Blood 214;124: VAF 3 42

8 Lower VAF at diagnosis and its interpretation mutations presen Targeted sequencing of genes at diagnosis of aplastic anaemia (n=96) at None of them had mutations with VAF > Cautious use of mutation calling in clini by sequencing laboratories Yoshizato et al. NEJM 215 Kulasekararaj et al, Unpublished

9 Frequency Aged versus disease clones Prevalence of somatic mutations according to age Age (years) Somatic mutations that drive clonal expansion are common in the elderly and most frequently involve DNMT3A, TET2 or ASXL1, and are associated with increased Jaiswal S et al. N Engl J Med 214;371:

10 Clonal eradication: spontaneous or therapy MSD, n=12, 1% UD, n=33, 9.5% King s College Hospital, 215 FCC regimen Babushok et al. BJH 216, Marsh et al unpublished

11 Clonal size and frequency Mutational profile of AA and its evolution following IST 1 MDS Clinical Significance IST AA Time and disease course Age related clonal haemopoiesis Normal haemopoietic stem cell (HSC) Overlap AA/MDS CHIP Hypoplastic MDS or Overlap AA/MDS (?) Progression to MDS?Consider early allogeneic Recovery of normal haemopoiesis transplantation Frequency of four commonly mutated genes in AA from two studies 2,3 Kulasekararaj Yoshizato et et al. al. Top four genes DNMT3A 8.3% 8.4% Dysplastic HSC Dysplastic HSC with single mutation *BCORL1 not analysed NA-PIGA not done for the whole cohort VAF-variant allele frequency ASXL1 8% 6.2% BCOR/BCORL 1 4%* 9.3% PIGA N/A 7.5% Dysplastic HSC with multiple mutations CHIP-Clonal haemopoiesis of indeterminate potential 1. Mufti GJ et al. N Engl J Med 215;373: ; 2. IST-immunosuppressive therapy Median Kulasekararaj VAF AG et al. 2% Blood 214;124: ; 9.3% 3.

12 Acknowledgements Rayne Institute Tony Pagliuca Robin Ireland Victoria Potter Donal McLornan Kavita Raj Hugues Delavallade Joop Gaken Alexander Smith Jie Jiang Azim Mohamedali Shreyans Gandhi Syed Mian Nik Lea Steve Best Shahram Kordasti SAAWP