Prospective, Multi-Institutional, Real-Time Next-Generation Sequencing of Pancreatic Cyst Fluid Reveals Diverse Genomic Alterations That Improve the Clinical Management of Pancreatic Cysts.

BACKGROUND & AIMS: Next-generation sequencing (NGS) of pancreatic cyst fluid is a useful adjunct in the assessment of patients with pancreatic cyst. However, previous studies have been retrospective or single institutional experiences. The aim of this study was to prospectively evaluate NGS on a multi-institutional cohort of patients with pancreatic cyst in real time. METHODS: The performance of a 22-gene NGS panel (PancreaSeq) was first retrospectively confirmed and then within a 2-year timeframe, PancreaSeq testing was prospectively used to evaluate endoscopic ultrasound-guided fine-needle aspiration pancreatic cyst fluid from 31 institutions. PancreaSeq results were correlated with endoscopic ultrasound findings, ancillary studies, current pancreatic cyst guidelines, follow-up, and expanded testing (Oncomine) of postoperative specimens. RESULTS: Among 1933 PCs prospectively tested, 1887 (98%) specimens from 1832 patients were satisfactory for PancreaSeq testing. Follow-up was available for 1216 (66%) patients (median, 23 months). Based on 251 (21%) patients with surgical pathology, mitogen-activated protein kinase/GNAS mutations had 90% sensitivity and 100% specificity for a mucinous cyst (positive predictive value [PPV], 100%; negative predictive value [NPV], 77%). On exclusion of low-level variants, the combination of mitogen-activated protein kinase/GNAS and TP53/SMAD4/CTNNB1/mammalian target of rapamycin alterations had 88% sensitivity and 98% specificity for advanced neoplasia (PPV, 97%; NPV, 93%). Inclusion of cytopathologic evaluation to PancreaSeq testing improved the sensitivity to 93% and maintained a high specificity of 95% (PPV, 92%; NPV, 95%). In comparison, other modalities and current pancreatic cyst guidelines, such as the American Gastroenterology Association and International Association of Pancreatology/Fukuoka guidelines, show inferior diagnostic performance. The sensitivities and specificities of VHL and MEN1/loss of heterozygosity alterations were 71% and 100% for serous cystadenomas (PPV, 100%; NPV, 98%), and 68% and 98% for pancreatic neuroendocrine tumors (PPV, 85%; NPV, 95%), respectively. On follow-up, serous cystadenomas with TP53/TERT mutations exhibited interval growth, whereas pancreatic neuroendocrine tumors with loss of heterozygosity of ≥3 genes tended to have distant metastasis. None of the 965 patients who did not undergo surgery developed malignancy. Postoperative Oncomine testing identified mucinous cysts with BRAF fusions and ERBB2 amplification, and advanced neoplasia with CDKN2A alterations. CONCLUSIONS: PancreaSeq was not only sensitive and specific for various pancreatic cyst types and advanced neoplasia arising from mucinous cysts, but also reveals the diversity of genomic alterations seen in pancreatic cysts and their clinical significance.

Targeting myeloperoxidase limits myeloid cell immunosuppression enhancing immune checkpoint therapy for pancreatic cancer.

Pancreatic ductal adenocarcinoma is a devastating disease characterized by an extreme resistance to current therapies, including immune checkpoint therapy. The limited success of immunotherapies can be attributed to a highly immunosuppressive pancreatic cancer microenvironment characterized by an extensive infiltration of immune suppressing myeloid cells. While there are several pathways through which myeloid cells contribute to immunosuppression, one important mechanism is the increased production of reactive oxygen species. Here, we evaluated the contribution of myeloperoxidase, a myeloid-lineage restricted enzyme and primary source of reactive oxygen species, to regulate immune checkpoint therapy response in preclinical pancreatic cancer models. We compared treatment outcome, immune composition and characterized myeloid cells using wild-type, myeloperoxidase-deficient, and myeloperoxidase inhibitor treated wild-type mice using established subcutaneous pancreatic cancer models. Loss of host myeloperoxidase and pharmacological inhibition of myeloperoxidase in combination with immune checkpoint therapy significantly delayed tumor growth. The tumor microenvironment and systemic immune landscape demonstrated significant decreases in myeloid cells, exhausted T cells and T regulatory cell subsets when myeloperoxidase was deficient. Loss of myeloperoxidase in isolated myeloid cell subsets from tumor-bearing mice resulted in decreased reactive oxygen species production and T cell suppression. These data suggest that myeloperoxidase contributes to an immunosuppressive microenvironment and immune checkpoint therapy resistance where myeloperoxidase inhibitors have the potential to enhance immunotherapy response. Repurposing myeloperoxidase specific inhibitors may provide a promising therapeutic strategy to expand therapeutic options for pancreatic cancer patients to include immunotherapies.

Pancreatectomy Induces Cancer-Promoting Neutrophil Extracellular Traps.

BACKGROUND: Neutrophil extracellular traps (NETs) occur when neutrophil chromatin is decondensed and extruded into the extracellular space in a web-like structure. Originally described as an anti-microbial function, this process has been implicated in the pathogenesis of pancreatic disease. In addition, NETs are upregulated during physiologic wound-healing and coagulation. This study evaluated how the inflammatory response to pancreatic surgery influences NET formation. METHODS: For this study, 126 patients undergoing pancreatectomy gave consent before participation. Plasma was collected at several time points (preoperatively and through the postoperative outpatient visit). Plasma levels of NET markers, including cell-free DNA (cfDNA), citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-6, and granulocyte colony-stimulating factor (G-CSF) were measured using enzyme-linked immunosorbent assay (ELISA). Patient clinical data were retrospectively collected from a prospectively maintained database. RESULTS: After pancreatic resection, NET markers (cfDNA and CitH3) were elevated, peaking on postoperative days 3 and 4. This increase in NETs was due to an inherent change in neutrophil biology. Postoperatively, NET-inducing cytokines (IL-8, IL-6, and G-CSF) were increased, peaking early in the postoperative course. The patients undergoing the robotic approach had a reduction in NETs during the postoperative period compared with those who underwent the open approach. The patients who experienced a pancreatic leak had an increase in NET markers during the postoperative period. CONCLUSIONS: Pancreatectomy induces cancer-promoting NET formation. The minimally invasive robotic approach may induce fewer NETs, although the current analysis was limited by selection bias. Pancreatic leak resulted in increased NETs. Further study into the potential for NET inhibition during the perioperative period is warranted.

A Combined DNA/RNA-based Next-Generation Sequencing Platform to Improve the Classification of Pancreatic Cysts and Early Detection of Pancreatic Cancer Arising From Pancreatic Cysts.

OBJECTIVE: We report the development and validation of a combined DNA/RNA next-generation sequencing (NGS) platform to improve the evaluation of pancreatic cysts. BACKGROUND AND AIMS: Despite a multidisciplinary approach, pancreatic cyst classification, such as a cystic precursor neoplasm, and the detection of high-grade dysplasia and early adenocarcinoma (advanced neoplasia) can be challenging. NGS of preoperative pancreatic cyst fluid improves the clinical evaluation of pancreatic cysts, but the recent identification of novel genomic alterations necessitates the creation of a comprehensive panel and the development of a genomic classifier to integrate the complex molecular results. METHODS: An updated and unique 74-gene DNA/RNA-targeted NGS panel (PancreaSeq Genomic Classifier) was created to evaluate 5 classes of genomic alterations to include gene mutations (e.g., KRAS, GNAS, etc.), gene fusions and gene expression. Further, CEA mRNA ( CEACAM5 ) was integrated into the assay using RT-qPCR. Separate multi-institutional cohorts for training (n=108) and validation (n=77) were tested, and diagnostic performance was compared to clinical, imaging, cytopathologic, and guideline data. RESULTS: Upon creation of a genomic classifier system, PancreaSeq GC yielded a 95% sensitivity and 100% specificity for a cystic precursor neoplasm, and the sensitivity and specificity for advanced neoplasia were 82% and 100%, respectively. Associated symptoms, cyst size, duct dilatation, a mural nodule, increasing cyst size, and malignant cytopathology had lower sensitivities (41-59%) and lower specificities (56-96%) for advanced neoplasia. This test also increased the sensitivity of current pancreatic cyst guidelines (IAP/Fukuoka and AGA) by >10% and maintained their inherent specificity. CONCLUSIONS: PancreaSeq GC was not only accurate in predicting pancreatic cyst type and advanced neoplasia but also improved the sensitivity of current pancreatic cyst guidelines.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Acute pancreatitis induces a transient hypercoagulable state in murine models.

BACKGROUND/OBJECTIVES: Although understudied, risk of venous thromboembolism (VTE) appears to be increased during acute pancreatitis (AP). We aimed to further characterize a hypercoagulable state associated with AP utilizing thromboelastography (TEG), a readily available, point of care test. METHODS: AP was induced in C57/Bl6 mice using l-arginine and caerulein. TEG was performed with citrated native samples. The maximum amplitude (MA) and coagulation index (CI), a composite marker of coagulability, were evaluated. Platelet aggregation was assessed using whole blood collagen-activated platelet impedance aggregometry. Circulating tissue factor (TF), the initiator of extrinsic coagulation, was measured with ELISA. A VTE model using IVC ligation followed by measurement of clot size and weight was evaluated. After IRB approval and consent, blood samples from patients hospitalized with a diagnosis of AP were evaluated by TEG. RESULTS: Mice with AP displayed a significant increase in MA and CI, consistent with hypercoagulability. Hypercoagulability peaked at 24 h after induction of pancreatitis, then returned to baseline by 72 h. AP resulted in significantly increased platelet aggregation and elevated circulating TF. Increased clot formation with AP was observed in an in vivo model of deep vein thrombosis. In a proof of concept, correlative study, over two thirds of patients with AP demonstrated an elevated MA and CI compared to the normal range, consistent with hypercoagulability. CONCLUSIONS: Murine acute pancreatitis results in a transient hypercoagulable state that can be assessed by TEG. Correlative evidence for hypercoagulability was also demonstrated in human pancreatitis. Further study to correlate coagulation measures to incidence of VTE in AP is warranted.

Tumor Size Differences Between Preoperative Endoscopic Ultrasound and Postoperative Pathology for Neoadjuvant-Treated Pancreatic Ductal Adenocarcinoma Predict Patient Outcome.

BACKGROUND & AIMS: The assessment of therapeutic response after neoadjuvant treatment and pancreatectomy for pancreatic ductal adenocarcinoma (PDAC) has been an ongoing challenge. Several limitations have been encountered when employing current grading systems for residual tumor. Considering endoscopic ultrasound (EUS) represents a sensitive imaging technique for PDAC, differences in tumor size between preoperative EUS and postoperative pathology after neoadjuvant therapy were hypothesized to represent an improved marker of treatment response. METHODS: For 340 treatment-naïve and 365 neoadjuvant-treated PDACs, EUS and pathologic findings were analyzed and correlated with patient overall survival (OS). A separate group of 200 neoadjuvant-treated PDACs served as a validation cohort for further analysis. RESULTS: Among treatment-naïve PDACs, there was a moderate concordance between EUS imaging and postoperative pathology for tumor size (r = 0.726, P < .001) and AJCC 8th edition T-stage (r = 0.586, P < .001). In the setting of neoadjuvant therapy, a decrease in T-stage correlated with improved 3-year OS rates (50% vs 31%, P < .001). Through recursive partitioning, a cutoff of ≥47% tumor size reduction was also found to be associated with improved OS (67% vs 32%, P < .001). Improved OS using a ≥47% threshold was validated using a separate cohort of neoadjuvant-treated PDACs (72% vs 36%, P < .001). By multivariate analysis, a reduction in tumor size by ≥47% was an independent prognostic factor for improved OS (P = .007). CONCLUSIONS: The difference in tumor size between preoperative EUS imaging and postoperative pathology among neoadjuvant-treated PDAC patients is an important prognostic indicator and may guide subsequent chemotherapeutic management.

Molecular pathogenesis and emerging targets of gastric adenocarcinoma.

Gastric adenocarcinoma (GC) is a devastating disease and is the third leading cause of cancer deaths worldwide. This heterogeneous disease has several different classification systems that consider histological appearance and genomic alterations. Understanding the etiology of GC, including infection, hereditary conditions, and environmental factors, is of particular importance and is discussed in this review. To improve survival in GC, we also must improve our therapeutic strategies. Here, we discuss new targets that warrant further exploration.

500 Minimally Invasive Robotic Pancreatoduodenectomies: One Decade of Optimizing Performance.

OBJECTIVES: This study aims to present the outcomes of our decade-long experience of robotic pancreatoduodenectomy and provide insights into successful program implementation. BACKGROUND: Despite significant improvement in mortality over the past 30 years, morbidity following open pancreatoduodenectomy remains high. We implemented a minimally invasive pancreatic surgery program based on the robotic platform as one potential method of improving outcomes for this operation. METHODS: A retrospective review of a prospectively maintained institutional database was performed to identify patients who underwent robotic pancreatoduodenectomy (RPD) between 2008 and 2017 at the University of Pittsburgh. RESULTS: In total, 500 consecutive RPDs were included. Operative time, conversion to open, blood loss, and clinically relevant postoperative pancreatic fistula improved early in the experience and have remained low despite increasing complexity of case selection as reflected by increasing number of patients with pancreatic cancer, vascular resections, and higher Charlson Comorbidity scores (all P<0.05). Operating room time plateaued after 240 cases at a median time of 391 minutes (interquartile rang 340-477). Major complications (Clavien >2) occurred in less than 24%, clinically relevant postoperative pancreatic fistula in 7.8%, 30- and 90-day mortality were 1.4% and 3.1% respectively, and median length of stay was 8 days. Outcomes were not impacted by integration of trainees or expansion of selection criteria. CONCLUSIONS: Structured implementation of robotic pancreatoduodenectomy can be associated with excellent outcomes. In the largest series of RPD, we establish benchmarks for the surgical community to consider when adopting this approach.

Outcomes of Neoadjuvant Chemotherapy Versus Chemoradiation in Localized Pancreatic Cancer: A Case-Control Matched Analysis.

BACKGROUND: Neoadjuvant therapy is increasingly used for patients with pancreatic ductal adenocarcinoma (PDAC). It is unknown whether neoadjuvant chemoradiotherapy is more effective than chemotherapy (NCRT vs. NAC). We aim to compare pathological and survival outcomes of NCRT and NAC in patients with PDAC. PATIENTS AND METHODS: Single-center analysis of PDAC patients treated with NCRT or NAC followed by resection between December 2008 and December 2018 was performed. Average treatment effect (ATE) was estimated after case-control matching using Mahalanobis distance nearest-neighbor matching. Inverse probability weighted estimates (IPWE)-based ATE was estimated for disease-free survival (DFS) and overall survival (OS). RESULTS: Among the 418 patients (mean age 66.8 years, 51% female) included in the study, 327 received NAC and 91 received NCRT. NCRT patients had higher rates of locally advanced disease, number of neoadjuvant chemotherapy cycles, more chemotherapy regimen crossover (gemcitabine and 5-FU based), and were more likely to undergo open surgical procedures and/or vascular resection (all p < 0.05). After matched analysis, NCRT was associated with a significant reduction in lymph node positive disease [ATE = (-)0.24, p = 0.007] and lymphovascular invasion [ATE = (-)0.20, p = 0.02]. While NCRT was associated with significantly improved DFS by 9.5 months (p = 0.006), it did not affect OS by IPWE-based ATE after adjusting for adjuvant therapy (ATE = 5.5 months; p = 0.32). CONCLUSION: Compared with NAC alone, NCRT is associated with improved pathologic surrogates and disease-free survival, but not overall survival in patients with PDAC.

Formal robotic training diminishes the learning curve for robotic pancreatoduodenectomy: Implications for new programs in complex robotic surgery.

INTRODUCTION: The learning curve associated with robotic pancreatoduodenectomy (RPD) is a hurdle for new programs to achieve optimal results. Since early analysis, robotic training has recently expanded, and the RPD approach has been refined. The purpose of this study is to examine RPD outcomes for surgeons who implemented a new program after receiving formal RPD training to determine if such training reduces the learning curve. METHODS: Outcomes for consecutive patients undergoing RPD at a single tertiary institution were compared to optimal RPD benchmarks from a previously reported learning curve analysis. Two surgical oncologists with formal RPD training performed all operations with one surgeon as bedside assistant and the other at the console. RESULTS: Forty consecutive RPD operations were evaluated. Mean operative time was 354 ± 54 min, and blood loss was 300 ml. Length of stay was 7 days. Three patients (7.5%) underwent conversion to open. Pancreatic fistula affected five patients (12.5%). Operative time was stable over the study and lower than the reported benchmark. These RPD operative outcomes were similar to reported surgeon outcomes after the learning curve. CONCLUSION: This study suggests formal robotic training facilitates safe and efficient adoption of RPD for new programs, reducing or eliminating the learning curve.

Impact of postoperative pancreatic fistula on long-term oncologic outcomes after pancreatic resection.

BACKGROUND: The short-term morbidity associated with post-operative pancreatic fistula (POPF) is well established, however data regarding the long-term impact are lacking. We aim to characterize long-term oncologic outcomes of POPF after pancreatic resection through a single institution, retrospective study of pancreatic resections performed for adenocarcinoma from 2009 to 2016. METHODS: Kaplan-Meier survival analysis, logistic regression, and multivariate analysis (MVA) were used to evaluate impact of POPF on overall survival (OS), disease free survival (DFS), and receipt of adjuvant chemotherapy (AC). RESULTS: 767 patients were included. 82 (10.6%) developed grade B (n = 67) or C (n = 15) POPF. Grade C POPF resulted in decreased OS when compared to no POPF (20.22 vs 26.33 months, p = 0.027) and to grade B POPF (20.22 vs. 26.87 months, p = 0.049). POPF patients were less likely to receive AC than those without POPF (59.5% vs 74.9%, p = 0.003) and grade C POPF were less likely to receive AC than all others (26.7% vs 74.2%, p = 0.0001). CONCLUSION: POPF patients are less likely to receive AC and more likely to have delay in time to AC. These factors are exacerbated in grade C POPF and likely contribute to decreased OS. These findings validate the clinical significance of the ISGPF definition of POPF.

Prognostic Value of the Systemic Immune-Inflammation Index (SII) After Neoadjuvant Therapy for Patients with Resected Pancreatic Cancer.

BACKGROUND: The systemic immune-inflammation index (SII), calculated using absolute platelet, neutrophil, and lymphocyte counts, has recently emerged as a predictor of survival for patients with pancreatic ductal adenocarcinoma (PDAC) when assessed at diagnosis. Neoadjuvant therapy (NAT) is increasingly used in the treatment of PDAC. However, biomarkers of response are lacking. This study aimed to determine the prognostic significance of SII before and after NAT and its association with the pancreatic tumor biomarker carbohydrate-antigen 19-9 (CA 19-9). METHODS: This study retrospectively analyzed all PDAC patients treated with NAT before pancreatic resection at a single institution between 2007 and 2017. Pre- and post-NAT lab values were collected to calculate SII. Absolute pre-NAT, post-NAT, and change in SII after NAT were evaluated for their association with clinical outcomes. RESULTS: The study analyzed 419 patients and found no significant correlation between pre-NAT SII and clinical outcomes. Elevated post-NAT SII was an independent, negative predictor of overall survival (OS) when assessed as a continuous variable (hazard ratio [HR], 1.0001; 95% confidence interval [CI] 1.00003-1.00014; p = 0.006). Patients with a post-NAT SII greater than 900 had a shorter median OS (31.9 vs 26.1 months; p = 0.050), and a post-NAT SII greater than 900 also was an independent negative predictor of OS (HR, 1.369; 95% CI 1.019-1.838; p = 0.037). An 80% reduction in SII independently predicted a CA 19-9 response after NAT (HR, 4.22; 95% CI 1.209-14.750; p = 0.024). CONCLUSION: Post-treatment SII may be a useful prognostic marker in PDAC patients receiving NAT.

Neutrophil extracellular traps in breast cancer and beyond: current perspectives on NET stimuli, thrombosis and metastasis, and clinical utility for diagnosis and treatment.

The formation of neutrophil extracellular traps (NETs), known as NETosis, was first observed as a novel immune response to bacterial infection, but has since been found to occur abnormally in a variety of other inflammatory disease states including cancer. Breast cancer is the most commonly diagnosed malignancy in women. In breast cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET-targeted therapies have shown success in preclinical cancer models and may prove valuable clinical targets in slowing or halting tumor progression in breast cancer patients. We will briefly outline the mechanisms by which NETs may form in the tumor microenvironment and circulation, including the crosstalk between neutrophils, tumor cells, endothelial cells, and platelets as well as the role of cancer-associated extracellular vesicles in modulating neutrophil behavior and NET extrusion. The prognostic implications of cancer-associated NETosis will be explored in addition to development of novel therapeutics aimed at targeting NET interactions to improve outcomes in patients with breast cancer.

Risk of Venous Thromboembolism for Patients with Pancreatic Ductal Adenocarcinoma Undergoing Preoperative Chemotherapy Followed by Surgical Resection.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is associated with a hypercoagulable state, resulting in a high risk of venous thromboembolism (VTE). Risk of VTE is well established for patients receiving chemotherapy for advanced disease and during the perioperative period for patients undergoing surgical resection. However, data are lacking for patients undergoing neoadjuvant treatment followed by resection, who may have a unique risk of VTE because of exposure to both chemotherapy and surgery. METHODS: The study included patients with PDA who underwent neoadjuvant therapy followed by surgery from 2007 to June 2017. Development of VTE was evaluated from the start of treatment through the 90-day postoperative period. Risk factors including demographic, treatment, and laboratory variables were evaluated. RESULTS: The study investigated 426 patients receiving neoadjuvant therapy before surgical resection. Of these patients, 20% had a VTE within 90 days postoperatively (n = 87), and 70% of the VTE occurred during the postoperative period. The VTE included pulmonary embolism (30%), deep vein thrombosis (33%), and thrombosis of the portal vein (PV)/superior mesenteric vein (SMV) (40%). A pretreatment hemoglobin level lower than 10 g/dL and a platelet count higher than 443 were independently associated with VTE during neoadjuvant treatment. The independent predictors of postoperative VTE were a body mass index higher than 35 kg/m2, a preoperative platelet-to-lymphocyte ratio higher than 260, resection with distal pancreatectomy with celiac axis resection/total pancreatectomy, PV/SMV resection, and longer operative times. Development of VTE was associated with worse overall and disease-free survival and an independent predictor of survival and decreased likelihood of receiving adjuvant chemotherapy. CONCLUSIONS: Venous thromboembolism during neoadjuvant therapy and the subsequent perioperative period is common and has a significant impact on outcome. Further study into novel thromboprophylaxis measures or protocols during neoadjuvant treatment and the perioperative period is warranted.

The platelet NLRP3 inflammasome is upregulated in a murine model of pancreatic cancer and promotes platelet aggregation and tumor growth.

Platelets are activated in solid cancers, including pancreatic ductal adenocarcinoma (PDA), a highly aggressive malignancy with a devastating prognosis and limited therapeutic options. The mechanisms by which activated platelets regulate tumor progression are poorly understood. The nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) inflammasome is a key inflammatory mechanism recently identified in platelets, which controls platelet activation and aggregation. In an orthotopic PDA mouse model involving surgical implantation of Panc02 murine cancer cells into the tail of the pancreas, we show that the NLRP3 inflammasome in circulating platelets is upregulated in pancreatic cancer. Pharmacological inhibition or genetic ablation of NLRP3 in platelets resulted in decreased platelet activation, platelet aggregation, and tumor progression. Moreover, interfering with platelet NLRP3 signaling significantly improved survival of tumor-bearing mice. Hence, the platelet NLRP3 inflammasome plays a critical role in PDA and might represent a novel therapeutic target.

Can post-hoc video review of robotic pancreaticoduodenectomy predict portal/superior mesenteric vein margin status in pancreatic adenocarcinoma?

BACKGROUND: Achieving margin negative resection is a significant determinant of outcome in pancreatic adenocarcinoma (PDA). However, because of the fibrotic nature of PDA, it can be difficult to discriminate fibrosis from active disease intra-operatively. We sought to determine if post-hoc video review of robotic pancreatico-duodenectomy (RPD) could predict the portal/superior mesenteric vein (PV/SMV) margin status on final pathology. METHODS: Experienced pancreatic surgeons, blinded to patient and operative variables, reviewed the PV/SMV margin for available RPD videos of consecutive PDA patients from 9/2012 through 6/2017. RESULTS: 107 RPD videos were reviewed. Of 76 patients (71%) predicted to have a negative vein margin on video review, 20 patients (26%) had a pathologic positive margin. 25 of 31 patients (81%) predicted to have positive margin on video review were positive on pathology. The specificity of video prediction was 90.3% with a sensitivity of 55.6% and an accuracy of 75.7%. CONCLUSION: Post-hoc video review prediction is unable to reliably predict a positive (R1) margin at the portal vein/SMV, suggesting that intra-operative clinical assessment may be suboptimal in determining the need for more extensive resections.

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps.

BACKGROUND: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. METHODS: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated. RESULTS: The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE. CONCLUSION: NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer. TRIAL REGISTRATION: This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Extracellular DNA promotes colorectal tumor cell survival after cytotoxic chemotherapy.

BACKGROUND: Inflammation promotes the growth and survival of malignant cells. Inflammation within the tumor microenvironment is a result of damage-associated molecular patterns released by dead or dying cells that provide survival signals to the surrounding cells. It has been proposed that extracellular DNA can act as a damage-associated molecular pattern given the association between circulating DNA and autoimmune diseases. Herein, we demonstrate a novel role for genomic extracellular DNA binding to the Toll-like receptor (TLR)-9 on tumor cells in response to cytotoxic insult. MATERIALS AND METHODS: The colorectal tumor cell line HCCT116 was used to study the role of DNA in tumor cell response to chemotherapy. Cell viability was assessed using CCK-8 assay. Cell death mechanisms were assessed by YOYO-1 and lactate dehydrogenase staining for necrosis and TUNEL staining for apoptosis. Autophagy was measured by LC3 punctate formation. TLR9-short hairpin RNA was used to knockdown TLR-9 and determine its role in tumor cell response to DNA. RESULTS: DNA is released from necrotic tumor cells after chemotherapy. Survival after cytotoxic insult is enhanced by the presence of extracellular DNA as a result of inhibition of apoptosis and enhanced autophagy. Knockdown of TLR-9 enhanced apoptosis, diminished autophagy, and decreased survival after cytotoxic insult in the presence or absence of extracellular DNA. CONCLUSIONS: DNA in the tumor microenvironment promotes survival through induction of autophagy via TLR-9 signaling. This work has important implications for targeting extracellular DNA, TLR-9, and autophagy during treatment with chemotherapy and enhances our understanding of the role of extracellular DNA in the tumor microenvironment.

Hepatic artery infusion of melphalan in patients with liver metastases from ocular melanoma.

BACKGROUND AND OBJECTIVES: Ocular melanoma has a predilection for liver metastases. Systemic treatment is ineffective and the optimal regional therapy approach is poorly defined. Isolated hepatic perfusion (IHP) with melphalan has emerged as a viable treatment option, however a subset of patients are not candidates for this treatment. We therefore sought to determine if melphalan could be safely administered via the hepatic artery for these patients. METHODS: A retrospective review of patients treated with hepatic artery infusion (HAI) of melphalan was undertaken. All patients had contraindications to IHP and were without other therapy options. Melphalan infusion was repeated every four weeks with consideration for dose escalation in the absence of toxicity or significant disease progression. RESULTS: Fourteen patients were treated with HAI of melphalan from 2010 to 2015. All patients had hepatic dysfunction or prohibitive tumor volume precluding IHP. There were no procedure-related complications. Three patients (21%) died within 30 days and the median survival was 2.9 months. Elevated baseline bilirubin > 2.5 mg/dL was associated with worse overall survival (0.93 vs 6.3 months, P < 0.05). CONCLUSION: HAI of melphalan is safe and feasible for patients with metastatic ocular melanoma. Further study to determine the optimal utilization of this treatment approach is warranted.