The Evolving (Epi)Genetic Landscape of Pancreatic Neuroendocrine Tumours

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Endocrine-Related C P Pipinikas et al. Evolution of PanNET genomics 26:9 R519–R544 Cancer REVIEW The evolving (epi)genetic landscape of pancreatic neuroendocrine tumours

Christodoulos P Pipinikas1, Alison M Berner1, Teresa Sposito1 and Christina Thirlwell1,2

1Medical Genomics Laboratory, University College London Cancer Institute, London, UK 2Neuroendocrine Tumour Unit and Department of Oncology, Royal Free Hospital, London, UK

Correspondence should be addressed to C Thirlwell: [email protected]

Abstract

Neuroendocrine neoplasms (NENs) are a relatively rare group of heterogeneous tumours Key Words originating from neuroendocrine cells found throughout the body. Pancreatic NENs ff pancreatic neuroendocrine (PanNENs) are the second most common pancreatic malignancy accounting for 1–3% of tumour all neoplasms developing in the pancreas. Despite having a low background mutation ff MEN1 rate, driver mutations in MEN1, DAXX/ATRX and mTOR pathway genes (PTEN, TSC1/2) are ff DAXX implicated in disease development and progression. Their increased incidence coupled ff ATRX with advances in sequencing technologies has reignited the interest in PanNEN research ff mTOR and has accelerated the acquisition of molecular data. Studies utilising such technological ff MUTYH advances have further enriched our knowledge of PanNENs’ biology through novel ff BRCA2 findings, including higher-than-expected presence of germline mutations in 17% of ff CHEK2 sporadic tumours of no familial background, identification of novel mutational signatures ff alternative lengthening of telomeres and complex chromosomal rearrangements and a dysregulated epigenetic machinery. ff chromatin remodelling. Integrated genomic studies have progressed the field by identifying the synergistic action between different molecular mechanisms, while holding the promise for deciphering disease heterogeneity. Although our understanding is far from being complete, these novel findings have provided the optimism of shaping the future of PanNEN research, ultimately leading to an era of precision medicine for NETs. Here, we recapitulate the existing knowledge on pancreatic neuroendocrine tumours (PanNETs) and discuss how recent, novel findings have furthered our understanding of these complex tumours. Endocrine-Related Cancer (2019) 26, R519–R544

Introduction

Neuroendocrine neoplasms (NENs) develop from cells of NENs are rare tumours previously described as ‘orphan’ neuroendocrine differentiation distributed throughout cancers (Kawasaki et al. 2018). However, this rarity may be the body, most commonly in the small intestine, lung due to variation in disease coding/nomenclature between and pancreas (PanNENs) (Dasari et al. 2017). PanNENs (inter)national cancer registries leading to inaccurate were originally called ‘islet cell tumours’ due to the belief incidence and prevalence rates (IR and PR, respectively) that they arise from islet cells. More recently, it has been (Niederle et al. 2010, Genus et al. 2017). Recent population- suggested that they may originate from pluripotent stem based studies indicate higher IRs than previously cells of the ductal-acinar system (Vortmeyer et al. 2004, described (Fraenkel et al. 2015) with a 3- to 5-fold increase Klöppel et al. 2007). during the last 3–4 decades both worldwide and in the

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United Kingdom (Halfdanarson et al. 2008, Yao et al. 2008, neuroendocrine carcinomas (PanNECs). While G1/G2 Dasari et al. 2017, Genus et al. 2017). Increased detection NETs can be either functional or non-functional, G3 rates are attributed to several factors including use of NECs are mainly non-functional due to loss of hormone more sensitive imaging/diagnostic modalities, refined secretion-associated genes (Sadanandam et al. 2015). This markers of neuroendocrine differentiation (for example, distinction in histological/morphological characteristics chromogranin A, synaptophysin, neuron-specific enolase, and clinical outcomes is also reflected in the molecular/ pancreatic polypeptide), establishment of accurate records, genetic landscape between the two entities (Yachida et al. improvements in training and increased awareness both 2012, Girardi et al. 2017). While TP53 and RB1 recurrent in the clinical field and the general population. mutations are found in NECs, they are rarely seen in NETs. The IR of PanNENs is currently 0.43/100,000 having However, reports indicate that 35–50% of G3 PanNECs been 0.17 in 1973–1977 (2003–2007 SEER) (Lawrence are well differentiated with similar clinical course and et al. 2011). Due to the indolent nature of some NENs the molecular profiles to G2 PanNETs Vélayoudom-Céphise( PR is higher than any other upper gastrointestinal or liver et al. 2013, Sorbye et al. 2014, Basturk et al. 2015, Tang et al. cancer, second only to colorectal cancer when considering 2016). The WHO classification criteria have been recently gastrointestinal malignancies (Yao et al. 2008, Dasari updated (Lloyd et al. 2017) to address this heterogeneity et al. 2017). PanNENs account for 1–3% of all pancreatic by further sub-dividing tumours into WD G3 PanNETs neoplasms and are the 2nd most common pancreatic and PD G3 PanNECs. malignancy (Bosman et al. 2010). Autopsy studies have PanNENs are highly heterogeneous and complex demonstrated that up to 10% of PanNENs remain tumours, which demonstrate diverse clinical behaviours clinically undetected, an observation that may impact (rate of development and progression, metastatic the reported IR/PR (Kimura et al. 1991, Halfdanarson et al. potential, treatment response/resistance and survival 2008, Fraenkel et al. 2012). Survival rates vary depending rates) that ultimately define tumour fate. Although various on grade and stage at diagnosis (Yao et al. 2008) with prognostic indicators and classification systems evolved 5-year survival rates post resection for well-differentiated over time have been clinically useful, they have their tumours of around 65% (de Wilde et al. 2012a). In own inherent limitations and, most importantly, they patients with distant unresectable disease (stage IV), most do not reflect the biological and clinical heterogeneity commonly liver metastases, 5-year survival is 16–46% of PanNETs. Such heterogeneity is more pronounced in (Mignon et al. 2000, Viúdez et al. 2016, Batukbhai et al. G1 and G2 tumours that, unlike the predictable lethality 2019) (Fig. 1A). of G3 NECs, have a variable clinical course ranging from Functionally, PanNENs are divided into functional and indolent to aggressive (Lloyd et al. 2017). non-functional tumours (F-PanNENs and NF-PanNENs, In this review, we provide an update on the evolving respectively). F-PanNENs are associated with various genomic and epigenomic landscape of G1/G2 PanNET clinical syndromes leading to earlier diagnosis due to development and progression and discuss recent novel clinical manifestation of hormone secretion-associated findings. As PD G3 NECs represent a separate tumour symptoms (Fig. 1A). In contrast, NF-PanNENs pose a entity, they are not discussed further. significant early-diagnostic challenge since symptoms only become evident later on in the disease course due to the growing tumour mass. At initial diagnosis, Genetic landscape of PanNETs approximately 60–80% of cases are detected with locally Cancer-predisposition syndromes advanced or metastatic disease (Baur et al. 2016, Viúdez et al. 2016), resulting in a worse prognosis. Although PanNETs are most commonly sporadic, they Classification of PanNENs, according to World Health also arise in the familial setting due to inherited germline Organization (WHO) criteria, is based on the degree mutations in tumour suppressor genes (TSG) that increase of cellular differentiation and fraction of proliferating organ susceptibility to cancer development. These cancer- neoplastic cells as a measure of tumour grade (determined predisposition syndromes (Fig. 1A) account for <10% by the mitotic count and the Ki-67 labelling index). of PanNETs (Marx et al. 2005) and are discussed below. PanNENs are classified into well-differentiated (WD), Recently, germline mutations in DNA damage repair genes, low-to-intermediate-grade (G1 and G2, respectively) such as CHEK2 and MUTYH, have been identified in 17% pancreatic neuroendocrine tumours (WD-PanNETs, >90%) of sporadic cases of apparently no familial background and poorly differentiated (PD), high-grade, G3 pancreatic (Scarpa et al. 2017).

Figure 1 Overview of the multi-layered pancreatic neuroendocrine tumours (PanNET) complexity. (A) Discrimination of PanNENs into those associated with a familial syndrome, due to germline mutations in tumour suppressor genes, and sporadic tumours further sub-divided into functional and non-functional. Epidemiological and survival rates (orange box) and tumour classification according to the WHO 2017 criteria (yellow boxes) are also shown. (B) Involvement of major molecular pathways/mechanisms in PanNET pathogenesis (numbered 1–6) and observed genomic and epigenomic alterations in various implicated genes reported in the literature. Disease mutational burden information in relation to pancreatic ductal adenocarcinomas is shown in the green box. Note: Numbers next to the various genes indicate the pathway/mechanism in which each gene belongs to. Pathways not indicated by a number are those previously implicated in PanNETs but their role needs further investigation to be fully elucidated. Panel A data from (1) Batukbhai et al. (2019), (2) Lloyd et al. (2017), (3) Lawrence et al. (2011), (4) de Wilde et al. (2012a), (5) Ries et al. (2007), (6) Fishbein & Nathanson (2015), (7) Zhang et al. (2013), (8) Franko et al. (2010), (9) Marx et al. (2005). Note: in (B), frequency of genetic events described according to studies discussed in the text. Amp, amplification; CA, chromosomal aberrations; CIMP, CpG island methylator phenotype; cnv, copy number variation; GF, gene fusion; GM, germline mutations; HyperM, hypermethylated, HypoM, hypomethylated; LoH, loss of heterozygosity; MI, Mitotic Index; PDAC, pancreatic ductal adenocarcinoma, PD, poorly differentiated; PTC, post-transcriptional control; re, rearrangement; SM, somatic mutations; WD, well differentiated. *More detailed description of miRNA involvement in PanNETs in Supplementary Table 1.

