Mark D. Girgis

Targeting CEA in Pancreas Cancer Xenografts with a Mutated scFv-Fc Antibody Fragment

BackgroundSensitive antibody-based tumor targeting has the potential not only to image metastatic and micrometastatic disease, but also to be the basis of targeted therapy. The vast majority of pancreas cancers express carcinoembryonic antigen (CEA). Thus, we sought to evaluate the potential of CEA as a pancreatic cancer target utilizing a rapidly clearing engineered anti-CEA scFv-Fc antibody fragment with a mutation in the Fc region [anti-CEA scFv-Fc H310A].MethodsImmunohistochemistry (IHC) with the antibody fragment was used to confirm expression of CEA on human pancreas cancer specimens. In vivo tumor targeting was evaluated by tail vein injection of I124-labeled anti-CEA scFv-Fc(H310A) into mice harboring CEA-positive and -negative xenografts. MicroPET/CT imaging was performed at successive time intervals. Radioactivity in blood and tumor was measured after the last time point. Additionally, unlabeled anti-CEA scFv-Fc(H310A) was injected into CEA-positive tumor bearing mice and ex vivo IHC was performed to identify the presence of the antibody to define the microscopic intratumoral pattern of targeting.ResultsModerate to strong staining by IHC was noted on 84% of our human pancreatic cancer specimens and was comparable to staining of our xenografts. Pancreas xenograft imaging with the radiolabeled anti-CEA scFv-Fc(H310A) antibody demonstrated average tumor/blood ratios of 4.0. Immunolocalization demonstrated peripheral antibody fragment penetration of one to five cell diameters (0.75 to 1.5 μm).ConclusionsWe characterized a preclinical xenograft model with respect to CEA expression that was comparable to human cases. We demonstrated that the anti-CEA scFv-Fc(H310A) antibody exhibited antigen-specific tumor targeting and shows promise as an imaging and potentially therapeutic agent.

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.

CA19-9 as a Potential Target for Radiolabeled Antibody-Based Positron Emission Tomography of Pancreas Cancer

Introduction. Sensitive and specific imaging of pancreas cancer are necessary for accurate diagnosis, staging, and treatment. The vast majority of pancreas cancers express the carbohydrate tumor antigen CA19-9. The goal of this study was to determine the potential to target CA19-9 with a radiolabeled anti-CA19-9 antibody for imaging pancreas cancer. Methods. CA19-9 was quantified using flow cytometry on human pancreas cancer cell lines. An intact murine anti-CA19-9 monoclonal antibody was labeled with a positron emitting radionuclide (Iodine-124) and injected into mice harboring antigen positive and negative xenografts. MicroPET/CT were performed at successive time intervals (72 hours, 96 hours, 120 hours) after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the images. Results. Antigen expression by flow cytometry revealed approximately 1.3 × 106 CA19-9 antigens for the positive cell line and no expression in the negative cell line. Pancreas xenograft imaging with Iodine-124-labeled anti-CA19-9 mAb demonstrated an average tumor to blood ratio of 5 and positive to negative tumor ratio of 20. Conclusion. We show in vivo targeting of our antigen positive xenograft with a radiolabeled anti-CA19-9 antibody. These data demonstrate the potential to achieve anti-CA19-9 antibody based positron emission tomography of pancreas cancer.

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.

Distinction of Risk Factors for Superficial vs Organ-Space Surgical Site Infections After Pancreatic Surgery

Importance Surgical site infection (SSI) rates are increasingly used as a quality metric. However, risk factors for SSI in pancreatic surgery remain undefined. Objective To stratify superficial and organ-space SSIs after pancreatectomy and investigate their modifiable risk factors. Design, Setting, and Participants This retrospective analysis included 201 patients undergoing pancreatic surgery at a university-based tertiary referral center from July 1, 2013, through June 30, 2015, and 10 371 patients from National Surgical Quality Improvement Program–Hepatopancreaticobiliary (NSQIP-HPB) Collaborative sites from January 1, 2014, through December 31, 2015. Main Outcomes and Measures Superficial, deep-incisional, and organ-space SSIs, as defined by NSQIP. Results Among the 201 patients treated at the single center (108 men [53.7%] and 93 women [46.3%]; median age, 48.6 years [IQR, 41.4-57.3 years]), 58 had any SSI (28.9%); 28 (13.9%), superficial SSI; 8 (4%), deep-incisional SSI; and 24 (11.9%), organ-space SSI. Independent risk factors for superficial SSI were preoperative biliary stenting (odds ratio [OR], 4.81; 95% CI, 1.25-18.56; P = .02) and use of immunosuppressive corticosteroids (OR, 13.42; 95% CI, 1.64-109.72; P = .02), whereas soft gland texture was the only risk factor for organ-space SSI (OR, 4.45; 95% CI, 1.35-14.66; P = .01). Most patients with organ-space infections also had grades B/C fistulae (15 of 24 [62.5%] vs 4 of 143 [2.8%] in patients with no SSI; P < .001). Organ/space but not superficial SSI was associated with an increased rate of sepsis (7 of 24 [29.2%] vs 4 of 143 [2.8%]; P < .001) and prolonged length of hospital stay (12 vs 8 days; P = .04). Among patients in the NSQIP-HPB Collaborative, 2057 (19.8%) had any SSI; 719 (6.9%), superficial SSI; 207 (2%), deep-incisional SSI; and 1287 (12.4%), organ-space SSI. Preoperative biliary stenting was confirmed as an independent risk factor for superficial SSI (OR, 2.07; 95% CI, 1.58-2.71; P < .001). In this larger data set, soft gland texture was an independent risk factor for superficial SSI (OR, 1.45; 95% CI, 1.14-1.85; P = .002) but was more strongly and significantly associated with organ-space SSI (OR, 2.32; 95% CI, 1.88-2.85; P < .001). Conclusions and Relevance Preoperative biliary stenting and coriticosteroid use increase superficial SSI, even in patients receiving perioperative piperacillin-tazobactam. Additional measures, including extended broad-spectrum perioperative antibiotic treatment, should be considered in these patients. Organ/space SSIs appear to be related to pancreatic fistulae, which are not modifiable. Reporting these different subtypes as a single, overall rate may be misleading.

Abstract 3070: Capture, isolation, and mutational analysis of single pancreatic circulating tumor cells using NanoVelcro technology

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Circulating tumor cells (CTCs) have been developed as a biomarker in several cancers, but not in pancreatic cancer (PDAC). The available clinical technology for CTC study, CellSearch, suffers from limited ability to perform molecular analysis. We sought to evaluate a novel microfluidic NanoVelcro technology coupled to laser micro-dissection (LMD), for CTC capture and single cell analysis of KRAS mutations to confirm tumor origin of captured CTCs. Methods: NanoVelcro utilizes a nano-spun polymer fiber, modified with streptavidin, to capture CTCs using biotinylated antibodies. The system was optimized with the CFPAC-1 (EpCAM+, KRAS G12 to V) cell line spiked into healthy donor blood. Patient samples were collected from 2 mL peripheral venous blood and run at the optimized flow rate of 1mL/hr. CTCs were defined by size (≥ 10 µm) and immuno-staining pattern (DAPI+/CK+/CD45-). Using LMD, single cells were isolated and subjected to whole genome amplification followed by PCR of KRAS exon 1 and subsequent Sanger Sequencing. Results: The NanoVelcro system allowed for detection of mutant KRAS in isolated CFPAC-1 cells. 6 CTCs were then isolated from a known KRAS G12 to V mutant patient and 3/6 (50.0%) captured CTCs were found to have KRAS G12 to V mutations while normal WBCs from the same patient contained wild-type KRAS. Conclusions: The microfluidic NanoVelcro technology demonstrated capture of pancreatic CTCs and may prove useful in the development of CTCs as a biomarker in PDAC. Application of LMD enabled single cell KRAS mutational analysis confirming cancer origin of isolated CTCs and supporting our current PDAC CTC identification criteria based on immuno-staining. This promising technology, combined with LMD, opens the door for molecular characterization of CTCs, thus providing insight into the biology of metastasis while potentially revealing molecular targets for therapeutics. Citation Format: Jacob S. Ankeny, Shuang Hou, Millicent Lin, Matthew Frias, Hank OuYang, Min Song, Matthew M. Rochefort, Mark D. Girgis, Hsian-Rong Tseng, James S. Tomlinson. Capture, isolation, and mutational analysis of single pancreatic circulating tumor cells using NanoVelcro technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3070. doi:10.1158/1538-7445.AM2014-3070

Targeting cancer with cys-diabody conjugated polymerized liposomal nanoparticles (PLNs)

Background: Polymerized liposomal nanoparticles (PLNs) are a promising new technology for delivery of chemotherapeutic drugs to cancer cells. They are easy to prepare, biocompatible, and can carry a wide variety of therapeutics. Additionally, PLNs have the potential to be conjugated to a tumor targeting agent in hopes of delivering a cytotoxic payload directly to the cancer cells, thus minimizing systemic toxicity. Cys-diabodies are the smallest tumor antibody fragment (55 Kd) that retain the bivalency of an intact antibody. The goal of this study was to determine our ability to conjugate various engineered cys-diabody fragments to PLNs and to evaluate the ability to specifically target tumor cells based on the specificity of the diabody-tumor antigen interaction. Methods: Cys-diabody constructs against tumor antigens (ALCAM, HER2/neu, Ca19.9) were engineered, expressed in NSO myeloma cells and purified using HPLC. A cysteine residue was inserted at the C-terminus to provide the free sulhydryl group necessary for the conjugation reaction. Using malemide chemistry, cys-diabodies were conjugated to PLNs. Immunofluorescence and flow cytometry were performed to evaluate targeting of conjugated PLNs to human pancreatic and breast cancer cell lines. Unconjugated PLNs and tumor antigen negative cell lines were used as controls. Results: Anti-HER2/neu, ALCAM, and CA19.9 cys-diabodies were characterized by flow cytometry showing specific binding to MCF7 (HER2/neu positive), HPAF (ALCAM positive), and BxPC3 (CA19.9 positive) cell lines, respectively. No binding was observed to antigen-negative cell lines nor with the unconjugated PLNs. Conjugation of cys-diabodies to PLNs was indicated by immunofluorescence showing specific binding of PLN-cys-diabody conjugates to breast and pancreatic cancer cells in vitro (Figure 1). These data were also confirmed by flow cytometry. Conclusions: We have demonstrated the ability to conjugate our PLN with engineered anti-tumor cys-diabodies. Furthermore, we have demonstrated that after conjugation, the PLNs demonstrate specific tumor-cell targeting based on the specificity of the diabody-tumor antigen affinity. These studies represent an important first step in the development of targeted PLNs with the ultimate goal of delivering high doses of a chemotherapeutic directly to the cancer cells.

Splenic artery pseudoaneurysm with hemosuccus pancreaticus requiring multimodal treatment

Abstract Termed hemosuccus pancreaticus by Sandblom in 1970, hemorrhage from the pancreatic duct into the gastrointestinal tract represents a rare and challenging problem. Patients present with repeated upper gastrointestinal bleeding that is intermittent but often self‐limited. In most cases, this pathophysiologic process is secondary to pancreatitis, chronic inflammation, and subsequent splenic artery pseudoaneurysm bleeding. Previously treated with open splenectomy and distal pancreatectomy, hemosuccus pancreaticus is now often managed with minimally invasive endovascular means. We describe an uncommon presentation of hemosuccus pancreaticus in the absence of prior pancreatitis, requiring open splenectomy, distal pancreatectomy, and celiac artery ligation after failed endovascular intervention.

Association of Hospital Length of Stay and Complications With Readmission After Open Pancreaticoduodenectomy

to such standards, however, remain unclear. Given our findings, we conclude that inmates have an unrecognized burden of morbidity and mortality secondary to surgical disease for which they lack adequate access to timely care. We hypothesize that these findings stem from mechanisms of underfunding and overcrowding, as well as outsourcing of prison health care. We also hypothesize that these trends are not unique to Florida and may be paralleled in other jurisdictions with high rates of incarceration.3 The establishment of a national quality surgical database that is specific to incarcerated persons may be an appropriate initiative for acute care surgeons. Further expansion of this work is required to better define barriers to surgical evaluation, access to surgical care, and long-term morbidity in surviving postoperative inmate cohorts.

Carcinoembryonic Antigen for Diagnosis of Colorectal Cancer Recurrence

A 78-year-old man with no significant medical or family history of cancer presented with 2 weeks of worsening abdominal pain and was found to have a cecal mass on computed tomography (CT) scan. Colonoscopy and biopsy revealed a partially obstructing cecal adenocarcinoma; colonoscope could not be passed beyond the mass. On further workup, his carcinoembryonic antigen (CEA) level was 11.0 ng/mL. CT scan of chest showed no evidence of metastatic disease. The patient underwent a right hemicolectomy with surgical pathology showing a 5.5-cm adenocarcinoma with 0 of 19 lymph nodes positive for cancer. A colonoscopy, which was performed 3 months after surgery because of an incomplete preoperative examination, did not show any remaining mass or synchronous lesions. Given stage IIA disease, the patient did not receive adjuvant chemotherapy and was scheduled for postoperative surveillance with CEA testing every 6 months and a CT scan and colonoscopy in 1 year. One year after surgery, testing showed the patient’s CEA level to be 5.1 ng/mL, then 6.1 ng/mL on repeat testing. The patient did not have any new symptoms and his physical examination was unremarkable. His serial CEA levels are shown in the Table.