Hyperpolarized [1-13C]-Pyruvate Differentiates Distinctive Molecular Phenotypes in Diffuse Intrinsic Pontine Gliomas
Ilwoo Park1, Rintaro Hashizume2, Joanna Phillips3,4, Sabine Mueller3,5, and Sarah Nelson1,6

1Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA, United States, 2Neurological Surgery, Northwestern University, Chicago, IL, United States, 3Neurological Surgery, University of California San Francisco, San Francisco, CA, United States, 4Pathology, University of California San Francisco, San Francisco, CA, United States, 5Pediatrics, University of California San Francisco, San Francisco, CA, United States, 6Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, United States

Synopsis

Diffuse intrinsic pontine gliomas (DIPGs) are one of the most difficult pediatric cancers to treat. This study investigated the feasibility of 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (HP) [1-13C]pyruvate for differentiating molecular characteristics of DIPGs. Differences in the lactate signal that were observed in two distinct biopsy-originated orthotopic DIPG tumors were associated with changes in the levels of LHDA and HIF-1α activity. This suggests that the non-invasive characterization of DIPGs using this new neuroimaging method may be helpful for assessing treatment response and tumor progression.

Introduction

Children with diffuse intrinsic pontine glioma (DIPG) continue to have a dismal prognosis with a median survival time of 9 month. Due to their brainstem location, surgical resection is impossible and biopsies have rarely been performed. Recent studies that used a combination of postmortem and biopsies samples have shed insight on genetic and molecular makeup of DIPGs1,2 and propelled the identification of potential treatment targets3,4. The in vivo characterization of tumor molecular properties would be beneficial for clinical management of the disease but there are currently no non-invasive methods for assessing tumor molecular characteristics. This study investigates the feasibility of using 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (HP) [1-13C]pyruvate to characterize DIPGs. The metabolism of two K27M-mutant, biopsy-originated DIPG xenografts, one with features of glioblastoma (SF8628) and one with features of WHO grade II infiltrative astrocytoma (SF7761), were investigated and compared with molecular features from immunohistochemical analysis.

Methods

Primary human DIPG cells were implanted into the brainstem of athymic rats (n=4 for SF8628 and n=4 for SF7761). All experiments were performed using a GE clinical 3T scanner with a custom-designed 1H/13C coil. 35μL of [1-13C]pyruvate (with 1.5mM gadolinium) was polarized using a HyperSense® DNP polarizer (Oxford Instruments, Abingdon, UK)5. Compressed sensing 13C 3D MRSI data6 were acquired at 20s from the start of the injection of 2.7mL hyperpolarized [1-13C]pyruvate through tail vein. The injection started ~10s after dissolution and lasted for 10s. The final dissolved solution had a concentration of 100 mM pyruvate and pH of 7.5. The lactate and pyruvate signals in the brainstem were normalized with respect to the relative signals in normal appearing brain in the supratentorial region. In addition, the ratio of lactate to pyruvate (Lac/Pyr) and lactate to total carbon (Lac/tC) were calculated. T2-weighted fast spin-echo (FSE) images (TE/TR=60/4000ms) and Gadolinium(Gd)-enhanced (0.2mmol/kg Gd-DTPA) T1-weighted spin-echo (SE) images (TE/TR=10/700ms) were acquired in axial plane. The brains of rats were resected and the following immunohistochemistry stains were performed: hematoxylin&eosin (H&E), lactate dehydrogenase-A (LDHA), carbonic anhydrase-IX (CA-IX) and isocitrate dehydrogenase-1 (IDH1).

Results and Discussion

HP 13C MRSI indicated that the SF8628 and SF7761 DIPG xenografts had distinct metabolic characteristics: The axial T2 FSE images showed regions of T2 hyperintensity for both tumors (Fig1). SF8628 had well-defined tumor margins (Fig1a), while SF7761 showed T2 lesion that spread throughout the entire brainstem (Fig1e). There was no contrast enhancement on T1 post-Gd images for either case (Fig1b, 1f). SF8628 produced elevated lactate signal (Fig1c) and high Lac/Pyr (Fig1d) in the region of T2 hyperintensity. In contrast, SF7761 did not show elevated levels of lactate (Fig1d, 1h). The mean Lac/Pyr, Lac/tC and normalized lactate (nLac) in SF8628 tumors were significantly higher than those in the contra-lateral brain tissue and in SF7761 tumors (Fig2). The level of lactate signal in SF7761 tumors was similar to that of contra-lateral normal brain tissue measured from rats with SF8628 tumors. The levels of normalized pyruvate (nPyr) were similar between SF8628 tumors, contra-lateral brain tissue and SF7761 tumors, highlighting that the amount of pyruvate delivered and taken up by these tissues were comparable between these different tissues.

Elevated lactate signal in SF8628 tumors was associated with increased staining for LDHA and CA-IX: Immunohistochemical analysis was used to further explain the findings from HP 13C MR imaging. This indicated that in comparison to SF7761, the SF8628 tumors had increased immunostaining for LDHA (Fig3), which is the enzyme that catalyzes pyruvate to lactate conversion. The SF8628 tumors also produced a high level of immunostaining for CA-IX (Fig3), which is an indicator of hypoxia-inducible factor 1α (HIF-1α) activity that in turn transcriptionally activates LDH expression. The differences in levels of LDHA and CA-IX staining are consistent with the levels of hyperpolarized 13C lactate signal for these two orthotopic DIPG tumors (Fig1, 2). A recent study reported that contrary to primary GBM tumors, IDH1 mutant oligoastrocytoma and oligodendroglioma did not produce significant amount of lactate from hyperpolarized pyruvate7. In our study, there was no difference in the level of IDH1 staining between the two types of tumor (Fig3).

Conclusions

Hyperpolarized 13C metabolic imaging is able to detect changes in metabolism that are associated with distinct molecular characteristics in two types of orthotopic human DIPG xenografts. The elevated lactate signal observed in DIPGs was associated with increased levels of LHDA and HIF-1α activity. The results of the study indicate that this novel neuroimaging method can be used to non-invasively characterize DIPGs and may be applied to assist in stratifying patients to receive the most appropriate treatments, and to assess treatment response and/or disease progression.

Acknowledgements

The first author was supported by a NCI training grant in translational brain tumor research (T32 CA151022) and Kure It Grant for Underfunded Cancer Research. The support for the research studies came from NIH grants R01EB007588, P41EB13598, R00 EB012064, R21CA170148 and R01CA154915.

References

1. Wu G, Broniscer A, McEachron TA et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet. Nat Genet. 2012;44(3):251-3.

2. Schwartzentruber J, Korshunov A, Liu XY, et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature. Nature. 2012;482(7384):226-31.

3. Grasso CS, Tang Y, Truffaux N, et al. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med. 2015;21(7):827.

4. Mueller S, Hashizume R, Yang X, et al. Targeting Wee1 for the treatment of pediatric high-grade gliomas. Nuero Oncol. 2014;16(3):352-60.

5. Ardenkjaer-Larsen JH, Fridlund B, Gram A, et al. Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR. Proc Natl Acad Sci USA. 2003;100(18):10158-63.

6. Park I, Hu S, Bok R, et al. Evaluation of heterogeneous metabolic profile in an orthotopic human glioblastoma xenograft model using compressed sensing hyperpolarized 3D 13C magnetic resonance spectroscopic imaging. Magn Reson Med. 2012;70(1):33-9.

7. Chaumeil M, Radoul M, Eriksson P, et al. The tumor exception that proves the rule: Hyperpolarized 13C MRS cannot be used to detect the presence of mutant IDH1 glioma or their responses to Temozolomide therapy. Proc. 23rd Intl. Soc. Mag. Reson. Med. 2015;322.

Figures

Figure 1. Examples of rats from two DIPG models. Both animals exhibited T2 hyperintensity with an absence of contrast enhancement, which are characteristics of DIPG. Hyperpolarized 13C MRSI data revealed that SF8628 produced a high level of lactate while SF7761 had negligible lactate.

Figure 2. Comparison of 13C metabolic parameters. The SF8628 tumors showed significantly higher levels of mean ratio of lactate to pyruvate (Lac/Pyr), lactate to total carbon (Lac/tC) and normalized lactate (nLac) compared to contra-lateral brain tissue and SF7761 tumors. The signal from normalized pyruvate (nPyr) was similar in all tissues.

Figure 3. H&E, LDHA, CA-IX and IDH1 stains of two orthotopic DIPG resected after 13C imaging. A clear increased level of LDHA and CA-IX staining can be seen in SF8628 compared to SF7761, which is consistent with the distinct level of lactate observed in these tumors from hyperpolarized 13C imaging.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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