Non-Invasive Glutamine PET Reflects Pharmacological Inhibition of BRAFV600E In Vivo

Abstract

Purpose: This study aimed to study whether cancer cells possess distinguishing metabolic features compared with surrounding normal cells, such as increased glutamine uptake. Given this, quantitative measures of glutamine uptake may reflect critical processes in oncology. Approximately, 10 % of patients with colorectal cancer (CRC) express BRAF ^V600E , which may be actionable with selective BRAF inhibitors or in combination with inhibitors of complementary signaling axes. Non-invasive and quantitative predictive measures of response to these targeted therapies remain poorly developed in this setting. The primary objective of this study was to explore 4-[¹⁸F]fluoroglutamine (4-[¹⁸F]F-GLN) positron emission tomography (PET) to predict response to BRAF^V600E-targeted therapy in preclinical models of colon cancer.

Procedures: Tumor microarrays from patients with primary human colon cancers (n = 115) and CRC liver metastases (n = 111) were used to evaluate the prevalence of ASCT2, the primary glutamine transporter in oncology, by immunohistochemistry. Subsequently, 4-[¹⁸F]F-GLN PET was evaluated in mouse models of human BRAF ^V600E -expressing and BRAF wild-type CRC.

Results: Approximately 70 % of primary colon cancers and 53 % of metastases exhibited positive ASCT2 immunoreactivity, suggesting that [¹⁸F]4-F-GLN PET could be applicable to a majority of patients with colon cancer. ASCT2 expression was not associated selectively with the expression of mutant BRAF. Decreased 4-[¹⁸F]F-GLN predicted pharmacological response to single-agent BRAF and combination BRAF and PI3K/mTOR inhibition in BRAF ^V600E -mutant Colo-205 tumors. In contrast, a similar decrease was not observed in BRAF wild-type HCT-116 tumors, a setting where BRAF^V600E-targeted therapies are ineffective.

Conclusions: 4-[¹⁸F]F-GLN PET selectively reflected pharmacodynamic response to BRAF inhibition when compared with 2-deoxy-2[¹⁸F]fluoro-D-glucose PET, which was decreased non-specifically for all treated cohorts, regardless of downstream pathway inhibition. These findings illustrate the utility of non-invasive PET imaging measures of glutamine uptake to selectively predict response to BRAF-targeted therapy in colon cancer and may suggest further opportunities to inform colon cancer clinical trials using targeted therapies against MAPK activation.

Keywords: BRAF; Cancer; Colon; Glutamine; Pet.

Fig. 1.

The glutamine transporter ASCT2 is highly expressed in primary colon tumors and colon cancer liver metastases. Representative images of IHC staining for ASCT2 protein in a tissue microarray (TMA) constructed from archival colon tissue sections collected from 115 colon cancer patients representing primary and 111 metastatic disease sections in the liver. a Representative micrograph of sections of normal colon tissue from a patient with colon cancer which stained negative for ASCT2. Representative micrographs are presented of b primary colon cancer sections and c matched liver metastases from a patient with colon cancer.

Fig. 2.

4-[¹⁸F]F-Gln uptake reflects molecular response to mutant BRAF therapy in vivo. Representative transverse and coronal a 4-[¹⁸F]F-Gln, and b [¹⁸F]FDG PET images of COLO-205 xenograft tumor-bearing vehicle or BEZ-235/PLX-4720-treated mice; tumors are denoted by white arrows. PET quantification of tissue %ID/g revealed a significant difference between vehicle and PLX-4720-single-agent and BEZ-235/PLX-4720 combination-treated xenografts only for c 4-[¹⁸F]F-Gln while d [¹⁸F]FDG PET was significantly reduced non-specifically in all the treated cohorts. PLX-4720 exposure led to decreased pERK levels in Colo- 205 xenografts while BEZ-235 exposure led to decreased pAKT. e Similarly, combination treatment led to decreases in both pERK and pAKT levels. d Changes in C0L0–205 tumor volume by the tenth day of treatment (N ≥ 8 for all cohorts), shown as percent change from day one baseline, revealed a significant reduction in size from vehicle-treated mice for PLX-470 and BEZ- 235/PLX-4720-treated tumors (p = 0.0001 and p = 0.0005, respectively).

Fig. 3.

4-[¹⁸F]F-Gln uptake is not affected by BRAF-targeted therapy in wild-type BRAF, HCT-116 colon cancer xenografts. Representative transverse and coronal a 4-[¹⁸F]F-Gln and b [¹⁸F]FDG PET images of HCT-116 xenograft tumor-bearing vehicle or BEZ-235/PLX-4720-treated mice; tumors are denoted by white arrows. PET quantification of tissue %ID/g revealed no significant difference between vehicle and any of the treated cohorts for c 4-[¹⁸F]F-Gln while d [¹⁸F]FDG PET was significantly reduced non-selectively in all the treated cohorts. e As expected, PLX-4720 exposure did not result in any difference in pERK levels in HCT-116 xenografts while single-agent BEZ-235 and BEZ-235/PLX-4720 combination treatment led to decreased pAKT. f Changes in HCT-116 tumor volume by the tenth day of treatment (N ≥ 8 for all cohorts), shown as percent change from day 1 baseline, revealed no significant reduction in size from vehicle-treated mice for any of the treated tumors.