Single-nuclei and bulk-tissue gene-expression analysis of pheochromocytoma and paraganglioma links disease subtypes with tumor microenvironment

Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors associated with autonomic nerves. Here we use single-nuclei RNA-seq and bulk-tissue gene-expression data to characterize the cellular composition of PCPG and normal adrenal tissues, refine tumor gene-expression subtypes and make clinical and genotypic associations. We confirm seven PCPG gene-expression subtypes with significant genotype and clinical associations. Tumors with mutations in VHL, SDH-encoding genes (SDHx) or MAML3-fusions are characterized by hypoxia-inducible factor signaling and neoangiogenesis. PCPG have few infiltrating lymphocytes but abundant macrophages. While neoplastic cells transcriptionally resemble mature chromaffin cells, early chromaffin and neuroblast markers are also features of some PCPG subtypes. The gene-expression profile of metastatic SDHx-related PCPG indicates these tumors have elevated cellular proliferation and a lower number of non-neoplastic Schwann-cell-like cells, while GPR139 is a potential theranostic target. Our findings therefore clarify the diverse transcriptional programs and cellular composition of PCPG and identify biomarkers of potential clinical significance.Magnus Zethoven, Luciano Martelotto, Andrew Pattison, Blake Bowen, Shiva Balachander, Aidan Flynn, Fernando J. Rossello, Annette Hogg, Julie A.Miller, Zdenek Frysak, Sean Grimmond, Lauren Fishbein, Arthur S. Tischler, Anthony J. Gill, Rodney J. Hicks, Patricia L. M. Dahia, Roderick Clifton-Bligh, Karel Pacak, Richard W. Tothil

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background inover one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and severalother tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlight-ing the importance of identifying SDHx mutations for patient management. Genetic variants of unknown signi-cance, where implications for the patient and family members are unclear, are a problem for interpretation. Forsuch cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB(SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatogra-phy–mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions providean alternative method. Here, we compare SDHB-IHC with metabolite proling in 189 tumours from 187 PPGLpatients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to estab-lish predictive models for interpreting metabolite data. Metabolite proling showed higher diagnostic specicitycompared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite proles improved predictive ability over that of the SFR, in particular forhard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, thecombination of metabolite proling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classied allbut one of the false negatives from metabolite proling strategies, while metabolite proling correctly classied allbut one of the false negatives/positives from SDHB-IHC. From 186 tumours with conrmed status of SDHx variantpathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benets ofboth strategies for patient management

Plasma gut hormone levels in 37 patients with pheochromocytomas

Pheochromocytomas are usually recognized by the effects of overproduction of catecholamines, but there are clinical features that cannot be ascribed to catecholamine excess that may be due to vasoactive peptides. We, therefore, measured blood levels of vasoactive intestinal peptides (VIP), substance P, somatostatin (SS), and motilin in 50 instances in 37 patients with pheochromocytomas-21 malignant, 10 benign intra-adrenal, and 6 ectopic (5 paracardial and 1 perirenal). Hormone levels were considered raised if the level was more than 3 S.D. above the mean value found in 52 healthy subjects. Of the 37 patients, 20 (54%) had an abnormality in 1 or more gut hormone levels. The most common abnormality was a raised SS in 9/37 (24%). In addition to these, however, 3 (8%) others had raised VIP, 5 (13.5%) raised motilin, and 3 (8%) raised substance P. Patients with benign adrenal adenomas had raised levels of SS and substance P. Ectopic pheochromocytomas produced only SS in addition to catecholamines, but malignant pheochromocytomas could secrete all 4 peptides, and more than 1 in the same patient. We conclude that pheochromocytomas may secrete multiple vasoactive peptides, and they are more likely to do so if malignant. Somatostatin is the most commonly secreted peptide and is found with benign adrenal and ectopic (paracardiac) tumors. If the level of more than 1 peptide is elevated, the likelihood of malignancy is significantly increased . Les phéochromocytomes sont généralement déceléspar les effets dûs à la surproduction de catécholamines, mais certains troubles ne peuvent être attribués à ce phénomène et relèvent peut être de l'action de peptides vasoactifs. Les auteurs se sont donc attachés à doser dans le sang le VIP, la substance P, la somatostatine (SS), et la motiline. Ces dosages furent pratiqués 50 fois chez 37 malades porteurs de phéochromocytomes: 21 malins, 10 bénins et 6 ectopiques (5 paracardiaque et 1 péri-rénal). Les taux des hormones furent considérés comme élevés lorsque leur niveau fut supérieur à plus de 3 fois le taux de 52 sujets sains. Sur les 37 malades 20 (54%) présentaient un excès d'une ou de plusieurs hormones digestives. L'anomalie constatée la plus fréquente fut l'élévation de la SS (9 fois sur 37 soit 24%). Ajoutée à ce fait fut l'élévation de la VIP chez 3 sujets (8%), de la motiline chez 5 (13.5%) et de la substance P chez 3 (8%). Les phéochromocytomes bénins surrénaliens présentaient à la fois une élévation du taux de la SS et de la substance P. Les phéochromocytomes ectopiques en revanche présentaient seulement une élévation de la SS. Les phéochromocytomes malins pouvaient sécréter les 4 peptides ou plus d'un chez le même malade. En conclusion les phéochromocytomes peuvent secréter de multiples peptides vasoactifs et plus particulièrement lorsqu'ils sont malins. La SS est la substance qui est la plus souvent secrétée et elle est trouvée dans les tumeurs bénignes surrénaliennes ou ectopiques. Si plus d'une de ces substances est produite en excès les risques de malignité de la tumeur sont significativement plus importants. Los feocromocitomas generalmente son diagnosticados por los efectos del exceso de producción de catecolaminas pero hay características clínicas que no pueden ser atribuidas al exceso de catecolaminas y que pueden ser más bien manifestación de péptidos vasoactivos. Hemos establecido los niveles sanguíneos del péptido intestinal vasoactivo (VIP), de la sustancia P, de la somatostatina (SS), y de la motilina en 50 determinaciones en 37 pacientes con feocromocitomas; 21 malignos, 10 benignos intra-adrenales, y 6 ectópicos (5 paracardiales y 1 perirrenal). Se consideró que los niveles hormonales estaban elevados cuando el nivel era de más de 3 de desviación estandar sobre el valor promedio en 52 individuos normales. De 37 pacientes, 20 (54%) presentaron un valor anormal en 1 o más determinaciones del nivel de hormonas intestinales. La anormalidad más común fue la elevación de la SS en 9/37 (24%). Además de esto, sinembargo, otros 3 (8%) presentaban elevación de VIP, 5 (13.5%) elevación de sustancia P. Los adenomas suprarrenales benignos exhibieron niveles elevados de SS y de sustancia P. Los feocromocitomas ectópicos demostraron producción sólo de SS además de catecolaminas, pero los feocromocitomas malignos demostraron ser capaces de secretar todos los 4 péptidos, y más de 1 en el mismo paciente. Hemos llegado a la conclusión de que los feocromocitomas pueden secretar múltiples peptidos vasoactivos y que ésto tiende a ocurrir cuando son malignos. La SS es el péptido más frecuentemente secretado y se lo encuentra en los tumores suprarrenales benigno y ectópico (paracardiacos). Si se encuentran niveles elevados de más de 1 péptido, la posibilidad de malignidad aparece significativamente aumentada.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41274/1/268_2005_Article_BF01655534.pd

Animal models of pheochromocytoma

Pheochromocytomas are neuroendocrine tumors of adrenal chromaffin cells. They are rare in all species except rats but occur with increased frequency in several human familial tumor syndromes. Concurrence of pheochromocytoma with other tumors sometimes parallels these human syndromes in rats, bovines, horses and dogs but a shared genetic basis for human and spontaneously occurring animal pheochromocytomas has thus far not been established. Pheochromocytomas are inducible in rats by a variety of non-genotoxic substances that may act indirectly by stimulating chromaffin cell proliferation. They are not known to be similarly inducible in other species but arise with increased frequency in transgenic and knockout mice that to varying degrees recapitulate human tumor syndromes. Preliminary evidence suggests that homologous somatic genetic changes might contribute to pheochromocytoma development in humans and some mouse models. The nerve growth factor-responsive PC12 cell line, established from a rat pheochromocytoma, has for almost 30 years served as a research tool for many aspects of neurobiology involving normal and neoplastic conditions. Recently developed pheochromocytoma cell lines from neurofibromatosis knockout mice supplement the PC12 line and have generated additional applications. Advantages of the mouse lines include expression of substantial levels of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase and expression of high levels of the receptor tyrosine kinase, Ret, which is characteristic of sporadic and familial human pheochromocytomas but not of PC12 cells. Disadvantages include an apparently less stable phenotype. It is difficult to establish pheochromocytoma cell lines from any species, although the tumor cells persist in culture for many months. Understanding of factors that permit pheochromocytoma cells to proliferate might itself provide important insights for tumor biology

MicroCT for high-resolution imaging of ectopic pheochromocytoma tumors in the liver of nude mice

Successful outcomes for patients with cancer often depend on the early detection of tumor and the prompt initiation of active therapy. Despite major advances in the treatment of many cancers, early-stage lesions often go undetected due to the suboptimal resolution of current anatomical and functional imaging modalities. This limitation also applies to preclinical animal tumor models that are crucial for the evaluation and development of new therapeutic approaches to cancer. We report a new mouse model of metastatic pheochromocytoma, generated using tail vein injection of the mouse pheochromocytoma cell (MPC) line that reproducibly generated multiple liver tumors in the animals. Furthermore, we show that in vivo microCT imaging enhanced using a hepatobiliary-specific contrast agent, glyceryl-2-oleyl-1,3-di- 7-(3-amino-2,4,6-triiodophenyl)-heptanoate (DHOG), detected tumors as small as 0.35 mm as early as 4 weeks after the injection of the tumor cells. This model may be useful for in vivo studies of tumor biology and for development of new strategies to treat metastatic pheochromocytoma