Princess Máxima Center

The fifth edition of the WHO Classification of Tumors of the Central Nervous System (CNS), published in 2021, is the sixth version of the international standard for the classification of brain and spinal cord tumors. Building on the 2016 updated fourth edition and the work of the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy, the 2021 fifth edition introduces major changes that advance the role of molecular diagnostics in CNS tumor classification. At the same time, it remains wedded to other established approaches to tumor diagnosis such as histology and immunohistochemistry. In doing so, the fifth edition establishes some different approaches to both CNS tumor nomenclature and grading and it emphasizes the importance of integrated diagnoses and layered reports. New tumor types and subtypes are introduced, some based on novel diagnostic technologies such as DNA methylome profiling. The present review summarizes the major general changes in the 2021 fifth edition classification and the specific changes in each taxonomic category. It is hoped that this summary provides an overview to facilitate more in-depth exploration of the entire fifth edition of the WHO Classification of Tumors of the Central Nervous System.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.

The classification of neuroendocrine neoplasms (NENs) differs between organ systems and currently causes considerable confusion. A uniform classification framework for NENs at any anatomical location may reduce inconsistencies and contradictions among the various systems currently in use. The classification suggested here is intended to allow pathologists and clinicians to manage their patients with NENs consistently, while acknowledging organ-specific differences in classification criteria, tumor biology, and prognostic factors. The classification suggested is based on a consensus conference held at the International Agency for Research on Cancer (IARC) in November 2017 and subsequent discussion with additional experts. The key feature of the new classification is a distinction between differentiated neuroendocrine tumors (NETs), also designated carcinoid tumors in some systems, and poorly differentiated NECs, as they both share common expression of neuroendocrine markers. This dichotomous morphological subdivision into NETs and NECs is supported by genetic evidence at specific anatomic sites as well as clinical, epidemiologic, histologic, and prognostic differences. In many organ systems, NETs are graded as G1, G2, or G3 based on mitotic count and/or Ki-67 labeling index, and/or the presence of necrosis; NECs are considered high grade by definition. We believe this conceptual approach can form the basis for the next generation of NEN classifications and will allow more consistent taxonomy to understand how neoplasms from different organ systems inter-relate clinically and genetically.

Abstract Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient–derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection. Significance: New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. Cancer Discov; 8(9); 1112–29. ©2018 AACR. See related commentary by Collisson, p. 1062. This article is highlighted in the In This Issue feature, p. 1047

Adrenocortical carcinoma (ACC) is a rare and in most cases steroid hormone-producing tumor with variable prognosis. The purpose of these guidelines is to provide clinicians with best possible evidence-based recommendations for clinical management of patients with ACC based on the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. We predefined four main clinical questions, which we judged as particularly important for the management of ACC patients and performed systematic literature searches: (A) What is needed to diagnose an ACC by histopathology? (B) Which are the best prognostic markers in ACC? (C) Is adjuvant therapy able to prevent recurrent disease or reduce mortality after radical resection? (D) What is the best treatment option for macroscopically incompletely resected, recurrent or metastatic disease? Other relevant questions were discussed within the group. Selected Recommendations: (i) We recommend that all patients with suspected and proven ACC are discussed in a multidisciplinary expert team meeting. (ii) We recommend that every patient with (suspected) ACC should undergo careful clinical assessment, detailed endocrine work-up to identify autonomous hormone excess and adrenal-focused imaging. (iii) We recommend that adrenal surgery for (suspected) ACC should be performed only by surgeons experienced in adrenal and oncological surgery aiming at a complete en bloc resection (including resection of oligo-metastatic disease). (iv) We suggest that all suspected ACC should be reviewed by an expert adrenal pathologist using the Weiss score and providing Ki67 index. (v) We suggest adjuvant mitotane treatment in patients after radical surgery that have a perceived high risk of recurrence (ENSAT stage III, or R1 resection, or Ki67 >10%). (vi) For advanced ACC not amenable to complete surgical resection, local therapeutic measures (e.g. radiation therapy, radiofrequency ablation, chemoembolization) are of particular value. However, we suggest against the routine use of adrenal surgery in case of widespread metastatic disease. In these patients, we recommend either mitotane monotherapy or mitotane, etoposide, doxorubicin and cisplatin depending on prognostic parameters. In selected patients with a good response, surgery may be subsequently considered. (vii) In patients with recurrent disease and a disease-free interval of at least 12 months, in whom a complete resection/ablation seems feasible, we recommend surgery or alternatively other local therapies. Furthermore, we offer detailed recommendations about the management of mitotane treatment and other supportive therapies. Finally, we suggest directions for future research.

Gliomas are the most frequent intrinsic tumours of the central nervous system and encompass two principle subgroups: diffuse gliomas and gliomas showing a more circumscribed growth pattern ('nondiffuse gliomas'). In the revised fourth edition of the WHO Classification of CNS tumours published in 2016, classification of especially diffuse gliomas has fundamentally changed: for the first time, a large subset of these tumours is now defined based on presence/absence of IDH mutation and 1p/19q codeletion. Following this approach, the diagnosis of (anaplastic) oligoastrocytoma can be expected to largely disappear. Furthermore, in the WHO 2016 Classification gliomatosis cerebri is not an entity anymore but is now considered as a growth pattern. The most important changes in the very diverse group of 'nondiffuse' gliomas and neuronal-glial tumours are the introduction of anaplastic pleomorphic xanthoastrocytoma, of diffuse leptomeningeal glioneuronal tumour and of RELA fusion-positive ependymoma as entities. In the last part of this review, after very briefly touching upon classification of neuronal, choroid plexus and pineal region tumours, some practical implications and challenges associated with the WHO 2016 Classification of gliomas are discussed.

There is a clear and unmet clinical need for biomarkers to predict responsiveness to chemotherapy for cancer. We developed an in vitro test based on patient-derived tumor organoids (PDOs) from metastatic lesions to identify nonresponders to standard-of-care chemotherapy in colorectal cancer (CRC). In a prospective clinical study, we show the feasibility of generating and testing PDOs for evaluation of sensitivity to chemotherapy. Our PDO test predicted response of the biopsied lesion in more than 80% of patients treated with irinotecan-based therapies without misclassifying patients who would have benefited from treatment. This correlation was specific to irinotecan-based chemotherapy, however, and the PDOs failed to predict outcome for treatment with 5-fluorouracil plus oxaliplatin. Our data suggest that PDOs could be used to prevent cancer patients from undergoing ineffective irinotecan-based chemotherapy.

BACKGROUND: Base substitution catalogues represent historical records of mutational processes that have been active in a cell. Such processes can be distinguished by various characteristics, like mutation type, sequence context, transcriptional and replicative strand bias, genomic distribution and association with (epi)-genomic features. RESULTS: We have created MutationalPatterns, an R/Bioconductor package that allows researchers to characterize a broad range of patterns in base substitution catalogues to dissect the underlying molecular mechanisms. Furthermore, it offers an efficient method to quantify the contribution of known mutational signatures within single samples. This analysis can be used to determine whether certain DNA repair mechanisms are perturbed and to further characterize the processes underlying known mutational signatures. CONCLUSIONS: MutationalPatterns allows for easy characterization and visualization of mutational patterns. These analyses willsupport fundamental research into mutational mechanisms and may ultimately improve cancer diagnosis and treatment strategies. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.

cIMPACT-NOW (the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy) was established to evaluate and make practical recommendations on recent advances in the field of CNS tumor classification, particularly in light of the rapid progress in molecular insights into these neoplasms. For Round 2 of its deliberations, cIMPACT-NOW Working Committee 3 was reconstituted and convened in Utrecht, The Netherlands, for a meeting designed to review putative new CNS tumor types in advance of any future World Health Organization meeting on CNS tumor classification. In preparatory activities for the meeting and at the actual meeting, a list of possible entities was assembled and each type and subtype debated. Working Committee 3 recommended that a substantial number of newly recognized types and subtypes should be considered for inclusion in future CNS tumor classifications. In addition, the group endorsed a number of principles-relating to classification categories, approaches to classification, nomenclature, and grading-that the group hopes will also inform the future classification of CNS neoplasms.

BACKGROUND: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma. METHODS: TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival. RESULTS: A total of 168 patients (86% with disease refractory to anti-programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P<0.001); 49% (95% CI, 38 to 60) and 21% (95% CI, 13 to 32) of the patients, respectively, had an objective response. Median overall survival was 25.8 months (95% CI, 18.2 to not reached) in the TIL group and 18.9 months (95% CI, 13.8 to 32.6) in the ipilimumab group. Treatment-related adverse events of grade 3 or higher occurred in all patients who received TILs and in 57% of those who received ipilimumab; in the TIL group, these events were mainly chemotherapy-related myelosuppression. CONCLUSIONS: In patients with advanced melanoma, progression-free survival was significantly longer among those who received TIL therapy than among those who received ipilimumab. (Funded by the Dutch Cancer Society and others; ClinicalTrials.gov number, NCT02278887.).

Organoids are in vitro-cultured three-dimensional structures that recapitulate key aspects of in vivo organs. They can be established from pluripotent stem cells and from adult stem cells, the latter being the subject of this review. Organoids derived from adult stem cells exploit the tissue regeneration process that is driven by these cells, and they can be established directly from the healthy or diseased epithelium of many organs. Organoids are amenable to any experimental approach that has been developed for cell lines. Applications in experimental biology involve the modeling of tissue physiology and disease, including malignant, hereditary, and infectious diseases. Biobanks of patient-derived tumor organoids are used in drug development research, and they hold promise for developing personalized and regenerative medicine. In this review, we discuss the applications of adult stem cell-derived organoids in the laboratory and the clinic.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically.