Multiple endocrine neoplasia Tuberous sclerosis complex 1/2 (TSC1/2) type 1 and 4 (MEN1/MEN4) Tuberous sclerosis (TS), linked to mutations in TSC1 MEN1 is an autosomal dominant syndrome caused and TSC2 genes (chr. 9 and 16, respectively), is a high- by germline mutations in the MEN1 gene (chr. 11q13). penetrance autosomal dominant syndrome associated Affected individuals often display loss of the WT with benign hamartomas, cognitive impairment and MEN1 allele (Larsson et al. 1988), resulting in PanNET epilepsy. Development of PanNETs is less frequent, ranging development in ~40% of cases. Loss of heterozygosity between 1.8 and 9% (Anlauf et al. 2007, Larson et al. 2012, (LoH) is found not only in MEN1-associated tumours, Koc et al. 2017). These are mainly due to germline TSC2 but also in patients without MEN1 (Friedman et al. mutations, consistent with the observation that sporadic 1989). Although most of the resulting tumours are non- PanNETs harbour recurrent somatic mutations mainly functional, ~10–25% of cases develop insulinomas (Anlauf in TSC2 (Jiao et al. 2011). LoH or large chromosome et al. 2007). Other common manifestations include 16 deletions subsequently inactivate the remaining, parathyroid tumours (with hyperparathyroidism occurring WT allele. The proteins encoded by TSC1 and TSC2, in 95% of patients), anterior pituitary tumours, duodenal hamartin and tuberin, dimerise to form a regulatory gastrinomas, adrenocortical and carcinoid tumours. protein upstream of phosphatidylinositol 3-kinase (PI3K) There are also cutaneous signs including angiomas and and mammalian target of rapamycin (mTOR) signalling lipomas (Vortmeyer et al. 1998). Germline mutations in pathway that regulates cell growth. Dysregulation of the MEN1 were also recently identified in sporadic PanNETs mTOR pathway is an intrinsic characteristic of PanNETs of apparently no familial history (Scarpa et al. 2017). It is, and has been successfully targeted by the drug everolimus therefore, important that any patient presenting with a (Larson et al. 2012, Koc et al. 2017). PanNET and a history of primary hyperparathyroidism or pituitary adenomas is referred for genetic testing. Neurofibromatosis type 1 NF1( ) PanNETs also develop in the context of multiple Germline mutations in the NF1 gene (chr. 17) result endocrine neoplasia type 4 (MEN4) due to CDKN1B in neurofibromatosis type 1 (NF1), a highly penetrant germline mutations. CDKN1B encodes the cyclin- autosomal dominant syndrome. NF1 encodes dependent kinase inhibitor p27 with mutations leading neurofibromin, both a negative regulator of the Ras-MAP to increased cell cycle progression and proliferation. The kinase pathway involved in cell growth and proliferation clinical spectrum of MEN4 is similar to MEN1, with ~3% (Evans et al. 2004, Brems et al. 2009) and an inhibitor of those suspected to have MEN1 actually harbouring of mTOR (Johannessen et al. 2005). NF1 patients have a CDKN1B mutation. CDKN1B has been previously distinctive cutaneous features of café au lait patches, found to be mutated in 8% of small intestinal NETs neurofibromas and axillary freckling. Patients may also (Francis et al. 2013). develop malignant tumours including optic or brain stem gliomas, phaeochromocytomas, gastroenteropancreatic Von Hippel-Lindau (VHL) (GEP) NETs, mainly of the small intestine and rarely VHL arises due to germline mutations in the VHL gene pancreatic somatostatinomas or insulinomas (Anlauf on chromosome 3p (Seizinger et al. 1988) and shows an et al. 2007). autosomal dominant mode of inheritance. Inactivation of the WT allele by somatic point mutations, LoH or Sporadic PanNETs loss of 3p results in a number of solid tumours such as haemangioblastomas, clear-cell renal carcinomas, Over the last 10 years, advances in sequencing phaechromocytomas (Kaelin 2002) and PanNETs in technologies have enabled unbiased genome-wide 12–17% of cases (Binkovitz et al. 1990, Libutti et al. analysis of PanNETs by several groups. Such studies have 1998). The VHL-encoded protein forms part of a highlighted the molecular heterogeneity of the disease, ubiquitination complex that targets and degrades driven by the crosstalk between various events (e.g. subunits of hypoxia inducible factor (HIF) 1 and 2. somatic mutations, variations in copy number and DNA Loss results in increased expression of HIF1/2 and other methylation changes) operating at distinct levels (genome factors, such as vascular endothelial growth factor and epigenome, including post-transcriptional control; (VEGF) and promotes formation of vascularised and Fig. 1B). Additionally, these studies have confirmed the cystic tumours (Kaelin 2002). role of previously identified alterations and revealed

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0175 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 06:30:47AM via free access Endocrine-Related C P Pipinikas et al. Evolution of PanNET genomics 26:9 R523 Cancer novel findings (e.g. involvement of germline mutations, Akt preventing its plasma membrane translocation and gene fusions and clinically relevant molecular subgroups) subsequently inhibits the PI3K/Akt/mTOR pathway adding further information to elucidate the molecular (Fig. 2A) (Wang et al. 2011). MEN1 alterations can, characterisation of PanNET development. therefore, trigger a chain of highly orchestrated events resulting in the dysregulation of downstream genes in several inter-connected pathways with catastrophic MEN1, histone modifications, chromatin remodelling consequences, the extent of which ultimately shape the and telomere maintenance fate of a pro-oncogenic cell. Somatic mutations in MEN1 are frequently detected in Furthermore, menin regulates the histone both functional and non-functional sporadic PanNETs methyltransferase activity of a nuclear protein complex and are commonly associated with LoH of the remaining including the mixed lineage leukaemia members 1 and 2 WT allele, consistent with its role as a TSG. Moore et al. (MLL1/2). This promotes trimethylation of histone H3 at (2001) reported that 26% of NF-PanNETs had inactivating lysine residue 4 (H3K4me3) and enhances transcriptional MEN1 mutations, while allelic loss of chromosome 11q13 activity (Agarwal & Jothi 2012). Menin-dependent histone (MEN1 locus) was observed in 70% of informative cases. methylation maintains in vivo the expression of cyclin- This frequency was confirmed in a subsequent study dependent kinase inhibitors (CDKis) p18Ink4c (CDKN2C) providing a link between MEN1 mutations, present in and p27Kip1 (CDKN1B) through active MLL recruitment 30% of cases and alterations in protein expression due to their promoters and coding regions. This increases to the fact that the majority of them (mainly truncating H3K4m3 and prevents islet cell tumour formation (Milne mutations) occur within regions of the gene associated et al. 2005). Dysregulation of this histone modification with subcellular protein localisation (Corbo et al. 2010). complex through menin or MLL loss reduces methylation More recently, Jiao et al. (2011) reported higher MEN1 levels and downregulates CDKis, resulting in uncontrolled mutational frequencies (44.1%) confirming its significant cell growth and tumour formation in Men1-mutant mice role in PanNET development. Most importantly, this study (Karnik et al. 2005). Collectively, these data highlight a provided the first evidence for recurrently inactivating key role for MEN1 in epigenetic control through histone mutations in two genes that had previously not been regulation of genes implicated in neuroendocrine cell implicated in disease pathogenesis. Approximately 43% growth and differentiation. Interestingly, this histone of tumours were found to carry mutations in DAXX (25%) complex is also known to impact the chromatin- or ATRX (17.6%). Within the cohort, 23.5% of tumours remodelling machinery. The involvement of other histone were found to harbour MEN1 mutations in the presence modifications in the aetiology of PanNETs is described in of either DAXX or ATRX, while 20.6% tumours had a more detail under the epigenetic section of this review. single MEN1 mutation. Collectively, 63.2% of tumours Mutations in ATRX are present in 17.6% of PanNETs had MEN1/DAXX/ATRX mutations. Interestingly, the (Jiao et al. 2011). ATRX is a nuclear protein of the SWI/ observed sequence alterations often resulted in truncating SNF complex of chromatin-remodelling genes and has a DAXX and ATRX mutations with loss of the corresponding vital role in maintaining genomic stability. Its depletion protein product through nonsense-mediated decay (Jiao increases local DNA damage and results in loss of structural et al. 2011, Fishbein & Nathanson 2015). The fact that integrity at telomeres and development of endocrine defects mutations in DAXX/ATRX occur in a mutually exclusive (Watson et al. 2013). Recurrent inactivating mutations fashion in the majority of cases indicates that both belong and chromosomal rearrangements in other chromatin- in the same pathway and is consistent with common remodelling genes (SETD2, ARID2, KMT2C (MLL3) and functional roles in apoptosis, chromatin remodelling and SMARCA4) were recently documented in PanNETs (Scarpa telomere maintenance (de Wilde et al. 2012b). et al. 2017) with SETD2 and ARID1A mutations present in Menin, encoded by MEN1, is a ubiquitously expressed 18 and 13% of advanced WD-PanNETs, respectively (Raj scaffold nuclear protein that interacts with >40 proteins et al. 2016). SETD2 mutations were exclusively present to control several vital processes including transcriptional in intermediate-to-high-grade tumours responding to regulation, DNA repair, genome stability, cell cycle temozolomide and capecitabine. Mutations in DAXX, control and miRNA biogenesis (Agarwal et al. 2005, a histone H3.3 chaperone recruited by ATRX, occur in Balogh et al. 2006, Matkar et al. 2013). It plays a key role 25% of PanNETs (Jiao et al. 2011). Once recruited by in coordinating chromatin-remodelling genes through ATRX, it forms a heterodimer guiding the H3.3 variant epigenetic regulation (Kim et al. 2003). MEN1 binds to into nucleosomes where it is incorporated at telomeres

