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Supplementary Figures - PDF file 259K, Figure S1. Comparison of 4T1-WT, NC and KD9 cell lines. (A) Oxylite Oxygen Consumption Rate; (B) Reactive oxygen species (ROS); (C) mitochondria level; (D) Primary tumor (~ 100 mm3) lactate concentration. Figure S2. Histology of small (100 mm3) primary NC and KD9 tumors (A). Representative small tumors show similar features: a few small areas of necrosis (H&E) and reduced blood flow (Hoechst). Scale bar represents 5 mm. Cell Mitochondria (B). NC and KD9 cells were grown on the cover slips and stained for mitochondria by Mitotracker red CMXRos. Fig. S3. Primary orthotopic tumor growth and development of lung metastases. Three weeks after tumor inoculation, the mice were sacrificed and the primary tumor volume and weight, as well as lung weight (reflecting the presence of lung metastases) were measured in NC (n=6) and KD9 (n=6) tumor bearing animals Fig. S4. Western blot analysis of LDH-A expression: The absence of puromycin in the culture media results in a reappearance of LDH-A expression in KD9 cells growing in vitro (columns 3 and 5)

Supplementary Methods - PDF file 158K, Supplemental methods contain both techniques and further data strengthening the manuscript. These include our Analysis of Breast Cancer Microarray Datasets, Generation of LDH-A knockdown and control cell lines and In vitro assays, and in vivo methods (MR measurements)

Purpose: To investigate the relationship between lactate dehydrogenase A (LDH-A) expression, lactate concentration, cell metabolism, and metastases in murine 4T1 breast tumors.Experimental Design: Inhibition of LDH-A expression and protein levels were achieved in a metastatic breast cancer cell line (4T1) using short hairpin RNA (shRNA) technology. The relationship between tumor LDH-A protein levels and lactate concentration (measured by magnetic resonance spectroscopic imaging, MRSI) and metastases was assessed.Results: LDH-A knockdown cells (KD9) showed a significant reduction in LDH-A protein and LDH activity, less acid production, decreased transwell migration and invasion, lower proliferation, reduced glucose consumption and glycolysis, and increase in oxygen consumption, reactive oxygen species (ROS), and cellular ATP levels, compared with control (NC) cells cultured in 25 mmol/L glucose. In vivo studies showed lower lactate levels in KD9, KD5, and KD317 tumors than in NC or 4T1 wild-type tumors (P < 0.01), and a linear relationship between tumor LDH-A protein expression and lactate concentration. Metastases were delayed and primary tumor growth rate decreased.Conclusions: We show for the first time that LDH-A knockdown inhibited the formation of metastases, and was accompanied by in vivo changes in tumor cell metabolism. Lactate MRSI can be used as a surrogate to monitor targeted inhibition of LDH-A in a preclinical setting and provides a noninvasive imaging strategy to monitor LDH-A–targeted therapy. This imaging strategy can be translated to the clinic to identify and monitor patients who are at high risk of developing metastatic disease. Clin Cancer Res; 19(18); 5158–69. ©2013 AACR.

The 2021 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) and recent smaller annual updates have shown that alterations in tumor genetics are essential to determining tumor diagnosis, biological activity, and potential treatment options. This review summarizes the most important mutations and oncometabolites, with a focus on the central role played by 2-hydroxyglutarate in isocitrate dehydrogenase mutant tumors, as well as their corresponding imaging counterparts using standard and advanced imaging techniques.

INTRODUCTION Treatment of brainstem tumors is often initiated without a tissue diagnosis due to the risk of biopsy. A subset of brainstem gliomas harbor an isocitrate dehydrogenase 1/2 (IDH) mutation, which predicts response to alkylator chemotherapy and IDH inhibitors; non-invasive diagnostic tests are needed to identify these mutations. METHODS We identified a cohort of patients with IDH-mutant brainstem gliomas through chart review of patients who underwent magnetic resonance spectroscopy (MRS) and/or brain biopsy. IDH mutation was established by biopsy, presence of a 2-hydroxyglutarate (2HG) peak on MRS, or identification on cerebrospinal fluid (CSF) sequencing of cell-free DNA (cfDNA). RESULTS We identified 15 patients with IDH-mutant brainstem gliomas, age range 5-48, 47% women. 15/15 were involving/abutting the brachium pontis, and 10/15 were non-enhancing at diagnosis. 13/15 patients were identified by biopsy, 1/15 by MRS and CSF, and 1/15 by MRS only. 10/13 patients with available data had a non-IDH1 R132H mutation in IDH1/2. 7/7 patients with MRS prior to radiation had a 2HG peak (MRS was concordant with tissue in all cases in which biopsy was obtained). 4/6 patients with MRS post-radiation retained a 2HG peak. 4 IDH-mutant tumors had sequencing of cfDNA and an IDH mutation was found in 2/4. Response data was available in 13/15 patients; all received radiation, 10 with concurrent temozolomide: best response was partial response in 8 and stable disease in 5. 4 patients received an IDH inhibitor: 2 patients after progression, and 2 as maintenance after radiation/temozolomide, achieving tumor control in all. For this cohort, median PFS was 71.4 months and median OS has not been reached. CONCLUSION IDH-mutant brainstem gliomas have a characteristic appearance, a high response rate to treatment, and a better overall prognosis than other brainstem tumors. MRS and CSF cfDNA sequencing allow for non-invasive diagnosis of IDH mutations in these patients.

To perform a survey among all European Society of Breast Imaging (EUSOBI) radiologist members to gather representative data regarding the clinical use of breast DWI.An online questionnaire was developed by two board-certified radiologists, reviewed by the EUSOBI board and committees, and finally distributed among EUSOBI active and associated (not based in Europe) radiologist members. The questionnaire included 20 questions pertaining to technical preferences (acquisition time, magnet strength, breast coils, number of b values), clinical indications, imaging evaluation, and reporting. Data were analyzed using descriptive statistics, the Chi-square test of independence, and Fisher's exact test.Of 1411 EUSOBI radiologist members, 275/1411 (19.5%) responded. Most (222/275, 81%) reported using DWI as part of their routine protocol. Common indications for DWI include lesion characterization (using an ADC threshold of 1.2-1.3 × 10The utilization of DWI in clinical practice among EUSOBI radiologists who responded to the survey is generally in line with international recommendations, with the main application being the differentiation of benign and malignant enhancing lesions, treatment response assessment, and prediction of response to chemotherapy. Report integration of qualitative and quantitative DWI data is not uniform.• Clinical performance of breast DWI is in good agreement with the current recommendations of the EUSOBI International Breast DWI working group. • Breast DWI applications in clinical practice include the differentiation of benign and malignant enhancing, treatment response assessment, and prediction of response to chemotherapy. • Report integration of DWI results is not uniform.

To investigate the diagnostic value of multiparametric MRI (mpMRI) including dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion-weighted imaging (DWI) in non-mass enhancing breast tumors.Patients who underwent mpMRI, who were diagnosed with a suspicious non-mass enhancement (NME) on DCE-MRI (BI-RADS 4/5), and who subsequently underwent image-guided biopsy were retrospectively included. Two radiologists independently evaluated all NMEs, on both DCE-MR images and high-b-value DW images. Different mpMRI reading approaches were evaluated: 1) with a fixed apparent diffusion coefficient (ADC) threshold (1.3 malignant, ≥1.3 benign) based on the recommendation by the European Society of Breast Imaging (EUSOBI); 2) with a fixed ADC threshold (1.5 malignant, ≥1.5 benign) based on recently published trial data; 3) with an ADC threshold adapted to the assigned BI-RADS classification using a previously published reading method; and 4) with individually determined best thresholds for each reader.The final study sample consisted of 66 lesions in 66 patients. DCE-MRI alone had the highest sensitivity for breast cancer detection (94.8-100 %), outperforming all mpMRI reading approaches (R1 74.4-87.1 %, R2 71.7-94.8 %) and DWI alone (R1 74.4 %, R2 79.4 %). The adapted approach achieved the best specificity for both readers (85.1 %), resulting in the best diagnostic accuracy for R1 (86.5 %) but a moderate diagnostic accuracy for R2 (77.2 %).mpMRI has limited added diagnostic value to DCE-MRI in the assessment of NME.

1H magnetic resonance spectroscopic imaging (MRSI) can improve the accuracy of target delineation for gliomas, but it lacks the anatomic resolution needed for image fusion. This paper presents a simple protocol for fusing simulation computer tomography (CT) and MRSI images for glioma intensity-modulated radiotherapy (IMRT), including a retrospective study of 12 patients. Each patient first underwent whole-brain axial fluid-attenuated-inversion-recovery (FLAIR) MRI (3 mm slice thickness, no spacing), followed by three-dimensional (3D) MRSI measurements (TE/TR: 144/1000 ms) of a user-specified volume encompassing the extent of the tumor. The nominal voxel size of MRSI ranged from 8 x 8 x 10 mm3 to 12 x 12 x 10 mm3. A system was developed to grade the tumor using the choline-to-creatine (Cho/Cr) ratios from each MRSI voxel. The merged MRSI images were then generated by replacing the Cho/Cr value of each MRSI voxel with intensities according to the Cho/Cr grades, and resampling the poorer-resolution Cho/Cr map into the higher-resolution FLAIR image space. The FUNCTOOL processing software was also used to create the screen-dumped MRSI images in which these data were overlaid with each FLAIR MRI image. The screen-dumped MRSI images were manually translated and fused with the FLAIR MRI images. Since the merged MRSI images were intrinsically fused with the FLAIR MRI images, they were also registered with the screen-dumped MRSI images. The position of the MRSI volume on the merged MRSI images was compared with that of the screen-dumped MRSI images and was shifted until agreement was within a predetermined tolerance. Three clinical target volumes (CTVs) were then contoured on the FLAIR MRI images corresponding to the Cho/Cr grades. Finally, the FLAIR MRI images were fused with the simulation CT images using a mutual-information algorithm, yielding an IMRT plan that simultaneously delivers three different dose levels to the three CTVs. The image-fusion protocol was tested on 12 (six high-grade and six low-grade) glioma patients. The average agreement of the MRSI volume position on the screen-dumped MRSI images and the merged MRSI images was 0.29 mm with a standard deviation of 0.07 mm. Of all the voxels with Cho/Cr grade one or above, the distribution of Cho/Cr grade was found to correlate with the glioma grade from pathologic finding and is consistent with literature results indicating Cho/Cr elevation as a marker for malignancy. In conclusion, an image-fusion protocol was developed that successfully incorporates MRSI information into the IMRT treatment plan for glioma.

SUMMARY: Two pediatric patients with diffuse pontine tumors underwent MR spectroscopic imaging pre- and postradiation. Choline/creatine (Cho/Cr) and Cho/N-acetylaspartate (NAA) ratios were elevated before treatment, with no MR imaging contrast enhancement. These ratios were further elevated at 2 posttreatment follow-up studies, despite signs of excellent clinical improvement at initial follow-up. This study suggests that MR spectroscopic imaging is more specific in assessing the aggressiveness of diffuse pontine tumors than conventional MR imaging and can serve as a valuable tool in early prognostication.