Showing papers by "Diane M. Simeone published in 2010"

Mechanism of radiosensitization by the Chk1/2 inhibitor AZD7762 involves abrogation of the G2 checkpoint and inhibition of homologous recombinational DNA repair

Abstract: The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM-mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G(2) checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent gamma-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G(2) checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer.

Glycosylation variants of mucins and CEACAMs as candidate biomarkers for the diagnosis of pancreatic cystic neoplasms.

Abstract: Background and Aims:Cystic lesions of the pancreas are increasingly being recognized due to the widespread use of high resolution abdominal imaging. Since certain cyst types are precursors to invasive cancer, this situation presents an opportunity to intervene prior to malignant progression. Effecti

Glycoprotein analysis using protein microarrays and mass spectrometry.

Abstract: Protein glycosylation plays an important role in a multitude of biological processes such as cell‐cell recognition, growth, differentiation, and cell death. It has been shown that specific glycosylation changes are key in disease progression and can have diagnostic value for a variety of disease types such as cancer and inflammation. The complexity of carbohydrate structures and their derivatives makes their study a real challenge. Improving the isolation, separation, and characterization of carbohydrates and their glycoproteins is a subject of increasing scientific interest. With the development of new stationary phases and molecules that have affinity properties for glycoproteins, the isolation and separation of these compounds have advanced significantly. In addition to detection with mass spectrometry, the microarray platform has become an essential tool to characterize glycan structure and to study glycosylation-related biological interactions, by using probes as a means to interrogate the spotted or captured glycosylated molecules on the arrays. Furthermore, the highthroughput and reproducible nature of microarray platforms have been highlighted by its extensive applications in the field of biomarker validation, where a large number of samples must be analyzed multiple times. This review covers a brief survey of the other experimental methodologies that are currently being developed and used to study glycosylation and emphasizes methodologies that involve the use of microarray platforms. This review describes recent advances in several options of microarray platforms used in glycoprotein analysis, including glycoprotein arrays, glycan arrays, lectin arrays, and antibody/ lectin arrays. The translational use of these arrays in applications related to characterization of cells and biomarker discovery is also included. # 2010 Wiley Periodicals, Inc., Mass Spec Rev 29:830–844, 2010

Quantitative proteomic profiling studies of pancreatic cancer stem cells.

Abstract: Analyzing subpopulations of tumor cells in tissue is a challenging subject in proteomic studies. Pancreatic cancer stem cells (CSCs) are such a group of cells that only constitute 0.2-0.8% of the total tumor cells but have been found to be the origin of pancreatic cancer carcinogenesis and metastasis. Global proteome profiling of pancreatic CSCs from xenograft tumors in mice is a promising way to unveil the molecular machinery underlying the signaling pathways. However, the extremely low availability of pancreatic tissue CSCs (around 10,000 cells per xenograft tumor or patient sample) has limited the utilization of currently standard proteomic approaches which do not work effectively with such a small amount of material. Herein, we describe the profiling of the proteome of pancreatic CSCs using a capillary scale shotgun technique by coupling offline capillary isoelectric focusing(cIEF) with nano reversed phase liquid chromatography(RPLC) followed by spectral counting peptide quantification. A whole cell lysate from 10,000 cells which corresponds to ∼1ug protein material is equally divided for three repeated cIEF separations where around 300ng peptide material is used in each run. In comparison with a non-tumorigenic tumor cell sample, among 1159 distinct proteins identified with FDR less than 0.2%, 169 differentially expressed proteins are identified after multiple testing corrections where 24% of the proteins are upregulated in the CSCs group. Ingenuity Pathway analysis of these differential expression signatures further suggests significant involvement of signaling pathways related to apoptosis, cell proliferation, inflammation and metastasis.

Spectral areas and ratios classifier algorithm for pancreatic tissue classification using optical spectroscopy.

Abstract: Pancreatic adenocarcinoma is one of the leading causes of cancer death, in part because of the inability of current diagnostic methods to reliably detect early-stage disease. We present the first assessment of the diagnostic accuracy of algorithms developed for pancreatic tissue classification using data from fiber optic probe-based bimodal optical spectroscopy, a real-time approach that would be compatible with minimally invasive diagnostic procedures for early cancer detection in the pancreas. A total of 96 fluorescence and 96 reflectance spectra are considered from 50 freshly excised tissue sites-including human pancreatic adenocarcinoma, chronic pancreatitis (inflammation), and normal tissues-on nine patients. Classification algorithms using linear discriminant analysis are developed to distinguish among tissues, and leave-one-out cross-validation is employed to assess the classifiers' performance. The spectral areas and ratios classifier (SpARC) algorithm employs a combination of reflectance and fluorescence data and has the best performance, with sensitivity, specificity, negative predictive value, and positive predictive value for correctly identifying adenocarcinoma being 85, 89, 92, and 80%, respectively.

Photon-tissue interaction model enables quantitative optical analysis of human pancreatic tissues.

Abstract: A photon-tissue interaction (PTI) model was developed and employed to analyze 96 pairs of reflectance and fluorescence spectra from freshly excised human pancreatic tissues. For each pair of spectra, the PTI model extracted a cellular nuclear size parameter from the measured reflectance, and the relative contributions of extracellular and intracellular fluorophores to the intrinsic fluorescence. The results suggest that reflectance and fluorescence spectroscopies have the potential to quantitatively distinguish among pancreatic tissue types, including normal pancreatic tissue, pancreatitis, and pancreatic adenocarcinoma.

The identification of auto-antibodies in pancreatic cancer patient sera using a naturally fractionated Panc-1 cell line

Abstract: The immunogenic nature of cancer can be explored to distinguish pancreatic cancer from related non-cancer conditions. We describe a liquid-based microarray approach followed by statistical analysis and confirmation for discovery of auto-immune biomarkers for pancreatic cancer. Proteins from the Panc-1 pancreatic cancer cell line were fractionated using a 2-D liquid separation method into over 1052 fractions and spotted onto nitrocellulose coated glass slides. The slides were hybridized with 37 pancreatic cancer sera, 24 chronic pancreatitis sera and 23 normal sera to detect elevated levels of reactivity against the proteins in spotted fractions. The response data obtained from protein microarrays was first analyzed by Wilcoxon Rank-Sum Tests to generate two lists of fractions that positively responded to the cancer sera and showed p-values less than 0.02 in the pairwise comparison between cancer specimens and normal and chronic pancreatitis specimens. The top 3 fractions with the lowest correlations were combined in receiver operating characteristic analyses. The area-under-the-curve (AUC) values are 0.813 and 0.792 for cancer vs. normal and cancer vs. pancreatitis respectively. Outlier-Sum statistics were then applied to the microarray data to determine the existence of outliers exclusive in cancer sera. The selected fractions were identified by LC-MS/MS. We further confirmed the occurrence of outliers with three proteins among cancer samples in a confirmation experiment using a separate dataset of 165 serum samples containing 48 cancer sera and 117 non-cancer controls. Phosphoglycerate kinase 1 (PGK1) elicited greater reactivity in 20.9% (10 in 48) of the samples in the cancer group, while no outlier was present in the non-cancer groups.

Methods for diagnosing the malignant potential of pancreatic cystic lesions

Abstract: A method of diagnosing the malignant potential of a pancreatic cystic lesion in a subject including: detecting a glycan alteration in MUC5AC in a sample of pancreatic cystic lesion fluid from a subject, determining whether the glycan alteration is differentially present in the sample, and diagnosing the malignant potential of the pancreatic cystic lesion. A method of diagnosing the malignant potential of a pancreatic cystic lesion in a subject including: (a) detecting a glycan alteration in MUC5AC in a sample of pancreatic cystic lesion fluid from a subject, (b) detecting CA 19-9 in the sample, (c) determining whether the glycan alteration and CA 19-9 are differentially present in the sample, and (d) diagnosing the malignant potential of the pancreatic cystic lesion. Related methods of treatment and kits also are included.

Quantitative optical spectroscopy for pancreatic cancer detection

Abstract: We report novel optical diagnostic algorithms for clinical pancreatic tissue classification, including a photon-tissue interaction model developed to extract biophysical parameters from reflectance and fluorescence spectra to distinguish pancreatic adenocarcinoma from normal tissue and pancreatitis.

Abstract LB-257: c-Met: a new cancer stem cell marker and therapeutic target for pancreatic cancer

Abstract: Pancreatic adenocarcinoma is a highly aggressive disease which is usually diagnosed in an advanced state and for which there are little/no effective therapies. The growth of many cancers depends on a subpopulation of self-renewing cells, termed cancer stem cells (CSC). We have previously identified such a CSC population in human pancreatic cancers which possess the cell surface marker phenotype CD44 + CD24 + ESA + . C-Met, a member of the receptor tyrosine kinases (RTKs) family and receptor for the hepatocyte growth factor (HGF) ligand, has been recently described as a marker of normal mouse pancreatic stem/progenitor cells. We hypothesized that c-Met may function as a cell surface marker and therapeutic target for human pancreatic CSCs. Studies were performed in low passage primary human pancreatic adenocarcinoma xenografts (n = 3) established in NOD-SCID mice. Self-renewal capacity of c-Met high pancreatic cancer cells was assessed using in vitro sphere assays and in vivo tumorigenicity of c-Met high pancreatic cancer cells was evaluated by cell implantation in NOD/SCID mice. In vitro studies demonstrated that c-Met high cells formed spheres in non-adherent cultures, while c-Met − cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with shRNA significantly inhibited tumor sphere formation. Pancreatic cancer cells expressing high c-Met had increased tumorigenic potential in vivo and cells expressing both CD44 and c-Met high , representing 0.5-5% of pancreatic cancer cells, demonstrated the highest tumorigenic potential of all populations studied to date. C-Met high CD44+ expressing pancreatic cancer cells demonstrated the capacity for self-renewal and production of differentiated progeny, properties that define CSCs. Further, c-Met inhibition of established primary human pancreatic tumors in NOD/SCID mice significantly inhibited tumor growth and depleted the pancreatic CSC population, when used alone or in combination with gemcitabine. Inhibition of c-Met also prevented the developmental of metastases using an intracardiac injection model. In this report, we define c-Met as a new marker for pancreatic CSC and define c-Met hi g h CD44 + cells as a highly tumorigenic pancreatic CSC population. Our data also indicate that targeting c-Met may be useful in eradicating a CSC population and inhibiting the development of metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-257.

Technical Advance Reliable Gene Expression Measurements from Fine Needle Aspirates of Pancreatic Tumors Effect of Amplicon Length and Quality Assessment

Abstract: Background and aims: Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRTPCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression. Methods: Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft. Results: RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance. Conclusions: The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens “informative” for qRT-PCR allowed accurate molecular assessment for the diagnosis of pan