Abstract Background Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing but highly metastatic, with most patients presenting metastases at diagnosis. Radical surgery remains the only potential curative option when feasible. Consequently, there is a critical need for novel therapeutic strategies that can limit tumor progression and enable more personalized treatment approaches in combination with current clinical practices. In this study, we evaluated the impact of metformin on SI-NET cell growth in vivo, characterized the associated microRNA expression profile, and identified potential driver genes modulated by metformin treatment. Methods A total of 22 SI-NET xenograft mouse models were established using CNDT2.5 and GOT1 cells. Mice were treated with metformin (2.56 mg/mL in drinking water) or water as control for 4 weeks. To explore the molecular impact of metformin, both small-RNA and total-RNA sequencing were performed on the dissected xenograft tumors. Proliferation and apoptosis were further evaluated by immunohistochemistry. Results In vivo treatment of SI-NET cells with metformin led to a reduction in tumor size in both CNDT2.5 and GOT1 xenograft models. Our sequencing analyses identified seven altered microRNAs and 1,776 differentially expressed genes in metformin-treated tumors compared to controls. To uncover potential driver genes in SI-NETs affected by metformin, we compared the differentially expressed genes from GOT1 xenograft model with those identified by comparing single-cell RNA profile of enterochromaffin cells to SI-NETs. This novel approach revealed a set of significantly regulated genes, including those involved in tumor proliferation, apoptosis, and metastasis, as well as genes related to voltage-gated calcium channels and signal transduction. Conclusions Our novel findings support further investigation of metformin as a potential therapeutic agent in clinical trials for SI-NET patients, and suggest that identified miRNAs should be assessed as potential predictive biomarkers for metformin treatment. This study highlights novel candidate driver genes affected by metformin, which are associated with key cellular processes and enterochromaffin cell’s function, offering insights into the underlying mechanisms in SI-NETs.

BACKGROUND:Several studies have attempted to identify the initiating drivers of small intestinal neuroendocrine tumor (SI-NET) development and the molecular mechanisms underlying their progression and metastatic spread. Previous gene expression studies have used bulk microarrays or RNA sequencing to compare tumor tissue with normal intestinal mucosa. However, the intestine comprises multiple distinct cell types, and bulk analyses are limited by this cellular heterogeneity, which can confound tumor-specific signals. METHODS:We performed single-cell RNA sequencing on primary SI-NETs and paired normal mucosa from two patients to directly compare tumor cells with their cells of origin, the enterochromaffin (EC) cells. To minimize type I errors, we applied a two-step validation strategy by overlapping differentially expressed genes with an external single-cell dataset and cross-referencing candidate genes for enteroendocrine expression in the Human Protein Atlas. RESULTS:For further distinction and characterization, ECs were subdivided into serotonergic and non-serotonergic clusters. This analysis revealed that the SI-NET cells are transcriptionally more similar to serotonergic ECs, consistent with serum metabolite profiles derived from clinical parameters. Our analyses uncovered a loss-of-expression program characterized by regulators of epithelial differentiation and in parallel, a gain-of-expression program displayed neuronal signaling gene induction, implicating functional reprogramming toward neuronal-like properties. Together, these specific losses and gains suggest that our patient-derived SI-NETs undergo adaptation through both loss of enteroendocrine functions and acquisition of neurobiological-promoting signaling pathways. CONCLUSIONS:These findings nominate candidate drivers for further functional validation and highlight potential therapeutic strategies in our patient cohort, including restoring suppressed Notch signaling and targeting aberrant neuronal signaling networks. However, even with a two-step validation procedure, the modest cohort size limits statistical power and generalizability, particularly for the proposed association to a serotonergic phenotype. Larger, multi-patient single-cell studies are required to confirm these mechanisms and establish their clinical relevance.

Small intestinal neuroendocrine tumors are often diagnosed at an advanced stage, with up to 70% of patients presenting with stage IV disease. While some guidelines recommend prophylactic resection of the primary tumor and mesenteric lymph node metastasis in patients without abdominal symptoms at diagnosis to prevent future abdominal complications, the benefit of this approach remains uncertain. This retrospective cohort study included 44 asymptomatic patients with stage IV small intestinal neuroendocrine tumors treated at Uppsala University Hospital between 2014 and 2019. Additional ten symptomatic patients who underwent at least two computed tomography scans before planned surgery were included in the analysis of mesenteric metastasis volume change and tumor growth rate. The primary outcomes were abdominal symptoms development requiring surgical intervention and the assessment of mesenteric metastasis size progression. During a 10-year follow-up, only four initially asymptomatic patients (9%) developed symptoms leading to surgery. Among all 54 patients, the median volume change in mesenteric metastases was -298 mm3 (IQR: -2,785-1,294), with no significant difference between baseline and most recent scans (P = 0.38). The median interval between scans was 29 months, and the median tumor growth rate was -0.6% per month (IQR: -3.6-1.9%). Similar results were observed in the asymptomatic group. These findings suggest that a non-operative management in stage IV patients without abdominal symptoms is associated with a low incidence of symptom development and limited progression of mesenteric metastases.

Objective:Primary aldosteronism (PA) is a common cause of hypertension. It entails elevated morbidity and mortality that do not sufficiently improve with conventional antihypertensive therapy. Screening for PA by plasma aldosterone-renin ratio (ARR) enables discovery and specific treatment of affected patients. By screening primary care patients with hypertension and evaluating them further according to the Endocrine Society guidelines, we aimed to assess the prevalence of PA, the factors affecting biochemical diagnostics, and the outcome of lateralization studies and of specific treatment of the discovered PA cases. Design patients and methods:Prospective evaluation of screening for PA was conducted in 1,181 patients. Screening by ARR was performed under current therapy, but without mineralocorticoid receptor antagonists (MRA), under normokalemia, and confirmed by the intravenous saline suppression test, SST#1. Those with results in a defined gray zone underwent therapy adjustment and then completed SST#2. Plasma aldosterone and ARR were compared under different stages of the diagnostic process. All patients with PA were offered adrenal venous sampling, or, in certain cases, adrenocortical-specific positron emission tomography. Lateralizing cases were offered laparoscopic adrenalectomy. Patients with bilateral disease were treated with MRA. Treatment results were assessed after a minimum of 6 months. Results:A total of 53 discovered cases of (mostly mild) PA corresponded to its prevalence of 4.5%. Initial seated ARR was higher than recumbent ARR before SST#1. At SST#2, initial ARR and final aldosterone were higher than at SST#1. Localizing studies (accepted by 45 patients) found 14 lateralized cases. Of the 11 operated cases, 4 had aldosterone-producing adenoma, and the remainder had micro- and macronodular histopathology. A total of 31 patients had bilateral PA. Both surgical and conservative treatments were well tolerated and led to improved blood pressure and higher renin, indicating risk amelioration. Conclusions:PA is prevalent among primary care patients with hypertension and can be screened for under current antihypertensive therapy. Aldosterone-producing adenoma was rare in this cohort. The study results support active screening of primary care patients with hypertension for PA in order to offer appropriate treatment options.

BackgroundPrimary aldosteronism (PA) is the principal cause of secondary hypertension; it leads to significantly elevated cardiovascular morbidity and mortality, but only a fraction of its cases ever get detected, partially due to diagnostic procedures that are difficult to perform and to interpret. More straightforward diagnostic methods are needed. Lateralized, or unilateral PA (uPA), is best treated by surgery. Bilateral PA (bPA) is treated medically.AimThe aim of our study was to explore microRNA (miRNA) in peripheral blood as markers of PA, uPA and bPA.MethodsIn groups of subjects with primary hypertension (HT, n = 11), bPA (n = 12), and uPA (n = 16), peripheral serum was used for isolation of total RNA, library preparation, and NGS sequencing to achieve a comparative analysis of miRNA expression. Five-fold cross-validation support vector machine learning (ML) models were employed to search for miRNA that could be used as markers of PA and its forms.ResultsIn our cohort of patients, the discovered combinations of miRNAs could, with a high level of accuracy, sensitivity, and specificity, characterize the difference between HT and PA, as well as between a combined group of HT + bPA vs. uPA. The differentiating parameters were moderately good for comparison of bPA vs. uPA.ConclusionWithin our patient cohort, and using ML, the study identified distinctly different miRNA profiles between HT and PA, as well as between bPA and uPA. Further validation studies may lead to the emergence of a new tool for clinical diagnostics of PA.

