The tumor specimen and the patient were previously reported (18). A right carotid body tumor was surgically resected for histology, genetics, and biochemistry analysis.

Genomic DNA was extracted from tumor tissue and patient blood using DNeasy Blood & Tissue kit (QIAGEN). Library preparation and exome enrichment were conducted using SureSelect V7 kit (Agilent). The sequencing was performed on a NovaSeq S4-300 chip. The resulting FASTQ data was aligned to the human reference genome for variant annotation.

Sanger sequencing was performed as previously described (19). Genomic DNA was amplified through polymerase chain reaction (PCR) using Herculase II Fusion DNA polymerase (Agilent). The amplicon was purified using Monarch PCR & DNA Cleanup Kit (New England Biolabs, Inc.) and sent for Sanger sequencing (Eurofin). The primers used for PCR and sequencing were: F: 5’-CAG AGA CAA GAG GAT GGC TAG G -3’ and R: 5’-TGT AAA ACG ACG GCC AGT GTC TGG CTG TGT TGT TGC TTG-3’. Sequence analysis was performed using Geneious Pro software (version 11.1.4).

Immunohistochemistry was performed as previously described. Tumor tissues were fixed and sectioned to 10-micron slices. The slices were labeled with primary antibodies and probed with Vectastain ABC kit (Vector laboratories). The slices were then exposed to 3,3′-Diaminobenzidine and mounted in Permount mounting medium (Fisher Chemical). The samples were then visualized by light microscopy (Olympus BX-43). The primary antibody used in this study was anti-IDH1/2 Mutant (R132/R172), clone MsMab-1 (1:200, MilliPore).

Mutagenesis was conducted as previously described (20). The pCMV6-IDH2 wild type plasmid (Origene) was used as a template. The coding sequence of IDH2 was transferred to pLenti-C-myc-DDK-IRES-puro plasmid (Origene). The mutagenesis reaction was performed using a Quikchange Mutagenesis Lightning kit (Agilent). The sequence of the mutant plasmid was confirmed by sequencing the entire coding region of IDH2. The plasmid was then used for lentivirus packaging. The hpheo-1 cells were kindly provided by Dr. Hans Ghayee (University of Florida). The hpheo-1 cells were infected with lentivirus with IDH2 coding sequence and selected by puromycin. The mutagenesis primers used were: 5’- CCA TGG GCG TGC CCG CCA ATG GTG ATG -3’; 5’- CAT CAC CAT TGG CGG GCA CGC CCA TGG-3’.

The tissue specimens or cell pellets were lysed with Radioimmunoprecipitation assay buffer (RIPA buffer, Thermo Fisher) and quantified using the DC Protein Assay (Bio-Rad). Protein samples were resolved on 4-12% Bis-tris gel (Thermo Fisher) and transferred to PVDF membrane (MilliPore). The membranes were labeled with primary antibodies and visualized through chemiluminescence kit (Bio-Rad). The primary antibodies used in this study include anti-IDH1/2 Mutant (R132/R172), clone MsMab-1 (1:2,000, MilliPore); Flag (1:5,000, Origene); and β-actin (1:5,000, Cell Signaling Technology).

Stock solutions of D- or L-2-hydroxyglutaric acid (D- or L-2-HG) disodium salt (10 mM), and the internal standards (IS) D or L-2-HG -¹³C₅ disodium salt (5 mM) were prepared in water and stored at -50°C until use. Calibration standard of D- and L-2-HG were prepared by serial dilutions of the stock with 80% methanol. The working IS mixture (1 µM each) was prepared by diluting the stock with 50% methanol. The TSPC (N-(p-toluenesulfonyl)-L-phenylalanyl chloride) derivatization reagent was prepared in acetonitrile (1 mg/mL). Tissues were homogenized in 80% methanol (15-75 mg tissue/mL solvent) using the Bead Rupter (Omni International). The supernatant was diluted with 80% methanol if needed to ensure the 2-HG level was within the linear range of the calibration curve. Samples were analyzed in triplicate. TSPC derivatization was carried out according to Cheng et al. (21) with slight modification. In a 0.5 mL micro-centrifuge tube, 30 µL of sample (or standard) were mixed with 10 µL of IS and then vacuum dried. Two µL of pyridine and 100 µL of TSPC were added to the dried samples. After 30 min, the reaction mixture was vacuum dried and reconstituted with 30 µL of 50% acetonitrile for LC/MS/MS. LC was performed with a Prominence 20AD system (Shimadzu). The injection volume was 2 µL. Separation was achieved at 35°C with a 2.1 x 100 mm, 2.7 µm Cortecs C18 column (Waters). Mobile phase A was 5 mM ammonium acetate in water and mobile phase B was methanol. The flow rate was 300 µL/min and peaks were eluted with a 5-min gradient from 25%-40%B. MS/MS was performed with a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher) operating at selected reaction monitoring mode with negative electrospray ionization. The TSPC derivatized peaks were detected using the following m/z precursor > product ions: 2-HG (448 > 318); 2-HG -¹³C₅ (453 > 318). D- and L-2-HG in the homogenates were determined by a 9-level calibration curve (0.05-25 µM) with linear regression (1/x weighting) using the Thermo Xcalibur software.

Statistical analysis was performed with GraphPad Prism software (Version 6.01). A student t-test was applied for statistical comparisons. All statistical tests were two-tailed. Results are shown as Mean ± SEM, and P < 0.05 was considered statistically significant.

The patient was a 57-year-old female presenting with a history of multinodular goiter and benign thyroid nodules through fine needle aspiration (FNA). The patient also has a family history of thyroid cancer. The patient presented approximately 2.5 x 1.5 cm mass at bifurcation of right common carotid artery with minimal vascularity. Whole-body CT and neck MRI revealed a 2.7 x 2.0 cm mass in the right neck suggestive of carotid body PGL ( Figures 1A, B ). The mass was also positive on ¹⁸F-fluorodopa (¹⁸F-FDOPA) PET/CT scan ( Figure 1C ). There was no evidence of any other pheochromocytomas (PHEOs) or PGLs on the aforementioned scans. A 2.8 x 1.8 x 1.1 cm tumor mass was surgically resected, and the operative findings and histopathology confirmed right carotid body PGL. Immunohistochemical studies also showed positivity for chromogranin A and synaptophysin. Six years after surgery, based on follow-up imaging with whole-body ¹⁸F-FDOPA ( Figure 1D ) and ⁶⁸Ga-DOTATATE PET/CT ( Figure 1E ), as well as whole-body CT and neck MRI, there was no evidence of any recurrent or new PGL.

This patient was enrolled under the protocol Diagnosis, Pathophysiology, and Molecular Biology of Pheochromocytoma and Paraganglioma (ClinicalTrials.gov Identifier: NCT00004847), which was approved by the Eunice Kennedy Shriver National Institutes of Child Health and Human Development and Institutional Review Board. Written informed consent was obtained from the patient.

Histopathological examination confirmed the typical PGL morphology ( Figure 2A ). The tumor tissue from the patient exhibited strong immunopositivity for the IDH2 mutant enzyme ( Figure 2B ). The IDH2 mutant immunopositivity was not seen in PGLs with other genetic backgrounds, such as NF2, SDHx or VHL. Further, we examined the expression of IDH2 mutant through immunoblotting. The IDH2 mutant was expressed in the protein lysate from the patient, but not in PGLs with other genetic backgrounds as described above ( Figure 2C ). The specificity of the IDH2 mutant antibody was confirmed by testing the ectopic expression of the IDH2 R172G variant in hPheo1 cell ( Figure 2C ).

Whole exome sequencing was performed based on DNA specimens from carotid body PGL and patient blood. A somatic change in IDH2 was identified at the location of arginine 172 residue. A follow-up Sanger sequencing confirmed the presence of IDH2 c.514A>G variant, which leads to R172G amino acid substitution ( Figure 2D ).

To confirm the catalytic function of the IDH2 mutant in cancer tissue, we performed the mass spec measurement on D-2-HG in frozen cancer tissue. We identified 15.39 pmol/µg protein, which was significantly higher than in non-IDH-mutant cancer tissue (0.0059 pmol/µg protein, Figure 2E , ****p<0.0001). The level of L-2-HG was found not significantly changed between the tested samples ( Figure 2E , IDH2^WT: 0.01436 pmol/µg protein, IDH2^R172G: 0.2407 pmol/µg protein).