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Case Reports
Except for UPN-2, all subjects had less than 10% blasts at evolution. In order to determine the presence of muta- tions within the malignant clone, we performed NGS on UPN-1, UPN-2, and UPN-4 samples from the time of myeloid malignancy diagnosis that had been enriched by flow cytometry sorting based on blast immunopheno- type (Table 1; Online Supplementary Table S1A). For UPN- 1, NGS of CD117+ peripheral blood mononuclear cells
(PBMC) at clonal evolution demonstrated mutations in NPM1 (Type A insertion) and NRAS. A custom ddPCR assay for the NPM1 mutation detected the variant in unsorted bone marrow taken 128 and 864 days prior to clonal evolution but not from samples collected prior to that (Figure 2A). In UPN-2, two variants in RUNX1 and one in ASXL1 were detected in sorted CD117+ from bone marrow mononuclear cells at clonal evolution. A custom
Table 1. Patient characteristics and details of clonal evolution.
Patient
Age at SAA diagnosis
Severity of SAA
Sex
Ethnicity
Initial Treatment
IST response at 6 months Relapse
PNH Clone* Telomere length
Time to clonal evolution
Diagnosis Blood counts
ANC (/mL) Platelet (/mL) Hemoglobin (g/dL) ARC (/mL)
Bone marrow
Cellularity (%) Blasts (%) Dysplasia
Immunophenotype by flow cytometry
Cytogenetics Treatment Outcomes molecular testing
RUNX1
NRAS NPM1
ASXL1
SETBP1
PHF6
UPN-1
35 years
VSAA Female White h-ATG/CsA/MMF Partial
No
- Normal
7.3 years
MDS EB-1
4,450 49,000 10.6 21,700
90-100
8 Megakaryocyte
and erythrocyte
NA; IHC (CD34 neg, CD117+, MPO+, TdT–, rare CD14+)
46, XY [20] MSD BMT Alive
UPN-2
29 years
SAA Male Hispanic h-ATG/CsA Partial No
No Normal
UPN-3
41 years
SAA
Male White h-ATG/CsA/EPAG Complete No
No Normal
UPN-4
59 years
SAA
Male White h-ATG/CsA/EPAG Partial
Yes
Yes Normal
5.1 years
MDS EB-1
1,920 115,000 12.5 116,100
40-50
5-6 Megakaryocyte
CD34+, CD117+,
HLADR+, CD33+,
CD13+, TDT+, MPO+ 45,X,-Y[20] MUD BMT Alive
6.3 years
AML
510 28,000 13.2 43,900
40-60 31 No
CD34+, CD117+, MPO+, TdT+
46, XY [20] MSD BMT Alive
5.0 years
MDS/MPN
9,360 175,000 14.7 182,100
60-70
3-4 Megakaryocyte
CD34+, CD117+, HLADR+, CD33+, CD13+
46, XY [20] Observation Alive
At clonal evolution
-
c.35G>C; p.G12A c.860_863dup; p.W288CfsTer12 -
- -
c.292del; p.L98SfsTer24 c.374del; p.P125QfsTer8 -
-
c.2067dup;
c.319C>T; p.R107C
- -
c.1900_1922del;
p.E635RfsTer15
c.2602G>A; D868N
c.821G>A; p.R274Q
c.768del; p.T257LfsTer54 c.507_508+5dup
-
-
-
- -
Detected variants in enriched blasts or unsorted bone marrow at clonal evolution
p.D690RfsTer28
- -
648
SAA: severe plastic anemia; IST: immunosuppressive treatment; PNH: paroxysmal nocturnal hemoglobinuria; ANC: absolute neutrophil count; ARC: absolute reticulocyte count; h-ATG: horse anti-thymocyte globulin; CsA: cyclosporine; EPAG: eltrombopag; MMF: Mycophenolate mofetil; UPN: unique patient number; IHC: immunohistochem- istry; MDS EB-1: myelodysplastic syndrome excess blast 1; AML: acute myeloid leukemia; MPN: myeloproliferative neoplasm; MSD: matched sibling donor; MUD: matched unrelated donor; BMT: bone marrow transplantation. VSAA (very severe aplastic anemia) was defined as ANC <200/mL. Complete response was defined as absolute ANC ≥1,000/mL, platelet≥100,000/mL, hemoglobin ≥10 g/dL. Partial response was defined as blood counts no longer meeting the standard “Camitta” criteria – ANC ≥500/mL, platelet ≥ 20,000/mL, absolute reticulocyte count ≥ 60,000/mL. *PNH clones >1% in granulocytes.
haematologica | 2021; 106(2)