Patient derived tumoroids of high grade neuroendocrine neoplasms for more personalized therapies.

There are no therapeutic predictive biomarkers or representative preclinical models for high-grade gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN), a highly aggressive, fatal, and heterogeneous malignancy. We established patient-derived (PD) tumoroids from biobanked tissue samples of advanced high-grade GEP-NEN patients and applied this model for targeted rapid ex vivo pharmacotyping, next-generation sequencing, and perturbational profiling. We used tissue-matched PD tumoroids to profile individual patients, compared ex vivo drug response to patients' clinical response to chemotherapy, and investigated treatment-induced adaptive stress responses.PD tumoroids recapitulated biological key features of high-grade GEP-NEN and mimicked clinical response to cisplatin and temozolomide ex vivo. When we investigated treatment-induced adaptive stress responses in PD tumoroids in silico, we discovered and functionally validated Lysine demethylase 5 A and interferon-beta, which act synergistically in combination with cisplatin. Since ex vivo drug response in PD tumoroids matched clinical patient responses to standard-of-care chemotherapeutics for GEP-NEN, our rapid and functional precision oncology approach could expand personalized therapeutic options for patients with advanced high-grade GEP-NEN.

3D virtual histopathology by phase-contrast X-ray micro-CT for follicular thyroid neoplasms.

Histological analysis is the core of follicular thyroid carcinoma (FTC) classification. The histopathological criteria of capsular and vascular invasion define malignancy and aggressiveness of FTC. Analysis of multiple sections is cumbersome and as only a minute tissue fraction is analyzed during histopathology, under-sampling remains a problem. Application of an efficient tool for complete tissue imaging in 3D would speed-up diagnosis and increase accuracy. We show that X-ray propagation-based imaging (XPBI) of paraffin-embedded tissue blocks is a valuable complementary method for follicular thyroid carcinoma diagnosis and assessment. It enables a fast, non-destructive and accurate 3D virtual histology of the FTC resection specimen. We demonstrate that XPBI virtual slices can reliably evaluate capsular invasions. Then we discuss the accessible morphological information from XPBI and their significance for vascular invasion diagnosis. We show 3D morphological information that allow to discern vascular invasions. The results are validated by comparing XPBI images with clinically accepted histology slides revised by and under supervision of two experienced endocrine pathologists.

External validation of a deep learning-based algorithm for detection of tall cells in papillary thyroid carcinoma: A multicenter study.

The tall cell subtype (TC-PTC) is an aggressive subtype of papillary thyroid carcinoma (PTC). The TC-PTC is defined as a PTC comprising at least 30% epithelial cells that are three times as tall as they are wide. In practice, this definition is difficult to adhere to, resulting in high inter-observer variability. In this multicenter study, we validated a previously trained deep learning (DL)-based algorithm for detection of tall cells on 160 externally collected hematoxylin and eosin (HE)-stained PTC whole-slide images. In a test set of 360 manual annotations of regions of interest from 18 separate tissue sections in the external dataset, the DL-based algorithm detected TCs with a sensitivity of 90.6% and a specificity of 88.5%. The DL algorithm detected non-TC areas with a sensitivity of 81.6% and a specificity of 92.9%. In the validation datasets, 20% and 30% TC thresholds correlated with a significantly shorter relapse-free survival. In conclusion, the DL algorithm detected TCs in unseen, external scanned HE tissue slides with high sensitivity and specificity without any retraining.

Pancreatic Neuroendocrine Microtumors (WHO 2022) Are Not Always Low-Grade Neoplasms: A Case with a Highly Increased Proliferation Rate.

Traditionally considered non-functional low proliferative benign neuroendocrine proliferations measuring less than 5 mm, pancreatic (neuro)endocrine microadenomas are now classified as pancreatic neuroendocrine microtumors in the 2022 WHO classification of endocrine and neuroendocrine tumors. This case report discussed the features of an incidentally identified 4.7-mm glucagon-expressing pancreatic neuroendocrine microtumor with MEN1 mutation only, chromosomally stable and an epigenetic alpha-like phenotype. The tumor was associated with an unexplained increased proliferation rate in Ki-67 of 15%. There was no associated DAXX/ATRX deficiency. The presented case challenges the conventional thought of a low proliferative disease of the so-called "pancreatic neuroendocrine microadenomas" and provides additional support to the 2022 WHO classification that also requires grading of these neoplasms. Despite exhibiting molecular features of less aggressive behavior, the case also underscores the biological complexity of pancreatic neuroendocrine microtumors. By recognizing the heterogenous spectrum of neuroendocrine neoplasms, the current case also contributes to ongoing discussions on how to optimize the clinical management of such tumors.

Syndromic MEN1 parathyroid adenomas consist of both subclonal nodules and clonally independent tumors.

Primary hyperparathyroidism with parathyroid tumors is a typical manifestation of Multiple Endocrine Neoplasia Type 1 (MEN1) and is historically termed "primary hyperplasia". Whether these tumors represent a multi-glandular clonal disease or hyperplasia has not been robustly proven so far. Loss of Menin protein expression is associated with inactivation of both alleles and a good surrogate for a MEN1 gene mutation. The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene is mutated in MEN4 and encodes for protein p27 whose expression is poorly studied in the syndromic MEN1 setting.Here, we analyzed histomorphology and protein expression of Menin and p27 in parathyroid adenomas of 25 patients of two independent, well-characterized MEN1 cohorts. The pattern of loss of heterozygosity (LOH) was assessed by fluorescence in situ hybridization (FISH) in one MEN1-associated parathyroid adenoma. Further, next-generation sequencing (NGS) was performed on eleven nodules of four MEN1 patients.Morphologically, the majority of MEN1 adenomas consisted of multiple distinct nodules, in which Menin expression was mostly lost and p27 protein expression reduced. FISH analysis revealed that most nodules exhibited MEN1 loss, with or without the loss of centromere 11. NGS demonstrated both subclonal evolution and the existence of clonally unrelated tumors.Syndromic MEN1 parathyroid adenomas therefore consist of multiple clones with subclones, which supports the current concept of the novel WHO classification of parathyroid tumors (2022). p27 expression was lost in a large fraction of MEN1 parathyroids and must therefore be used with caution in suggesting MEN4.

[Practical application of immunohistochemistry in pancreatic neuroendocrine neoplasms : Tips and pitfalls]. / Praktische Anwendung von Immunhistochemie in pankreatischen neuroendokrinen Neoplasien : Tipps und Pitfalls.

Pancreatic neuroendocrine neoplasms (PanNEN) are rather rare entities. Morphology, combined with immunohistochemistry, allows typing and grading, thereby leading therapeutic decisions. Depending on tumor stage and differential diagnosis, a broad diagnostic panel may be required. The present work summarizes the minimal diagnostic, prognostic, and predictive markers in PanNEN.Markers of choice for defining a neuroendocrine phenotype are synaptophysin, chromogranin A, and INSM1. The proliferation fraction Ki67 is indispensable for grading, while p53 and Rb1 can help in the differentiation from neuroendocrine carcinoma (NEC). Transcription factors, such as cdx2, TTF­1, and Islet­1, can indicate the site of a primary tumor in the setting of a cancer of unknown primary (CUP). DAXX/ATRX immunohistochemistry has mainly prognostic value. Molecular pathology studies currently have little practical value in the diagnosis of PanNEN.An important pitfall in routine diagnostics is the wide spectrum of differential diagnoses mimicking neuroendocrine neoplasms. An expanded immunohistochemical panel is strongly recommended in case of doubt.

Impact of template-based synoptic reporting on completeness of surgical pathology reports.

Synoptic reporting increases completeness and standardization of surgical pathology reports and thereby contributes to an increased quality of clinical cancer care. Nevertheless, its widespread practical implementation remains a challenge, which is in part related to the effort required for setup and maintenance of database structures. This prompted us to assess the effect of a simple template-based, database-free system for synoptic reporting on completeness of surgical pathology reports. For this purpose, we analyzed 200 synoptic reports (100 colon and 100 lung cancer resections each) for completeness as required by the pertinent College of American Pathologists (CAP) protocols and compared these to a control dataset of 200 narrative reports. Introduction of template-based synoptic reporting resulted in improved completeness (98% of mandatory data elements) as compared to narrative reports (77%). Narrative reports showed a high degree of completeness for data elements covered by previously existing dictation templates. In conclusion, template-based synoptic reporting without underlying database structure can be a useful transitory phase in the implementation of synoptic reporting. It can result in a similar degree of completeness as reported in the literature for database solutions and provides other benefits of synoptic reporting while facilitating its implementation.

European Neuroendocrine Tumour Society (ENETS) 2023 guidance paper for nonfunctioning pancreatic neuroendocrine tumours.

This ENETS guidance paper for well-differentiated nonfunctioning pancreatic neuroendocrine tumours (NF-Pan-NET) has been developed by a multidisciplinary working group, and provides up-to-date and practical advice on the management of these tumours. Using the extensive experience of centres treating patients with NF-Pan-NEN, the authors of this guidance paper discuss 10 troublesome questions in everyday clinical practice. Our many years of experience in this field are still being verified in the light of the results of new clinical, which set new ways of proceeding in NEN. The treatment of NF-Pan-NEN still requires a decision of a multidisciplinary team of specialists in the field of neuroendocrine neoplasms.

Phase II study of everolimus and temozolomide as first-line treatment in metastatic high-grade gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: The optimal treatment for metastatic high-grade gastroenteropancreatic (GEP) neuroendocrine neoplasms when Ki-67 ≤55% is unknown. A prospective multi-centre phase 2 study was performed to evaluate the efficacy and safety of everolimus and temozolomide as first-line treatment for these patients. METHODS: Patients received everolimus 10 mg daily continuously and temozolomide 150 mg/m2 for 7 days every 2 weeks. Endpoints included response, survival, safety and quality of life (QoL). Histopathological re-evaluation according to the 2019 WHO classification was performed. RESULTS: For 37 eligible patients, the primary endpoint with 65% disease control rate (DCR) at 6 months (m) was reached. The response rate was 30%, the median progression-free survival (PFS) 10.2 months and the median overall survival (OS) 26.4 months. Considering 26 NET G3 patients, 6 months DCR was 77% vs. 22% among nine NEC patients (p = 0.006). PFS was superior for NET G3 vs. NEC (12.6 months vs. 3.4 months, Log-rank-test: p = 0.133, Breslow-test: p < 0.001). OS was significantly better for NET G3 (31.4 months vs. 7.8 months, p = 0.003). Grade 3 and 4 toxicities were reported in 43% and 38%. QoL remained stable during treatment. CONCLUSION: Everolimus and temozolomide may be a treatment option for selected GEP-NET G3 patients including careful monitoring. Toxicity did not compromise QoL. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NTC02248012).

Müller matrix polarimetry for pancreatic tissue characterization.

Polarimetry is an optical characterization technique capable of analyzing the polarization state of light reflected by materials and biological samples. In this study, we investigate the potential of Müller matrix polarimetry (MMP) to analyze fresh pancreatic tissue samples. Due to its highly heterogeneous appearance, pancreatic tissue type differentiation is a complex task. Furthermore, its challenging location in the body makes creating direct imaging difficult. However, accurate and reliable methods for diagnosing pancreatic diseases are critical for improving patient outcomes. To this end, we measured the Müller matrices of ex-vivo unfixed human pancreatic tissue and leverage the feature-learning capabilities of a machine-learning model to derive an optimized data representation that minimizes normal-abnormal classification error. We show experimentally that our approach accurately differentiates between normal and abnormal pancreatic tissue. This is, to our knowledge, the first study to use ex-vivo unfixed human pancreatic tissue combined with feature-learning from raw Müller matrix readings for this purpose.

Pathologist Computer-Aided Diagnostic Scoring of Tumor Cell Fraction: A Swiss National Study.

Tumor cell fraction (TCF) estimation is a common clinical task with well-established large interobserver variability. It thus provides an ideal test bed to evaluate potential impacts of employing a tumor cell fraction computer-aided diagnostic (TCFCAD) tool to support pathologists' evaluation. During a National Slide Seminar event, pathologists (n = 69) were asked to visually estimate TCF in 10 regions of interest (ROIs) from hematoxylin and eosin colorectal cancer images intentionally curated for diverse tissue compositions, cellularity, and stain intensities. Next, they re-evaluated the same ROIs while being provided a TCFCAD-created overlay highlighting predicted tumor vs nontumor cells, together with the corresponding TCF percentage. Participants also reported confidence levels in their assessments using a 5-tier scale, indicating no confidence to high confidence, respectively. The TCF ground truth (GT) was defined by manual cell-counting by experts. When assisted, interobserver variability significantly decreased, showing estimates converging to the GT. This improvement remained even when TCFCAD predictions deviated slightly from the GT. The standard deviation (SD) of the estimated TCF to the GT across ROIs was 9.9% vs 5.8% with TCFCAD (P < .0001). The intraclass correlation coefficient increased from 0.8 to 0.93 (95% CI, 0.65-0.93 vs 0.86-0.98), and pathologists stated feeling more confident when aided (3.67 ± 0.81 vs 4.17 ± 0.82 with the computer-aided diagnostic [CAD] tool). TCFCAD estimation support demonstrated improved scoring accuracy, interpathologist agreement, and scoring confidence. Interestingly, pathologists also expressed more willingness to use such a CAD tool at the end of the survey, highlighting the importance of training/education to increase adoption of CAD systems.

European Neuroendocrine Tumor Society 2023 guidance paper for functioning pancreatic neuroendocrine tumour syndromes.

This ENETS guidance paper aims to provide practical advice to clinicians for the diagnosis, treatment and follow-up of functioning syndromes in pancreatic neuroendocrine tumours (NET). A NET-associated functioning syndrome is defined by the presence of a clinical syndrome combined with biochemical evidence of inappropriately elevated hormonal levels. Different hormonal syndromes can be encountered in pancreatic NET patients, including insulinoma, gastrinoma as well as the rare glucagonoma, VIPoma, ACTHoma, PTHrPoma, carcinoid syndrome, calcitoninoma, GHRHoma and somatostatinoma. The recommendations provided in this paper focus on the biochemical, genetic and imaging work-up as well as therapeutic management of the individual hormonal syndromes in well-differentiated, grade 1-3, functioning NET with the primary tumour originating in the pancreas, and for specific subtypes also in the duodenum.

Germline pathogenic variants in patients with high-grade gastroenteropancreatic neuroendocrine neoplasms.

High-grade gastroenteropancreatic (HG-GEP) neuroendocrine neoplasms (NENs) are highly aggressive cancers. The molecular etiology of these tumors remains unclear, and the prevalence of pathogenic germline variants in patients with HG-GEP NENs is unknown. We assessed sequencing data of 360 cancer genes in normal tissue from 240 patients with HG-GEP NENs; 198 patients with neuroendocrine carcinomas (NECs) and 42 with grade 3 neuroendocrine tumors (NET G3). Applying strict criteria, we identified pathogenic germline variants and compared the frequency with previously reported data from 33 different cancer types. We found a recurrent MYOC variant in three patients and a recurrent MUTYH variant in two patients, indicating that these genes may be important underlying risk factors for HG-GEP NENs when mutated. Further, germline variants were found in canonical tumor-suppressor genes, such as TP53, RB1, BRIP1 and BAP1. Overall, we found that 4.5% of patients with NEC and 9.5% of patients with NET G3 carry germline pathogenic or highly likely pathogenic variants. Applying identical criteria for variant classification in silico to mined data from 33 other cancer types, the median percentage of patients carrying pathogenic or highly likely pathogenic variants was 3.4% (range: 0-17%). The patients with NEC and pathogenic germline variants had a median overall survival of 9 months, similar to what is generally expected for metastatic GEP NECs. A patient with NET G3 and pathogenic MUTYH variant had much shorter overall survival than expected. The fraction of HG-GEP NENs with germline pathogenic variants is relatively high, but still <10%, meaning that that germline mutations cannot be the major underlying cause of HG-GEP NENs.

Characterisation of hepatic lipid signature distributed across the liver zonation using mass spectrometry imaging.

Background & Aims: Lipid metabolism plays an important role in liver pathophysiology. The liver lobule asymmetrically distributes oxygen and nutrition, resulting in heterogeneous metabolic functions. Periportal and pericentral hepatocytes have different metabolic functions, which lead to generating liver zonation. We developed spatial metabolic imaging using desorption electrospray ionisation mass spectrometry to investigate lipid distribution across liver zonation with high reproducibility and accuracy. Methods: Fresh frozen livers from healthy mice with control diet were analysed using desorption electrospray ionisation mass spectrometry imaging. Imaging was performed at 50 µm × 50 µm pixel size. Regions of interest (ROIs) were manually created by co-registering with histological data to determine the spatial hepatic lipids across liver zonation. The ROIs were confirmed by double immunofluorescence. The mass list of specific ROIs was automatically created, and univariate and multivariate statistical analysis were performed to identify statistically significant lipids across liver zonation. Results: A wide range of lipid species was identified, including fatty acids, phospholipids, triacylglycerols, diacylglycerols, ceramides, and sphingolipids. We characterised hepatic lipid signatures in three different liver zones (periportal zone, midzone, and pericentral zone) and validated the reproducibility of our method for measuring a wide range of lipids. Fatty acids were predominantly detected in the periportal region, whereas phospholipids were distributed in both the periportal and pericentral zones. Interestingly, phosphatidylinositols, PI(36:2), PI(36:3), PI(36:4), PI(38:5), and PI(40:6) were located predominantly in the midzone (zone 2). Triacylglycerols and diacylglycerols were detected mainly in the pericentral region. De novo triacylglycerol biosynthesis appeared to be the most influenced pathway across the three zones. Conclusions: The ability to accurately assess zone-specific hepatic lipid distribution in the liver could lead to a better understanding of lipid metabolism during the progression of liver disease. Impact and Implications: Zone-specific hepatic lipid metabolism could play an important role in lipid homoeostasis during disease progression. Herein, we defined the zone-specific references of hepatic lipid species in the three liver zones using molecular imaging. The de novo triacylglycerol biosynthesis was highlighted as the most influenced pathway across the three zones.

Cribriform Morular Thyroid Carcinoma - Ultimobranchial Pouch-Related? Deep Molecular Insights of a Unique Case.

A 44-year-old female patient with a familial adenomatous polyposis (FAP) was diagnosed with a cribriform morular thyroid carcinoma (CMTC). We observed within the very necrotic tumor a small but distinct poorly differentiated carcinomatous component. As expected, next generation sequencing of both components revealed a homozygous APC mutation and in addition, a TERT promoter mutation. A TP53 mutation was found exclusively in the CMTC part, while the poorly differentiated component showed a clonal evolution, harboring an activating PIK3CA mutation and copy number gains of BRCA2, FGF23, FGFR1, and PIK3CB-alterations which are typically seen in squamous cell carcinoma. The mutational burden in both components was low, and there was no evidence for microsatellite instability. No mutations involving the mitogen-activated protein kinase (MAPK) pathway, typically seen in papillary thyroid carcinomas, were detected. Immunohistochemically, all tumor parts were negative for thyroglobulin, providing further evidence that this entity does not belong to the follicular epithelial cell-derived thyroid carcinoma group. CD5 was negative in the poorly differentiated component, making a relation to intrathyroidal thymic carcinoma rather unlikely. However, since this marker was seen in the morules, a loss in the poorly differentiated component and a relation to the ultimobranchial body cannot be excluded either. After total thyroidectomy and radioiodine ablation, the patient was disease-free with no residual tumor burden on 2-year follow-up.

Insulinomatosis: new aspects.

Endogenous hyperinsulinemic hypoglycemia (EHH) is a rare condition with an incidence of approximately 4-6 per million person-years and comprises a group of disorders causing hyperinsulinemic hypoglycemia without exogenous administration of insulin or its secretagogues. In adults, most cases (approximately 90%) are secondary to a single insulinoma. Other causes include insulinoma in the context of multiple endocrine neoplasia type 1 (approximately 5% of cases) and non-insulinoma pancreatogenous hypoglycemia syndrome, which is estimated to account for 0.5-5% of all cases. Recently, an entity called insulinomatosis has been described as a novel cause of EHH in adults. The characteristic feature of insulinomatosis is the synchronous or metachronous occurrence of multiple pancreatic neuroendocrine tumors expressing exclusively insulin. While most cases arise sporadically, there is recent evidence that autosomal dominant inheritance of mutations in the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) gene can cause a familial form of insulinomatosis. In these families, EHH is paradoxically associated with the occurrence of diabetes mellitus within the same family. This review summarizes the current clinical, biochemical, imaging and genetic knowledge of this disease.

Mutation Spectrum in Liquid Versus Solid Biopsies From Patients With Advanced Gastroenteropancreatic Neuroendocrine Carcinoma.

PURPOSE: Gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC) are rare and have a poor prognosis. Most GEP-NEC are diagnosed with metastatic disease, with only minor biopsies available for molecular diagnostics. We assessed the applicability of liquid biopsies for molecular profiling of GEP-NEC. MATERIALS AND METHODS: We performed massive parallel sequencing of 76 cancer-related genes in circulating tumor DNA from 50 patients with advanced GEP-NEC and compared findings to previous analyses of solid tumor biopsies from the same patients. Plasma samples were collected before therapy, and the median time span between blood and tissue sampling was 25 days. RESULTS: We detected 178 somatic mutations in the liquid biopsies, 127 (71%) were also detected in the solid biopsies, whereas 51 (29%) were unique to the liquid biopsies. In the same 76 genes, we previously detected 199 somatic mutations (single nucleotide variants) in solid biopsies, of which 127 (64%) were also now detected in liquid biopsies. In exploratory subgroup assessments, concordance was higher in patients with liver metastases (P = 1.5 × 10-5) and increasing with level of liver involvement (P = 1.2 × 10-4). The concordance was similar between GEP-NEC with different primary sites, except being lower in esophageal cases (P = .001). Concordance was not associated with tumor mutation burden. Tumor tissue mutations also detected in liquid biopsies was lower for MSI (40%) versus MSS tumors (70%; P = 7.8 × 10-4). We identified potentially targetable mutations in plasma of 26 (52%) of patients with GEP-NEC; nine patients (18%) had potentially targetable mutation detected only in liquid biopsies. CONCLUSION: Liquid biopsy analyses may be an applicable alternative to solid biopsies in GEP-NEC. Liquid biopsies may add additional mutations compared with tumor biopsies alone and could be useful for biomarker assessment in clinical trials for these patients.

Spatially restricted tumour-associated and host-associated immune drivers correlate with the recurrence sites of pancreatic cancer.

OBJECTIVE: Most patients with pancreatic ductal adenocarcinoma (PDAC) will experience recurrence after resection. Here, we investigate spatially organised immune determinants of PDAC recurrence. DESIGN: PDACs (n=284; discovery cohort) were classified according to recurrence site as liver (n=93/33%), lung (n=49/17%), local (n=31/11%), peritoneal (n=38/13%) and no-recurrence (n=73/26%). Spatial compartments were identified by fluorescent imaging as: pancytokeratin (PanCK)+CD45- (tumour cells); CD45+PanCK- (leucocytes) and PanCK-CD45- (stromal cells), followed by transcriptomic (72 genes) and proteomic analysis (51 proteins) for immune pathway targets. Results from next-generation sequencing (n=194) were integrated. Finally, 10 tumours from each group underwent immunophenotypic analysis by multiplex immunofluorescence. A validation cohort (n=109) was examined in parallel. RESULTS: No-recurrent PDACs show high immunogenicity, adaptive immune responses and are rich in pro-inflammatory chemokines, granzyme B and alpha-smooth muscle actin+ fibroblasts. PDACs with liver and/or peritoneal recurrences display low immunogenicity, stemness phenotype and innate immune responses, whereas those with peritoneal metastases are additionally rich in FAP+ fibroblasts. PDACs with local and/or lung recurrences display interferon-gamma signalling and mixed adaptive and innate immune responses, but with different leading immune cell population. Tumours with local recurrences overexpress dendritic cell markers whereas those with lung recurrences neutrophilic markers. Except the exclusive presence of RNF43 mutations in the no-recurrence group, no genetic differences were seen. The no-recurrence group exhibited the best, whereas liver and peritoneal recurrences the poorest prognosis. CONCLUSIONS: Our findings demonstrate distinct inflammatory/stromal responses in each recurrence group, which might affect dissemination patterns and patient outcomes. These findings may help to inform personalised adjuvant/neoadjuvant and surveillance strategies in PDAC, including immunotherapeutic modalities.