Christopher J. Tignanelli

UV activation of polymeric high aspect ratio microstructures: Ramifications in antibody surface loading for circulating tumor cell selection

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ~3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymers damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the devices cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens.

House staff–led interdisciplinary morbidity and mortality conference promotes systematic improvement

BACKGROUND Improvements in patient safety are critical to improving clinical outcomes. We present a resident-led interdisciplinary morbidity and mortality (M&M) conference utilizing postconference task forces to identify unique system issues, classify key contributors to interdisciplinary complications, and implement systems solutions. The conference also served to facilitate resident involvement in quality improvement projects. MATERIALS AND METHODS Members of the UNC Housestaff Council designed and implemented a hospital-wide M&M conference. Cases involving two or more service lines and resulting from systematic failures were selected for presentation by an interdisciplinary group of residents involved in the patients care. Postconference task forces addressed problems and developed initiatives to improve care. RESULTS Of the 15 cases presented, 60% were attributable to an error in judgment, 26% to an error in diagnosis, and 13% to an error in technique. Communication (67%), coordination/care utilization (47%), poor process/workflow (40%), and inadequate training (33%) were the main associated contributing factors. Poor communication contributed to all complications resulting from an error in judgment. Inadequate training and poor workflow were the most common contributing factors with an error in technique. Poor utilization of care and inadequate processes were most common with an error in diagnosis. Postconference task forces identified system-based improvement projects in 73% (11 of 15) of cases with 82% (9 of 11) of projects successfully implemented or in process. CONCLUSIONS House staff-led interdisciplinary M&M conference utilizing postconference task forces is an ideal setting to identify unique system issues and implement system-based improvement strategies.

Outcomes of acute kidney injury in patients with severe ARDS due to influenza A(H1N1) pdm09 virus

Background The incidence and long‐term outcomes of acute kidney injury in patients with severe acute respiratory distress syndrome (ARDS) due to influenza A(H1N1) pdm09 virus (pH1N1) have not been examined. Objective To assess long‐term renal recovery in patients with acute kidney injury and severe ARDS due to pH1N1. Methods A retrospective observational cohort study of adults with severe pH1N1‐associated ARDS admitted to a tertiary referral center. Baseline characteristics, acute kidney injury stage, continuous renal replacement therapy (CRRT), intermittent hemodialysis, extracorporeal membrane oxygenation, survival, and renal recovery (defined as dialysis independence) were evaluated. Results Fifty‐seven patients, most with stage 3 acute kidney injury, were included. The 53% mortality rate among the 38 patients requiring CRRT was significantly higher than the 0% mortality rate among the 19 patients not requiring CRRT or intermittent hemodialysis. Increased duration of CRRT was not significantly associated with decreased survival. Fifteen CRRT patients required transition to intermittent hemodialysis. Of the CRRT patients who survived, 94% experienced renal recovery. Extracorporeal membrane oxygenation was instituted in 17 patients; 15 of these patients required CRRT. Conclusions Acute kidney injury is common in patients with severe ARDS caused by pH1N1 infection. CRRT is a significant risk factor for increased mortality, but most patients who survived experienced full renal recovery.

Are Predictive Energy Expenditure Equations in Ventilated Surgery Patients Accurate

Background: While indirect calorimetry (IC) is the gold standard used to calculate specific calorie needs in the critically ill, predictive equations are frequently utilized at many institutions for various reasons. Prior studies suggest these equations frequently misjudge actual resting energy expenditure (REE) in medical and mixed intensive care unit (ICU) patients; however, their utility for surgical ICU (SICU) patients has not been fully evaluated. Therefore, the objective of this study was to compare the REE measured by IC with REE calculated using specific calorie goals or predictive equations for nutritional support in ventilated adult SICU patients. Materials and Methods: A retrospective review of prospectively collected data was performed on all adults (n = 419, 18-91 years) mechanically ventilated for >24 hours, with an Fio 2 ≤ 60%, who met IC screening criteria. Caloric needs were estimated using Harris-Benedict equations (HBEs), and 20, 25, and 30 kcal/kg/d with actual (ABW), adjusted (ADJ), and ideal body (IBW) weights. The REE was measured using IC. Results: The estimated REE was considered accurate when within ±10% of the measured REE by IC. The HBE, 20, 25, and 30 kcal/kg/d estimates of REE were found to be inaccurate regardless of age, gender, or weight. The HBE and 20 kcal/kg/d underestimated REE, while 25 and 30 kcal/kg/d overestimated REE. Of the methods studied, those found to most often accurately estimate REE were the HBE using ABW, which was accurate 35% of the time, and 25 kcal/kg/d ADJ, which was accurate 34% of the time. This difference was not statistically significant. Conclusion: Using HBE, 20, 25, or 30 kcal/kg/d to estimate daily caloric requirements in critically ill surgical patients is inaccurate compared to REE measured by IC. In SICU patients with nutrition requirements essential to recovery, IC measurement should be performed to guide clinicians in determining goal caloric requirements.