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

A signature event for organoids Human cancer genomes harbor cryptic mutational signatures that represent the cumulative effects of DNA damage and defects in DNA repair processes. Knowledge of how specific signatures originate could have a major impact on cancer diagnosis and prevention. One approach to address this question is to reproduce the signatures in experimental systems by genetic engineering and then match the signatures to those found in naturally occurring cancers. Drost et al. used CRISPR-Cas9 to delete certain DNA repair enzymes from human colon organoids. In a proof-of-concept study, they show that deficiency in base excision repair is responsible for a mutational signature previously identified in cancer genome sequencing projects. Science , this issue p. 234

In recent years, major advances have been made in cancer immunotherapy. This has led to significant improvement in prognosis of cancer patients, especially in the hematological setting. Nonetheless, translation of these successes to solid tumors was found difficult. One major mechanism through which solid tumors can avoid anti-tumor immunity is the downregulation of major histocompatibility complex class I (MHC-I), which causes reduced recognition by- and cytotoxicity of CD8+ T-cells. Downregulation of MHC-I has been described in 40–90% of human tumors, often correlating with worse prognosis. Epigenetic and (post-)transcriptional dysregulations relevant in the stabilization of NFkB, IRFs, and NLRC5 are often responsible for MHC-I downregulation in cancer. The intrinsic reversible nature of these dysregulations provides an opportunity to restore MHC-I expression and facilitate adaptive anti-tumor immunity. In this review, we provide an overview of the mechanisms underlying reversible MHC-I downregulation and describe potential strategies to counteract this reduction in MHC-I antigen presentation in cancer.

Recent advances have increased survival rates of children and adults suffering from cancer thanks to effective anti-cancer therapy, such as chemotherapy. However, during treatment and later in life they are frequently confronted with the severe negative side-effects of their life-saving treatment. The occurrence of numerous features of accelerated aging, seriously affecting quality of life, has now become one of the most pressing problems associated with (pediatric) cancer treatment. Chemotherapies frequently target and damage the DNA, causing mutations or genome instability, a major hallmark of both cancer and aging. However, there are numerous types of chemotherapeutic drugs that are genotoxic and interfere with DNA metabolism in different ways, each with their own biodistribution, kinetics, and biological fate. Depending on the type of DNA lesion produced (e.g., interference with DNA replication or RNA transcription), the organ or cell type inflicted (e.g., cell cycle or differentiation status, metabolic state, activity of clearance and detoxification mechanisms, the cellular condition or micro-environment), and the degree of exposure, outcomes of cancer treatment can largely differ. These considerations provide a conceptual framework in which different classes of chemotherapeutics contribute to the development of toxicities and accelerated aging of different organ systems. Here, we summarize frequently observed side-effects in (pediatric) ex-cancer patients and discuss which types of DNA damage might be responsible.

•This updated ESMO Clinical Practice Guideline provides key recommendations on the management of adrenal cancer.•Authorship includes a multidisciplinary group of experts from different institutions and countries in Europe.•Detailed guidance on diagnosis, classification, treatment and follow-up is provided for both adrenocortical carcinoma and malignant phaeochromocytoma.•Recommendations are provided in the text, including levels of evidence and grades of recommendation, where applicable.•Due to the rarity of the adrenocortical carcinoma and malignant phaeochromocytoma, there is a clear recommendation to consult expert centres. Two different primary malignancies can arise from the adrenal gland: adrenocortical carcinoma (ACC) from the adrenal cortex and malignant phaeochromocytoma from the adrenal medulla. Both malignancies are rare. ACC has an estimated incidence of ∼0.5-2 new cases per million people per year.1Kerkhofs T.M. Verhoeven R.H. Van der Zwan J.M. et al.Adrenocortical carcinoma: a population-based study on incidence and survival in the Netherlands since 1993.Eur J Cancer. 2013; 49: 2579-2586Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar,2Kebebew E. Reiff E. Duh Q.Y. et al.Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress?.World J Surg. 2006; 30: 872-878Crossref PubMed Scopus (318) Google Scholar Phaeochromocytomas are catecholamine-producing neuroendocrine tumours arising from chromaffin cells of the adrenal medulla or extra-adrenal paraganglia. The latter are usually called paraganglioma, leading to the combined term phaeochromocytomas and paragangliomas (PPGLs). The detected incidence of PPGLs is commonly reported at 2-8 per million per year3Pacak K. Eisenhofer G. Ahlman H. et al.Pheochromocytoma: recommendations for clinical practice from the First International Symposium. October 2005.Nat Clin Pract Endocrinol Metab. 2007; 3: 92-102Crossref PubMed Scopus (517) Google Scholar (supplementary information, available at https://doi.org/10.1016/j.annonc.2020.08.2099). All patients with suspected and proven ACC or PPGL should be discussed in a multidisciplinary expert team meeting, at least at the time of initial diagnosis (ideally before surgery) and in case of progressive disease. Every patient with (suspected) ACC or PPGL should undergo careful clinical assessment, including case history, clinical examination for symptoms and signs of adrenal hormone excess. For more details on this topic, we refer to more comprehensive guidelines and reviews.3Pacak K. Eisenhofer G. Ahlman H. et al.Pheochromocytoma: recommendations for clinical practice from the First International Symposium. October 2005.Nat Clin Pract Endocrinol Metab. 2007; 3: 92-102Crossref PubMed Scopus (517) Google Scholar, 4Fassnacht M. Dekkers O.M. Else T. et al.European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors.Eur J Endocrinol. 2018; 179: G1-G46Crossref PubMed Scopus (403) Google Scholar, 5Lenders J.W. Duh Q.Y. Eisenhofer G. et al.Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.J Clin Endocrinol Metab. 2014; 99: 1915-1942Crossref PubMed Scopus (1471) Google Scholar, 6Amar L. Servais A. Gimenez-Roqueplo A.P. et al.Year of diagnosis, features at presentation, and risk of recurrence in patients with pheochromocytoma or secreting paraganglioma.J Clin Endocrinol Metab. 2005; 90: 2110-2116Crossref PubMed Scopus (283) Google Scholar, 7Fassnacht M. Libe R. Kroiss M. Allolio B. Adrenocortical carcinoma: a clinician's update.Nat Rev Endocrinol. 2011; 7: 323-335Crossref PubMed Scopus (302) Google Scholar, 8Mansmann G. Lau J. Balk E. et al.The clinically inapparent adrenal mass: update in diagnosis and management.Endocr Rev. 2004; 25: 309-340Crossref PubMed Scopus (602) Google Scholar The aims of hormonal evaluation are multiple: (i) hormone assays provide orientation to the nature of the adrenal mass and can be useful to assess presence of malignancy; (ii) a phaeochromocytoma should always be ruled out, because these tumours can induce life-threatening crises, requiring specific management before any intervention; (iii) massive adrenocortical steroid excess can impact short-term survival and quality of life (QoL), requiring specific treatments to block impacts of hormonal excess; (iv) abnormal hormone secretions may serve as biological markers for the follow-up of patients; (v) in case of large bilateral adrenal masses, a systematic assessment of adrenal function is recommended to rule out adrenal insufficiency. Precise hormone assays have been detailed in recent guideline statements,4Fassnacht M. Dekkers O.M. Else T. et al.European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors.Eur J Endocrinol. 2018; 179: G1-G46Crossref PubMed Scopus (403) Google Scholar,5Lenders J.W. Duh Q.Y. Eisenhofer G. et al.Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.J Clin Endocrinol Metab. 2014; 99: 1915-1942Crossref PubMed Scopus (1471) Google Scholar,9Plouin P.F. Amar L. Dekkers O.M. et al.European Society of Endocrinology Clinical Practice Guideline for long-term follow-up of patients operated on for a phaeochromocytoma or a paraganglioma.Eur J Endocrinol. 2016; 174: G1-G10Crossref PubMed Scopus (251) Google Scholar,10Fassnacht M. Arlt W. Bancos I. et al.Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors.Eur J Endocrinol. 2016; 175: G1-G34Crossref PubMed Scopus (853) Google Scholar and are summarised in Table 1.Table 1Diagnostic work-up of (suspected) adrenal- or paraganglioma-related malignanciesSpecific questionAssaysIndication(Suspected) ACC Exclusion of glucocorticoid excess?1 mg dexamethasone suppression testAll adrenal masses with no overt Cushing (clinically) Characterisation of glucocorticoid excess?1 mg dexamethasone suppression testFree cortisol in 24-h urineBasal ACTH (plasma)Adrenal masses with clinical signs of Cushing or pathological 1 mg dexamethasone test Sex steroids and steroid precursors excess?DHEA-S17-OH progesteroneAndrostenedioneTestosterone (only in women)17-beta-oestradiol (only in men and postmenopausal women)11-deoxycortisol (if available)Any adrenal mass suspected to be an ACC Mineralocorticoid excess?PotassiumAldosterone/renin ratioAny adrenal masses with hypertension and/or hypokalaemia Extension of the adrenal tumour and evidence for metastases?CT or MRI of abdomen, pelvis and chest (or FDG-PET/CT including full-dose CT)All suspected ACCs Evidence of cerebral metastases?Cerebral MRTOnly if cerebral metastases are suspected Evidence of bone metastasesFDG-PET/CT, bone scan, bone CT or bone MRIOnly if skeletal metastases are suspected(Suspected) PPGL Catecholamine excess?Fractionated metanephrines in 24-h urine or plasma-free metanephrines and methoxytyramineAll adrenal masses and all paraganglioma Extension of the adrenal tumour?CT or MRI of abdomenAll biochemically confirmed phaeochromocytoma Evidence of thoracic metastases?Chest CT (or PET/CT including full-dose CT)All PPGL patients with a 'high risk of metastases'aThe authors suggest being at a 'high risk of metastases' applies to all patients who fulfil one or more of the following criteria: adrenal phaeochromocytoma ≥5 cm or any extra-adrenal paraganglioma or known SDHB germline mutation or plasma methoxytyramine more than threefold above the upper reference limit. Evidence of cerebral metastases?Cerebral MRTOnly if cerebral metastases are suspected Evidence of bone metastasesFDG-PET/CT, DOTATATE-PET/CT, bone scan, bone CT or bone MRIOnly if skeletal metastases are suspectedbSome authors are in favour of carrying out functional imaging to detect bone metastases in all PPGL patients. Additional functional imaging?At least one functional whole-body imaging (i.e. FDG-PET, DOTATATE-PET, MIBG scintigraphy)In all PPGL patients with a 'high risk of metastases'aThe authors suggest being at a 'high risk of metastases' applies to all patients who fulfil one or more of the following criteria: adrenal phaeochromocytoma ≥5 cm or any extra-adrenal paraganglioma or known SDHB germline mutation or plasma methoxytyramine more than threefold above the upper reference limit. Radionuclide therapy possible?MIBG scintigraphy and somatostatin-based imaging (e.g. DOTATATE-PET/CT)In all PPGL patients with evidence for metastases17-OH, 17-hydroxy; ACC, adrenocortical carcinoma; ACTH, adrenocorticotropic hormone; CT, computed tomography; DHEA-S, dehydroepiandrosterone sulfate; FDG-PET, 2-fluoro-2-deoxy-d-glucose-positron electron tomography; MIBG, meta-iodobenzylguanidine; MRI, magnetic resonance imaging; MRT, magnetic resonance tomography; PPGL, phaeochromocytoma and paraganglioma; SDHB, succinate dehydrogenase complex iron sulfur subunit B.a The authors suggest being at a 'high risk of metastases' applies to all patients who fulfil one or more of the following criteria: adrenal phaeochromocytoma ≥5 cm or any extra-adrenal paraganglioma or known SDHB germline mutation or plasma methoxytyramine more than threefold above the upper reference limit.b Some authors are in favour of carrying out functional imaging to detect bone metastases in all PPGL patients. Open table in a new tab 17-OH, 17-hydroxy; ACC, adrenocortical carcinoma; ACTH, adrenocorticotropic hormone; CT, computed tomography; DHEA-S, dehydroepiandrosterone sulfate; FDG-PET, 2-fluoro-2-deoxy-d-glucose-positron electron tomography; MIBG, meta-iodobenzylguanidine; MRI, magnetic resonance imaging; MRT, magnetic resonance tomography; PPGL, phaeochromocytoma and paraganglioma; SDHB, succinate dehydrogenase complex iron sulfur subunit B. For all adrenal masses, the diagnosis of phaeochromocytoma should be systematically assessed by measuring plasma-free or urinary-fractionated metanephrines [V, A].5Lenders J.W. Duh Q.Y. Eisenhofer G. et al.Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.J Clin Endocrinol Metab. 2014; 99: 1915-1942Crossref PubMed Scopus (1471) Google Scholar,10Fassnacht M. Arlt W. Bancos I. et al.Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors.Eur J Endocrinol. 2016; 175: G1-G34Crossref PubMed Scopus (853) Google Scholar Additional measurements of plasma methoxytyramine, a biomarker now increasingly available, provide useful information to assess the likelihood of malignancy.11Eisenhofer G. Lenders J.W. Siegert G. et al.Plasma methoxytyramine: a novel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status.Eur J Cancer. 2012; 48: 1739-1749Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar In cases of suspected ACC, an extensive steroid hormone work-up is recommended, assessing gluco-, mineralo-, sex- and precursor-steroids ([V, B] (Table 1)).12Arlt W. Biehl M. Taylor A.E. et al.Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors.J Clin Endocrinol Metab. 2011; 96: 3775-3784Crossref PubMed Scopus (305) Google Scholar, 13Taylor D.R. Ghataore L. Couchman L. et al.A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocortical carcinoma.Clin Chem. 2017; 63: 1836-1846Crossref PubMed Scopus (66) Google Scholar, 14Hines J.M. Bancos I. Bancos C. et al.High-resolution, accurate-mass (HRAM) mass spectrometry urine steroid profiling in the diagnosis of adrenal disorders.Clin Chem. 2017; 63: 1824-1835Crossref PubMed Scopus (63) Google Scholar, 15Schweitzer S. Kunz M. Kurlbaum M. et al.Plasma steroid metabolome profiling for the diagnosis of adrenocortical carcinoma.Eur J Endocrinol. 2018; 180: 117-125Crossref Scopus (40) Google Scholar, 16Kerkhofs T.M. Kerstens M.N. Kema I.P. et al.Diagnostic value of urinary steroid profiling in the evaluation of adrenal tumors.Horm Cancer. 2015; 6: 168-175Crossref PubMed Scopus (65) Google Scholar For best patient care, adequate visualisation of the tumour and potential metastases is essential. For differential diagnosis of an adrenal mass, computed tomography (CT) and magnetic resonance imaging (MRI) are both effective [IV, A]. Although these methods cannot determine the exact entity of the mass, both are able to correctly diagnose a subset of benign tumours—at least when carried out according to state-of-the-art criteria. The single best criterion to diagnose a benign tumour (e.g. adenoma) remains Hounsfield units ≤10 in an unenhanced CT.17Dinnes J. Bancos I. Ferrante di Ruffano L. et al.Management of endocrine disease: imaging for the diagnosis of malignancy in incidentally discovered adrenal masses: a systematic review and meta-analysis.Eur J Endocrinol. 2016; 175: R51-R64Crossref PubMed Scopus (113) Google Scholar However, other imaging criteria, such as rapid washout in 10- or 15-min delayed contrast-enhanced CT, signal intensity loss using opposed-phase MRI, and low 2-fluorine-18 [18F]fluoro-2-deoxy-d-glucose (FDG) uptake in [18F]FDG-positron electron tomography (PET)/CT are also suggestive of a benign tumour. Most ACCs show an inhomogeneous appearance in CT or MRI with irregular margins and irregular enhancement of solid components after intravenous injection of contrasted agent. Detection of local invasion or tumour extension into the inferior vena cava, as well as lymph node or other metastases—including lung and liver—is mandatory before planning any surgery. Therefore, cross-sectional imaging of the chest, abdomen and pelvis is required preoperatively [V, A]. For PPGLs, conventional radiological imaging can be important to determine the presence of metastases. However, neither CT nor MRI can be used to determine whether PPGLs are benign or malignant. Malignancy can only be determined from the presence of metastatic lesions at sites where chromaffin cells are normally absent. Without such evidence, all PPGLs should be considered potentially malignant, with risk dependent on several factors as outlined below. There are a number of functional imaging modalities available for patients with PPGLs (supplementary File, available at https://doi.org/10.1016/j.annonc.2020.08.2099). The indication is twofold: (i) best tumour staging in patients with suspected metastases (e.g. by conventional imaging) or with presumably high risk for metastases; (ii) to evaluate the option of a radionuclide-based therapy in patients with nonresectable PPGL. For assessments of metastatic risk for the first indication the authors suggest the presence of one or more of the following criteria:•tumour size ≥5 cm;•any extra-adrenal paraganglioma;•known succinate dehydrogenase complex iron sulfur subunit B (SDHB) germline mutation; or•plasma methoxytyramine more than threefold above the upper cut-offs of reference intervals. For imaging-based diagnosis of metastatic PPGLs, it is important to avoid confusing metastases with multiple primary tumours that often occur in patients with hereditary PPGL syndromes. Additionally, local recurrences should not be misdiagnosed as metastases. Biopsy of adrenal tumours is usually contraindicated because of the risk of tumour spillage, poor diagnostic power to discriminate benign from malignant adrenocortical tumours and risk of hypertensive crises in phaeochromocytoma. However, a biopsy might be indicated in an adrenal mass without any hormone excess in patients with a history of extra-adrenal cancers to exclude or prove an adrenal metastasis of an extra-adrenal malignancy, and in patients in whom tumour sequencing is desired. The pathological differential diagnosis of adrenal neoplasias in both biopsied and resected specimens is primarily based on morphological features requiring an experienced pathologist [IV, A]. Preferably, a panel of immunohistochemical markers should be applied to aid diagnosis; for example, steroidogenesis factor 1 (SF1) or, alternatively, inhibin-alpha, calretinin and melan-A for identification of adrenocortical tumours and chromogranin A for identification of PPGL [IV, A]. Staining for tyrosine hydroxylase and synaptophysin may also be helpful to highlight PPGL, but positive results for synaptophysin are also possible in adrenocortical tumours. The differential diagnosis between ACC and adenoma may be challenging as no single marker indicates malignancy. The most widely used diagnostic score has been introduced by Weiss18Weiss L.M. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors.Am J Surg Pathol. 1984; 8: 163-169Crossref PubMed Scopus (768) Google Scholar,19Weiss L.M. Medeiros L.J. Vickery Jr., A.L. Pathologic features of prognostic significance in adrenocortical carcinoma.Am J Surg Pathol. 1989; 13: 202-206Crossref PubMed Scopus (653) Google Scholar and includes nine parameters (supplementary Table S1, available at https://doi.org/10.1016/j.annonc.2020.08.2099) [IV, A]. A score of ≥3 suggests malignancy. In addition, the Ki-67 labelling index, as a marker of proliferative activity, may be useful and is very helpful for prognostic purposes (supplementary Tables S2 and S3, available at https://doi.org/10.1016/j.annonc.2020.08.2099). For phaeochromocytomas, the situation is similarly demanding. Tumour size, SDHB mutation status, extra-adrenal location and plasma methoxytyramine may all be used to indicate risk of metastasis. However, there is no single histological or immunohistochemical parameter that can predict the clinical behaviour of PPGL. Nevertheless, several histological scoring systems have been developed.20Thompson L.D. Pheochromocytoma of the Adrenal gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 cases.Am J Surg Pathol. 2002; 26: 551-566Crossref PubMed Scopus (494) Google Scholar, 21Kimura N. Takayanagi R. Takizawa N. et al.Pathological grading for predicting metastasis in phaeochromocytoma and paraganglioma.Endocr Relat Cancer. 2014; 21: 405-414Crossref PubMed Scopus (193) Google Scholar, 22Koh J.M. Ahn S.H. Kim H. et al.Validation of pathological grading systems for predicting metastatic potential in pheochromocytoma and paraganglioma.PLoS One. 2017; 12: e0187398Crossref PubMed Scopus (45) Google Scholar, 23Pierre C. Agopiantz M. Brunaud L. et al.COPPS, a composite score integrating pathological features, PS100 and SDHB losses, predicts the risk of metastasis and progression-free survival in pheochromocytomas/paragangliomas.Virchows Arch. 2019; 474: 721-734Crossref PubMed Scopus (19) Google Scholar All these scores have value but none of them has reached general acceptance. Therefore, according to the current World Health Organization (WHO) classification, all PPGLs should be considered to have some malignant potential.24Lloyd R.V. Osamura R.Y. Klöppel G. Rosai J. WHO Classification of Tumours of Endocrine Organs. IARC, Lyon, France2017Google Scholar A definitive diagnosis of malignancy can only be made by the presence of metastases at sites where chromaffin tissue is normally absent (e.g. liver, bone, lungs or lymph nodes), assessed by pathology or imaging, in particular functional imaging. Molecular characterisation of ACC (supplementary File, available at https://doi.org/10.1016/j.annonc.2020.08.2099) and PPGL is an active area of ongoing research. The gene encoding subunit B of the SDHB complex is by far the most important molecular contributor to malignant PPGL, with at least 40% of all cases of metastatic PPGLs carrying mutations of this gene.25Brouwers F.M. Eisenhofer G. Tao J.J. et al.High frequency of SDHB germline mutations in patients with malignant catecholamine-producing paragangliomas: implications for genetic testing.J Clin Endocrinol Metab. 2006; 91: 4505-4509Crossref PubMed Scopus (256) Google Scholar Inactivation of SDHB reduces function of the succinate dehydrogenase complex, leading to activation of the hypoxia-inducible pathway and a pseudohypoxic state characterised by increased angiogenesis, growth and expression of mitogenic factors,26Jochmanova I. Yang C. Zhuang Z. Pacak K. Hypoxia-inducible factor signaling in pheochromocytoma: turning the rudder in the right direction.J Natl Cancer Inst. 2013; 105: 1270-1283Crossref PubMed Scopus (126) Google Scholar but also to DNA hypermethylation, which is believed to provide a further drive to metastatic progression.27Letouze E. Martinelli C. Loriot C. et al.SDH mutations establish a hypermethylator phenotype in paraganglioma.Cancer Cell. 2013; 23: 739-752Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar At least 35% of PPGLs result from germline mutations of over 18 tumour-susceptibility genes identified to date,28Dahia P.L. Pheochromocytoma and paraganglioma pathogenesis: learning from genetic Rev Cancer. 2014; PubMed Scopus Google Scholar, Jr., T. et of the practice in the diagnosis and management of Relat Cancer. 2018; 25: PubMed Scopus Google Scholar, J. Pacak K. on pheochromocytoma and paraganglioma: a molecular Rev. 2017; PubMed Scopus Google Scholar, N. A. et on diagnostic of hereditary phaeochromocytomas and Rev Endocrinol. 2017; 13: PubMed Scopus Google Scholar, L. J. K. update on the of it to 2019; PubMed Scopus Google Scholar, J. A. S. et in pheochromocytoma and paraganglioma.Endocr Relat Cancer. 2019; 26: PubMed Scopus (63) Google Scholar with in of hypoxia-inducible factors and and carrying a risk of metastatic disease than to other mutations (supplementary Table available at https://doi.org/10.1016/j.annonc.2020.08.2099). is in patients with PPGL to mutations in the SDHB L. J. E. et in pheochromocytoma or functional paraganglioma.J Clin 2005; 23: PubMed Scopus Google Scholar The of all metastatic PPGLs, including disease to SDHB from or PPGLs with Nevertheless, a of of metastatic PPGLs from adrenal tumours that are characterised by of as by increased plasma or urinary M. et of of SDHB mutation in metastatic J Endocrinol. 2015; PubMed Scopus Google Scholar metastatic disease may only after the primary tumour is and in some patients of disease the of recommendations for follow-up of all patients with resected P.F. Amar L. Dekkers O.M. et al.European Society of Endocrinology Clinical Practice Guideline for long-term follow-up of patients operated on for a phaeochromocytoma or a paraganglioma.Eur J Endocrinol. 2016; 174: G1-G10Crossref PubMed Scopus (251) Google Scholar patients with an adrenal tumour suspected to be an ACC or a phaeochromocytoma should undergo careful clinical assessment for signs of adrenal hormone patients with suspected and proven ACC or PPGL should be discussed in a multidisciplinary expert team meeting, at least at the time of initial diagnosis and in case of progressive patients with suspected ACC a comprehensive endocrine work-up to potential excess of and adrenocortical steroid hormone precursors [IV, imaging for patients with suspected ACC includes at least CT or MRI and chest CT (or [IV, of suspected ACC are usually not and should be ACC is by a score ≥3 [IV, A]. diagnosis should be carried out by an experienced endocrine Ki-67 to the risk of patients with suspected PPGL the of plasma or urinary metanephrines [V, A]. of disease is primarily based on (i) the presence of symptoms or other of excess; (ii) adrenal or (iii) to high risk with hereditary or history of all patients with 'high risk of metastases' a chest CT and at least one functional whole-body imaging FDG-PET, DOTATATE-PET, is recommended in to of suspected PPGL are contraindicated in most PPGLs are by the presence of metastasis. However, all PPGLs are considered to have some malignant In the assessment of disease the authors the metastasis by the European Network for the Study of Adrenal Tumours (supplementary Table available at https://doi.org/10.1016/j.annonc.2020.08.2099) [IV, M. S. M. et prognostic value of the International Cancer staging for adrenocortical carcinoma: for a PubMed Scopus (503) Google Scholar because this to be to other staging systems and is by the for International Cancer and R.V. Osamura R.Y. Klöppel G. Rosai J. WHO Classification of Tumours of Endocrine Organs. IARC, Lyon, France2017Google Scholar indicated above for at least a CT of the chest, abdomen and pelvis (or FDG-PET/CT including full-dose is suggest that lymph node with than R. I. et factors in adrenocortical an European Network for the Study of Adrenal 2015; 26: Full Text Full Text PDF PubMed Scopus Google Scholar the of ACC is However, suggest that in patients with of a tumour survival can be as high as M. S. W. et survival in patients with adrenocortical carcinoma by Clin Endocrinol Metab. PubMed Scopus Google Scholar In metastatic ACC, survival is However, in this there is a of patients with long-term M. S. M. et prognostic value of the International Cancer staging for adrenocortical carcinoma: for a PubMed Scopus (503) Google R. I. et factors in adrenocortical an European Network for the Study of Adrenal 2015; 26: Full Text Full Text PDF PubMed Scopus Google Scholar is well established that disease and are the most important prognostic factors in For the European Society of Endocrinology a comprehensive for prognostic factors has been carried out and only the marker Ki-67 and glucocorticoid excess a with [IV, (supplementary Table available at M. Dekkers O.M. Else T. et al.European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors.Eur J Endocrinol. 2018; 179: G1-G46Crossref PubMed Scopus (403) Google T. M. G. Dekkers O.M. of hormonal functional on survival in adrenocortical carcinoma: systematic review and meta-analysis.Eur J Endocrinol. 2018; 179: PubMed Scopus Google Scholar other markers for and survival have been but clinical use In the WHO a tumour staging for PPGL (supplementary Table available at R.V. Osamura R.Y. Klöppel G. Rosai J. WHO Classification of Tumours of Endocrine Organs. IARC, Lyon, France2017Google Scholar Although this staging remains to be the authors