CD73-Dependent Adenosine Signaling through Adora2b Drives Immunosuppression in Ductal Pancreatic Cancer.

The microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing targets for effective prevention and therapy. Here, we interrogated differential molecular mechanisms dependent on cell of origin and subtype that promote immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell-of-origin-dependent epithelial gene signatures revealed that Nt5e/CD73, a cell-surface enzyme required for extracellular adenosine generation, is one of the top 10% of genes overexpressed in murine tumors arising from the ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by IHC and high-performance liquid chromatography. Analysis in human PDAC subtypes indicated that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC squamous and basal subtypes, considered to have the highest immunosuppression and worst prognosis. Multiplex immunofluorescent analysis showed that activated CD8+ T cells in the PDAC tumor microenvironment express high levels of CD73, indicating an opportunity for immunotherapeutic targeting. Delivery of CD73 small-molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. In addition, the adenosine receptor Adora2b was a determinant of adenosine-mediated immunosuppression in PDAC. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment elevated in the ductal cell of origin, linking biology with subtype classification, critical components for PDAC immunoprevention and personalized approaches for immunotherapeutic intervention. SIGNIFICANCE: Ductal-derived pancreatic tumors have elevated epithelial and CD8+GZM+ T-cell CD73 expression that confers sensitivity to small-molecule inhibition of CD73 or Adora2b to promote CD8+ T-cell-mediated tumor regression. See related commentary by DelGiorno, p. 977.

Assessment of the Murine Tumor Microenvironment by Multiplex Immunofluorescence.

We describe the staining methods used for simultaneous detection of tumor microenvironment components as well as the automated quantification methodologies. This method uses mouse formalin-fixed paraffin-embedded tissues and multiplex immunofluorescence (Multiplex IF) followed by multispectral imaging. Currently, this methodology has shown to have a valuable role in murine immunoprofiling, and can be useful when evaluating the changes incurred on the tumor microenvironment upon various immunopreventive strategies.

Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.

Phase 2 study of vismodegib, a hedgehog inhibitor, combined with gemcitabine and nab-paclitaxel in patients with untreated metastatic pancreatic adenocarcinoma.

BACKGROUND: The Hedgehog (Hh) signalling pathway is overexpressed in pancreatic ductal adenocarcinoma (PDA). Preclinical studies have shown that Hh inhibitors reduce pancreatic cancer stem cells (pCSC), stroma and Hh signalling. METHODS: Patients with previously untreated metastatic PDA were treated with gemcitabine and nab-paclitaxel. Vismodegib was added starting on the second cycle. The primary endpoint was progression-free survival (PFS) as compared with historical controls. Tumour biopsies to assess pCSC, stroma and Hh signalling were obtained before treatment and after cycle 1 (gemcitabine and nab-paclitaxel) or after cycle 2 (gemcitabine and nab-paclitaxel plus vismodegib). RESULTS: Seventy-one patients were enrolled. Median PFS and overall survival (OS) were 5.42 months (95% confidence interval [CI]: 4.37-6.97) and 9.79 months (95% CI: 7.85-10.97), respectively. Of the 67 patients evaluable for response, 27 (40%) had a response: 26 (38.8%) partial responses and 1 complete response. In the tumour samples, there were no significant changes in ALDH + pCSC following treatment. CONCLUSIONS: Adding vismodegib to chemotherapy did not improve efficacy as compared with historical rates observed with chemotherapy alone in patients with newly diagnosed metastatic pancreatic cancer. This study does not support the further evaluation of Hh inhibitors in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01088815.

Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes.

Most patients diagnosed with resected pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a minor subset survives longer. Here, we dissect the role of the tumor microbiota and the immune system in influencing long-term survival. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition in PDAC patients with short-term survival (STS) and long-term survival (LTS). We found higher alpha-diversity in the tumor microbiome of LTS patients and identified an intra-tumoral microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from STS, LTS, or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. Our study demonstrates that PDAC microbiome composition, which cross-talks to the gut microbiome, influences the host immune response and natural history of the disease.

Tumor Spheres Quantification with Smoothed Euclidean Distance Transform.

Tumor sphere quantification plays an important role in cancer research and drugs screening. Even though the number and size of tumor spheres can be found manually, this process is time-consuming, prone to making errors, and may not be viable when the number of images is very large. This manuscript presents a method for automated quantification of spheres with a novel segmentation technique. The segmentation method relies on initial watershed algorithm which detects the minima of the distance transform and finds a tumor sphere for each minimum. Due to the irregular edges of tumor spheres, the distance transform matrix has often more number of minima than the true number of spheres. This leads to the over segmentation problem. The proposed approach uses the smoothed form of the distance transform to effectively eliminate superfluous minima and then seeds the watershed algorithm with the remaining minima. The proposed method was validated over pancreatic tumor spheres images achieving high efficiency for tumor spheres quantification.

Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells.

BACKGROUND & AIMS: Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to pancreatic interepithelial neoplasia (PanIN) initiation and progression. We investigated the effects that IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. METHODS: We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KCiMist;G) and Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KCiMist mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of DCLK1 and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or short hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using The Cancer Genome Atlas database. RESULTS: PanIN cells from KCiMist;G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17-neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern and had significantly decreased expression of DCLK1 and POU2F3, which regulate tuft cell development. KCiMist mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and mitogen-activated protein kinase signaling and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells) compared with cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 after incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C was higher in DCLK1-positive PanIN cells from mice compared with DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 were also associated with patient survival time. CONCLUSIONS: In studies of mouse and human pancreatic tumors and precursors, we found that immune cell-derived IL17 regulated development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.

An examination of competition and efficiency for hospital industry in Turkey.

The two particular reforms that have been undertaken under the Health Transformation Program in Turkey are enhancing efficiency and increasing competition. However, there is a lack of information about the relationship between competition and hospital efficiency. The purpose of this paper is to analyze the effect of competition on technical efficiency for the hospital industry in Turkey. The target population included all public and private general hospitals that were open in 2010 in Turkey (n = 1,224). From these, 1,103 hospitals met the selection criteria and were included in the study. Data were obtained from the Turkish Statistical Institute, the Ministry of Health, and through a field survey. Technical efficiency of hospitals was estimated using Data Envelopment Analysis with five outputs and five inputs. The intensity of competition among hospitals was measured by objective and subjective measures. Objective competition was measured using the Hirschman-Herfindahl Index, and subjective competition was measured based on the perceptions of top level hospital managers. Multivariate Tobit regression was used to investigate the relationship between competition and efficiency while controlling the effects of demand and supply characteristics of the market and the hospital traits. Efficiency results showed that 17% of hospitals were technically efficient. Regression analyses portrayed that the degree of competition among general hospitals did not have a statistically significant relationship with hospitals' technical efficiency. To conclude, hospital efficiency in Turkey does not seem to be affected by the intensity of competition among hospitals.

Financial catastrophe and poverty impacts of out-of-pocket health payments in Turkey.

OBJECTIVE: To determine the prevalence of catastrophic health payments, examine the determinants of catastrophic expenditures, and assess the poverty impact of out-of-pocket (OOP) payments. METHODS: Data came from the 2004 to 2010 Household Budget Survey. Catastrophic health spending was defined by health payments as percentage of household consumption expenditures and capacity to pay at a set of thresholds. The poverty impact was evaluated by poverty head counts and poverty gaps before and after OOP health payments. RESULTS: The percentage of households that catastrophically spent their consumption expenditure and capacity to pay increased from 2004 to 2010, regardless of the threshold used. Households with a share of more than 40% health spending in both consumption expenditure and capacity to pay accounted for less than 1% across years. However, when a series of potential confounders were taken into account, the study found statistically significantly increased risk for the lowest threshold and decreased risk for the highest threshold in 2010 relative to the base year. Household income, size, education, senior and under 5-year-old members, health insurance, disabled members, payment for inpatient care and settlement were also statistically significant predictors of catastrophic health spending. Overall, poverty head counts were below 1%. Poverty gaps reached a maximum of 0.098%, with an overall increase in 2010 compared to 2004. CONCLUSIONS: Catastrophe and poverty increased from 2004 to 2010. However, given that the realization of some recent policies will affect the financial burden of OOP payments on households, the findings of this study need to be replicated.

Registration of 2D point sets by complex translation and rotation operations.

