Defining the Magnetic Resonance Features of Renal Lesions and Their Response to Everolimus in a Transgenic Mouse Model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an inherited genetic disorder characterized by mutations in TSC1 or TSC2 class of tumor suppressers which impact several organs including the kidney. The renal manifestations are usually in the form of angiomyolipoma (AML, in 80% of the cases) and cystadenomas. mTOR inhibitors such as rapamycin and everolimus have shown efficacy in reducing the renal tumor burden. Early treatment prevents the progression of AML; however, the tumors regrow upon cessation of therapy implying a lifelong need for monitoring and management of this morbid disease. There is a critical need for development of imaging strategies to monitor response to therapy and progression of disease which will also facilitate development of newer targeted therapy. In this study we evaluated the potential of multiparametric 1H magnetic resonance imaging (mpMRI) to monitor tumor response to therapy in a preclinical model of TSC, the transgenic mouse A/J Tsc2+/- . We found 2-dimensional T2-weighted sequence with 0.5 mm slice thickness to be optimal for detecting renal lesions as small as 0.016 mm3. Baseline characterization of lesions with MRI to assess physiological parameters such as cellularity and perfusion is critical for distinguishing between cystic and solid lesions. Everolimus treatment for three weeks maintained tumor growth at 36% from baseline, while control tumors displayed steady growth and were 70% larger than baseline at the end of therapy. Apparent diffusion coefficient, T1 values and normalized T2 intensity changes were also indictive of response to treatment. Our results indicate that standardization and implementation of improved MR imaging protocols will significantly enhance the utility of mpMRI in determining the severity and composition of renal lesions for better treatment planning.

False positive PSMA PET for tumor remnants in the irradiated prostate and other interpretation pitfalls in a prospective multi-center trial.

PURPOSE: Readers need to be informed about potential pitfalls of [68Ga]Ga-PSMA-11 PET interpretation. METHODS: Here we report [68Ga]Ga-PSMA-11 PET findings discordant with the histopathology/composite reference standard in a recently published prospective trial on 635 patients with biochemically recurrent prostate cancer. RESULTS: Consensus reads were false positive in 20 regions of 17/217 (8%) patients with lesion validation. Majority of the false positive interpretations (13 of 20, 65%) occurred in the context of suspected prostate (bed) relapse (T) after radiotherapy (n = 11); other false positive findings were noted for prostate bed post prostatectomy (T, n = 2), pelvic nodes (N, n = 2), or extra pelvic lesions (M, n = 5). Major sources of false positive findings were PSMA-expressing residual adenocarcinoma with marked post-radiotherapy treatment effect. False negative interpretation occurred in 8 regions of 6/79 (8%) patients with histopathology validation, including prostate (bed) (n = 5), pelvic nodes (n = 1), and extra pelvic lesions (n = 2). Lesions were missed mostly due to small metastases or adjacent bladder/urine uptake. CONCLUSION: [68Ga]Ga-PSMA-11 PET at biochemical recurrence resulted in less than 10% false positive interpretations. Post-radiotherapy prostate uptake was a major source of [68Ga]Ga-PSMA-11 PET false positivity. In few cases, PET correctly detects residual PSMA expression post-radiotherapy, originating however from treated, benign tissue or potentially indolent tumor remnants. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifiers: NCT02940262 and NCT03353740.

Glutamate Is a Noninvasive Metabolic Biomarker of IDH1-Mutant Glioma Response to Temozolomide Treatment.

Although lower grade gliomas are driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene and are less aggressive than primary glioblastoma, they nonetheless generally recur. IDH1-mutant patients are increasingly being treated with temozolomide, but early detection of response remains a challenge and there is a need for complementary imaging methods to assess response to therapy prior to tumor shrinkage. The goal of this study was to determine the value of magnetic resonance spectroscopy (MRS)-based metabolic changes for detection of response to temozolomide in both genetically engineered and patient-derived mutant IDH1 models. Using 1H MRS in combination with chemometrics identified several metabolic alterations in temozolomide-treated cells, including a significant increase in steady-state glutamate levels. This was confirmed in vivo, where the observed 1H MRS increase in glutamate/glutamine occurred prior to tumor shrinkage. Cells labeled with [1-13C]glucose and [3-13C]glutamine, the principal sources of cellular glutamate, showed that flux to glutamate both from glucose via the tricarboxylic acid cycle and from glutamine were increased following temozolomide treatment. In line with these results, hyperpolarized [5-13C]glutamate produced from [2-13C]pyruvate and hyperpolarized [1-13C]glutamate produced from [1-13C]α-ketoglutarate were significantly higher in temozolomide-treated cells compared with controls. Collectively, our findings identify 1H MRS-detectable elevation of glutamate and hyperpolarized 13C MRS-detectable glutamate production from either pyruvate or α-ketoglutarate as potential translatable metabolic biomarkers of response to temozolomide treatment in mutant IDH1 glioma. SIGNIFICANCE: These findings show that glutamate can be used as a noninvasive, imageable metabolic marker for early assessment of tumor response to temozolomide, with the potential to improve treatment strategies for mutant IDH1 patients.

Simultaneous Metabolic and Perfusion Imaging Using Hyperpolarized 13C MRI Can Evaluate Early and Dose-Dependent Response to Radiation Therapy in a Prostate Cancer Mouse Model.

PURPOSE: To investigate use of a novel imaging approach, hyperpolarized (HP) 13C magnetic resonance imaging (MRI) for simultaneous metabolism and perfusion assessment, to evaluate early and dose-dependent response to radiation therapy (RT) in a prostate cancer mouse model. METHODS AND MATERIALS: Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (n = 18) underwent single-fraction RT (4-14 Gy steep dose across the tumor) and were imaged serially at pre-RT baseline and 1, 4, and 7 days after RT using HP 13C MRI with combined [1-13C]pyruvate (metabolic active agent) and [13C]urea (perfusion agent), coupled with conventional multiparametric 1H MRI including T2-weighted, dynamic contrast-enhanced, and diffusion-weighted imaging. Tumor tissues were collected 4 and 7 days after RT for biological correlative studies. RESULTS: We found a significant decrease in HP pyruvate-to-lactate conversion in tumors responding to RT, with concomitant significant increases in HP pyruvate-to-alanine conversion and HP urea signal; the opposite changes were observed in tumors resistant to RT. Moreover, HP lactate change was dependent on radiation dose; tumor regions treated with higher radiation doses (10-14 Gy) exhibited a greater decrease in HP lactate signal than low-dose regions (4-7 Gy) as early as 1 day post-RT, consistent with lactate dehydrogenase enzyme activity and expression data. We also found that HP [13C]urea MRI provided assessments of tumor perfusion similar to those provided by 1H dynamic contrast-enhanced MRI in this animal model. However, apparent diffusion coefficien , a conventional 1H MRI functional biomarker, did not exhibit statistically significant changes within 7 days after RT. CONCLUSION: These results demonstrate the ability of HP 13C MRI to monitor radiation-induced physiologic changes in a timely and dose-dependent manner, providing the basic science premise for further clinical investigation and translation.

Elevated Tumor Lactate and Efflux in High-grade Prostate Cancer demonstrated by Hyperpolarized 13C Magnetic Resonance Spectroscopy of Prostate Tissue Slice Cultures.

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) 13C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-13C]pyruvate to [1-13C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP 13C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP 13C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. 31P spectra demonstrated good viability and dynamic HP 13C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-13C]pyruvate MR.

Hyperpolarized in vivo pH imaging reveals grade-dependent acidification in prostate cancer.

There is an unmet clinical need for new and robust imaging biomarkers to distinguish indolent from aggressive prostate cancer. Hallmarks of aggressive tumors such as a decrease in extracellular pH (pHe) can potentially be used to identify aggressive phenotypes. In this study, we employ an optimized, high signal-to-noise ratio hyperpolarized (HP) 13C pHe imaging method to discriminate between indolent and aggressive disease in a murine model of prostate cancer. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice underwent a multiparametric MR imaging exam, including HP [13C] bicarbonate MRI for pHe, with 1H apparent diffusion coefficient (ADC) mapping and HP [1-13C] pyruvate MRI to study lactate metabolism. Tumor tissue was excised for histological staining and qRT-PCR to quantify mRNA expression for relevant glycolytic enzymes and transporters. We observed good separation in pHe between low- and high-grade tumor regions, with high-grade tumors demonstrating a lower pHe. The pHe also correlated strongly with monocarboxylate transporter Mct4 gene expression across all tumors, suggesting that lactate export via MCT4 is associated with acidification in this model. Our results implicate extracellular acidification as an indicator of indolent-to-aggressive transition in prostate cancer and suggest feasibility of HP pHe imaging to detect high-grade, clinically significant disease in men as part of a multiparametric MRI examination.

PI3K/mTOR inhibition of IDH1 mutant glioma leads to reduced 2HG production that is associated with increased survival.

70-90% of low-grade gliomas and secondary glioblastomas are characterized by mutations in isocitrate dehydrogenase 1 (IDHmut). IDHmut produces the oncometabolite 2-hydroxyglutarate (2HG), which drives tumorigenesis in these tumors. The phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway represents an attractive therapeutic target for IDHmut gliomas, but noninvasive indicators of drug target modulation are lacking. The goal of this study was therefore to identify magnetic resonance spectroscopy (MRS)-detectable metabolic biomarkers associated with IDHmut glioma response to the dual PI3K/(mTOR) inhibitor XL765. 1H-MRS of two cell lines genetically modified to express IDHmut showed that XL765 induced a significant reduction in several intracellular metabolites including 2HG. Importantly, examination of an orthotopic IDHmut tumor model showed that enhanced animal survival following XL765 treatment was associated with a significant in vivo 1H-MRS detectable reduction in 2HG but not with significant inhibition in tumor growth. Further validation is required, but our results indicate that 2HG could serve as a potential noninvasive MRS-detectable metabolic biomarker of IDHmut glioma response to PI3K/mTOR inhibition.

Assessing high-intensity focused ultrasound treatment of prostate cancer with hyperpolarized 13 C dual-agent imaging of metabolism and perfusion.

The goal of the study was to establish early hyperpolarized (HP) 13 C MRI metabolic and perfusion changes that predict effective high-intensity focused ultrasound (HIFU) ablation and lead to improved adjuvant treatment of partially treated regions. To accomplish this a combined HP dual-agent (13 C pyruvate and 13 C urea) 13 C MRI/multiparametric 1 H MRI approach was used to measure prostate cancer metabolism and perfusion 3-4 h, 1 d, and 5 d after exposure to ablative and sub-lethal doses of HIFU within adenocarcinoma of mouse prostate tumors using a focused ultrasound applicator designed for murine studies. Pathologic and immunohistochemical analysis of the ablated tumor demonstrated fragmented, non-viable cells and vasculature consistent with coagulative necrosis, and a mixture of destroyed tissue and highly proliferative, poorly differentiated tumor cells in tumor tissues exposed to sub-lethal heat doses in the ablative margin. In ablated regions, the intensity of HP 13 C lactate or HP 13 C urea and dynamic contrast-enhanced (DCE) MRI area under the curve images were reduced to the level of background noise by 3-4 h after treatment with no recovery by the 5 d time point in either case. In the tissues that received sub-lethal heat dose, there was a significant 60% ± 12.4% drop in HP 13 C lactate production and a significant 30 ± 13.7% drop in urea perfusion 3-4 h after treatment, followed by recovery to baseline by 5 d after treatment. DCE MRI Ktrans showed a similar trend to HP 13 C urea, demonstrating a complete loss of perfusion with no recovery in the ablated region, while having a 40%-50% decrease 3-4 h after treatment followed by recovery to baseline values by 5 d in the margin region. The utility of the HP 13 C MR measures of perfusion and metabolism in optimizing focal HIFU, either alone or in combination with adjuvant therapy, deserves further testing in future studies.

Dynamic UltraFast 2D EXchange SpectroscopY (UF-EXSY) of hyperpolarized substrates.

In this work, we present a new ultrafast method for acquiring dynamic 2D EXchange SpectroscopY (EXSY) within a single acquisition. This technique reconstructs two-dimensional EXSY spectra from one-dimensional spectra based on the phase accrual during echo times. The Ultrafast-EXSY acquisition overcomes long acquisition times typically needed to acquire 2D NMR data by utilizing sparsity and phase dependence to dramatically undersample in the indirect time dimension. This allows for the acquisition of the 2D spectrum within a single shot. We have validated this method in simulations and hyperpolarized enzyme assay experiments separating the dehydration of pyruvate and lactate-to-pyruvate conversion. In a renal cell carcinoma cell (RCC) line, bidirectional exchange was observed. This new technique revealed decreased conversion of lactate-to-pyruvate with high expression of monocarboxylate transporter 4 (MCT4), known to correlate with aggressive cancer phenotypes. We also showed feasibility of this technique in vivo in a RCC model where bidirectional exchange was observed for pyruvate-lactate, pyruvate-alanine, and pyruvate-hydrate and were resolved in time. Broadly, the technique is well suited to investigate the dynamics of multiple exchange pathways and applicable to hyperpolarized substrates where chemical exchange has shown great promise across a range of disciplines.

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy.

The impact of high-resolution magic angle spinning (HR-MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n = 20) or radical prostatectomy surgery (n = 20), snap-frozen on dry ice, and stored at -80°C until use. Twenty-one samples (n = 11 biopsy, n = 10 surgical) underwent HR-MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n = 9 biopsy, n = 10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14-µm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (H&E) and used to target areas of predominantly epithelial tissue on the remaining slides for mRNA integrity and cDNA microarray analysis. Histopathologic integrity was graded from 1 (best) to 5 (worst) by two 'blinded' pathologists. Histopathologic integrity scores were not significantly different for post-surgical tissues (HR-MAS vs controls); however, one pathologist's scores were significantly lower for biopsy tissues following HR-MAS while the other pathologist's scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR-MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over- or under-expressed genes, including 14 housekeeping genes, between HR-MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR-MAS analysis; however, biopsy tissues are more fragile than surgical tissues.