Small intestinal neuroendocrine tumours (SI-NETs) are the most common malignancy of the small bowel. Curative treatment is surgical, with exploratory laparotomy considered the standard approach. This study aimed to assess the outcomes of minimally invasive surgery compared to open approach for SI-NETs at the Endocrine surgical unit at Uppsala University Hospital. This retrospective cohort study included patients who underwent surgery for SI-NET between 2013 and 2023 at Uppsala University Hospital. Variables such as operative time, length of hospital stay, use of analgesia and radicality were compared between groups of patients operated on before and after 2019, when hand-port assisted laparoscopic surgery (HALS) for SI-NETs was introduced at our unit. Outcomes were further compared between open and hand-port assisted laparoscopic approaches. The primary outcome was the rate of radicality achieved for stage II-III patients. Secondary outcomes included operative time, the length of hospital stay and the use of epidural and patient-controlled analgesia. Of 97 patients, 58 (59.8

Primary aldosteronism (PA) is a common, potentially reversible, cause of hypertension. Distinguishing unilateral from bilateral PA is critical when deciding who should be offered surgery (unilateral adrenalectomy). Recent studies have shown that PET/CT with [11C]metomidate can accurately identify unilateral PA, with localization of the causative aldosterone-producing adenoma (APA). However, the availability of [11C]metomidate is limited to centers with an on-site cyclotron. Here, we report an early-phase human study with the 18F-labeled analog, para-chloro-2-[18F]fluoroethyletomidate ([18F]CETO). Methods: We conducted a phase I/IIa, single-center, open-label, microdosing study. The primary objective was to evaluate the safety of up to 2 administrations of [18F]CETO in 6 patients with PA (3 unilateral disease, 3 bilateral disease) and 5 healthy volunteers. Safety evaluation included assessment of adrenal function after the first [18F]CETO administration. The biodistribution of [18F]CETO was assessed in a 90-min dynamic PET acquisition. In patients with PA, the effect of pretreatment with oral dexamethasone on [18F]CETO uptake by normal adrenal tissue and APAs was also assessed. Results: Eleven participants were recruited to the trial, including 6 patients and 5 healthy volunteers. No subjects experienced serious adverse events or reactions, and all participants had normal adrenal function after [18F]CETO administration. [18F]CETO demonstrated high selectivity for the adrenal glands with low uptake in other tissues. Visualization of APAs was enhanced after dexamethasone pretreatment, which suppressed [18F]CETO uptake by normal adrenal tissue. Conclusion: [18F]CETO is a safe radiopharmaceutical for PET imaging of the adrenal glands, with no observed adverse reactions or impairment of adrenal function in this study. [18F]CETO demonstrates selective high affinity for adrenal tissue, particularly APAs. Distinction between APAs and normal adrenal tissue is enhanced by dexamethasone pretreatment to suppress [18F]CETO uptake by normal glands. This positions [18F]CETO as a promising imaging tool for evaluation in the context of PA.

Primary aldosteronism (PA) is a common cause of hypertension. Compared to patients with essential hypertension, untreated PA is associated with a two- to fourfold greater risk of cardiovascular disease, renal failure, and death. PA is caused by increased secretion of aldosterone from one adrenal gland in 30% of the patients and both adrenal glands in 70%. Patients with unilateral PA can be cured by adrenalectomy, whereas patients with bilateral PA should be treated with mineralocorticoid receptor antagonists. With these disease-specific treatments, the long-term prognosis is generally favorable. A major challenge in the management of patients with PA is, however, subtype differentiation, that is, to determine whether the disease is unilateral or bilateral. Adrenal imaging using CT or MRI misclassifies more than one third of patients, and clinical variables-including blood pressure, aldosterone, and renin concentrations-provide limited guidance, underscoring the need for more accurate diagnostic approaches. Adrenal vein sampling is currently considered the gold standard for this purpose, but it is a technically challenging procedure performed only in specialized centers. After adrenalectomy for unilateral PA, histopathological assessment and subtyping are crucial for determining postoperative prognosis. This process is complex and relies on detailed functional analyses that are not always straightforward to interpret. Therefore, new approaches for subtyping PA are required. In this review, we summarize recent advances in the management of patients with PA, with particular focus on the limitations of current approaches to subtype differentiation and the emerging roles of novel techniques, including positron emission tomography, steroidomics, proteomics, and transcriptomics.

