Oncogenic pathway signatures predict the risk of progression and recurrence in well-differentiated pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (pNETs) are genomically diverse tumors. The management of newly diagnosed well-differentiated pNETs is limited by a lack of sensitivity of existing biomarkers for prognostication. Our goal was to investigate the potential utility of genetic markers as a predictor of progression-free survival (PFS) and recurrence-free survival (RFS). METHODS: Whole-exome sequencing of resected well-differentiated, low and intermediate-grade (G1 and G2) pNETs and normal adjacent tissue from patients who underwent resection from 2005 to 2015 was performed. Genetic alterations were classified using pan-genomic and oncogenic pathway classifications. Additional samples with genetic and clinicopathologic data available were obtained from the publicly available International Cancer Genome Consortium (ICGC) database and included in the analysis. The prognostic relevance of these genomic signatures on PFS and RFS was analyzed. RESULTS: Thirty-one patients who underwent resection for pNET were identified. Genomic analysis of mutational, copy number, cytogenetic, and complex phenomena revealed similar patterns to prior studies of pNETs with relatively few somatic gene mutations but numerous instances of copy number changes. Analysis of genomic and clinicopathologic outcomes using the combined data from our study as well as the ICGC pNET cohort (n = 124 patients) revealed that the recurrent pattern of whole chromosome loss (RPCL) and metastatic disease were independently associated with disease progression. When evaluating patients with local disease at the time of resection, RPCL and alterations in the TGFß oncogenic pathway were independently associated with the risk of recurrence. CONCLUSIONS: Well-differentiated pNETs are genomically diverse tumors. Pathway signatures may be prognostic for predicting disease progression and recurrence.

Patient and hospital factors influence surgical approach in treatment of acute cholecystitis.

BACKGROUND: Minimally invasive (MIS) cholecystectomies have become standard due to patient and hospital advantages; however, this approach is not always achievable. Acute and gangrenous cholecystitis increase the likelihood of conversion from MIS to open cholecystectomy. This study aims to examine patient and hospital factors underlying differential utilization of MIS vs open cholecystectomies indicated for acute cholecystitis. METHODS: This is a retrospective, observational cohort study of patients with acute cholecystitis who underwent a cholecystectomy between 2016 and 2018 identified from the California Office of Statewide Health Planning and Development database. Univariate analysis and multivariable logistic regression models were used to analyze patient, geographic, and hospital variables as well as surgical approach. RESULTS: Our total cohort included 53,503 patients of which 98.4% (n = 52,673) underwent an initial minimally invasive approach and with a conversion rate of 3.3% (n = 1,759). On multivariable analysis advancing age increased the likelihood of either primary open (age 40 to < 65 aOR 2.17; ≥ 65 aOR 3.00) or conversion to open cholecystectomy (age 40 to < 65 aOR 2.20; ≥ 65 aOR 3.15). Similarly, male sex had higher odds of either primary open (aOR 1.70) or conversion to open cholecystectomy (aOR 1.84). Hospital characteristics increasing the likelihood of either primary open or conversion to open cholecystectomy included teaching hospitals (aOR 1.37 and 1.28, respectively) and safety-net hospitals (aOR 1.46 and 1.33, respectively). CONCLUSIONS: With respect to cholecystectomy, it is well-established that a minimally invasive surgical approach is associated with superior patient outcomes. Our study focused on the diagnosis of acute cholecystitis and identified increasing age as well as male sex as significant factors associated with open surgery. Teaching and safety-net hospital status were also associated with differential utilization of open, conversion-to-open, and MIS. These findings suggest the potential to create and apply strategies to further minimize open surgery in the setting of acute cholecystitis.

Robotic versus open pancreaticoduodenectomy in elderly patients: a propensity score-matched analysis.