Alignment of two sets containing two dimensional vectors (2D points) constitutes an important problem in medical imaging, remote sensing, and computer vision. We assume that the points in one set, called the transformed set, are constructed by translating and rotating the points in the other set, called the original set. The points in both sets are represented by complex numbers. In order to translate and then rotate a point, we add a complex constant and then multiply by a complex exponential respectively. We construct a cost function which tries to achieve the least-squares differences between a given transformed set and the set containing transformed points with respect to optimization parameters. We implement the Newton-Raphson optimization algorithm with polynomial line search in order to minimize this cost function. Simulation results with multiple datasets demonstrate that the proposed method aligns two sets efficiently and reliably.

A method for subsample fetal heart rate estimation under noisy conditions.

In this paper, we consider a new approach for estimating the fundamental period in fetal ECG waveforms. The fundamental period contains information that is indicative of the physiological condition of the fetus such as hypoxia and acidemia. Our method is based on the minimization of a cost function that measures the differences between the discrete Fourier transform (DFT) of the fetal ECG waveform and the DFTs of its circularly shifted forms. By using the linear phase shift property of the DFT, we show that the minimization of this cost function is equivalent to finding the cosine waveform that matches best to the ECG power spectrum. The optimal cosine waveform is then used to estimate the fundamental period. We expand this method and discuss estimation of the fundamental period with subsample precision. Subsample estimates may be useful especially when a low sampling rate is used for a long period of monitoring. Comparison of performance of this method with Cepstrum and average mIn this paper, we consider a new approach for estimating the fundamental period in fetal ECG waveforms. The fundamental period contains information that is indicative of the physiological condition of the fetus such as hypoxia and acidemia. Our method is based on the minimization of a cost function that measures the differences between the discrete Fourier transform (DFT) of the fetal ECG waveform and the DFTs of its circularly shifted forms. By using the linear phase shift property of the DFT, we show that the minimization of this cost function is equivalent to finding the cosine waveform that matches best to the ECG power spectrum. The optimal cosine waveform is then used to estimate the fundamental period. We expand this method and discuss estimation of the fundamental period with subsample precision. Subsample estimates may be useful especially when a low sampling rate is used for a long period of monitoring. Comparison of performance of this method with Cepstrum and average magnitude difference function methods shows that our approach achieves very accurate period estimation results for both simulated and real fetal EGC waveforms that are taken at different stages of the gestation under noisy conditions.agnitude difference function methods shows that our approach achieves very accurate period estimation results for both simulated and real fetal EGC waveforms that are taken at different stages of the gestation under noisy conditions.

Measurement of efficiency of the dialysis sector in Turkey using data envelopment analysis.

OBJECTIVES: Limited prior work exists on the efficiency of the dialysis sector characterized by steady increase in the dialysis population, providers, and dialysis expenditures in Turkey. The study aimed to examine technical efficiency among producers of dialysis treatment in Turkey. METHODS: Cross-sectional data were used from the 2008 Turkish Statistical Yearbook of Dialysis for total 830 dialysis facilities. Efficiency was measured using data envelopment analysis technique. RESULTS: Results drawn from an input-oriented variable-returns-to-scale model showed technical efficiency from only 3% of facilities, as well as 50% reduction in inputs. Mean efficiency score was found higher among facilities that were freestanding, private, affiliated with international chains, older, and located in Istanbul. CONCLUSION: Efficiency enhancement in dialysis production in Turkey should be placed on the priority agenda, along with careful evaluation of impact of the already-inserted changes on efficiency.

A least square DFT method for sub-sample fetal heart rate estimation under noisy conditions.

In this paper, we consider fundamental period estimation problem for fetal ECG waveforms. By means of a signal separation algorithm, a fetal ECG waveform can be obtained from abdominal ECG of a pregnant woman which contains both maternal and fetal ECG waveforms. We develop a new fundamental period estimator based on minimization of a cost function which measures the least square differences between the discrete Fourier transform (DFT) of the original fetal ECG waveform and the DFT of its circularly shifted form. This cost function attains to its minimum when the circular shift corresponds to integer multiples of the fundamental period. We minimize this cost function to obtain an integer fundamental period estimate. We also minimize this cost function with a gradient descent method to achieve sub-sample precision in fundamental period estimation. With two examples, we demonstrate applications of this method to fetal ECG waveforms and compare its performance to the well-known generalized correlation method.