Serum 2-Hydroxyglutarate Level Can Predict IDH2 Mutation in Myeloid Sarcoma

Myeloid sarcoma (MS) is defined as a tumor mass consisting of myeloid blasts with or without maturation occurring at an anatomical site other than bone marrow (BM). MS may occur before, concurrently or after a characterized acute myeloid leukemia (AML). Cytogenetic abnormalities are found in 50% of the cases but molecular alterations are less well described and involved FLT3 and/or NPM1 mutations. Mutations in IDH1 and IDH2 genes are found in 15% to 20% of patients with AML but have never been described in MS. Mutated IDH enzymes produce in vast excess D-2-hydroxyglutarate (2-HG) in leukemic cells, which can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations. As availability of DNA sequencing techniques on paraffin samples are limited, molecular characterization of MS remained difficult. We asked whether in MS, serum 2-HG would predict the presence of IDH1/2 mutations at diagnosis, and could provide a biomarker for follow up.

Tissue samples and serum samples from 8 patients with a MS diagnosis were analyzed. High quality genomic DNA was extracted from frozen MS samples using conventional phenol/chloroforme extraction procedures. Exon 4 of IDH1 and IDH2 genes (IDH1/R132 and IDH2/R140 and /R172 codons) was amplified by PCR using HotStar Taq polymeraze (Qiagen) and primers. Direct sequencing was performed using the Sanger method as previously described. In case of MS relapse or AML evolution, IDH1 and IDH2 genes were analyzed in the same way from frozen tissue sample or bone marrow sample.

Serum samples at MS diagnosis were analyzed for total 2-HG, D-2-HG and L-2-HG by reverse-phase liquid chromatography coupled to mass spectrometry. In case of myeloid sarcoma with IDH1/2 mutation, 2-HG values were compared to ¹⁸F-FDG-PET results when available during remission phase and at relapse.

Three patients (3/8; 37.5%) had an IDH2 R140Q mutation at diagnosis of MS localized to lymph node, soft tissue, skin or pharynx. At MS diagnosis, serum total 2-HG, D-2-HG and ratio D/L-2-HG were significantly higher in case of myeloid sarcoma with IDH2 R140Q mutation compared to patients with no IDH mutation (Table 1). Serum total 2-HG level ≥2µM or D-2-HG level ≥1.8µM or ratio D/L 2-HG >2.5 were significantly associated with the presence of IDH2 mutation (Fisher's exact test P≤0.02).

All 3 patients with IDH2 R140Q mutated MS received intensive chemotherapy treatment and achieved complete remission (CR). Two patients relapsed: one experienced isolated extramedullary relapse (thigh muscle); one had a bone marrow relapse. IDH2 R140Q mutation was found at the site of relapse in both cases.

When available, serum 2-HG values and ¹⁸F-fluorodeoxyglucose-positron-emission tomography (FDG-PET) were compared at different time points (at diagnosis, remission and relapse; Table 2).

Serum 2-HG values were in accordance with FDG-PET interpretations except in patient #1 who presented a transient hypermetabolic splenic nodule (SUVmax 9.6) without serum 2-HG increase. Patient #2 remained in CR but had recently increased 2-HG values without overt relapse. Patient #3 presented relapse as a refractory anemia with excess of blast without extra-medullary localization. FDG-PET didn't find any abnormality contrary to the persistent increased value of serum 2-HG (total 2-HG: 16.7µM).

These data show that myeloid sarcoma can be associated with IDH2 R140Q mutation and suggest that 2-HG measurement in the serum predicts the presence of IDH1/2 mutations at diagnosis. During follow-up, serum 2-HG values could be representative of the disease status. Because of IDH inhibitors promising results in AML, 2-HG screening at MS diagnosis could be useful.