Background

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. In recent years, more and more somatic mutations and their clinical significance were identified in AML. Most AML patients carry multiple gene mutations and the repertoire of mutations changing during the disease process, which determine the patient's unique clinical manifestations. Herein we recommend using the novel word ”mutaome¡± for representing the repertoire of somatic gene mutations in a specific tumor tissue, and aimed to establish a panel of mutation profiling protocol and retrospective profiling using archived bone marrow smear for clinical use in AML.

Methods and Cases

Mutation profiling protocol for CEBPA, DNMT3A, FLT3-ITD/TKD, IDH1, IDH2, KIT, NPM1, PHF6 and TET2 by PCR and Sanger sequencing was established, with the detection sensitivity about 15% to 20%. Bone Marrow (BM) or peripheral blood (PB) was collected form patients with newly diagnosed or relapsed. Archived BM smear on glass slides at the time of newly diagnosed were used for retrospective mutation analysis.

Results

1) Totally 61 archived smear, 106 fresh BM or PB and 2 paraffin-embedded pathological specimens from 157 patients were analyzed. Age ranged from 1 to 77 years old, with the median age of 27 years old.

2) 60.4% (102/169) samples carrying at least one mutation, 51.0% (53/102) of them carrying 2 or more mutations, 30.4% (31/102) carrying mutations within 2 or more different genes. The number of mutated sample for each gene is 33 for CEBPA¡¢9 DNMT3A¡¢35 FLT3¡¢6 IDH1¡¢7 IDH2¡¢7 KIT¡¢19 NPM1¡¢8 PHF6¡¢and 21 TET2.

3) Paired archived smear and relapsed samples were analyzed for 11 cases, 9 of them showing difference, detailed results shown in Table 1. 

4) Totally 53 patients come to our hospital after certain treatment, with the tumor cells below 15% and without AML gene mutation results. For these patients, archived BM smear samples were used for retrospective mutation profiling and then to guide targeted therapy and stratified evaluation.

Conclusions

Panel testing for gene mutations is effective method for detection of AML molecular markers. For the patients came with partial remission and without gene mutation result, archived bone marrow smear sample can be used for retrospective mutation analysis and then help guiding targeted therapy and stratified evaluation.

Table 1

gene mutation results in paired archived bone marrow smear and relapsed samples

Patient No. mutations in archived bone marrow smear mutation at relapse
P13  NPM1 W288CfsX12;TET2 [T759N+Q1606X+L719F+Q1527H]  (-)  
P27  NPM1 W288CfsX12  IDH2 R140Q; NPM1 W288CfsX12; TET2 A347V  
P56  IDH2 R172K  (-)  
P59  FLT3-ITD 102bp  FLT3 D835V  
P63  FLT3 N841K; WT1 K404X  FLT3 N841K  
P75  FLT3 H811Q; WT1 R458P; IDH1 A51D; CEBPA A295T  FLT3 D839G; WT1 R458P  
P78  CEBPA K313dup; TET2 Q1624K  CEBPA K313dup  
P82  CEBPA [H24RfsX137+Q312dup]  (-)  
P85  CEBPA [H24AfsX84+K304_Q305insL]  CEBPA [H24AfsX84+K304_Q305insL]  
P113  FLT3 G822W; TET2 [P208H+R447S]  (-)  
P127  (-)  (-)  
Patient No. mutations in archived bone marrow smear mutation at relapse
P13  NPM1 W288CfsX12;TET2 [T759N+Q1606X+L719F+Q1527H]  (-)  
P27  NPM1 W288CfsX12  IDH2 R140Q; NPM1 W288CfsX12; TET2 A347V  
P56  IDH2 R172K  (-)  
P59  FLT3-ITD 102bp  FLT3 D835V  
P63  FLT3 N841K; WT1 K404X  FLT3 N841K  
P75  FLT3 H811Q; WT1 R458P; IDH1 A51D; CEBPA A295T  FLT3 D839G; WT1 R458P  
P78  CEBPA K313dup; TET2 Q1624K  CEBPA K313dup  
P82  CEBPA [H24RfsX137+Q312dup]  (-)  
P85  CEBPA [H24AfsX84+K304_Q305insL]  CEBPA [H24AfsX84+K304_Q305insL]  
P113  FLT3 G822W; TET2 [P208H+R447S]  (-)  
P127  (-)  (-)  
Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.