Local ablative therapies and the effect on antitumor immune responses in pancreatic cancer - A review.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, projected to rank as the second most prevalent cause of cancer-related mortality by 2030. Despite significant progress in advances in surgical techniques and chemotherapy protocols, the overall survival (OS) remains to be less than 10 % for all stages combined. In recent years, local ablative techniques have been introduced and utilized as additional therapeutic approaches for locally advanced pancreatic cancer (LAPC), with promising results with respect to local tumor control and OS. In addition to successful cytoreduction, there is emerging evidence that local ablation induces antitumor immune activity that could prevent or even treat distant metastatic tumors. The enhancement of antitumor immune responses could potentially make ablative therapy a therapeutic option for the treatment of metastatic PDAC. In this review, we summarize current ablative techniques used in the management of LAPC and their impact on systemic immune responses.

Work-Life Integration for Academic Surgeons.

This Viewpoint discusses the time surgeons spend juggling work and life responsibilities by presenting a brief survey of academic surgeons who tracked their time use for 1 week.

Pressure-Enabled Drug Delivery (PEDD) of a class C TLR9 agonist in combination with checkpoint inhibitor therapy in a murine pancreatic cancer model.

BACKGROUND: Systemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma. This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent preclinical cancer models and early-phase clinical trials have demonstrated the potential of toll-like receptor 9 agonists, including the synthetic CpG oligonucleotide SD-101, to stimulate a wide range of immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure-Enabled Drug Delivery via Pancreatic Retrograde Venous Infusion of toll-like receptor 9 agonist would improve responsiveness to systemic anti-programmed death receptor-1 checkpoint inhibitor therapy in a murine orthotopic pancreatic ductal adenocarcinoma model. METHODS: Murine pancreatic ductal adenocarcinoma (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to one of the following treatment groups: Pancreatic Retrograde Venous Infusion delivery of saline, Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist, systemic anti-programmed death receptor-1, systemic toll-like receptor 9 agonist, or the combination of Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist and systemic anti-programmed death receptor-1 (Combo). Fluorescently labeled toll-like receptor 9 agonist (radiant efficiency) was used to measure uptake of the drug on day 1. Changes in tumor burden were evaluated by necropsy at 2 different time points, 7 and 10 days after toll-like receptor 9 agonist treatment. Blood and tumors were collected at necropsy 10 days after toll-like receptor 9 agonist treatment for flow cytometric analysis of tumor-infiltrating leukocytes and plasma cytokines. RESULTS: All mice analyzed survived to necropsy. Site of tumor fluorescence measurements revealed 3-fold higher intensity fluorescence in Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist compared to systemic toll-like receptor 9 agonist mice. Tumor weights were significantly lower in the Combo group compared to Pancreatic Retrograde Venous Infusion delivery of saline. Flow cytometry of the Combo group demonstrated significantly increased overall T-cell number, specifically CD4+ T-cells, and a trend toward increased CD8+ T-cells. Cytokine analysis showed significantly decreased IL-6 and CXCL1. CONCLUSION: Pressure-Enabled Drug Delivery of toll-like receptor 9 agonist by Pancreatic Retrograde Venous Infusion with systemic anti-programmed death receptor-1 demonstrated improved pancreatic ductal adenocarcinoma tumor control in a murine pancreatic ductal adenocarcinoma model. These results support study of this combination therapy in pancreatic ductal adenocarcinoma patients and expansion of ongoing Pressure-Enabled Drug Delivery clinical trials.

Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model.

BACKGROUND: Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab). METHODS: KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses. RESULTS: The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4-6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups. CONCLUSIONS: IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.

Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by an immunosuppressive tumor microenvironment containing few tumor infiltrating lymphocytes (TILs) and an insensitivity to checkpoint inhibitor immunotherapies. Gains in the PTK2 gene encoding focal adhesion kinase (FAK) at Chr8 q24.3 occur in ∼70% of HGSOC tumors, and elevated FAK messenger RNA (mRNA) levels are associated with poor patient survival. Herein, we show that active FAK, phosphorylated at tyrosine-576 within catalytic domain, is significantly increased in late-stage HGSOC tumors. Active FAK costained with CD155, a checkpoint receptor ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), in HGSOC tumors and a selective association between FAK and TIGIT checkpoint ligands were supported by patient transcriptomic database analysis. HGSOC tumors with high FAK expression were associated with low CD3 mRNA levels. Accordingly, late-stage tumors showed elevated active FAK staining and significantly lower levels of CD3+ TILs. Using the KMF (Kras, Myc, FAK) syngeneic ovarian tumor model containing spontaneous PTK2 (FAK) gene gains, the effects of tumor intrinsic genetic or oral small molecule FAK inhibitior (FAKi; VS-4718) were evaluated in vivo. Blocking FAK activity decreased tumor burden, suppressed ascites KMF-associated CD155 levels, and increased peritoneal TILs. The combination of FAKi with blocking TIGIT antibody (1B4) maintained elevated TIL levels and reduced TIGIT+ T regulatory cell levels, prolonged host survival, increased CXCL13 levels, and led to the formation of omental tertiary lymphoid structures. Collectively, our studies support FAK and TIGIT targeting as a rationale immunotherapy combination for HGSOC.

The Landmark Series: Locally Advanced Pancreatic Cancer and Ablative Therapy Options.

Locally advanced pancreatic cancer (LAPC) is a challenging disease to treat. There is consensus that systemic chemotherapy should be the first line of therapy for most patients. However, there is no consensus on how to manage those patients who do not have sufficient response to become candidates for resection but also do not have distant progression after weeks or months of systemic therapy. Radiation therapy is the most commonly used and best-studied local ablative therapy. One recent randomized controlled trial (LAP-07) failed to demonstrate an overall survival benefit for conventional chemoradiation therapy after induction chemotherapy versus chemotherapy alone. This study had several limitations, and ongoing studies are re-evaluating the role of chemoradiation after more effective chemotherapy regimens as well as more advanced radiation techniques. In parallel, there has been increasing interest in other thermal and non-thermal methods of ablation. In particular, irreversible electroporation has gained traction for treatment of LAPC, with at least one ongoing randomized controlled trial designed to address its role compared with systemic chemotherapy alone. Multiple preclinical and clinical studies are investigating combinations of local ablation and immunotherapy with the goal of generating immune responses that will meaningfully improve outcomes.

Optimizing Patient Selection for Irreversible Electroporation of Locally Advanced Pancreatic Cancer: Analyses of Survival.

BACKGROUND: Irreversible electroporation (IRE) has emerged as a viable consolidative therapy after induction chemotherapy, in which this combination has improved overall survival of locally advanced pancreatic cancer (LAPC). Optimal timing and patient selection for irreversible electroporation remains a clinically unmet need. The aim of this study was to investigate preoperative factors that may assist in predicting progression-free and overall survival following IRE. METHODS: A multi-institutional, prospectively maintained database was reviewed for patients with LAPC treated with induction chemotherapy followed by open-technique irreversible electroporation from 7/2015-5/2019. RECIST 1.1 criteria were used to assess tumor response and radiological progression. Overall survival (OS) and progression-free survival (PFS) were recorded. Survival analyses were performed using Kaplan Meier and Cox multivariable regression analyses. RESULTS: 187 LAPC patients (median age 62 years range, 21 - 91, 65% men, 35% women) were treated with IRE. Median PFS was 21.7 months and median OS from diagnosis was 25.5 months. On multivariable analysis, age ≤ 61 (HR 0.41, 95%CI 0.21-0.78, p<0.008) and no prior radiation (HR 0.49, 95%CI 0.26-0.94, p=0.03) were positive predictors of OS after IRE. Age ≤ 61(HR 0.53, 95%CI, 0.28-.99, p=0.046) and FOLFIRINOX followed by gemcitabine/abraxane induction chemotherapy (HR 0.37,95%CI 0.15-0.89, p=0.027) predicted prolonged PFS after IRE. Abnormal CA19-9 values at the time of surgery negatively impacted both OS (HR 2.46, 95%CI 1.28-4.72, p<0.007) and PFS (HR 2.192, 95%CI 1.143-4.201, p=0.018) following IRE. CONCLUSIONS: Age, CA 19-9 response, avoidance of pre-IRE radiation, and FOLFIRINOX plus gemcitabine/abraxane induction chemotherapy are prominent factors to consider when referring or selecting LAPC patients to undergo IRE.

Pressure-enabled delivery of gemcitabine in an orthotopic pancreatic cancer mouse model.

BACKGROUND: We describe the use of pancreatic retrograde venous infusion in an orthotopic murine model of pancreatic ductal adenocarcinoma and hypothesize that pancreatic retrograde venous infusion delivery of gemcitabine will increase concentrations of gemcitabine in the tumor and the subsequent tumor response to treatment. METHODS: Murine pancreatic ductal adenocarcinoma (KPC4580P) was transplanted onto the pancreatic tail of C57BL/6J mice. Groups (n = 15) of mice were assigned to sham laparotomy and 100 mg/kg intraperitoneal infusion of gemcitabine (systemic gemcitabine), pancreatic venous isolation with pancreatic retrograde venous infusion of 100 mg/kg gemcitabine, or pancreatic retrograde venous infusion with saline infusion. Tumor pressures were recorded during pancreatic retrograde venous infusion. Mice were killed at 1 hour or 7 days after infusion. RESULTS: Baseline tumor pressures were 45 ± 8 mm Hg, and pancreatic retrograde venous infusion increased tumor pressures by 29 ± 6 mm Hg (P < .01). Pancreatic retrograde venous infusion gemcitabine mice had greater tumor gemcitabine concentrations compared with systemic gemcitabine (127 vs 19 ng/mg; P < .01) and lesser tumor volumes compared with both systemic gem and pancreatic retrograde venous infusion with saline (274 vs 857 vs 629 mm3; P < .01). CONCLUSION: Pancreatic retrograde venous infusion increased tumor pressures greater than baseline, improved gemcitabine delivery, and increased the treatment response. These findings suggest that pressurized, regional delivery overcomes the increased pressure barrier in pancreatic ductal adenocarcinoma. Additional preclinical studies with cytotoxic and immunotherapeutic agents and clinical trials using pressure-enabled drug delivery with pancreatic retrograde venous infusion devices are underway.

Anti-KIT DNA Aptamer for Targeted Labeling of Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is characterized by KIT protein overexpression, and tumors are frequently driven by oncogenic KIT mutations. Targeted inhibition of KIT revolutionized GIST therapy and ushered in the era of precision medicine for the treatment of solid malignancies. Here, we present the first use of a KIT-specific DNA aptamer for targeted labeling of GIST. We found that an anti-KIT DNA aptamer bound cells in a KIT-dependent manner and was highly specific for GIST cell labeling in vitro Functionally, the KIT aptamer bound extracellular KIT in a manner similar to KIT mAb staining, and was trafficked intracellularly in vitro The KIT aptamer bound dissociated primary human GIST cells in a mutation agnostic manner such that tumors with KIT and PDGFRA mutations were labeled. In addition, the KIT aptamer specifically labeled intact human GIST tissue ex vivo, as well as peritoneal xenografts in mice with high sensitivity. These results represent the first use of an aptamer-based method for targeted detection of GIST in vitro and in vivo.

Irreversible Electroporation Combined with Checkpoint Blockade and TLR7 Stimulation Induces Antitumor Immunity in a Murine Pancreatic Cancer Model.

Irreversible electroporation (IRE) is a nonthermal ablation technique that is used clinically in selected patients with locally advanced pancreatic cancer, but most patients develop recurrent distant metastatic disease. We hypothesize that IRE can induce an in situ vaccination effect by releasing tumor neoantigens in an inflammatory context. Using an immunocompetent mouse model, we demonstrated that IRE alone produced complete regression of subcutaneous tumors in approximately 20% to 30% of mice. IRE was not effective in immunodeficient mice. Mice with complete response to IRE demonstrated prophylactic immunity and remained tumor free when rechallenged with secondary tumors on the contralateral flank. CD8+ T cells from IRE-responsive mice were reactive against peptides representing model-inherent alloantigens and conferred protection against tumor challenge when adoptively transferred into immunocompromised, tumor-naïve mice. Combining IRE with intratumoral Toll-like receptor-7 (TLR7) agonist (1V270) and systemic anti-programmed death-1 receptor (PD)-1 checkpoint blockade resulted in improved treatment responses. This combination also resulted in elimination of untreated concomitant distant tumors (abscopal effects), an effect not seen with IRE alone. These results suggest that the systemic antitumor immune response triggered by IRE can be enhanced by stimulating the innate immune system with a TLR7 agonist and the adaptive immune system with anti-PD-1 checkpoint blockade simultaneously. Combinatorial approaches such as this may help overcome the immunosuppressive pancreatic cancer microenvironment.

