Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.

One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.

Three-dimensional cardiac models: a pre-clinical testing platform.

Major advancements in human pluripotent stem cell (hPSC) technology over recent years have yielded valuable tools for cardiovascular research. Multi-cell type 3-dimensional (3D) cardiac models in particular, are providing complementary approaches to animal studies that are better representatives than simple 2-dimensional (2D) cultures of differentiated hPSCs. These human 3D cardiac models can be broadly divided into two categories; namely those generated through aggregating pre-differentiated cells and those that form self-organizing structures during their in vitro differentiation from hPSCs. These models can either replicate aspects of cardiac development or enable the examination of interactions among constituent cell types, with some of these models showing increased maturity compared with 2D systems. Both groups have already emerged as physiologically relevant pre-clinical platforms for studying heart disease mechanisms, exhibiting key functional attributes of the human heart. In this review, we describe the different cardiac organoid models derived from hPSCs, their generation methods, applications in cardiovascular disease research and use in drug screening. We also address their current limitations and challenges as pre-clinical testing platforms and propose potential improvements to enhance their efficacy in cardiac drug discovery.

Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well-characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the nematode Ascaris to study the timing of PDE breaks and examine the DSBs and their end processing. Using END-seq, we characterize the DSB ends and demonstrate that DNA breaks are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3' overhangs before the addition of telomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends in Ascaris may be due to the sequestration of the eliminated DNA into micronuclei, preventing their ends from telomere healing. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for nematode PDE.

A Machine Learning Algorithm Improves the Diagnostic Accuracy of the Histologic Component of Antibody Mediated Rejection (AMR-H) in Cardiac Transplant Endomyocardial Biopsies.

BACKGROUND: Pathologic antibody mediated rejection (pAMR) remains a major driver of graft failure in cardiac transplant patients. The endomyocardial biopsy remains the primary diagnostic tool but presents with challenges, particularly in distinguishing the histologic component (pAMR-H) defined by 1) intravascular macrophage accumulation in capillaries and 2) activated endothelial cells that expand the cytoplasm to narrow or occlude the vascular lumen. Frequently, pAMR-H is difficult to distinguish from acute cellular rejection (ACR) and healing injury. With the advent of digital slide scanning and advances in machine deep learning, artificial intelligence technology is widely under investigation in the areas of oncologic pathology, but in its infancy in transplant pathology. For the first time, we determined if a machine learning algorithm could distinguish pAMR-H from normal myocardium, healing injury and ACR. MATERIALS AND METHODS: A total of 4,212 annotations (1,053 regions of normal, 1,053 pAMR-H, 1,053 healing injury and 1,053 ACR) were completed from 300 hematoxylin and eosin slides scanned using a Leica Aperio GT450 digital whole slide scanner at 40X magnification. All regions of pAMR-H were annotated from patients confirmed with a previous diagnosis of pAMR2 (>50% positive C4d immunofluorescence and/or >10% CD68 positive intravascular macrophages). Annotations were imported into a Python 3.7 development environment using the OpenSlide™ package and a convolutional neural network approach utilizing transfer learning was performed. RESULTS: The machine learning algorithm showed 98% overall validation accuracy and pAMR-H was correctly distinguished from specific categories with the following accuracies: normal myocardium (99.2%), healing injury (99.5%) and ACR (99.5%). CONCLUSION: Our novel deep learning algorithm can reach acceptable, and possibly surpass, performance of current diagnostic standards of identifying pAMR-H. Such a tool may serve as an adjunct diagnostic aid for improving the pathologist's accuracy and reproducibility, especially in difficult cases with high inter-observer variability. This is one of the first studies that provides evidence that an artificial intelligence machine learning algorithm can be trained and validated to diagnose pAMR-H in cardiac transplant patients. Ongoing studies include multi-institutional verification testing to ensure generalizability.

Septic Arthritis of the Sternoclavicular Joint.

Introduction: Sternoclavicular joint (SCJ) septic arthritis is a rare but rapidly fatal joint infection. Without proper medical or surgical management, it can progress to osteomyelitis, chest wall abscess, mediastinitis, or myositis. Case Report: A 57-year-old male with a past history of intravenous drug use presented to the emergency department (ED) with chest tenderness of one week duration. Vital signs were unremarkable, and exam was notable for tender, raised right SCJ without any fluctuance. On point-of-care ultrasound we noted fluid collection and capsular distention along the SCJ, which aided in rapidly diagnosing septic arthritis. The patient was immediately started on antibiotics and taken to the operating room for excision and debridement. Conclusion: While computed tomography is routinely used in the emergency department for diagnosing septic arthritis, ultrasound offers a rapid and safe alternative for diagnosis.

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

A QTL on Maize Chromosome 5 is Associated with Root-Knot Nematode Resistance.

This study provides the first report of a quantitative trait locus (QTL) in maize (Zea mays) for resistance to the southern root-knot nematode (SRKN) (Meloidogyne incognita). The SRKN can feed on the roots of maize in the U.S. Southern Coastal Plain region and can cause yield losses of 30% or greater in heavily infested fields. Increases in SRKN density in the soil may reduce the yield for subsequently planted susceptible crops. The use of maize hybrids with resistance to SRKN could prevent an increase in SRKN density, yet no genetic regions have been identified that confer host resistance. In this study, a B73 (susceptible) x Ky21 (resistant) S5 recombinant inbred line (RIL) population was phenotyped for total number of eggs (TE) and root weight. This population has been previously genotyped using single nucleotide polymorphisms (SNPs). By utilizing the SNP data with the phenotype data, a single QTL was identified on chromosome 5 that explained 15% of the phenotypic variation (PV) for the number of eggs and 11% of the PV for the number of eggs per g of root (EGR). Plants that were homozygous for the Ky21 allele for the most associated marker PZA03172.3 had fewer eggs and fewer EGR than the plants that were homozygous or heterozygous for the B73 allele. Thus, the first QTL for SRKN resistance in maize has been identified and could be incorporated into maize hybrids.

Doxycycline Sclerodesis-"Rhinodesis"-for Enhanced Soft Tissue Adhesion in Rhinoplasty: A Preliminary Study.

The use of doxycycline as a sclerosing agent is well-established. Given the clinical efficacy of doxycycline sclerosant therapy, we embarked upon a study to evaluate the efficacy of small-volume liquified doxycycline particularly in thick skinned rhinoplasty patients to promote re-adhesion of the nasal skin-soft tissue envelope (SSTE) thereby minimizing surgical dead space and enhancing surface contour, to improve the eventual outcome of surgery.We present two clinical case series using rhinodesis. All patients were treated with the same rhinodesis protocol that included conventional splinting and taping. The first series consisted of 102 consecutive primary rhinoplasties with medium to thick nasal skin treated via open rhinoplasty. Doxycycline solution at a concentration of 20 mg/mL was applied beneath the skin flap using a 14-gauge angiocath inserted through small gaps in the marginal suture line following closure, retained for 2 to 3 minutes, and then expressed from the dead space. Firm manual compression of the SSTE was maintained for at least 1 additional minute, and the splint was then applied. The second series consisted of 25 thick-skinned primary rhinoplasties that were also treated with open rhinoplasty using the same rhinodesis protocol. However, the second group was evaluated with serial postoperative ultrasonography to characterize the soft-tissue response to rhinodesis, particularly within the tip and supra-tip regions.Results revealed enhanced skin adherence in nearly all patients when compared to traditional taping and splinting alone. Ultrasonic examination demonstrated enhanced adherence of the subcutaneous tissue to the nasal framework and suggests that rhinodesis is effective at minimizing dead space in majority of thick-skinned rhinoplasty patients. No complications were observed. Doxycycline can be used easily and safely to seal the surgical dead space post-rhinoplasty and minimize degradation of nasal contour with excellent outcome.

Chimeric antigen receptor T cells to target CD79b in B-cell lymphomas.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas. METHODS: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models. RESULTS: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion. CONCLUSION: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.

Surgical Inflammation Alters Immune Response to Intraoperative Photodynamic Therapy.

Surgical cytoreduction for patients with malignant pleural mesothelioma (MPM) is used for selected patients as a part of multi-modality management strategy. Our group has previously described the clinical use of photodynamic therapy (PDT), a form of non-ionizing radiation, as an intraoperative therapy option for MPM. Although necessary for the removal of bulk disease, the effects of surgery on residual MPM burden are not understood. In this bedside-to-bench study, Photofrin-based PDT introduced the possibility of achieving a long-term response in murine models of MPM tumors that were surgically debulked by 60% to 90%. Thus, the addition of PDT provided curative potential after an incomplete resection. Despite this success, we postulated that surgical induction of inflammation may mitigate the comprehensive response of residual disease to further therapy. Utilizing a previously validated tumor incision (TI) model, we demonstrated that the introduction of surgical incisions had no effect on acute cytotoxicity by PDT. However, we found that surgically induced inflammation limited the generation of antitumor immunity by PDT. Compared with PDT alone, when TI preceded PDT of mouse tumors, splenocytes and/or CD8+ T cells from the treated mice transferred less antitumor immunity to recipient animals. These results demonstrate that addition of PDT to surgical cytoreduction significantly improves long-term response compared with cytoreduction alone, but at the same time, the inflammation induced by surgery may limit the antitumor immunity generated by PDT. These data inform future potential approaches aimed at blocking surgically induced immunosuppression that might improve the outcomes of intraoperative combined modality treatment. Significance: Although mesothelioma is difficult to treat, we have shown that combining surgery with a form of radiation, photodynamic therapy, may help people with mesothelioma live longer. In this study, we demonstrate in mice that this regimen could be further improved by addressing the inflammation induced as a by-product of surgery.

SARS-CoV-2 omicron spike simulations: broad antibody escape, weakened ACE2 binding, and modest furin cleavage.

The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the BA1 and BA2 omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is a significant reduction of antibody (Ab) binding strength corresponding to escape, the omicron spikes pay a cost in terms of weaker receptor binding as measured by interfacial hydrogen bonds (H-bond). The furin cleavage domain (FCD) is the same or weaker binding than the delta variant, suggesting lower fusogenicity resulting in less viral load and disease intensity than the delta variant. IMPORTANCE The BA1 and BA2 and closely related BA2.12.2 and BA.5 omicron variants of SARS-CoV-2 dominate the current global infection landscape. Given the high number of mutations, particularly those which will lead to antibody escape, it is important to establish accurate methods that can guide developing health policy responses that identify at a fundamental level whether omicron and its variants are more threatening than its predecessors, especially delta. The importance of our work is to demonstrate that simple in silico simulations can predict biochemical binding details of the omicron spike protein that have epidemiological consequences, especially for binding to the cells and for fusing the viral membrane with the cells. In each case, we predicted weaker binding of the omicron spike, which agreed with subsequent experimental results. Future virology experiments will be needed to test these predictions further.

Global Meta-Analysis of Organoid and Organ-on-Chip Research.

Organoids and cells in organ-on-chip platforms replicate higher-level anatomical, physiological, or pathological states of tissues and organs. These technologies are widely regarded by academia, the pharmacological industry and regulators as key biomedical developments. To map advances in this emerging field, a meta-analysis based on a quality-controlled text-mining algorithm is performed. The analysis covers titles, keywords, and abstracts of categorized academic publications in the literature and preprint databases published after 2010. The algorithm identifies and tracks 149 and 107 organs or organ substructures modeled as organoids and organ-on-chip, respectively, stem cell sources, as well as 130 diseases, and 16 groups of organisms other than human and mouse in which organoid/organ-on-chip technology is applied. The meta-analysis illustrates changing diversity and focus in organoid/organ-on-chip research and captures its geographical distribution. The downloadable dataset provided is a robust framework for researchers to interrogate with their own questions.

The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

Beneficial effects of chronic mexiletine treatment in a human model of SCN5A overlap syndrome.

AIMS: SCN5A mutations are associated with various cardiac phenotypes, including long QT syndrome type 3 (LQT3), Brugada syndrome (BrS), and cardiac conduction disease (CCD). Certain mutations, such as SCN5A-1795insD, lead to an overlap syndrome, with patients exhibiting both features of BrS/CCD [decreased sodium current (INa)] and LQT3 (increased late INa). The sodium channel blocker mexiletine may acutely decrease LQT3-associated late INa and chronically increase peak INa associated with SCN5A loss-of-function mutations. However, most studies have so far employed heterologous expression systems and high mexiletine concentrations. We here investigated the effects of a therapeutic dose of mexiletine on the mixed phenotype associated with the SCN5A-1795insD mutation in HEK293A cells and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: To assess only the chronic effects on trafficking, HEK293A cells transfected with wild-type (WT) SCN5A or SCN5A-1795insD were incubated for 48 h with 10 µm mexiletine followed by wash-out, which resulted in an increased peak INa for both SCN5A-WT and SCN5A-1795insD and an increased late INa for SCN5A-1795insD. Acute re-exposure of HEK293A cells to 10 µm mexiletine did not impact on peak INa but significantly decreased SCN5A-1795insD late INa. Chronic incubation of SCN5A-1795insD hiPSC-CMs with mexiletine followed by wash-out increased peak INa, action potential (AP) upstroke velocity, and AP duration. Acute re-exposure did not impact on peak INa or AP upstroke velocity, but significantly decreased AP duration. CONCLUSION: These findings demonstrate for the first time the therapeutic benefit of mexiletine in a human cardiomyocyte model of SCN5A overlap syndrome.

Moving beyond audit: driving system learning using a novel mortality classification system in a tertiary training hospital in Kenya.

Clinical classification systems have proliferated since the APGAR score was introduced in 1953. Numerical scores and classification systems enable qualitative clinical descriptors to be transformed into categorical data, with both clinical utility and ability to provide a common language for learning. The clarity of classification rubrics embedded in a mortality classification system provides the shared basis for discussion and comparison of results. Mortality audits have been long seen as learning tools, but have tended to be siloed within a department and driven by individual learner need. We suggest that the learning needs of the system are also important. Therefore, the ability to learn from small mistakes and problems, rather than just from serious adverse events, remains facilitated.We describe a mortality classification system developed for use in the low-resource context and how it is 'fit for purpose,' able to drive both individual trainee, departmental and system learning. The utility of this classification system is that it addresses the low-resource context, including relevant factors such as limited prehospital emergency care, delayed presentation, and resource constraints. We describe five categories: (1) anticipated death or complication following terminal illness; (2) expected death or complication given clinical situation, despite taking preventive measures; (3) unexpected death or complication, not reasonably preventable; (4) potentially preventable death or complication: quality or systems issues identified and (5) unexpected death or complication resulting from medical intervention. We document how this classification system has driven learning at the individual trainee level, the departmental level, supported cross learning between departments and is being integrated into a comprehensive system-wide learning tool.

Venous Invasion in Pancreatic Neuroendocrine Tumors Is Independently Associated With Disease-free Survival and Overall Survival.

In this study, we evaluated venous invasion and its association with survival in patients with resected pancreatic neuroendocrine tumor (PanNET). Surgical Pathology Archives were searched for pancreatectomies performed for PanNET between October 1, 2005, and December 31, 2019. Hematoxylin and eosin (H&E)-stained slides were evaluated for venous invasion, and Movat's stain was performed in all cases with no venous invasion detected on H&E stains. Pathology reports and electronic medical records were also reviewed. Venous invasion was identified in 23 of 145 (15.9%) cases on H&E stains, and Movat's stain identified additional 34 cases with venous invasion (39.3% overall). Orphan arteries with adjacent well-defined tumor nodules or subtle hyalinizing nodules in hyalinizing tumors are highly specific for venous invasion. In stage I-III cases (n=122), venous invasion was associated with larger tumor size, higher World Health Organization (WHO) tumor grade, perineural invasion, extrapancreatic extension, lymph node metastasis, and liver metastasis ( P <0.05). In univariate analyses, tumor size, WHO grade, venous invasion, perineural invasion, T stage, and lymph node metastasis all correlated with disease-free survival; however, only venous invasion was associated with worse disease-free survival in multivariate analyses ( P <0.01). In all-stage cases, venous invasion was the only attributor associated with worse overall survival in multivariate analyses ( P =0.03). In summary, venous invasion in PanNET can be histologically subtle, and Movat's stain can greatly increase the detection rate. More importantly, enhanced venous invasion by Movat's stain correlates independently with disease-free survival in patients with stage I-III tumors and overall survival in all-stage patients.