Hospital Based Nutrition Support: A Review of the Latest Evidence

Meeting appropriate nutritional demands in the inpatient setting is a fundamental aspect of optimal patient care. Optimizing nutrition delivery and preventing malnutrition can have a significant positive effect on clinical outcomes and costs of care. Despite extensive research, many questions remain regarding the delivery of nutrients to hospitalized patients, especially in the critically ill. Recent advances have been made over the past decade, and landmark studies have yielded an end to many controversial topics, such as the broad utilization of immunonutrition. However, there are still many questions that remain unanswered, for example how do we objectively define malnutrition? Cutting edge research in the areas of morphomics and metabolomics are raising new questions which are poised to revolutionize how we will answer today’s questions. In this review, we summarize the historical pedagogy underlying nutritional practice alongside contemporary evidence supporting current practice guidelines. Furthermore, we identify and explore key barriers preventing the rapid identification and treatment of malnutrition. We introduce two emerging technologies foremost in nutritional research that may eventually disrupt current barriers. And finally, we discuss key populations at specifically high risk for the development of malnutrition.

Abstract B71: Multiplexed kinase inhibitor beads identify multiple pathways of resistance to PI3K inhibition facilitating the rational selection of novel combination therapies in pancreatic cancer

KRAS mutational activation plays a critical role in tumorigenesis, but exactly which downstream KRAS effector pathway is critical for this role remains less clear. One of the most studied downstream pathways is the phosphatidylinositol-3-kinase (PI3K) pathway which mediates cellular metabolism, growth, and survival. We have previously shown using validated pancreatic ductal adenocarcinoma (PDAC) patient derived xenograft (PDX) mouse models that treatment with BKM120 (a pan-class 1 PI3K inhibitor, currently in Phase I/II clinical trials) resulted in tumor growth inhibition (p = 0.017) but not regression. In this study, we evaluated possible mechanisms of resistance to BKM120 therapy. We developed a novel multiplex inhibitor bead/mass spectrometry (MIB/MS) assay to measure the activation state of the kinome. We have recently shown that kinome response to kinase inhibitor therapy (i.e. kinome reprogramming) is a potential mechanism of resistance in triple negative breast cancer in response to MEK inhibition and in drug-resistant leukemia in response to imatinib. We hypothesized that kinome reprogramming may play a role in resistance to PI3K inhibition in PDAC and used MIB/MS to identify second targets that may be used in combination with PI3K inhibitors. We found kinome reprogramming in response to PI3K inhibition through both previously known as well as less studied pathways. For instance, we found MEK1 and MEK2 activation in response to BKM120 treatment. MEK activation has previously been implicated in resistance to PI3K inhibition and combined MEK and PI3K inhibition has been shown to be synergistic in PDAC. However, our results suggest that resistance to PI3K may be mediated through many more pathways than MEK alone. We found ErbB1, ErbB2 and ErbB3 activation in response to BKM120 treatment in both cell lines and PDX tumors. ErbB2 and ErbB3 activation has previously been noted in response to PI3K inhibition in breast cancer. However, no studies have evaluated this combination in PDAC. Thus we evaluated the effect of combined PI3K and pan-ErbB inhibition in a panel of 10 PDAC cell lines using BKM120 and dacomitinib (a pan-ErbB inhibitor currently in Phase III clinical trials). Combined treatment with BKM120 and dacomitinib inhibited proliferation in 10 of 10 PDAC cell lines evaluated. This combination showed impressive synergy across all cell lines with a mean combination index of 0.24 (0.00245 – 0.49). MIB/MS is a powerful unbiased approach to identify second targets for combination therapy. We identified both known and novel kinase pathways that may mediate resistance to PI3K inhibition in PDAC. Our results suggest that pan-ErbB inhibition may be a promising second target in combination with PI3K inhibition in PDAC. Combination studies in PDX models are ongoing. Pan-ErbB and PI3K inhibition in PDAC may be more effective than either single agent alone and should be considered in clinical trials. Citation Format: Christopher J. Tignanelli, Jeran Stratford, Richard Moffitt, Rachel Reuther, Gary L. Johnson, Jen Jen Yeh. Multiplexed kinase inhibitor beads identify multiple pathways of resistance to PI3K inhibition facilitating the rational selection of novel combination therapies in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B71.