22.05 The Use of Smart Phone Thermal Imaging for Assessment of Peripheral Perfusion in Vascular Patients

Posted on January 18, 2016

G. Wallace1, E. Quiroga1, N. Tran1 1University Of Washington,Vascular Surgery,Seattle, WA, USA

Introduction: Ankle brachial index (ABI) is a reliable quantitative determination of extremity perfusion but required specialized training and could cause patient’s discomfort or not feasible in trauma patients with mangled extremities. Smart phone based thermal imaging utilizing forward looking infrared imaging (FLIR) can easily determine temperature of the extremity. This study reports on the feasibility of using smart phone based FLIR to assess peripheral perfusion by measurement of extremity temperature.

Methods: Patients seen in a vascular clinic with ABI’s measured by an ICAVL certified vascular lab were selected. Patients then underwent imaging using a smart phone based FLIR. Color spectrograph was taken and surface temperature of each extremity was determined and recorded. A single operator who was blinded to ABI’s results obtained all images. Comparison between ABI and extremity’s temperature was performed. Thermal ankle brachial index (TABI) was calculated by dividing the temperature of the lower extremity by that of the upper extremity. Temperature accuracy of FLIR was determined against known standard. Statistical analysis was done using Pearson’s correlation and Bland-Altman plot.

Results: 23 patients had both ABI and extremity temperature recorded on the same day resulting in 45 lower extremities analyzed (one patient has a BKA). ABI ranged from 0.33 to 1.46 with mean (IQR) of 0.92 (0.35). Lower extremity temperature ranged from 60.67 to 96.87°F with mean of 83.41 (14.10). Upper extremity range was 81.86 to 94.60°F with mean of 91.03 (3.44). Positive correlation between ABI and FLIR temperature was seen with Pearson’s correlation coefficient r = 0.66 p = .001, n = 45. Using ABI and the newly described TABI, positive correlation was also seen with r = 0.83, p < .0001, n = 45 (Fig 1). Thermal sensitivity of FLIR was determined at 0.18°F. When comparing against known standard, thermal imaging has an average of 3.16% error across temperature range of 34 to 144°F.

Conclusion: Smart phone based FLIR imaging can be used to reliably determine extremity perfusion as an alternative to traditional ABI measurement. FLIR can be easily done, portable, and has a variety of potential applications from intraoperative assessment of revascularization to evaluation of the injured extremity in trauma.

21.10 Risk Factors of Mortality Following Abdominal Aortic Aneurysm Repair using Analytic Morphomics

Posted on January 18, 2016

A. A. Mazurek1, J. F. Friedman1, A. Hammoud1, C. Inglis1, J. Haugen1, A. Hallway1, J. Lawton1, J. Ruan1, B. Derstine1, J. S. Lee1, S. C. Wang1, M. J. Englesbe1, N. H. Osborne1 1University Of Michigan,Morphomic Analysis Group, Department Of Surgery,Ann Arbor, MI, USA

Introduction:
Risk stratification for patients undergoing open or endovascular abdominal aortic aneurysm (AAA) repair has focused primarily on utilizing patient comorbidities as predictors of operative outcomes. However, there is often minimal variation in the burden of comorbid disease among patients with AAAs. Analytic morphomics is a novel method of risk-stratification that uses cross-sectional images to quantitatively measure domains of patient health. The utility of morphometric measurements as predictors of surgical outcome has been validated in several patient populations. Previously, AAA mortality after open repair has been associated with total psoas area. This study sought to further understand the role of both core muscle size and adiposity on the risk of mortality following open and endovascular AAA repair.

Methods:
A total of 795 patients underwent open or endovascular AAA repair between 2000 and 2012. 722 patients (91%) had preoperative abdominal CT scans available for analysis. After excluding patients with incomplete medical records, manual chart review was used to identify patient demographics and comorbidities. Validated methods of analytic morphomics, previously described, were used to measure cross-sectional areas and densities of psoas muscle, dorsal (paraspinous) muscle groups and subcutaneous and visceral fat at the vertebral levels T12 through L5. Univariate and multivariate analyses were used to determine which morphometric variables were significant predictors of mortality, controlling for traditional patient factors.

Results:
A total of 610 patients were identified; 322 patients underwent open repairs and 288 underwent endovascular repairs between the years 2000 and 2012. Following open repair, overall mortality ranged from 7% at 90 days to 21.1% at 3 years. Following endovascular repair, overall mortality ranged from 4% at 90 days to 21.3% at 3 years. Morphometric variables associated with mortality between 90 and 1095 days included measures of subcutaneous fat, visceral fat and both dorsal muscle group and psoas muscle density and area. After controlling for patient comorbidities and type of repair, a composite score of morphometric variables continued to be highly associated with mortality between 90 days and 1095 days; important variables included subcutaneous fat density at L2 (OR 1.09, p<0.001) and visceral fat density at L3 (OR 1.08, p<0.001).

Conclusion:
Morphometric measurements of adiposity and muscle mass correlate strongly with intermediate and late-term mortality after both open and endovascular AAA repair. Traditional methods of risk stratification in patients undergoing AAA repair may be augmented using analytic morphomics. These objective measures of frailty may aid in patient decision-making and provide insight into domains of health that clinicians and patients can work to optimize preoperatively in order to maximize positive outcomes for the patient.

21.12 Lymphovascular Invasion is Associated with Compromised Survival for Papillary Thyroid Cancer

Posted on January 18, 2016

L. N. Pontius1, L. M. Youngwirth1, S. M. Thomas1, R. P. Scheri1, S. A. Roman1, J. A. Sosa1 1Duke University Medical Center,Durham, NC, USA

Introduction: Data regarding the association between lymphovascular invasion for survival for papillary thyroid cancer (PTC) are limited. This study sought to examine lymphovascular invasion as an independent prognostic factor for patients with PTC undergoing total thyroidectomy.

Methods: The National Cancer Data Base (2010-2011) was queried for all patients with a diagnosis of PTC undergoing total thyroidectomy. Patients were classified into two groups based on the presence/absence of lymphovascular invasion. Demographic, clinical, and pathologic features at the time of diagnosis were evaluated for all patients. A Cox proportional hazards model was developed to identify factors associated with survival.

Results: In total, 40,324 patients met inclusion criteria; 12.5% had lymphovascular invasion. Patients with lymphovascular invasion were more likely to have larger tumors (2.8 cm vs 1.6 cm, p<0.01), metastatic lymph nodes (75.1% vs 34.1%, p<0.01), and distant metastases (3.1% vs 0.5%, p<0.01). They also were more likely to receive radioactive iodine when compared to patients without lymphovascular invasion (70.2% vs 48.7%, p<0.01). Unadjusted overall survival was reduced for patients with lymphovascular invasion compared to patients without it (log-rank p<0.01), with 5-year survival rates of 86.1% and 94.2%, respectively. After adjustment, increasing patient age (HR=1.06, p<0.01), male gender (HR=1.63, p<0.01), presence of metastatic lymph nodes (HR=1.73, p<0.01), presence of distant metastases (HR=4.90, p<0.01), and presence of lymphovascular invasion (HR=1.99, p<0.01) all were associated with compromised survival. Treatment with radioactive iodine was protective in both patients with lymphovascular invasion (HR=0.42, p<0.01) and patients without lymphovascular invasion (HR=0.48, p<0.01).

Conclusion: The presence of lymphovascular invasion among patients undergoing total thyroidectomy for PTC is independently associated with compromised survival. Patients with PTC and lymphovascular invasion should be considered higher risk, and providers should consider aggressive surgical and adjuvant treatment measures to maximize patient outcomes.

22.01 Interferon Regulatory Factor 1 Promotes Hepatocyte Exosome Secretion through Induction of Rab27a

Posted on January 18, 2016

M. Yang1, Q. Du1, P. R. Varley1, Z. Liang1, B. Chen1, C. Heres1, D. B. Stolz2, S. C. Watkins2, D. A. Geller1 1Thomas E. Starzl Transplantation Institure,Department Of Surgery, University Of Pittsburgh School Of Medicine,Pittsburgh, PA, USA 2Center Of Biologic Imaging,University Of Pittsburgh,Pittsburgh, PA, USA

Introduction: Exosomes play an important role in cell communication, including tumorigenesis, cancer metastases, and immune regulation. Rab27a is a GTPase that has been shown to promote exosome secretion. However, mechanisms controlling cell exosome secretion in response to pathologic conditions are not well-defined. Interferon regulatory factor 1 (IRF-1) is a transcription factor regulating immunity, carcinogenesis, and hepatic I/R injury. The role of IRF-1 in regulating Rab27a and exosome secretion is unknown. Since IRF-1 can regulate expression of some GTPases, we hypothesized that IRF-1 might regulate Rab27a expression and increase exosome secretion.

Methods: Primary human hepatocytes (hHC) and human hepatoma cell line Huh-7 were treated with Interferon-γ (IFNγ). PCR and Western blot were used to detect IRF-1 and Rab27a mRNA and proteins. Exosomes secreted by hHC and Huh-7 cells were isolated with ultracentrifugation. Isolated exosomes were confirmed by transmission electron microscopy (TEM) and surface markers. Exosome proteins secreted by these cells were quantitated with bicinchoninic acid assay (BCA). The sizes of the exosomes were measured by nanoparticle tracking analysis (NTA). The binding sites of IRF-1 in promoter region of Rab27a were predicted with PROMO bioinformatics software. IRF-1 expression plasmid and Rab27a promoter-driven luciferase reporter were used to determine the effect of IRF-1 on Rab27a transcription. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) were used to verify IRF-l binding in the Rab27a gene promoter region in vivo and in vitro.

Results: IFNγ increased IRF-1 and Rab27a mRNA and protein in primary hHC in a dose-dependent manner (Fig. A). Deflated soccer-ball like exosomes, which were verified by TEM (Fig. B), were secreted more by hHC and Huh7 after IFNγ treatment (Fig. C, p < 0.05). IFNγ did not alter the exosome size (data not shown). Multi-vesicle bodies (MVB) are precursors of exosomes and were markedly increased by IFNγ stimulation in Huh7 cells (Fig. D, CD63 staining red). Transfection of HepG2 cells with IRF-1 expression plasmid increased Rab27a promoter activity 4.9-fold. Using PROMO software, we found ten putative IRF-1 binding motifs upstream in the Rab27a promoter region. ChIP and EMSA verified five IRF-1 binding sites in the Rab27a promoter region (data not shown).

Conclusion: These novel findings indicate that IFNγ induces IRF-1 which promotes exosome secretion through increasing the expression of Rab27a. These results provide important insights into fundamental cellular signaling pathways regulating exosome secretion under inflammatory conditions in hepatocytes and hepatocellular carcinoma.

21.07 Ultrasound is a Sensitive Adjunct to Plain Radiographs in Management of Necrotizing Enterocolitis

Posted on January 18, 2016

S. E. Horne3, S. M. Cruz1,3, S. Nuthakki2, P. E. Lau1,3, D. A. Lazar1,3, S. E. Welty2, O. O. Olutoye1,3 1Baylor College Of Medicine,Department Of Surgery,Houston, TX, USA 2Texas Children’s Hospital,Pediatrics,Houston, TX, USA 3Texas Children’s Hospital,Pediatric Surgery,Houston, TX, USA

Introduction:

Necrotizing Enterocolitis (NEC) is the most common gastrointestinal emergency of the preterm infant with an incidence of 5-10%. It is traditionally diagnosed with a combination of physical examination and plain radiographs of the abdomen. The diagnostic role for ultrasound in NEC is uncertain. We hypothesized that ultrasound (US) is as sensitive as plain radiographs in the diagnosis and management of NEC.

Methods:

The medical records of all infants with NEC in a single pediatric tertiary center from January 2006- January 2013 were reviewed. In order to factor in NEC’s rapid rate of changes in pathologic findings, patients that underwent US within four hours of abdominal XR were included in the analysis. Bell’s Criteria were utilized to stage each patient during his/her course. Clinical, radiologic, surgical and pathological findings were reviewed. Statistical analysis was performed using Student's t-test and Mann-Whitney U test for continuous variables and Fisher's exact for categorical variables.

Results:

During this period, 186 neonates were diagnosed with NEC, of which 26 met inclusion criteria. It was noted that US was done for confirmatory purposes in these 26 patients after plain radiographs did not agree with clinical findings. Plain radiographs and ultrasound were taken within an average of 2.46 ± 1.17 hrs of each other. At the time of XR and US, the Bell’s staging of the patients was Stage 1 in 27% of the cases (n=7), Stage 2 in 42% (n=11), and Stage 3 in 31% (n=8). There were 92% preterm infants, 38.5% had congenital anomalies (i.e. omphalocele, congenital neck mass, and Congenital High Airway Obstruction Syndrome), 19% had cardiac abnormalities, and 23% had patent ductus arteriosus. The survival rate of our NEC population was 65% (n=17). Surgical intervention was undergone in 65% (n=17) of our patients in which the median time between imaging findings and time of surgery was 2 (0-59) days. When comparing both modality for reliability in detecting intestinal ischemia and/or perforation with surgical findings, US had a sensitivity of 72% with a positive predictive value (PPV) of 93% while plain radiographs had a sensitivity of 42% with a PPV of 100%. In all cases that required surgical drainage (n=4), ultrasound findings of complex fluid collections guided the decision for bedside surgical drainage where plain radiograph did not suggest free fluid.

Conclusion:

In this study, ultrasound proved to be helpful in assessing the need for surgical interventions in neonates where diagnosis of advanced NEC is ambiguous. US appeared to be more sensitive in reliably detecting intestinal perforation/ischemia in comparison to plain radiographs. However, both plain radiographs and ultrasound play a key role in the diagnosis and should be considered conjunctively during the management and treatment of NEC.

21.08 Cells from In Vivo Models of Heterotopic Ossification Exhibit Increased Osteogenic Properties

Posted on January 18, 2016

J. Drake1, S. Agarwal1, K. Shigemori1, S. Loder1, C. Hwang1, S. Li1, Y. Mishina1, S. Wang1, B. Levi1 1University Of Michigan,Ann Arbor, MI, USA

Introduction: Large burns and high-energy trauma can lead to heterotopic ossification (HO), a process by which pathologic, ectopic bone forms within soft tissue. Management of HO is limited by efficacy of available treatments, difficulty identifying at-risk patients, and high recurrence rates following surgical excision. The cellular and molecular basis of HO is unknown. Here we investigate whether the cells themselves or their environment drive HO formation. We demonstrate that human and mouse cells isolated from sites of HO retain increased osteogenic capacity when cultured outside of an inflammatory environment.

Methods: Human cells were cultured from HO and surrounding normal bone. Mouse cells were obtained from two models including trauma-induced and genetic HO. Cells from the trauma induced model were isolated from the tendon transection site of mice which had undergone a dorsal burn with tendon transection (burn/tenotomy) at 1, 2, and 3 weeks after injury, a model that reliably produces HO. In the genetic HO model (Nfatc1-cre/caACVR1fl/wt) normal and HO-derived osteoblasts were isolated from 1, 2, and 3 week old mice. Osteogenic differentiation was assessed for by alkaline phosphatase production, alizarin red stain for mineral deposition, RNA expression, and protein expression. Cell proliferation was also assessed.

Results: Human HO cells showed increased osteogenic signaling compared to human osteoblasts from non-HO bone (Fig. 1). Cells isolated from the burn/tenotomy mice 2 and 3 weeks after injury demonstrated significantly increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with controls. Similarly, HO-derived cells from our genetic HO model in 2 or 3 week old mice exhibited increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with non-HO osteoblasts from the same mice (Fig. 2). Finally, targeting these cells with inhibitors of smad5 phosphorylation (LDN-193189) decreased osteogenic capacity by alkaline phosphatase and alizarin red quantification (p<0.05), consistent with its effect on HO formation in our trauma model.

Conclusion: In vitro analysis demonstrates significant differences in cellular behavior with regard to proliferation and osteogenic differentiation in HO models when cells are cultured separate from their in vivo environment. This data suggest that changes in cell behavior drive the process of HO as cell characteristics are preserved after they are removed from their environment. Furthermore, the striking differences in these HO-derived cells from normal cells suggests that they may be used for in vitro assays to study potential therapies targeting HO development.

21.09 Surgical and Post-Operative Risk Factors for Lymphedema Following Lymphadenectomy for Melanoma

Posted on January 18, 2016

S. J. Diljak1, R. D. Kramer1, R. J. Strobel1, B. Sunkara1, D. J. Mercante1, J. S. Jehnsen1, J. F. Friedman1, A. Durham2, M. Cohen1 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of Dermatology,Ann Arbor, MI, USA

Introduction: Secondary lymphedema (SLE) is a significant complication following lymphadenectomy in melanoma patients, with a reported incidence between 9-25% for axillary dissection (ALND) and as high as 24-44% for inguinal dissections (ILND). While radiation therapy and tumor burden are positive predictors for arm lymphedema in breast cancer, this has not been as well defined in the melanoma population using large cohorts. The purpose of this study is to identify unique post-operative and surgical risk factors for SLE in patients with regionally metastatic melanoma using the largest cohort to date compiled from a prospectively collected database of melanoma patients following an ALND or ILND.

Methods: From a prospectively collected, IRB-approved database we identified 688 melanoma patients receiving a complete lymphadenectomy (June 2005-June 2015) with 557 patients having either an ALND or ILND. Patients having iliac or bilateral dissections, or pre-op chemotherapy were excluded. Demographic, clinical, and post-op data were reviewed from the electronic medical record (EMR). SLE was defined as being mentioned in more than one post-operative note or a documented referral to the lymphedema clinic. Univariate statistical analysis and odds ratios (OR) with 95% confidence intervals (CI) were used to determine independent post-op and surgical predictors of SLE.

Results: Of the cohort of 557 melanoma patients, 119 (21.4%) developed SLE following lymph node dissection. The cohort was split between ALND (N=322 (57.8%); 10.9% with SLE) and ILND (N=235 (42.2%); 35.7% with SLE). On univariate logistic regression [Table], having an ILND (OR=4.58; CI: 2.95-7.11), post-operative adjuvant (OR=1.61 CI: 1.07-2.42) or radiation therapy (OR=1.81 CI: 1.02-3.22), and developing non-SLE complications (e.g. hematoma, infection, DVT) (OR=1.84 CI: 1.21-2.80), were each significantly associated with an increased risk of developing SLE. Non-SLE post-op complications increased the risk of SLE only in the first 2 months after surgery (OR=2.25 CI: 1.35-3.74). Use of an energy device during surgery, number of nodes removed, blood loss, and operative times were not significantly associated with risk of SLE. The average post-op time to develop SLE was 103 ± 126 days and follow-up was 1.9 ± 2.2 years.

Conclusion: This is the largest study to date evaluating surgical/post-op risk factors for SLE in melanoma patients after ALND or ILND. Post-operative factors significantly increasing the risk of SLE include adjuvant or radiation therapy, having an ILND, or non-SLE post-op complications. We believe that this work, combined with further evaluation of patient pre-op characteristics, will enhance informed clinical decision-making and risk assessment.

21.04 Sarcopenia as a Prognostic Factor in Emergency Abdominal Surgery

Posted on January 18, 2016

R. C. Dirks1, B. L. Edwards1, E. Tong1, B. Schaheen1, F. E. Turrentine1, A. L. Shada2, P. W. Smith1 1University Of Virginia,General Surgery,Charlottesville, VA, USA 2University Of Wisconsin,Surgery,Madison, WI, USA

Introduction: Sarcopenia, a loss of skeletal muscle mass associated with aging, is a practical measure of frailty and has been previously identified as a predictor of outcomes in surgical cohorts including cancer resection and elderly patients. We hypothesize that sarcopenia, as measured by preoperative CT scans of the psoas muscle, predicts mortality and morbidity in emergent laparotomy.

Methods: Institutional NSQIP data were queried for adult patients who underwent open emergency abdominal surgery between 2008 and 2013. Patient demographics, clinical variables, and outcomes were extracted from NSQIP. Patients with abdominal CT scans within 30 days prior to surgery were included and the cross sectional areas of the psoas muscles at vertebral level L4 were summed and normalized by patient height. Patients were assigned to sex-stratified tertiles based on this normalized total psoas area (TPA) for analysis, with the lowest tertile being classified as sarcopenic. Kaplan Meier curves were constructed to compare survival between TPA tertiles. Cox Proportional Hazards models stratified by sex and controlling for ASA score, ascites, International Normalized Ratio (INR), functional dependency and work Relative Value Units as a proxy for surgery complexity were used to evaluate the influence of TPA on postoperative mortality.

Results: NSQIP revealed 781 patients undergoing emergent open abdominal surgery and 593 of these (75.9%) had appropriate preoperative CT scans. Median patient age was 61 years (IQR 50-72), median TPA was 1719 mm2 (IQR 1341-2293), and median BMI was 26.7kg/m2 (22.9-33). Bivariable analysis demonstrated that TPA was significantly associated with total postoperative morbidity (p=0.013), increased length of stay (p<0.0001) and 90-day mortality (p=0.0008) but not 30-day mortality (p=0.26). Kaplan Meier curves demonstrated significantly decreased 90-day survival in lowest TPA tertile. A Cox proportional hazards model demonstrated that the impact of TPA was overwhelmed by previously validated predictors of mortality, most notably ASA score.

Conclusion: Sarcopenia, as measured by TPA, is significantly associated with increased 90-day mortality, length of stay, and total morbidity in patients undergoing open emergency abdominal surgery. Since many patients undergoing emergent abdominal surgery have already undergone CT scanning, TPA is readily available to the practicing surgeon at no added risk or cost. As such, sarcopenia is a convenient additional tool for preoperative risk assessment and risk counseling.

21.05 Changes in Liver Allograft Steatosis and its Impact on Early Graft Function and Long Term Survival

Posted on January 18, 2016

J. Davis1, S. Fuller1, S. Kubal1, J. Fridell1, A. J. Tector1, R. S. Mangus1 1Indiana University School Of Medicine,Transplant Division, Dept Of Surgery,Indianapolis, IN, USA

Introduction:
Deceased organ donor liver transplant allografts with steatosis have an increased risk of primary non-function and initial poor function post-transplant. A large percentage of donor livers have significant steatosis. Previous research suggests improvement in steatosis in the immediate post-transplant period. This study compares reperfusion and early post-transplant surveillance biopsies, and correlates the results with initial graft function and long-term outcomes.

Methods:
Records of all liver transplants (LTs) performed at a single center over a 14-year period were reviewed. The original biopsies were reviewed by experienced liver pathologists. Liver biopsies are obtained at the time of transplant and 3 days after transplant. Total steatosis is calculated as the sum of both micro- and macrovesicular steatosis, and is categorized into four study groups: (1) none (0%), (2) mild (<10%), (3) moderate (10-20%) and (4) severe (>20%). For this analysis, change in liver steatosis is calculated as moving from one study group to another. Early post transplant liver function is assessed by biochemical analysis of liver enzymes (alanine aminotransferase (ALT); liver injury), total bilirubin (TB; excretion), international normalized ratio (INR; synthesis). Long-term survival is assessed using Cox regression analysis.

Results:
Data were available for 1572 adult subjects. Among the patients with steatosis, there was a significant and rapid decrease in steatosis. The median group change was greatest for severe steatosis groups (Group 3, >20%: -1.54; Group 2, 10 to 20%: -0.93; Group 1, 1 to 10%: -0.47 (p<0.001). Moderate and severe steatosis was associated with more acute liver injury (p<0.05 for days 1 to 6), and delayed graft function (higher TB and INR (p<0.05 on days 1, 3)). These values decreased for all study groups until they were similar by day 7 (ALT) and day 14 (TB and INR). Systemically, steatotic groups demonstrated an acute decrease in glomerular filtration rate (GFR) from 1 to 3 days post transplant, ranging from -12 to -22% change, compared to only a -5% change for the nonsteatotic group. Graft survival was worse at all time periods for moderate and severe steatosis livers. Subgroup analysis was employed to identify groups that have a more dynamic decrease in steatosis. Those groups with better clearance of severe steatosis included recipients who were younger, more obese, male, and those with fatty liver disease.

Conclusion:
These results confirm a marked post-transplant decrease in allograft steatosis that occurs within 3 days of transplant. Subgroup analysis suggests that younger male patients who are obese or have fatty liver disease are more able to clear steatosis in this period. Allografts with moderate to severe steatosis have worse early injury, delayed graft function and worse early and late survival. Steatotic grafts are associated with a substantial acute decrease in renal function early post transplant.

21.06 Are NSQIP Hospitals Unique? A Description of Hospitals Participating in ACS NSQIP.

Posted on January 18, 2016

C. R. Sheils1,2, A. R. Dahlke1, A. Yang1, K. Bilimoria1 1Northwestern University,Department Of Surgery,Chicago, IL, USA 2University Of Rochester,School Of Medicine,Rochester, NY, USA

Introduction: The American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) is a well-recognized program for surgical quality measurement. Given the widespread use of ACS NSQIP in research and recent calls for it to become a platform for national public reporting and pay-for-performance initiatives, it is important to understand which types of hospitals elect to participate in the program. Our objective was to compare the characteristics of ACS NSQIP-participating hospitals to non-participating hospitals in the United States.

Methods: Using the 2013 American Hospital Association data on hospital characteristics, hospitals participating in ACS NSQIP were compared to non-participating hospitals. The 2013 Healthcare Cost Report Information System (HCRIS) dataset was used to calculate hospital operating margin as a measure of financial health. The CMS 2013 Inpatient Prospective Payment System (IPPS) Final Rule Impact File was used to abstract the Medicare and Medicaid Services Value Based Purchasing (VBP) and Disproportionate Share adjustment scores, which were used as proxies for hospital quality and patient population, respectively.

Results: Of 3,872 total U.S. general medical and surgical hospitals, 475 (12.3%) participated in ACS NSQIP. ACS NSQIP hospitals performed 29.0% of operations in the U.S, with a slightly greater share of inpatient operations (32.4%) and a smaller share of outpatient operations (27.1%). Compared to non-participating hospitals, ACS NSQIP hospitals had a higher mean annual inpatient surgical case volume (6,426 vs 1,874; p<0.001), a larger number of hospital beds (420 vs 167; p<0.001), were more often academic affiliates (35.2% vs 4.1%; p<0.001), were more often accredited by JCAHO and CoC (p<0.001), and had higher mean operating margins (p<0.05). ACS NSQIP hospitals were less likely to be designated as critical access hospitals (p<0.001). No significant differences in VBP or Disproportionate Share adjustment scores were found. States with the highest percentage of hospitals participating in ACS NSQIP were states with established surgical quality improvement collaboratives (Figure 1).

Conclusion: Hospitals that participate in ACS NSQIP represent 12% of all U.S. hospitals performing inpatient surgery, yet they perform nearly 30% of all surgeries done in the U.S. ACS NSQIP disproportionately includes larger, accredited, and academic-affiliated hospitals with more financial resources. These findings should be taken into account in research studies using ACS NSQIP, and more importantly, indicate that additional efforts are needed to address barriers to enrollment in order to facilitate participation in surgical quality improvement programs by all hospitals.

21.01 Roles of Mentorship and Research in Surgical Career Choice: Longitudinal Study of Medical Students.

Posted on January 18, 2016

A. Berger1, J. Giacalone1, P. Barlow2, M. Kapadia3, J. Keith3 1University Of Iowa,College Of Medicine,Iowa City, IA, USA 2University Of Iowa,Department Of Internal Medicine,Iowa City, IA, USA 3University Of Iowa,Department of Surgery,Iowa City, IA, USA

Introduction: A medical student’s choice to pursue a career in surgery is influenced by many evolving factors and considerations, and likely by experiences during education. We hypothesize that quantifying these variables will reveal trends influencing choice of medical specialty. Our goal is to ascertain trends and factors that can be used to increase interest in surgery, and attract students to pursue a surgical career.

Methods: A questionnaire-based, longitudinal prospective study was conducted at a university program. Surveys were administered to medical students in the class of 2017 prior to the start of the first, second, and third years. The questions cover topics including specialty preferences, debt, mentorship, research, and factors deemed desirable or important in a future career. Residency choices were classified as surgical and nonsurgical. Z-tests for population proportions and odds ratios were calculated using SAS/STAT software.

Results: Data was collected from 143, 139, and 144 students at the beginning of their first, second, and third medical school year, respectively, and response rates were >90%. Students with any research experience prior to medical school were 64% less likely (p=0.016) to have surgical career interests in the first survey. By the third year, however, students expressing interest in surgery were 2.5 times more likely to be actively involved in research (p=0.029) and 8.4 times more likely to have a surgery-related research focus (p=0.0001), as compared to non-surgery classmates. At the beginning of their third year, students with an interest in surgical specialties were 2.2 times more likely to have a self-maintained surgeon-mentor relationship (p=0.031), as compared to students interested in non-surgical specialties. With near significance, students initially expressing interest in surgical specialties are 2.4 times more likely to change their interest to ‘undecided’ in future surveys (p=0.06). Students without both research experience and active surgeon-mentor relationships are 9 times more likely to switch to ‘undecided’ or non-surgical specialties (0.02619). Furthermore, students involved in research, but without a mentor, are 20 times more likely to change their interests to ‘undecided’ or non-surgical specialties (p=0.0012), compared to those with both research and active surgeon-mentor relationships.

Conclusion: Students involved in surgery-specific research are significantly more likely to continue expressing interest in a surgical career if they have a mentor. In the absence of a mentor, students involved in research are more likely to change interests than those not involved in research at all. Our preliminary conclusion is that while research attracts students to consider surgical specialties, meaningful surgeon-mentor relationships are essential to maintain a student’s interest.

21.02 Amitriptyline Treatment Improves Survival After Trauma and Hemorrhage

Posted on January 18, 2016

H. He1,2, P. L. Jernigan1,2, R. S. Hoehn1,2, A. L. Chang1,2, L. Friend1,2, R. Veile1,2, T. Johannigman1,2, A. T. Makely1,2, M. D. Goodman1,2, T. A. Pritts1,2 1University Of Cincinnati,Trauma And Critical Care,Cincinnati, OH, USA 2University Of Cincinnati,Institute For Military Medicine,Cincinnati, OH, USA

Introduction: Hemorrhagic shock is the leading cause of potentially preventable death after trauma. The optimal treatment for hemorrhagic shock is to reverse circulatory losses, metabolic acidosis, and cellular hypoxia with blood products, but these are often not available for immediate administration. Alternative approaches to initial resuscitation are needed, especially in resource poor environments. Amitriptyline is a serotonin-norepinephrine reuptake inhibitor with anti-inflammatory effects. We have previously demonstrated that treatment of blood products with amitriptyline leads to decreased lung injury after hemorrhage, but the direct effect of amitriptyline in treatment of hemorrhage is unknown. We hypothesized that administration of amitriptyline after trauma and hemorrhage would improve survival in a non-resuscitation injury model.

Methods: Healthy C57/BL6 male mice underwent laparotomy to induce tissue trauma and hemorrhage via femoral artery cannulation to a mean arterial pressure of 25±5mmHg for 60 minutes. After laparotomy closure and decannulation, mice received amitriptyline (0.1 mg/kg) or an equivalent volume of vehicle (50 uL of normal saline) via intraperitoneal injection. For survival analysis, mice underwent the above treatment (n=10/group) and were monitored for 24 hours. For hemodynamic and blood chemistry analysis, mice (n=5/group) underwent the same injuries and treatments as described above and were sacrificed 60 minutes after treatment.

Results: Administration of amitriptyline after trauma and hemorrhage significantly increased 24-hour survival in mice (Figure 1; 70% survival with amitriptyline vs 0% with vehicle; p<0.001). Sixty minutes after trauma and hemorrhage, mice treated with amitriptyline also had significantly increased mean arterial blood pressure (p=0.04). Blood gas analysis revealed that mice treated with amitriptyline had statistically significant improvements in base deficit and serum bicarbonate, consistent with decreased metabolic acidosis.

Conclusion: Our results demonstrate that administration of amitriptyline significantly improves survival in mice after trauma and hemorrhage, even in the absence of resuscitation. We found that amitriptyline increases mean arterial blood pressure and decreases metabolic acidosis after injury. Administration of amitriptyline following traumatic injury could potentially be useful in situations with limited access to blood products.

21.03 Hospital Teaching Status and Medicare Expenditures for Complex Surgery

Posted on January 18, 2016

J. C. Pradarelli1, C. P. Scally1, H. Nathan1, J. R. Thumma1, J. B. Dimick1 1University Of Michigan,Ann Arbor, MI, USA

Introduction:
Several emerging payment policies penalize hospitals for higher costs. Teaching hospitals may be at a disadvantage given the perception that they deliver care less efficiently.

Methods:
We studied Medicare patients who underwent abdominal aortic aneurysm (AAA) repair (n=74,767), colectomy (n=288,378), or pulmonary resection (n=94,629) from 2009 to 2012. Patients’ hospitals were categorized into quintiles of teaching intensity (very major, major, minor, very minor, and non-teaching hospitals) based on the resident-to-bed ratio. Risk-adjusted 30-day Medicare payments were price-standardized to account for social subsidies and regional variation in costs. Risk-adjusted perioperative outcomes were also assessed.

Results:
Comparing risk-adjusted Medicare payments per episode of surgery, very major teaching hospitals were $13,947 more expensive than non-teaching hospitals for AAA repair ($45,632 vs. $31,685; p<0.001), $19,315 more expensive for colectomy ($52,199 vs. $32,884; p<0.001), and $9,788 more expensive for pulmonary resection ($39,513 vs. $29,725; p<0.001). However, after accounting for social subsidies and regional variation in Medicare payments, very major teaching hospitals were paid only $1,811 more than were non-teaching hospitals for AAA repair ($30,030 vs. $28,219; p=0.35), $4,701 more for colectomy ($35,182 vs. $30,480; p<0.001), and $1,424 less for pulmonary resection ($25,373 vs. $26,796; p=1.00). Very major teaching hospitals generally had higher risk-adjusted rates of serious complications and readmissions, but lower risk-adjusted rates of failure to rescue and 30-day mortality than did non-teaching hospitals.

Conclusion:
After price-standardization to account for intended differences in payments, risk-adjusted Medicare payments for an episode of surgical care were similar at teaching hospitals and non-teaching hospitals for three inpatient operations.

02.19 Hypoxia Inducible Factor-1α Modulates Pro-Tumorigenic Macrophage Activation Induced by Cancer Cells

Posted on January 18, 2016

J. Yi1, E. E. Moore1,2, R. D. Schulick1, B. Edil1, K. C. El Kasmi1, C. C. Barnett1,2 1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA

Introduction: Alternative activation of tumor-associated macrophages is pro-tumorigenic and associated with poor prognosis. Similarly, upregulation of tumor-expressed hypoxia inducible factor-1α (HIF1α) augments tumor progression. We hypothesize that tumor derived mediators activate macrophages towards an alternative activation phenotype. Further, inhibition of macrophage HIF1α signaling will mitigate cancer cell-induced alternative macrophage activation.

Methods: Pan02 pancreatic and MC38 colon adenocarcinoma murine cell lines were grown under uniform conditions to create cancer-conditioned media (CCM) rich in tumor-derived factors. YC1, a HIF1α inhibitor, and dimethyloxalylglycine (DMOG), a HIF1α inducer, were used to modulate HIF1α activity of RAW 264.7 murine macrophages. Macrophages were pretreated with YC1 or DMOG for 6 hours prior to 18 hours of CCM exposure and compared to vehicle controls. Polymerase chain reaction was used to phenotype macrophages based on expression of canonical activation genes (Socs1=classical activation, Socs3=alternative activation). Expression of HIF1α target gene vascular endothelial growth factor (Vegfa) was used to determine HIF1α modulation.

Results: CCM exposure induced mild Socs1 expression in macrophages, representing minimal classical/pro-inflammatory activation (Pan02 1.55±0.02 fold-increase, p<0.0001; MC38 2.09±0.19 fold-increase, p<0.0001). CCM exposure greatly induced Socs3 expression (Pan02 11.34 ±0.29 fold-increase, p<0.0001; MC38 962.90±53.88 fold-increase, p<0.0001). YC-1 pretreatment reduced Socs3 expression (Pan02 0.19±0.03 fold-decrease, p<0.0001; MC38 0.13±0.01 fold-decrease, p<0.0001), while DMOG pretreatment increased Socs3 expression (Pan02 6.09±2.80 fold-increase, p=0.0118; MC38 8.73±0.71 fold-increase, p=0.0006). Correspondingly, Vegfa expression increased with DMOG (Pan02 1.75±0.11 fold-increase, p<0.0001) and decreased with YC1 pre-treatment among CCM-exposed macrophages (Pan02 0.44±0.02 fold-decrease, p<0.0001) as compared to CCM alone. (See Figure)

Conclusion: Conditioned media from pancreatic and colon adenocarcinoma induces alternative activation as represented by increased Socs3 expression. Pretreatment with the HIF1α inhibitor YC1 mitigates CCM-induced alternative activation, while pretreatment with the HIF1α inducer DMOG augments alternative activation. This was dependent upon HIF1α activity, as shown by the modulation in expression of HIF1α target gene Vegfa among treated macrophages. These data demonstrate that targeting HIF1α effectively modulates pro-tumorigenic alternative macrophage activation.

02.20 RON Kinase Isoforms Demonstrate Variable Cell Motility in Normal Cells

Posted on January 18, 2016

A. Greenbaum1, G. Wan1, A. Rajput1 1University Of New Mexico,Department Of Surgery,Albuquerque, NEW MEXICO, USA

Introduction: Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility.

Methods: Cell lines with stable expression of wtRON were generated by inserting the coding region of RON into pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON165, RON160, and RON155) were generated by creating a mutagenesis-based wtRON-pTagRFP plasmid. These expression plasmids (RON-pTagRFP, RON165-pTagRFP, RON160-pTagRFP and RON155-pTagRFP) were then stably transfected into HEK293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants.

Results: Figure 1. All RON transfected cells increased cell motility compared to HEK293 control cells (RFP). WtRON, RON155 and RON 165 demonstrated increased cell motility compared to the RON 160 isoform at 12 hours.

Conclusion: RON tyrosine kinase isoforms have variable cell motility. This could reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

02.21 Can Cancer Cell Lines Clarify Molecular Mechanisms of Hereditary Non-Polyposis Colorectal Cancer?

Posted on January 18, 2016

H. L. Roberts1, M. McClain1, J. Rice1, J. Carter1, J. Burton1, S. Galandiuk1 1University Of Louisville School Of Medicine,Department Of Surgery, Division Of Colorectal Surgery,Louisville, KY, USA

Introduction:
Sporadic colon cancer (CC) is commonly caused by chromosomal instability, while hereditary non-polyposis colorectal cancer (HNPCC) is characterized by microsatellite instability (MSI). HNPCC is associated with different clinical manifestations and improved survival but respond poorly to 5-FU based chemotherapy compared to sporadic CC patients.

HCT116 is a widely used experimental Dukes’ D CC cell line not commonly known to be derived from an HNPCC patient. A molecular characteristic of HNPCC is inherited mutations in DNA mismatch repair (MMR) leading to MSI. We have demonstrated different microRNA (miR) expression patterns between HCT116 and other sporadic CC cell lines with respect to miR-99a. This miR is an established inhibitor of mammalian Target of Rapamycin (mTOR). Increases in mTOR protien have been shown to increase cell growth, proliferation, migration, invasion and decrease apoptosis. Thus miR-99a is a potential pathway for CC treatment and metasistis. We hypothesize the influence of miR-99a on the mTOR pathway, with respect to cell proliferation and motility, is dissimilar between MMR proficient (MMR+) and deficient (MMR-) CC cell lines.

Methods:
Dukes C and Dukes D MMR- (HCT15, HCT116), MMR+ (HT29, T84) and normal colon epithelium (CCD841) cell lines (ATCC®) were transfected with miR-99a mimic (99M) and a negative control (M). Transfection efficiency was verified via qPCR. mRNA and protein substrates of both mTOR complexes were analyzed by qPCR and western blot, respectively. Functional assays were performed measuring cell migration and invasion.

Results:
All cell lines were successfully transfected and showing significant upregulation of miR-99a (p<0.001). mRNA levels of proteins of interest were unchanged for all cell lines measured irrespective of transfection group. After transfection with 99M, total mTOR protein was decreased as compared to M- for all cell lines. Migration decreased after transfection with 99M for all cell lines as compared to M- except for the HCT116 cell line (Figure 1). Invasion assays showed no difference in either transfection group.

Conclusion:
HCT116 showed increased migration following transfection with 99M as compared to M- whereas all other cell lines exhibited decreased migration regardless of MMR status and CC stage of the cell line. We intend to investigate further to identify different pathways involved in HNPCC that may permit development of more effective adjuvant therapy for MMR deficient cancer patients with advanced disease.

02.16 Sphere Formation by Neuroblastoma Stem-like Cells is Blocked by 13-cis-Retinoic Acid

Posted on January 18, 2016

B. T. Craig1, E. J. Rellinger1, A. L. Alvarez1, J. Qiao1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA

Introduction: Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, the mechanisms that are responsible for sphere formation remain poorly understood. We hypothesize that a dedifferentiation program mediates neuroblastoma sphere formation and that more stem cell-like cell lines will form spheres at a higher rate than their more differentiated counterparts.

Methods: Four human neuroblastoma cell lines (BE(2)-C, I-type, MYCN amplified; IMR-32, N-type, MYCN amplified; SK-N-SH, S-type, MYCN single copy; SK-N-AS, S-type, MYCN single copy) were examined for intrinsic frequency of sphere formation by limiting dilution analysis in serum-free media supplemented with EGF (20 ng/ml) and bFGF (40 ng/ml). 13-cis-retinoic acid (RA, 5 µM) is clinically used as a differentiating agent for high-risk neuroblastoma. Protein levels were assessed by SDS-PAGE. Multiple group comparisons were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Two group comparisons were analyzed by two-tailed unpaired Student’s t test. In all cases a p value of <0.05 was considered significant.

Results: Both MYCN amplified and MYCN single copy cell lines formed spheres, contrary to previous reports that this phenotype depended on MYCN amplification. BE(2)-C cells demonstrated the highest frequency of sphere formation and significantly differed from the other 3 cell lines (6.58% vs. 0.7%, 1.5% and 2.26%, p<0.05). bFGF regulated sphere formation in a dose-dependent fashion while EGF did not (20-60 ng/ml, 3-5x fold change, p<0.05). Serial passage in sphere culture conditions increased frequency of sphere formation two fold (p <0.05), and increased expression of the stem cell factor Oct4. The related factor Nanog did not change expression from baseline. 13-cis-RA (5 µM) nearly completely blocked sphere formation in the BE(2)-C cell line (6.58% vs. 0.57%, p<0.05).

Conclusion: BE(2)-C cells are I-type neuroblastoma cells that have previously been shown to exhibit stem cell features. BE(2)-C is the most avid sphere-forming cell line, and this phenotype is inhibited by induced differentiation with 13-cis-RA. Furthermore, sphere culture affected the expression level of a critical stem cell factor in a manner consistent with previous reports. Hence, the in vitro model system of sphere formation correlates with known parameters of stem cell-like behavior in neuroblastoma and is therefore a valid system for the study of neuroblastoma stem cells.

02.17 Lung-Selective Delivery of PAN-PI3K Inhibitor-Loaded Nanoparticles as Treatment for CRC Metastasis

Posted on January 18, 2016

P. Rychahou1,2, Y. Bae1,4, Y. Zaytseva1, E. Y. Lee1,3, H. L. Weiss1, B. M. Evers1,2 1University Of Kentucky,Markey Cancer Center,Lexington, KY, USA 2University Of Kentucky,Department Of Surgery,Lexington, KY, USA 3University Of Kentucky,Department Of Pathology And Laboratory Medicine,Lexington, KY, USA 4University Of Kentucky,Department Of Pharmaceutical Sciences,Lexington, KY, USA

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is important for CRC progression and metastasis; inhibitors have been developed that are being evaluated in clinical trials with limited success due to systemic toxicity. The purpose of our study was to: (i) determine expression of pAkt (Ser473), Akt1 and Akt2 in primary and metastatic CRCs, and (ii) develop an effective nanocarrier for lung-selective delivery of pan-PI3K inhibitors as targeted therapy of CRC lung metastasis.

Methods: (1) To determine the expression of PI3K/Akt pathway components, we obtained primary CRCs (n=12) and CRC lung metastases (n=10). All samples were tested for pAkt (Ser473), Akt1 and Akt2 expression by immunohistochemistry (IHC) and blindly scored by a pathologist. (2) Polymeric nanoparticles were constructed and loaded with either fluorescent dye (Alexa 547) or pan-PI3K inhibitors (either PX-866 or wortmannin). Lung selective accumulation of fluorescently-labeled nanoparticles was confirmed by confocal imaging of frozen tissue sections from lung, liver and spleen. Selective PI3K inhibition in lung tissue was confirmed by western blot of protein extracts from lung, liver, spleen and kidney after intravenous administration of PX-866-loaded nanoparticles.

Results: (1) Increased pAkt (Ser473) expression was detected in 80% of primary CRCs and CRC lung metastases; 60% of the samples demonstrated markedly elevated expression of Akt2. (2) Treatment with PX-866, an irreversible pan-PI3K inhibitor currently in the clinic, inhibited cell cycle progression and induced apoptosis in patient-derived CRC organotypic cultures, CRC stem cell lines and pik3ca mutant CRC cells. (3) Importantly, in vivo treatment with pan-PI3K-loaded nanoparticles demonstrated a marked suppression of lung metastasis growth using a clinically-relevant CRC lung metastasis model.

Conclusion: We demonstrate, for the first time, safe and efficient delivery of drug-loaded nanocarriers to lung metastases, suggesting that lung selective PI3K inhibition is a viable treatment strategy for CRC lung metastasis.

02.18 Novel Small Molecule ML327 Sensitizes Colon Cancer Cells to the TRAIL Ligand

Posted on January 18, 2016

C. Padmanabhan1, C. W. Lindsley5,6, A. G. Waterson5,6, R. D. Beauchamp1,2,3,4 1Vanderbilt University Medical Center,Surgery,Nashville, TN, USA 2Vanderbilt University Medical Center,Cell And Developmental Bioogy,Nashville, TN, USA 3Vanderbilt University Medical Center,Cancer Biology,Nashville, TN, USA 4Vanderbilt University Medical Center,Ingram Cancer Center,Nashville, TN, USA 5Vanderbilt University Medical Center,Chemistry,Nashville, TN, USA 6Vanderbilt University Medical Center,Pharmacology,Nashville, TN, USA

Introduction:

Colorectal cancer (CRC) is the third most common cancer diagnosis and the third most common cause of cancer related death in the United States. The inability of chemotherapy to eradicate quiescent cancer stem cells is a leading hypothesis for cancer recurrence. New therapies that do not rely on cellular proliferation for cytotoxic effect must be developed. The tumor necrosis factor related apoptosis inducing ligand (TRAIL), which selectively induces apoptosis in cancer cells irrespective of cell proliferation, was thought to be such a therapy but the anti-cancer effect identified in preclinical models was not realized in clinical trials due to resistance. Our lab has identified a novel small molecule (ML327) that sensitizes colorectal cancer cell lines to TRAIL. We hypothesize this effect is due to loss of the anti-apoptotic protein c-FLIP, a potent inhibitor of the extrinsic apoptosis pathway initiated by TRAIL.

Methods:

CRC cell lines were pre-treated with ML327 or vehicle control at a 10-μM concentration for 24 hours. TRAIL was then added at 100 ng/mL for 4 hours. Cells were lysed and Western blot was performed with antibodies against cFLIP and cleaved caspase 3. Cells were also fixed with 70% ethanol and stained with propidum iodide for FACS analysis. Cells were then treated with ML327 at a 10-μM concentration for up to 4 hours and lysed at hourly increments. Western blot was performed with antibodies against cFLIP.

Results:

24 hours of ML327, but not vehicle, pre-treatment increased TRAIL-induced cleaved caspase 3 protein levels (Fig. A). cFLIP protein level was abundant in vehicle treated cells but was nearly undetectable after 24 hour ML327 treatment (Fig. B). These findings were associated with increased cell death after TRAIL exposure as confirmed by a statistically significant increase (p = 0.002) in the Sub G0 population on FACS analysis (Fig. C). ML327-induced reductions in cFLIP protein occurred quickly, with reduced levels evident by 1 hour and complete loss by 4 hours (Fig. D).

Conclusion:

ML327 is a novel small molecule that sensitizes CRC cells to the TRAIL ligand as evident by increased apoptosis and subsequent cell death. This sensitization is associated with loss of cFLIP. Ongoing analysis will determine whether this is a cause and effect association and further elucidate the exact mechanism of cFLIP loss. It will be of interest to determine the in vivo efficacy of ML327 induced TRAIL sensitization. With these experiments, we hope to develop a novel therapeutic that will induce cell death in cancer cells irrespective of cell proliferation.

02.12 Notch-2 and -4 May Mediate Vemurafenib Drug Resistance in Melanoma

Posted on January 18, 2016

J. Sheldon1, G. Khaushik1, P. Dandawante1, S. Anant1, J. M. Mammen1 1University Of Kansas,Surgery,Kansas City, KS, USA

Introduction:
The incidence of melanoma has increased dramatically over the last few decades making it one of the fastest growing malignancies in the United States. Melanoma expresses a plastic and aggressive phenotype, lacking the majority of regulatory mechanisms due to the aberrant activation of various signaling pathways. In this context, aberrant activation of the Notch signaling pathway in melanoma has also been reported by our laboratory as well as by others. Also of importance, the oncogenic mutation predominantly at codon 600 in the BRAF gene (present in nearly 40–50% of melanoma patients) has changed treatment options for melanoma over the past decade. Unfortunately, recent studies report that the majority of patients treated with the BRAF inhibitors vemurafenib and dabrafenib eventually develop drug resistance. The mechanisms of resistance are still poorly understood. In the present study, we explore the role of Notch signaling in vemurafenib induced drug resistance in melanoma cells.

Methods:

For our experiments, we used various melanoma cell lines (SKMEL-28, UACC275 and A2058 cells). The hexoseaminidase assay was used to determine cell proliferation and to calculate the IC50 (drug concentration causing 50% growth inhibition) in drug sensitive and drug resistant cell lines. Drug-resistant cells were developed by repeatedly growing cells in culture media with increasing doses of drug with time. The surviving daughter resistant cells were compared to the parental sensitive cells using proliferation assay. The IC50 for these paired cell lines was used to determine the increase in resistance. Protein expression studies in cells were done by using standard immunoblotting techniques.

Results:
We observed an increased IC50 value of vemurafenib in drug resistant UACC275 cells. IC50 values at the 72hr time point for UACC-RV-10, UACC-RV-10(1), UACC-RV-15(1) and UACC-RV-15(2) (UACC275 isogenic cells) were ~15, 14, 25 and 22 µM respectively as compared to the IC50 value for parental wild type UACC275 cells of ~0.50 µM. We observed a similar pattern of increased IC50 values in other melanoma cell lines (B16/F10, SKMEL-28, A2058, M14, and melanoma patient derived cells). After several passage of culture in vemurafenib, we also noted changes in cell morphology with cells becoming small and round compared to their usual elongated and stretched appearance. We further evaluated the expression pattern of various Notch receptors in drug sensitive cells as compared to drug resistant isogenic cell lines. Levels of cleaved Notch-2 (10 fold) and -4 (3 fold) were significantly higher in drug resistant cells.(n=5)

Conclusion:

A reductionist model of vemurafenib resistance can be developed using the UACC275 cell line. Both Notch-2 and -4 levels are higher in vemurafenib resistant melanoma cells in this model. Notch signaling may directly or indirectly play an important role in the development of vemurafenib drug resistance in melanoma cells.

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