Primary aldosteronism (PA) is a common cause of hypertension. Compared to patients with essential hypertension, untreated PA is associated with a two- to fourfold greater risk of cardiovascular disease, renal failure, and death. PA is caused by increased secretion of aldosterone from one adrenal gland in 30% of the patients and both adrenal glands in 70%. Patients with unilateral PA can be cured by adrenalectomy, whereas patients with bilateral PA should be treated with mineralocorticoid receptor antagonists. With these disease-specific treatments, the long-term prognosis is generally favorable. A major challenge in the management of patients with PA is, however, subtype differentiation, that is, to determine whether the disease is unilateral or bilateral. Adrenal imaging using CT or MRI misclassifies more than one third of patients, and clinical variables—including blood pressure, aldosterone, and renin concentrations—provide limited guidance, underscoring the need for more accurate diagnostic approaches. Adrenal vein sampling is currently considered the gold standard for this purpose, but it is a technically challenging procedure performed only in specialized centers. After adrenalectomy for unilateral PA, histopathological assessment and subtyping are crucial for determining postoperative prognosis. This process is complex and relies on detailed functional analyses that are not always straightforward to interpret. Therefore, new approaches for subtyping PA are required. In this review, we summarize recent advances in the management of patients with PA, with particular focus on the limitations of current approaches to subtype differentiation and the emerging roles of novel techniques, including positron emission tomography, steroidomics, proteomics, and transcriptomics.

Small intestinal neuroendocrine tumors (SI-NETs) represent the most frequent neoplasms of the small intestine, with loss of chromosome 18 as the most common genetic aberration. To date, no highly frequently mutated genes have been identified on chromosome 18 or elsewhere. This study aimed to explore the potential role of methyl-CpG binding domain protein 2 (MBD2), located at 18q21.2, as a tumor suppressor gene in SI-NETs. By immunohistochemistry, we found undetectable or very low levels of MBD2 expression in 64% of the analyzed SI-NETs, and overall, very low levels of mRNA and protein expression were observed. Overexpression of MBD2 in the SI-NET cell line, GOT1, reduced cell growth and induced apoptosis, though cell migration remained unaffected. In addition, MBD2 expression decreased cell proliferation and migration capacity of the neuroendocrine cells NCI-H727 but showed no effect on apoptosis. Furthermore, MBD2 expression in both cell lines was found to upregulate E-cadherin but downregulate the levels of N-cadherin and vimentin in GOT1 and NCI-H72, respectively, as determined by western blot analysis. These results suggest that MBD2 plays a role in inhibiting the epithelial-mesenchymal transition process and may function as a potential tumor suppressor gene in SI-NETs. Future studies to elucidate the underlying mechanisms are warranted.

Several studies have attempted to find the initiating drivers of small intestinal neuroendocrine tumors (SI-NET) development and the molecular mechanisms driving progression and metastatic spread. For gene expression studies using bulk microarrays and RNA sequencing, researchers commonly use normal intestinal mucosa as a control. The intestine is made up of several different cell types, and using bulk RNA-seq may generate findings that reflect factors other than the tumor transformation. This could potentially contribute to the lack of discoverable treatments and prevention strategies for SI-NETs. We performed scRNA-seq on tissue from two patients that had tumor resection surgery and separated the EC cells from the normal intestinal mucosa and used specific markers to compare them against SI-NET tumors cells. We pinpointed new genes associated with chr18 haploinsufficiency but also others with loss or gain of expression that have not previously been associated with SI-NETs, and which could be potential targets for further functional genomic studies. ### Competing Interest Statement The authors have declared no competing interest.

