Isocitrate dehydrogenase (IDH) gene mutations, frequent in gliomas, cause increased levels of 2-hydroxyglutarate (2-HG). 2-HG levels were found to be elevated in the urine of patients with IDH-mutant versus IDH-wild-type glioma; no significant differences in 2-HG levels were observed in the serum or the small set of cerebrospinal fluid samples obtained. 2-HG may represent a future surrogate, noninvasive biomarker to aid in diagnosis, prognosis, and management of IDH-mutant glioma.
Keywords: Isocitrate dehydrogenase, 2-Hydroxyglutarate, Glioma, Novel biomarkers, Targeted therapies
Abstract
Background.
Recurrent mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, which are frequent in gliomas, result in marked accumulation of the metabolic by-product 2-hydroxyglutarate (2-HG) within tumors. In other malignancies, such as acute myeloid leukemia, presence of IDH mutation is associated with elevated 2-HG levels in serum or urine compartments. Circulating 2-HG in patients with glial malignancies has not been thoroughly investigated.
Methods.
In this study, we analyzed 2-HG levels in the serum and urine of a large set of patients with IDH-mutant and IDH-wild-type glioma, and the cerebrospinal fluid (CSF) from a subset of this cohort.
Results.
We found that 2-HG was elevated in the urine of patients with IDH-mutant versus IDH-wild-type glioma, although no significant differences in 2-HG levels were observed in the serum or the small set of CSF samples obtained. Among patients with IDH-mutant glioma, 2-HG levels did not differ based on the histopathologic grade, genetic subtype (TP53 mutant or 1p/19q codeleted), presence of a canonical (IDH1 R132H) or noncanonical (any other IDH variant) mutation, or treatment type.
Conclusion.
Our finding suggests that urinary 2-HG is increased among patients with IDH-mutant gliomas, and may represent a future surrogate, noninvasive biomarker to aid in diagnosis, prognosis, and management.
Implications for Practice:
Patients with glioma who harbor mutations in isocitrate dehydrogenase genes showed selective elevation of the oncometabolite 2-hydroxyglutarate in the urine. Similar elevations were not identified in the serum or cerebrospinal fluid. 2-Hydroxyglutarate may serve as a useful, noninvasive biomarker to stratify patients newly diagnosed with glioma with regard to prognosis and management.
Introduction
Recurrent, somatic mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes have garnered significant interest in human malignancy, with the recent discovery of cancer genetic alterations impacting canonical metabolic enzymes. IDH mutations are identified in a significant subset of patients with gliomas and are associated with younger age and highly characteristic radiographic features [1, 2]. Intriguingly, patients with IDH mutations have significantly better survival compared with patients with wild-type IDH [1, 3–5]. IDH mutations are also found in the majority of lower-grade glial malignancies and are the earliest known somatic alterations in the pathogenic development of gliomas [2, 6, 7]. IDH mutations are found in approximately 10% of World Health Organization (WHO) grade 4 (glioblastoma [GBM]), approximately 70% of grade 3 (anaplastic) tumors, and approximately 90% of grade 2 (low-grade) gliomas [3, 8].
IDH1 and IDH2 mutations are mutually exclusive when present, with the latter being much less commonly detected and predominantly occurring in oligodendroglial tumors [8, 9]. In gliomas, approximately 85% of IDH alterations occur within a “hotspot” in codon 132 of one allele of the IDH1 gene and result in heterozygous substitution of arginine to histidine (R132H). Mutations in IDH2 impact the analogous amino acid R172. IDH mutations are missense at hotspot loci and are heterozygous; therefore, these alterations likely lead to a phenotypic “gain of function” [3, 10–12].