Figure 2 Main pathways and mechanisms implicated in PanNET development and progression. (A) Schematic representation of the interactions between menin/ MEN1, PI3K/AKT/mTOR, Ras-Raf-MAPK-ERK and p53 pathways. A phosphorylation cascade initiated by IRS-1 activates PI3K, which in turn results in downstream AKT activation through PDK1-mediated phosphorylation. Subsequently, AKT inhibits TSC2 through phosphorylation resulting in mTOR activation and phosphorylation of the S6K and 4E-BP1 proteins to increase cellular proliferation and survival. PTEN is a negative regulator of PI3K which reduces its activation by dephosphorylating PIP3 to PIP2. TSC2 contains a GAP (GTPase-activating protein) domain that enhances the conversion of Rheb into its GDP-bound inactive state, thus inhibiting mTORC1. In PanNETs, the negative regulators of the mTOR pathway, PTEN, TSC1/2, MEN1 and DEPDC5 are frequently mutated resulting in increased cellular growth. Menin also inhibits the Ras-Raf-MAPK-ERK by preventing the RAS-SOS interaction and indirectly the mTOR pathway by preventing the RAS-mediated activation of PI3K. Also shown, is the p53 pathway. Its function is tightly controlled by the negative regulators MDM2, MDM4 and WIP1. WIP1 also inactivates p53 through dephosphorylation of its upstream activators (ATM, CHK1/2). Upregulation of miR-21 and miR-196 results in PTEN inhibition and AKT regulation, respectively. Approved drugs (sunitinib, everolimus and somatostatin receptor analogues) are also shown. (B) Interaction between the chromatin remodellers ATRX and DAXX in maintenance of telomere integrity and genome stability. Inactive heterochromatin at both telomeric and pericentric regions, typically reflected in the methylation status of lysine residues on histone 3, is recognized by the N-terminal domain of the ATRX protein containing a DNA-binding domain. DAXX is a histone H3.3 chaperone recruited by ATRX to form a heterodimer. The resulting heterodimer guides the H3.3 variant into nucleosomes where it is incorporated at telomeres and pericentric heterochromatin and other repressed areas of the genome. The chromatin-bound deposition complex might also contain the histone chaperone DEK and the histone deacetylase HDACII. Successful H3.3 incorporation into the nucleosomes allows deposition of a histone tail modification that maintains compact chromatin structure to stabilise telomere length. This interaction is inactive in the presence of loss-of-function, mutually exclusive mutations in ATRX and DAXX observed in approximately 43% of PanNETs and leads to the activation of the ALT phenotype. ALT is a telomerase-independent telomere elongation mechanism associated with an altered DNA damage response/repair mechanism involving homologous recombination (HR). ALT-positive cells show uncontrolled expression of telomere motifs (TTAGGG)n responsible for telomeres of heterogeneous length. Also shown is the HIRA complex that mediates deposition of H3.3 at promoters, transcribed regions and facultative heterochromatin in senescent cells. Panel A data from Stevenson et al. (2018), Oberg et al. (2013), Stolz et al. (2011), Hu et al. (2010). Panel B data from Elsaesser & Allis (2010).

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0175 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 06:30:47AM via free access Endocrine-Related C P Pipinikas et al. Evolution of PanNET genomics 26:9 R525 Cancer and centromeres to mediate chromatin remodelling and patient stratification. Multivariate analysis demonstrated stabilise telomere length (Lewis et al. 2010) (Fig. 2B). that ATRX/DAXX status was an independent prognostic MEN1 has also been shown to play an important role predictor of disease relapse alongside tumour grade and in the biology of telomeres. Lack of telomerase activity stage (Marinoni et al. 2014). Tumour grade was the only and expression of its protein component, hTERT, has been independent predictor of tumour-specific survival. Of associated with the gradual shortening of chromosomal note, ALT status was not tested in either multivariate ends (telomeres) leading eventually to cell senescence analyses. However, a recent study identified ALT positivity (Lin et al. 2003). It has been previously demonstrated through multivariate analysis as an independent risk that menin interacts directly with the hTERT promoter factor for liver metastases (Pea et al. 2018). to negatively regulate its expression. In contrast, hTERT reactivation due to menin protein loss or MEN1 mutations results in increased telomerase activity and telomere PI3K/Akt/mTOR pathway length. In addition, menin depletion results in extended Mutations in the PI3K/Akt/mTOR pathway occur in cellular lifespan and immortalisation due to the ability of 14.7% of PanNETs (Jiao et al. 2011). This pathway is a cells to escape senescence (Lin et al. 2003). A subsequent master cancer regulator involved in several vital cellular study, however, has questioned the role of menin in processes including angiogenesis, cell cycle progression, regulating telomerase activity in normal and cancer cells DNA repair, apoptosis, gene expression and epigenetic (Hashimoto et al. 2008). In fact, a subset of tumours use regulation (Fig. 2A). These processes are facilitated alternative means to maintain telomere lengths (Lovejoy through crosstalk between upstream regulatory proteins et al. 2012). Consistent with an important role for (PTEN, PI3K) and downstream effectors (MDM2, FOXO, telomere biology in NETs, DAXX and ATRX mutations GSK-3β) that are, in turn, under the control of various lead to alternative lengthening of telomeres (ALT; Fig. 2B). compensatory signalling pathways (Ersahin et al. 2015). ALT is a telomerase-independent telomere elongation The first evidence suggesting disruption of the mTOR mechanism associated with an altered DNA damage pathway in NF-PanNET pathogenesis was presented in a response/repair mechanism involving homologous study describing a metastatic primary case with a PTEN recombination (HR) (Lovejoy et al. 2012). Mutations mutation and LoH of the remaining WT allele resulting in genes involved in HR DNA repair have recently in loss of protein expression (Perren et al. 2000). Frequent been identified in PanNETs Scarpa( et al. 2017) and are TSC2 and PTEN gene expression downregulation leading described in the following section. It has been previously to protein level alterations has also been documented shown that PanNETs with DAXX or ATRX mutations and/ in ~80% of NF-PanNETs with more pronounced losses or protein loss show 100% concordance with the ALT correlating with liver metastases, shorter time to phenotype (Heaphy et al. 2011a, Jiao et al. 2011). The ALT progression and shorter disease-free and overall survival phenotype has been described in other human cancers (OS) (Missiaglia et al. 2010). Further evidence implicating including neuroblastomas, paediatric glioblastomas, the PI3K/Akt/mTOR pathway in disease pathogenesis oligodendrogliomas and medulloblastomas (Heaphy et al. stems from the first whole-exome sequencing (WES) study 2011b, Schwartzentruber et al. 2012). of 68 sporadic PanNETs (Jiao et al. 2011) where 14.7% of While neuroendocrine microadenomas, a potential cases (10/68) had recurrent inactivating (loss-of-function) sporadic PanNET precursor lesion, display dysregulated TSC2 (8.8%) and PTEN (7.3%) mutations. One PTEN- menin expression associated with somatic mutations, mutant case also harboured a PIK3CA mutation in a region they lack DAXX/ATRX mutations and are ALT negative. (described as an ‘oncogenic hotspot’) involved in the Therefore, menin/MEN1 alterations are considered as early activation of the kinase domain of the encoded protein events in the pathogenesis of PanNETs (de Wilde et al. (Samuels et al. 2004). Mutations in DEPDC5, another gene 2012b, Hackeng et al. 2016). ATRX/DAXX loss/mutations in the mTOR pathway, were recently identified (Scarpa and ALT positivity have been associated with larger et al. 2017). Although PTEN, TSC1, TSC2 and DEPDC5 tumours (>2–3 cm, G2, T3/T4), chromosomal instability, mutations commonly occur in a mutually exclusive metastatic disease and reduced relapse-free and tumour- manner, a tumour carrying both PTEN/TSC2 mutations specific survival Marinoni( et al. 2014, Pea et al. 2018) has been reported (Jiao et al. 2011). suggesting that these changes are a later event in tumour Collectively, these findings highlight that evolution. This established association may, therefore, dysregulation of the PI3K/Akt/mTOR pathway is an innate have a potential value as a marker of aggressiveness for characteristic of a significant proportion of PanNETs.

This may explain improved survival rates observed in CHK2-mediated increase in phosphorylation of NEK6 a subset of advanced tumours treated with the mTOR and CDK–cyclin complexes is also known to result in inhibitor everolimus (Yao et al. 2011). However, there is G2/M cell cycle arrest (Donzelli & Draetta 2003, Lee currently insufficient evidence to suggest PI3K pathway et al. 2008). In addition, following DNA damage, CHK2 mutations as a biomarker of response (Yao et al. 2019). phosphorylates BRCA1 to restore survival, while it also Indeed, current treatment guidelines are based on clinical regulates DNA repair through BRCA2 transcriptional judgement, while predictive biomarkers of disease stimulation and enhancement of RAD51 interactions recurrence or metastatic progression and treatment with chromatin (Venkitaraman 2001, Jang & Lee 2004). response/resistance are still lacking. Recently, a SNP Germline mutations are generally associated with breast substituting an arginine (R) for glycine (G) in codon 388 cancers, where they confer moderate risk increases, and of FGFR4 (G388R) was linked to PanNETs of higher stage, other carcinomas including sarcomas and brain tumours risk of liver metastases and reduced response to everolimus (Desrichard et al. 2011). MUTYH maintains genome (Serra et al. 2012). integrity by preventing oxidative damage-induced mutations and coordinates error-free base excision repair (BER), thus preventing tumour development (Mazzei et al. DNA damage repair 2013). Germline mutations have been previously linked Single-base excision and HR DNA double-strand break to MUTYH-associated polyposis (MAP), an autosomal repair systems are both part of the DNA damage repair recessive colorectal polyposis syndrome (Sieber et al. machinery. Several lines of evidence support the role of 2003) associated with the accumulation of multiple bowel the HR DNA repair complex in the development of the ALT polyps and increased predisposition to colorectal cancer phenotype. In telomerase-negative yeasts, genes encoding (Al-Tassan et al. 2002). Around 1 in 100 of the general the HR proteins (such as Rad50, Rad51 and Rad52) are population carry a pathogenic mutation but in both essential for their survival (Cesare et al. 2010). In humans, colorectal cancer and PanNETs, evidence suggests that this complex is also vital for telomere length maintenance two germline mutations are required for pathogenesis. and includes several HR proteins such as ATM, CHEK2, Given that NETs have a very low mutational background, BRCA 1 and 2, RAD50 and RAD51. ATM is an important it is interesting that a significant proportion of patients cell cycle checkpoint serine/threonine kinase, which has harbour germline defects in DNA repair pathways. a genome-wide surveillance role and regulates, upon sensing double-strand DNA breaks, various downstream Copy number alterations proteins such as p53 and BRCA1, CHEK2 checkpoint Chromosomal alterations, implicated as potential drivers kinase and other RAD checkpoint proteins required for of PanNET development and progression, have been genome stability. ATM has been found mutated in ~5% studied using various approaches such as fluorescence of PanNETs (Corbo et al. 2012), while germline mutations in situ hybridisation (FISH), comparative genome in BRCA2, CHEK2 and MUTYH, another gene involved hybridisation (CGH) and genome-wide, high-density SNP in single-base repair, were recently identified in PanNETs arrays. Several themes have emerged from these studies, (Scarpa et al. 2017). which are summarised below: Germline BRCA2 mutations are associated with familial breast and ovarian cancer, prostate and PDACs. 1. Multiple chromosomal alterations are an inextricable BRCA2 acts with RAD51 to enable double-strand DNA part of the complex genetic landscape of PanNETs. break repair and mutations predispose to cancers 2. There is a progressive accumulation of chromosomal harbouring multiple structural genomic rearrangements alterations in PanNETs – the extent of such events (Chapman et al. 2012, Stoppa-Lyonnet 2016). CHK2, a correlates with higher tumour stage and disease serine/threonine protein kinase encoded by CHEK2, is a progression: critical cell cycle checkpoint regulator which is activated a. Functional PanNETs (insulinomas and gastrinomas) in response to double-strand DNA breaks (Stolz et al. have the lowest number of chromosomal changes, 2011). It has several tumour suppressor functions and its while some degree of overlap exists between dysregulation may result in chromosomal instability. It has insulinomas capable of metastatic dissemination been implicated in G1 cell cycle arrest and regulation of and non-functional PanNETs. NF-PanNETs have the apoptosis through p53 stabilisation and phosphorylation, highest number of such alterations (Jonkers et al. respectively (Ou et al. 2005, Cai et al. 2009, Roeb et al. 2012). 2007, Capurso et al. 2012).

b. Compared to primary tumours, metastases have a the association between sex chromosome losses and higher frequency of chromosomal alterations (Hu characteristics of disease aggressiveness (higher grade, et al. 2010). advanced stage, local invasion and metastasis and worse 3. Losses are more frequently detected in non-metastatic clinical outcome) (Missiaglia et al. 2002). In agreement tumours, while gains in metastatic disease (Pea et al. with the confirmed role ofATRX in maintaining telomere 2018). integrity and genome stability, it has been previously 4. Recurrent chromosomal changes are often associated suggested that genes on X chromosome may act as with widespread CN-LoH events in other chromosomes negative regulators of cell proliferation through induction which may result in CIN, an intrinsic feature of of senescence (Missiaglia et al. 2002). Collectively, these aggressive cancers (Pea et al. 2018). findings highlight a potential pathogenic function of X 5. The high detection frequency of recurrent alterations chromosome losses in determining disease development at certain chromosomal regions, implicates them and progression. as a potential mechanism, alternative to somatic LoH at 17p13 (TP53 locus) is limited to metastatic mutations, that may drive disease development and NF-PanNETs lacking TP53 mutations (Beghelli et al. 1998), progression. suggesting the presence of other TSGs in the region. The p53 pathway, regulated by both positive and negative Several studies have confirmed the presence of multiple regulators, has a vital role in maintaining genomic chromosomal alterations, generally occurring in stability (Fig. 2A). Among the negative p53 regulators, broad genomic regions which can encompass entire aberrant activity of MDM2, MDM4 and WIP1 impairs p53 chromosomes (Pea et al. 2018). Specific regions associated function, which is a significant step in tumourigenesis. with recurrent gains and losses and LoH are summarised MDM2 (an E3 ubiquitin ligase) binds to and degrades in Table 1. p53, while MDM4, required for MDM2 optimal activity, As previously described, MEN1 is inactivated early interacts and negatively regulates p53 function (Bond in the pathogenesis of PanNETs through somatic et al. 2005, Marine et al. 2007). WIP1 (a serine/threonine mutations in one allele and loss of the remaining WT phosphatase) inactivates p53 either directly or indirectly allele. Therefore, detection of recurrent chromosome through dephosphorylation of p53 upstream activators 11q losses in smaller tumours <2 cm is not unexpected (ATM, Chk1, Chk2), while it is also known to induce p53 (Speel et al. 2001). It has been suggested that both DAXX degradation through MDM2 stabilisation (Lu et al. 2007). (6q21.2) and ATRX (Xq21.1) are inactivated through Although <3% of PanNETs harbour TP53 mutations (Scarpa mutations in one allele and either loss or epigenetic et al. 2017), TP53 is a haploinsufficient gene; the smallest silencing of the remaining allele in the case of DAXX deviation from its normal functions can have a huge (6q21.2) or X chromosome inactivation of the second impact on tumourigenesis (Malkin et al. 1990). MDM2 ATRX allele (Xq21.1) indicating a tumour suppressor role (22%), MDM4 (30%) and WIP1 (51%) amplification, for both genes. In fact, copy number changes involving correlating with mRNA and protein expression, occurs in the DAXX and ATRX locus have been found in 53% and PanNETs (Hu et al. 2010) in a mutually exclusive fashion to 19% of cases, respectively (Scarpa et al. 2017). The notion TP53 mutations (Marine et al. 2007). Furthermore, a high regarding the involvement of an alternative mechanism, frequency functional MDM2 promoter SNP inducing its such as epigenetic silencing, was recently supplemented expression and resulting in p53 attenuation has also been through data showing that DAXX and ATRX-mutant identified in PanNETs Hu( et al. 2010). Collectively, these PanNETs are epigenetically more dysregulated compared observations demonstrate a highly impaired p53 PanNET to WT tumours (Pipinikas et al. 2015). In addition, pathway and highlight the potential value of MDM more pronounced genome-wide epigenetic changes inhibitors in reversing p53 inactivation and sensitising were present in DAXX mutants and in higher grade, G2 PanNETs to other therapies. Chromosomal alterations tumours. In the case of ATRX, 10/98 tumours analysed by included as part of multi-layered data integration studies Scarpa et al. (2017) were found to harbour inactivating are described later in this review. point mutations. Biallelic ATRX inactivation by LoH was exclusively detected in three out of four female patients. Earlier reports have highlighted a higher frequency of Novel findings Xq loss in PanNETs of females (45%) compared to males The most comprehensive genomic study of PanNETs to (5%) (Speel et al. 1999) and have provided evidence for date identified five, whole-genome-derived mutational

Table 1 Chromosomal aberrations identified in PanNETs.

Broad genomic regions commonly associated with recurrent chromosomal losses and gains1-2,a Frequency (%) Losses Putative tumour suppressor genes NF-PanNETs F-PanNETs 1p CDKN2C (1p32.3), RUNX3 (1p36.1), RIZ (1p36.21), 28 3–27 TP73 (1p36.32) 1q RABGAP1L (1q25.1), IL24/MDA7 (1q32.1), PHLDA3 24 10–20 (1q32.1) 3p SFMBT1 (3p21.1), RASSF1 (3p21.31), β-catenin 27 20 (3p22.1), hMLH1 (3p22.2), RARβ (3p24.2), VHL (3p25.3) 6q CCNC (6q16.2), AIM1 (6q21), PTPRK (6q22.33) 38 3–70 10q PTEN (10q23.31), MGMT (10q26.3) 26 3–23 11p WT1 (11p13) 34 3–23 11q MEN1 (11q13.1), ATM (11q22.3), TSG11 (11q23), 39 13–23 SDHD (11q23.1) Gains Putative oncogenes 7p EGFR (7p11.2) 28 6–37 7q HGF (7q21.11), c-MET (7q31) 36 12–47 9p CDKN2A (9p21.3), RRAGA (9p22.1), OVC (9p24), JAK2 19 6–29 (9p24.1) 9q CDK9 (9q34.11), ABL1 (9q34.12), VAV2 (9q34.2), 27 12–43 LMX1B (9q33.3), NOTCH1 (9q34.3) 17q ERRB2 (17q12) 41 5–57 20q AURKA (20q13.2) 31 6–43 Gene-specific changes due to CNVs or biallelic inactivation through LoH2-3,b Frequency (%) Chromosomal event Gene CNV LoH Losses MUTYH (1p34.1) 47 5 CHEK2 (22q12.1) 49 2 BRCA2 (13q13.1) 9 1 SETD2 (3p21.31) 51 4 MLL3/KMT2C (7q36.1) 10 n/a MEN1 (11q13.1) 33–70 40 DAXX (6p21.32) 53 20 ATRX (Xq21.1) 19 3 PTEN (10q23.31) 40 7 DEPDC5 (22q12.2-3) 49 2 TSC1 (9q34) 17 n/a TSC2 (16p13.3) 43 2 PHLDA3 (1q32.1)c n/a 75 EYA1 (8q13.3) n/a n/a Gains AKT2 (19q13.2) 25 n/a ERBB2 (17q12) 25 n/a ULK1 (12q24.33) 31 n/a PSPN (19p13.3) 21 n/a Prevalent chromosomal aberrations associated with metastatic disease3-5,d Frequency (%) Losses 2pq, 3pq, 6pq, 10p, 11pq, 16pq, 22pq 25–75 Gains 4pq, 5pq, 7pq, 9q, 12q, 14q, 17pq, 18q, 19pq, 20q 25–71

[1] Capurso et al. 2012; [2] Scarpa et al. 2017, [3] Pea et al. 2018, [4] Speel et al. 2001, [5] Zhao et al. 2001. aFunctional PanNETs (F-PanNETs) in the study by Capurso et al. (2012) included gastrinomas, insulinomas and metastatic insulinomas. Metastatic insulinomas had the highest observed frequencies in the range indicated for each chromosomal event and were similar to those observed in NF-PanNETs. b91 G1/G2 NF-PanNETs included in the study by Scarpa et al. (2017). Genetic analyses in Pea et al. (2018) included 48 primary WD-PanNETs (<3 cm) of which 24 developed liver metastases. cPHLDA3, a novel TSG, is inactivated in up to 75% of PanNETs through LoH and DNAme in the second allele (Ohki et al. 2014). Its loss inhibits AKT-driven suppression of the mTOR pathway and has been implicated in disease progression. dHigher frequency of chromosomal gains in metastasising tumours, while losses were more prevalent in non-metastatic. Chromosomal gains frequently associated with recurrent CN-LOH events.

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0175 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 06:30:47AM via free access Endocrine-Related C P Pipinikas et al. Evolution of PanNET genomics 26:9 R529 Cancer signatures from 98 G1/G2 PanNETs (Scarpa et al. 2017). Interestingly, Vandamme et al. (2019) identified mutational While two of the signatures, ‘APOBEC’ and ‘age’ were hotspots in DAXX and CYFIP2, a novel proapoptotic significantly enriched in 4 and 5 cases respectively, 69 gene, and multiple low-abundance recurrent mutations cases were found to have the unknown ‘signature 5’, associated with another hotspot in the PTCH2 gene, which has been previously reported in tumours with involved in Hedgehog signalling. Mutations in SMAD4, FHIT gene deletions (Alexandrov et al. 2013, Volinia et al. previously described in small intestinal NETs, were also 2017). LoH at 3p14.2, the FHIT locus, was previously detected in three tumours (Vandamme et al. 2019). documented in 40% of NF-PanNETs (Beghelli et al. 1998). Another novel finding, previously observed in A fourth signature, ‘BRCA’ was enriched in 15 cases of 2–3% of all cancers and in up to 25% of bone tumours which one found to carry a pathogenic BRCA2 germline (Stephens et al. 2011), is the first ever documentation of mutation coupled with LoH in the remaining allele. chromothripsis in PanNETs (Scarpa et al. 2017). Copy However, the most striking finding was the identification number changes and structural variations, compatible of a previously unknown mutational signature (‘novel’) with chromothripsis, were identified in nine PanNETs. in five sporadic tumours enriched for inactivating Four tumours were associated with complex structural germline mutations in the MUTYH gene. Driven by the rearrangements on 11q13 resulting in complete MEN1 detection of germline mutations, Scarpa et al. (2017) inactivation in two of them (Scarpa et al. 2017). In screened the entire cohort for germline mutations in addition, 17 tumours had less complex rearrangements DNA damage repair genes (MUTYH, BRCA2, CHEK2) resulting in inactivation of TSGs: ARID2 (5), MTAP (4), and genes linked to familial PanNETs (MEN1, VHL, SMARCA4 (3), KMT2C/MLL3 (3), SETD2 (1) and CDKN2A CDKN1B). MUTYH germline mutations associated with (1). Of note, in three cases, rearrangements resulted in the same ‘novel’ signature were also identified through oncogenic gene fusions of the EWSR1 with BEND2 and targeted sequencing in a validation cohort of 62 cases. In FLI1. EWSR1 fusions have been proposed as being part total, ~18% of tumours were found to harbour germline of a novel mTOR activation mechanism. Both complex mutations at a higher-than-expected frequency: 8 MUTYH, genomic rearrangements and EWSR1 gene fusions were 6 MEN1, 4 CHEK2 and 1 in each of BRCA2, VHL and associated with shorter telomeres supporting the idea that CDKN1B. However, their penetrance among PanNET cases chromothripsis may arise as result of telomere exhaustion. is currently unknown and warrants further investigation A key limitation in validating novel findings in possibly within a clinical genetic referral context. This list PanNET research is the lack of reliable cell lines and in vivo has recently been expanded through the identification of models, with BON1 and QGP1 cell lines still in use despite novel non-synonymous germline mutations in PIF1 (n = 2), increasing evidence that they are not representative of the involved in transcriptional regulation and chromatin disease phenotype (Boora et al. 2015, Vandamme et al. remodelling, MAPKBP1 (n = 1), a member of the MAPK/ERK 2015). Rip-Tag mouse models for MEN1 and insulinoma signalling pathway (Vandamme et al. 2019) known to have been successfully produced (Bertolino et al. be co-activated with the PI3K/Akt/mTOR pathway in 2003, Crabtree et al. 2003, Olson et al. 2011); however, neuroendocrine tumours through PI3K (Carracedo et al. creating DAXX/ATRX-knockout mice has proved more 2008), and identification of a pathogenic germline variant challenging. There are currently ongoing efforts to leading to somatic loss of ATM (Lawrence et al. 2018). produce neuroendocrine tumour organoids, which, when Although no somatic alterations in MAPK/ERK have been validated would provide a much awaited resource. previously reported in PanNETs, recent identification of novel, high-abundance MAP4K2 and MAPKBP1 mutations in ~16% of tumours potentially implicates the pathway Integrated multi-omic data approaches in disease pathogenesis and highlights a rationale for a Recently, extensive chromosomal alterations involving combined MAPK-mTOR inhibition (Vandamme et al. whole chromosome arm losses or gains were used 2019). The use of MEK inhibitor PD0325901 was shown to classify tumours into four clusters with distinct to enhance the antiproliferative effect of the PI3K-mTOR mutational profiles and ALT status Scarpa( et al. 2017). inhibitor BEZ235 in BON-derived mouse xenografts Cluster 1 characterised by a recurrent pattern of whole (Valentino et al. 2014). Similarly, the antitumoural chromosomal losses (RPCL) in regions previously reported effect of mTORC1 inhibition by rapamycin both in vitro to be affected by loss (1, 3, 6, 8, 10, 11) as well as losses in and in a xenograft mouse model was enhanced additional chromosomal regions (2, 15, 16 and 22). This through MAPK inhibition (Carracedo et al. 2008). cluster included mainly G2 tumours and was enriched for

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Cluster 2, including G1 and G2 tumours, was 2017) and is similar to the frequency of mutations in characterised by limited copy number alterations confined MEN1 and other chromatin-remodelling genes (ARID1A mainly to chromosome 11 and mutations limited and PSIP1) reported for lung carcinoids (Fernandez- to MEN1. Approximately 83% of the cases were ALT Cuesta et al. 2014). Since these mutations affect the negative with 4/6 ALT+ cases carrying a DAXX mutation. epigenetic machinery, the epigenome is likely to be a Cluster 3 included polyploid tumours with gains in all causative tumorigenic mechanism, acting synergistically chromosomes and the highest somatic mutational rates. with the genome to provide somatic cells a survival and Tumours in this cluster were mainly ALT negatives and growth advantage. The epigenetic machinery (from the generally mutationally silent for MEN1, DAXX/ATRX and Greek prefix ε‘ πι’ meaning ‘superimposed upon’ genetics) mTOR genes. Cluster 4 included aneuploid tumours represents an additional layer of stable information with predominantly whole chromosome gains affecting inherited through mitotic division that affects gene several chromosomes. Interestingly, a 100% concordance function without changing the DNA sequence and has between ALT+ and DAXX/ATRX mutations was observed. a fundamental role in cancer development (Jones & Despite the novelty of the described findings, deducting Baylin 2002). useful information regarding their potential prognostic value is not straightforward. However, in this study DNA methylation series, G2 tumours (coinciding with the RPCL cluster) The most studied and best understood epigenetic mark is harbouring mutations in DAXX/ATRX and correlating DNA methylation (DNAme), the covalent addition of a with mutations in mTOR genes were associated with methyl group to cytosines preceding guanines in a CpG poor prognosis. dinucleotide. CpG islands (CGIs) are CpG-rich regions Another recent targeted/whole-genome sequencing present at high frequency in gene promoter-regulatory study reported three CN-based clusters (Lawrence et al. regions. Although CGIs have low methylation levels in 2018) similar to those identified in the Scarpa study. normal cells, cancers are characterised by aberrant CGI- Correlation with clinical data demonstrated that tumours promoter hypermethylation resulting in gene silencing. in cluster 1 had the poorest prognosis, cluster 2 tumours On the other hand, extensive methylation within the had more favourable clinical/pathological outcomes, gene body has an opposite gene expression effect (Yang while those in cluster 3 had variable clinical outcomes. et al. 2014). Overall, cancers are globally hypomethylated, Similar clusters have also been reported by Pea et al. a phenomenon that has been linked to chromosomal (2018). Supplementary Figure 1A, B and C (see section instability, another hallmark of cancer (Eden et al. 2003, on supplementary data given at the end of this article) Fraga et al. 2004). DNA hypermethylation is an early summarises the findings from these three studies. Recently, event in cancer initiation and dictates the rate of disease integration of DNA methylation and gene expression development and progression. profiles demonstrated the presence of two separate The first studies of DNAme in NETs were candidate clusters of WD-PanNETs, with one consisting exclusively gene driven. However, recent development of genome- of MEN1/DAXX/ATRX-mutant cases (Chan et al. 2018) wide DNAme approaches has provided a more detailed enriched for a gene expression signature characteristic of insight into the importance of the mechanism in disease the α-cells of pancreatic islets. In contrast, an earlier study pathogenesis (Pipinikas et al. 2015, Karpathakis et al. identified mature isletβ -cells as the possible cell of origin 2016, 2017). of MEN1/DAXX/ATRX WT PanNETs (Sadanandam et al. Hypermethylation of the RASSF1A gene promoter, 2015; Supplementary Fig. 1D). which is regulated by p53 and DAXX (Zhang et al. 2013), is the most commonly epigenetically dysregulated region observed in 75% of NF-PanNETs (House et al. 2003) and is most frequent in metastatic compared to non-metastatic Epigenetic landscape of PanNETs tumours (Malpeli et al. 2011) and less frequent in PDACs PanNETs are generally mutationally silent compared (Dammann et al. 2003). Functional RASSF1A is known to their exocrine counterpart, pancreatic ductal to suppress miR-21 (discussed in the next section),

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0175 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 06:30:47AM via free access Endocrine-Related C P Pipinikas et al. Evolution of PanNET genomics 26:9 R531 Cancer which is associated with liver metastases (Roldo et al. Post-transcriptional control 2006, Ram et al. 2014). It also leads to p53 stabilisation (microRNAs and lncRNAs) and cell cycle arrest upon DNA damage through Non-coding RNAs (ncRNAs) are post-transcriptional disruption of the DAXX–MDM2 axis (Song et al. 2008). gene regulators implicated in cancer development CDKN2A, TIMP3 and MGMT promoter hypermethylation and progression (Gibb et al. 2011). They are classified has been reported in 40–44% of PanNETs and is into small/short and long non-coding RNAs (sncRNAs associated with more aggressive tumours and worse and lncRNAs, respectively). miRNAs are sncRNAs (~22 prognosis (House et al. 2003, Wild et al. 2003). MGMT nucleotides long) with a post-transcriptional RNA promoter hypermethylation leading to loss of protein interference role leading to gene silencing through their expression has been suggested as a predictive biomarker imperfect 5’-base pairing with target mRNA sequences of temozolomide response (Kulke et al. 2009). Increased (Ambros 2004). Under specific conditions, however, DNAme across multiple CpG loci, known as CIMP they also induce gene expression (Vasudevan et al. 2007, (CpG island methylator phenotype) and LINE1 and Ørom et al. 2008) indicating a dual role in switching Alu hypomethylation, correlating with early recurrence on/off gene expression and have, therefore, both and disease aggressiveness, have also been identified in oncogenic and tumour suppressing properties. Up to 1000 PanNETs (Arnold et al. 2007, Choi et al. 2007, Stricker et al. miRNAs present in the human genome regulate ~30% of 2012). Finally, PanNET subgroups with distinct global the transcriptome with a single miRNA targeting or being hypomethylation and gene-specific hypermethylation the target of hundreds of mRNAs (Berezikov et al. 2005, profiles associated with different clinical outcomes have Holley & Topkara 2011). Their differential expression not been described (Stefanoli et al. 2014). Genes aberrantly only between different cancer types but also between methylated in PanNETs are summarised in Table 2. subtypes of the same tumour and correlation with various clinical characteristics coupled with their stability in Histone modification (acetylation and methylation) circulation, makes them promising candidates for the Several histone modifications, including methylation, development of non-invasive diagnostic/prognostic acetylation and demethylation, have been described. tools. Long non-coding RNAs (>200 nucleotides long) Histone methylation of genes encoding other CDKis, regulate gene expression, both transcriptionally and post- such as p16Ink4a (CDKN2A) and p14ARF, has been identified transcriptionally (splicing and translation) (Hauptman as an early driver in NET pathogenesis. Enhanced & Glavač 2013), while the involvement of individual H3K9me3 activity through menin–Daxx interactions lncRNAs in chromatin remodelling suggests an active leads to epigenetic inhibition of a pro-proliferative gene, role in moulding the epigenome of cancer cells (Gupta Mme, and results in NET suppression in mice (Patel et al. et al. 2010). 2018), while Men1−/− mouse embryonic fibroblasts have reduced global methylation of histone H3K9 and H3K4. In MicroRNAs addition, menin interacts with the SUV39H1 (suppressor One of the earliest studies of miRNAs in PanNET of variegation 3-9 homolog protein family) and mediates development was a study comparing their regulation in H3K9 methylation. This menin-dependent recruitment normal pancreas, F-/NF-PanNETs and pancreatic acinar cell of SUV39H1 is essential for chromatin remodelling and carcinomas (PACCs) (Roldo et al. 2006). Identified miRNAs is reduced through introduction of patient-derived MEN1 were able to discriminate tumours from normal pancreas, mutations into the SUV39H1 interaction domain (Yang with those shared in common between PanNETs and PACCs et al. 2013). Furthermore, downregulation of MEG3 and having a possible involvement in pancreatic tumourigenesis HOX genes has been associated with development of non- (Supplementary Table 1). Similarly, miRNAs upregulated functional pituitary adenomas and parathyroid tumours, in PanNETs in comparison to PACCs were suggested as respectively (Shen et al. 2008, Cheunsuchon et al. 2011). having a possible role in either normal neuroendocrine A study in mouse embryonic stem cells demonstrated differentiation or NET pathogenesis. Compared to normal that both the Meg3 and Hox loci are regulated by menin pancreas, miR-103 and miR-107 were upregulated and in a H3K4me3-dependent manner. Loss of menin results miR-155 downregulated in PanNETs, while miR-204, in H3K4me3 loss and gene downregulation and gives rise miR-211 and miR-203 were upregulated in insulinomas to MEN1-like sporadic pancreatic tumours (Agarwal & compared to NF-PanNETs. With regards to clinical Jothi 2012). presentation, miR-21 (targets and down-regulates PTEN) was

Table 2 Genes with aberrant DNA methylation in sporadic, non-functional pancreatic neuroendocrine tumours (NF-PanNETs).

DNAme DNAme pattern Function/pathway Gene Chromosome frequency Disease association References HyperM Cell cycle regulation/apoptosis RASSF1A 3p21.31 75–80% Larger tumours and [1], [2], [3] presence of metastatic disease Cell cycle regulation/apoptosis CDKN2A 9p21.2 0%, 17%, 40%, Early tumour recurrence, [1], [2], [4], (p16INKA4a) 52% metastasis and reduced OS [5], [6], Cell cycle regulation/apoptosis P73 1p36.32 17% Higher DNAme frequency in [2] larger tumours associated with metastasis Cell cycle regulation/apoptosis APC 5q22.2 21–48% Increased proliferation [2], [4] and CIN Cell cycle regulation/apoptosis HIC-1 17p13.3 93% Worse prognosis (as part of [4] CIMP) Inhibition of tumour invasion E-cadherin 16q22.1 23% Higher DNAme frequency in [2] larger tumours associated with metastasis Inhibition of tumour invasion TIMP3 22q12.3 0–57% Increased risk of metastatic [2], [4], [7], disease (LNs and liver) [8], [9] Gene expression regulation RAR-β 3p24.2 25% Higher DNAme frequency in [2] larger tumours associated with metastasis Genome integrity/DNA repair hMLH1 3p22.2 0–23% Higher DNAme frequency in [2], [4] larger tumours associated with metastasis Genome integrity/DNA repair O-6-MGMT 10q26.3 17%, 40% Hypermethylation possibly [2], [4] predicts response to temozolamide Angiogenesis - Hypoxia VHL 3p25.3 6% Reduced DFS [10] pathway (HIF) activation Multiple functions and MEN1 11q13 19% Worse prognosis (as part of [4] pathway interactions CIMP) Regulation of apoptosis, RUNX3 1p36.11 7% Worse prognosis (as part [4] angiogenesis, EMT, cell of CIMP) migration/invasion CpG Island Methylator CIMP – 29–83% Correlation with higher Ki67 [4] Phenotype and poor prognosis Regulation of AKT activity, cell PHLDA3 1q32.1 100% Disease progression and [11] proliferation and apoptosis poor prognosis Cellular differentiation HOPX 4q12 G1: 7%, G2: Higher grade and worse [12] 12%, G3: 67% prognosis Cytoskeletal protein with α-internexin 10q24.33 N/A Advanced stage, metastasis, [13] potential role in tumour recurrence and reduced OS initiation-progression HypoM Transposable element LINE1 – Variable Higher tumour grade, LN [9], [14], methylation metastasis and worse [15] levelsa prognosis Transposable element Alu – Variable Higher tumour stage and [14] methylation poor prognosis levels

LINE1 methylation levels <58% observed in 21% of tumours were associated with worse prognosis (Stefanoli et al. 2014). In Arnold et al. (2007), overall DNAme frequencies were given across different PanNET subtypes (benign and malignant functional and non-functional PanNETs). [1]Dammann et al. 2003; [2] House et al. 2003; [3] Malpeli et al. 2011; [4] Arnold et al. 2007; [5] Muscarella et al. 1998; [6] Serrano et al. 2000; [7] Wild et al. 2003; [8] Arnold et al. 2008; [9] Stefanoli et al. 2014; [10] Schmitt et al. 2009; [11] Ohki et al. 2014; [12] Ushiku et al. 2016; [13] Liu et al. 2014; [14] Choi et al. 2007; [15] Stricker et al. 2012. aLINE1 median methylation levels were 66% in G1 (range 61–72%) and 63% in G2 (range 56–64%) PanNETs compared to a median methylation level of 73% in normal pancreas (Stricker et al. 2012). Alu, short interspersed element (Arthrobacter LUteus Homologues); CIN, chromosomal instability; DFS, disease-free survival; EMT, epithelial-to- mesenchymal transition; LINE1, long interspersed element; LN, lymph node; NF-PanNETs, non-functional PanNETs; OS, overall survival.

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0175 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 06:30:47AM via free access Endocrine-Related C P Pipinikas et al. Evolution of PanNET genomics 26:9 R533 Cancer strongly associated with both higher Ki67 and metastatic through regulation of the p38/ERK/Akt and Wnt/β- disease. PDCD4 mRNA expression, a tumour suppressor catenin pathways. The MEG3/miR183/BRI3 axis may, gene and putative target of miR-21, was downregulated in therefore, play a significant role in PanNET development metastatic tumours. Grolmusz et al. (2018) subsequently (Zhang & Feng 2017). Furthermore, MEG3 directly targets used this dataset to identify prognostic miRNAs correlating the c-MET proto-oncogene with human MEN1-associated with altered proliferation, tumour stage and survival rates. PanNETs demonstrating MEG3 downregulation and Hierarchical miRNA clustering identified two clusters: C1 c-MET upregulation. In human sporadic insulinomas, this including 82% of G1 tumours and C2 including mainly discordant MEG3–c-MET expression is associated with G2 (11/18) and further confirmed miR-21 upregulation in MEG3 promoter hypermethylation, while demethylating metastatic disease. In a follow-up study, positive correlation agents reactivate MEG3 and suppress cell proliferation with Ki-67 and metastatic disease was demonstrated in mouse insulinoma cells. Therefore, epigenetic MEG3 for miR-642 and miR-210, respectively, but not miR-21 reactivation and c-MET suppression may induce a MEN1- (Thorns et al. 2014). As miR-210 is induced by HIF-1a, it dependent anti-tumour response and represents an has been suggested to contribute to tumour adaptation to attractive therapeutic approach worth evaluating (Modali hypoxia-related stress and to determine disease initiation et al. 2015). and metastatic potential of PanNETs (Huang et al. 2009). Two lncRNAs, the HOX antisense intergenic RNA Overexpression of miR-196 (regulates AKT signalling and (HOTAIR; 12q12.13) and the metastasis-associated lung HOX gene expression) and miR-27b has also been linked to adenocarcinoma transcript 1 (MALAT1; 11q13), were more aggressive tumours, disease recurrence and decreased recently implicated in PanNETs, with both tumour- OS. miR-196 was also associated with shorter DFS suggestive promoting (Hajjari & Salavaty 2015, Zhao et al. 2018) and of a potential value in patient stratification Lee( et al. 2015). tumour-suppressing (Cao et al. 2016, Kwok et al. 2018) Recently, a 30-miRNA signature demonstrated the presence overexpression observed in various human cancers. Their of three clusters matching with mRNA-based disease tumour-promoting overexpression is due to activation subtypes with distinct clinical characteristics (Sadanandam of downstream pathways including Wnt/β-catenin et al. 2015). miR-Cluster-1 tumours were enriched in (Hirata et al. 2015) and ERK/MAPK (Wu et al. 2014) and the MEN1-like/intermediate mRNA subtype, composed induction of epithelial-mesenchymal transition (EMT) of NF-PanNETs associated with a moderate metastatic through epigenetic regulation of genes such as E-cadherin potential. miR-Cluster-2 was enriched in the metastasis- downregulation and induction of gene members of the like primaries (MPL) mRNA subtype and included tumours MMP family (Hajjari & Salavaty 2015, Hirata et al. 2015). In with high metastatic potential. miR-Cluster-3 included PDACs, overexpression of both lncRNAs is associated with mainly insulinomas, of which none was associated with more aggressive disease and poorer disease-free survival metastatic disease and was enriched in the ‘islet/insulinoma’ (Kim et al. 2013, Pang et al. 2015). HOTAIR is a chromatin- (IT) mRNA cluster. Distinct gene mutational rates in mTOR, modifying lncRNA involved in re-programming the MEN1/DAXX/ATRX and DNA damage pathways were global chromatin state of cancer cells (Valastyan et al. associated with each cluster (Supplementary Fig. 1D). 2012). It has been previously implicated in prostate cancer Additional studies have investigated miRNA regulation neuroendocrine differentiation (Chang et al. 2018), while in PanNETs for a variety of purposes (Li et al. 2013, Zhou its overexpression results in alterations in H3K27me and et al. 2016, Zimmermann et al. 2018, Panarelli et al. 2019). increases the metastatic potential of breast cancer cells Supplementary Table 1 provides a summary of miRNAs (Gupta et al. 2010). Targeting HOTAIR has been shown to implicated in PanNETs. reduce cell invasiveness through induction of apoptosis and cell cycle arrest (Kim et al. 2013). However, a recent Long non-coding RNAs study on 83 primary GEP-NENs (72% G1, 20% G2, 8% G3) Studies of lncRNAs in PanNETs are scarce with most of which 52% developing in the pancreas, demonstrated having evaluated MEG3, which is activated by menin in an that upregulation of both lncRNAs is associated with H3K4me3-dependent manner and CpG hypomethylation less aggressive disease and better prognosis (Chu et al. at the promoter region (Agarwal & Jothi 2012). In BON1 2019). Another lncRNA, telomeric repeat-containing cells, MEG3 upregulation demonstrated an anti-tumour RNA (TERRA), which regulates telomerase activity and effect that was eliminated in the presence of miR-183 heterochromatin formation, is suppressed in the presence overexpression targeting BRI3 to promote tumourigenesis of active ATRX (Cusanelli & Chartrand 2015).

Clinical implications (Strosberg et al. 2017) demonstrated the efficacy of Lu-177- dotatate in midgut NETs and results of this and other Molecular targeted therapies retrospective studies in PanNETs (Ramage et al. 2018) have The most successful targeted therapies in PanNETs are led to its FDA approval in advanced GEP-NETs. However, those against the somatostatin receptor (SSR), known to date, it has not been possible to drug all of the pathways to be overexpressed in approximately 70% of cases identified as PanNET drivers. The two drugs that have (Grozinsky-Glasberg et al. 2008). The CLARINET study been the most successful for therapy target the mTOR demonstrated a significantly improved PFS with the and VEGF pathways. Everolimus, an oral mTOR serine- somatostatin receptor analogue (SSA) lanreotide over threonine kinase inhibitor, is now used routinely to treat placebo (Caplin et al. 2014) in PanNETs, while efficacy pancreatic and intestinal NETs along with typical/atypical of octreotide was extrapolated from the PROMID trial bronchial NETs (Yao et al. 2011). Sunitinib, an oral tyrosine in midgut NETs (Rinke et al. 2009). SSAs result mostly kinase and potent inhibitor of VEGF and platelet derived in stable disease and have become standard first-line growth factor (PDGF) receptors (Faivre et al. 2006), has treatment for slow growing, G1 or G2 PanNETs and those proved effective in targeting activated angiogenic PanNET with a functional syndrome (Pavel et al. 2016). Peptide pathways (Raymond et al. 2011, Faivre et al. 2017). Results receptor radiotherapy (PRRT) targets the SSR using an are awaited from an ongoing study, which aims to select SSA attached to a radioactive isotope such as Lu 177 or between everolimus and sunitinib depending on whether Y 90 and is effective in those tumours overexpressing tumours harbour predominantly mutations in the the SSR on radionuclide imaging. The NETTER-1 study mTOR or angiogenesis pathways (Neychev et al. 2015).

Figure 3 Timeline of key molecular, therapeutic and disease model developments in neuroendocrine tumour research with a special focus on PanNETs, and future perspectives. [1] Oberndorfer (1907)*, [2] Oberndorfer (1929)*, [3] Maher et al. (2011), Davison et al. (1936), [4] Wermer (1954), [5] Hanahan (1985), [6] Larsson et al. (1988), [7] Pless (2005), [8] Latif et al. (1993), [9] Chadrasekharappa et al. (1997), [10] Crabtree et al. (2003), [11] Pellegata et al. (2006), [12] Missiaglia et al. (2010), [13] Jiao et al. (2011), [14] Heaphy et al. (2011a), [15] Blumenthal et al. (2012), [16] Stefanoli et al. (2014), [17] Mohamed et al. (2014), [18] Sadanandam et al. (2015), [19] Pipinikas et al. (2015), [20] Scarpa et al. (2017), [21] Lloyd et al. (2017), [22] Guo et al. (2017), [23] https://www. fda.gov/drugs/resources-information-approved-drugs/fda-approves-lutetium-lu-177-dotatate-treatment-gep-nets; accessed 05/05/2019, [24] Kunz et al. (2018), [25] Chamberlain et al. (2018). (a) Original references (1) and (2) from Modlin et al. (2004); (b) 2018 – E2211 trial shows temozolomide and capecitabine achieved the longest PFS in advanced low or intermediate-grade PanNETs. ALT, alternative lengthening of telomeres; CIMP, CpG island methylator phenotype; cfDNA, cell-free DNA; CTCs, circulating tumour cells; FDA, Food and Drug Administration; iPSC, induced pluripotent stem cells; PDX, patient-derived xenografts.

However, preliminary results suggest that only 7.7% of Immunotherapy tumours had actionable mutations in either pathway. A deeper understanding of the genomic PanNET Combination therapies targeting both mTOR and VEGF landscape will greatly improve patient stratification for pathways or using targeted agents in combination with immunotherapies. Phase Ib (KEYNOTE-028) and phase somatostatin analogues (SSAs) have not shown clinically II (KEYNOTE-158) trials of PD1 inhibitor pembrolizumab meaningful benefits and have been associated with several in PanNET patients have shown modest activity limited adverse events (Kulke et al. 2015, 2017). mainly to patients with PD-L1 >1%, with the best response Inhibition of mTOR results in increased PI3K-Akt being stable disease (Mehnert et al. 2017, Strosberg et al. signalling and, therefore, dual targeting of these pathways 2019). Disease control in WD-PanNETs was shown in has been explored, with some success, in cell lines and an ongoing trial for the PD1 inhibitor spartalizumab pre-clinical models (Antonuzzo et al. 2017). However, (PDR001) (Yao et al. 2018), while PD-L1 positivity and the phase II trial of the PI3K mTOR inhibitor BEZ235 tumour mutational burden (TMB) are currently evaluated combined with downstream mTOR inhibition resulted in as biomarkers of efficacy as part of a trial for the PD1 non-significant improvement in survival and high adverse inhibitor sintilimab (IBI308) (Jia et al. 2018). TMB is a side effects rates (Salazar et al. 2018), while the pan-Akt recognised biomarker for immunotherapy because of inhibitor MK-2206 (phase II trial) also showed modest its relationship to neoantigen burden, which in turn benefits (Reidy-Lagunes et al. 2012). Resistance can also stimulates T-cell proliferation and increases immune develop following VEGF inhibition through HIF-α and reactivity (McGranahan et al. 2016). Although PanNETs c-MET activation (Sennino et al. 2012). Cabozantanib, generally have a low TMB (Yao et al. 2019), it is possible a multi-tyrosine kinase inhibitor targeting VEGF and that immunotherapy may still prove effective for rare cases c-MET, has shown responses in early-phase trials, though with advanced disease and high TMB or other biomarker, final results are currently awaited Chan( et al. 2017). The when conventional therapies have failed. CDK4/6 inhibitors, palbociclib and ribociclib, commonly Due to modest responses to single-agent checkpoint used in breast cancer, are currently being trialled in inhibition, dual blockade is being considered. The DUNE PanNETs due to the observed overexpression of CDKs phase II trial recruits a range of NET patients including (Tang et al. 2012, Pavel & Sers 2016). G1-G3 PanNETs for treatment with anti-CTLA4 and anti- PD-L1 combination therapy (Hernando-Cubero et al. 2018). Another option to improved checkpoint inhibition Epigenome-based targeted therapies response may be addition of metronomic chemotherapy Given the high CIMP prevalence in PanNETs (Stefanoli (Chen et al. 2017) to promote inflammation and induce et al. 2014) and DNMT activation following MEN1 loss mutations to increase neoantigen presentation. There (Yuan et al. 2016), studies have examined the efficacy have also been promising results from early studies in of DNA methyltransferase inhibitors (DNMTis). Despite oncolytic virus therapy and CAR-T cell therapy (Vedvyas success in cell lines, these drugs have not yet been et al. 2016, Yamamoto et al. 2017). trialled in humans (Alexander et al. 2010). Interest has also been shown in histone deacetylase inhibitors (HDACis) due to the involvement of MEN1 in chromatin Conclusions remodelling. Although response rates for the HDACi panobinostat were low, stable disease was demonstrated Over 100 years have passed since Siegfried Oberndorfer in a proportion of patients (Jin et al. 2016). Inhibitors first used the term ‘Karzinoid’ to describe this rare, of the bromo and extraterminal domain (BET) protein unusual tumour entity. Figure 3 illustrates some of the family, which interact with acetylated histone residues, most important findings in PanNET research. Although have also shown success in pre-clinical studies. One our understanding surrounding NET biology is far from compound, JQ1, reduced growth and induced apoptosis being complete, recent PanNET studies have benefited in both cell lines and mouse PanNET models (Lines et al. from sequencing advances and have started to provide 2017). RRx-001, inhibiting both DNAme and HDACs, is a deeper understanding of their complex molecular being trialled to induce global epigenetic changes and landscape. However, tumour heterogeneity remains resensitise patients to chemotherapy (Carter et al. 2015, poorly defined and has delayed the development of better Padda et al. 2018). diagnostic/prognostic tools and novel, more efficacious

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Received in final form 29 May 2019 Accepted 26 June 2019 Accepted Preprint published online 26 June 2019