A prospective, multi-institution assessment of irreversible electroporation for treatment of locally advanced pancreatic adenocarcinoma: initial outcomes from the AHPBA pancreatic registry.

BACKGROUND: The optimal treatment and management of locally advanced pancreatic cancer (LAPC) remains unclear and controversial. This study aimed to report the initial outcomes of the AHPBA Registry and evaluate the reproducibility of existing evidence that the addition of Irreversible Electroporation (IRE), a nonthermal ablative treatment, confers survival benefits beyond standard therapeutic options for patients with LAPC. METHODS: From December 2015 to October 2017, patients with LAPC were treated with open-technique IRE following the AHPBA Registry Protocols. Patient demographics, long-term outcomes, and adverse events were recorded. Survival analyses were performed using Kaplan-Meier (KM) curves for overall survival (OS), progression free survival (PFS) and time to progression (TTP). RESULTS: A total of 152 patients underwent successful IRE. Morbidity and mortality were 18% and 2% respectively, with 19 (13%) patients experiencing severe adverse events. Nine (6%) patients presented with local recurrence. Median TTP, PFS, and OS from diagnosis were 27.3 months, 22.8 months, and 30.7 months respectively. CONCLUSION: The combination of IRE with established multiagent therapy is safe and demonstrates encouraging survival among patients with LAPC. IRE is associated with a low rate of serious adverse events and has been optimized for more widespread adoption through the standardized protocols available through the AHPBA registry.

Polymer-Mediated Inhibition of Pro-invasive Nucleic Acid DAMPs and Microvesicles Limits Pancreatic Cancer Metastasis.

Nucleic acid binding polymers (NABPs) have been extensively used as vehicles for DNA and RNA delivery. More recently, we discovered that a subset of these NABPs can also serve as anti-inflammatory agents by capturing pro-inflammatory extracellular nucleic acids and associated protein complexes that promote activation of toll-like receptors (TLRs) in diseases such as lupus erythematosus. Nucleic-acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here, we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic-acid-mediated activation of TLRs and invasion of PC tumor cells in vitro, but can also directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

Clinical Management: Resectable Disease.

Despite the identification of more active systemic therapy combinations for pancreatic cancer, cures remain elusive and feasible only in patients with localized, operable disease. When examining outcome data from phase III adjuvant trials conducted during the past decade, the survival for patients with localized disease has improved, likely owing to a combination of factors including more active adjuvant therapy and improved surgical and perioperative care. Perhaps the greatest recent change in the care of patients with localized pancreatic cancer has been the extension of surgery to tumors previously thought to be inoperable because of involvement of major blood vessels. These so-called "borderline resectable pancreatic cancers" have now been objectively defined, and their management is being studied in randomized trials. This has been made feasible by the availability of more active systemic therapy combinations that are increasingly being used in the neoadjuvant setting. Given the increasing activity of systemic regimens, the challenges in delivering such therapy in the postoperative setting, and the numerous novel agents in late stages of clinical development, it is reasonable to hypothesize that the neoadjuvant setting may eventually become the standard of care for patients with resectable disease.

Cell-SELEX Identifies a "Sticky" RNA Aptamer Sequence.

Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface.

Aptamers as Therapeutics.

Aptamers are single-stranded nucleic acid molecules that bind to and inhibit proteins and are commonly produced by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers undergo extensive pharmacological revision, which alters affinity, specificity, and therapeutic half-life, tailoring each drug for a specific clinical need. The first therapeutic aptamer was described 25 years ago. Thus far, one aptamer has been approved for clinical use, and numerous others are in preclinical or clinical development. This review presents a short history of aptamers and SELEX, describes their pharmacological development and optimization, and reviews potential treatment of diseases including visual disorders, thrombosis, and cancer.

TGF-ß-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-ß (TGF-ß) induces the expression of CYR61 in PSCs through canonical TGF-ß-ALK5-Smad2/3 signaling. Activation of TGF-ß signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-ß-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9.

The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs) that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery.