Serum D‐2‐hydroxyglutarate and the ratio of D‐2HG/L‐2HG predict IDH mutation in acute myeloid leukemia

Abstract This study investigates whether serum D‐2HG (D‐2‐hydroxyglutarate) produced by the mutated isocitrate dehydrogenase (IDH) can predict IDH mutations in acute myeloid leukemia (AML) at diagnosis. D‐2HG and L‐2HG are measured by liquid chromatography‐tandem mass spectrometry. D‐2HG, total 2HG and the D/L ratio (D‐2HG/L‐2HG) are significantly higher in IDH mutated cases than in IDH wild cases. The optimal cutoff values to predict IDH mutations at 100% sensitivity (specificity 91%–94%) are >588 ng/mL for D‐2HG and >2.33 for the D/L ratio. Our study indicates that elevated serum D‐2HG and the D/L ratio may serve as noninvasive biomarkers of IDH mutation in AML.


INTRODUCTION
Mutations in genes encoding isocitrate dehydrogenase (IDH1 and IDH2) in acute myeloid leukemia (AML) result in loss-of-function for the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and confer a novel catalytic activity that reduces α-KG to the D-enantiomer of 2-hydroxyglutarate (D-2HG) [1][2][3][4][5][6][7]. All known IDH mutations involve arginine (R), in the codon 132 of IDH1 or codon 140 or 172 of IDH2. The oncogenic property of D-2HG appears to stem from its ability to inhibit α-KG-dependent dioxygenases, including histone demethylases, Ten-eleven translocation hydroxylases and prolyl hydroxylases, thus initiating dysregulated epigenetic programming and hypoxia inducible transcription factor (HIF)-1α pathway. The determination of IDH mutation status in AML has clinical relevance in treating AML patients harboring the IDH mutations by the IDH inhibitor and monitoring response to conventional as well as IDH-targeted therapy. Chelsea  are two enantiomers of 2-hydroxyglutarate (2HG). They are normal endogenous metabolites that can be oxidized back to α-KG by their respective 2-hydroxyglutarate dehydrogenases (D-2HG dehydrogenase and L-2HG dehydrogenase, respectively). As such, D-2HG and L-2HG levels are at homeostasis but could be affected by abnormal enzymatic activities with associated alterations in these genes.
Notably, IDH mutations only produce D-2HG. Several studies have found that elevated 2HG in plasma or serum (either total 2HG [D-2-HG + L-2HG] or without specifying enantiomer type) could serve as a biomarker of IDH mutations [8][9][10][11][12]. However, only rare studies reported the ratio of D-2HG/L-2HG (referred to as the D/L ratio) and reporting on D-2HG is largely absent.
Our study aims to systematically investigate whether increased D-2HG, total 2HG, and the D/L ratio can predict IDH mutations in AML at diagnosis. Our results indicate at the optimal cut-offs with 100% eJHaem. 2023;4:723-727.
wileyonlinelibrary.com/journal/jha2 sensitivity, D-2HG, total 2HG, the D/L ratio can predict IDH mutations with a high specificity (91%-94%). Additionally, we report unusual and novel findings, two cases with increased D-2HG but lacking IDH mutation and the first case of elevated L-2HG in AML. The significance of these findings will be discussed.

METHODS
Peripheral blood specimens (53 cases collected in 2015) were obtained from newly diagnosed or refractory AML patients at the Hospitals The levels of two enantiomers, D-2HG and L-2HG, were measured by liquid chromatography-tandem mass spectrometry [13]. All data were analyzed by Prism (Version 9). A p value less than 0.05 was considered significant. Of 20 patients with refractory AML, most patients underwent standard chemotherapy (7 + 3). While the levels of D-2HG are expected to be affected due to blast count changes following chemotherapy, the key results and conclusion are not expected to be affected because the inclusion criteria were AML with the blast counts generally at the leukemia range, and there is no report of standard chemotherapy agents specifically targeting IDH genes.

3.2
High D-2HG, total 2HG, and the D/L ratio to predict IDH mutations (establishment of cut-off values) D-2HG, total 2HG, and the D/L ratio were significantly higher in IDH m cases (median of 3815 ng/mL, 3852 ng/mL, and 80.6, respectively) than in IDH w cases (67 ng/mL, 138 ng/mL, and 1.27, respectively, p < 0.001; Table 2, Figure 1). There was no difference in these values among the IDH mutation types. The L-2HG levels were not different between the two groups.
The optimal diagnostic cut-off values for D-2HG, total 2HG, or the D/L ratio that ensure maximum specificity at 100% sensitivity to predict IDH mutation were determined by area under the receiver operating characteristic curve (ROC AUC). The cut-off values were > 588 ng/mL for D-2HG (with a specificity of 94% in this dataset, AUC 0.996), >623 ng/mL for total 2HG (with a specificity of 92%, AUC 0.996), and >2.33 for the D/L ratio (with a specificity of 91% in this dataset, AUC 0.994) (Table 2 and Figure 2). These results indicated that elevated serum D-2HG, total 2HG, and the D/L ratio could be used as highly sensitive and specific biomarkers (91-94% specificity at 100% sensitivity) of IDH mutation in AML.

Unusual cases with discordancy between IDH mutation and the D/L ratio/2HG levels
Several notable findings in this study merit further description (Table 3) Understanding these apparently discordant results between IDH mutation status and the D/L ratio and 2HG levels may provide insights into the significance of these biomarkers.

DISCUSSION
Our study demonstrates that elevated serum D-2HG (>588 ng/mL), total 2HG (>623 ng/mL), and the D/L ratio (>2.33) are highly sensitive and specific biomarkers (91%-94% specificity at 100% sensitivity) of IDH mutation in AML at diagnosis. These cut-off values are generally comparable to the levels established in other studies, such as the D/L ratio of 1.9-2.5 in two European studies [8,9] and 2HG level of ∼500-600 ng/mL (without specifying enantiomer type) in two US studies [10,12], but a much lower 2HG level of ∼300 ng/mL in one European study [8]. Additionally, our study seems to be the first to report the D-2HG threshold. Comparing the D/L ratio of 2.33 from our F I G U R E 2 Diagnostic performances of D-2HG, total 2HG and the D/L ratio to predict IDH mutations indicated by the ROC curves. The optimal diagnostic cut-off values for D-2HG, total 2HG and the D/L ratio that ensure maximum specificity at 100% sensitivity to predict IDH mutation are determined by area under the receiver operating characteristic curve (ROC AUC). The cut-off values are >588 ng/mL for D-2HG (with a specificity of 94%, AUC 0.996), >623 ng/mL for total 2HG (with a specificity of 92%, AUC 0.996) and >2.33 for the D/L ratio (with a specificity of 91%, AUC 0.994). study to those reported, a cut-off of 1.9 will maintain a 100% sensitivity but reduce the specificity from 91% to 79% while a cut-off of 2.5 will result in a similar sensitivity and specificity. As D-2HG and Given IDH mutation producing only D-2HG and not L-2HG, the diagnostic performance of the enantiomeric ratio may be superior to that of D-2HG especially at low mutant allele burden. This was suggested by two other studies [8,9], and the findings in case 1 in our cohort in which only the D/L ratio met the diagnostic cut-off with IDH2 R140Q mutation at 3% VAF. This better performance may stem from the D/L ratio being technically more reliable than measuring D-2-HG and L-2HG concentrations since its quantification does not require the addition of an internal standard. Future study on a large cohort is needed to evaluate the performance of the D/L ratio in monitoring therapeutic response to conventional and IDH-targeted therapy.
While there is a general association of serum D-2HG level and IDH mutation status in AML, the correlation is not perfect. A small subset of AML patients (two cases in our cohort and a small subset of AML cases in other two studies [10,11]) had a high level of 2HG, yet no IDH mutation was identified by employing moderately high sensitivity molecular assays including NGS. The mechanism underlying this apparent discordancy remains to be investigated, including exploring other IDH mutation hot spots not covered by routine NGS testing, altered glutamine pathway to provide excess α-KG, and disruption of D-2HG dehydrogenase in AML [11,14]. Indeed, additional D-2HG-producing IDH1 mutations have been identified in non-AML tumors, including IDH1 R100 in glioma, IDH1 G97 in colon cancer, and IDH1 Y139 in glioblastoma [14].
Finally, we report the first case of increased L-2HG without IDH mutation, which has not yet been described in AML. Increased L-2HG has been reported in clear cell renal cell carcinoma due to loss or dysfunction of L-2HG dehydrogenase (L2HGDH) to convert L-2HG to α-KG, which is largely associated with gene deletion on 14q, a region coding for L2HGDH [15]. While the relationship between increased L2HG and AML remains unclear, L-2HG is likely an oncometabolite as L-2HG is also an inhibitor of α-KG-dependent dioxygenases. The mechanism underlying such a highly increased L-2HG in AML remains to be explored. No mutation in L2HGDH was detected in 10 AML patients with increased total 2HG [11]. From perspective of biomarkers, this rare finding further supports the superior diagnostic performance of D-2HG and the D/L ratio to total 2HG as increased L-2HG could be confounding.
In summary, our study demonstrates that elevated serum D-2HG, total 2HG, and the D/L ratio may serve as noninvasive biomarkers of IDH mutation in AML. Moreover, diagnostic performance of D-2HG and the D/L ratio may also be superior to total 2HG, particularly the D/L ratio at low mutant allele burden. Identification of elevated D-2HG and L-2HG in IDH unmutated AML should prompt investigations into novel mechanisms associated with altered metabolic activity.

FUNDING INFORMATION
The authors received no specific funding for this work.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
This study was approved by the institutional review board (IRB 122013-023). Informed consent was not required for this study.