Hijacking the neuronal NMDAR signaling circuit to promote tumor growth and invasion.

Glutamate and its receptor N-methyl-D-aspartate receptor (NMDAR) have been associated with cancer, although their functions are not fully understood. Herein, we implicate glutamate-driven NMDAR signaling in a mouse model of pancreatic neuroendocrine tumorigenesis (PNET) and in selected human cancers. NMDAR was upregulated at the periphery of PNET tumors, particularly invasive fronts. Moreover, elevated coexpression of NMDAR and glutamate exporters correlated with poor prognosis in cancer patients. Treatment of a tumor-derived cell line with NMDAR antagonists impaired cancer cell proliferation and invasion. Flow conditions mimicking interstitial fluid pressure induced autologous glutamate secretion, activating NMDAR and its downstream MEK-MAPK and CaMK effectors, thereby promoting invasiveness. Congruently, pharmacological inhibition of NMDAR in mice with PNET reduced tumor growth and invasiveness. Therefore, beyond its traditional role in neurons, NMDAR may be activated in human tumors by fluid flow consequent to higher interstitial pressure, inducing an autocrine glutamate signaling circuit with resultant stimulation of malignancy.

Hypoxia exosome derived CEACAM5 promotes tumor-associated macrophages M2 polarization to accelerate pancreatic neuroendocrine tumors metastasis via MMP9.

Exosomes play significant roles in the communications between tumor cells and tumor microenvironment. However, the specific mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic neuroendocrine tumors (pNETs) are not well understood. This study aims to investigate these mechanisms and made several important discoveries. We found that hypoxic exosomes derived from pNETs cells can activate tumor-associated macrophages (TAM) to the M2 phenotype, in turn, the M2-polarized TAM, facilitate the migration and invasion of pNETs cells. Further investigation revealed that CEACAM5, a protein highly expressed in hypoxic pNETs cells, is enriched in hypoxic pNETs cell-derived exosomes. Hypoxic exosomal CEACAM5 was observed to induce M2 polarization of TAM through activation of the MAPK signaling pathway. Coculturing pNETs cells with TAM or treated with hypoxic exosomes enhanced the metastatic capacity of pNETs cells. In conclusion, these findings suggest that pNETs cells generate CEACAM5-rich exosomes in a hypoxic microenvironment, which in turn polarize TAM promote malignant invasion of pNETs cells. Targeting exosomal CEACAM5 could potentially serve as a diagnostic and therapeutic strategy for pNETs.

Expression patterns of novel immunotherapy targets in intermediate- and high-grade lung neuroendocrine neoplasms.

BACKGROUND: Advancements in immunotherapeutic approaches only had a modest impact on the therapy of lung neuroendocrine neoplasms (LNENs). Our multicenter study aimed to investigate the expression patterns of novel immunotherapy targets in intermediate- and high-grade LNENs. METHODS: The expressions of V-domain Ig suppressor of T cell activation (VISTA), OX40L, Glucocorticoid-induced TNF receptor (GITR), and T cell immunoglobulin and mucin domain 3 (TIM3) proteins were measured by immunohistochemistry in surgically resected tumor samples of 26 atypical carcinoid (AC), 49 large cell neuroendocrine lung cancer (LCNEC), and 66 small cell lung cancer (SCLC) patients. Tumor and immune cells were separately scored. RESULTS: Tumor cell TIM3 expression was the highest in ACs (p < 0.001), whereas elevated tumor cell GITR levels were characteristic for both ACs and SCLCs (p < 0.001 and p = 0.011, respectively). OX40L expression of tumor cells was considerably lower in ACs (vs. SCLCs; p < 0.001). Tumor cell VISTA expression was consistently low in LNENs, with no significant differences across histological subtypes. ACs were the least immunogenic tumors concerning immune cell abundance (p < 0.001). Immune cell VISTA and GITR expressions were also significantly lower in these intermediate-grade malignancies than in SCLCs or in LCNECs. Immune cell TIM3 and GITR expressions were associated with borderline prognostic significance in our multivariate model (p = 0.057 and p = 0.071, respectively). CONCLUSIONS: LNEN subtypes have characteristic and widely divergent VISTA, OX40L, GITR, and TIM3 protein expressions. By shedding light on the different expression patterns of these immunotherapy targets, the current multicenter study provides support for the future implementation of novel immunotherapeutic approaches.

Primary Cilia as a Tumor Marker in Pituitary Neuroendocrine Tumors.

Pituitary neuroendocrine tumors (PitNETs) account for approximately 15% of all intracranial neoplasms. Although they usually appear to be benign, some tumors display worse behavior, displaying rapid growth, invasion, refractoriness to treatment, and recurrence. Increasing evidence supports the role of primary cilia (PC) in regulating cancer development. Here, we showed that PC are significantly increased in PitNETs and are associated with increased tumor invasion and recurrence. Serial electron micrographs of PITNETs demonstrated different ciliation phenotypes (dot-like versus normal-like cilia) that represented PC at different stages of ciliogenesis. Molecular findings demonstrated that 123 ciliary-associated genes (eg, doublecortin domain containing protein 2, Sintaxin-3, and centriolar coiled-coil protein 110) were dysregulated in PitNETs, representing the upregulation of markers at different stages of intracellular ciliogenesis. Our results demonstrate, for the first time, that ciliogenesis is increased in PitNETs, suggesting that this process might be used as a potential target for therapy in the future.

Changes in tumor-to-blood ratio as a prognostic marker for progression-free survival and overall survival in neuroendocrine tumor patients undergoing PRRT.

BACKGROUND: Historically, patient selection for peptide receptor radionuclide therapy (PRRT) has been performed by virtue of somatostatin receptor scintigraphy (SRS). In recent years, somatostatin receptor positron emission tomography (SSTR-PET) has gradually replaced SRS because of its improved diagnostic capacity, creating an unmet need for SSTR-PET-based selection criteria for PRRT. Tumor-to-blood ratio (TBR) measurements have shown high correlation with the net influx rate Ki, reflecting the tumor somatostatin receptor expression, to a higher degree than standardized uptake value (SUV) measurements. TBR may therefore predict treatment response to PRRT. In addition, changes in semiquantitative SSTR-PET parameters have been shown to predate morphological changes, making them a suitable metric for response assessment. METHODS: The institutional database of the Department of Nuclear Medicine (University Hospital Essen) was searched for NET patients undergoing ≥ 2 PRRT cycles with available baseline and follow-up SSTR-PET. Two blinded independent readers reported the occurrence of new lesions quantified tumor uptake of up to nine lesions per patient using SUV and TBR. The association between baseline TBR and changes in uptake/occurrence of new lesions with progression-free survival (PFS) and overall survival (OS) was tested by use of a Cox regression model and log-rank test. RESULTS: Patients with baseline TBR in the 1st quartile had a shorter PFS (14.4 months) than those in the 3rd (23.7 months; p = 0.03) and 4th (24.1 months; p = 0.02) quartile. Similarly, these patients had significantly shorter OS (32.5 months) than those with baseline TBR in the 2nd (41.8 months; p = 0.03), 3rd (69.2 months; p < 0.01), and 4th (42.7 months; p = 0.03) quartile. Baseline to follow-up increases in TBR were independently associated with shorter PFS when accounting for prognostic markers, e.g., RECIST response (hazard ratio = 2.91 [95%CI = 1.54-5.50]; p = 0.01). This was confirmed with regard to OS (hazard ratio = 1.64 [95%CI = 1.03-2.62]; p = 0.04). Changes in SUVmean were not associated with PFS or OS. CONCLUSIONS: Baseline TBR as well as changes in TBR were significantly associated with PFS and OS and may improve patient selection and morphological response assessment. Future trials need to assess the role of TBR for therapy monitoring also during PRRT and prospectively explore TBR as a predictive marker for patient selection.

Altered biodistribution of [68Ga]Ga-DOTA-TOC during somatostatin analogue treatment.

PURPOSE: The need for an interval between the administration of long-acting Somatostatin Receptor Analogues (SSA) and the [68Ga]Ga-DOTA-TATE PET has been questioned based on recent literature in the new EANM guidelines. Here an earlier studies showed that SSA injection immediately before SSTR PET had minimal effect on normal organ and tumor uptake (1). However, data are scarce and there are (small) differences between [68Ga]Ga-DOTA-TATE and [68Ga]Ga-DOTA-TOC binding affinity, and it remains unknown whether these findings can be directly translated to scans with [68Ga]Ga-DOTA-TOC as well. The purpose of this study was to assess the effect of SSA use on the biodistribution in a subsequent [68Ga]Ga-DOTA-TOC PET/CT and compare this intra-individually across several cycles of SSA treatments. METHODS: Retrospectively, 35 patients with NENs were included. [68Ga]Ga-DOTA-TOC PET at staging and after the 1st and 2nd cycle of SSA were included. SUVmean and SUVmax of blood, visceral organs, primary tumor and two metastases were determined. Also, the interval between SSA therapy and the PET scan was registered. RESULTS: Treatment with SSA resulted in a significantly higher bloodpool activity and lower visceral tracer uptake. This effect was maintained after a 2nd cycle of SSA therapy. Furthermore, there was an inverse relationship between bloodpool tracer availability and visceral tracer binding and a positive correlation between bloodpool tracer availability and primary tumor tracer uptake. With an interval of up to 5 days, there was a significantly higher bloodpool activity than at longer intervals. CONCLUSION: Absolute comparison of the SUV on [68Ga]Ga-DOTA-TOC PET should be done with caution as the altered biodistribution of the tracer after SSA treatment should be taken into account. We recommend not to perform a scan within the first 5 days after the injection of lanreotide.

Dosimetry and pharmacokinetics of [177Lu]Lu-satoreotide tetraxetan in patients with progressive neuroendocrine tumours.

PURPOSE: To evaluate the dosimetry and pharmacokinetics of the novel radiolabelled somatostatin receptor antagonist [177Lu]Lu-satoreotide tetraxetan in patients with advanced neuroendocrine tumours (NETs). METHODS: This study was part of a phase I/II trial of [177Lu]Lu-satoreotide tetraxetan, administered at a median cumulative activity of 13.0 GBq over three planned cycles (median activity/cycle: 4.5 GBq), in 40 patients with progressive NETs. Organ absorbed doses were monitored at each cycle using patient-specific dosimetry; the cumulative absorbed-dose limits were set at 23.0 Gy for the kidneys and 1.5 Gy for bone marrow. Absorbed dose coefficients (ADCs) were calculated using both patient-specific and model-based dosimetry for some patients. RESULTS: In all evaluated organs, maximum [177Lu]Lu-satoreotide tetraxetan uptake was observed at the first imaging timepoint (4 h after injection), followed by an exponential decrease. Kidneys were the main route of elimination, with a cumulative excretion of 57-66% within 48 h following the first treatment cycle. At the first treatment cycle, [177Lu]Lu-satoreotide tetraxetan showed a median terminal blood half-life of 127 h and median ADCs of [177Lu]Lu-satoreotide tetraxetan were 5.0 Gy/GBq in tumours, 0.1 Gy/GBq in the bone marrow, 0.9 Gy/GBq in kidneys, 0.2 Gy/GBq in the liver and 0.8 Gy/GBq in the spleen. Using image-based dosimetry, the bone marrow and kidneys received median cumulative absorbed doses of 1.1 and 10.8 Gy, respectively, after three cycles. CONCLUSION: [177Lu]Lu-satoreotide tetraxetan showed a favourable dosimetry profile, with high and prolonged tumour uptake, supporting its acceptable safety profile and promising efficacy. TRIAL REGISTRATION: NCT02592707. Registered October 30, 2015.

Lysine Methyltransferase 5A Promotes the Progression of Growth Hormone Pituitary Neuroendocrine Tumors through the Wnt/ß-Catenin Signaling Pathway.

INTRODUCTION: Growth hormone (GH) secreting pituitary adenoma is considered one of the most harmful types of Pituitary Neuroendocrine Tumors (PitNETs). Our previous research has found that high expression of Lysine methyltransferase 5A (KMT5A) is closely related to the proliferation of PitNETs. The aim of this study was to investigate the role and molecular mechanism of KMT5A in the progression of GH PitNETs. METHODS: Immunohistochemistry, qRT-PCR, and Western blot (WB) were used to assess the expression levels of KMT5A in human normal pituitary and GH PitNETs, as well as in rat normal pituitary and GH3 cells. Additionally, we utilized RNA interference technology and treatment with a selective KMT5A inhibitor to decrease the expression of KMT5A in GH3 cells. CCK-8, EdU, flow cytometry (FCM), clone formation, and WB assay were further employed to evaluate the impact of KMT5A on the proliferation of GH3 cells in vitro. A xenograft model was established to evaluate the role of KMT5A in GH PitNETs progression in vivo. RESULTS: KMT5A was highly expressed in GH PitNETs and GH3 cells. Moreover, the reduction of KMT5A expression led to inhibited growth of GH PitNETs and increased apoptosis of tumor cells, as indicated by the findings from CCK-8, EdU, clone formation, and FCM assays. Additionally, WB analysis identified the Wnt/ß-catenin signaling pathway as a potential mechanism through which KMT5A promotes GH PitNETs progression. CONCLUSION: Our research suggests that KMT5A may facilitate the progression of GH PitNETs via the Wnt/ß-catenin signaling pathway. Therefore, KMT5A may serve as a potential therapeutic target and molecular biomarker for GH PitNETs.

The interplay between autophagy and ferroptosis presents a novel conceptual therapeutic framework for neuroendocrine prostate cancer.

In American men, the incidence of prostate cancer (PC) is the highest among all types of cancer, making it the second leading cause of mortality associated with cancer. For advanced or metastatic PC, antiandrogen therapies are standard treatment options. The administration of these treatments unfortunately carries the potential risk of inducing neuroendocrine prostate cancer (NEPC). Neuroendocrine differentiation (NED) serves as a crucial indicator of prostate cancer development, encompassing various factors such as phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR), Yes-associated protein 1 (YAP1), AMP-activated protein kinase (AMPK), miRNA. The processes of autophagy and ferroptosis (an iron-dependent form of programmed cell death) play pivotal roles in the regulation of various types of cancers. Clinical trials and preclinical investigations have been conducted on many signaling pathways during the development of NEPC, with the deepening of research, autophagy and ferroptosis appear to be the potential target for regulating NEPC. Due to the dual nature of autophagy and ferroptosis in cancer, gaining a deeper understanding of the developmental programs associated with achieving autophagy and ferroptosis may enhance risk stratification and treatment efficacy for patients with NEPC.

Data-independent acquisition combined with liquid chromatography mass spectrometry technique to detect prognostic protein markers in type I gastric neuroendocrine neoplasm.

RATIONALE: This study used proteomics-based data-independent acquisition (DIA) technology with the aim of screening for differential expression proteins in type I gastric neuroendocrine neoplasm (g-NEN). METHODS: Differential expression proteins in type I g-NEN and peritumoral tissues were screened using DIA with liquid chromatography/tandem mass spectrometry (DIA-LC/MS/MS). The identified proteins were then functionally analysed using bioinformatics methods. We selected the three most highly expressed proteins, combined with patients' clinical data, for prognostic analysis. RESULTS: Compared with peritumoral tissues, 224 proteins were up-regulated, and 70 were down-regulated. The most significantly enriched biological processes and pathways were vacuolar proton-transporting V-type ATPase complex assembly and metabolism-related pathways. PCSK1, FBXO2, ACSL1, IRS2, and PTPRZ1 expression was markedly up-regulated in type I g-NENs. High IRS2 expression significantly correlated with a shorter time to recurrence. CONCLUSIONS: Our study provides a comprehensive proteomic signature based on DIA-LC/MS/MS and highlights high IRS2 expression as a potential prognostic marker for type I gNENs.

Digital image analysis of Ki67 hotspot detection and index counting in gastroenteropancreatic neuroendocrine neoplasms.

The Ki-67 proliferative index plays a pivotal role in the subclassification of neuroendocrine neoplasm (NEN) according to the WHO Classification of Digestive System Tumors (5th edition), which designates neuroendocrine tumor (NET) grades 1, 2, and 3 for Ki-67 proliferative index of <3 %, 3-20 %, and >20 %, respectively. Proliferative index calculation must be performed in the hotspot, traditionally selected by visual scanning at low-power magnification. Recently, gradient map visualization has emerged as a tool for various purposes, including hotspot selection. This study includes 97 cases of gastrointestinal neuroendocrine neoplasms, with hotspots selected by bare eye and gradient map visualization (GM). Each hotspot was analyzed using three methods: eye estimation (EE), digital image analysis (DIA), and manual counting. Of the NENs studied, 91 % were NETs (26 % for G1, 55 % for G2, and 10 % for G3). Only 9 cases were neuroendocrine carcinoma (NEC). Between two hotspot selection methods, GM resulted in a higher grade in 14.77 % of cases, primarily upgrading from NET G1 to G2. Among the counting methods, DIA demonstrated substantial agreement with manual counting, both for pathologist and resident. Grading by other methods tended to result in a higher grade than MC (26.99 % with EE and 8.52 % with DIA). Given its clinical and statistical significance, this study advocates for the application of GM in hotspot selection to identify higher-grade tumors. Furthermore, DIA provides accurate grading, offering time efficiency over MC.

INSM1 is a useful neuroendocrine marker to differentiate pancreatic serous cystadenoma from pancreatic well-differentiated neuroendocrine tumors in cytology and surgical specimens.

INTRODUCTION: Differentiating pancreatic serous cystadenoma (SCA) from well-differentiated neuroendocrine tumors (WDNETs) based on histomorphology is critical yet challenging, particularly in small biopsy samples. Our study aimed to examine the expression profile of INSM1 in cytologic and surgical resection specimens from pancreatic SCA to evaluate its potential as a discriminative marker against pancreatic WDNET. METHODS: We characterized INSM1 immunohistochemistry in 34 patients with pancreatic SCA, comprising 23 surgical resections and 11 cytology specimens. As a control, we used 28 cytology specimens from pancreatic WDNET. Clinical information was retrieved through a review of electronic medical records. RESULTS: All 11 pancreatic SCA cytology specimens and 15 of 23 pancreatic SCA surgical resections exhibited absent INSM1 immunostaining. Each of the remaining eight surgical resection specimens demonstrated 1 % immunoreactivity. In contrast, 27 out of 28 (96 %) pancreatic WDNET cytology specimens were positive for INSM1 immunostaining, with a median immunoreactivity of 90 % and a range of 30-90 %. Overall, INSM1 immunostains perform similarly to chromogranin and synaptophysin in pancreatic SCA. CONCLUSIONS: The results indicate that INSM1 immunohistochemistry staining may serve as a useful neuroendocrine marker to differentiate pancreatic SCA from pancreatic WDNET in clinical practice. To our knowledge, this represents the first large-scale study to evaluate INSM1 immunostaining in surgical and cytology specimens from pancreatic SCA.

[Acidophil stem cell pituitary neuroendocrine tumors/adenoma: a clinicopathological analysis of five cases].

Objective: To investigate the clinicopathological characteristics of acidophil stem cell pituitary neuroendocrine tumors (PitNET)/adenoma. Methods: Five cases of acidophil stem cell PitNET/adenoma were diagnosed between May 2022 and July 2023 at the Second Hospital of Hebei Medical University, Shijiazhuang, China. The clinicopathological features of the tumor were analyzed by using histology, immunohistochemistry, and electron microscopy. The relevant literature was reviewed. Results: There were 1 male and 4 females, aged from 23 to 69 years. Patient 3 was 55 years old at the time of diagnosis and first surgery, and relapsed 5 years later. The patients' median age was 32 years. Patients 1 and 5 showed elevated blood prolactin, with various degrees of hormonal symptoms except Patient 3, who showed only tumor compression symptoms. Imaging studies showed that all cases involved the sellar floor. The tumors of Patients 1, 2 and 5 were closely related to the cavernous sinus segment of the internal carotid artery. The tumors exhibited a diffuse growth pattern with chromophobic to slightly acidophilic cytoplasm. A few of tumor cells showed chromophobic cytoplasm. The nucleoli were conspicuous. Intranuclear inclusion bodies and variably-sized clear vacuoles were observed occasionally. Under electron microscope, marked mitochondrial abnormalities were observed, including increased mitochondria number, expanded hypertrophy, and absence of mitochondrial ridge fracture. Some mitochondrial matrices were dense, while some were vacuolated. Conclusions: Acidophil stem cell PitNET/adenoma is a rare type of pituitary adenomas/PitNETs. It often has a more clinically aggressive manner with immature cells, diffuse expression of PIT1, prolactin, and varying degrees of growth hormone expression. Because of the obvious diversity of their clinical hormone status and hormone immune expression, the diagnosis of this type tumor is still a challenge.

[Characteristics of gastric neuroendocrine tumors and the PDX-1 transcription factor expression]. / Kharakteristika neiroendokrinnykh opukholei zheludka i ekspressiya v nikh transkriptsionnogo faktora PDX-1.

OBJECTIVE: To study the features of gastric neuroendocrine tumors (NETs) and the diagnostic and prognostic significance of PDX-1 expression in them. MATERIAL AND METHODS: 207 NETs identified in 56 men and 115 women (59 had multiple NETs), and 94 cases of gastric cancer (comparison group) were studied morphologically and immunohistochemically. RESULTS: In more than half of the cases (54.93%), NETs were localized in the body of the stomach; the cardiac and antral parts of the stomach accounted for 8.64% and 11.73%, respectively. NETs of the cardiac region predominated in men, and of the body and antrum - in women. NETs of the cardiac region predominated in men, and of the body and antrum - in women. The vast majority of NETs were highly differentiated (89.20%), of which Grade 1, 2 and 3 were 55.41%, 40.76% and 3.82%, respectively. Neuroendocrine carcinomas (NEC) accounted for 10.80% of all NET cases. NECs were more often localized in the cardiac part of the stomach and accounted for 35.71% of all NETs in the cardiac part. The share of NEC among all NETs of the antrum was 15.79%, of the body of the stomach - only 3.37%. Metastases were found in 17.90% of NETs. Expression of PDX-1 was detected in 44.73% of NETs, 70% of NECs and 74.50% of gastric cancers. CONCLUSION: PDX-1 is involved in the mechanisms of precancerous and cancerous lesions of the stomach and its overexpression is detected in the majority of the most malignant NETs and gastric cancers.

EGFR is a potential therapeutic target for highly glycosylated and aggressive pancreatic neuroendocrine neoplasms.

pNENs are relative indolent tumors with heterogeneous clinical presentation at diagnosis. It is important to establish aggressive subgroups of pNENs and identify potential therapeutic targets. Patients with pNEN (322 cases) were included to examine the association between glycosylation biomarkers and clinical/pathological traits. The molecular and metabolic features stratified by glycosylation status were assessed by RNA-seq/whole exome sequencing and immunohistochemistry. A considerable proportion of patients had elevated glycosylation biomarkers (carbohydrate antigen [CA] 19-9, 11.9%; CA125, 7.5%; carcinoembryonic antigen [CEA], 12.8%). CA19-9 (hazard ratio [HR] = 2.26, P = .019), CA125 (HR = 3.79, P = .004) and CEA (HR = 3.16, P = .002) were each independent prognostic variables for overall survival. High glycosylation group, defined as pNENs with elevated level of circulating CA19-9, CA125 or CEA, accounted for 23.4% of all pNENs. High glycosylation (HR = 3.14, P = .001) was an independent prognostic variable for overall survival and correlated with G3 grade (P < .001), poor differentiation (P = .001), perineural invasion (P = .004) and distant metastasis (P < .001). Epidermal growth factor receptor (EGFR) was enriched in high glycosylation pNENs using RNA-seq. EGFR was expressed in 21.2% of pNENs using immunohistochemistry and associated with poor overall survival (P = .020). A clinical trial focusing on EGFR expressed pNENs was initiated (NCT05316480). Thus, pNEN with aberrant glycosylation correlates with a dismal outcome and suggests potential therapeutic target of EGFR.

Tissue- and cell-specific properties of enterochromaffin cells affect the fate of tumorigenesis toward nonendocrine adenocarcinoma of the small intestine.

Small intestinal neuroendocrine tumors (SI-NETs) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. However, EC cell-derived tumorigenesis remains poorly understood. Here, we examined whether the gain of Myc and the loss of RB1 and Trp53 function in EC cells result in SI-NET using tryptophan hydroxylase 1 (TPH1) Cre-ERT2-driven RB1fl Trp53fl MycLSL (RPM) mice. TPH1-Cre-induced gain of Myc and loss of RB1 and Trp53 function resulted in endocrine or neuronal tumors in pancreas, lung, enteric neurons, and brain. Lineage tracing indicated that the cellular origin for these tumors was TPH1-expressing neuroendocrine, neuronal, or their precursor cells in these organs. However, despite that TPH1 is most highly expressed in EC cells of the small intestine, we observed no incidence of EC cell tumors. Instead, the tumor of epithelial cell origin in the intestine was exclusively nonendocrine adenocarcinoma, suggesting dedifferentiation of EC cells into intestinal stem cells (ISCs) as a cellular mechanism. Furthermore, ex vivo organoid studies indicated that loss of functions of Rb1 and Trp53 accelerated dedifferentiation of EC cells that were susceptible to apoptosis with expression of activated MycT58A, suggesting that the rare dedifferentiating cells escaping cell death went on to develop adenocarcinomas. Lineage tracing demonstrated that EC cells in the small intestine were short-lived compared with neuroendocrine or neuronal cells in other organs. In contrast, EC cell-derived ISCs were long-lasting and actively cycling and thus susceptible to transformation. These results suggest that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation, affect the fate and rate of tumorigenesis induced by genetic alterations and provide important insights into EC cell-derived tumorigenesis.NEW & NOTEWORTHY Small intestinal neuroendocrine tumors are of putative enterochromaffin (EC) cell origin and are the most common malignancy in the small intestine, followed by adenocarcinoma. However, the tumorigenesis of these tumor types remains poorly understood. The present lineage tracing studies showed that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation affect the fate and rate of tumorigenesis induced by genetic alterations toward a rare occurrence of adenocarcinoma.

PDZ-binding kinase aggravates pancreatic neuroendocrine neoplasm progression by activating the AKT/mTOR pathway.

The therapeutic effects of existing drug regimens against pancreatic neuroendocrine neoplasms (pNENs) remain limited, and identifying ideal therapeutic targets is warranted. PDZ binding kinase (PBK) may play an oncogenic role in most solid tumors. However, its function in pNEN remains unclear. In this study, pNEN samples and International Cancer Genome Consortium data were used to determine the clinical significance of PBK. Cell counting and CCK8 assays were used to assess cell proliferation. Flow cytometry was used to assess drug-induced apoptosis and cell cycle arrest. An in vivo PBK-targeting experiment was performed in mice bearing pNENs. Western blotting, quantitative PCR, and immunohistochemistry were performed to assess the molecular mechanisms. PBK was significantly upregulated in pNEN tissues compared with paracancerous tissues. Additionally, PBK was a poor prognostic factor for pNEN patients. PBK was found to promote the proliferation of pNEN cells by activating the AKT/mTOR pathway. Furthermore, PBK inhibition combined with everolimus treatment had enhanced antitumour effects on pNEN via inhibiting AKT/mTOR pathway and inducing G0/G1 phase cell cycle arrest. This study highlights that PBK plays an oncogenic role in and is a promising therapeutic target for pNEN.

Practice patterns for reporting digestive system neuroendocrine neoplasms: results from a large, comprehensive international survey.

AIMS: Criteria for the interpretation of digestive system neuroendocrine neoplasms (NENs) continue to evolve. Although there are some literature recommendations regarding workup and diagnosis of these lesions, different practice patterns exist among pathologists when signing out these specimens. The aim of this study was to assess practice trends among pathologists worldwide when reporting these neoplasms. METHODS AND RESULTS: We created an online survey with multiple questions pertaining to digestive NENs. The results were analysed based on type of practice setting, years of sign-out experience, and practice location. Respondents included 384 practicing pathologists: 70% academic, 30% private practice; 63% gastrointestinal (GI) pathology-subspecialised, 37% not; 39% North American, 42% European, 19% others; 45% with ≤10 years in practice; 55% with >10 years. Some question responses were chosen by the majority (e.g. 85% use both mitotic count and Ki67 index for grading NENs, 82% complete a synoptic, and Ki67 stain even for small incidental appendiceal neuroendocrine tumours [NETs], and 96% utilize the diagnosis of grade 3 NET). However, some questions showed varying responses, including counting mitotic figures, Ki67 stain interpretation, and pancreatic grade 3 NEN workup. Pathologists also had some variability in interpreting regional metastatic foci of small bowel NETs and in choosing blocks for Ki67 staining in multifocal lesions. CONCLUSION: There existed scenarios wherein practice patterns varied despite recommendations in the literature, and there were also scenarios lacking clear guidelines wherein pathologists used varying judgement. This survey highlights current key grey areas in digestive system NEN evaluation, leading to variation in practice patterns.

Somatostatin receptor activity assessed by 68Ga-DOTATOC PET can preoperatively predict DAXX/ATRX loss of expression in well-differentiated pancreatic neuroendocrine tumors.

PURPOSE: To evaluate the role of 68Ga-DOTATOC PET parameters in predicting DAXX/ATRX loss of expression in patients with Pancreatic neuroendocrine tumors (PanNET) candidate to surgery. METHODS: This retrospective study included 72 consecutive patients with PanNET (January 2018-March 2022) who underwent to 68Ga-DOTATOC PET for preoperative staging. Image analysis: qualitative assessment and extraction of SUVmax, SUV mean, somatostatin receptor density (SRD), and total lesion somatostatin receptor density (TLSRD) from primary PanNET. Radiological diameter and biopsy information (grade, Ki67) were collected. Loss of expression (LoE) of DAXX/ATRX was assessed by immunohistochemistry on surgical specimen. Student t-test, univariate and multivariate logistic regression and ROC curves have been used to investigate the predictive value of PET parameters on DAXX/ATRX LoE. RESULTS: Forty-two/72 patients had a G1, 28/72 a G2, and 2/72 a G3 PanNET. Seven/72 patients had DAXX LoE, 10/72 ATRX LoE, and 2/72 DAXX/ATRX LoE. SRD and TLSRD could predict DAXX LoE (p = 0.002, p = 0.018, respectively). By evaluating SRD in combination with radiological diameter, only SRD maintained statistical significance (multivariate logistic regression: p = 0.020, OR = 1.05), providing the best prediction (AUC-ROC = 79.01%; cut-off = 46.96; sensitivity = 77.78%; specificity = 88.89%). In the sub-analysis performed on 55 patients with biopsy availability, SRD demonstrated its role in providing useful and additional information (multivariate logistic regression: SRD p = 0.007; grade p = 0.040). CONCLUSION: SRD has a predictive role on DAXX LoE in PanNETs, with higher probability of LoE at increasing SRD values. SRD provides complementary/additional information to grade assessed on biopsy material, and the combined use of these approaches might support patients' management by preoperatively identifying subjects with more aggressive diseases.

DAXX, ATRX, and MSI in PanNET and Their Metastases: Correlation with Histopathological Data and Prognosis.

INTRODUCTION: Studies on pancreatic neuroendocrine tumors (PanNETs) regarding loss of ATRX, DAXX, or frequency of microsatellite instability (MSI) show inconclusive results. So far, data on corresponding metastaseshave not been published. METHODS: We performed immunohistochemistry (IHC) of ATRX, DAXX, MSH2, MSH6, MLH1, and PMS2 on 74 PanNETs and 19 metastases. ATRX- and DAXX-negative PanNETs were further sequenced for mutations. We used polymerase chain reaction for MSI on cases with IHC loss of MSH2, MSH6, MLH1, and PMS2. RESULTS: Immunohistochemical loss of DAXX and ATRX was observed in 8/74 (11%) and 6/74 (8%) PanNETs. Loss of DAXX immunoreactivity was statistically associated with higher tumor grade and showed a tendency toward a decreased overall survival. Sequencing of DAXX- (7/11 [64%]) and ATRX-negative (5/11 [45%]) PanNETs revealed a mutation in 6/7 (86%) and 2/5 (40%). The specificity of immunohistochemical loss of DAXX and ATRX for mutation was 80% and 67%, respectively. The expression status of DAXX compared to primary tumor differs in 2/12 (17%) lymph node metastases. We further identified 3/74 (4%) tumors as MSI, associated with a poor prognosis. DISCUSSION/CONCLUSION: Our study supports the hypothesis that a loss of DAXX immunoreactivity can identify a more aggressive subtype of PanNET with high confidence, while ATRX loss is a weaker indicator. Our results also strengthen the role of DAXX immunolabeling as a prognostic marker. We could show that ATRX might be less suitable as a surrogate for sequencing. Our results indicate that IHC of DAXX and ATRX may identify PanNET subtypes as targets for more aggressive therapy.