In vivo metabolic signature of Reed Syndrome Ruth T. Casey1, 2, Mary McLean3, Benjamin G Challis2, Terri P McVeigh4, Anne Y Warren5, Lee Mendil3, Richard Houghton3, Stefano De Sanctis5, Vasilis Kosmoliaptsis 6, Richard N Sandford1, Ferdia A Gallagher3, 7*, Eamonn R Maher1* 1. Department of Medical Genetics, University of Cambridge and NIHR Cambridge Biomedical Research Centre and Cancer Research UK Cambridge Centre, CB2 OQQ, United Kingdom. 2. Department of Endocrinology, Cambridge University NHS Foundation Trust, Cambridge, CB2 OQQ, United Kingdom. 3. Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK 4. Cancer Genetics Unit, Royal Marsden NHS Foundation Trust, London, SW3 6JJ 5. Department of Histopathology Cambridge University NHS Foundation Trust and Cancer Research UK Cambridge Centre Cambridge, CB2 0QQ, United Kingdom. 6. Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge University Hospitals NHS Foundation Trust, Cambridge, CB2 OQQ, United Kingdom. 7. Department of Radiology, Cambridge University NHS Foundation Trust, CB2 OQQ, United Kingdom. *= joint last author Corresponding author: Dr Ruth Casey, Department of Medical Genetics, University of Cambridge and NIHR Cambridge Biomedical Research Centre and Cancer Research UK Cambridge Centre, CB2 OQQ, United Kingdom. Email: rc674@medschl.cam.ac.uk The authours have no conflict of interest to declare Disclosures BGC is an employee of AstraZeneca. Word count: 2700, Figures: 3, Tables 1, References 18 Translational Relevance In this study we describe the utility of MR spectroscopy to detect fumarate in vivo for the first time. This has translational utility in the early detection of a hereditary metabolic neoplastic syndrome as demonstrated with Reed syndrome here. Lessons learned from this study could be applied to other metabolically-driven tumours. Summary Purpose: Inherited pathogenic variants in genes encoding the metabolic enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) predispose to tumour development through accumulation of oncometabolites (succinate and fumarate respectively) (1). Non-invasive in vivo detection of tumour succinate by proton magnetic resonance spectroscopy (1H-MRS) has been reported in SDH-deficient tumours but the potential utility of this approach in the management of patients with hereditary leiomyomatosis and renal cell cancer syndrome or Reed syndrome is unknown. Experimental design: Magnetic resonance spectroscopy (1H-MRS) was performed on three cases and correlated with germline genetic results and tumour immunohistochemistry when available. Results:  Here, we have demonstrated a proof-of-principle that 1H-MRS can provide a non-invasive diagnosis of hereditary leiomyomatosis and renal cell cancer syndrome or Reed syndrome through detection of fumarate accumulation  in vivo. Conclusion: This study demonstrates that in vivo detection of fumarate could be employed as a functional biomarker Introduction In the past two decades, loss of function mutations in genes that encode components of the citric acid cycle enzymes SDH and FH have been demonstrated to predispose to a range of benign and malignant tumours (2)(3)(4)(5)(6). The tumour risk for patients with SDHX mutations (SDHB, SDHD, SDHC, SDHA, SDHAF2) varies according to the specific gene involved but the most frequent tumours overall are phaeochromocytomas, paragangliomas, head and neck paragangliomas, renal cell carcinomas (RCC), gastrointestinal stromal tumours and rarely, pituitary tumours (7). The clinical phenotype associated with FH mutations is hereditary leiomyomas and renal cell carcinoma syndrome (HLRCC) or Reed syndrome and comprises cutaneous and uterine leiomyomas, type 2 papillary RCCs (5)(8) and occasionally phaeochromocytomas/paragangliomas (6). Tumours from patients with inherited FH and SDHX mutations demonstrate biallelic inactivation of the relevant gene and this results in loss of SDH or FH activity, which in turn causes pathological accumulation of the metabolites succinate and fumarate respectively (1)(9). Accumulation of succinate and fumarate causes competitive inhibition of 2-oxoglutarate dependent enzymes (e.g. prolyl hydroxylase and DNA and histone demethylase enzymes) that drive tumourigenesis through epigenetic and gene expression alterations (9)(10)(11). Targeted molecular imaging has served as a key adjunct to morphological cross sectional imaging studies, for the diagnosis and management of cancer for decades. Positron Emission Tomography with the glucose analogue 18F-fluorodeoxyglucose in conjunction with Computed Tomography (18F-FDG PET/CT) serves as a paradigm for metabolic imaging in clinical oncology by measuring glucose uptake and phosphorylation in tumour cells. Despite the great sensitivity and wide clinical applications for PET imaging, the method does not distinguish individual metabolites or their cellular compartmentalisation and provides no direct information on glycolytic flux or mitochondrial oxidative metabolism. In contrast, although 1H-MRS is many orders of magnitude less sensitive than PET, it can non-invasively distinguish endogenous metabolites in vivo without the use of ionising radiation. This ability to characterise the metabolic phenotype or tumour signature and detect its genotype is particularly relevant for metabolically-driven tumours, which often demonstrate high endogenous metabolite concentrations (12). The ability to measure fumarate in vivo as a functional biomarker has a number of important potential clinical applications including the early identification of FH-deficient tumours, which can enable tailored patient surveillance and facilitate timely cascade family screening. In vivo detection of fumarate could also non-invasively verify the pathogenicity of genetic variants in the era of next generation sequencing. Methods This study was performed in accordance with the Declaration of Hellsinki. Informed consent was obtained from all participants in accordance with the Declaration of Hellsinki. All participants gave written informed consent and the study was approved by South Birmingham Research Ethics Committee (REC reference number: 5175). Genotyping DNA was extracted from peripheral blood samples according to standard protocols. Next generation sequencing of a clinical gene panel was performed for cases 1 and 2 at Cambridge University Hospital NHS Foundation Trust using the TrusightONE sequencing panels (Illumina Inc., UK) and included: SDHA, SDHB, SDHC, SDHD, SDHAF2, MAX, TMEM127, VHL, RET, FH. An average coverage depth of >20 fold was achieved for 98% of the regions sequenced. Single gene sequencing of all coding exons of the FH gene was undertaken for case 3, in West Midlands Regional Genetics Laboratory. 1H-MRS 1H-MRS studies were performed on a 3T MRI system (MR750, GE Healthcare, Waukesha, WI), with body coil transmission and 32-channel reception coils. T1 and T2-weighted images were acquired and a single voxel was prescribed within the tumour in each case. Automated adjustment of transmitter frequency, power and magnetic field homogeneity was performed on all voxels prior to acquisition. Spectra at an echo time of 144 ms were acquired from the tumour with and without chemical shift selective (CHESS) water suppression pulses. Acquisition parameters were: Case 1: TR 1.5s, 256 averages, 6:48 acquisition, voxel size 337 ml; Case 2: TR 2 s, 256 averages, 9:04, voxel size 39 ml, Case 3: variable TR (respiration triggered), 128 averages, 90.8 ml. The full width at half maximum height (FWHM) of the water peak in Hz was measured as an additional data quality metric. The chemical shift of the peak assigned as choline was 3.22 ppm and for fumarate was 6.54 ppm. The SAGE (GE Healthcare, Waukesha, WI) spectroscopy analysis program was used to reconstruct, analyze, and display spectra. The concentrations of choline and fumarate were calculated based on an assumed water concentration of 35 M and the following relaxation constants for pelvic tumour at 3 T (13): T1 water 1.6 s, T1 choline 1.1s, T2 water 109 ms, T2 choline 220 ms (Table 1). Since relaxation measurements have never been performed in vivo for fumarate, they were assumed to be the same as for choline. For the purpose of this pilot study, MRS was regarded as a technical failure if choline was not detected because it was assumed that choline should be detectable in a metabolically active tumour. Immunohistochemistry A rabbit polyclonal anti-2SC antibody (Cambridge Research Biochemicals LTD; dilution 1:400) was used to detect 2-succinyl cysteine (2SC), a product of succination secondary to excess fumarate accumulation. A second rabbit polyclonal antibody directed against the fumarate hydratase (FH) protein (Abcam, ab95947; dilution 1:4000) was also employed. 2SC and FH immunohistochemistry was performed on 4 µm sections of formalin-fixed paraffin-embedded tissue, after appropriate selection of tissue blocks by an experienced pathologist. Liquid chromatography mass spectroscopy (LC-MS): Paraffin embedded tissue from the retroperitoneal mass excised from case 2 was analysed. A xylene/ethanol method was used to remove the paraffin wax from the tissue sample. Once the wax was removed the remaining tissue was homogenised in water to a concentration of 33.3 mg/mL. 10 µL of homogenate was taken for extraction by protein precipitation. The sample was then analysed using LC-MS. The calibration range for fumarate was 18.8-6006 nmol/g. Results Case series Clinical phenotype of patients Case 1 A female presented at the age of 25 years with a skin lesion on her back, subsequently diagnosed as a leiomyoma following a skin biopsy. Her family history revealed that multiple family members including father, paternal grandfather and three siblings had similar skin lesions. At the age of 38 years, the patient re-presented to the dermatology clinic with two new painful skin lesions, both of which were confirmed histologically as leiomyomas. During this episode, the patient was also investigated for menorrhagia, and diagnosed with renal cysts and a 10 cm uterine fibroid (Figure 1A). Analysis of the uterine fibroid using 1H-MRS demonstrated an elevated fumarate peak at 6.54 ppm (Figure 1B, Table 1). Case 2 A 45-year old female, presented to the dermatology clinic with multiple cutaneous leiomyomas. Past medical history was significant for a previous hysterectomy for uterine fibroids and a family history of uterine fibroids. CT imaging of the abdomen and pelvis was performed to further investigate abdominal pain and incidentally demonstrated a large 7.5 x 5.7 cm right sided heterogeneous retroperitoneal mass. A review of historic imaging studies, determined that this mass was present in 2008, when it measured 4.1 x 3.3 cm. At that time a biopsy of the lesion suggested a diagnosis of reactive lymphadenopathy. As the retroperitoneal mass had almost doubled in size over a 10 year period, the differential diagnosis included a sarcoma, paraganglioma or haematological malignancy. Plasma metanephrines were normal thereby making a secretory paraganglioma unlikely (normetanephrine 440 pmol/l, normal range (NR) <1000 pmol/l; metanephrine 323pmol/l, NR <900 pmol/l; and 3-methoxytyramine levels <75 pmol/l, NR <180 pmol/l). An 18F-FDG PET/CT demonstrated heterogeneous FDG uptake in the mass (maximum standardised uptake value, SUVmax 4.7; Figure 2C) with no evidence of metastatic or distant disease. 1H-MRS was performed on the retroperitoneal mass and despite the excellent spectral acquisition, there was no evidence of a fumarate peak (Table 1, Figure 2B and 2C). Following a multi-disciplinary review, surgical excision of the retroperitoneal mass was recommended. Case 3 A 43 year old male patient first presented to a dermatology clinic with painful widespread biopsy- proven cutaneous leiomyomas, requiring laser treatment, and nifedipine for pain control. This man was subsequently diagnosed, with a left sided renal tumour (Figure 3A) on surveillance imaging and a biopsy confirmed a low grade renal cell carcinoma. Germline genetic testing identified an FH variant of uncertain clinical significance (c.1370C>A, p.Thr457Lys). Analysis of parental samples confirmed maternal inheritance. The patient had no family history of renal cell carcinoma or uterine fibroids, but reported that a maternal uncle had similar skin lesions; as this relative was not living in the UK, clinical and molecular confirmation was not possible. Insufficent tissue was available to perform immunohistochemistry for FH or 2SC on the renal biopsy or skin samples. 1H-MRS was performed on the left sided renal mass in order to gain further functional evidence to support the pathogenicity of the identified FH variant given the suspicious phenotype. Unfortunately, the quality of the spectra was affected by the presence of adjacent renal calcification (Figure 3B) and the study was classified as a technical failure as neither choline nor fumarate were detected in the acquired tumour spectra (Figure 3C). The renal tumour is currently under close surveillance and has not yet been excised. Histology review Case 2 Skin incision biopsy: An ill-defined collection of elongated, eosinophilic, spindled cells, arranged in intersecting fascicles in the dermis was observed. Features were in keeping with a pilar leiomyoma (Figure 2G). Immunohistochemistry demonstrated loss of FH protein expression (Figure 2H) and positive 2-succinyl cysteine expression (Figure 2I). Retroperitoneal mass biopsy: Well circumscribed lymphoid tissue with focal peripheral sinus but no paracortical sinuses was identified (Figure 2D).  The morphology and immunohistochemical profile of this lesion was diagnostic of Hyaline Vascular Castleman Disease. Immunohostorchemistry demonstrated preserved FH protein expression (Figure 2E) and absent 2-succinyl cysteine expression (Figure 2F). Case 3 Skin incision biopsy: Two lesions were sampled and both show features of benign pilar leiomyomas. Left sided renal tumour biopsy: The core renal biopsy demonstrated nests and tubules of eosinophilic epitheliod cells with mild variation in nuclear size and occasional nucleoli. No necrosis was evident. The immunohistochemistry and morphology was non-specific,so a diagnosis of low grade renal cell carcinoma was made but further sub-classification was not possible on the biopsy specimen. iii) Germline genetic testing A pathogenic missense variant in exon 8 of the FH gene (c.1189G>A, p.Gly397Arg) was identified in patient 1, a likely pathogenic missense variant in exon 7 of the FH gene (c.956A>G, p.Asp319Gly) was present in case 2, and a variant of uncertain clinical significance in exon 9 of the FH gene (c.1370C>A, p.Thr457Lys) was identified in case 3. iv) Ex vivo metabolomics using LC-MS No fumarate was detected in the retroperitoneal mass from case 2 ex vivo, correlating with the in vivo findings and immunohistochemistry results, suggesting an intact FH enzyme and therefore ruling out a causative role of the germline FH variant in the pathogenesis of the retroperitoneal tumour in case 2. Discussion Reed syndrome, the co-occurrence of uterine and skin leiomyomas, was first described in 1973 (8). However, three decades later the clinical observation that renal cell carcinoma co-segregated with cases of uterine and cutaneous leiomyomas prompted a renaming of the syndrome to hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) (14). The morbidity and mortality associated with this syndrome is accounted for by the predisposition to an aggressive form of renal carcinoma: HLRCC associated renal tumo urs predominately exhibit a type 2 papillary morphology, typically present during the fourth decade, and possess metastatic potential, which is independent of tumour size (14)(15)(16). A clinical diagnosis of HLRCC is based on the presence of cutaneous smooth muscle tumours, but it is notable that almost 30-50% of patients with FH gene mutations may not show evidence of cutaneous leiomyomas (15)(17). Therefore, HLRCC may be under-reported due to the bias for patients presenting with cutaneous leiomyomas, highlighting the need for confirmatory genetic testing or sensitive biomarkers to make a definitive diagnosis of HLRCC (16). In the cases we describe here, cutaneous and uterine leiomyomas raised the possibility of a diagnosis of HLRCC. Early diagnosis of HLRCC in patients presenting with common benign tumours such as uterine and skin leiomyomas enables targeted screening for RCC in probands and prompts family genetic screening and surveillance of other mutation carriers in the family, therefore helping to reduce the morbidity associated with this syndrome. However, not infrequently, rare FH missense substitutions in patients with possible HLRCC may be difficult to categorise as pathogenic or benign. Though immunohistochemistry for protein succinylation is a helpful adjunct for assessing variant pathogenicity (18), this can only be performed after surgery or biopsy. Given the prevalence of uterine leiomyomas and the rarity of HLRCC syndrome, the availability of a non-invasive diagnostic tool to assess the likelihood that a lesion is HLRCC-related is an important advance and could be considered in clinical practice for those women presenting with uterine leiomyomas at a young age and or a family or personal history of cutaneous leiomyoma, RCC or uterine leiomyomas. Here, we have demonstrated a proof-of-principle that 1H-MRS can provide a non-invasive diagnosis of HLRCC through detection of pathological fumarate accumulation in vivo. Furthermore, 1H-MRS can also assist in non-invasively determining whether an intrabdominal mass in a patient with HLRCC is likely to be a component of the syndrome or coincidental: this is an important consideration given the high incidence of incidental findings on surveillance imaging. In Case 2 this was particularly helpful in demonstrating that the retroperitoneal mass was not a non-functional paraganglioma. Given that 1H-MRS can also be used to detect succinate as well as fumarate within a paraganglioma, both SDH and FH-deficiency could be assessed simultaneously. Type 2 papillary RCC associated with germline FH mutations, is generally highly aggressive and has a poor prognosis (17). Advances in our understanding of the molecular mechanisms of tumorigenesis in HLRCC, such as activation of hypoxic gene response pathways, DNA hypermethylation, defective DNA repair and increased sensitivity to poly-ADP ribose polymerase (PARP) inhibitors suggests a range of personalised experimental therapeutics that could be applied in patients with metastatic disease (19). Functional imaging of FH-deficient RCC by 1H-MRS could be used as a sensitive tool to determine tumour response to therapy as we have previously described in SDH-deficient tumours in which reductions in tumour succinate levels occurred before changes in tumour morphology (12). However, important limitations of in vivo metabolomic analysis using 1H-MRS have been demonstrated in this study and earlier studies (12); for example, spectral quality was poor in case 3 due to the relatively small tumour size and close proximity of renal calcification. We also speculate that spectroscopic detection of metabolites may have been affected by the earlier tumour biopsy, suggesting that the timing of the 1H-MRS in relation to a diagnostic biopsy is an important consideration due to the risk of haemorrhage, necrosis or subsequent calcification affecting spectral quality. In an earlier study investigating succinate accumulation in vivo, our group has reported a technical failure rate of 26%, which is similar to the failure rate reported in earlier reports using 1H-MRS (12). Therefore, further studies are required to determine the most appropriate selection criteria when considering in vivo fumarate analysis using 1H-MRS in a clinical setting. Based on the findings of this pilot study, we recommend that 1H-MRS is performed on tumours greater than 1.5 cm in size, or greater than 2.5cm if the tumour location is deep since coil sensitivity decreases with distance, and, when possible, 1H-MRS should be performed prior to tumour biopsy or alternatively several weeks later if a surgical excision is not being considered. In addition to investigating appropriate patient selection criteria, future validation studies should also include repeatability analysis as either repeated analysis of the same tumour or comparison between primary and metastatic lesions to confirm how robust this method is for the in vivo detection of fumarate . In conclusion, this study demonstrates that in vivo detection of fumarate using 1H-MRS could be employed as a functional biomarker of metabolic derangement in patients suspected of having HLRCC. In the future it could be used as a treatment response biomarker for targeted therapies. Table 1: Results of in vivo metabolomics using 1H-MRS Case TumourFWHM of water peakCholine detected Fumarate detected Choline concentrationFumarate concentration1 Uterine fibroid12 HzYesYes13.3 mM6.9 mM2 Retroperitoneal tumour 14 HzYesNo1.6 mMND3Left sided renal tumour12HzNoNoNDND ND= not detected Figure legends Figure1: A: axial T2-weighted MRI from case 1; the red arrow highlights the 10 cm uterine leiomyoma. B: 1H-MRS acquired from the uterine fibroid; the red arrow demonstrates the pathological fumarate peak at 6.54 ppm. Figure 2: A: axial T2-weighted MR image from case 2 with the red arrow demonstrating the large heterogeneous retroperitoneal mass. B: 1H-MRS acquired from this mass with evidence of choline but no fumarate as depicted by the absence of a peak at 6.54 ppm marked by the red arrow. C: coronal maximum intensity projection (MIP) PET image and coronal fused 18F-FDG PET/CT image demonstrating heterogeneous FDG uptake (SUVmax = 4.7) in the retroperitoneal mass as highlighted by the red arrow. D: pathological appearance on haematoxylin and eosin of the retroperitoneal mass excised in Case 2, showing a morphology diagnostic of Hyaline Vascular Castleman Disease. E: shows preservation of FH protein expression in the retoperitoneal mass on immunohistochemistry. F: demonstrates absent 2SC staining in the same tumour. G: haematoxylin and eosin appearance of the skin leiomyoma. H: demonstrates loss of FH immunoexpression in the skin leiomyoma. I: demonstrates positive 2SC staining in the same tumour. Figure 3: A: coronal CT image from case 3 with the red arrow highlighting the left-sided renal tumour. B: a axial CT image from case 3 showing the renal calcification as demonstrated by the red arrow. C: 1H-MR spectra acquired from the renal tumour. The red arrow demonstrates the expected spectral location of the fumarate signal at 6.54 ppm. No choline or fumarate was detected in this spectra and the scan was classified as a ‘technical failure’. References 1. Morin A, Letouzé E, Gimenez-Roqueplo A-P, Favier J. Oncometabolites-driven tumorigenesis: From genetics to targeted therapy. Int J Cancer [Internet]. 2014 Nov 15;135(10):2237–48. Available from: http://doi.wiley.com/10.1002/ijc.29080 2. Astuti D, Latif F, Dallol A, Dahia PL, Douglas F, George E, Sköldberg F, Husebye ES, Eng C ME. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. 3. Gimenez-Roqueplo A-P, Favier J, Rustin P, Rieubland C, Crespin M, Nau V, et al. Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas. Cancer Res [Internet]. 2003 Sep 1;63(17):5615–21. Available from: http://www.ncbi.nlm.nih.gov/pubmed/14500403 4. Boikos SA, Pappo AS, Killian JK, LaQuaglia MP, Weldon CB, George S, et al. Molecular Subtypes of KIT/PDGFRA Wild-Type Gastrointestinal Stromal Tumors. JAMA Oncol [Internet]. 2016 Jul 1;2(7):922. Available from: http://oncology.jamanetwork.com/article.aspx?doi=10.1001/jamaoncol.2016.0256 5. Tomlinson IPM, Alam NA, Rowan AJ, Barclay E, Jaeger EEM, Kelsell D, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet [Internet]. 2002 Apr;30(4):406–10. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11865300 6. Clark GR, Sciacovelli M, Gaude E, Walsh DM, Kirby G, Simpson MA, et al. Germline FH Mutations Presenting With Pheochromocytoma. J Clin Endocrinol Metab [Internet]. 2014 Oct;99(10):E2046–50. Available from: https://academic.oup.com/jcem/article-lookup/doi/10.1210/jc.2014-1659 7. Evenepoel L, Papathomas TG, Krol N, Korpershoek E, de Krijger RR, Persu A, et al. Toward an improved definition of the genetic and tumor spectrum associated with SDH germ-line mutations. Genet Med [Internet]. 2015 Aug 13;17(8):610–20. Available from: http://www.nature.com/articles/gim2014162 8. Reed WB, Walker R, Horowitz R. Cutaneous leiomyomata with uterine leiomyomata. Acta Derm Venereol [Internet]. 1973;53(5):409–16. Available from: http://www.ncbi.nlm.nih.gov/pubmed/4127477 9. Yang M, Soga T, Pollard PJ, Adam J. The emerging role of fumarate as an oncometabolite. Front Oncol [Internet]. 2012;2. Available from: http://journal.frontiersin.org/article/10.3389/fonc.2012.00085/abstract 10. Dahia PLM, Ross KN, Wright ME, Hayashida CY, Santagata S, Barontini M, et al. A HIF1α Regulatory Loop Links Hypoxia and Mitochondrial Signals in Pheochromocytomas. PLoS Genet [Internet]. 2005;1(1):e8. Available from: http://dx.plos.org/10.1371/journal.pgen.0010008 11. Letouzé E, Martinelli C, Loriot C, Burnichon N, Abermil N, Ottolenghi C, et al. SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer Cell [Internet]. 2013 Jun 10;23(6):739–52. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23707781 12. Casey RT, McLean MA, Madhu B, Challis BG, ten Hoopen R, Roberts T, et al. Translating In Vivo Metabolomic Analysis of Succinate Dehydrogenase–Deficient Tumors Into Clinical Utility. JCO Precis Oncol [Internet]. 2018 Mar;(2):1–12. Available from: http://ascopubs.org/doi/10.1200/PO.17.00191 13. McLean MA, Barrett T, Gnanapragasam VJ, Priest AN, Joubert I, Lomas DJ, et al. Prostate cancer metabolite quantification relative to water in 1H-MRSI in vivo at 3 Tesla. Magn Reson Med [Internet]. 2011 Apr;65(4):914–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21413057 14. Launonen V, Vierimaa O, Kiuru M, Isola J, Roth S, Pukkala E, et al. Inherited susceptibility to uterine leiomyomas and renal cell cancer. Proc Natl Acad Sci U S A [Internet]. 2001 Mar 13;98(6):3387–92. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11248088 15. Bhola PT, Gilpin C, Smith A, Graham GE. A retrospective review of 48 individuals, including 12 families, molecularly diagnosed with hereditary leiomyomatosis and renal cell cancer (HLRCC). Fam Cancer [Internet]. 2018;17(4):615–20. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29423582 16. Toro JR, Nickerson ML, Wei M-H, Warren MB, Glenn GM, Turner ML, et al. Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. Am J Hum Genet [Internet]. 2003 Jul;73(1):95–106. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12772087 17. Muller M, Ferlicot S, Guillaud-Bataille M, Le Teuff G, Genestie C, Deveaux S, et al. Reassessing the clinical spectrum associated with hereditary leiomyomatosis and renal cell carcinoma syndrome in French FH mutation carriers. Clin Genet [Internet]. 2017 Dec;92(6):606–15. Available from: http://doi.wiley.com/10.1111/cge.13014 18. Joseph NM, Solomon DA, Frizzell N, Rabban JT, Zaloudek C, Garg K. Morphology and Immunohistochemistry for 2SC and FH Aid in Detection of Fumarate Hydratase Gene Aberrations in Uterine Leiomyomas From Young Patients. Am J Surg Pathol [Internet]. 2015 Nov;39(11):1529–39. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26457356 19. Sulkowski PL, Sundaram RK, Oeck S, Corso CD, Liu Y, Noorbakhsh S, et al. Krebs-cycle-deficient hereditary cancer syndromes are defined by defects in homologous-recombination DNA repair. Nat Genet [Internet]. 2018 Aug;50(8):1086–92. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30013182 Funding: We thank the following funding organisations; GIST Support UK (RC), NIHR (RC), Cambridge Experimental Cancer Medicine Centre, Addenbrooke’s Charitable Trust, National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, Cancer Research UK CRUK (FAG), CRUK Cambridge Centre (MM, FAG), the Uni Funding: We thank the following funding organisations; GIST Support UK (RC), NIHR (RC), Cambridge Experimental Cancer Medicine Centre, Addenbrooke’s Charitable Trust, National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, Cancer Research UK CRUK (FAG), CRUK Cambridge Centre (MM, FAG), the University of Cambridge, and Hutchison Whampoa Ltd (MM), NIHR Senior Investigator Award (ERM), European Research Council Advanced Researcher Award (ERM), CRUK and Engineering and Physical Sciences Research Council (EPSRC) Imaging Centre in Cambridge and Manchester (FAG). The University of Cambridge has received salary support in respect of EM from the NHS in the East of England through the Clinical Academic Reserve. The views expressed are those of the authors and not necessarily those of the NHS or Department of Health. Acknowledgements: