Blood Of Pancreatic Cancer Patients Research Articles

Ultrasensitive Detection of Cancer Biomarkers Using Photonic-Crystal-Enhanced Single-Molecule Imaging.

The rapid and sensitive quantification of low-abundance protein markers holds immense significance in early disease diagnosis and treatment. Single-molecule fluorescence imaging exhibits very high detection sensitivity and thus has great application potential in this area. The single-molecule signal, however, is often susceptible to interference from background noise due to its inherently weak intensity. A variety of signal amplification techniques based on cascading reactions have been developed to improve the signal-to-noise ratio of single-molecule imaging. Nevertheless, the operation of these methods is typically complicated and time-consuming, which limits the clinical application. Herein, we introduce an enzyme-free, photonic-crystal-based single-molecule (PC-SM) biochip for cost-effective, time-efficient, and ultrasensitive detection of disease markers. The PC-SM biochip can enhance the signal-to-noise ratio of single molecules by nearly 3-fold compared with unamplified samples, through coupling of the single-molecule photon energy with the optical band gap of the photonic crystal. We used the PC-SM biochip to detect the low-abundance leukemia inhibitory factor in the blood of pancreatic cancer patients and healthy people and achieved a detection limit of 2.0 pg/L and an AUC of 0.9067. The method exhibits exceptional sensitivity and specificity, showing great application potential in various clinical settings.

Abstract 6900: Patient-derived xenografts from circulating cancer stem cells and their potential utility in personalized medicine of pancreatic cancer

Abstract Background: Pancreatic cancer carries a poor prognosis in the majority of patients, with an overall 5-year survival rate of less than 10%. Therefore, the development of new therapies and preclinical models is crucial and urgently needed. Circulating cancer stem cells (cCSCs) are a subset of circulating cancer cells that have stem cell-like properties and play a significant role in tumor progression, metastasis, and treatment resistance. Patient-derived xenografts (PDX) reflect the genotypic and phenotypic landscape of patients' cancers and are useful for drug screening, biomarker development, and preclinical evaluation of personalized medicine strategies. In this study, we describe a fast, simple, and cost effective method to generate PDX on Chorioallantoic Membrane (CAM) from circulating cancer stem cells. Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays from 30 patients with newly diagnosed pancreatic cancer. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form patient- derived xenografts. Results: For the identification of cCSCs from peripheral blood of pancreatic cancer patients, an innovative in vitro sphere-forming assay was established. The number of tumorspheres was correlated with clinicopathological parameters like tumor size, lymph node involvement and grading status of the primary tumor. Implantation of tumorspheres onto the CAM was successful in ~90 % of the applied samples. The histological analysis suggests that the PDX tumors were morphologically and pathologically identical to primary tumor tissue. Conclusions: The number of tumorspheres was associated with clinicopathological parameters. Further follow-up is needed to evaluate the prognostic significance of tumorsphere detection in pancreatic cancer. Generation of PDX from cCSCs on CAM has a high success rate and may become an integral part of the drug development arena, including drug screening and biomarker development. Citation Format: Monika Pizon, Dorothea Schott, Andreas Ettner-Sittner, Benedikt J. Wagner, Katharina Pachmann, Thiha Aung, Christina Hackl, Silke Haerteis. Patient-derived xenografts from circulating cancer stem cells and their potential utility in personalized medicine of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6900.

Abstract 3381: Establishment of a novel multi-omic biomarker panel in cyst fluid and blood for stratifying patient risk of pancreatic cancer

Abstract Introduction: Pancreatic cancer was responsible for almost 500,000 deaths globally in 2020 according to GLOBOCAN. Pancreatic cystic lesions (PCLs) are fluid-filled protrusions either on or inside the pancreas and can either be benign or pre-malignant. Current clinical guidelines to risk stratify PCL patients are imperfect. Multi-omic profiling of pancreatic cyst fluid (PCF) could aid in the identification of a novel biomarker panel to improve PCL risk stratification. Methods: PCF was collected from 40 patients by EUS-FNA, with matched serum collected prior to EUS. Patients were stratified using the 2018 European evidence-based guidelines into low-risk (n=15), high risk (n=15) and no-risk or pseudocyst (n=10). PCF was sonicated and subsequently processed using an SP3 paramagnetic bead protocol prior to LC-MS. MS-generated LFQ intensity data were analyzed in Perseus (v1.6.13.0) and STRING (v11.5). HTG microRNA whole transcriptome sequencing was run on whole PCF. MiRNA sequencing data were analyzed using HTG EdgeSeq Reveal (v3.1.0). Spearman correlations were generated using R packages ‘Hmisc’ (v4.5-0) and ‘corrplot’ (v0.90). Results: MS-analysis of PCF revealed 8 proteins to be significantly upregulated in high-risk compared to low-risk (p<0.05, FDR=0.05, s0=0.1). All 8 proteins had significantly positive correlations with patient risk and expression of the other seven. LCN-2, REG1A, LGALS3, PIGR and S100A8 have been shown to be elevated in the blood of pancreatic cancer patients. PRSS8 is known to be elevated in the serum of ovarian cancer patients, while MUC6 and TCN1 have not been shown to be differentially expressed in the circulation of cancer patients. STRING analysis revealed 11.8% and 6.8% of the proteins identified to be involved in the innate and adaptive immune responses, respectively. Significant positive correlations were found between 11 immune-associated proteins and patient risk classification (p<0.05). Whole transcriptome sequencing revealed 3 miRNA (miR-216a-5p, miR-216b-5p and HK_SKORA66) to be significantly upregulated in high-risk PCF compared to low-risk, and 5 miRNA (miR-197-5p, miR-6741-5p, miR-3180-3p, miR-3180 and miR-6782-5p) to be significantly upregulated in matched high-risk serum compared to low-risk (adj-p<0.05, FDR=0.05, s0=0.1). Unsupervised hierarchical clustering of patients using the 8 differentially expressed proteins and 3 miRNA from the PCF gave a clustering accuracy of 95.8%, with just 1/24 patients being misclassified. Conclusion: We have identified a putative multi-omic biomarker panel for PCL patient risk stratification. Practically, further refinement of this panel through the inclusion of additional biological compartments is required. These data will be validated in a larger patient cohort, with the aim of generating a less invasive blood-based panel that will aid in the improvement of risk stratification. Citation Format: Laura E. Kane, Gregory S. Mellotte, Rebecca G. Lyons, Eimear Mylod, Simone Marcone, Paul F. Ridway, Finbar MacCarthy, Kevin C. Conlon, Joanne Lysaght, Barbara M. Ryan, Stephen G. Maher. Establishment of a novel multi-omic biomarker panel in cyst fluid and blood for stratifying patient risk of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3381.

Modulation of Early Neutrophil Granulation: The Circulating Tumor Cell-Extravesicular Connection in Pancreatic Ductal Adenocarcinoma.

Simple SummaryCirculating tumor cells (CTCs) found in the blood of pancreatic cancer patients show a worse prognosis to therapy if they are seen in clusters of cells with neutrophils or platelets or with other cell types than when they are seen as singlets. We wanted to investigate if there is a secondary mode of communication between the CTCs and neutrophils that causes them to associate. We describe for the first time an extravesicular (EV) mediated communication between CTCs and neutrophils that modulates early transcriptome changes that can cause neutrophils to partially degranulate and form associations. We also identify the protein cargo carried in such EVs and how when added to naïve neutrophils, they can modulate transcriptomic changes in neutrophils partially disarming them to form clusters rather than undergo specialized cell death, which is characterized by release of condensed chromatin (NETs) and granular contents termed as NETosis.Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tumor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tissue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on companion cells in clusters.

Peripheral natural killer cell activity is associated with poor clinical outcomes in pancreatic ductal adenocarcinoma.

We aimed to measure the natural killer (NK) cell activity and pro-inflammatory cytokine levels in the peripheral blood of pancreatic cancer patients and investigate the correlation of NK cell activity and cytokines with cancer status and clinical outcomes. We prospectively enrolled patients who were pathologically diagnosed with pancreatic ductal adenocarcinoma (PDAC) between 2016 and 2017 at a tertiary hospital in Seoul, South Korea. As a control group, healthy participants were enrolled by mobile application recruitment. A total of 203 patients were enrolled for this study (PDAC, n=102; healthy participants, n=101). The peripheral blood NK cell activity of PDAC patients was significantly lower than that of healthy participants (median level, 95pg/mL vs 2000pg/mL, P<0.001), and decreased NK cell activity was correlated to poor clinical outcome in terms of response to chemotherapy, tumor progression, and survival. The pro-inflammatory cytokine interleukin-6 had a strong negative correlation with NK cell activity. In pancreatic cancer patients, NK cell activity decreased as cancer progressed, and decreased NK cell activity was associated with poor clinical outcomes.