Small intestinal neuroendocrine tumors (SI-NETs) are the most common tumor of the small intestine. Despite typically being metastatic at the time of diagnosis, the tumors are usually indolent and associated with a relatively long survival. Nevertheless, some patients fare worse and succumb to the disease soon after diagnosis despite similar stage and grade. To date, molecular prognostic markers are lacking, and our understanding of the disease biology is incomplete. Only CDKN1B is recurrently mutated in a subset of cases. Copy number alterations, including loss of chromosome 18, are common. However, the oncogenic mediators of these alterations remain unknown. The aim of this study was to identify molecular derangements in SI-NETs and identify potential prognostic markers. Forty tumor samples (including ten paired metastatic and primary tumors) from thirty patients with unusually long or unusually short survival after diagnosis were included for whole genome sequencing. Twenty of the samples were also included for RNA Sequencing. We validate known gene mutations and copy number alterations, as well as previously described molecular subgroups. We identify potential prognostic markers based on gene expression. Finally, we identify genes whose expression is affected by the most common copy number alterations in these tumors. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by Lions Cancerforskningsfond, and grants by Cancerfonden to Peter Stalberg, and by grants from Region Uppsala (ALF). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The regional ethical review board in Uppsala gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Primary aldosteronism (PA) is prevalent among hypertensive patients, and associated with worsened cardiovascular outcomes compared to essential hypertension (HT). Screening and diagnostics for PA are currently complicated and invasive, why new methods are needed. Unilateral PA (uPA) is best treated surgically, and bilateral PA (bPA) - medically. No validated proteomic diagnostic test has been found yet. Our aim was to explore proteomic markers in peripheral serum to discriminate between HT, PA, uPA and bPA. Eighty-eight hypertensive individuals were evaluated for PA, and diagnosed with HT (n = 30); bPA (n = 29); and uPA (n = 29). Serum samples from these study groups were analyzed by Olink® Explore 384 Cardiometabolic Panel. A machine learning model based on ridge logistic regression with a stratified 5-fold cross-validation was used to identify HT, PA, bPA and uPA. In the study groups, 56 circulating proteins were significantly different, and some of them specifically: 4 between PA vs. HT; 3 between bPA vs. uPA; 1 between bPA vs. HT; 9 between uPA vs. HT; 1 between HT vs. bPA vs. uPA. Three proteins with strongest differentiation (Coagulation factor IX for PA vs. HT; dipeptidyl peptidase 4 for uPA vs. HT and bPA; heat shock protein B1 for bPA vs. uPA) were validated by enzyme-linked immunosorbent assay. Our machine learning model could successfully identify 95

Abstract Introduction Primary aldosteronism (PA) is common, clinically elusive, and causes considerable morbidity and mortality. The current praxis of diagnosing PA is cumbersome, invasive, and depends on arbitrary thresholds. Very few cases are clinically discovered; new diagnostic methods are needed. Method Hypertensive patients were evaluated for PA in accordance with the current guidelines. Those diagnosed with PA underwent adrenal vein catheterization and/or adrenocortical positron emission tomography. Three groups were included: primary hypertension (HT), lateralized or unilateral PA (uPA), and bilateral PA (bPA). Serum from 11 HT, 16 uPA, and 12 bPA patients was subjected to microRNA extraction and sequencing. Serum from 29 HT, 26 uPA, and 29 bPA was subjected to a proximity extension assay on a cardiometabolic 384 protein panel (Olink® Explore 384). Result Several differentially expressed miRNAs were identified between the groups HT versus PA, but also uPA versus bPA. To validate our results in an external cohort, a machine learning model was developed and applied to an earlier published primary aldosteronism dataset (Decmann et al., 2019). This model could discriminate uPA from bPA successfully in 83% of the cases. Numerous significantly different proteins were observed between the groups. Interestingly, one of the coagulation factors had a distinct elevated expression in both uPA and bPA versus HT, which could be confirmed by an ELISA assay. Validation using an external cohort is underway. Discussion Further studies of serum biomarkers as a tool for identifying PA and discriminating between uPA and bPA may aid in the diagnosis and subsequently improve the treatment outcomes of PA.

Abstract Background [11C]metomidate, a methyl ester analogue of etomidate, is used for positron emission tomography of adrenocortical cancer, and has been tested in recent clinical trials for lateralization in primary aldosteronism (PA). However, in PA, visualization as well as uptake quantification are hampered by the tracer’s rather high non-specific liver uptake, and its overall clinical usefulness is also limited by the short 20-minute half-life of carbon-11. Therefore, we evaluated para-chloro-2-[18F]fluoroethyl-etomidate, [18F]CETO, a fluorine-18 (T1/2=109.8 min) analogue, as a potential new adrenocortical PET tracer. The aim of this study was to assess radiation dosimetry of [18F]CETO. Results [18F]CETO showed a high uptake in adrenal glands, still increasing at 5 h post injection. Adrenal glands (absorbed dose coefficients 0.100 ± 0.032 mGy/MBq in males and 0.124 ± 0.013 mGy/MBq in females) received the highest absorbed dose. The effective dose coefficient was 20 µSv/MBq. Conclusions [18F]CETO has a favourable biodistribution in humans for adrenal imaging. The effective dose for a typical clinical PET examination with 200 MBq [18F]CETO is 4 mSv. Trial registration ClinicalTrials.gov, NCT05361083 Retrospectively registered 29 April 2022. at, URL: https://clinicaltrials.gov/ct2/show/NCT05361083.

Tumour evolution with acquisition of more aggressive disease characteristics is a hallmark of disseminated cancer. Metastatic pancreatic neuroendocrine tumours (PanNETs) in particular, show frequent progression from a low/intermediate to a high-grade disease. To understand the molecular mechanisms underlying this phenomenon, we performed multi-omics analysis of 32 longitudinal samples from six metastatic PanNET patients. Following MEN1 inactivation, PanNETs exhibit genetic heterogeneity on both spatial and temporal dimensions with parallel and convergent tumuor evolution involving the ATRX/DAXX and mTOR pathways. Following alkylating chemotherapy treatment, some PanNETs develop mismatch repair deficiency and acquire a hypermutator phenotype. This DNA hypermutation phenotype was only found in cases that also showed transformation into a high-grade PanNET. Overall, our findings contribute to broaden the understanding of metastatic PanNET, and suggests that therapy driven disease evolution is an important hallmark of this disease.

Abstract Aims Small intestinal neuroendocrine tumor (SI-NETs) is the most common malignancy of the small bowel. Curative treatment is exclusively operative and explorative laparotomy is still considered to be the standard procedure. The purpose of this retrospective cohort study was to assess the outcome of minimally invasive surgery in patients with SI-NETs, at our endocrine surgery unit in 2019, compared to the open approach. Methods Patients who underwent surgery for SI-NETs between 2013 and 2023 were included. Descriptive variables such as operative time, length of hospital stay, use of analgesia and radiciality were compared primarily between groups of patients operated before and after 2019 and secondarily between open and hand-port assisted laparoscopic surgery (HALS). Results Of 97 patients with SI-NET included in the study, 58 (59.8 %) had an open and 39 (40.2 %) minimal invasive surgery. While difference in the operative time, 121 min (91.3-150.3) vs 108 min (83-141), the length of hospital stay, 6 days (4-7) vs 5 days (4-8) and surgical radicality in patients with stage II-III, 79.3% vs 85%, (p-value 0.313) was not significant, 86.2% of patients with explorative laparotomy required epidural analgesia compared to 23.1% with HALS (p-value = <0.001). Conclusion Hand-port assisted laparoscopic surgery of SI-NETs was assessed to be a feasible approach without compromising radical resection and enhancing postoperative recovery with lower amount of required analgesia postoperatively.

Tumour evolution with acquisition of more aggressive disease characteristics is a hallmark of disseminated cancer. Metastatic pancreatic neuroendocrine tumours (PanNETs) in particular may progress from a low/intermediate to a high-grade disease. The aim of this work was to understand the molecular mechanisms underlying metastatic progression as well as PanNET transformation from a low/intermediate to a high-grade disease. We performed multi-omics analysis (genome/exome sequencing, total RNA-sequencing and methylation array) of 32 longitudinal samples from six patients with metastatic low/intermediate grade PanNET. The clonal composition of tumour lesions and underlying phylogeny of each patient were determined with bioinformatics analyses. Findings were validated in post-alkylating chemotherapy samples from 24 patients with PanNET using targeted next generation sequencing. We validate the current PanNET evolutionary model with MEN1 inactivation that occurs very early in tumourigenesis. This was followed by pronounced genetic diversity on both spatial and temporal levels, with parallel and convergent tumour evolution involving the ATRX/DAXX and mechanistic target of the rapamycin (mTOR) pathways. Following alkylating chemotherapy treatment, some PanNETs developed mismatch repair deficiency and acquired a hypermutational phenotype. This was validated among 16 patients with PanNET who had high-grade progression after alkylating chemotherapy, of whom eight had a tumour mutational burden >50 (50%). In comparison, among the eight patients who did not show high-grade progression, 0 had a tumour mutational burden >50 (0%; odds ratio ‘infinite’, 95% confidence interval 1.8 to ‘infinite’, p  = 0.02). Our findings contribute to broaden the understanding of metastatic/high-grade PanNETs and suggests that therapy driven disease evolution is an important hallmark of this disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.