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Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

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.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.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.This pilot study provides an effective assessment tool for promoting operative competency in robot-assisted donor nephrectomy among transplant fellows.

This investigation described clinicopathological features and outcomes of extraskeletal myxoid chondrosarcoma (EMC) patients.EMC patients were identified from the United States Sarcoma Collaborative database between 2000 and 2016. Overall survival (OS) and recurrence-free survival (RFS) were calculated, and prognostic factors were analyzed.Sixty individuals with a mean age of 55 years were included, and 65.0% (n = 39) were male. 73.3% (n = 44) had a primary tumor. A total of 41.6% (n = 25) developed tumor relapse following resection. The locoregional recurrence rate was 30.0% (n = 18/60), and mean follow-up was 42.7 months. The 5-year OS was 71.0%, while the 5-year RFS was 41.4%. On multivariate analysis for all EMC, chemotherapy (hazard ratio [HR], 6.054; 95% confidence interval [CI], 1.33-27.7; p = 0.020) and radiation (HR, 5.07, 95% CI, 1.3-20.1; p = 0.021) were independently predictive of a worse RFS. Among patients with primary EMC only, the 5-year OS was 85.3%, with a 30.0% (n = 12) locoregional recurrence rate, though no significant prognostic factors were identified.Long-term survival with EMC is probable, however there exists a high incidence of locoregional recurrence. While chemotherapy and radiation were associated with a worse RFS, these findings were likely confounded by recurrent disease as significance was lost in the primary EMC-only subset.

Soft tissue sarcomas are a heterogenous group of neoplasms without well-validated biomarkers. Cancer-related inflammation is a known driver of tumor growth and progression. Recent studies have implicated a high circulating neutrophil-lymphocyte ratio as a surrogate marker for the inflammatory tumor microenvironment and a poor prognosticator in multiple solid tumors, including colorectal and pancreatic cancers. The impact of circulating neutrophil-lymphocyte ratio in soft tissue sarcomas has yet to be elucidated.We performed a retrospective analysis of patients undergoing curative resection for primary or recurrent extremity soft tissue sarcomas at academic centers within the US Sarcoma Collaborative. Neutrophil-lymphocyte ratio was calculated retrospectively in treatment-naïve patients using blood counts at or near diagnosis.A high neutrophil-lymphocyte ratio (≥4.5) was associated with worse survival on univariable analysis in patients with extremity soft tissue sarcomas (hazard ratio 2.07; 95% confidence interval, 1.54-2.8; P.001). On multivariable analysis, increasing age (hazard ratio 1.03; 95% confidence interval, 1.02-1.04; P.001), American Joint Committee on Cancer T3 (hazard ratio 1.89; 95% confidence interval, 1.16-3.09; P = .011), American Joint Committee on Cancer T4 (hazard ratio 2.36; 95% confidence interval, 1.42-3.92; P = .001), high tumor grade (hazard ratio 4.56; 95% confidence interval, 2.2-9.45; P.001), and radiotherapy (hazard ratio 0.58; 95% confidence interval, 0.41-0.82; P = .002) were independently predictive of overall survival, but a high neutrophil-lymphocyte ratio was not predictive of survival (hazard ratio 1.26; 95% confidence interval, 0.87-1.82; P = .22).Tumor inflammation as measured by high pretreatment neutrophil-lymphocyte ratio was not independently associated with overall survival in patients undergoing resection for extremity soft tissue sarcomas.

Allogeneic cancer vaccines are designed to induce antitumor immune responses with the goal of impacting tumor growth. Typical allogeneic cancer vaccines are produced by expansion of established cancer cell lines, transfection with vectors encoding immunostimulatory cytokines, and lethal irradiation. More than 100 clinical trials have investigated the clinical benefit of allogeneic cancer vaccines in various cancer types. Results show limited therapeutic benefit in clinical trials and currently there are no FDA-approved allogeneic cancer vaccines. We used recently developed bioinformatics tools including the pVACseq suite of software tools to analyze DNA/RNA-sequencing data from the The Cancer Genome Atlas to examine the repertoire of antigens presented by a typical allogeneic cancer vaccine, and to simulate allogeneic cancer vaccine clinical trials. Specifically, for each simulated clinical trial, we modeled the repertoire of antigens presented by allogeneic cancer vaccines consisting of three hypothetical cancer cell lines to 30 patients with the same cancer type. Simulations were repeated ten times for each cancer type. Each tumor sample in the vaccine and the vaccine recipient was subjected to human leukocyte antigen (HLA) typing, differential expression analyses for tumor-associated antigens (TAA), germline variant calling, and neoantigen prediction. These analyses provided a robust, quantitative comparison between potentially beneficial TAAs and neoantigens versus distracting antigens present in the allogeneic cancer vaccines. We observe that distracting antigens greatly outnumber shared TAAs and neoantigens, providing one potential explanation for the lack of observed responses to allogeneic cancer vaccines. This analysis provides additional rationale for the redirection of efforts toward a personalized cancer vaccine approach. Significance: A comprehensive examination of allogeneic cancer vaccine antigen repertoire using large-scale genomics datasets highlights the large number of distracting antigens and argues for more personalized approaches to immunotherapy that leverage recent strategies in tumor antigen identification.

Purpose: FOLFIRINOX has demonstrated promising results for patients with pancreatic ductal adenocarcinoma (PDAC). Chemotherapy-induced immunogenic cell death can prime antitumor immune responses. We therefore performed high-dimensional profiling of immune cell subsets in peripheral blood to evaluate the impact of FOLFIRINOX on the immune system. Experimental Design: Peripheral blood mononuclear cells (PBMC) were obtained from treatment-naïve (n = 20) and FOLFIRINOX-treated patients (n = 19) with primary PDAC tumors at the time of resection. PBMCs were characterized by 36 markers using mass cytometry by time of flight (CyTOF). Results: Compared with treatment-naïve patients, FOLFIRINOX-treated patients showed distinct immune profiles, including significantly decreased inflammatory monocytes and regulatory T cells (Treg), increased Th1 cells, and decreased Th2 cells. Notably, both monocytes and Treg expressed high levels of immune suppression-associated CD39, and the total CD39+ cell population was significantly lower in FOLFIRINOX-treated patients compared with untreated patients. Cellular alterations observed in responders to FOLFIRINOX included a significantly decreased frequency of Treg, an increased frequency of total CD8 T cells, and an increased frequency of CD27−Tbet+ effector/effector memory subsets of CD4 and CD8 T cells. Conclusions: Our study reveals that neoadjuvant chemotherapy with FOLFIRINOX enhances effector T cells and downregulates suppressor cells. These data indicate that FOLFIRINOX neoadjuvant therapy may improve immune therapy and clinical outcome in patients with PDAC.

BACKGROUND: Postoperative wound complications are challenging in patients with localized extremity soft-tissue sarcomas. Various factors have been associated with wound complications, but there is no individualized predictive model to allow providers to counsel their patients and thus offer methods to mitigate the risk of complications and implement appropriate measures. QUESTIONS/PURPOSES: We used data from multiple centers to ask: (1) What risk factors are associated with postoperative wound complications in patients with localized soft-tissue sarcomas of the extremity? (2) Can we create a predictive nomogram that will assess the risk of wound complications in individual patients after resection for soft-tissue sarcoma? METHODS: From 2000 to 2016, 1669 patients undergoing limb-salvage resection for a localized primary or recurrent extremity soft-tissue sarcoma with at least 120 days of follow-up at eight participating United States Sarcoma Collaborative institutions were identified. Wound complications included superficial wounds with or without drainage, deep wounds with drainage because of dehiscence, and intentional opening of the wound within 120 days postoperatively. Sixteen variables were selected a priori by clinicians and statisticians as potential risk factors for wound complications. A univariate analysis was performed using Fisher’s exact tests for categorical predictors, and Wilcoxon’s rank-sum tests were used for continuous predictors. A multiple logistic regression analysis was used to train the prediction model that was used to create the nomogram. The prediction performance of the datasets was evaluated using a receiver operating curve, area under the curve, and calibration plot. RESULTS: After controlling for potential confounding factors such as comorbidities, functional status, albumin level, and chemotherapy use, we found that increasing age (odds ratio 1.02; 95% confidence interval, 1.00-1.03; p = 0.008), BMI (OR 1.05; 95% CI, 1.02-1.09; p = 0.004), lower-extremity location (OR 6; 95% CI, 2.87-12.69; p < 0.001), and neoadjuvant radiation (OR 2; 95% CI, 1.47-3.16; p < 0.001) were associated with postoperative wound complications (area under the curve 69.2% [range 62.8%-75.6%]). CONCLUSIONS: We found that age, BMI, tumor location, and timing of radiation are associated with the risk of wound complications. Based on these factors, a validated nomogram has been established that can provide an individualized prediction of wound complications in patients with a resected soft-tissue sarcoma of the extremity. This may allow for proactive management with nutrition and surgical techniques, and help determine the delivery of radiation in patients with a high risk of having these complications. LEVEL OF EVIDENCE: Level III, therapeutic study.

OBJECTIVES: The “Surgery for Abdomino-thoracic ViolencE (SAVE)” animate lab engages surgical residents in the management of complex penetrating injuries. We hypothesized that residents will improve their understanding of the management of trauma patients and will perform skills that they have not previously performed in training. DESIGN: Pre- and post-lab assessments were reviewed from surgical residents participating in the SAVE lab over two years (2017-2018). Residents of varying levels were grouped and reviewed “real-life” trauma scenarios with supplemental imaging. Seniors were tasked with creating injuries while juniors performed as primary surgeons under supervision. Each successive scenario increased in difficulty, from hollow viscus injury and solid organ disruption, to great vessel and cardiac injuries with the goal to “SAVE” the patient. Assessments included a pre- and post-lab multiple-choice questionnaire of trauma management knowledge and a survey of completed technical skills. SETTING: Academic General Surgery residency program PARTICIPANTS: General, Vascular, Urology, and Plastic Surgery PGY1-5 residents RESULTS: 119 residents participated in the SAVE lab in 2017 and 2018. PGY1-PGY4 residents showed significant improvement in knowledge of trauma management on matched pre- and post-lab assessments. The most significant improvement was seen in the PGY1 and PGY2 residents, with scores increasing by 21% (p

The postoperative outcomes of elderly patients undergoing resection of retroperitoneal sarcomas (RPS) have not been widely studied.Patients undergoing surgical resection for primary or recurrent RPS between 2000 and 2015 at participating US Sarcoma Collaborative institutions were identified. Patient demographics, perioperative morbidity, mortality, length of stay, discharge to home, disease-specific survival, and disease-free survival were compared between elderly (≥70 y, n = 171) and nonelderly (70 y, n = 494) patients.There was no difference in perioperative morbidity (total and major complications elderly versus nonelderly: 39% versus 35%; P = 0.401 and 18% versus 17%; P = 0.646, respectively) or mortality between elderly and nonelderly patients with each group experiencing a 1% 30-d mortality rate. Length of stay and 30-d readmission rates were similar (elderly versus nonelderly; 7 d interquartile range [IQR: 5-9] versus 6 d [IQR: 4-9], P = 0.528 and 11% versus 12%, P = 0.667). Elderly patients were more likely to be discharged to a skilled nursing or rehabilitation facility (elderly versus nonelderly; 19% versus 7%, P 0.001). There was no difference in 3-y disease-free survival between the elderly and nonelderly patients (41% versus 43%, P = 0.65); however, elderly patients had a lower 3-y disease-specific survival (60% versus 76%, P 0.001). In elderly patients, the presence of multiple comorbidities and high-grade tumors were most predictive of outcomes.Advanced age was not associated with an increased risk of perioperative morbidity and mortality following resection of RPS in this multi-institutional review. Although short-term oncologic outcomes were similar in both groups, the risk of death after sarcoma recurrence was higher in elderly patients and may be related to comorbidity burden and tumor histology.

Background The "Surgery for Abdomino-thoracic ViolencE (SAVE)" animate lab engages surgical residents in the management of penetrating injuries in a team setting. Senior residents, representing postgraduate year (PGY) 3-5, assume the role of team leader and facilitate the junior residents, PGY1-2, in operative management of simulated penetrating wounds. Residents completed five scenarios with increasing level of difficulty within set time limits. Senior residents were evaluated on their team's ability to "SAVE" their patient within the time allotted, as well as their communication and leadership skills. Methods General, vascular, urology, and plastic surgery residents (n = 79) were divided into 25 teams of three to four residents by "resident scores" (R scores, the sum of the team members' PGY) to create balanced teams with comparable years of clinical experience. Residents completed assessments of their senior resident's leadership ability and style. Results Evaluation of a resident's desired learning style changed across PGY with junior residents preferring more hands-on guidance compared with senior residents preferring only verbal correction. Resident leadership evaluations demonstrated that team leaders of varied resident years achieved the highest scores. Greater differences in the mismatch between autonomy provided to and desired by junior residents correlated to greater junior resident discomfort in expressing their opinion, confidence, and leadership ratings of senior residents. However, greater autonomy mismatch also correlated to more rapid time to task completion. Conclusion Different from our expectations, clinical experience alone did not define team leader success. Leadership is a powerful influence on the outcome of team performance and may be a skill, which can transcend overall clinical experience. A match between desired and provided resident autonomy and team cohesion may demonstrate a stronger effect on team success in stressful operative situations, such as trauma resuscitation. Enhancement of leadership skills early in residency training may represent an important focus for trauma surgery education.

Background Gun violence (GV) is a complex public health issue, and the management of GV as a disease engages the surgeon in technical and nontechnical skills. The Anatomy of Gun Violence (AGV) curriculum was developed to teach surgical trainees these seemingly disparate skills, training residents to manage the multiple aspects of firearm injury. Study Design The AGV curriculum was delivered over 6 weeks in the 2017–2018 and 2018–2019 academic years (AY), and used multiple educational methods including didactic lectures, mock oral examinations, a Bleeding Control training session, a GV survivor’s personal story, a Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) training session, and the Surgery for Abdominal-thoracic ViolencE (SAVE) simulation lab. As surgical residents were involved over both AYs, components of the curriculum were available every other year to provide variety. As proof of concept, this novel curriculum was objectively evaluated by residents’ improvement in knowledge and overall experience using pre- and post-surveys. Results Sixty surgical residents participated in the AGV curriculum in both AYs, with 41 and 36 residents completing the survey regarding their experiences with the curriculum. The curriculum was well received by residents overall in both AYs (median ± IQR 5 ± 0 and 5 ± 0.1, respectively), with the SAVE simulation lab being the most highly favored portion. Additionally, residents had an average 7.5% improvement in knowledge attributed to the curriculum, with a larger effect seen in the junior residents. Conclusions This novel AGV curriculum created a well-received learning experience involving the technical and nontechnical skills necessary to care for GV victims. This comprehensive approach to GV may represent a unique opportunity to engage surgical trainees in both the treatment and prevention of firearm injury.

ObjectiveChemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated CCR2+ macrophages (TAM) alone improves antitumour immunity in preclinical models. However, a compensatory influx of an alternative myeloid subset may result in a persistent immunosuppressive TME and promote therapeutic resistance. Here, we show CCR2 and CXCR2 combined blockade reduces total tumour-infiltrating myeloids, promoting a more robust antitumour immune response in PDAC compared with either strategy alone.MethodsBlood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy.ResultsA systemic increase in CXCR2+ TAN correlates with poor prognosis in PDAC, and patients receiving CCR2i showed increased tumour-infiltrating CXCR2+ TAN following treatment. In an orthotopic PDAC model, CXCR2 blockade prevented neutrophil mobilisation from the circulation and augmented chemotherapeutic efficacy. However, depletion of either CXCR2+ TAN or CCR2+ TAM resulted in a compensatory response of the alternative myeloid subset, recapitulating human disease. This was overcome by combined CCR2i and CXCR2i, which augmented antitumour immunity and improved response to FOLFIRINOX chemotherapy.ConclusionDual targeting of CCR2+ TAM and CXCR2+ TAN improves antitumour immunity and chemotherapeutic response in PDAC compared with either strategy alone.

Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the capacity to deliver small molecules as drug cargoes. We utilized the sigma-2-based drug delivery concept to convert Erastin, a clinically underperforming drug, into a potent pancreatic cancer therapeutic. The Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to sigma-2 ligand SV119 to create SW V-49. Conjugation increased the killing capacity of dm-Erastin by nearly 35-fold in vitro and reduced the size of established tumors and doubled the median survival in syngeneic and patient-derived xenograft models when compared to non-targeted dm-Erastin. Mechanistic analyses demonstrated that cell death was associated with robust reactive oxygen species production and could be efficiently antagonized with antioxidants. Mass spectrometry was employed to demonstrate selective uptake into pancreatic cancer cells. Thus, targeted delivery of dm-Erastin via conjugation to the sigma-2 ligand SV119 produced efficient tumor control and prolonged animal survival with minimal off-target toxicities, and SW V-49 represents a promising new therapeutic with the potential to advance the fight against pancreatic cancer.

FOLFIRINOX is the first line treatment option for patients with pancreatic ductal adenocarcinoma (PDAC). Neoadjuvant chemotherapy has a beneficial effect on overall survival in PDAC in comparison with upfront surgery and adjuvant therapy. However, chemotherapy has not yet been successfully combined with immune therapy in PDAC. Herein, we characterize the immunologic effect of FOLFIRINOX and thus provide a rationale for future combination with immune therapy in PDAC. Peripheral Blood Mononuclear Cells were obtained from treatment-naïve (n=20) and FOLFIRINOX-treated patients (n=15) with primary PDAC tumors. Immune cell subset composition was assessed by using mass cytometry (CyTOF). Response to FOLFIRINOX was defined as a reduction in tumor size by more than 30%. 8 of 15 patients were considered responders with a range from 32 to 60% reduction in tumor volume. Responders to FOLFIRINOX showed a significantly higher proportion of CD8+ T cells and lower proportion of monocytes in their peripheral blood compared to treatment naïve patients (both p

BACKGROUND The 8th edition AJCC staging system for truncal/extremity soft tissue sarcoma (STS) offers significant changes from the 7th. However the complexity of both limits their clinical utility. METHODS Patients with truncal/extremity STS undergoing resection from 2000 to 2016 at seven institutions of the US Sarcoma Collaborative were analyzed. The proposed staging system was externally validated using the National Cancer Database (NCDB). RESULTS Of 1318 patients, mean age was 59 years, and 54% were male. Median tumor size was 9 cm; 72% were high grade. Applying 8th edition staging, there was no differentiation between stages IA/IB ( P = 0.92), and clinically similar outcomes between stages II/IIIA. Receiver operating characteristic (ROC) analysis identified 7.5 cm as the ideal tumor size discriminating 5-year OS for high-grade tumors. Therefore, a simplified staging system defining all low-grade tumors as stage I, high-grade 7.5 cm as stage III, and metastatic disease as stage IV improved stratification (all P

Myeloid derived suppressor cells (MDSCs) are a heterogeneous group of bone marrow derived immature myeloid cells that are expanded in patients with cancer, infection, trauma, or autoimmune diseases (1). MDSC originate from a common myeloid progenitor cell and develop along two separate lineages, monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC), but never develop the characteristics of mature monocytes, macrophages, or neutrophils (1).

Chemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2Blood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy.A systemic increase in CXCR2Dual targeting of CCR2

Objective Fundamentals of laparoscopic surgery (FLS) was developed by the Society of American Gastrointestinal and Endoscopic Surgeons to teach the physiology, fundamental knowledge, and technical skills required for basic laparoscopic surgery. We hypothesize that residents are doing more laparoscopic surgery earlier in residency, and therefore would benefit from an earlier assessment of basic laparoscopic skills. Here, we examine FLS test results and ACGME case logs to determine whether it is practical to administer FLS earlier in residency. Design FLS test results were reviewed for the 42 residents completing FLS between July 2011 and July 2016. ACGME case logs for current and former residents were reviewed for laparoscopic cases logged by each postgraduate year. Basic and complex laparoscopic cases were determined by ACGME General Surgery Defined Category and Minimums Report. Descriptive statistics were used for analysis. Setting Academic general surgery residency, Washington University in St. Louis School of Medicine. Participants Current and former general surgery residents. Results A total of 42 residents took and passed FLS between July 2011 and July 2016. All residents successfully passed the FLS knowledge and skills examinations on the first attempt regardless of their postgraduate year (PGY 3 n = 13, PGY 4 n = 15, and PGY 5 n = 14). Total laparoscopic case volume has increased over time. Residents who graduated in 2012 or 2013 completed 229 laparoscopic cases compared to 267 laparoscopic cases for those who graduated from 2014 to 2016 (p = 0.02). Additionally, current residents completed more laparoscopic cases in the first 2 years of residency than residents who graduated from 2012 to 2016 (median current = 38; former = 22; p Examining laparoscopic case numbers for current residents by PGY demonstrated that the number of total and complex laparoscopic cases increased in each of the first 3 years of residency with the largest increase occurring between the PGY 2 and PGY 3 years. In the PGY 4 and PGY 5 years, most laparoscopic cases were complex. Conclusion Increased use of laparoscopic surgery has led to a corresponding increase in laparoscopic case volume among general surgery residents. We would advocate for FLS testing to serve as an early assessment of laparoscopic knowledge and skill and should be performed before a significant increase in complex laparoscopic surgery during training.

Operative management at our institution for all colon injuries have followed a defined algorithm (ALG) based on risk factors originally identified for penetrating injuries. The purpose of this study was to evaluate the applicability of the ALG to blunt colon injuries.Patients with blunt colon injuries during 13 years were identified. As per the ALG, nondestructive (ND) injuries are treated with primary repair. Patients with destructive wounds (serosal tear of ≥50% colon circumference, mesenteric devascularization, and perforations) and concomitant risk factors (transfusion of6 U packed red blood cells and/or presence of significant comorbidities) are diverted, while patients with no risk factors undergo resection plus anastomosis (RA). Outcomes included suture line failure (SLF), abscess, and mortality. Stratification analysis was performed to determine additional risk factors in the management of blunt colon injuries.A total 151 patients were identified: 76 with destructive injuries and 75 with ND injuries. Of those with destructive injuries, 44 (59%) underwent RA and 29 (39%) underwent diversion. All ND injuries underwent primary repair. Adherence to the ALG was 95%: three patients with destructive injuries underwent primary repair, and five patients with risk factors underwent RA. There were three SLFs (2%) (one involved deviation from the ALG) and eight abscesses (5%). Colon-related mortality was 2.1%. Stratification analysis based on mesenteric involvement, degree of shock, and need for abbreviated laparotomy failed to identify additional risk factors for SLF following RA for blunt colon injuries.Adherence to an ALG, originally defined for penetrating colon injuries, simplified the management of blunt colon injuries. ND injuries should be primarily repaired. For destructive wounds, management based on a defined ALG achieves an acceptably low morbidity and mortality rate.Prognostic/epidemiologic study, level III; therapeutic study, level IV.

Summary Tumor-associated macrophages (TAMs) are essential components of the cancer microenvironment and play critical roles in the regulation of tumor progression. Optimal therapeutic intervention requires in-depth understanding of the sources that sustain macrophages in malignant tissues. In this study, we investigated the ontogeny of TAMs in murine pancreatic ductal adenocarcinoma (PDAC) models. We identified both inflammatory monocytes and tissue-resident macrophages as sources of TAMs. Unexpectedly, significant portions of pancreas-resident macrophages originated from embryonic development and expanded through in situ proliferation during tumor progression. Whereas monocyte-derived TAMs played more potent roles in antigen presentation, embryonically derived TAMs exhibited a pro-fibrotic transcriptional profile, indicative of their role in producing and remodeling molecules in the extracellular matrix. Collectively, these findings uncover the heterogeneity of TAM origin and functions and could provide therapeutic insight for PDAC treatment.

Background Multi-institutional collaborations provide granularity lacking in epidemiologic data sets to enable in-depth study of rare diseases. For patients with superficial, high-grade soft tissue sarcomas of the trunk and extremity, the value of radiation therapy (RT) is not clear. We aimed to use the 7-institution US Sarcoma Collaborative (USSC) and the National Cancer Database (NCDB) to investigate this issue. Study Design All adult patients with superficial truncal and extremity high-grade soft tissue sarcomas who underwent primary curative-intent resection from 2000 to 2016 at USSC institutions or were included in the NCDB from 2004 to 2013 were analyzed. Propensity score matching was performed. End points were locoregional recurrence-free survival (LRFS), overall survival (OS), and disease-specific survival (DSS). Results Of 4,153 patients in the USSC, 169 patients with superficial high-grade tumors underwent primary curative-intent resection, 38% of which received RT. On multivariable Cox-regression analysis, RT was not associated with improved LRFS (p = 0.56), OS (p = 0.31), or DSS (p = 0.20). On analysis of 51 propensity score-matched pairs, RT was still not associated with increased LRFS, OS, or DSS. Analysis of 631 propensity score-matched pairs in the NCDB demonstrated improved 5-year OS rate associated with RT (80% vs 70%; p = 0.02). The LRFS and DSS rates were not evaluable. Conclusions Granular data afforded by collaborative research enables in-depth analysis of patient outcomes. The NCDB, although powered with large numbers, cannot assess many relevant outcomes (eg recurrence, DSS, or complications). In this study, the approaches yielded conflicting results. The USSC data suggested no value of radiation and the NCDB demonstrated improved OS, contradicting all randomized controlled trials in sarcoma. The pros and cons of either approach must be considered when applying results to clinical practice, and underscore the importance of randomized controlled trials.

Introduction: The sigma 2 receptor is overexpressed in pancreatic cancer and has been an attractive target for development of cancer selective therapies. However, the mechanism of sigma 2 mediated cancer cell death remains elusive. The sigma 2 receptor has been recently identified as TMEM97, a lysosomal/endoplasmic reticulum protein involved in regulation of cholesterol export in conjunction with NPC1. Methods: Uptake of SW43 in pancreatic cancer cells was visualized using a NBD fluorophore conjugated ligand. Intracellular cholesterol staining was visualized using filipin staining. Protein expression was quantified by both Western blot and RT qPCR. Cell death was assayed in triplicate using Titer-Glo viability assay. Results: We hypothesized that sigma 2 ligands may kill pancreatic cancer cells through disruption of lysosomal cholesterol export. Using ASPC1 pancreatic cancer cells, we first demonstrate that sigma 2 ligand SW43 is rapidly taken up and targets to endo-lysosomal compartments, and that treatment with SW43 causes a rapid and dose dependent sequestration of cholesterol in lysosomal compartments as visualized by filipin cholesterol staining. This correlated to an increase in TMEM97 and corresponding decrease in NPC1 expression following SW43 treatment. We next demonstrate that this cholesterol sequestration phenotype is required for cell death, and might be related to the free amine side chain of SW43, as its N-phthalimide derivative fails to induce both lysosomal cholesterol sequestration and cell death despite high doses of ligand used. Lysosomal cholesterol sequestration from sigma 2 ligand treatment sensitizes cells to further targeting of cholesterol metabolism. Using low doses of simvastatin and SW43, we demonstrate that targeting both cholesterol export and cholesterol synthesis displays significant synergism in killing ASPC1 pancreatic cancer cells. Conclusions: These findings provide further insight into how the sigma 2 receptor/ligand interaction mediates cancer cell death and provides the basis for rationally combined targeted therapy regimens to be tested in vivo. This work was supported by NIH R01CA163764 and NIH T32CA009621-26A1 and the SUS Foundation Resident Research Award. Citation Format: Linda X. Jin, Suwanna Vangveravong, Narendra V. Sankpal, Darren R. Cullinan, Peter Goedegebuure, Dirk Spitzer, William G. Hawkins. Sigma 2 ligands kill pancreatic cancer cells via disruption of lysosomal cholesterol transport [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 877.

Introduction: Cancer neoantigens result from mutations accumulated in an individual tumor. These mutations are usually private and yield high-affinity T-cells as they are not limited by mechanisms of self-tolerance. Neoantigens presented in the context of MHC class I can be recognized by anti-tumor CD8+ T-cells. Methods: Three consecutive patients with confirmed pancreatic ductal adenocarcinoma (PDAC) had DNA and RNA isolated from areas of high tumor cellularity. Comparative normal DNA was isolated from peripheral blood mononuclear cells (PBMC). Nonsynonymous mutations were identified by tumor/normal whole exome sequencing and expression in the tumor was verified by cDNA capture. HLA typing was inferred from exome sequencing. Candidate neoantigens were identified using multiple epitope prediction algorithms for MHC class I. Matched patient PBMC were cultured in serum-free media for 48 hours with synthetic mutated short peptides to determine the baseline immune response by interferon-gamma (IFNγ) enzyme-linked immunospot assay. Results: A KRAS driver mutation was identified in all three patients. Using KRAS variant allele frequency (VAF) as a surrogate for tumor purity, we achieved greater than 40% tumor purity for all three samples. This provides an adequate level of purity to proceed with neoantigen identification. In these samples, the mean number of nonsynonymous mutations identified was 27 (range: 18-32; DNA VAF cutoff 10%). Of these nonsynonymous mutations, a mean of 8.6 mutations (range: 7-10) were expressed (RNA VAF cutoff >10% and FPKM >1). Following confirmation of mutation expression, a mean of 5 mutations (range: 4-6) were predicted to bind to MHC class I using multiple prediction algorithms (binding affinity Conclusion: Utilizing advanced genomic and bioinformatics tools we have successfully characterized the cancer neoantigens in a series of three consecutive patients with PDAC. From this data, we plan to open a phase I clinical trial to assess the safety and immunogenicity of a neoantigen DNA vaccine. Citation Format: Darren R. Cullinan, Michael McLellan, Xiuli Zhang, Timothy M. Nywening, Chris A. Miller, S. Peter Goedegebuure, William G. Hawkins, William E. Gillanders. Development of a personalized pancreatic cancer vaccine [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 5640.

331 Background: Pancreatic cancer is a devastating disease that is poorly responsive to traditional systemic therapies. Cancer-selective drug delivery can improve survival and reduce systemic toxicities. We have developed a pancreatic cancer drug delivery platform based on sigma-2 receptor ligands conjugated to small molecule drug cargos, which improves delivery and efficacy through efficient drug internalization. However, the mechanism of drug internalization via the sigma-2 receptor/ligand interaction remains unclear. Methods: Uptake of fluorescently conjugated sigma-2 ligand SW120 was studied in ASPC-1 human pancreatic cancer cells. Uptake was measured at 37C, 4C, after competitive inhibition with sigma-2 ligand SW43, and after pretreatment with Pitstop 2 (Abcam), an inhibiter of clathrin-mediated endocytosis. Uptake was visualized using live-cell imaging using A1Rsi confocal laser scanning microscope (Nikon). Image analysis was performed using FIJI (NIH) and Matlab (Mathworks). Results: SW120 (10 nM) was rapidly internalized into ASPC-1 cells at 37C with maximal fluorescence at 12 minutes. As a negative control, incubation of ASPC-1 cells with the unconjugated flourophore NBD Cl demonstrated no uptake after 15 minutes, indicating that uptake of SW120 depends on the specific sigma-2 receptor/ligand interaction. Fluorescence at 15 minutes was reduced by 85% in ASPC-1 cells incubated at 4C, indicating uptake of SW120 into cells is an energy dependent process. Pretreatment of cells with Pitstop 2 decreased total flourescence at 15 minutes by 76%, suggesting an important role of clathrin-mediated endocytosis in sigma-2 receptor uptake, while pretreatment with competitive inhibitor SW43 reduced uptake by 84%, suggesting that sigma-2 ligands are internalized via a specific receptor capable of saturation. Conclusions: Sigma-2 receptor ligands are rapidly internalized into pancreatic cancer cells via a specific, targetable, energy-dependent pathway that appears to rely on clathrin-mediated endocytosis. Further understanding of sigma-2 mediated drug internalization can help optimize targeted drug development and delivery for pancreatic cancer patients.

Background Multi-visceral resection for extra-pancreatic carcinoma is an uncommon procedure that may offer palliation and potential cure but must be balanced against the risk for morbidity and mortality. Methods A retrospective analysis was made of patients who had undergone multi-visceral resection of non-pancreatic carcinoma. Factors influencing this procedure included histology, pathologic confirmation of pancreaticoduodenal invasion, tumor clearance, peri-operative morbidity and outcome. Results Sixteen patients had en bloc resection including a Whipple procedure (6 patients) and a distal resection (10). Primary pathology mostly originated from the stomach and adenocarcinoma was predominately histological. An R0 resection was made in 13 patients, and actual cancer invasion or abutment into the pancreas or duodenum was confirmed pathologically in 11 patients. Twelve patients suffered from at least one complication. Ten patients required therapeutic intervention for complications. There were 2 in-hospital deaths. The median survival of deceased patients was 7.5 months. Six patients are alive at a median of 21 months, and 4 patients have no evidence of disease to the present. Conclusions Multi-visceral resections for extra-pancreatic carcinoma are associated with substantial morbidity that requires therapeutic intervention. Clinical determination of pancreaticoduodenal abutment and achievement of tumor clearance is excellent. Survival with and without recurrent disease is often limited, supporting that it is necessary to cautiously perform the aggressive procedures in consideration of neoadjuvant therapy.

Background For more than a decade, operative decisions (resection plus anastomosis vs diversion) for colon injuries, at our institution, have followed a defined management algorithm based on established risk factors (pre- or intraoperative transfusion requirements of more than 6 units packed RBCs and/or presence of significant comorbid diseases). However, this management algorithm was originally developed for patients managed with a single laparotomy. The purpose of this study was to evaluate the applicability of this algorithm to destructive colon injuries after abbreviated laparotomy (AL) and to determine whether additional risk factors should be considered. Study Design Consecutive patients over a 17-year period with colon injuries after AL were identified. Nondestructive injuries were managed with primary repair. Destructive wounds were resected at the initial laparotomy followed by either a staged diversion (SD) or a delayed anastomosis (DA) at the subsequent exploration. Outcomes were evaluated to identify additional risk factors in the setting of AL. Results We identified 149 patients: 33 (22%) patients underwent primary repair at initial exploration, 42 (28%) underwent DA, and 72 (49%) had SD. Two (1%) patients died before re-exploration. Of those undergoing DA, 23 (55%) patients were managed according to the algorithm and 19 (45%) were not. Adherence to the algorithm resulted in lower rates of suture line failure (4% vs 32%, p=0.03) and colon-related morbidity (22% vs 58%, p=0.03) for patients undergoing DA. No additional specific risk factors for suture line failure after DA were identified. Conclusions Adherence to an established algorithm, originally defined for destructive colon injuries after single laparotomy, is likewise efficacious for the management of these injuries in the setting of AL.

Introduction: Recent reports suggest that the mutational landscape predicts response to checkpoint blockade therapy in non-small cell lung cancer and colorectal cancer. Neoantigens, identified by exome and RNA sequencing using immune prediction algorithms, have been successfully targeted by dendric cell vaccination in melanoma. The relative abundance of non-malignant cells within the stroma has made Identification of neoantigens in pancreatic cancer (PC) challenging. Here we report successful isolation and high quality sequencing of PC neoplastic ductal cells specifically, resulting in the identification of immunogenic mutations. Methods: Specimens from freshly resected PC tumors were collected in the operating room and immediately frozen in OCT. Laser microdissection was performed by a pathologist to isolate PC malignant cells from the stromal compartment at >90% purity. DNA and RNA were isolated from the purified tumors with nonsynonymous mutations identified by tumor/normal exome sequencing and expression verified by RNA sequencing. HLA typing was inferred from exome sequencing and verified by standard HLA typing techniques. Candidate neoantigen binding scores were determined via NetMHC. Tumor infiltrating lymphocyte (TIL) culture was carried out over 12-14 days by serial reduction of tumor concentration as the TIL proliferated in the presence of IL-2 containing media (50 units/mL). Results: Patient #567 was confirmed to express HLA-A3 and HLA-A23. 105 nonsynonymous mutations were identified by tumor/normal exome sequencing, all of which were single nucleotide polymorphisms. Candidate neoantigens were identified and prioritized based on predicted binding of the mutant amino acid sequence to MHC class I. 30 candidate neoantigens were prioritized for further evaluation based on a binding score of < 500 nM, and superior predicted binding associated with the mutant sequence compared to the wildtype sequence. Mutant allele expression was verified by a fragments per kilobase of exon per million fragments mapped (FKPM) >1, with a cut off for RNA variant allele frequency (VAF) greater than 20%. Using this stringent criteria 10 candidate epitopes were identified. Next, we validated this process in 17 consecutive PC patients. DNA and RNA were successfully isolated from 13 patients (76.5%), the remaining four had no viable tumor at pathology. The mean RNA integrity number (RIN) for the isolated samples was 7.6 (Range 5-8.7). Additionally, to test for existing immune response to the predicted epitopes TIL were successfully cultured from 6 of 8 (75%) attempted patients with a mean yield of 3.0x106 (Range 1x105 – 7.2x106). Conclusion: Obtaining high quality, reliable sequencing data from PC patients is possible using rigorous tumor purification techniques to specifically select malignant cells. Candidate neoantigens can be identified using a combination of epitope prediction algorithms. Expansion of TIL populations in vitro may offer functional studies to further refine the optimal selection of immunogenic neoantigens and optimize personalized vaccination strategies in PC. Citation Format: Darren R. Cullinan, S. Peter Goedegebuure, Chris A. Miller, Timothy M. Nywening, Samuel Kim, Elaine R. Mardis, William G. Hawkins, William E. Gillanders.{Authors}. Identification of pancreatic cancer neoantigens by exome and RNA sequencing analysis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A70.

Introduction: Ovarian cancer (OC) expresses the tumor associated antigen mesothelin and contains a relative abundance of T-cells. However, ovarian cancer is also infiltrated by immunosuppressive tumor associated macrophages (TAM) that dominate the tumor microenvironment (TME). The CCL2/CCR2 chemokine axis is co-opted by various human malignancies to facilitate the recruitment of bone marrow (BM) derived inflammatory monocytes (IM) to the TME where they become immunosuppressive TAMs. Herein, we explore the rationale for combination of a CCR2 inhibitor (CCR2i) with a mesothelin peptide vaccine. Methods: Monocyte counts were obtained from preoperative CBCs under IRB approval. Mice were vaccinated with a dual eight-mer peptide (50 nM/vaccination) on days 0 and 7 with an irradiated peptide pulsed dendritic cell boost on day 14. Mice were challenged with 4 million syngeneic OC cells (ID8) on day 15. CCR2 inhibitor (Tocris) and CCR2 KO mice were used. Results: Preoperative monocyte counts of human ovarian cancer patients were stratified into low (>1 SD below mean), mid (within 1 SD of mean), and high (>1 SD above the mean) groups. Patients with a high monocyte count (n = 15) had a significantly decreased median survival of 1.2 years compared to 4.8 years in the low monocyte group (n = 15). The mid group (n = 69) had a median survival of 3.5 years (p 0.001). The hazard ratio between the low and high groups was 0.24 (0.05-0.39). Flow cytometry of peripheral blood from these patients demonstrated that the majority of these monocytes were CCR2+ inflammatory monocytes. Human OC overexpresses CCL2 compared to normal ovarian tissue and analysis of the TME from resected human OC patients revealed an abundance of CCR2+ TAM, which greatly outnumbered tumor infiltrating lymphocytes (TIL). In a murine ID8 tumor model, which recapitulates features of human OC, CCR2i prevented IM egress from the bone marrow with a resultant decrease in TAM at the primary tumor site. Furthermore, there was an increase in TIL infiltrate following CCR2 blockade. Addition of vaccine to CCR2i caused an improvement of effector to suppressor ratio and prolonged survival compared to vaccine (p = 0.02) or CCR2i alone (p = 0.02) and control (p Conclusion: Thus far vaccination has not provided durable patient responses in OC. Therapies targeting the immunosuppressive TME are an attractive treatment modality to enhance vaccination and facilitate anti-tumor immunity in OC. Citation Format: Darren Cullinan, Pratibha Binder, Timothy Nywening, Ivy Wilkinson-Ryan, Brian Belt, Peter Goedegebuure, David Linehan, Matthew Powell, William Hawkins. Vaccination enhances anti-tumor immunity in ovarian cancer following repolarization of the tumor microenvironment with CCR2 blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2350.