Detection of circulating tumor cells in blood of pancreatic ductal adenocarcinoma patients.

Aim: Previous studies suggest that circulating tumor cells (CTC) are present at very low frequencies in blood of pancreatic cancer (PC) patients. However, no technique has proven efficient for their detection, in part due to the lack of accurate tumor markers. Here, we evaluated the potential utility of two marker candidates - Mucin 16 (MUC16) and Tetraspanin 1 (TSPAN1) - identified through a detailed review of the literature.Methods: To evaluate the pattern of expression of both markers in pancreatic tumor cells vs. normal blood, we used cell lines derived from pancreatic cancer patients and blood from healthy adults.Results: Antibodies against both MUC16 and TSPAN1 showed expression in three pancreatic cancer (PC) cell lines while they were absent in blood cells. To evaluate the efficiency of isolating tumor cells from blood, PC cell lines were spiked at different frequencies in blood, sequentially stained with biotin-conjugated TSPAN1 and MUC16 antibodies and a streptavidin ferrofluids, followed by immunomagnetic enrichment. The recovery of spiked TSPAN1+ tumor cells was high with limited contamination by leukocytes. In contrast, no PC cells were isolated when the biotin MUC16 reagent was used because the biotin-conjugated clone did not recognize PC cells.Conclusion: The combination of MUC16, TSPAN1, and epithelial cell adhesion molecule (EpCAM) antibodies will likely increase the efficiency of capturing circulating tumor cell in blood of pancreatic ductal adenocarcinoma. To further develop a protocol for isolation of circulating tumor cell in blood of PC patients, high amounts of antibodies (5-10 mg) against EpCAM, MUC16, and TSPAN1 will be needed.

Abstract 3963: Deep serial analysis of the TCR/BCR CDR3 adaptome repertoire in peripheral blood of pancreatic cancer patients in a randomized study of neoadjuvant chemotherapy with or without autophagy inhibition

Abstract Pancreatic cancer has a paucity of dendritic cells and almost no T lymphocytes. Here, we used Next Generation Sequencing following dimer avoidance multiplexed PCR (DAM-PCR) immune repertoire to study the characteristics and diversity of the TCR repertoire among PDAC (Pancreatic Ductal Adenocarcinoma) patients who underwent chemotherapy with or without Hydroxycholoroquine followed by surgical extirpation. We sequenced the TCR α,β,γ, and δ chains and the B-cell receptor (BCR) heavy chain (IgH) complementary-determining region 3 (CDR3) repertoire in peripheral blood mononuclear cells (PBMCs) from 10 PDAC patients before (Pre-chemo) and after chemotherapy (Post-chemo) and following operation (Postop). The distribution of CDR3, VD indel, and DJ indel lengths were similar among the Prechemo, Postchemo and Postop groups. The TCR repertoire diversity of PDAC patients was much lower compared to healthy donors (HD). The diversity index (D50) of TCR α and β’s in PDAC patients who received HCQ during chemotherapy was less than in patients without HCQ (p&amp;lt;.05). TRBV expression increased and some public sequences diminished at individual time points following chemotherapy and postop. Full analysis of an ongoing study will be presented. Citation Format: Yue Wang, Michael T. Lotze. Deep serial analysis of the TCR/BCR CDR3 adaptome repertoire in peripheral blood of pancreatic cancer patients in a randomized study of neoadjuvant chemotherapy with or without autophagy inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3963.

Prognostic significance of plasma matrix metalloprotease-2 in pancreatic cancer patients.

Background & objectives:Pancreatic cancer has a propensity for wide stromal invasion. Matrix metalloprotease-2 (MMP-2) is a protease that degrades the peri-tumoural tissue and helps in tumour dissemination. Thus, this study was aimed to assess any association of plasma MMP-2 levels with clinicopathological parameters and survival of patients with pancreatic cancer.Methods:Plasma samples from 127 pancreatic cancer patients were analyzed for MMP-2 levels by ELISA. Survival and other clinicopathological parameters of patients were analyzed for any correlation with plasma MMP-2 levels.Results:The mean MMP-2 levels in pancreatic cancer patients were 560.3±222.0 ng/ml which were significantly elevated compared to chronic pancreatitis patients (P<0.001) and healthy individuals (P<0.05). The plasma levels of MMP-2 significantly correlated with tissue expression of this protease (P=0.004). However, MMP-2 levels did not exhibit any association either with clinicopathological parameters or with survival.Interpretation & conclusions:Elevated MMP-2 levels were observed in blood of pancreatic cancer patients which correlated with its tissue expression. However, these levels did not associate with survival or any clinicopathological parameters of patients. Further studies need to be done to confirm the prognostic/clinical significance of MMP-2 in cancer patients before and after surgery.

Prevalence stratification of malignancy in resected intraductal papillary mucinous neoplasms (IPMN) involving main duct: is the 10 mm Wirsung diameter an adequate cutoff?

The detection of cancer-specific DNA in peripheral blood, known as a liquid biopsy, has been reported recently. Most such studies have used plasma as a sample; however, whether or not serum can be used as effectively is unclear. We attempted to clarify suitable samples for detecting KRAS mutations in circulating DNA in the blood of pancreatic cancer patients using droplet digital polymerase chain reaction (PCR).DNA was extracted from the tissue, plasma, and serum of 40 pancreatic cancer patients. The presence of KRAS mutations G12D, G12V, and G12R was analyzed by droplet digital PCR.The amount of DNA isolated from the serum was much higher than that from plasma (1.0- to 42.0-fold). At least 1 KRAS mutation was observed in 93% of cancer tissues, whereas we detected the mutations in only 48% of the serum and plasma DNA samples. The G12D mutation was the most prevalent of the three mutations, followed by the G12V mutation. The presence of the G12D KRAS mutation in the plasma, serum, or tissue did not correlate to the overall survival; however, the prognosis of the patients with a KRAS mutation at G12V in the plasma or serum was significantly poorer than that of the patients without the mutation (P < 0.01).Serum and plasma were found to be good materials for detecting cancer-specific DNA in the peripheral blood and the presence of KRAS mutations in blood-derived DNA may be used as a prognostic biomarker for patients with pancreatic cancer.

Circulating and disseminated tumor cells in pancreatic cancer and their role in patient prognosis: a systematic review and meta-analysis.

BackgroundDisseminated tumor cells (DTCs) and circulating tumor cells (CTCs) have been postulated to seed metastases and contribute to poorer patient outcomes in many types of solid cancer. To date, no systematic reviews have examined the role of both DTCs and CTCs in pancreatic cancer. We aimed to determine the prognostic value of DTCs/CTCs in pancreatic cancer using a systematic review and meta-analysis.Materials and MethodsA comprehensive literature search identified studies examining DTCs and CTCs in the bone marrow and blood of pancreatic cancer patients at diagnosis with follow-up to determine disease-free/progression-free survival (DFS/PFS) and overall survival (OS). Statistical analyses were performed to determine the hazard ratio (HR) of DTCs/CTCs on DFS/PFS and OS.ResultsThe literature search identified 16 articles meeting the inclusion criteria. The meta-analysis demonstrated statistically significant HR differences in DFS/PFS (HR = 1.93, 95% CI 1.19–3.11, P = 0.007) and OS (HR = 1.84, 95% CI 1.37–2.45, P =< 0.0001), indicating patients with detectable DTCs/CTCs at diagnosis have worse prognoses. Subgroup analyses suggested CTCs in the peripheral blood (HR =2.03) were more indicative of poor OS prognosis than DTCs in the bone marrow (HR = 1.91), although the difference between these was not statistically significant. Positivity of the CellSearch detection method for DTC/CTC had the highest correlation with decreased OS (HR = 2.79) while immunodetection (HR = 1.91) and RT-PCR (HR = 1.25) were less effective in determining prognosis.ConclusionThe detection of DTCs/CTCs at diagnosis is associated with poorer DFS/PFS and OS in pancreatic cancer.

MicroRNA-186 affects the proliferation of tumor cells via yes-associated protein 1 in the occurrence and development of pancreatic cancer.

The present study aimed to determine the expression of microRNA (miRNA or miR)-186 in tumor tissues and peripheral blood of patients with pancreatic cancer (PC), as well as its mechanism of regulation. A total of 65 patients with PC who underwent surgery between June 2013 and October 2015 were included. In addition, 59 healthy subjects were recruited as controls. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of mRNA and miRNA. Western blotting and enzyme-linked immunosorbent assay were used to determine protein expression. Bioinformatics was employed for the prediction of the target gene of miR-186, whereas dual luciferase reporter assay was performed to identify whether miR-186 directly bound to YAP1 mRNA. Human pulmonary aortic endothelial cells (HPACs) were transfected with ago-miR-186. YAP1 expression in HPACs was silenced by siRNA. MTT assay was used to evaluate the viability of HPACs. YAP1 mRNA and protein expression levels were elevated in PC. In addition, expression levels of miR-186 in PC were downregulated. miR-186 regulated the expression of YAP1 by binding with the 3′-untranslated region of YAP1. Elevated expression of miR-186 inhibited the proliferation of HPACs by downregulating the expression of YAP1. Decreased expression of YAP1 by siRNA reduced the viability of HPACs. The present study demonstrates that YAP1 is upregulated in the tumor tissues and blood of PC patients, and this may be associated with the downregulation of miR-186. In addition, miR-186 may affect the occurrence and development of PC by controlling the proliferation of PC cells via YAP1.

REG3\u03b2 modifies cell tumor function by impairing extracellular vesicle uptake

Extracellular vesicles (EVs), including exosomes and microvesicles, are nano-sized membrane vesicles containing proteins and nucleic acids, which act as intercellular messengers. They play an important role in a variety of physiological processes, as well as in pathological situations such as inflammation or cancer. Here, we show that in the case of pancreatic ductal adenocarcinoma (PDAC), the healthy pancreatic tissue surrounding the tumor releases REG3β, a lectin that binds to the glycoproteins present in the surface of EVs, thus interfering with their uptake and internalization by target cells. In vitro, the disruption of the signaling mediated by EVs due to the presence of REG3β, prevents the EV-induced phenotypic switch in macrophages, inhibits the increased cell migration of cancer cells and reverses a number of metabolomic changes promoted by EVs. In vivo, the uptake of REG3β+ EVs by tumor cells is significantly impaired. Furthermore, it results in an increase of circulating REG3β+ EVs in blood of pancreatic cancer patients. Our findings highlight the effect of a lectin released by the healthy pancreatic tissue surrounding the tumor in modulating the EV-mediated interactions between different cell types in PDAC.

Clinical significance of monitoring KRAS in the blood of pancreatic cancer patients.

e23042 Background: KRAS monitoring provides valuable information for early diagnosis and prediction of treatment outcome in colorectal cancer. KRAS mutation is observed in only half of colon cancer patients, whereas it is detected in 90% of pancreatic cancer patients. Therefore, investigating tumor DNA in blood by KRAS monitoring may be even more valuable in pancreatic cancer patients. In this study, we elucidated the clinical significance of KRAS monitoring in pancreatic cancer patients during treatment. Methods: KRAS in tumor tissues was analyzed for mutations by Scorpion ARMS or RASKET in 38 patients with pancreatic tumors. KRAS in blood was analyzed for mutations (G12D, G12V, G12C, G12A, G12S, G12R, G13D, Q61L, and Q61H) using droplet digital polymerase chain reaction in 23 patients who underwent chemotherapy and who had KRAS mutation in their tissues. Results: KRAS mutation in tumor tissues was detected in 27 of 38 patients (71.1%). These 27 patients showed significantly poorer prognosis (24.4 months median overall survival time [MST]) than the six patients without mutation (p = 0.04), who were all alive. Patients with mutation of 12D in tumor tissues showed better prognosis than patients with other types of mutation; thus, mutation of 12D may have a different biological effect to other mutations. Monitoring of KRAS in blood revealed KRAS mutation in 14 of 23 patients (60.9%). These 14 patients exhibited significantly poorer treatment outcomes than patients without mutation. The MST of patients with KRAS mutation in their blood was significantly shorter than that of patients without KRAS mutation (17.7 months vs. 44.3 months, p = 0.03). Conclusions: KRAS mutation in blood could be a valuable predictive and prognostic biomarker in pancreatic cancer patients who undergo chemotherapy. Additionally, assessment of KRAS in tumor tissues may provide information about individual tumor biology.

A Novel Microfluidic Device for Isolation of Circulating Tumor Cells from Pancreatic Cancer Blood Samples.

Enumeration of circulating tumor cells (CTCs) can provide valuable prognostic information to guide cancer treatment as well as help monitor disease progression. Analysis of these rare malignant cells has the potential to further our understanding of cancer metastasis by gaining insights into CTC characteristics and properties. Microfluidics presents a unique platform to isolate and study CTCs. In this chapter, we describe the detailed procedures for the fabrication and use of a microfluidic device to detect CTCs from the blood of pancreatic cancer patients.

Utility of serum DNA as a marker for KRAS mutations in pancreatic cancer tissue

Background/ObjectivesThe detection of cancer-specific DNA in peripheral blood, known as a liquid biopsy, has been reported recently. Most such studies have used plasma as a sample; however, whether or not serum can be used as effectively is unclear. We attempted to clarify suitable samples for detecting KRAS mutations in circulating DNA in the blood of pancreatic cancer patients using droplet digital polymerase chain reaction (PCR). MethodsDNA was extracted from the tissue, plasma, and serum of 40 pancreatic cancer patients. The presence of KRAS mutations G12D, G12V, and G12R was analyzed by droplet digital PCR. ResultsThe amount of DNA isolated from the serum was much higher than that from plasma (1.0- to 42.0-fold). At least 1 KRAS mutation was observed in 93% of cancer tissues, whereas we detected the mutations in only 48% of the serum and plasma DNA samples. The G12D mutation was the most prevalent of the three mutations, followed by the G12V mutation. The presence of the G12D KRAS mutation in the plasma, serum, or tissue did not correlate to the overall survival; however, the prognosis of the patients with a KRAS mutation at G12V in the plasma or serum was significantly poorer than that of the patients without the mutation (P < 0.01). ConclusionsSerum and plasma were found to be good materials for detecting cancer-specific DNA in the peripheral blood and the presence of KRAS mutations in blood-derived DNA may be used as a prognostic biomarker for patients with pancreatic cancer.

Abstract 1595: Analysis of circulating epithelial and EMT-like CTCs in pancreatic cancer using a sensitive microfluidic CTC capture device

Abstract Background &amp; Aims: A critical factor in the poor outcomes in pancreatic cancer is due to it's silent nature until late in the disease process, so early detection has remained a major issue in this type of cancer. Detection, quantification, and molecular characterization of Epithelial circulating tumor cells (CTCs) as well as Epithelial-to-Mesenchymal Transition (EMT)-like CTCs in peripheral blood have been recognized as a means of “liquid biopsy” technique in the diagnosis of cancer and metastasis. Methods: A modified version of CTC chip, CTC Carpet chip was used to investigate the inextricable relationship between circulating epithelial and EMT-like CTCs in 40 pancreatic cancer patients at both protein and RNA levels. Results: The recovery from whole blood spiked with different cancer cell line was validated, achieving a mean efficiency of ∼100% for both HT-29 and Panc-1 cell lines compared to &amp;lt;80% for PC-3 cell line. Out of 40 patients, 38 patients had ≥5 CK+ CTCs with a mean of 21.84 per mL and all 40 samples had ≥ 15 Vim+ CTCs with mean of 89.77 per mL of whole blood. Up to 67 CK+ epithelial and 257 Vim+ EMT like CTCs were detected in these set of samples. The results indicated the significant differences between the numbers of Epithelial CTCs vs. EMT-like CTCs in both stage I-II and IV of these 40 pancreatic cancer patients’ samples. Conclusion: Although the isolation of CTCs from pancretic cancer is possible nowadays, investigation of their clinical utility has proven less successful than the other cancer types, due to poor sensitivity of many CTC assays for patients with PDAC. Herein, we present an integrated microfluidic technology- and biology-based translational approach using bioengineering tools to identify and study the biological relevance of rare CTCs including both circulating epithelial and EMT-like tumor cells simultaneously from the peripheral blood of pancreatic cancer patients by using multiple markers of interest. Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathius Hafner, Shimeng Shao, Ebrahim Azizi, Max S. Wicha, Kyle Cuneo, Diane M. Simeone, Sunitha Nagrath. Analysis of circulating epithelial and EMT-like CTCs in pancreatic cancer using a sensitive microfluidic CTC capture device. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2015-1595

SELEX aptamer used as a probe to detect circulating tumor cells in peripheral blood of pancreatic cancer patients.

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15+/CD45- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.

Players of epithelial-to-mesenchymal transition (EMT) are expressed in the peri-tumoral stroma and in blood of pancreatic cancer patients.

Giuseppe Celesti , Andrea Gallioli , Matteo Tacelli , Francesca Gavazzi , Paola Spaggiari , Francesco Novelli , Massimo Roncalli , Alessandro Zerbi , Alberto Malesci , Luigi Laghi i Humanitas Research Hospital, Laboratory of Molecular Gastroenterology, Italy Humanitas Research Hospital, Department of General Surgery, Laboratory of Molecular Gastroenterology, University of Milan, Italy Humanitas Research Hospital, Laboratory of Molecular Gastroenterology, University of Milan, Italy Humanitas Research Hospital, Department of General Surgery, Italy Humanitas Research Hospital, Department of Pathology, Italy f CRMS Azienda San Giovanni Battista, University of Torino, Department of Medicine and Experimental Oncology, Italy Humanitas Research Hospital, Department of Pathology, University of Milan, Italy Humanitas Research Hospital, Department of Gastroenterology, University of Milan, Italy i Humanitas Research Hospital, Department of Gastroenterology, Laboratory of Molecular Gastroenterology, Italy

Potentials of Plasma NGAL and MIC-1 as Biomarker(s) in the Diagnosis of Lethal Pancreatic Cancer

Pancreatic cancer (PC) is lethal malignancy with very high mortality rate. Absence of sensitive and specific marker(s) is one of the major factors for poor prognosis of PC patients. In pilot studies using small set of patients, secreted acute phase proteins neutrophil gelatinase associated lipocalin (NGAL) and TGF-β family member macrophage inhibitory cytokine-1 (MIC-1) are proposed as most potential biomarkers specifically elevated in the blood of PC patients. However, their performance as diagnostic markers for PC, particularly in pre-treatment patients, remains unknown. In order to evaluate the diagnostic efficacy of NGAL and MIC-1, their levels were measured in plasma samples from patients with pre-treatment PC patients (n = 91) and compared it with those in healthy control (HC) individuals (n = 24) and patients with chronic pancreatitis (CP, n = 23). The diagnostic performance of these two proteins was further compared with that of CA19-9, a tumor marker commonly used to follow PC progression. The levels of all three biomarkers were significantly higher in PC compared to HCs. The mean (± standard deviation, SD) plasma NGAL, CA19-9 and MIC-1 levels in PC patients was 111.1 ng/mL (2.2), 219.2 U/mL (7.8) and 4.5 ng/mL (4.1), respectively. In comparing resectable PC to healthy patients, all three biomarkers were found to have comparable sensitivities (between 64%-81%) but CA19-9 and NGAL had a higher specificity (92% and 88%, respectively). For distinguishing resectable PC from CP patients, CA19-9 and MIC-1 were most specific (74% and 78% respectively). CA19-9 at an optimal cut-off of 54.1 U/ml is highly specific in differentiating resectable (stage 1/2) pancreatic cancer patients from controls in comparison to its clinical cut-off (37.1 U/ml). Notably, the addition of MIC-1 to CA19-9 significantly improved the ability to distinguish resectable PC cases from CP (p = 0.029). Overall, MIC-1 in combination with CA19-9 improved the diagnostic accuracy of differentiating PC from CP and HCs.

Multimarker Gene Analysis of Circulating Tumor Cells in Pancreatic Cancer Patients: A Feasibility Study

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients.