Introduction

Tumors may reprogram their metabolism to meet the demands of rapid cell-proliferation. Nucleotides for DNA and RNA-biosynthesis and lipids for membrane-synthesis are the major requirements of an aggressively proliferating cell. Glutamine (Gln) is the precursor for de-novo synthesis of pyrimidine-nucleotides while citrate (Cit), a TCA cycle intermediate, is a major precursor for fatty acid or lipid-synthesis. A mass-spectroscopy study in cancer-cell-lines showed increased Gln-metabolism ¹. Cit has been shown to be elevated in high-grade gliomas ². We hypothesized that a subset of malignant-gliomas will reprogram the Gln and Cit metabolism to meet the demands of rapid cell-proliferation. Reductive-carboxylation of Gln will remodel the TCA-cycle towards increased Cit production for enhanced lipid synthesis. Cit, whose resonance is centered at 2.6 ppm, is not measureable by MRS in- vivo in healthy-brain owing to its low concentration and spectral-overlap with neighboring-resonances from N-Acetylaspartate (NAA-Asp) and aspartate (Asp) around 2.6 ppm. Here, we have reliably measured Cit together with Gln, 2-Hydroxyglutarate (2HG) and other tumor relevant metabolites in glioma patients in vivo at 3T using an in house customized PRESS TE 97 ms ¹H MRS-sequence. The association of Gln and Cit elevation with clinical MR-features of malignancy such as ‘enhancement on T1-weighted post gadolinium-images’ was also evaluated.

Methods

A PRESS sequence was tailored for Cit detection using volume localized density-matrix simulations ³. The PRESS sequence included a 9.8-ms 90° pulse (bandwidth, 4.2 kHz) and two 13.2-ms 180° RF-pulses (bandwidth, 1.3 kHz) at an RF field-intensity of 13.5 µT. In-vivo 1H MR measurements were performed in 20 high-grade glioma-patients on a whole-body 3T scanner (Philips Medical Systems). PRESS spectra were obtained from tumors identified by T2-weighted fluid-attenuated inversion-recovery (T2-FLAIR) imaging. The T1-weighted Post-gadolinium images were obtained at the 3T scanners at Roger’s Cancer Imaging Center. The MRS voxel-size was 3-8 mL depending on the tumor volume. MRS acquisition parameters included TR=2s, sweep width=2.5 kHz, sampling-points=2048 with signal-averages of 128-256 depending on the voxel-size. Unsuppressed-water was obtained with a short-TE (14 ms) STEAM-sequence with TR=20s as a reference for metabolite-quantification. Spectral-fitting was performed with LCModel-software ⁴ using numerically-calculated basis-spectra of 22-metabolites. Metabolite concentrations were calculated by setting the mean total-Creatine estimate of the medial-occipital brain from healthy-subjects at 8 mM. Proliferative-index and Isocitrate-dehydrogenase (IDH) mutations were identified through immunohistochemistry using antibodies against MIB-1 and IDH respectively as performed previously ^{5, 6}.

Results and Discussion

The PRESS TE 97 ms sequence provided well discernible signal of Gln at 2.4 ppm, a negative polarity signal of Cit at 2.6 ppm without overlap from adjoining positive polarity signals of NAA-Asp and Asp, and 2HG at 2.25 ppm (Fig.2). Of the 20 patients, 18 had biopsy-proven Anaplastic-gliomas (grade 3) or glioblastoma (grade 4), while the other 2 patients had brain-stem glioma. Of the 18 high-grade gliomas, concurrent elevation of Gln and Cit was estimated in 15 of the gliomas. These tumors with elevated-levels of Gln and Cit presented with enhancement on the T1-w Post contrast-image indicative of broken blood-brain-barrier (Fig. 3A). These 15 gliomas were grouped as subset-1 while the other 3 gliomas that had elevated Cit without elevation in Gln (Fig. 3B) were grouped as subset-2 (Fig. 4A). Cit was not significantly different between the two-subset of gliomas (1.4±0.5 vs. 1.6±0.4 mM). However, subset-1 had significantly high total-choline (tCho) (4.7±1.8 vs 1.6±0.3 mM, p<0.01) together with high (13%) MIB-1 labeling index of proliferation compared to subset-2. Since tCho is considered to be a cellularity marker, a finding of high tCho and high MIB-1 in the tumors with elevated Gln and Cit indicates that the tumors have high tumor-cellularity and cell-multiplication competence: typical of high-grade tumors. 13 of the 18 gliomas were IDH-mutant with high level of 2HG (Fig. 4B). Elevated levels of Gln and Cit in IDH-mutant gliomas were not significantly different compared to IDH wild-types (Fig. 4B). Noticeably, of the two brain-stem gliomas, one presented with elevated Gln and Cit while other had low Cit with normal Gln levels (Fig. 5). The brain-stem glioma with elevated Gln and Cit progressed within 6-months and showed enhancement on the T1w post-gadolinium images but the other brain-stem remained stable for almost 7-years without enhancement or progression.

Conclusion

A subset of high-grade tumors undergoes a metabolic-rearrangement of concurrent elevation in Gln and Cit to attain malignant characters such as high-cellularity, uncontrolled proliferation and blood-brain-barrier breakdown. IDH mutant and wildtype gliomas may share a common metabolic rearrangement of glutamine-mediated-citrate-elevation to attain malignancy. Measurement of elevated Gln and Cit together may serve as a potential ‘imaging-biomarker’ for identifying malignant-gliomas.

Acknowledgements

This study was supported by National Cancer Institute of the National Institutes of Health under Award Number R01CA184584 and by a Cancer Prevention Research Institute of Texas grant RP130427