BACKGROUND: Pancreaticoduodenectomy (PD) is complex procedure with high morbidity in the elderly. This retrospective study aimed to compare post-operative outcomes in patients ≥75 years of age who underwent robot-assisted (RA)PD and open PD. METHODS: We analyzed 2502 patients ≥75 years of age who underwent PD from 2015 to 2018 in the National Surgical Quality Improvement Program (NSQIP) database. RAPD and open PD patients were propensity score matched 1:5 to assess the 30-day outcomes of interest: postoperative complications, length of stay, discharge destination, and readmissions. RESULTS: Of 725 matched patients, 110 underwent RAPD, 615 OPD, and 12 were converted to an open operation. Post-operative outcomes were largely similar between cohorts. RAPD was associated a shorter length of stay (median 8 days, interquartile range [IQR] 6 to 11) than OPD (median 8 days, IQR 7 to 13) (p = 0.003). However, RAPD was associated with more readmissions (28.1% vs. 17.7%; p = 0.02). CONCLUSIONS: RAPD in patients ≥75 years of age appears to be safe and has a similar complication profile to open PD. Randomized or well-designed prospective matched studies are needed to confirm these findings.

Coupling Nanostructured Microchips with Covalent Chemistry Enables Purification of Sarcoma-Derived Extracellular Vesicles for Downstream Functional Studies.

Tumor-derived extracellular vesicles (EVs) play essential roles in intercellular communication during tumor growth and metastatic evolution. Currently, little is known about the possible roles of tumor-derived EVs in sarcoma because the lack of specific surface markers makes it technically challenging to purify sarcoma-derived EVs. In this study, a specific purification system is developed for Ewing sarcoma (ES)-derived EVs by coupling covalent chemistry-mediated EV capture/ release within a nanostructure-embedded microchip. The purification platform-ES-EV Click Chip-takes advantage of specific anti-LINGO-1 recognition and sensitive click chemistry-mediated EV capture, followed by disulfide cleavage-driven EV release. Since the device is capable of specific and efficient purification of intact ES EVs with high purity, ES-EV Click Chip is ideal for conducting downstream functional studies of ES EVs. Absolute quantification of the molecular hallmark of ES (i.e., EWS rearrangements) using reverse transcription Droplet Digital PCR enables specific quantification of ES EVs. The purified ES EVs can be internalized by recipient cells and transfer their mRNA cargoes, exhibiting their biological intactness and potential role as biological shuttles in intercellular communication.

Cancer Modeling-on-a-Chip with Future Artificial Intelligence Integration.

Cancer is one of the leading causes of death worldwide, despite the large efforts to improve the understanding of cancer biology and development of treatments. The attempts to improve cancer treatment are limited by the complexity of the local milieu in which cancer cells exist. The tumor microenvironment (TME) consists of a diverse population of tumor cells and stromal cells with immune constituents, microvasculature, extracellular matrix components, and gradients of oxygen, nutrients, and growth factors. The TME is not recapitulated in traditional models used in cancer investigation, limiting the translation of preliminary findings to clinical practice. Advances in 3D cell culture, tissue engineering, and microfluidics have led to the development of "cancer-on-a-chip" platforms that expand the ability to model the TME in vitro and allow for high-throughput analysis. The advances in the development of cancer-on-a-chip platforms, implications for drug development, challenges to leveraging this technology for improved cancer treatment, and future integration with artificial intelligence for improved predictive drug screening models are discussed.

A novel multimarker assay for the phenotypic profiling of circulating tumor cells in hepatocellular carcinoma.

