The indications for robotic surgery in transplant and HPB procedures are rapidly expanding. However, there is growing concern about the adequacy of robotic training during fellowship in preparing trainees for the evolving demands of modern transplant surgery. This is a single center, retrospective review of the robotic experience of 9-abdominal transplant fellows (ASTS) over a 7-year period (2017–2024) at a high-volume center. A 10-question survey was administered to elicit fellow perspective on their training. The 9 fellows participated in 692 of the total 860 robotic cases (80

This review establishes current training pathways for HPB surgeons with a focus on the benefits of abdominal transplant experience for HPB-minded trainees. Abdominal transplant training is increasingly relevant to HPB surgery. Clinically, time spent on liver transplant services facilitates increases exposure to oncotransplant, a budding therapy increasingly utilized for various HPB malignancies. Technically, transplant regularly exposes trainees to retrohepatic anatomy, challenging porta dissections, and vascular anastomoses, skills beneficial for the advanced resections often performed today. Presently, trainees pursuing a career in HPB surgery have multiple means by which to gain transplant exposure, including select fellowships that offer joint HPB-transplant accreditation and institutions that participate in fellowship exchanges. Abdominal transplant surgery training confers unique clinical and technical expertise relevant to today’s HPB surgeon. Although currently HPB trainees may seek out abdominal transplant experience during fellowship, in the future this may be a standardized aspect of HPB fellowship.

We investigated the use of robotic objective performance metrics (OPM) to predict number of cases to proficiency and independence among abdominal transplant fellows performing robot-assisted donor nephrectomy (RDN). 101 RDNs were performed by 5 transplant fellows from September 2020 to October 2023. OPM included fellow percent active control time (

Abstract Background Neoantigen vaccines can induce or enhance highly specific antitumor immune responses with minimal risk of autoimmunity. We have developed a neoantigen DNA vaccine platform capable of efficiently presenting both HLA class I and II epitopes and performed a phase 1 clinical trial in triple-negative breast cancer patients with persistent disease on surgical pathology following neoadjuvant chemotherapy, a patient population at high risk of disease recurrence. Methods Expressed somatic mutations were identified by tumor/normal exome sequencing and tumor RNA sequencing. The pVACtools software suite of neoantigen prediction algorithms was used to identify and prioritize cancer neoantigens and facilitate vaccine design for manufacture in an academic GMP facility. Neoantigen DNA vaccines were administered via electroporation in the adjuvant setting (i.e., following surgical removal of the primary tumor and completion of standard of care therapy). Vaccines were monitored for safety and immune responses via ELISpot, intracellular cytokine production via flow cytometry, and TCR sequencing. Results Eighteen subjects received three doses of a neoantigen DNA vaccine encoding on average 11 neoantigens per patient (range 4–20). The vaccinations were well tolerated with relatively few adverse events. Neoantigen-specific T cell responses were induced in 14/18 patients as measured by ELISpot and flow cytometry. At a median follow-up of 36 months, recurrence-free survival was 87.5% (95% CI: 72.7–100%) in the cohort of vaccinated patients. Conclusion Our study demonstrates neoantigen DNA vaccines are safe, feasible, and capable of inducing neoantigen-specific immune responses. Clinical trial registration number NCT02348320.

Introduction: Despite considerable interest in robotic surgery, successful incorporation of robotics into transplant programs has been challenging. Lack of a dedicated OR team with expertise in both robotics and transplant is felt to be a major barrier. This paper assesses the impact of a dedicated robotic transplant team (DART) on program growth and fellowship training at one of the largest robotic transplant programs in North America. Methods: This is a single center, retrospective review of all robotic operations performed on the transplant surgery service from October 2017 to October 2022. DART was incorporated in February 2020 and included transplant first assists (RFAs), scrub technologists and circulating nurses who received robotic training. Robotic experience before and after DART was compared to assess its impact on program growth and training. Results: Four hundred and two robotic cases were performed by five transplant surgeons: 63 pre-DART and 339 post-DART. 40% of cases were transplant-related and 59.5%, HPB. There was a significant increase in case volume (2.5-10.6 cases/month, p <.0001) and complexity (36.5% vs. 70.3% high complexity cases, p <.0001) post-DART. RFA case coverage increased from 17% to 95%, and participation of transplant fellows as primary surgeons increased from 17% to 95% post-DART period (both p <.05). Conversion rates (9.5% vs. 4.1%) and room turn-around-times (TAT) (58.4 vs. 40.3 min) were lower post-DART (p <.05). There were no emergent conversions, conversions in transplant patients, or robot-related complications in either group. Conclusion: OR teams with expertise in robotics and transplant surgery can accelerate growth of robotic transplant programs while maintaining patient safety.

BACKGROUND:An increasing number of transplant centers have adopted robot-assisted living donor nephrectomy. Thus, a transplant fellow assessment tool is needed for promoting operative independence in an objective and safe manner.METHODS:In this pilot study, data was prospectively collected on both fellow performance with focus on technique, efficiency, and communication ("overall RO-SCORE"), and operative steps ("operative steps RO-SCORE"). Robotic user performance metrics were analyzed from the da Vinci Xi system, including fellow percent active control time (ACT) and handoff counts.RESULTS:From July 2020 to February 2021, twenty-one robot-assisted donor nephrectomies were performed. In regression analysis, fellow performance (based on both RO-SCOREs and robot % ACT) was significantly associated with both time and case number, with time-to-independence modelled at 8.4-14.2 months, and case number-to-independence estimated at 15-22 cases. Robot user metrics provided valid objective measures alongside RO-SCOREs.CONCLUSIONS:This pilot study provides an effective assessment tool for promoting operative competency in robot-assisted donor nephrectomy among transplant fellows.

ABSTRACTPURPOSECancer neoantigens are important targets of cancer immunotherapy. Neoantigen vaccines have the potential to induce or enhance highly specific antitumor immune responses with minimal risk of autoimmunity. We have developed a neoantigen DNA vaccine platform capable of efficiently presenting both HLA class I and II epitopes. To test the safety, feasibility and efficacy of this platform, we performed a phase 1 clinical trial in triple negative breast cancer patients with persistent disease following neoadjuvant chemotherapy, a patient population at high risk of disease recurrence.EXPERIMENTAL DESIGNExpressed somatic mutations were identified by tumor/normal exome sequencing and tumor RNA sequencing. The pVACtools software suite was used to identify and prioritize cancer neoantigens. Neoantigen DNA vaccines were designed and manufactured in an academic GMP facility at Washington University School of Medicine. Neoantigen DNA vaccines were administered via electroporation following completion of standard of care therapy. Safety was measured by clinical and laboratory evaluation. Immune responses were assessed by ELISPOT, flow cytometry and TCR sequencing.RESULTS18 subjects received three doses of a personalized neoantigen DNA vaccine encoding on average 11 neoantigens per patient (range 4-20). The vaccinations were well tolerated with limited adverse events, primarily related to injection site reactions. Neoantigen-specific immune responses were induced in 16/18 patients as measured by ELISPOT and flow cytometry. At a median follow-up of 36 months, progression-free survival was 87.5% (95% CI: 72.7-100%) in the cohort of vaccinated patients compared to 49% (95% CI: 36.4-65.9%) in a cohort of institutional historical control patients (p=0.011).CONCLUSIONSNeoantigen DNA vaccines are safe, feasible, and capable of inducing a neoantigen-specific immune response. There is preliminary evidence of improved disease-free survival compared to historical controls.

Conventional type 1 dendritic cells (cDC1)1 are thought to perform antigen cross-presentation, which is required to prime CD8+ T cells2,3, whereas cDC2 are specialized for priming CD4+ T cells4,5. CD4+ T cells are also considered to help CD8+ T cell responses through a variety of mechanisms6-11, including a process whereby CD4+ T cells 'license' cDC1 for CD8+ T cell priming12. However, this model has not been directly tested in vivo or in the setting of help-dependent tumour rejection. Here we generated an Xcr1Cre mouse strain to evaluate the cellular interactions that mediate tumour rejection in a model requiring CD4+ and CD8+ T cells. As expected, tumour rejection required cDC1 and CD8+ T cell priming required the expression of major histocompatibility class I molecules by cDC1. Unexpectedly, early priming of CD4+ T cells against tumour-derived antigens also required cDC1, and this was not simply because they transport antigens to lymph nodes for processing by cDC2, as selective deletion of major histocompatibility class II molecules in cDC1 also prevented early CD4+ T cell priming. Furthermore, deletion of either major histocompatibility class II or CD40 in cDC1 impaired tumour rejection, consistent with a role for cognate CD4+ T cell interactions and CD40 signalling in cDC1 licensing. Finally, CD40 signalling in cDC1 was critical not only for CD8+ T cell priming, but also for initial CD4+ T cell activation. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of antigen processing and priming for both CD4+ and CD8+ T cells and of the direct orchestration of their cross-talk that is required for optimal anti-tumour immunity.

BACKGROUND:The objective of this study was to determine if there is an impact of surgical delay on 5-year overall survival (OS) from early stage colon cancer, and if so, to define how long surgery can safely be postponed. METHODS:Using the NCDB, we compared early (14-30 days) and delayed surgery (31-90 days) in patients with Stage I/II colon cancer. Outcomes included OS at five years and odds of death. RESULTS:Delayed resection conferred a decreased 5-year OS of 73.0% (95% CI, 72.6-73.4), compared to early resection 78.3% (95% CI, 77.9-78.8). When time to surgery was divided into one-week intervals, there was no difference in the odds of death with delay up to 35-41 days (6 weeks), but odds of death increased by 9% per week thereafter. CONCLUSIONS:These data support that definitive resection for early stage colon cancer may be safely delayed up to 6 weeks.

Appendiceal neuroendocrine tumors (A-NETs) are rare neoplasms of the GI tract. They are typically managed according to tumor size; however, the impact of surgical strategy on the short- and long-term outcomes is unknown. All patients who underwent resection of A-NET at 8 institutions from 2000 to 2016 were analyzed retrospectively. Patient clinicopathologic features and outcomes were stratified according to resection type. Of 61 patients identified with A-NET, mean age of presentation was 44.7 ± 16.0 years and patients were predominantly Caucasian (77%) and female (56%). Mean tumor size was 1.2 ± 1.3 cm with a median of 0.8 cm. Thirty-one patients (51%) underwent appendectomy and 30 (49%) underwent colonic resection. The appendectomy group had more T1 tumors (87% vs 42%, p < 0.01) than the colon resection group. Of patients in the colon resection group, 27% had positive lymph nodes and 3% had M1 disease. R0 resections were achieved in 90% of appendectomy patients and 97% of colon resection patients. Complications occurred with a higher frequency in the colon resection group (30%) compared with those in the appendectomy group (6%, p = 0.02). The colon resection group also had a longer length of stay, higher average blood loss, and longer average OR time. Median RFS and OS were similar between groups. A-NET RFS and OS are equivalent regardless of surgical strategy. Formal colon resection is associated with increased length of stay, OR time, higher blood loss, and more complications. Further study is warranted to identify patients that are likely to benefit from more aggressive surgery.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.

Cross-presentation allows for the presentation of exogenous viral and tumor antigens to CD8+ T cells by XCR1+ classical dendritic cells (cDC1s). Previously, the role of cross-presentation in cytotoxic T cell responses in vivo was unclear due to the lack of mouse models specifically deficient in cross-presentation. We developed a CRISPR-Cas9 screen to find novel regulators of cross-presentation and found that the protein WDFY4 is essential for cross-presentation of cell-associated antigens but not for direct presentation or presentation on MHCII. WDFY4-deficient mice had normal cDC1 development and were able to survive infections with Toxoplasma gondii through production of IL-12. However, WDFY4-deficient mice failed to prime CD8+ T cells to viral antigens and were completely unable to control immunogenic tumors. WDFY4 interacts with proteins related to vesicular formation and trafficking such as clathrin and HSP90ab1 and localizes near the cell surface, suggesting its involvement in vesicular targeting after antigen uptake during cross-presentation.

During the process of cross-presentation, viral or tumor-derived antigens are presented to CD8+ T cells by Batf3-dependent CD8α+/XCR1+ classical dendritic cells (cDC1s). We designed a functional CRISPR screen for previously unknown regulators of cross-presentation, and identified the BEACH domain-containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1s in mice. However, WDFY4 was not required for major histocompatibility complex class II presentation, nor for cross-presentation by monocyte-derived dendritic cells. In contrast to Batf3 -/- mice, Wdfy4 -/- mice displayed normal lymphoid and nonlymphoid cDC1 populations that produce interleukin-12 and protect against Toxoplasma gondii infection. However, similar to Batf3 -/- mice, Wdfy4 -/- mice failed to prime virus-specific CD8+ T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in antiviral and antitumor immunity.