Introduction: Pancreatic cancer (PC) is characterized by a dense tumor stroma with a heavy leukocytic infiltrate, comprised predominately of immunosuppressive bone marrow (BM) derived cells. We have previously demonstrated in a phase Ib clinical trial that CCR2 inhibition (CCR2i) prevents inflammatory monocyte (IM) recruitment from the BM and results in a significant reduction of tumor associated macrophages (TAM) and an increase in treatment efficacy. However, granulocytic myeloid derived suppressor cells (G-MDSC) remain in the tumor microenvironment (TME) following CCR2i. Herein, we explored the impact of targeting G-MDSC recruitment to PC tumors both alone and in combination with CCR2i. Methods: Human BM, blood, and tumor was collected under an IRB approved protocol. A tissue microarray (TMA) from resected PC patients was analyzed for immune infiltrate. Mice were injected orthotopically with 2.5×106 syngeneic PC cells. CXCR2 and CCR2 inhibitors (Tocris) were given twice daily. Tumor growth was assessed and specimens obtained for analysis by flow cytometry, RNAseq, and IHC. Results: Human PC overexpresses CXCL5 and CXCL8, corresponding with an abundance of tumor infiltrating CXCR2+ G-MDSC. Furthermore, the ratio of CD8 to G-MDSC correlates with survival in human PC patients. In an orthotopic murine model that recapitulates human disease, ΣCXCL ligands were also increased. Either Ly6G depletion or targeted blockade with a CXCR2 inhibitor decreased G-MDSC and reduced tumor burden. Intriguingly, blockade of IM from the BM did not reduce G-MDSC and paradoxically resulted in a modest increase in this population within the tumors from human patients following CCR2i. Thus, we explored the combination of CCR2/CXCR2 blockade both with and without FOLFIRINOX chemotherapy. This resulted in a synergistic impact when both BM derived populations were targeted and dual therapy was further enhanced by FOLFIRINOX. RNAseq analysis of tumors following monotherapy or dual inhibition revealed alterations in the TME favoring an anti-tumor immune response. To test the hypothesis that this effect was mediated by restoration of anti-tumor immunity we analyzed the tumor infiltrating lymphocyte (TIL) populations and found a significant increase in the relative and absolute numbers of CD8+ and C4+ TIL. Analysis of the activation status of these cells demonstrated an increase in effector CD8+ T-cell phenotype (IFNγ+, CD69+, CD44+). Using Nur77GFP T-cell receptor reporter mice, we showed an increase in GFP expressing CD8+ TIL following dual blockade. CD8 depletion resulted in a loss of therapeutic efficacy of myeloid blockade, further confirming our hypothesis. Conclusion: These findings suggest that combinatorial blockade strategies preventing tumor infiltration by myeloid cells may restore anti-tumor immunity in PC. Citation Format: Timothy M. Nywening, Brian A. Belt, Roheena Z. Panni, Darren Cullinan, Dominic E. Sanford, Ryan C. Fields, William G. Hawkins, David G. DeNardo, William E. Gillanders, Peter Goedegebuure, David C. Linehan. Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4150.

Background To date, work-hour restrictions have not been imposed on attending surgeons in the United States. The purpose of this study was to investigate the impact of working an overnight trauma shift on outcomes of general surgery operations performed the next day by the post-call attending physician. Study Design Consecutive patients over a 3.5-year period undergoing elective general surgical procedures were reviewed. Procedures were limited to hernia repairs (inguinal and ventral), cholecystectomies, and intestinal operations. Any operations that were performed the day after the attending surgeon had taken an overnight trauma shift were considered post-call (PC) cases; all other cases were considered nonpost-call (NP). Outcomes from the PC operations were compared with those from the NP operations. Results There were 869 patients identified; 132 operations were performed PC and 737 were NP. The majority of operations included hernia repairs (46%), followed by cholecystectomies (35%), and intestinal procedures (19%). Overall, the PC operations did not differ from the NP operations with respect to complication rate (13.7% vs 13.5%, p = 0.93) or readmission within 30 days (5% vs 6%, p = 0.84). Additionally, multivariable logistic regression failed to identify an association between PC operations and the development of adverse outcomes. Follow-up was obtained for an average of 3 months. Conclusions Performance of general surgery operations the day after an overnight in-hospital trauma shift did not affect complication rates or readmission rates. At this time, there is no compelling evidence to mandate work-hour restrictions for attending general surgeons.