Faculty Bibliography

Hepatocyte nuclear factor 1A (HNF1A) is a transcription factor essential to normal pancreas and liver development and homeostasis. HNF1A has been shown to be upregulated in pancreatic cancer stem cells (CSC), a proposed key driver of pancreatic ductal adenocarcinoma (PDAC), relative to other pancreatic cancer cells. A CSC gene signature consisting of 50 upregulated genes, including HNF1A, was previously identified by a microarray. This signature predicts worse survival compared to HNF1A expression alone. This research aims to establish an HNF1A-dependent gene signature that could potentially be used as a biomarker for pancreatic cancer. Using quantitative reverse transcriptase PCR, multiple CSC signature genes were shown to respond to HNF1A knockdown and overexpression. Responsive genes varied across cell lines, suggesting a complex pathway significantly affected by HNF1A transcriptional control

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.

OBJECTIVES: Definitive chemoradiotherapy for unresectable pancreatic cancer has traditionally involved 5-fluorouracil-based chemotherapy. Our institution has a long history of combining gemcitabine and radiotherapy (RT), and performed a retrospective review of all patients treated in this manner. MATERIALS AND METHODS: We reviewed the records of 180 patients treated from 1999 to 2012. Mean RT dose was 40.9 Gy in 2.2-Gy fractions, and targeted only radiographically apparent disease. Ninety-six percent of patients received full-dose gemcitabine-based chemotherapy with RT. Kaplan-Meier was used to analyze time-to-event endpoints, and Cox regression models were used to assess significant prognostic variables. RESULTS: Eighty-nine percent of patients completed RT without a toxicity-related treatment break. Median follow-up was 10.2 months. Twenty-nine percent of patients had a radiographic decrease in primary tumor size following treatment. Median overall survival was 11.8 months, time to distant metastasis (TDM) was 6.7 months, and time to local recurrence (TLR) was 8.3 months. On multivariate analysis, male sex, lower performance status, and higher posttreatment CA 19-9 level predicted for worse overall survival. Posttreatment, CA 19-9 was also associated with TDM and TLR, and radiographic tumor response was associated with better TLR. CONCLUSION: Definitive chemoradiation using full-dose gemcitabine is well tolerated and achieves survival outcomes comparable to reported trials in the literature.

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations.

OBJECTIVE: We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue. DESIGN: Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors. RESULTS: Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth. CONCLUSIONS: Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach.

The extent of inter- and intra-tumor cell heterogeneity observed in patient tumors appears to be directly associated with patient prognosis. Moreover, studies indicate that targeting distinct subpopulations of tumor cells may be more relevant to successfully managing cancer metastasis. The ability to distinguish and characterize unique tumor cell subpopulations within a given sample is thus exigent. Existing platforms separate cells binarily, based on some threshold level of phenotypic characteristics without consideration of the continuum levels of biomarker expression and the associated implications. Herein we describe how specific tumor cell groups have been immunomagnetically enriched according to a continuum of EpCAM surface marker expression levels. Even among a relatively homogenous group of cells such as the PANC-1 cell line, cells could be separated according to their EpCAM levels into low, moderate and high expression. To physiologically assess each subpopulation, a wound healing assay was performed which revealed distinct invasive potentials among each subset. Furthermore, the clinical relevance of the approach was demonstrated by isolating pancreatic cancer CTCs from the same patient sample based on their EpCAM levels. We demonstrate a robust method of isolating CTCs according to their varying protein levels, which enables extensive studies on tumor cell heterogeneity. Interestingly, 5 of 6 samples had CTCs that could be recovered at all three levels of EpCAM expression though the majority of CTCs were recovered as low expression events. Preliminary studies that compare tumor cell subpopulations in this continuum manner can potentially increase our understanding of the dynamic nature of cell heterogeneity and how it relates to patient outcomes. Ultimately further investigation may yield therapeutic targets against virulent cell subpopulations.

OBJECTIVES: Current pancreatic cancer diagnostics cannot reliably detect early disease or distinguish it from chronic pancreatitis. We test the hypothesis that optical spectroscopy can accurately differentiate cancer from chronic pancreatitis and normal pancreas. We developed and tested clinically compatible multimodal optical spectroscopy technology to measure reflectance and endogenous fluorescence from human pancreatic tissues. METHODS: Freshly excised pancreatic tissue specimens (39 normal, 34 chronic pancreatitis, 32 adenocarcinoma) from 18 patients were optically interrogated, with site-specific histopathology representing the criterion standard. A multinomial logistic model using principal component analysis and generalized estimating equations provided statistically rigorous tissue classification. RESULTS: Optical spectroscopy distinguished pancreatic cancer from normal pancreas and chronic pancreatitis (sensitivity, 91%; specificity, 82%; positive predictive value, 69%; negative predictive value, 95%; area under receiver operating characteristic curve, 0.89). Reflectance alone provided essentially the same classification accuracy as reflectance and fluorescence combined, suggesting that a rapid, low-cost, reduced-footprint, reflectance-based device could be deployed without notable loss of diagnostic power. CONCLUSIONS: Our novel, clinically compatible, label-free optical diagnostic technology accurately characterizes pancreatic tissues. These data provide the scientific foundation demonstrating that optical spectroscopy can potentially improve diagnosis of pancreatic cancer and chronic pancreatitis.

BACKGROUND: Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity. OBJECTIVE: The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. METHODS: Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells (mostly myeloid cell population) were depleted by diphtheria toxin treatment during tumour initiation or in established tumours. RESULTS: Depletion of myeloid cells prevented KrasG12D-driven pancreatic cancer initiation. In pre-established tumours, myeloid cell depletion arrested tumour growth and in some cases, induced tumour regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T-cell anti-tumour activity by inducing the expression of programmed cell death-ligand 1 (PD-L1) in tumour cells in an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent manner. CONCLUSION: Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer.

Background Cabozantinib and gemcitabine improve tumor control in pancreatic ductal adenocarcinoma (PDAC) in preclinical models through c-Met inhibition. We sought to determine the maximum tolerated dose (MTD) of this combination in patients with advanced PDAC. Methods Patients with 25 % for all dose levels tested, and thus an MTD was not determined. DLTs included grade 3 ALT/AST elevations and thrombocytopenia. Three patients had partial responses, but each discontinued therapy due to toxicity. Median PFS and OS were 4.7 (95 % CI: 1.4-9.7) and 10.1 months (95 % CI: 3.6-20.6). Exploratory biomarker analysis showed correlation of c-Met and VEGF levels with response. Conclusions An MTD for the combination was not established. Cabozantinib and gemcitabine appear impractical for further development due to DLT at low doses and continuing toxicities with ongoing therapy. Acknowledging the small sample size, responses were seen suggesting further investigation of c-Met inhibition in PDAC may be warranted.

OBJECTIVE: This study aimed to determine if the improved pain response to endoscopic retrograde cholangiopancreatogrphy (ERCP) and pancreatic stent placement (EPS) predicts pain response in patients with chronic pancreatitis after modified lateral pancreaticojejunostomy (LPJ). METHODS: A multi-institutional, retrospective review of patients who underwent successful EPS before LPJ between 2001 and 2010 was performed. The primary outcome was narcotic independence (NI) within 2 months after ERCP or LPJ. RESULTS: A total of 31 narcotic-dependent patients with chronic pancreatitis underwent successful EPS before LPJ. Ten (32%) achieved post-LPJ NI (median follow-up, 8.5 months; interquartile range [IQR], 2-38 months). Eight (80%) of 10 patients with NI post-ERCP achieved NI post-LPJ. Two (10%) without NI post-ERCP achieved NI post-LPJ. Narcotic independence post-EPS was associated strongly with NI post-LPJ with an odds ratio of 38 (P = 0.0025) and predicted post-LPJ NI with a sensitivity, specificity, positive predictive value, and negative predictive value of 80%, 90.5%, 80%, and 90.5%, respectively. CONCLUSIONS: Narcotic independence after EPS is associated with NI after LPJ. Failure to achieve NI post-ERCP predicts failure to achieve NI post-LPJ. These results support the need for larger studies to confirm the predictive value of pancreatic duct stenting for better selection of chronic pancreatitis patients who will benefit from LPJ.