1. Clearance of ‘Driver-COSMIC’ mutations post-CR1 with persisting RUNX1_L56S is unlikely to contribute towards disease progression
L . Rai1, T. Boneva1, D. Brazma1, R. Dunn1, C. Grace1, E. Nacheva1,2
1 Department of OncoGenomics, HSL Analytics LLP, London UK
2 Cancer Institute, University College London, London UK
Introduction
Clinical significance of gene variants in AML is well established (Papaemmanuil E et al, NEJM 2016) and is increasingly being implemented into routine diagnostic algorithms. Although 80% of patients achieve morphological remission after induction chemotherapy, long-term relapse free survival is a meagre 50% (Walter RB et al, JCO 2010). Monitoring of disease kinetics, is therefore, very critical.
Results
summARY
A median of 4 mutations per patient was observed in each clinical subgroup. Driver mutation was identified in 38 patients. Patient characteristics are described in Table 1 and examples of gene variant kinetics in the three clinical subgroups is illustrated in Figure 2.
D-C mutations persisted in 85.7% of relapse patients compared to only 11% of patients in remission [P-value: 0.001, figure 3(i)]. Additionally, in intermediate risk cytogenetics group D-C mutations were retained in all 13 relapse patients while complete clearance was observed in all 6 patients with sustained remission [P-value: 0.001, Figure 3(ii)].
Eight of 9 patients who remained in morphological remission post-induction had a persistent D-C/S mutation, while all D-C disappeared. This suggests that driver mutations with both COSMIC and SNP reference may not always contribute towards disease progression. Further investigation of persisting exonic D-C/S mutations in the remission cohort revealed that 4 patients had persistent DNMT3A-p.R693H, 1 had DNMT3A-p.R693C and two had RUNX1-p.L56S 1 (Fig 4). These mutations were seen in both myeloid and lymphoid compartments of remission bone marrow samples in all 7 patients (Fig.4iii)
As DNMT3A mutations are considered to occur in pre-leukemic stem cells contributing to clonal hematopoiesis (Shlush et al, Nature 2014) we studied the distribution of RUNX1 gene variants in 119 additional AML diagnostic samples. 34 patients (21%) harboured RUNX1 mutations, of which 5 had RUNX1_L56S.
Finally, we evaluated kinetics of D-C in 40 MDS cases (Fig 5) of which 34 had chronic MDS and 6 had secondary AML. No significant difference was observed in the no. of patients with persistent D-C mutation in the two subgroups (P-value: 1.000).
AML Cohort (Total no. of patients: 45)
Clinical Outcome
Morphological remission
AML relapse
Refractory disease
No. of patients in each clinical outcome category 19 21 5
Mean Age (Range) (in years)
55.8 (32-71)
57.3 (30-77)
63.8 (43-82)
No. of patients ≤60 years
10 of 19
10 of 21
2 of 5
Time to clinical end-point(Range) (in months)
5.2 (3-15)
17.8 (3-66)
4 ( )
Cytogenetics
Good 2 1
Intermediate 16 17
Poor 3
Table 1: AML patient characteristics
(i)
(ii)
(iii)
dg AML Remission Relapse
Objectives
To study the kinetics of gene variants post-clinical remission (post-CR1) in patients with Acute Myeloid Leukaemia.
To ascertain the prognostic relevance of gene variants, especially in intermediate cytogenetic risk group.
dg AML Refractory disease
Figure 2: Graph illustrating typical gene variant kinetics post CR1 in AML patients in; (i) morphological remission, (ii) relapse, (iii) refractory disease
P-value: 0.001
P-value: 0.001
(ii)
(i)
Methods
130 follow-up samples from 45 de novo AML patients were screened for gene variants using TruSight Myeloid panel (Illumina, CA, USA) covering 54 genes with relevance in myeloid diseases. Bone marrow (BM) or peripheral blood (PB) was obtained at presentation (BM:44; PB:1) and follow-up (BM-130).
All AML patients had standard induction chemotherapy followed by personalized treatment options. Gene variants kinetics was compared between patients in morphological remission (MR), relapse (R ) and refractory disease (RD).
Gene variants in 95 samples from 40 MDS patients were also evaluated for progression to secondary AML.
Gene variants at Variant allele frequency (VAF) of ≥10% at diagnosis and VAF of ≥1.5% during follow-up; both with target coverage of ≥300 reads were considered.
Public databases-GRCh37/hg19, Catalogue of Somatic Mutations In Cancer (COSMIC), dbSNP and 1000 genome (>2%) were used to classify somatic gene variants as either
[i] driver with COSMIC reference (D-C)
[ii] driver with both COSMIC and SNP reference (D-C/S)
P-value was generated with 2-tailed Fisher Exact (GraphPad Software, Inc, USA).
Conclusions
Regardless of patient age at presentation and post-induction treatment regimen followed, with the exception of allogeneic SCT; persistence of ‘D-C’ mutations post-CR1 is associated with high risk of relapse. This is especially obvious in AML with intermediate risk cytogenetics.
DNMT3A-p.R693H, DNMT3A-p.R693C and RUNX1L56S are not associated with disease progression.
Targeted NGS offers a cost effective method of monitoring the kinetics of somatic variants in AML with an ability to capture disease progression.
Figure 3: Graph (i) compares no. of AML patients with persistent D-C mutations between clinical sub-groups.
Graph (ii) compares no. of AML intermediate risk cytogenetics patients with persistent D-C mutations between clinical sub-groups
(i)
(ii)
(iii)
Gene Variant
Case No.
Present in Granulocytes (Y/N)
Present in MNC (Y/N)
DNMT3A-p.R693H
1682 Y
16125
161025
DNMT3A-p.R693C
16667
RUNX1-p.L56S
161070
161398
17140
CBL p.? N
DNMT3A-p.R693H
DNMT3A-p.R693C
RUNX1-p.L56S
CBL p.? (intronic, splice-region variant)
References
Papaemmanuil E et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med 2016; Jun 9;374(23): Walter RB et al. Effect of complete remission and responses less than complete remission on survival in acute myeloid leukemia: a combined Eastern Cooperative Oncology Group, Southwest Oncology Group, and M. D. Anderson Cancer Center Study. JCO 2010; Apr 1;28(10):
Shlush LI et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature Feb 20;506(7488):
Figure 4: Graph (i) compares no. of AML patients with persistent D-C/S mutations between clinical sub-groups; figure (ii) lists the D-C/S mutations in the remission sub-group that persist after CR1; Table (iii) demonstrates presence of a gene variant in the granulocyte and/or mononuclear compartment of bone marrow samples from AML patients in remission
P-value:
CONTACT INFORMATION
Figure 5: Graph compares no. of patients with persistent D-C mutations between those with chronic MDS and those with secondary AML
Figure1: Graph illustrating no. of diagnostic and follow-up samples from de novo AML and MDS patients included in this study