Abstract Introduction: Dendritic cells (DCs) are professional antigen-presenting cells with two major subsets, the classical DC type 1 and 2 (cDC1 and cDC2). The cDC1, also known as the CD8α+ DC, is especially equipped for the process of cross-presentation in which dead cell antigen is presented on MHC-I to prime cytotoxic T cells. This process is relevant to anti-cancer immunity, as mice deficient in the transcription factor BATF3, which specifically lack the cDC1, succumb to highly immunogenic tumors that are normally rejected and cleared in wild-type mice. The mechanism of cross-presentation by the cDC1 is poorly understood. In this study, we developed a focused CRISPR/Cas9 screen to identify novel genes that endow the cDC1 with the ability to cross-prime cytotoxic T cells. Methods: A curated list of target cDC1 genes was constructed using the Immunological Genome Project Database as the primary resource (www.immgen.org). 120 genes specifically expressed by the cDC1 in comparison to other murine immune cells were selected. Guide RNAs (gRNAs) were designed to early exon sequences using the MIT CRISPR Design Tool to minimize off-target effects (www.crispr.mit.edu), and 2-3 different gRNAs were evaluated in tandem for each candidate. These gRNAs were transduced via retroviral vector into lineage depleted c-Kithi bone marrow cells isolated from C57BL/6J Cas9 knock-in mice. These cells were differentiated in vitro for 8 days in the presence of the cytokine Flt3L. Transduced DCs were sorted by FACS and co-cultured in the presence of a source of dead cell antigen (heat-killed L. monocytogenes expressing ovalbumin, or HKLM-OVA) as well as CFSE-labelled transgenic CD8+ T cells with an ovalbumin peptide-specific T cell receptor (OT-1). The proliferation of OT-1 cells was measured by CFSE dilution. Results: To validate our CRISPR/Cas9-based screening approach, we targeted the first exon of the beta-2 microglobulin gene (B2M), which is required for cell surface expression of MHC-I. B2M knockout DCs were co-cultured in the presence of HKLM-OVA and CSFE-labelled OT-1 cells for 3 days. Controls included DCs transduced with a scramble gRNA with or without antigen. As expected, targeting B2M in DCs led to near-complete reduction in OT-1 proliferation compared to scramble control (3.7% vs. 20.6%, p=0.003), similar to the absence of antigen (3.7% vs. 0.96%, p=0.99). We then expanded our assay to our curated list. Promising candidate genes have been discovered and are currently being validated in murine knockout models. Conclusions: The CRISPR/Cas9 system can be adapted to screen the cDC1 for novel genes involved in cross-priming cytotoxic T cells. Validation of candidates in vivo, including tumor assays, will be presented at the meeting. These findings may have important implications for adaptive immunity against cancer. Citation Format: Jesse T. Davidson, Derek J. Theisen, Carlos G. Briseño, Vivek Durai, William E. Gillanders, Theresa L. Murphy, Kenneth M. Murphy. A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3781.

694 Background: Intestinal neuroendocrine tumors (NETs) can often be indolent, with survival commonly exceeding 10 years. However, a subset can be more aggressive. The search for a prognostic test based on preoperative lab results would be of benefit. An elevated NLR has been found to be predictive of diminished overall survival (OS) in several cancers, and we hypothesized that this same trend would hold true for intestinal NETs. Methods: Utilizing the 8 member institutions of the retrospective U.S. NET Study Group, patients undergoing resection of intestinal NETs were included. After excluding patients undergoing emergent surgery, those with preoperative sepsis or distant mets, and those with missing neutrophil/lymphocyte lab values, 281 patients were included for analysis. The cutoff values for NLR were determined using receiver operator curve (ROC) analysis. Univariate analysis was performed based on these groups. A Kaplan-Meier survival curve was created based off optimal NLR from ROC analysis. OS was calculated as date of surgery to date of death/ last follow-up. SPSS 23.0 was used for analysis. Results: Of the 281 patients identified, 9% were appendiceal, 9% colonic, 21% duodenal, 5% rectal, and 56% jejunal/ileal. Using a NLR of >2.47, the ROC area under the curve was 0.609 ( p=0.002). Median survival for the 130 patients in the <2.47 group was 159.8 months, versus 115.63 months in the ≥2.47 group (p =0.009). When comparing the NLR <2.47 and ≥2.47 groups, mean age (57.5 vs. 59.0 years, respectively, p=0.642), ASA status (89% ASA 2 /3 in both groups, p=0.314), tumor Ki-67 status (Ki-67>3% in 30% vs. 26%, respectively, p=0.57), and post-op complications (36 vs. 42%, respectively, p=0.310) were similar between groups. A significantly greater number of patients in the NLR high group had positive lymph nodes (60 vs. 47%, p =0.002). Conclusions: For patients undergoing curative resection of intestinal NETs with no distant mets, a preoperative NLR of ≥2.47 was significantly predictive of decreased OS, with those in the elevated NLR group having an OS 3.7 years less than those in the <2.47 group. Future prospective data is warranted to further validate these findings.

BACKGROUND:Staging laparoscopy (SL) is used to avoid resection failure and thus increase the curative resection rate. SL utilization in extra-hepatic biliary tumors (EHBT) is variable.METHODS:Data from 1090 patients with potentially resectable EHBT including gallbladder (GBC), distal (DC), and hilar (HC) subtypes were retrospectively collected from 10 academic centers (2000-2015).RESULTS:The SL utilization rate increased over time and was significantly higher in GBC than DC and HC. SL yield was 16.8% and did not differ between groups or over time. In patients undergoing attempted resection with prior SL, the curative resection rate did not differ between subtypes. In patients undergoing attempted resection without prior SL, the curative resection rate was less in GBC compared with DC or HC. After matching cohorts by inverse probability weighting, prior SL was associated with curative resection in GBC only (odds ratio [OR], 2.41, 95% CI, 1.36-4.27). On multivariable regression analysis, elevated carbohydrate antigen 19-9 (CA 19-9), low serum albumin, and GBC were strong predictors of distant disease on SL. After categorizing patients undergoing SL into low, intermediate, and high-risk groups based on these parameters, SL yield improved progressively from 10.0% to 19.6% to 52.6%.CONCLUSIONS:We recommend routine SL for patients with GBC, particularly with elevated CA19-9 level and/or decreased serum albumin.

CD4 + T follicular helper (T FH ) cells support germinal center (GC) reactions promoting humoral immunity. Dendritic cell (DC) diversification into genetically distinct subsets allows for specialization in promoting responses against several types of pathogens. Whether any classical DC (cDC) subset is required for humoral immunity is unknown, however. We tested several genetic models that selectively ablate distinct DC subsets in mice for their impact on splenic GC reactions. We identified a requirement for Notch2 -dependent cDC2s, but not Batf3 -dependent cDC1s or Klf4 -dependent cDC2s, in promoting T FH and GC B cell formation in response to sheep red blood cells and inactivated Listeria monocytogenes . This effect was mediated independent of Il2ra and several Notch2 -dependent genes expressed in cDC2s, including Stat4 and Havcr2 . Notch2 signaling during cDC2 development also substantially reduced the efficiency of cDC2s for presentation of MHC class II-restricted antigens, limiting the strength of CD4 T cell activation. Together, these results demonstrate a nonredundant role for the Notch2 -dependent cDC2 subset in supporting humoral immune responses.

The receptor Flt3 and its ligand Flt3L are both critical for dendritic cell (DC) development, but DC deficiency is more severe in Flt3l−/− mice than in Flt3−/− mice. This has led to speculation that Flt3L binds to another receptor that also supports DC development. However, we found that Flt3L administration does not generate DCs in Flt3−/− mice, arguing against a second receptor. Instead, Flt3−/− DC progenitors matured in response to macrophage colony–stimulating factor (M-CSF) or stem cell factor, and deletion of Csf1r in Flt3−/− mice further reduced DC development, indicating that these cytokines could compensate for Flt3. Surprisingly, Flt3−/− DC progenitors displayed enhanced M-CSF signaling, suggesting that loss of Flt3 increased responsiveness to other cytokines. In agreement, deletion of Flt3 in Flt3l−/− mice paradoxically rescued their severe DC deficiency. Thus, multiple cytokines can support DC development, and the discrepancy between Flt3−/− and Flt3l−/− mice results from the increased sensitivity of Flt3−/− progenitors to these cytokines.

Background: Surgical resection is the cornerstone of curative-intent therapy for patients with hilar cholangiocarcinoma (HC). The role of vascular resection (VR) in the treatment of HC in western centres is not well defined. Methods: Utilizing data from the U.S. Extrahepatic Biliary Malignancy Consortium, patients were grouped into those who underwent resection for HC based on VR status: no VR, portal vein resection (PVR), or hepatic artery resection (HAR). Perioperative and long-term survival outcomes were analyzed. Results: Between 1998 and 2015, 201 patients underwent resection for HC, of which 31 (15%) underwent VR: 19 patients (9%) underwent PVR alone and 12 patients (6%) underwent HAR either with (n = 2) or without PVR (n = 10). Patients selected for VR tended to be younger with higher stage disease. Rates of postoperative complications and 30-day mortality were similar when stratified by vascular resection status. On multivariate analysis, receipt of PVR or HAR did not significantly affect OS or RFS. Conclusion: In a modern, multi-institutional cohort of patients undergoing curative-intent resection for HC, VR appears to be a safe procedure in a highly selected subset, although long-term survival outcomes appear equivalent. VR should be considered only in select patients based on tumor and patient characteristics.