The altered forms of the IDH enzymes are impaired in their ability to catalyze the normal conversion of isocitrate to α-ketoglutarate (α-KG). Instead, they catalyze an NADPH-dependent reduction of α-KG to the R enantiomer of 2-hydroxyglutarate (2-HG) [12, 13]. 2-HG, a trace metabolic by-product in normal cells, is markedly elevated in IDH-mutant glioma tumors, which display nearly 100-fold accumulation compared with IDH-wild-type (IDH-WT) gliomas [13]. The pathogenesis and transformation of IDH-mutant cancers may be related to a block in differentiation, and 2-HG may play an important role in this process. 2-HG, considered an oncometabolite, is structurally homologous to α-KG and, therefore, may interact with α-KG-requiring enzymes essential to epigenetic modulation and HIF-1α regulation [14–22].
2-HG levels are elevated in various IDH1/2-mutant malignancies in tissue and blood compared with healthy control subjects [12, 23–25]. Serum or urine levels of 2-HG have been studied as biomarkers of disease activity in various IDH-mutant malignancies [23, 26]. Studies in acute myeloid leukemia have detected elevated circulating 2-HG levels during treatment [23]. To date, circulating 2-HG in patients with glial malignancies has not been extensively investigated. A study of 16 grade 2/3 glioma samples, of which 10 demonstrated IDH1 R132 mutations, found no correlation between serum 2-HG levels and the presence of IDH mutations [27]. Another, more recently published study of 38 patients with IDH1 R132H mutations and 46 patients with IDH-WT did not detect a difference in plasma or urine 2-HG levels but, interestingly, reported a significantly higher plasma-to-urine 2-HG ratio in patients with IDH1 mutations [28]. Given the prevalence and prognostic value of IDH mutations in gliomas, we sought to better characterize 2-HG levels in the serum, urine, and cerebrospinal fluid (CSF) in our larger patient population with IDH1 mutations.
Methods
Patients 18 years of age or older and identified with a diagnosis of a brain glioma were considered for enrollment in this study. Peripheral blood and urine samples were obtained from patients with active disease, but at various stages in their disease course (i.e., immediately postoperative, postradiation, during temozolomide chemotherapy, stable disease in observation, at the time of progression). CSF samples were obtained preoperatively from patients presumed to have an IDH-mutant glioma, based on highly characteristic radiographic features [2], and IDH status was confirmed postoperatively. Clinical and demographic information was available and collected from patients in this study. Tumor volumes were measured on the T2/FLAIR brain magnetic resonance images obtained near the time of 2-HG sampling, by a neuroradiologist using semi-automated segmentation software (OsiriX 6.0; Pixmeo SARL, Bernex, Switzerland, http://pixmeo.pixmeo.com). This study was approved by the Dana-Farber/Harvard Cancer Center scientific review committee and institutional review board. Informed consent was obtained according to the Declaration of Helsinki.
Peripheral venous blood was obtained via venipuncture preoperatively or postoperatively, as indicated. Urine samples were obtained in the clinic, or after Foley catheter placement perioperatively. Cerebrospinal fluid was obtained at the time of surgery using a 10-mL syringe. Measurement of 2-HG levels was performed by Agios Pharmaceuticals (Cambridge, MA, http://www.agios.com), as previously described [20, 21]. In brief, metabolites were extracted using 80% aqueous methanol. For serum, urine, and CSF extraction, 1 mL of sample was mixed with 4 mL of −80°C methanol. All extracts were spun at 13,000 rpm at 4°C to remove precipitate, dried at room temperature, and stored at −80°C. 2-HG levels were then determined by ion-paired reverse-phase liquid chromatography coupled to negative-ion mode electrospray triple-quadrupole mass spectrometry using multiple reaction monitoring. Integrated elution peaks were compared with metabolite standard curves for absolute quantification.
Fluorescence in situ hybridization analysis to assess the status of the 1p and 19q chromosomes was performed as described previously [29]. IDH1/2 and TP53 mutational assays were performed for all patients, using single-base extension sequencing by the SNaPshot assay (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com), as described previously [30]. Immunohistochemistry for IDH1 R132H variant expression was performed as described previously [31].
All statistical analyses were performed using SAS software (SAS Institute, Cary, NC, https://www.sas.com). The Wilcoxon rank-sum test was used to compare 2-HG levels in the indicated biospecimens between the various groups tested. To test the significance of 2-HG biomarker levels as IDH-mutation classifiers, a logistic regression model was fitted and the area under the curve (AUC) of the resulting receiver operating characteristic (ROC) curve was assessed. The Spearman statistic was used to measure correlation between tumor volume and 2-HG levels.
Results
A total of 60 patients with glioma were studied, of whom 16 had IDH-WT and 44 harbored IDH1/2-mutated glial tumors. Of the IDH-mutant cases, the large majority (42 of 44; 95.5%) were IDH1 mutant, consisting of 33 IDH1-R132H-, 5 IDH1-R132C-, 2 IDH1-R132S-, and 2 IDH1-R132G-mutant cases. Two patients harbored IDH2-R172K alterations. Demographic information, histologic type, and WHO grade of the tumors, as well as the 1p/19q chromosome status, TP53 mutational status, and prior treatment exposures are provided in Table 1. Samples were collected across a range of histologic subtypes and grades, including grade 2 and grade 3 oligodendrogliomas, oligoastrocytomas, and astrocytomas, as well as glioblastomas (grade 4). As expected, and reported in previous literature, a greater proportion of IDH-WT patients had GBM.
Table 1.
Patient characteristics
Absolute levels of 2-HG in the serum were not significantly higher in patients with IDH-mutant glioma versus IDH-WT glioma (IDH-mutant median, 132 ng/mL, range, 33–283 ng/mL; IDH-WT median, 126 ng/mL, range, 35–277 ng/mL; p = .70) (Fig. 1A). However, urine 2-HG levels were significantly higher among patients with IDH-mutant gliomas than among those with IDH-WT disease (IDH-mutant median, 2,780 ng/mL, range, <1,000–14,600 ng/mL; IDH-WT median, 1,625 ng/mL, range, <1,000–4,160 ng/mL; p = .03) (Fig. 1B). Baseline serum creatinine values were available for the majority of patients, and a urine 2-HG to serum creatinine ratio was therefore calculated to account for renal clearance of 2-HG. The urine 2-HG to creatinine ratio was also significantly increased among those with IDH1/2 mutations versus those with IDH-WT disease (IDH-mutant median, 2,926 ng/mL, range, 1,020–20,000 ng/mL; IDH-WT median 1,694 ng/mL, range, 820–5,474 ng/mL; p = .005) (Fig. 1C). We also calculated the ratio of serum to urine 2-HG and found the ratio to be significantly higher among patients with IDH-WT glioma (mean, 0.09 vs. 0.06; p = .03).
Figure 1.
2-HG levels were elevated in the urine of patients with IDH-mutant glioma. (A, B): Bar graphs comparing median 2-HG levels (ng/mL; ±SEM) in the serum (A) and urine (B) of patients with glioma with and without underlying IDH mutation. (C): 2-HG adjusted for serum creatinine is also provided. Wilcoxon rank-sum test p values are given.
Abbreviations: 2-HG, 2-hydroxyglutarate; Cr, creatinine; IDH, isocitrate dehydrogenase gene; WT, wild type.
As an exploratory analysis, we fitted a logistic regression model with urinary 2-HG as the predictive variable for IDH1/2 mutation. The resulting ROC curve from this model had a calculated AUC of 0.684 (p = .0148) (supplemental online Fig. 1). The sensitivity and specificity combinations for all biomarker levels are provided in supplemental online Table 1. Of particular interest was a urinary 2-HG value of 1,720 ng/mL, which corresponds to a reasonable preliminary sensitivity and specificity combination of 79% and 50%, respectively.
We then compared the serum and urine 2-HG values by histologic grade or genetic subtype (1p/19q codeleted vs. TP53 mutant) [32–34] within patients with IDH-mutant glioma. We did not detect differences in serum or urine 2-HG levels between grade 2 versus grade 3 versus grade 4 IDH-mutant glioma, between low-grade (grade 2; n = 21) and high-grade (grades 3 and 4) IDH-mutant gliomas (n = 23) (serum, p = .23; urine, p = .19), nor between lower-grade gliomas (grades 2 and 3; n = 41) and glioblastoma (grade 4; n = 3) (serum, p = .61; urine, p = .33). In addition, no difference in serum or urine 2-HG levels was detected between the 1p/19q codeleted and TP53-mutant subtypes of IDH-mutant gliomas (Table 2). We compared the levels of serum 2-HG, urine 2-HG, and creatinine-adjusted urine 2-HG to tumor volume (T2/FLAIR) at the time of 2-HG sampling. We found no significant correlation between tumor volume and 2-HG levels in either compartment (Spearman r = .2 and .04 for serum and urine, respectively; p = .19 and .79, respectively).
Table 2.
Median 2-HG levels by histopathologic grade, treatment status, and genetic subtype
Although all of the variants of IDH1 at the R132 position produce 2-HG, the rate of NADPH-dependent reduction of α-ketoglutarate may differ between the R132 variants [13]. Therefore, we compared the serum and urine 2-HG levels of patients with IDH1 R132H with that of patients with other IDH1 R132 variants. Interestingly, serum 2-HG levels were significantly higher in the patients with IDH1 R132H mutant gliomas than in patients with other IDH1 R132 variants (median, 157 ng/mL vs. 91 ng/mL; range, p = .02). Paradoxically, no significant difference in urine 2-HG levels was observed between the two groups, even when normalized to creatinine levels (IDH1 R132H mutant gliomas: median 2,963 ng/mL vs. other IDH1 R132 variants: median 2,477 ng/mL; p = .48).
In addition, we examined whether serum or urine 2-HG levels differed based on receipt of adjuvant treatment. Twelve of 44 patients (27%) with IDH1/2-mutant glioma received no adjuvant therapy at the time of serum or urine collection. Of the remaining patients, 11 had previously received adjuvant radiation, eight received chemotherapy only (all received temozolomide), and 13 received chemoradiation (12 received concurrent temozolomide and radiation and one received radiation followed by procarbazine, lomustine, and vincristine chemotherapy). Interestingly, urine 2-HG levels were higher in the group that received any prior adjuvant therapy compared with no adjuvant therapy (urine 2-HG levels: no adjuvant therapy: median, 1,230 ng/mL, range, 1,000–12,300 ng/mL vs. prior adjuvant therapy: median, 2,855 ng/mL, range, 1,000–14,600 ng/mL, p = .017; creatinine-adjusted urine 2-HG levels: no adjuvant therapy: median, 1,892 ng/mL, range, 1,020–18,636 ng/mL vs. prior adjuvant therapy: median, 3,085 ng/mL, range, 1,385–20,000 ng/mL, p = .03). There was no difference in serum 2-HG levels between the two groups, nor were there differences in serum or urine 2-HG levels between treatment arms (Table 2).
In an exploratory analysis, we examined the CSF of four patients with IDH-mutant gliomas and compared the absolute 2-HG levels to that in CSF from three patients with IDH-WT gliomas. All CSF samples were obtained before tumor resection. We detected relatively high 2-HG levels in one patient with IDH-mutant glioma. 2-HG levels from the other three patients were undetectable, less elevated, or near the lower limit of detection (Table 3).
Table 3.
CSF 2-HG level by IDH status
Discussion
Although elevation in circulating 2-HG levels has been detected in patients with various IDH-mutant malignancies, to date, there are no such data to support this in gliomas. Here, we describe a large series of patients with IDH-mutant glioma, characterizing serum, urine, and CSF 2-HG levels. We found that patients with IDH-mutant gliomas did not generally have a higher serum 2-HG level compared with those with IDH-WT tumors. However, urinary 2-HG levels were significantly higher among our patients with IDH-mutant compared with patients with IDH-WT gliomas, with and without correction for renal function. In addition, we measured 2-HG levels in a subset of CSF samples from patients with IDH-mutant and some with IDH-WT gliomas, and, intriguingly, one IDH1-mutant sample displayed a markedly elevated 2-HG level.
Although IDH mutations result in a marked elevation of 2-HG level within glial tumors [13], it is unclear whether 2-HG is detectable outside of the tumor in these patients. In a recently described, patient-derived, IDH1-mutant glioma orthotopic xenograft mouse model, 2-HG was detected in the serum of xenografted mice [35]. However, to date, 2-HG levels have not been found to be elevated in the serum of patients with IDH-mutant gliomas. In our study, the mean serum 2-HG level was higher in IDH-mutant cases but did not reach statistical significance, a similar finding to a recent report of 16 serum samples (9 IDH1-R132H, 1 IDH2-R172K mutant, and 6 IDH-WT cases) [27]. Another recently published series [28] of 38 patients with IDH1 R132-mutant disease and 56 patients with IDH-WT mutations similarly did not detect a difference in plasma 2-HG levels, and absolute levels of circulating 2-HG were similar. However, their additional results did not correspond to ours, in that they did not note a significant increase in urine 2-HG levels among IDH-mutant patients, but did report a significant difference when they compared the ratio of plasma to urine 2-HG levels, with patients with IDH1 mutations displaying a significantly higher ratio, despite no difference in plasma 2-HG levels. Given these results, we calculated the serum to urine 2-HG ratio for our patients, and, not surprisingly, given the higher urine 2-HG results, we detected a lower serum to urine 2-HG ratio among our patients with IDH1 mutations.
Although it would appear intuitive that urine 2-HG levels would be comparatively elevated, as we have found, among a population of patients with IDH-mutant disease, the reason for the discordance with a previous report that observed lower urinary 2-HG levels in patients with IDH1-mutant compared with IDH-WT gliomas is unclear [28]. However, when comparing absolute 2-HG levels in both the circulating and urinary compartments, a significant difference in absolute urine 2-HG levels in patients with IDH-WT disease is noted between the two studies. The absolute plasma 2-HG values reported by Lombardi et al. [28] (97.0 ng/mL for IDH-WT and 97.2 ng/mL for IDH-mutant) were similar to values we detected in the serum, as were the creatinine-corrected median urinary 2-HG values in IDH1/2-mutant patients (4.6 µg/mL in Lombardi et al. [28] vs. 2.926 µg/mL in our study). However, there is a large disparity in absolute urinary 2-HG values in patients with IDH-WT glioma, with the prior study reporting a median value of 7.3 µg/mL versus 1.694 µg/mL in our study. It is possible that patient variation in the IDH-WT populations between the two studies could account for the discrepant conclusions. In our study, only three of the 16 patients had disease that was progressing at the time of sample collection, and the others were either immediately postoperative or had stable tumor after completion of adjuvant chemoradiation. The former study did not report the stage of disease (e.g., postresection, stable, recurrent) when most of the samples were collected from either the IDH-WT or IDH-mutant population. These factors, among others, could affect circulating or compartment 2-HG levels.
The lack of significant 2-HG accumulation in sera and CSF of all patients with IDH-mutant cancer may be related to an organ-specific 2-HG production effect, local clearance of 2-HG, or site-specific accumulation, all of which may impact sampling. 2-HG levels in different compartments may also vary based on the status of the tumor (i.e., unresected vs. gross totally resected tumors, stable vs. progressive tumors, and untreated tumors vs. tumors treated with radiation or chemotherapy). Here, we enrolled patients with tumors at any stage in a pilot analysis. We did not observe a correlation between tumor size and 2-HG levels; however, intriguingly, we found that patients who had received prior adjuvant therapy had higher levels of urine 2-HG than those who received no adjuvant therapy. It is possible that the patients who received adjuvant therapy represented more aggressive disease; however, nine of the 12 untreated patients had either grade 3 or progressive grade 2 disease, and seven went on to have treatment within 3 months of serum/urine collection. Therefore, selection bias remains a possible but unlikely reason for this difference. Further studies stratifying patients based on receipt of adjuvant therapy will inform whether these factors affect circulating 2-HG levels in patients with glioma. Nevertheless, urine 2-HG levels, with and without adjustment for creatinine, were significantly higher in IDH1/2-mutant compared with IDH-WT cases in the entire glioma cohort. The reasons for the apparent accumulation of 2-HG levels in the urine of patients with IDH-mutant glioma remain to be elucidated.
In one case, the CSF 2-HG level was elevated in the CSF of a patient with IDH1-R132H grade 2 oligoastrocytoma (4,660 ng/mL). All other CSF samples were near or below the lower levels of 2-HG detection. A recent metabolic profiling study of CSF from patients with IDH-mutant and IDH-WT gliomas detected significant differences in a number of metabolites, including pyruvate, oxaloacetate, lactate, citrate, and isocitrate, but 2-HG was not detected in any patient sample [36]. Thus, a larger study to characterize 2-HG accumulation in the central nervous system is warranted.
Our pilot finding that urine 2-HG level is elevated among patients with IDH-mutant glioma has potential for clinical utility if validated in larger studies with more defined patient populations. Obtaining tumor tissue from patients with glioma for diagnostic and molecular analysis requires invasive neurosurgical procedures, and a noninvasive means to detect 2-HG, a potential surrogate biomarker for IDH mutation, could serve as a valuable aid in diagnosis, prognostication, and, potentially, assessment of therapeutic response. Noninvasive methods such as imaging methods to detect and quantify 2-HG levels in glioma tumors are already under investigation [37, 38]. Furthermore, evidence is emerging that patients with IDH-mutant glioma survive longer with greater extent of resection at diagnosis [39]; therefore, knowledge of whether a tumor harbors a IDH mutation before surgery may impact therapeutic decisions and survival.
Conclusion
We found that urine 2-HG levels are elevated among patients with IDH-mutant glioma when compared with that of patients with IDH-WT disease. A noninvasive method for detecting and quantifying 2-HG levels would have clinical impact in patients with IDH-mutant glioma, and our findings suggest further investigation of urinary 2-HG is warranted.
See http://www.TheOncologist.com for supplemental material available online.
This article is available for continuing medical education credit at CME.TheOncologist.com.
Supplementary Material
Acknowledgments
We thank Agios Pharmaceuticals for 2-HG quantification support. This work had no specific funding source.
Author Contributions
Conception/Design: Amir T. Fathi, Brian V. Nahed, Seth A. Wander, A. John Iafrate, Darrell R. Borger, Alona Muzikansky, Andrew S. Chi
Provision of study material or patients: Amir T. Fathi, Brian V. Nahed, Ashley M. Perry, Andrew S. Chi
Collection and/or assembly of data: Amir T. Fathi, Brian V. Nahed, Seth A. Wander, A. John Iafrate, Christelle P. Joseph, Andrew S. Chi
Data analysis and interpretation: Amir T. Fathi, Brian V. Nahed, Seth A. Wander, Darrell R. Borger, Ranliang Hu, Ashraf Thabet, Daniel P. Cahil, Ashley M. Perry, Alona Muzikansky, Andrew S. Chi
Manuscript writing: Amir T. Fathi, Brian V. Nahed, Seth A. Wander, Alona Muzikansky, Andrew S. Chi
Final approval of manuscript: Amir T. Fathi, Brian V. Nahed, Seth A. Wander, A. John Iafrate, Darrell R. Borger, Ranliang Hu, Ashraf Thabet, Daniel P. Cahill, Ashley M. Perry, Christelle P. Joseph, Alona Muzikansky, Andrew S. Chi
Disclosures
Amir T. Fathi: Agios (C/A); A. John Iafrate: ArcherDx (C/A, OI, IP). The other authors indicated no financial relationships.
(C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board
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