Current clinicopathologic staging systems and serum biomarkers poorly discriminate tumor biology in hepatocellular carcinoma (HCC), with high recurrence rates following curative-intent surgical resection and liver transplantation (LT). Identification of accurate biomarkers for improved prognostication and treatment selection is a critical unmet need. We sought to develop a novel "liquid-biopsy" assay capable of detecting HCC circulating tumor cells (CTCs) and characterizing phenotypic subpopulations with prognostic significance. Using HCC cell lines, a tissue microarray, and human blood samples, an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), glypican-3, and epithelial cell adhesion molecule was optimized for HCC CTC capture using the NanoVelcro CTC Assay. The ability of HCC CTCs and vimentin (VIM)-positive CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival (OS) was evaluated in a prospective study of 80 patients. Multimarker capture detected greater numbers of CTCs than any individual antibody alone for both cell line and patient samples (P < 0.001). HCC CTCs were identified in 59/61 (97%) patients, and HCC (median, 6 CTCs) and non-HCC patients (median, 1 CTC; area under the receiver operating characteristic curve [AUROC] = 0.92; P < 0.001; sensitivity = 84.2%; specificity = 88.5%) were accurately discriminated. VIM-positive CTCs accurately discriminated early-stage, LT eligible patients (median, 0 CTCs) from locally advanced/metastatic, LT ineligible patients (median, 6 CTCs; AUROC = 0.89; P = 0.001; sensitivity = 87.1%; specificity = 90.0%), and predicted OS for all patients (hazard ratio [HR], 2.21; P = 0.001), and faster recurrence after curative-intent surgical or locoregional therapy in potentially curable early-stage HCC (HR, 3.14; P = 0.002). In conclusion, we developed a novel multimarker CTC enrichment assay that detects HCC CTCs with high efficiency and accuracy. A phenotypic subpopulation of VIM-positive CTCs appears to signify the presence of aggressive underlying disease and occult metastases and may have important implications for treatment selection. Liver Transplantation 24 946-960 2018 AASLD.

Circulating Tumor Cells Predict Occult Metastatic Disease and Prognosis in Pancreatic Cancer.

BACKGROUND: Occult metastatic tumors, below imaging thresholds, are a limitation of staging systems that rely on cross-sectional imaging alone and are a cause of the routine understaging of pancreatic ductal adenocarcinomas (PDACs). We investigated circulating tumor cells (CTCs) as a preoperative predictor of occult metastatic disease and as a prognostic biomarker for PDAC patients. EXPERIMENTAL DESIGN: A total of 126 patients (100 with cancer, 26 with benign disease) were enrolled in our study and CTCs were identified and enumerated from 4 mL of venous blood using the microfluidic NanoVelcro assay. CTC enumeration was correlated with clinicopathologic variables and outcomes following both surgical and systemic therapies. RESULTS: CTCs were identified in 78% of PDAC patients and CTC counts correlated with increasing stage (ρ = 0.42, p < 0.001). Of the 53 patients taken for potentially curative surgery, 13 (24.5%) had occult metastatic disease intraoperatively. Patients with occult disease had significantly more CTCs than patients with local disease only (median 7 vs. 1 CTC, p < 0.0001). At a cut-off of three or more CTCs/4 mL, CTCs correctly identified patients with occult metastatic disease preoperatively (area under the receiver operating characteristic curve 0.82, 95% confidence interval (CI) 0.76-0.98, p < 0.0001). CTCs were a univariate predictor of recurrence-free survival following surgery [hazard ratio (HR) 2.36, 95% CI 1.17-4.78, p = 0.017], as well as an independent predictor of overall survival on multivariate analysis (HR 1.38, 95% CI 1.01-1.88, p = 0.040). CONCLUSIONS: CTCs show promise as a prognostic biomarker for PDAC patients at all stages of disease being treated both medically and surgically. Furthermore, CTCs demonstrate potential as a preoperative biomarker for identifying patients at high risk of occult metastatic disease.

Determination of hepatocellular carcinoma grade by needle biopsy is unreliable for liver transplant candidate selection.

The objective of this article is to evaluate the utility of preoperative needle biopsy (PNB) grading of hepatocellular carcinoma (HCC) as a biomarker for liver transplantation (LT) candidate selection. Given the prognostic significance of HCC tumor grade, PNB grading has been proposed as a biomarker for LT candidate selection. Clinicopathologic characteristics of HCC LT recipients (1989-2014) with a PNB were analyzed, and the concordance of PNB grade to explant grade and vascular invasion was assessed to determine whether incorporation of PNB grade to accepted transplant criteria improved candidate selection. Of 965 patients undergoing LT for HCC, 234 (24%) underwent PNB at a median of 280 days prior to transplant. Grade by PNB had poor concordance to final explant pathology (κ = 0.22; P = 0.003), and low sensitivity (29%) and positive predictive value (35%) in identifying poorly differentiated tumors. Vascular invasion was predicted by explant pathologic grade (rs= 0.24; P < 0.001) but not PNB grade (rs = -0.05; P = 0.50). Increasing explant pathology grade (P = 0.02), but not PNB grade (P = 0.65), discriminated post-LT HCC recurrence risk. The incorporation of PNB grade to the established radiologic Milan criteria (MC) did not result in improved prognostication of post-LT recurrence (net reclassification index [NRI] = 0%), whereas grade by explant pathology resulted in significantly improved reclassification of risk (NRI = 19%). Preoperative determination of HCC grade by PNB has low concordance with explant pathologic grade and low sensitivity and positive predictive value in identifying poorly differentiated tumors. PNB grade did not accurately discriminate post-LT HCC recurrence and had no utility in improving prognostication compared with the MC alone. Incorporation of PNB to guide transplant candidate selection appears unjustified. Liver Transplantation 23 1123-1132 2017 AASLD.

Precision oncology using a limited number of cells: optimization of whole genome amplification products for sequencing applications.

BACKGROUND: Sequencing analysis of circulating tumor cells (CTCs) enables "liquid biopsy" to guide precision oncology strategies. However, this requires low-template whole genome amplification (WGA) that is prone to errors and biases from uneven amplifications. Currently, quality control (QC) methods for WGA products, as well as the number of CTCs needed for reliable downstream sequencing, remain poorly defined. We sought to define strategies for selecting and generating optimal WGA products from low-template input as it relates to their potential applications in precision oncology strategies. METHODS: Single pancreatic cancer cells (HPAF-II) were isolated using laser microdissection. WGA was performed using multiple displacement amplification (MDA), multiple annealing and looping based amplification (MALBAC) and PicoPLEX. Quality of amplified DNA products were assessed using a multiplex/RT-qPCR based method that evaluates for 8-cancer related genes and QC-scores were assigned. We utilized this scoring system to assess the impact of de novo modifications to the WGA protocol. WGA products were subjected to Sanger sequencing, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) to evaluate their performances in respective downstream analyses providing validation of the QC-score. RESULTS: Single-cell WGA products exhibited a significant sample-to-sample variability in amplified DNA quality as assessed by our 8-gene QC assay. Single-cell WGA products that passed the pre-analysis QC had lower amplification bias and improved aCGH/NGS performance metrics when compared to single-cell WGA products that failed the QC. Increasing the number of cellular input resulted in improved QC-scores overall, but a resultant WGA product that consistently passed the QC step required a starting cellular input of at least 20-cells. Our modified-WGA protocol effectively reduced this number, achieving reproducible high-quality WGA products from ≥5-cells as a starting template. A starting cellular input of 5 to 10-cells amplified using the modified-WGA achieved aCGH and NGS results that closely matched that of unamplified, batch genomic DNA. CONCLUSION: The modified-WGA protocol coupled with the 8-gene QC serve as an effective strategy to enhance the quality of low-template WGA reactions. Furthermore, a threshold number of 5-10 cells are likely needed for a reliable WGA reaction and product with high fidelity to the original starting template.

Gender as a risk factor for adverse intraoperative and postoperative outcomes of elective pancreatectomy.

BACKGROUND AND OBJECTIVES: Patient selection remains paramount when developing and adopting quality-based assessment and reimbursement models, and enhanced recovery protocols. Gender is a patient characteristic known before surgery which can inform risk stratification. Our aim was to evaluate the effect of gender on intraoperative blood transfusions, operative time, length of hospital stay, estimated blood loss (EBL) as well as postoperative surgical site infections (SSIs), and mortality. METHODS: Patients undergoing elective pancreatectomy from 2005 to 2013 were identified in the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) and Northwestern institutional databases. Multivariable analyses were conducted to identify the association between gender and these outcomes. RESULTS: Analyses demonstrated that male gender was independently associated with blood transfusion (OR 1.23), operative time >6 hr (OR 1.76), length of stay greater than 11 days (OR 1.17), and all-type SSIs (OR 1.17), especially superficial SSIs (OR 1.15) and organ space SSIs (OR 1.18). Analysis of the institutional cohort found that male gender was independently associated with increased odds of EBL > 1 L for Whipple procedures (OR 2.85). CONCLUSIONS: Male gender is a significant predictor of increased operative time, length of stay, transfusions, EBL > 1L, as well as postoperative organ space surgical site infections in these patients. J. Surg. Oncol. 2017;115:131-136. © 2016 Wiley Periodicals, Inc.

A prognostic mutation panel for predicting cancer recurrence in stages II and III colorectal cancer.

BACKGROUND AND OBJECTIVES: Approximately 20-40% of stage II/III colorectal cancer (CRC) patients develop relapse. Clinicopathological factors alone are limited in detecting these patients, resulting in potential under/over-treatment. We sought to identify a prognostic tumor mutational profile that could predict CRC recurrence. METHODS: Whole-exome sequencing data were obtained for 207 patients with stage II/III CRC from The Cancer Genome Atlas. Mutational landscape in relapse-free versus relapsed cohort was compared using Fisher's exact test, followed by multivariate Cox regression to identify genes associated with cancer recurrence. Bootstrap-validation was used to examine internal/external validity. RESULTS: We identified five prognostic genes (APAF1, DIAPH2, NTNG1, USP7, and VAV2), which were combined to form a prognostic mutation panel. Patients with ≥1 mutation(s) within this five-gene panel had worse prognosis (3-yr relapse-free survival [RFS]: 53.0%), compared to patients with no mutation (3-yr RFS: 84.3%). In multivariate analysis, the five-gene panel remained prognostic for cancer recurrence independent of stage and high-risk features (hazard ratio 3.63, 95%CI [1.93-6.83], P < 0.0001). Furthermore, its prognostic accuracy was superior to the American Joint Commission on Cancer classification (concordance-index: 0.70 vs 0.54). CONCLUSIONS: Our proposed mutation panel identifies CRC patients at high-risk for recurrence, which may help guide adjuvant therapy and post-operative surveillance protocols.

Circulating Tumor Cells in Gastrointestinal Cancer: Current Practices and Future Directions.

GI cancers are the leading cause of cancer-related death worldwide primarily due to a combination of late presentation and aggressive biology. The lack of adequate biomarkers for screening, diagnosis, staging, and prognosis confounds clinical decision-making and delays potentially effective therapies. Circulating tumor cells (CTCs) are a new biomarker with particular promise in GI cancers, potentially offering clinicians and researchers real-time access to tumor tissue in a reliable, safe, and cost-effective manner. Preliminary studies have investigated the potential clinical utility of CTCs for all GI cancer types with promising results. Furthermore, advances in single cell analytics have been successfully applied to CTCs, allowing for exciting new clinical and research applications. In this chapter, we will review the current state of CTC research in GI cancers as well as the potential future applications that are currently being developed.

Pancreatic cancer patients with lymph node involvement by direct tumor extension have similar survival to those with node-negative disease.

BACKGROUND: Lymph node (LN) involvement is a well-known poor prognostic factor in patients with pancreatic ductal adenocarcinoma (PDAC). However, there have been conflicting results on the significance of the mechanism of LN involvement, "direct" tumor invasion versus "metastatic," disease on patient survival. METHODS: Clinicopathologic records from all patients who underwent resection for PDAC from 1990 to 2014 at a single-institution were reviewed. RESULTS: Of the 385 total patients, there was tumor invasion outside of the pancreas in 289 (75.1%) patients. Overall, 239 (62.1%) had node-positive disease: 220 (92.0%) by "metastatic" involvement, 14 (5.9%) by "direct" tumor extension, and five (2.1%) by a mix of "metastatic" and "direct". There were no significant differences in clinicopathologic factors associated with PDAC survival between "metastatic" and "direct" LN patients. The median overall survival for the whole cohort was 31.1 months. Compared to overall survival in patients with LN-negative disease (median 40.7 months), those with LNs involved by "metastatic" spread was significantly shorter (median 25.7 months, P < 0.001), yet "direct" LN extension was similar (median 48.1 months, P = 0.719). CONCLUSIONS: The mechanism of LN involvement affects PDAC prognosis. Patients with LNs involved by direct extension have similar survival to those with node-negative disease.

Metabolic exploitation of the sialic acid biosynthetic pathway to generate site-specifically labeled antibodies.

Lack of a universal site-specific conjugation methodology for antibodies limits their potential to be developed as tumor-specific imaging agents or targeted therapeutics. A potential mechanism for site-specific conjugation involves utilization of the conserved N-glycosylation site in the CH2 domain. We sought to develop an antibody with an altered azido-sugar at this site whereby site-specific label could be added. The HB8059 hybridoma was cultured with peracetylated N-azidoacetlymannosamine (Ac4ManNAz). The resulting azido-sugar antibody was conjugated to phosphine-polyethylene glycol (PEG3)-biotin via a modified Staudinger reaction. Biochemical and functional characterization of the biotinylated antibody was performed. The azido-sugar antibody was also labeled with DyLight-650-Phosphine and injected into mice harboring pancreatic cancer xenografts. The tumors were dissected and imaged utilizing an IVIS fluorescent camera. The antibody was successfully produced in 100 µM Ac4ManNAz. The biotinylated antibody demonstrated a 50 kDa heavy and 25 kDa light chain on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but demonstrated a single band at 50 kDa on western blot. Treatment with a N-linked glycosidase extinguished the band. Flow cytometry demonstrated antigen-specific binding of CA19-9-positive cells and the antibody localized to the antigen-positive tumor in vivo. We successfully produced an antibody with an azido-sugar at the conserved CH2 glycosylation site. We were able to utilize this azide to label the antibody with biotin or fluorescent label and demonstrate that the label is added in a site-specific manner to the heavy chain, N-linked glycosylation site. Finally, we demonstrated functionality of our antibody for in vitro and in vivo targeting of pancreatic cancer cells.

Postoperative complications reduce adjuvant chemotherapy use in resectable pancreatic cancer.

OBJECTIVE: To assess the impact of postoperative complications on the receipt of adjuvant chemotherapy. BACKGROUND: Randomized trials have demonstrated that adjuvant chemotherapy is associated with improved long-term survival. However, pancreatic surgery is associated with significant morbidity and the degree to which complications limit subsequent treatment options is unknown. METHODS: Patients from the American College of Surgeons National Surgical Quality Improvement Program and the National Cancer Data Base who underwent pancreatic resection for cancer were linked (2006-2009). The associations between complications and adjuvant chemotherapy use or treatment delay (≥ 70 days from surgery) were assessed using multivariable regression methods. RESULTS: From 149 hospitals, 2047 patients underwent resection for stage I-III pancreatic adenocarcinoma of which 23.2% had at least 1 serious complication. Overall adjuvant chemotherapy receipt was 57.7%: 61.8% among patients not experiencing any complication and 43.6% among those who had a serious complication. Serious complications increased the likelihood of not receiving adjuvant therapy over twofold [odds ratio (OR) = 2.20, 95% confidence interval (CI): 1.73-2.80]. Specific complications associated with adjuvant chemotherapy omission were reintubation (OR = 7.79, 95% CI: 3.59-16.87), prolonged ventilation (OR = 5.92, 95% CI: 3.23-10.86), pneumonia (OR = 2.83, 95% CI: 1.63-4.90), sepsis/shock (OR = 2.76, 95% CI: 2.02-3.76), organ space/deep surgical site infection (OR = 2.19, 95% CI: 1.53-3.13), venous thromboembolism (OR = 1.92, 95% CI: 1.08-3.43), and urinary tract infection (OR = 1.61, 95% CI: 1.02-2.54). Serious complications also doubled the likelihood of delaying adjuvant treatment administration (OR = 2.08, 95% CI: 1.42-3.05). Sensitivity analysis in a younger, healthier patient cohort demonstrated similar associations. CONCLUSIONS: Postoperative complications are common following pancreatic surgery and are associated with adjuvant chemotherapy omission and treatment delays.

Impact of tumor grade on pancreatic cancer prognosis: validation of a novel TNMG staging system.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) patients demonstrate highly variable survival within each stage of the American Joint Committee on Cancer (AJCC) staging system. We hypothesize that tumor grade is partly responsible for this variation. Recently our group developed a novel tumor, node, metastasis, grade (TNMG) classification system utilizing Surveillance Epidemiology and End Results (SEER) data in which the presence of high tumor grade results in advancement to the next higher AJCC stage. This study's objective was to validate this TNMG staging system utilizing single-institution data. METHODS: All patients with PDAC who underwent resection at UCLA between 1990 and 2009 were identified. Clinicopathologic data reviewed included age, sex, node status, tumor size, grade, and stage. Grade was redefined as a dichotomous variable. The impact of grade on survival was assessed by Cox regression analysis. Disease was restaged into the TNMG system and compared to the AJCC staging system. RESULTS: We identified 256 patients who underwent resection for PDAC. Patients with low-grade tumors experienced a 13-month improvement in median survival compared to those with high-grade tumors. On multivariate analysis, tumor grade was the strongest predictor of survival with a hazard ratio of 2.02 (p = 0.0005). Restaging disease according to the novel TNMG staging system resulted in improved survival discrimination between stages compared to the current AJCC system. CONCLUSIONS: We were able to demonstrate that grade is one of the strongest independent prognostic factors in PDAC. Restaging with our novel TNMG system demonstrated improved prognostication. This system offers an effective and convenient way of adding grade to the current AJCC staging system.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles.

BACKGROUND: Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer. METHODS: The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images. RESULTS: Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro. CONCLUSIONS: Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.

Circulating Fatty Objects and Their Preferential Presence in Pancreatic Cancer Patient Blood Samples.

Human cancers are often complicated with increased incidences of blood vessel occlusion, which are mostly insensitive to anticoagulation therapy. We searched for causal factors of cancer-associated embolism. A total of 2,017 blood samples was examined for visible abnormalities. Examined were peripheral blood samples from cancer patients who were about to undergo surgical treatment for genitourinary, breast, gastrointestinal or abdominal tumors. Samples from ambulatory patients being treated for recurrent or castration-resistant prostate cancers were included in the study. The lipid-rich nature was studied with lipophilic stains and lipid panel analysis, while surface membrane was assessed with specific staining and antibody detection. We identified a new entity, lipid droplet-like objects or circulating fatty objects (CFOs), visible in the blood samples of many cancer patients, with the potential of causing embolism. CFOs were defined as lipid-rich objects with a membrane, capable of gaining in volume through interaction with peripheral blood mononuclear cells in ex vivo culture. Blood samples from pancreatic cancer patients were found to have the highest CFO incidence and largest CFO numbers. Most noticeably, CFOs from many pancreatic cancer samples presented as large clusters entangled in insoluble fiber networks, suggestive of intravascular clotting. This study identifies CFO as an abnormal entity in cancer patient blood, and a contributory factor to intravascular embolism during cancer development and progression.

Coupling Lipid Labeling and Click Chemistry Enables Isolation of Extracellular Vesicles for Noninvasive Detection of Oncogenic Gene Alterations.

Well-preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry-based EV capture platform ("Click Beads"), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT-dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression.