Role of Diagnostic Laparoscopy During Pancreatic Cancer Surgery in the Modern Era.

INTRODUCTION: Despite improvements in preoperative image resolution, approximately 10% of curative-intent resection attempts for pancreatic ductal adenocarcinoma are aborted at the time of operation. To avoid nontherapeutic laparotomy, many surgeons perform intraoperative diagnostic laparoscopy (DL) to identify radiographically occult metastatic disease. There are no consensus guidelines regarding DL in pancreatic cancer. The goal of this study is to investigate the efficacy of same-procedure DL at avoiding nontherapeutic laparotomy. METHODS: A single-institution retrospective review was performed from 2016 to 2022, identifying 196 patients with pancreatic ductal adenocarcinoma who were taken to the operating room for open curative-intent resection. Patient demographic, tumor characteristic, treatment, and outcome data were abstracted. Univariate and multivariate Cox hazard ratio analysis was performed to investigate risk factors for overall survival and recurrence-free survival. Number needed to treat (NNT) was calculated to identify number of DLs necessary to avoid one nontherapeutic laparotomy. RESULTS: Curative-intent resection was achieved in 161 (82.1%) patients. One hundred twenty six (64.0%) patients received DL prior to resection and DL identified metastatic disease in three (2.4%) patients with an NNT of 42. NNT of DL in a subgroup analysis performed on clinically high-risk patients (defined by preoperative or preneoadjuvant therapy carbohydrate antigen 19-9 > 500 U/mL) is 11. Receipt of DL did not prolong operative times in patients receiving pancreaticoduodenectomy when accounting for completed versus aborted resection. CONCLUSIONS: Although intraoperative DL is a short procedure with minimal morbidity, these data demonstrate that same-procedure DL has potential efficacy in avoiding nontherapeutic laparotomy only in a subgroup of clinically high-risk patients. Focus should remain on optimizing preoperative patient selection and further investigating novel diagnostic markers predictive of occult metastatic disease.

Divergent Learning-Related Transcriptional States of Cortical Glutamatergic Neurons.

Experience-dependent gene expression reshapes neural circuits, permitting the learning of knowledge and skills. Most learning involves repetitive experiences during which neurons undergo multiple stages of functional and structural plasticity. Currently, the diversity of transcriptional responses underlying dynamic plasticity during repetition-based learning is poorly understood. To close this gap, we analyzed single-nucleus transcriptomes of L2/3 glutamatergic neurons of the primary motor cortex after 3 d motor skill training or home cage control in water-restricted male mice. "Train" and "control" neurons could be discriminated with high accuracy based on expression patterns of many genes, indicating that recent experience leaves a widespread transcriptional signature across L2/3 neurons. These discriminating genes exhibited divergent modes of coregulation, differentiating neurons into discrete clusters of transcriptional states. Several states showed gene expressions associated with activity-dependent plasticity. Some of these states were also prominent in the previously published reference, suggesting that they represent both spontaneous and task-related plasticity events. Markedly, however, two states were unique to our dataset. The first state, further enriched by motor training, showed gene expression suggestive of late-stage plasticity with repeated activation, which is suitable for expected emergent neuronal ensembles that stably retain motor learning. The second state, equally found in both train and control mice, showed elevated levels of metabolic pathways and norepinephrine sensitivity, suggesting a response to common experiences specific to our experimental conditions, such as water restriction or circadian rhythm. Together, we uncovered divergent transcriptional responses across L2/3 neurons, each potentially linked with distinct features of repetition-based motor learning such as plasticity, memory, and motivation.

Gastroenteropancreatic neuroendocrine carcinoma tumor spheroid drug screen reveals vulnerability to tyrosyl-DNA phosphodiesterase 1 inhibitors.

BACKGROUND: Gastroenteropancreatic neuroendocrine carcinomas are rare neoplasms with no effective treatments and poor prognosis. Few reliable preclinical models exist for the study of gastroenteropancreatic neuroendocrine carcinomas, limiting investigation of novel treatments. We used tumor spheroids from our recently established gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models to systematically screen for compounds with diverse structures to identify potential new categories of therapeutic agents that can target gastroenteropancreatic neuroendocrine carcinomas. METHODS: Tumor spheroids were derived from our NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models. Gastroenteropancreatic neuroendocrine carcinoma spheroids were screened against a library of 885 compounds from the National Cancer Institute Diversity Set VII collection. Cell viability was measured via AlamarBlue assay. After identification of potential therapeutic compounds, synergy screening of a selected group with temozolomide and doxorubicin was performed, and these combinations were further analyzed for γH2AX and phosphorylated-ERK proteins. RESULTS: We identified 16 compounds that inhibit over 75% of gastroenteropancreatic neuroendocrine carcinoma spheroid survival. Seven are inhibitors of tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme working closely with the topoisomerase I complex. When combined with temozolomide or doxorubicin, the tyrosyl-DNA phosphodiesterase 1 inhibitor cytarabine increased the cytotoxic effects of these drugs on NEC1452 cells which was further evidenced by increasing γH2AX and decreasing phosphorylated-ERK in combination treatment compared to temozolomide alone. CONCLUSION: Both NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma spheroid lines are useful preclinical models for drug testing. Our library screen revealed these gastroenteropancreatic neuroendocrine carcinoma spheroids are highly sensitive to a novel class of anti-cancer drugs that target nuclear genome stability.

A luminescent sensor for investigating serotonin metabolism in neuroendocrine cancer.

BACKGROUND: Serotonin is emerging as a promising therapeutic target in tryptophan hydroxylase 1-positive tumors, but further mechanistic studies are needed to effectively target dysregulated serotonin metabolism. One challenge is a lack of methods for studying the dynamic nature of serotonin metabolism. Here, we report the development of a genetically encoded luminescent biosensor, termed iSero-Rluc, for the real-time detection of serotonin in live cells. METHODS: The engineered serotonin binding domain (iSero) and Renilla luciferase (Rluc) reporter were cloned into yeast and mammalian expression vectors to create a fusion protein that could act as a biosensor to detect endogenous serotonin levels in live cells. The iSero-Rluc biosensor was stably expressed in the BON cell line and luciferase assays, mass spectroscopy, immunofluorescence, and Western blotting were used to study serotonin metabolism under different cell culture conditions. RESULTS: The iSero-Rluc sensor detected exogenous serotonin in a yeast model. When stably expressed in the BON cell line, iSero-Rluc revealed that serotonin biosynthesis is increased in an anchorage-independent growth state and is induced upon serum starvation. CONCLUSION: The iSero-Rluc biosensor is a powerful tool in the study of tumor serotonin metabolism. It enabled real-time detection of alterations in serotonin synthesis in living cells under various growth conditions and has the potential to provide greater insight into serotonin metabolism in different stages of tumor progression and to identify therapeutic strategies to target cancer metastases and carcinoid crises.

Chemokine Receptor CXCR4 Radioligand Targeted Therapy Using 177Lutetium-pentixather for Pulmonary Neuroendocrine Cancers.

Intermediate to high-grade lung neuroendocrine tumors (NETs; i.e., atypical carcinoid tumors) and neuroendocrine carcinomas (NECs) are currently difficult to cure. These tumors were found to express the CXCR4 G-protein coupled receptor that can be targeted with radioligands. PCR and flow cytometric analysis of lung NET and NEC cell lines using an anti-CXCR4 antibody demonstrated that all cell lines tested expressed CXCR4. PET/CT imaging with 68Galium-pentixafor in mouse xenografts of NETs and NECs verified tumor targeting that was blocked by a CXCR4 agonist. Clonogenic survival analysis demonstrated a more than additive enhancement of killing when 1 µM auranofin (a thioredoxin reductase inhibitor) was used as a radiosensitizer in combination with 177Lu-pentixather (10 µCi). DMS273 small cell lung cancer xenografts in female nude mice treated with 25 µCi/g 177Lu-pentixather induced inhibition of tumor growth and resulted in an increase in overall survival without causing unacceptable normal tissue toxicities. Immunohistochemical staining of 95 retrospective human samples (containing 90 small cell lung carcinomas) demonstrated 84% CXCR4 positivity. In a multivariable analysis of this cohort that included age, gender, stage, primary site, SSTR2 status, and CXCR4 status, Cox regression models determined that only distant metastasis at presentation (P < 0.01) and a CXCR4 H-score >30 (P = 0.04) were significantly associated with reduced survival. Prospective clinical testing of patient tumors identified CXCR4-positivity in 76% of 21 NECs, 67% of 15 lung NETs (including 8 of 10 atypical carcinoids), and 0% of 25 non-lung NETs (including 5 NETS G3s). These data support the hypothesis that CXCR4-targeted theranostics can be utilized effectively for select NETs and NECs.

Prioritization of Fluorescence In Situ Hybridization (FISH) Probes for Differentiating Primary Sites of Neuroendocrine Tumors with Machine Learning.

Determining neuroendocrine tumor (NET) primary sites is pivotal for patient care as pancreatic NETs (pNETs) and small bowel NETs (sbNETs) have distinct treatment approaches. The diagnostic power and prioritization of fluorescence in situ hybridization (FISH) assay biomarkers for establishing primary sites has not been thoroughly investigated using machine learning (ML) techniques. We trained ML models on FISH assay metrics from 85 sbNET and 59 pNET samples for primary site prediction. Exploring multiple methods for imputing missing data, the impute-by-median dataset coupled with a support vector machine model achieved the highest classification accuracy of 93.1% on a held-out test set, with the top importance variables originating from the ERBB2 FISH probe. Due to the greater interpretability of decision tree (DT) models, we fit DT models to ten dataset splits, achieving optimal performance with k-nearest neighbor (KNN) imputed data and a transformation to single categorical biomarker probe variables, with a mean accuracy of 81.4%, on held-out test sets. ERBB2 and MET variables ranked as top-performing features in 9 of 10 DT models and the full dataset model. These findings offer probabilistic guidance for FISH testing, emphasizing the prioritization of the ERBB2, SMAD4, and CDKN2A FISH probes in diagnosing NET primary sites.

Multiomic sequencing of paired primary and metastatic small bowel carcinoids.

Background: Small bowel carcinoids are insidious tumors that are often metastatic when diagnosed. Limited mutation landscape studies of carcinoids indicate that these tumors have a relatively low mutational burden. The development of targeted therapies will depend upon the identification of mutations that drive the pathogenesis and metastasis of carcinoid tumors. Methods: Whole exome and RNA sequencing of 5 matched sets of normal tissue, primary small intestine carcinoid tumors, and liver metastases were investigated. Germline and somatic variants included: single nucleotide variants (SNVs), insertions/deletions (indels), structural variants, and copy number alterations (CNAs). The functional impact of mutations was predicted using Ensembl Variant Effect Predictor. Results: Large-scale CNAs were observed including the loss of chromosome 18 in all 5 metastases and 3/5 primary tumors. Certain somatic SNVs were metastasis-specific; including mutations in ATRX, CDKN1B, MXRA5 (leading to the activation of a cryptic splice site and loss of mRNA), SMARCA2, and the loss of UBE4B. Additional mutations in ATRX, and splice site loss of PYGL, leading to intron retention observed in primary and metastatic tumors. Conclusions: We observed novel mutations in primary/metastatic carcinoid tumor pairs, and some have been observed in other types of neuroendocrine tumors. We confirmed a previously observed loss of chromosome 18 and CDKN1B. Transcriptome sequencing added relevant information that would not have been appreciated with DNA sequencing alone. The detection of several splicing mutations on the DNA level and their consequences at the RNA level suggests that RNA splicing aberrations may be an important mechanism underlying carcinoid tumors.

Systemic Therapy for Tumor Control in Metastatic Well-Differentiated Gastroenteropancreatic Neuroendocrine Tumors: ASCO Guideline.

PURPOSE: To develop recommendations for systemic therapy for well-differentiated grade 1 (G1) to grade 3 (G3) metastatic gastroenteropancreatic neuroendocrine tumors (GEP-NETs). METHODS: ASCO convened an Expert Panel to conduct a systematic review of relevant studies and develop recommendations for clinical practice. RESULTS: Eight randomized controlled trials met the inclusion criteria for the systematic review. RECOMMENDATIONS: Somatostatin analogs (SSAs) are recommended as first-line systemic therapy for most patients with G1-grade 2 (G2) metastatic well-differentiated GI-NETs. Observation is an option for patients with low-volume or slow-growing disease without symptoms. After progression on SSAs, peptide receptor radionuclide therapy (PRRT) is recommended as systematic therapy for patients with somatostatin receptor (SSTR)-positive tumors. Everolimus is an alternative second-line therapy, particularly in nonfunctioning NETs and patients with SSTR-negative tumors. SSAs are standard first-line therapy for SSTR-positive pancreatic (pan)NETs. Rarely, observation may be appropriate for asymptomatic patients until progression. Second-line systemic options for panNETs include PRRT (for SSTR-positive tumors), cytotoxic chemotherapy, everolimus, or sunitinib. For SSTR-negative tumors, first-line therapy options are chemotherapy, everolimus, or sunitinib. There are insufficient data to recommend particular sequencing of therapies. Patients with G1-G2 high-volume disease, relatively high Ki-67 index, and/or symptoms related to tumor growth may benefit from early cytotoxic chemotherapy. For G3 GEP-NETs, systemic options for G1-G2 may be considered, although cytotoxic chemotherapy is likely the most effective option for patients with tumor-related symptoms, and SSAs are relatively ineffective. Qualifying statements are provided to assist with treatment choice. Multidisciplinary team management is recommended, along with shared decision making with patients, incorporating their values and preferences, potential benefits and harms, and other characteristics and circumstances, such as comorbidities, performance status, geographic location, and access to care.Additional information is available at www.asco.org/gastrointestinal-cancer-guidelines.

Inhibition of serotonin biosynthesis in neuroendocrine neoplasm suppresses tumor growth in vivo.

Small bowel neuroendocrine tumors (SBNETs) originate from enterochromaffin cells in the intestine which synthesize and secrete serotonin. SBNETs express high levels of tryptophan hydroxylase 1 (Tph1), a key enzyme in serotonin biosynthesis. Patients with high serotonin level may develop carcinoid syndrome, which can be treated with somatostatin analogues and the Tph1 inhibitor telotristat ethyl in severe cases. Although the active drug telotristat can efficiently reduce serotonin levels, its effect on tumor growth is unclear. This study determined the effect of serotonin inhibition on tumor cell growth in vitro and in vivo . The levels of Tph1 in various neuroendocrine neoplasms (NENs) were determined and the biological effects of Tph1 inhibition in vitro and in vivo using genetic and pharmacologic approaches was tested. Gene and protein expression analyses were performed on patient tumors and cancer cell lines. shRNAs targeting TPH1 were used to create stable knockdown in BON cells. Control and knockdown lines were assessed for their growth rates in vitro and in vivo , angiogenesis potential, serotonin levels, endothelial cell tube formation, tumor weight, and tumor vascularity. TPH1 is highly expressed in SBNETs and many cancer types. TPH1 knockdown cells and telotristat treated cells showed similar growth rates as control cells in vitro . However, TPH1 knockdown cells formed smaller tumors in vivo and tumors were less vascularized. Although Tph1 inhibition with telotristat showed no effect on tumor cell growth in vitro , Tph1 inhibition reduced tumor formation in vivo . Serotonin inhibition in combination with other therapies is a promising new avenue for targeting metabolic vulnerabilities in NENs.

Peritoneal Metastases After Intraductal Papillary Mucinous Neoplasm Resection: How Common are They?

INTRODUCTION: Peritoneal metastases (PMs) following resection of pancreatic intraductal papillary mucinous neoplasms (IPMNs) are rare. Consequently, prevalence, risk factors, and prognosis are not well known. We reviewed our institution's experience and published literature to further characterize the scope of this phenomenon. METHODS: All pancreatectomy cases (556 patients) performed at a tertiary care center between 2010 and 2020 were reviewed to identify IPMN diagnoses. Patients with adenocarcinoma not arising from IPMN, or a history of other malignancies were excluded. RESULTS: Seventy-eight patients underwent pancreatectomy with IPMN on final pathology at our institution; 51 met inclusion criteria. Of these, there were five cases of PMs (4:1 females:males). Four had invasive carcinoma arising from IPMN and one had high-grade dysplasia at the index operation. Female sex and invasive histology were significantly associated with PM (P < 0.05). PM rates by sex were 3% (95% confidence interval [CI]: 0.5-15) in males and 22% (95% CI: 9-45) in females. Rates by histology were 2.9% (95% CI: 0.5-15) for noninvasive IPMN, and 23.5% (95% CI: 9.5-47) for invasive carcinoma arising from IPMN. Median interval from surgery to PMs was 7 mo (range: 3-13). CONCLUSIONS: PMs following IPMN resection are rare but may be more common in patients with invasive histology. Although rare, PMs can arise in patients with noninvasive IPMNs. Further studies on pathophysiology and risk factors of PM following IPMN resection are needed and may reinforce adherence to guidelines recommending long-term surveillance.

Surgical Management of G3 Gastroenteropancreatic Neuroendocrine Neoplasms: A Systematic Review and Meta-analysis.

BACKGROUND: Grade 3 (G3) gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) are rare, aggressive tumors with poor prognosis. The World Health Organization 2017 and 2019 classifications further subdivided G3 NENs into G3 neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs). Current guidelines favor medical management in most of these patients, and the role of surgical management is not well defined. We performed a systematic literature review and meta-analysis of surgical management versus nonsurgical management for G3 GEP NENs. MATERIALS AND METHODS: A PRISMA-compliant systematic review of the MEDLINE, Embase, Scopus, and Cochrane Library databases (end-of-search date: 16 July 2021) was conducted. Individual patient survival data were reconstructed, and random-effects meta-analyses were performed. RESULTS: Fourteen studies comprising 1810 surgical and 910 nonsurgical patients were systematically reviewed. Publication bias adjusted meta-analysis of 12 studies (1788 surgical and 857 nonsurgical patients) showed increased overall survival (OS) after surgical compared with nonsurgical management for G3 GEP NENs [hazard ratio (HR) 0.40, 95% confidence interval (CI) 0.31-0.53]. Subgroup meta-analyses showed increased OS after surgical management for both pancreatic and gastrointestinal primary sites separately. In another subgroup meta-analysis of G3 GEP NETs (not NECs), surgical management was associated with increased OS compared with nonsurgical management (HR 0.26, 95% CI 0.11-0.61). CONCLUSIONS: Surgical management of G3 GEP NENs may provide a potential survival benefit in well-selected cases. Further research is needed to define which patients will benefit most from surgical versus nonsurgical management. The current literature is limited by inconsistent reporting of survival outcomes in surgical versus nonsurgical groups, tumor grade, differentiation, primary tumor site, and selection criteria for surgical and nonsurgical management.

Preclinical Models of Neuroendocrine Neoplasia.

Neuroendocrine neoplasia (NENs) are a complex and heterogeneous group of cancers that can arise from neuroendocrine tissues throughout the body and differentiate them from other tumors. Their low incidence and high diversity make many of them orphan conditions characterized by a low incidence and few dedicated clinical trials. Study of the molecular and genetic nature of these diseases is limited in comparison to more common cancers and more dependent on preclinical models, including both in vitro models (such as cell lines and 3D models) and in vivo models (such as patient derived xenografts (PDXs) and genetically-engineered mouse models (GEMMs)). While preclinical models do not fully recapitulate the nature of these cancers in patients, they are useful tools in investigation of the basic biology and early-stage investigation for evaluation of treatments for these cancers. We review available preclinical models for each type of NEN and discuss their history as well as their current use and translation.

High-Contrast Detection of Somatostatin Receptor Subtype-2 for Fluorescence-Guided Surgery.

Dye design can influence the ability of fluorescently labeled imaging agents to generate tumor contrast and has become an area of significant interest in the field of fluorescence-guided surgery (FGS). Here, we show that the charge-balanced near-infrared fluorescent (NIRF) dye FNIR-Tag enhances the imaging properties of a fluorescently labeled somatostatin analogue. In vitro studies showed that the optimized fluorescent conjugate MMC(FNIR-Tag)-TOC bound primarily via somatostatin receptor subtype-2 (SSTR2), whereas its negatively charged counterpart with IRDye 800CW had higher off-target binding. NIRF imaging in cell line- and patient-derived xenograft models revealed markedly higher tumor contrast with MMC(FNIR-Tag)-TOC, which was attributed to increased tumor specificity. Ex vivo staining of surgical biospecimens from primary and metastatic tumors, as well as involved lymph nodes, demonstrated binding to human tumors. Finally, in an orthotopic tumor model, a simulated clinical workflow highlighted our unique ability to use standard preoperative nuclear imaging for selecting patients likely to benefit from SSTR2-targeted FGS. Our findings demonstrate the translational potential of MMC(FNIR-Tag)-TOC for intraoperative imaging and suggest broad utility for using FNIR-Tag in fluorescent probe development.

Is There a Role for Surgical Resection of Grade 3 Neuroendocrine Neoplasms?

BACKGROUND: Grade 3 (G3) gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) are aggressive tumors with poor survival outcomes for which medical management is generally recommended. This study sought to evaluate outcomes of surgically treated G3 GEP-NEN patients. METHODS: A single-institutional prospective NEN database was reviewed. Patients with G3 GEP-NENs based on World Health Organization (WHO) 2019 definitions included well-differentiated neuroendocrine tumors (G3NET) and poorly differentiated neuroendocrine carcinomas (G3NEC). Clinicopathologic factors were compared between groups. Overall survival from G3 diagnosis was assessed by the Kaplan-Meier method. RESULTS: Surgical resection was performed for 463 patients (211 G1, 208 G2, 44 G3). Most had metastatic disease at presentation (54% G1, 69% G2, 91% G3; p < 0.001). The G3 cohort included 39 G3NETs and 5 G3NECs, 22 of pancreatic and 22 of midgut origin. Median overall survival (mOS; in months) was 268.1 for G1NETs, 129.9 for G2NETs, 50.5 for G3NETs, and 28.5 for G3NECs (p < 0.001). Over the same period, 31 G3 patients (12 G3NETs, 19 G3NECs) were treated non-surgically, with mOS of 19.0 for G3NETs and 12.4 for G3NECs. CONCLUSIONS: Surgical resection of G3 GEP-NENs remains controversial due to poor prognosis, and surgical series are rare. This large, single-institutional study found significantly lower mOS in patients with resected G3NENs than those with G1/G2 tumors, reflecting more aggressive tumor biology and a higher proportion with metastatic disease. The mOS for resected G3NETs and G3NECs exceeded historical non-surgical G3NEN series (mOS 11-19 months), suggesting surgery should be considered in carefully selected patients with G3NENs, especially those with well-differentiated tumors.

FOCAD Indel in a Family With Juvenile Polyposis Syndrome.

Juvenile polyposis syndrome (JPS) is a childhood polyposis syndrome with up to a 50% lifetime risk of gastrointestinal cancer. Germline pathogenic variants in BMPR1A and SMAD4 are responsible for around 40% of cases of JPS, but for the majority of individuals, the underlying genetic cause is unknown. We identified a family for which polyposis spanned four generations, and the proband had a clinical diagnosis of JPS. Next-generation sequencing was conducted, followed by Sanger sequencing confirmation. We identified an internal deletion of the FOCAD gene in all family members tested that altered the reading frame and is predicted to be pathogenic. We conclude that inactivation of the FOCAD gene is likely to cause JPS in this family.