PaCa Patients Research Articles

Abstract 5202: Comparison of [124I]-FIAC and [18F]-FAC as potential biomarkers to predict response to gemcitabine therapy in pancreatic cancers

Abstract Pancreatic cancer (PaCa) is one of the most lethal kinds of cancers because it is virtually symptom free in early stages, metastasizes rapidly and is difficult to resect. Gemcitabine (GEM) as a single agent or in conjunction with radiation is the most commonly used in the management of PaCa. But GEM has shown efficacy only in a small but select group of patients. While the role of key transporters and enzymes has been implicated in sensitizing or conferring resistance to gemcitabine, no clear correlation has been establishe. Currently, there is no a priori way to determine invasively or non-invasively which PaCa patients stand to benefit from GEM therapy. Any biomarker that can act as a prognostic indicator of GEM response will greatly aid in optimizing treatment regimen, reducing unnecessary side effects and subjecting resistant patients to aggressive therapies. Our goal is to develop a PET imaging based biomarker to predict response to GEM therapy in pancreatic cancer patients non-invasively. [18F]-FAC was evaluated as biomarker for GEM response in leukemia models but the short half life (t1/2 = 110 min) combined with background gut activity can complicate image analysis. Therefore our goal was to develop a longer lived analog of GEM that can act as a surrogate marker. Based on modeling studies and ability to label with longer lived isotope (Iodine-124; t1/2 = 4.18 d) we concentrated our efforts on evaluating I-124 labeled 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodocytosine ([124I]-FIAC) as a positron emission tomography (PET) biomarker of GEM response. [124I]-FIAC was synthesized by iodination of 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-cytosine (FAC) with [124I]-NaI using chloramine-T as an oxidant. In vitro phosphorylation efficacy studies were carried out on GEM, FAC and FIAC using recombinant deoxycytidine kinase (dCK) enzyme. In vitro kinetic uptake assay was performed on representattive pancreatic cancer cells lines AsPc-1 and MiaPaCa-2. Subcutaneous tumor xenografts were established in athymic nude mice. [124I]-FIAC was administered intravenously via tail vein and its uptake was studied by PET imaging and sacrificial biodistribution studies at 4, 24, 48 and 72 h. Imaging and biodistribution studies using [18F]-FAC were also performed in tumor bearing mice at 1 and 4 h. [124I]-FIAC was synthesized in >17% yields with >99% radiochemical purity. The efficiency of phosphorylation was in the following order GEM ∼ FAC > FIAC. Cellular uptake assays showed that [124I]-FIAC was taken up preferentially (4X) by AsPc-1 in comparison to MiaPaCa-2. [124I]-FIAC and [18F]-FAC were synthesized and compared for their efficacy to serve as potential biomarker for gemcitabine response in pancreatic cancer models in vitro and in vivo. Preliminary studies indicated that [124I]-FIAC and [18F]-FAC behave differently in these models with former showing preferential uptake in AsPC-1 cells and xenografts. Citation Format: Kishore K. Gangangari, Blesida Punzalan, Brendan Huang, NagaVaraKishore Pillarsetty. Comparison of [124I]-FIAC and [18F]-FAC as potential biomarkers to predict response to gemcitabine therapy in pancreatic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5202.

Do founder mutations characteristic of some cancer sites also predispose to pancreatic cancer?

Understanding of the etiology and risk of pancreatic cancer (PaCa) is still poorly understood. This study evaluated the prevalence of 10 Polish founder mutations in four genes among PaCa patients and assessed their possible association with the risk of disease in Poland. In the study 383 PaCa patients and 4,000 control subjects were genotyped for founder mutations in: BRCA1 (5382insC, 4153delA, C61G), CHEK2 (1100delC, IVS2 + 1G > A, del5395, I157T), NBS1 (657del5) and PALB2 (509_510delGA, 172_175delTTGT). A statistically significant association between the 657del5 mutation and an increased risk of pancreatic cancer was observed for NBS1 gene. The Slavic NBS1 gene mutation (657delACAAA) was detected in 8 of 383 (2.09%) unselected cases compared with 22 of 4,000 (0.55%) controls (OR: 3.80, p = 0.002). The PALB2 509_510delGA and 172_175delTTGT mutations combined were seen in 2 (0.52%) unselected cases of PaCa and in 8 (0.20%) of 4,000 controls (OR: 2.61, p = 0.49). For BRCA1, the three mutations combined were detected in 4 of 383 (1.04%) PaCa patients and in 17 of 4,000 (0.42%) controls (OR: 2.46, p = 0.20). CHEK2 mutations were not associated with the risk of pancreatic cancer (OR: 1.11, p = 0.72). The founder mutation in NBS1 (657del5) was associated with an increased risk of PaCa in heterozygous carriers, indicating that this mutation appears to predispose to cancer of the pancreas. By identifying pancreatic cancer risk groups, founder mutation testing in Poland should be considered for people at risk for PaCa.

MicroRNA-183-5p promotes the proliferation, invasion and metastasis of human pancreatic adenocarcinoma cells.

