Circulating oncometabolite 2-hydroxyglutarate is a potential surrogate biomarker in patients with isocitrate dehydrogenase-mutant intrahepatic cholangiocarcinoma

Abstract

Purpose

Mutations in the IDH1 and IDH2 (IDH1/2) genes occur in ~20% of intrahepatic cholangiocarcinoma (ICC) and lead to accumulation of 2-hydroxyglutarate (2HG) in the tumor tissue. However, it remains unknown whether IDH1/2 mutations can lead to high levels of 2HG circulating in the blood and whether serum 2HG can be used as a biomarker for IDH1/2 mutational status and tumor burden in ICC.

Experimental Design

We initially measured serum 2HG concentration in blood samples collected from 31 ICC patients in a Screening cohort. Findings were validated across 38 resected ICC patients from a second cohort with tumor volume measures. Circulating levels of 2HG were evaluated relative to IDH1/2 mutational status, tumor burden and a number of clinical variables.

Results

Circulating levels of 2HG in the Screening cohort were significantly elevated in patients with IDH1/2-mutant (median 478 ng/ml) versus IDH1/2-wild-type (median 118 ng/ml) tumors (p<0.001). This significance was maintained in the Validation cohort (343 ng/ml vs 55 ng/ml, p<0.0001) and levels of 2HG directly correlated with tumor burden in IDH1/2-mutant cases (p<0.05). Serum 2HG levels ≥170 ng/ml could predict the presence of an IDH1/2 mutation with a sensitivity of 83% and a specificity of 90%. No differences were noted between the allelic variants IDH1 or IDH2 in regards to the levels of circulating 2HG.

Conclusions

This study indicates that circulating 2HG may be a surrogate biomarker of IDH1 or IDH2 mutation status in ICC and that circulating 2HG levels may correlate directly with tumor burden.

Introduction

Intrahepatic cholangiocarcinoma (ICC) represents a unique clinical entity with significant challenges. As a subset of biliary tract cancers, it represents the second most common form of liver malignancy, and incidence as well as mortality rates have steadily increased (1). Surgical resection represents the only curative treatment. However, only 10-20% of patients present with early-stage disease that is amenable to curative surgery (1, 2). Even with the standard treatment of gemcitabine and cisplatin combination chemotherapy, the median survival of patients with locally advanced or metastatic disease remains less than one year (3). Therefore, molecularly-targeted therapies may provide an important means of improving patient outcomes.

Our understanding of the molecular mechanisms underlying the pathogenesis of ICC has been expanding (4, 5). Recently, mutations in the genes encoding for isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) have been identified as a significant molecular feature in ~20% of ICC cases (6-8). The prevalence of this genetic signature may suggest an important pathophysiological mechanism in ICC, which may offer novel insights on how to develop targeted approaches against this treatment-refractory disease.

IDH1 and IDH2 (IDH1/2) normally function to catalyze the oxidative carboxylation of isocitrate to α-ketoglutarate. The recurrent cancer mutations in these enzymes confer neomorphic activity through the reduction of α-ketoglutarate to the metabolite R(–)-2-hydroxyglutarate (2HG), resulting in 2HG accumulation in the tumor tissue (9, 10). High intracellular levels of 2HG have recently been shown to be sufficient for promoting the tumorigenic effects of mutant IDH activity that are associated with enhanced proliferation and impaired differentiation (11).

While elevated levels of circulating 2HG have been identified in the blood of patients with IDH1/2-mutant acute myeloid leukemia (12-14), the clinical utility of circulating 2HG has not yet been clearly established in solid tumors (15). This has partly been hampered by the prevalence of IDH1/2 mutations confined primarily to a small number of cancer types, which is then further limited when considering a relatively rare malignancy such as ICC. We have previously identified elevated levels of 2HG in the tumor tissue of IDH1- and IDH2-mutant ICC (6). Since 2HG has been shown to be a membrane diffusible metabolite (9, 10, 16), it is of interest to determine whether 2HG can be detected in the serum of ICC patients carrying a somatic IDH1 or IDH2 mutation. Accurate detection and quantification of serum 2HG could potentially serve as an efficient and less-invasive method of assessing a patient's response to IDH-targeted therapies, once promising drugs currently undergoing preclinical evaluation enter into clinical testing (17, 18). In this study, we therefore sought to characterize serum 2HG levels as a biomarker of IDH1/2 mutational status and its association with tumor burden in IDH1/2-mutant ICC tumors.

Patients and Methods

Patients and samples

An initial Screening cohort consisted of 31 patients diagnosed with ICC that were treated at the Massachusetts General Hospital from 2008-2012. Subjects were selected based on the availability of tumor mutational profiling (performed as part of their clinical management) and paired blood samples (collected previously with informed consent for research banking purposes). However, many of the banked blood samples available for this cohort were obtained after the initiation of treatment. To increase the number of evaluable samples and to overcome any bias associated with obtaining blood samples post-treatment, a second Validation cohort from Queen Mary Hospital (QMH) at The University of Hong Kong was evaluated. This group consisted of 38 patients with blood samples that were collected prior to tumor resection and treatment. Tumor burden measurements were recorded for each patient in the Validation cohort based on linear dimensions measured along perpendicular axes from resected tumors. The tumor volume was calculated using an ellipsoid formula. Clinical data was extracted from patient records, including age, grade, stage, treatment, recurrence, and survival. Institutional review board approval was obtained for this study.

Genotype analysis

Mutational profiling was performed using nucleic acids that were extracted from resected ICC tumor tissue for each patient. Specific hotspot mutations in the IDH1 gene at nucleotide positions c.394 and c.395 (amino acid position p.R132) were identified using a multiplexed mutational profiling platform that has been previously described and clinically implemented (6, 19). Rare IDH1 mutations that have been reported in other tumor types were not evaluated (20). Sanger sequencing was used to identify mutations in the IDH2 gene at exon 4 (including mutations at codons p.140 and p.172) using methods and polymerase chain reaction primers that have been previously reported (6). Labeled PCR products were separated using an ABI PRISM 3730 DNA Analyzer, and the data were interpreted with GeneMapper Analysis Software (Life Technologies/Applied Biosystems).

Circulating 2-Hydroxyglutarate analysis

Serum was isolated from whole blood, aliquoted, and stored at –80°C until analysis. Circulating levels of 2HG were measured at Agios Pharmaceuticals (Cambridge, MA) using lLC-MS/MS analysis (AB Sciex 4000, Framingham, MA) operating in negative electrospray mode. MRM data was acquired for each compound using the following transitions: 2HG (146.9/128.8 amu), 13C5-2HG (151.9/133.8 amu) & 3HMG (160.9/98.9 amu). Chromatographic separation was performed using an ion-exchanged column (Bio-rad Fast Acid analysis, 9 μm, 7.8 mm × 100 mm; Bio-rad). The flow rate was 1 ml/min of 0.1% formic acid in water with a total run time of 4 minutes. 30 μl of sample was extracted by adding 30 μl of internal standard (ISTD) in water followed by 200 μl of acetonitrile. The sample was vortex mixed, centrifuged and 100 μL of supernatant transferred to a clean 96-well plate. The supernatant was diluted with 100 μl of deionized water and 25 μl injected on column.

Statistical analysis

The comparison of biomarkers with respect to IDH1/2 mutational status or study site was performed using exact Mann-Whitney-Wilcoxon test. Median and interquartile ranges are provided as descriptive statistics. Correlations were quantified as Spearman's correlation coefficients and tested with the Spearman's test. P-values of <0.05 were considered statistically significant.

Results

Patient Characteristics

The patient characteristics of the Screening and Validation cohorts are summarized in Table 1. A total of 31 diagnosed ICC patients with clinically-determined IDH1 and IDH2 gene mutational status, and with available banked whole blood, comprised the Screening cohort. The median age of this group was 57 years, and approximately 65% of these patients presented with stage IV disease. These characteristics are consistent with patients that normally undergo clinical mutational profiling in an effort to identify alternative treatment courses after failing standard of care.

Table 1.

Characteristics of patients with intrahepatic cholangiocarcinoma across cohorts.

Characteristics	Screening Cohort	Validation Cohort
Total Number of Cases:	31	38
Age at Diagnosis (Years):
Median	57	64
Range	36-78	25-79
Sex:
Male	17	22
Female	14	16
Baseline CA19-9 Level (U/ml):
Median	34	34
Range	<1-3175	<2-7520
Unknown	5	18
Stage of Disease at Diagnosis:
I	4	19
II	4	5
III	3	14
IV	20	0

In order to expand analysis of ICC patients, a second Validation cohort consisting of 38 ICC patients who underwent surgical resection was then identified and retrospectively genotyped to identify IDH1/2 mutations. The age, sex and CA19-9 blood levels of this Validation cohort were comparable to those in the Screening cohort (Table 1). However, the Validation group patients spanned early disease stages, including stage I (50%), stage II (~13%) and stage III (37%), and did not include stage IV patients.

IDH1 and IDH2 mutational status

In the Screening cohort, IDH1 mutations were found in 11 out of 31 patients with ICC, for an overall incidence of 35%. These included point mutations in IDH1 p.R132C (n=7), p.R132L (n=3) and p.R132G (n=1) (Table 2 and Supplemental Table 1). No IDH2 mutations were identified. The ICC resected tumor samples from the Validation cohort were genotyped using the same clinical genotyping platform. We identified mutations in IDH1 in 4 out of 38 patients (10%) and IDH2 in 3 out of 38 patients (8%), for a combined frequency of IDH1/2 mutations in 18% of the cohort. The point mutations included IDH1 p.R132C (n=2) and p.R132L (n=2), as well as IDH2 p.R172W (n=2) and p.R172K (n=1). The frequency of IDH1 mutations was statistically higher in the Screening cohort, and the frequency of IDH2 mutations was significantly greater in the Validation cohort (p=0.04). It is not certain whether these differences are related to regional differences or the distinct tumor stage characteristics between the 2 cohorts. Unlike what has been previously reported (7), there was no correlation between the presence of IDH1/2 mutations and either the grade of tumor or the histological clear cell changes (Table 3).

Table 2.

Characteristics of /DH1-mutant or /DH2-mutant intrahepatic cholangiocarcinoma patients.

Screening Cohort
Pt ID	/DH1 or /DH2 Mutation	Serum 2HG (ng/ml)	CA19-9 (U/ml)	Stage	Sex	Age
R9	/DH1c.394C>G (p.R132G)	1093	653	IV	F	56
R10	/DH1c.394C>T (p.R132C)	173	n/a	IV	M	48
R11	/DH1c.394C>T (p.R132C)	174	n/a	IV	M	58
R14	/DH1c.394C>T (p.R132C)	97	<1	IV	M	46
R23	/DH1c.394C>T (p.R132C)	330	24	IV	F	51
R25	/DH1c.394C>T (p.R132C)	107	n/a	II	F	64
R26	/DH1c.394C>T (p.R132C)	509	19	IV	M	64
R27	/DH1c.394C>T (p.R132C)	25570	74	IV	M	56
R22	/DH1c.395G>T (p.R132L)	500	35	IV	F	60
R4	/DH1c.395G>T (p.R132L)	777	382	IV	F	77
R20	/DH1c.395G>T (p.R132L)	478	n/a	IV	F	42

Validation Cohort
Pt ID	IDH1 or IDH2 Mutation	Serum 2HG (ng/ml)	CA19-9 (U/ml)	Stage	Sex	Age
P14	/DH1c.394C>T (p.R132C)	174	45	I	M	56
P15	/DH1c.394C>T (p.R132C)	545	11	II	F	50
P3	/DH1c.395G>T (p.R132L)	108	<2.0	I	M	70
P30	/DH1c.395G>T (p.R132L )	343	n/a	I	F	76
P2	/DH2c.514A>T (p.R172W)	211	n/a	I	F	55
P36	/DH2c.514A>T (p.R172W)	648	n/a	I	F	57
P23	/DH2c.515G>A (p.R172K)	1212	n/a	I	F	73

Table 3.

Histological features of IDH1I2-mutant versus /DH1/2-wild-type intrahepatic cholangiocarcinoma in 33 evaluable patients from the Validation cohort.

IDH1I2 Mutational Status	Number of cases	Clear cell change	Grade of tumor
			Grade 1	Grade 2	Grade 3
/DH1/2 Mutant	7	0 (0%)	1 (14%)	5 (72%)	1 (14%)
/DH1/2 Wild-Type	26	7 (27%)	4 (15%)	16 (62%)	6 (23%)

