Natalie Cook

The pancreas cancer microenvironment

Pancreatic ductal adenocarcinoma (PDA) is a common and lethal malignancy resulting in more than 250,000 deaths per year worldwide. Despite extensive efforts, cytotoxic and targeted therapies have provided only limited efficacy for patients with PDA to date. One contributing factor to the failure of systemic therapies may be the abundant tumor stromal content that is the characteristic of PDA. The PDA stroma, aptly termed the tumor microenvironment, occupies the majority of the tumor mass, and consists of a dynamic assortment of extracellular matrix components and nonneoplastic cells including fibroblastic, vascular, and immune cells. Recent work has revealed that the PDA stroma supports tumor growth and promotes metastasis and simultaneously serves as a physical barrier to drug delivery. Accordingly, methods that alter stromal composition or function, for instance interference with the vasculature via Notch/Hedgehog pathway inhibition or relief of vascular compression by hyaluronidase, are under active investigation. Here, we will review our current understanding of the PDA tumor microenvironment, and highlight opportunities for further exploration that may benefit patients. Clin Cancer Res; 18(16); 4266–76. ©2012 AACR.

Stromal biology and therapy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is an almost uniformly lethal disease. One explanation for the devastating prognosis is the failure of many chemotherapies, including the current standard of care therapy gemcitabine. Although our knowledge of the molecular events underlying multistep carcinogenesis in PDA has steadily increased, translation into more effective therapeutic approaches has been inefficient over the last several decades. Evidence for this innate resistance to systemic therapies was recently provided in an accurate mouse model of PDA by the demonstration that chemotherapies are poorly delivered to PDA tissues because of a deficient vasculature. This vascular deficiency correlated with the presence of a dense stromal matrix that is a prominent histological hallmark of PDA tumours. Therapeutic targeting of stromal cells decreased the stroma from pancreatic tumours, resulting in increased intratumoral perfusion and therapeutic delivery of gemcitabine. Stromal cells contained within the PDA tumour microenvironment therefore represent an additional constituent to neoplastic cells that should be critically evaluated for optimal therapeutic development in preclinical models and early clinical trials.

nab-Paclitaxel Potentiates Gemcitabine Activity by Reducing Cytidine Deaminase Levels in a Mouse Model of Pancreatic Cancer

UNLABELLED Nanoparticle albumin-bound (nab)-paclitaxel, an albumin-stabilized paclitaxel formulation, demonstrates clinical activity when administered in combination with gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma (PDA). The limited availability of patient tissue and exquisite sensitivity of xenografts to chemotherapeutics have limited our ability to address the mechanistic basis of this treatment regimen. Here, we used a mouse model of PDA to show that the coadministration of nab-paclitaxel and gemcitabine uniquely demonstrates evidence of tumor regression. Combination treatment increases intratumoral gemcitabine levels attributable to a marked decrease in the primary gemcitabine metabolizing enzyme, cytidine deaminase. Correspondingly, paclitaxel reduced the levels of cytidine deaminase protein in cultured cells through reactive oxygen species-mediated degradation, resulting in the increased stabilization of gemcitabine. Our findings support the concept that suboptimal intratumoral concentrations of gemcitabine represent a crucial mechanism of therapeutic resistance in PDA and highlight the advantages of genetically engineered mouse models in preclinical therapeutic trials. SIGNIFICANCE This study provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer.

Crosstalk between the canonical NF-κB and Notch signaling pathways inhibits Pparγ expression and promotes pancreatic cancer progression in mice

The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-κB pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of κB kinase 2 (Ikk2), a component of the canonical NF-κB signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-α stimulated canonical NF-κB signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-α stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Pparγ. Thus, crosstalk between Tnf-α/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.

Cathepsin B promotes the progression of pancreatic ductal adenocarcinoma in mice

Objective The lysosomal protease cathepsin B is upregulated in human pancreatic ductal adenocarcinoma (PDA) and represents a potential therapeutic target. Loss of cathepsin B delays tumour progression in mouse models of islet, mammary and intestinal carcinoma and decreases invasion and metastasis. This study examines the role of cathepsin B in the initiation, progression and metastasis of PDA. Methods Cathepsin B germline knockout mice were crossed with animals expressing an endogenous KrasG12D allele in the pancreas, and mice were aged to evaluate the role of cathepsin B in pancreatic intraepithelial neoplasia (PanIN). A survival study was also performed with mice carrying an additional heterozygous conditional Trp53R172H allele. Cell lines derived from tumours were used to investigate the role of cathepsin B in vitro, and subcutaneous allografts investigated the cell autonomous and non-cell autonomous roles of cathepsin B in pancreatic cancer. Results Constitutive cathepsin B loss resulted in delayed progression of both PanIN and PDA and a significant survival advantage in mice. Cathepsin B-deficient PDA cells and PanIN showed decreased proliferation and mitogen-activated protein (MAP) kinase signalling. The reconstitution of deficient cells with cathepsin B reversed these findings, which correlated with decreased levels of the active forms of the related protease cathepsin L. Conversely, acute ablation of cathepsin L activated the MAP kinase cascade in PDA cells. Conclusions These results confirm that cathepsin B plays an important cell autonomous role in the progression of PDA and suggest that the regulation of cathepsin L by cathepsin B may be a means of stimulating cell proliferation in neoplasia.

Direct histological processing of EUS biopsies enables rapid molecular biomarker analysis for interventional pancreatic cancer trials

OBJECTIVE Current practice to diagnose pancreatic cancer is accomplished by endoscopic ultrasound guided fine needle aspiration (EUS-FNA) using a cytological approach. This method is time consuming and often fails to provide suitable specimens for modern molecular analyses. Here, we compare the cytological approach with direct formalin fixation of pancreatic EUS-FNA micro-cores and evaluate the potential to perform molecular biomarker analysis on these specimen. METHODS 130 specimens obtained by EUS-FNA with a 22G needle were processed by the standard cytological approach and compared to a separate cohort of 130 specimens that were immediately formalin fixed to preserve micro-cores of tissue prior to routine histological processing. RESULTS We found that direct formalin fixation significantly shortened the time required for diagnosis from 3.6 days to 2.9 days (p<0.05) by reducing the average time (140 vs 33 min/case) and number of slides (9.65 vs 4.67 slides/case) for histopathological processing. Specificity and sensitivity yielded comparable results between the two approaches (82.3% vs 77% and 90.9% vs 100%). Importantly, EUS-FNA histology preserved the tumour tissue architecture with neoplastic glands embedded in stroma in 67.89% of diagnostic cases compared to 27.55% with the standard cytological approach (p < 0.001). Furthermore, micro-core samples were suitable for molecular studies including the immunohistochemical detection of intranuclear Hes1 in malignant cells, and the laser-capture microdissection-mediated measurement of Gli-1 mRNA in tumour stromal myofibroblasts. CONCLUSIONS Direct formalin fixation of pancreatic EUS-FNA micro-cores demonstrates superiority regarding diagnostic delay, costs, and specimen suitability for molecular studies. We advocate this approach for future investigational trials in pancreatic cancer patients.

Early phase clinical trials to identify optimal dosing and safety

The purpose of early stage clinical trials is to determine the recommended dose and toxicity profile of an investigational agent or multi‐drug combination. Molecularly targeted agents (MTAs) and immunotherapies have distinct toxicities from chemotherapies that are often not dose dependent and can lead to chronic and sometimes unpredictable side effects. Therefore utilizing a dose escalation method that has toxicity based endpoints may not be as appropriate for determination of recommended dose, and alternative parameters such as pharmacokinetic or pharmacodynamic outcomes are potentially appealing options. Approaches to enhance safety and optimize dosing include improved preclinical models and assessment, innovative model based design and dose escalation strategies, patient selection, the use of expansion cohorts and extended toxicity assessments. Tailoring the design of phase I trials by adopting new strategies to address the different properties of MTAs is required to enhance the development of these agents. This review will focus on the limitations to safety and dose determination that have occurred in the development of MTAs and immunotherapies. In addition, strategies are proposed to overcome these challenges to develop phase I trials that can more accurately define the recommended dose and identify adverse events.

K-Ras-driven pancreatic cancer mouse model for anticancer inhibitor analyses.

Genetically engineered mouse (GEM) models of cancer have progressively improved in technical sophistication and accurately recapitulating the cognate human condition and have had a measurable impact upon our knowledge of tumorigenesis. However, the application of such models toward the development of innovative therapeutic and diagnostic approaches has lagged behind. Our laboratory has established accurate mouse models of early and advanced ductal pancreatic cancer by conditionally expressing mutant K-ras and Trp53 alleles from their endogenous promoters in pancreatic progenitor cells. These K-Ras-dependent preclinical models provide valuable information on the cell types and pathways involved in the development of pancreatic cancer. Furthermore, they can be used to investigate the molecular, cellular, pharmacokinetic, and radiological characteristics of drug response to classical chemotherapeutics and to targeted agents. This chapter reviews the methods used to explore issues of drug delivery, imaging, and preclinical trial design in our GEM models for pancreatic cancer. We hypothesize that results of our preclinical studies will inform the design of clinical trials for pancreatic cancer patients.

Chemotherapy vs supportive care alone for relapsed gastric, gastroesophageal junction, and oesophageal adenocarcinoma: a meta-analysis of patient-level data.

Background:Second-line chemotherapy treatment of patients with relapsed gastric and oesophageal cancers in comparison with supportive care (SC) alone has been supported by recent phase 3 clinical trials, but a meta-analysis of patient-level data is lacking.Methods:We searched Medline, the Cochrane Central Register of Controlled Trials (CENTRAL), and the Web of Science for phase 3 clinical trials that compared second-line chemotherapy with SC alone for gastric and oesophageal cancers. A meta-analysis of the comprehensive patient-level data from the three identified trials was performed.Results:A total of 410 patients with gastric (n=301), gastroesophageal junction (n=76), or oesophageal (n=33) adenocarcinoma were identified. In all, 154 patients received single-agent docetaxel and 84 patients received single-agent irinotecan, each with SC. SC alone was given to 172 patients. Chemotherapy significantly reduced the risk of death (hazard ratio (HR)=0.63, 95% confidence interval (CI)=0.51–0.77, P<0.0001). This effect was observed for treatment with docetaxel (HR=0.71, 95% CI=0.56–0.89, P=0.003) and irinotecan (HR=0.49, 95% CI=0.36–0.67, P<0.001). Overall survival (OS) benefit was greatest for patients who progressed 3–6 months following first-line chemotherapy (HR=0.39, 95% CI=0.26–0.59, P<0.0001). Performance status (PS) 0–1 compared with PS 2 (HR=0.66, 95% CI=0.46–0.94, P=0.02), locally advanced disease compared with metastatic disease (HR=0.41, 95% CI=0.25–0.67, P=0.0004) and older age (HR=0.94 per 5 years, 95% CI=0.90–0.99, P=0.01) were significant predictors of improved OS. Progression of disease during first-line treatment (HR=1.24, 95% CI=0.96–1.59) or within the first 3 months of completion of first-line treatment (HR=1.42, 95% CI=1.09–1.83) were predictors of an increased risk of death compared with progression between 3 and 6 months (P=0.03). Health-related quality of life outcomes were reported in only one of the three trials, precluding meta-analysis of these parameters.Conclusions:This meta-analysis of patient-level data confirms that second-line chemotherapy treatment results in significantly better OS compared with SC alone in patients with platinum and fluoropyrimidine refractory gastric and oesphageal adenocarcinoma. Health-related quality of life outcomes should be included in future trials in this setting.

Anti-tumour efficacy of capecitabine in a genetically engineered mouse model of pancreatic cancer.

Capecitabine (CAP) is a 5-FU pro-drug approved for the treatment of several cancers and it is used in combination with gemcitabine (GEM) in the treatment of patients with pancreatic adenocarcinoma (PDAC). However, limited pre-clinical data of the effects of CAP in PDAC are available to support the use of the GEMCAP combination in clinic. Therefore, we investigated the pharmacokinetics and the efficacy of CAP as a single agent first and then in combination with GEM to assess the utility of the GEMCAP therapy in clinic. Using a model of spontaneous PDAC occurring in KrasG12D; p53R172H; Pdx1-Cre (KPC) mice and subcutaneous allografts of a KPC PDAC-derived cell line (K8484), we showed that CAP achieved tumour concentrations (∼25 µM) of 5-FU in both models, as a single agent, and induced survival similar to GEM in KPC mice, suggesting similar efficacy. In vitro studies performed in K8484 cells as well as in human pancreatic cell lines showed an additive effect of the GEMCAP combination however, it increased toxicity in vivo and no benefit of a tolerable GEMCAP combination was identified in the allograft model when compared to GEM alone. Our work provides pre-clinical evidence of 5-FU delivery to tumours and anti-tumour efficacy following oral CAP administration that was similar to effects of GEM. Nevertheless, the GEMCAP combination does not improve the therapeutic index compared to GEM alone. These data suggest that CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies for advanced pancreatic cancer.