The aim of the current study was to investigate the potential role of microRNA-183-5p (miR-183-5p) in the proliferation, invasion and metastasis of pancreatic cancer, and to identify promising target genes of oncogenic miR-183-5p. Western blotting and quantitative polymerase chain reaction (qPCR) were used to investigate whether these oncogenic microRNAs may be useful as biomarkers in pancreatic carcinoma (PaCa). Potential target genes were verified using miRDB, PicTar and TargetSCAN, and qPCR was used to detect the expression of miR-183 and suppressor of cytokine signaling 6 (SOCS-6; a potential target of miR-183) in PANC-1 PaCa cells and in the HPDE6-C7 pancreatic ductal cell line for comparison. The function of miR-183 in cell proliferation, wound healing, invasion and migration was also investigated using a miR-183 inhibitor. Western blot analysis was used to confirm SOCS-6 as a tumor suppressor and qPCR was used to detect and confirm that this potential target gene is directly regulated by miR-183. The results indicated that the expression of miR-183 in PANC-1 cells was upregulated compared with that in HPDE6-C7 cells, whilst the expression of SOCS-6 was downregulated. SOCS-6 expression was also significantly lower in PaCa tissues compared with that in matched normal pancreatic tissues from PaCa patients. Furthermore, expression of miR-183 was inversely correlated with that of SOCS-6. miR-183 knockdown decreased cell growth and motility in pancreatic cancer cells and significantly increased the expression of SOCS-6. These data suggest that oncogenic miR-183 may be useful as a pancreatic cancer biomarker. In addition, inhibition of miR-183 expression may be beneficial as PaCa treatment. SOCS-6 is a potential target gene of miR-183.

Abstract 1782: Human tumor explants are better predictors of clinical trial outcome than cell line xenografts for the KSP inhibitor ARRY-520

Abstract Historically, human tumor cell line cultures and xenografts have served as standard models for identifying and optimizing anticancer drugs. Despite wide use, cell line models are poor predictors of clinical activity of drugs, particularly cytotoxics. Primary human tumor explant models have significant potential to inform clinical direction. These models have not undergone genetic drift or selection for growth in culture. Furthermore, low-passage explants retain the morphology and aspects of the tumor microenvironment reflecting the primary cancer in patients. These models further represent patients who have been treated with current chemotherapeutics. ARRY-520 is a novel kinesin spindle protein inhibitor which has been investigated in clinical studies in both solid and hematological cancers. In cell line xenografts, ARRY-520 has significant activity in solid tumors and even greater activity in hematological cell lines both in vitro and in vivo. In clinical studies, while ARRY-520 has demonstrated significant single-agent activity in multiple myeloma, activity in solid tumors has been modest. Thus, while preclinical data in hematological models has correlated with clinical activity in myeloma, the impressive solid tumor data has not translated to clinical activity in solid tumors. To probe the disparity of preclinical and clinical activity in solid tumors, we have investigated the ability of preclinical models to inform clinical success by retrospective analysis of the activity of ARRY-520 in solid tumor cell line and explant models, as compared to clinical data of this molecule in these indications. ARRY-520 shows potent activity in human tumor cell lines both in vitro, (IC50 0.5-14nM) and in vivo (50% tumor regression in 4/5 cell line xenografts CRC and PaCa including 100% regression in 2 models). By contrast, in 6 primary CRC and PaCa explants, ARRY-520 showed minimal activity (transient stable disease in 1/6 models and progressive disease in 5/6 models). Phase 1 solid tumor data on ARRY-520 was reported at ASCO 2010. In this study, 30 patients with solid tumors, treated at or above the MTD, were evaluable for response with best response of SD>12 weeks in 2 patients (7%). Within this study, there were 4 CRC and 3 PaCa patients, all of whom experienced progressive disease within 12 weeks of initiation. Further investigation of ARRY-520 in solid tumor explants is ongoing. Analysis of responsive and non-responsive models will be used to identify solid tumor types for further clinical development. In summary, in this study, human solid tumor explant xenografts more accurately predicted the actual clinical results from a Phase 1 solid tumor trial. These data suggest that solid tumor explant models may have significant utility in informing clinical decisions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1782. doi:1538-7445.AM2012-1782

Abstract 4907: Methylated cell-free DNA in plasma as an early biomarker of pancreatic cancer

Abstract Background: Presently there is an urgent need for better biomarkers for early pancreatic cancer (PaCa) diagnosis. Hypermethylation of several genes is known to correlate with presence of neoplasia in pancreatic tissue, and tumor DNA is known to be present in bodily fluids. With this background, we wanted to develop and validate a panel of methylated gene markers that would provide clinically relevant information when applied to plasma from those undergoing diagnostic procedures for pancreatic cancer. Methods: Cell-free DNA was extracted from plasma obtained from 11 PaCa patients, 15 pancreatitis patients and 15 unaffected age-matched controls using either the automated EZ-1 (Qiagen, Valencia, CA) or the manual Charge Switch (Invitrogen, Carlsbad, CA) methods. The DNA was bisulfite treated using EZ DNA Methylation-Gold (Zymo Research, Orange, CA) then analyzed using a panel of quantitative real-time PCR assays to discriminate the methylated C or T that exists after bisulfite conversion, and normalizing the signal to the amplification level of a housekeeping gene (Col2A) to assess methylation of status of 13 genes known to be differentially methylated in pancreatic tissues. Several different combinations of 6 methylated gene markers (3-OST-2, ARF/p14, FOXE1, CDKN2/p16, ppENK, and RASSF1A) showed good discrimination between the 3 groups of patients to warrant further study with an additional 53 plasma specimens (from 21 PaCa patients, 17 pancreatitis patients, and 15 unaffected controls). Receiver operator curves were generated to examine the predictive probability between marker methylation and disease, and reliability of the regression model was evaluated by using the Hosmer-Leme show goodness-of-fit test. Results: Statistical analysis of the 6 methylated gene markers for all 94 plasma specimens yielded an AUC of 0.638 (95% CI: 0.50-0.78) to differentiate PaCa from pancreatitis, and the goodness-of-fit for the model was good (p=0.697). Conclusions: Cell-free DNA extracted from plasma is an appropriate sample for the study of gene methylation in PaCa. We utilized real-time assays to detect methylation in 6 target genes and found a good correlation between the methylation status of genes and the presence of PaCa. Further studies are needed to define the clinical utility of this approach. Funding was provided by a grant from the American Cancer Society - Illinois Division (#07-06) and by funds from the Department of Pathology and Laboratory Medicine at NorthShore. 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 4907.

CXC‐chemokine/CXCR2 biological axis promotes angiogenesis in vitro and in vivo in pancreatic cancer

Angiogenesis is essential for tumor growth and metastasis. Although ELR(+)-CXC-chemokines and their corresponding receptor, CXC-receptor 2 (CXCR2), are known mediators of angiogenesis, little is known about their role in pancreatic cancer (PaCa). The aim of our study was to determine the role of ELR(+)-CXC-chemokine/CXCR2 biological axis in promoting PaCa angiogenesis. We prospectively collected secretin-stimulated exocrine pancreatic secretions (SSEPS) from normal individuals (NP) and PaCa patients. We showed that summed concentrations of ELR(+)-CXC-chemokines in SSEPS from PaCa patients were significantly higher than in those from NP (p = 0.002). We measured ELR(+)-CXC-chemokine levels in supernatants from multiple PaCa cell lines and confirmed that BxPC-3, Colo-357 and Panc-28 had significantly higher expression compared with an immortalized human pancreatic ductal epithelial (HPDE) cell line. After confirming lack of autocrine effects of ELR(+)-CXC-chemokines on PaCa cells (due to absence of CXCR2 expression), we investigated paracrine effects of these chemokines on human umbilical vein endothelial cells (HUVEC). Both recombinant ELR(+)-CXC-chemokines and co-culturing with BxPC-3 significantly enhanced proliferation, invasion, and tube formation of HUVEC (p < 0.05). These biological effects were significantly inhibited by treatment with a neutralizing antibody against CXCR2 (anti-CXCR2 Ab) (p < 0.05). Finally, anti-CXCR2 Ab significantly reduced tumor volume (p < 0.05), Ki-67 proliferation index (p = 0.043) and Factor VIII(+) microvessel density (p = 0.004) in an orthotopic nude mouse PaCa model. Our results show that ELR(+)-CXC-chemokines promote PaCa tumor-associated angiogenesis through CXCR2, suggesting that CXCR2 is an anti-angiogenic target in PaCa.

In vitro evidence for genetic predisposition in some human pancreatic adenocarcinoma.

A numerical alteration in chromosome complement in human dermal fibroblast cultures, hyperdiploidy with a normal occurrence of tetraploidy (IVH) has been reported to be associated with some hereditary single tumors, including pancreatic adenocarcinoma (PaCa). The incidence of IVH was compared in cultures derived from 34 PaCa patients and 39 clinically normal subjects without a family tumor history by three assay systems: (a) percentage of numerically altered metaphases in chromosome preparations (MA); (b) percentage of cells from high-density, stationary cultures with a DNA index (DI) greater than 1 (FCMs); (c) percentage of cells with a DI greater than 2 from cultures of cells undergoing division (FCMd). The last two measurements were obtained by flow cytometric measurement of propidium idodide (PI) stained cells. Concordance was observed between the three assays. There was a linear relationship between the percentage of hyperdiploid metaphases assayed in the chromosome preparation and the percentage of cells with a DI greater than 1 by FCMs and a DI greater than 2 by FCMd. IVH was considered to be present (IVH+) by the MA technique if the percentage of numerically altered metaphases was over 4% exclusive of tetraploids, by the FCMs technique if DI greater than 1 was greater than 10%, and by FCMd if DI greater than 2 was greater than 7%. The assayed group could be divided into two IVH groups on the basis of all three assays, individually or collectively.(ABSTRACT TRUNCATED AT 250 WORDS)