Circulating 2-hydroxyglutarate levels and correlation with tumor burden

For our Screening cohort, blood had been obtained from ICC patients at variable times in regards to the initiation and response to treatment. For patients with a somatic IDH1/2 mutation, the radiological tumor burden and response to therapy at the time of 2-HG measurement are listed in Supplemental Table 2. Despite the very different blood sampling schedules, serum 2HG levels were still detected at significantly elevated levels in IDH1-mutant (median 478 ng/ml; interquartile range 174-643 ng/ml) versus IDH1-wild-type ICC patients (median 118 ng/ml; interquartile range 68-160 ng/ml) (p<0.001) (Figure 1). There were only two IDH1-mutant patients with 2HG levels less than 170 ng/ml in the Screening cohort, where blood was obtained after a clinical response to treatment (Table 2). The first of these two patients (R25) had resected stage II ICC, where the blood sample was obtained about two months postoperatively with no evidence of disease. The second patient (R14) had stage IV ICC where the blood sample was obtained 6 months after receiving gemcitabine, oxaliplatin and panitumumab on clinical study and when a partial radiologic response was documented. The ability to detect elevated 2HG in the blood samples collected post-treatment from other IDH1/2-mutant cases was most likely attributed to the presence of metastatic disease and an incomplete response to treatment.

Figure 1.

The distribution of circulating 2HG levels in blood samples collected across the two cohorts. Serum 2HG levels were measured and compared between patients with IDH1-mutant or IDH2-mutant (IDH-Mut) versus IDH-wild-type (IDH-WT) ICCs in the A) Screening cohort (n=31) and B) in the Validation cohort (n=38). For each column, the dark grey box represents the 2HG values in the 75^th quartile and the light grey box represents values in the 50^th quartile. The lines extending vertically from the boxes indicate the variability outside the upper and lower quartiles. A significant elevation of circulating 2HG levels was seen in IDH-Mut ICC patients compared to IDH-WT ICC patients (p<0.001).

To address the limitations of evaluating 2HG in samples drawn after treatment initiation, a second Validation cohort was pursued where all blood samples were collected prior to surgical resection and chemotherapy treatment (n=38). The serum 2HG levels in the Validation cohort were also significantly elevated in the IDH1/2-mutant (median 343 ng/ml; interquartile range 192-596 ng/ml) versus IDH1/2-wild-type (median 55 ng/ml; interquartile range 42-82 ng/ml) (p<0.0001) ICC patients (Figure 1). Surprisingly, there were no notable differences between 2HG levels in patients with IDH1/2 mutant ICC across the two cohorts (p=0.68), supporting the robustness of 2HG as a potential means of identifying IDH1/2-mutant versus wild-type ICC patients in a clinical setting. These data combined indicate that a threshold of serum 2HG set at >170 ng/ml can discriminate the presence of an IDH1/2-mutant from an IDH1/2-wild-type ICC tumor using patient blood, with a sensitivity of 83% and a specificity of 90%. Within the context of our limited cohort size and using Cox proportional hazards model, baseline 2HG was not found to be predictive of overall survival or recurrence free survival in the Validation cohort (p=0.737 and 0.756, respectively).

While in vitro differences have been reported (21), it has not been clearly established whether the various mutant forms of IDH1 or IDH2 can produce variable levels of 2HG in vivo. When evaluating blood collected across all of our ICC patients, albeit a limited sample size, there were no significant differences in the levels of circulating 2HG in patients with a tumor mutant for IDH1 versus IDH2 (p=0.29). However, the high variability that we noted in circulating 2HG levels across the IDH1/2-mutant ICC patients could have reduced the ability to identify more subtle differences. This also raised the question of whether the wide range of circulating 2HG could be attributed to differences in tumor burden. Since all blood samples from the Validation group were collected prior to surgical resection, levels of circulating 2HG levels were evaluated relative to tumor volume (Supplemental Table 3). While tumor weight was not recorded, tumor linear dimensions measured along perpendicular axes from these resected tumors were recorded and the tumor volume was calculated using an ellipsoid formula. As shown in Figure 2, 2HG levels indeed correlated directly with tumor burden (Spearman's ρ 0.89; p=0.0123) in patients with IDH1/2-mutant ICC.

Figure 2.

Correlation between serum 2HG level and tumor volume measured at surgery in IDH1-mutant and IDH2-mutant ICC patients from the Validation cohort.

Discussion

The treatment of advanced ICC remains an unmet need, where new strategies are desperately needed. In this study, we confirmed an increasing body of evidence that ICCs have a relatively high frequency of IDH1 and IDH2 mutation (6-8). Newly-developed IDH1 and IDH2 inhibitors have shown early evidence of activity in preclinical studies using IDH1-mutant glioma and IDH2-mutant leukemic model systems, respectively (17, 18), and suggest a potentially viable treatment strategy in ICC. As these IDH-targeted therapies begin to enter early clinical testing, there will be a need for robust pharmacodynamic markers that can be used to evaluate on-target drug effects.

There have been tremendous advances in our understanding of how mutant IDH1 and IDH2 drive the tumorigenic process(22). It has recently been demonstrated that 2HG in itself is sufficient to disrupt differentiation and promote growth factor independence in leukemic cells (11). Therefore, 2HG may very well be the primary oncogenic effector of mutant IDH1 and IDH2 activity. The intracellular accumulation of this metabolite has been shown to disrupt normal functioning of multiple α-ketoglutarate-dependent dioxygenases (23). Key targets include TET2 and jumonji-domain containing proteins that function in epigenetic regulation, likely serving as the primary mechanisms of aberrant DNA and histone methylation that has been identified in IDH-mutant tumors (11, 17, 23-28). Another target of 2HG is the EglN prolyl 4-hydroxylases that promote proteasomal degradation of the HIF (hypoxia-inducible factor) transcription factor (11, 24, 29). Given the diversity of these mechanisms, it will be of interest in determining the precise role of 2HG in driving the carcinogenic process in ICC.

The neomorphic activity of mutant IDH1 and IDH2 in the production of the cell-permeable 2HG oncometabolite lends itself for use as a circulating biomarker. Acute myeloid leukemia (AML) has so far provided the model of how 2HG can be used to monitor treatment effects and disease activity. Levels of 2HG in the serum and urine of IDH1 or IDH2-mutant AML patients have been found to decrease throughout the course of conventional therapy, in a manner concordant with decreases in blast counts (13). Furthermore, the inability to achieve a normal level of serum 2HG at remission has been associated with worse overall survival in AML (12).

Given the rarity of ICC relative to AML, there is difficulty in obtaining sufficient patient samples for meaningful evaluation, and our study reports on a collaborative effort between two institutions where samples were collected over several years. Unlike what has been recently reported in glioma (15), we have identified for the first time a correlation between circulating 2HG and tumor IDH mutation status in a solid tumor. Not surprisingly, the concentration of blood 2HG was significantly lower in IDH-mutant ICC patients (i.e., a solid tumor) compared to what has been reported in IDH-mutant AML patients (i.e., a blood malignancy) (12, 13). From our small cohort analysis, a threshold of circulating 2HG levels >170 ng/ml could predict the presence of an IDH1/2 mutation in an ICC patient with a sensitivity of 83% and a specificity of 90%. We have also provided preliminary data suggesting a correlation between circulating 2-HG levels and tumor burden in IDH mutant cholangiocarcinoma, raising the question as to whether serial 2HG monitoring in the blood can serve as a potential surrogate marker of treatment response in IDH mutant cholangiocarcinoma. While this study is an important first step, larger prospective studies are needed to confirm the relationship between levels of circulating 2HG and tumor burden. Furthermore, it is unknown whether serum 2HG can serve as a prognostic marker for IDH1- or IDH2-mutant cholangiocarcinoma patients.

In summary, our study suggests that circulating 2HG may serve as a surrogate biomarker of IDH1 or IDH2 mutation status in ICC that is related to tumor burden. Future studies will be needed to determine how well 2HG correlates with changes in tumor volume over the course of treatment. With the highly anticipated entrance of new IDH1 and IDH2 targeted therapies in early-phase clinical trials, it will be of interest to determine the utility of 2HG as a pharmacodynamic marker of treatment response in IDH-mutant ICC patients.

STATEMENT OF TRANSLATIONAL RELEVANCE.

Mutations in the IDH1 and IDH2 (IDH1/2) genes represent a new genetic signature in intrahepatic cholangiocarcinoma (ICC). While accumulation of 2-hydroxyglutarate (2HG) has been previously detected in the tumor, it remains unknown whether IDH1/2 mutations can lead to high levels of 2HG circulating in the blood. Our exploratory study has provided evidence that elevated circulating levels of 2HG in IDH1/2 mutant ICC patients may serve as a surrogate biomarker of IDH1/2 mutation status in ICC patients. Furthermore, preliminary evidence of circulating 2HG correlating with tumor burden further indicates the potential for evaluating serum 2HG as a pharmacodynamic biomarker of treatment response in IDH1/2-mutant ICC patients.