30.06 Node-negative Rectal Cancer after neoadjuvant therapy: How many lymph nodes should be removed?

Posted on December 22, 2014

M. Raoof1, V. N. Nfonsam1, J. Warneke1, R. Krouse1,2  1University Of Arizona College Of Medicine,Surgical Oncology,Tucson, AZ, USA 2Southern Arizona Veterans Affairs Health Care System,Surgical Care Line,Tucson, AZ, USA

Introduction:  Neoadjuvant therapy for locally advanced rectal cancer reduces the yield of lymph node (LN) sampling.  In the setting of pathologically negative LNs after neoadjuvant therapy, the significance of the number of LNs retrieved is unclear. Studies in the literature have reported conflicting results. We hypothesized that the number of negative lymph nodes harvested is related to long-term survival.

Methods:  This is a study of a contemporary cohort from NCI’s SEER database over a 7-year period (Jan, 2004- Nov, 2010).  Patients with locally advanced rectal adenocarcinoma who underwent neoadjuvant therapy and had pathologically negative LNs in resected specimens were included. The collaborative staging fields in the SEER database record worst T-stage (i.e. clinical T-stage unless pathological T-stage was worse, in which case pathological T-stage is recorded). This allowed a unique opportunity to distinguish T-stage progression vs. T-stage stable disease or regression. Pathologic LN stage was available for all patients and by inclusion criteria were N0. Clinical LN status was available for 73% of patients. Survival analysis was performed using the Kaplan-Meier method. Inferential statistics were performed using a Cox-proportional hazard model.

Results: Of the total 3864 patients that met the strict inclusion criteria, 65% were males, 82% were white and 51% were ≥60 years of age. Majority had T3 (T1, 9%; T2, 19%; T3, 67%, T4 5%), clinical LN positive (Positive, 44%; Negative, 29%; Unknown, 27%) and moderately differentiated (G1, 6%; G2, 71%; G3, 10%; G4, 0.5%) tumors. Median lymph nodes retrieved were 12 (IQR 7-16). Median follow-up was 28 months (IQR 12-47). Using the minimum p-value approach we identified a cut-off of 7 LNs provides oprtimal stratification of patients in terms of overall survival (Figure). A multivariate Cox-proportional hazard model adjusted for age, race, T-stage, grade, clinical LN status and T-stage response to neoadjuvant therapy demonstrated that patients who have >7 LNs examined have a better overall (hazard ratio 0.66; 95% CI 0.53-0.81; p < .0001) and cancer-specific survival (hazard ratio 0.64; 95% CI 0.50-0.83; p = 0.001) compared with patients who had ≤7 LNs examined.

Conclusion: This is the largest study to date that demonstrates the prognostic significance of LN sampling in patients with rectal cancer after neoadjuvant therapy.  Patients with less than seven LNs harvested have worse survival and should be considered for more aggressive adjuvant therapy and follow-up strategy.
 

30.07 Increased Malignancy Rates in Surgical Patients with Incidentally-Discovered Thyroid Nodules

Posted on December 22, 2014

A. R. Marcadis1, M. Rodriguez1, S. Liu1, B. Wang1, J. I. Lew1  1University Of Miami Miller School Of Medicine,Division Of Endocrine Surgery,Miami, FL, USA

Introduction: With the advent of better imaging technology and its widespread utilization in the clinic setting, incidental thyroid nodules are often discovered while evaluating patients for unrelated disease. If the risk of underlying thyroid malignancy in such incidental nodules is common, further evaluation is required. However, if thyroid cancers are exceedingly rare in such incidentally discovered thyroid nodules, further costly evaluations can be avoided. The purpose of this study is to compare the rate of malignancy in incidentally-discovered thyroid nodules (IDTN) by imaging to nonincidentally-discovered thyroid nodules (NDTN) in surgical patients.

Methods: A retrospective review of prospectively collected data of 1370 patients who underwent thyroidectomy at a single institution was performed. Before surgical resection, all patients underwent surgeon-performed ultrasound (SUS) and fine-needle aspiration (FNA). Patients who had IDTN by imaging studies for unrelated reasons (n=536) and patients who had NDTN (n=834) were further stratified according to age, gender, FNA results, SUS characteristics and final pathology. Rates of malignancy in IDTN and NDTN were calculated and statistical significance was determined by  two-tailed Z-test.

Results: Of 1370 patients, 536 presented with IDTN by imaging and 306 (57%) were found to have malignancy on final pathology. Of patients with IDTN found to have malignancy, 92% (n=283) had papillary thyroid cancer, 5% (n=14) medullary thyroid cancer, and 3% (n=9) follicular thyroid cancer. Of the patients with NDTN, 401 (48%) were found to have malignancy following surgery with 44% (n=366) papillary thyroid cancer (PTC), 2.2% (n=18) medullary thyroid cancer, 2% (n=14) follicular thyroid cancer, and <1% anaplastic thyroid cancer (n=3). The rate of PTC on final pathology for IDTN was statistically higher than the rate of PTC found in NDTN (p<0.05). Patients with IDTN had statistically higher rates of microcalcifications (34%), hypoechogenicity (60%), and irregular borders (35%) by SUS compared to patients with NDTN (p<0.05). There was also a significantly higher rate of patients with IDTN who had all three SUS features together (14%) compared to patients with NDTN (10%) (p<0.05). There was no significant difference in percentage of malignant FNA results between patients with IDTN and NDTN.

Conclusion: For IDTN, malignancy rates may be higher than expected in surgical patients. Furthermore, certain characteristics of SUS may help predict malignancy in IDTN. The high rate of malignancy suggests that total thyroidectomy by an experienced surgeon should be strongly considered when managing such patients with IDTN.

 

30.08 Stoma Creation and Reversal after Cytoreductive Surgery with Heated Intraperitoneal Chemotherapy

Posted on December 22, 2014

A. N. Doud1, N. Fitzgerald2, E. Levine1, P. Shen1, J. Stewart1, K. Votanopoulos1  1Wake Forest University,Department Of General Surgery, Surgical Oncology Service,Winston-Salem, NC, USA 2Wake Forest University,Division Of Public Health Sciences, Department Of Biostatistical Sciences,Winston-Salem, NC, USA

Introduction:  

Stoma creation is often included as a part of Cytoreductive Surgery with Heated Intraperitoneal Chemotherapy (CRS/HIPEC).  Herein, we evaluate the indications for stoma creation during CRS/HIPEC procedures as well as the morbidity and mortality associated with stoma reversal after CRS/HIPEC procedures.

Methods:  

A retrospective analysis of a prospective database of 1149 CRS/HIPEC procedures was performed.  Patient demographics, type of malignancy, comorbidities, Clavien-graded morbidity, mortality and overall survival were abstracted.  Patients were grouped into those with and without stoma creation and documented indications for stoma creation and reversal were recorded and analyzed.

Results: 

16% (186/1149) of CRS/HIPEC procedures included stoma creation, while 1.1% (11/963) of patients without stoma creation during CRS/HIPEC developed anastomotic leaks requiring reoperation with stoma formation.  This resulted in 197 stomas after CRS/HIPEC, of which 37.6% (74/197) were end ileostomies, 27.9% (55/197) were loop ileostomies, 23.9% (47/197) were end colostomies and 8.6% (17/197) were loop colostomies.  Patients requiring stoma at initial operation had worse preoperative performance status (ECOG 0/1: 77.2% vs 86.11%, p=0.002), worse preoperative nutrition (mean albumin 3.69 vs 3.78, p=0.03), greater burden of disease (PCI 18.57 vs. 12.90, p<0.0001) and were more likely to have R2 resections (74.19% vs 48.69%, p<0.0001) than those without stoma creation.  18.6% (36/197) of the stomas were intended to be permanent.  Of the 161 patients with potentially reversible ostomies, only 26.7% (43/161) had their ostomies reversed.   Patients undergoing reversal were more likely to have complete cytoreductions than those not undergoing reversal (41.9% vs 23.7%, p=0.0007).  The most common reasons for failure to reverse were disease progression (43/161, 26.7%) and death (40/161, 24.8%).  After reversal, 27.9% (12/43) suffered a Clavien I/II minor morbidity, 27.9% (12/43) suffered Clavien III/IV major morbidity, and 30 day mortality was 4.7% (2/43). Anastomotic leak occurred after 9% (3/33) of ileostomy and 10% (1/10) of colostomy reversals.

Conclusion: 

Stoma creation during CRS/HIPEC is more common in patients with higher burden of disease, incomplete CRS and poor functional status.  Less than half of the patients with complete CRS will have their stomas reversed.  Stoma reversal after CRS/HIPEC has a significant risk of major morbidity that should be taken into consideration especially if postoperative systemic chemotherapy is part of the treatment strategy.  Patients with high burden of disease and poor functional status should be counseled prior to CRS/HIPEC as to their increased risk of stoma creation and their low likelihood of successful reversal.

 

30.09 The Utility of Afirma Gene Expression Classifier in the Management of Indeterminate Thyroid Nodules

Posted on December 22, 2014

L. Zhou1, K. Patel1  1New York University School Of Medicine,Endocrine Surgery,New York, NY, USA

Introduction:
The Afirma® Gene Expression Classifier (AGEC) has been designed to identify benign thyroid nodules among those classified as cytologically indeterminate with a negative predictive value of 94%–95%. This test therefore has the potential of avoiding unnecessary surgery on cytologically indeterminate nodules which are likely to be benign. The objective of this study was to examine the impact of AGEC on the management of thyroid nodules at a single institution and appreciate the importance of how institutional malignancy rates may affect clinical decision making and patient counseling.

Methods:
A retrospective cohort analysis of all patients treated by the authors with thyroid nodules classified as Bethesda category III or IV, who had AGEC testing. Only patients with cytologic diagnosis, AGEC and final pathology were included. Cytology was categorized according to the Bethesda classification system. The dataset was analyzed to determine the benefit of AGEC testing in our patient population.

Results:
A total of 54 patients were included in the study. 44 had initial cytologic diagnosis categorized as Bethesda III and 10 were categorized as Bethesda IV. Of the 54 GECs performed, 15 were benign and 39 were suspicious. 13/15 (86.7%) patients with benign GEC testing opted for close observation and 2/15 (13.3%) opted for surgery, both had benign disease on final pathology. Surgery was avoided in 13/54 (24%) patients. 23/39 (59.0%) patients with suspicious GEC testing have had surgery and their pathology is as follows: 11/23 (47.8%) benign, 5/23 (21.7%) papillary thyroid cancer (PTC), and 7/23 (30.4%) follicular variant PTC. The PPV of AGEC “suspicious” in our patient population is 52.2%. The historical prevalence of malignancy amongst cytologically indeterminate lesions (Bethesda III and IV) at NYU is 35%. Based on this, the NPV of AGEC at our institution is closer to 91%.

Conclusion:
Malignancy rates in cytologically indeterminate thyroid nodules vary widely across institutions. Utilization of the AGEC helped avoid surgery in 24% of patients with cytologically indeterminate thyroid nodules. However, given our center’s higher prevalence of malignancy in indeterminate nodules, slightly higher PPV and slightly lower NPV were observed compared to previously published rates. This information must be taken into consideration when making clinical decisions and counseling patients in regards to AGEC testing.
 

30.10 Signet Ring Carcinoma of the Pancreas: A Case Series of 621 Patients

Posted on December 22, 2014

K. Mahendraraj1,2, J. Chabot2, M. Kluger2  1Saint Barnabas Medical Center,Department Of Surgery,Livingston, NJ, USA 2Columbia University College Of Physicians And Surgeons,Department Of Surgery,New York, NY, USA

Introduction:
Signet ring carcinoma of the pancreas (SRC) is a rare and aggressive mucin-producing exocrine pancreatic malignancy, comprising less than 1% of all pancreatic carcinoma. Clinical information relating to this disease is scant and derived primarily from several case reports. This study sexamines a large cohort of SRC in order to determine characteristic demographic and pathologic factors of SRC and compare them to the relatively more common pancreatic ductal adenocarcinoma (PDAC). 

Methods:
Demographic and clinical data on 103,341 patients with pancreatic cancer was abstracted from the SEER database (1973-2010). 621 SRC and 102,720 PDAC patients formed the study groups. Data was analyzed using standard statistical methodoloy. 

Results:
SRC comprised 0.6% of all pancreatic cancers identified. The mean age of the SRC and PDAC patients were similar (67.5±12 vs. 68.2±11.7). Both SRC and PDAC were more common in males (54.1% and 51.1% (PDAC), p=0.08), Caucasians (74.1% and 74.7% (PDAC), p=0.005) and in the head of the pancreas (48.8% and 50.9% (PDAC), p<0.005). SRC was more often poorly differentiated (81.3%), undifferentiated (7.1%), had more lymph node positivity (50.4%), and presented more often with metastatic disease (68.1%), p<0.001. The majority of SRC and PDAC patients were not treated at all (70% and 71.8% (PDAC)), and surgery was used more often than radiotherapy to treat SRC (14% vs. 10.4%). Mean survival for SRC was significantly lower than PDAC (0.47±0.1 years vs. 0.85±0 years; p<0.05). Furthermore, mean survival for SRC patients treated with primary surgical therapy or combination surgery and radiotherapy were also significantly inferior to PDAC despite receiving equivalent therapy, p<0.001. However, SRC patients benefited more from primary radiotherapy than PDAC patients (1.05±0.17 vs. 0.93±0.02 years, p<0.001). Multivariate analysis identified tumor size over 2 cm (OR 1.4), metastatic disease (OR 1.8), and poor or undifferentiated grade (OR 2.3) as risk factors for mortality for SRC, p<0.005. A survival advantage for SRC was seen in patients treated with surgical resection (OR 0.36), p<0.005

Conclusion:
Pancreatic SRC is a rare malignancy that presents with larger tumor size, a more undifferentiated histology, greater lymph node positivity, and a higher rate of metastatic disease than PDAC, features which were associated with increased mortality. Despite these aggressive features, surgical resection with or without radiotherapy was associated with the longest survival for SRC. Surgical resection and radiotherapy should be considered in all SRC patients with resectable disease.

32.02 Clinicopathologic Features and Time Interval Analysis of Contralateral Breast Cancers

Posted on December 22, 2014

E. L. Liederbach1, R. Piro1, R. Watkin1, K. Hughes1, C. Wang2, C. Pesce1, D. J. Winchester1, K. Yao1  1Northshore University Health System,Surgery,Evanston, IL, USA 2Northshore University Health System,Center For Biomedical Research Informatics,Evanston, IL, USA

Introduction: Multiple studies have shown that contralateral prophylactic mastectomy improves survival, but follow-up for most of these studies are five years or less and provide little data on tumor characteristics of the contralateral breast cancer (CBC). We hypothesized that most CBCs develop after five years and that these CBCs have favorable tumor characteristics.

Methods: This is a single institution retrospective review of 323 patients who were diagnosed with CBCs from 1990 to 2014. CBCs were diagnosed at least one year after the primary cancer diagnosis. BRCA mutation carriers were not excluded. Utilizing chi-square tests and one-way ANOVA tests, we examined the time interval and pathological features between the primary and contralateral cancer.

Results: The average time interval between the primary and CBC was 7.15 years (median: 6.2, range: 1.01-23.0), with 60.4% of patients having a time interval of >5 years. Older patients ≥70 yo developed a CBC sooner than patients <70 yo (5.0 and 7.6 years respectively, p<.001). A majority of stage III patients (69.2%) developed a CBC within 5 years compared to 51.9% of stage 0, 39.3% of stage I, and 36.6% of stage II patients (p=.039). On average, patients with ILC developed their CBC in 9.2 years compared to 7.1 years for IDC patients, 6.6 years for mixed histology patients, and 5.9 years for DCIS patients (p=.016).  Factors that had no influence on the time interval between CBCs were race, body mass index, menopausal status, use of hormone replacement therapy, family history of breast/ovarian cancer, estrogen receptor (ER) status, BRCA status, tumor grade, and presence of lymphatic vascular invasion. In comparison to the first primary breast cancer, a higher proportion of CBCs were stage I (51.0% vs. 36.2%), T1 tumors (72.1% vs. 59.1%), node negative (67.5% vs. 62.2%), and ER(+) tumors (68.7% vs. 51.7%).  Of the 252 patients with available tumor size information for both breast cancers, 54 (21.4%) patients developed a CBC that was >1cm larger than their original primary, and only 25 (9.9%) patients developed a CBC that was >2cm larger than their original primary. There were 201 (62.2%) patients with node negative disease for their first breast cancer, and only 28 (13.9%) of these patients developed a node positive CBC. Of the 300 patients with stage information, 85 (28.3%) patients had a higher stage CBC compared to their first primary. Of the 67 patients with an ER(-) primary, 44 (62.7%) developed an ER(+) CBC. 

Conclusion: A majority of CBCs develop >5 years after the diagnosis of the first primary breast cancer. CBCs have more favorable tumor characteristics than the primary tumor because they tend to be smaller, less aggressive, and lower in stage compared to the primary breast cancer. Patient age, stage, and tumor histology significantly influence the time interval from primary to CBCs. It is unlikely that CBC would affect survival at five years of follow-up given this data.
 

32.03 Increasing Incidence of Duodenal Neuroendocrine Tumors: Incidental Discovery of Indolent Disease?

Posted on December 22, 2014

S. Dennis1, S. D. Kachare1, N. Vohra1, E. E. Zervos1, T. L. Fitzgerald1  1East Carolina University Brody School Of Medicine,Greenville, NC, USA

Introduction: An exponential increase in gastroenteropancreatic neuroendocrine tumors (GEP-NT), including pancreatic, gastric, colorectal and small bowel primaries has been clearly documented. Studies often combine duodenal neuroendocrine tumors (D-NET) with other small bowel GEP-NTs, as such the natural history and clinical ramifications of this diagnosis are poorly understood. We sought to better define the epidemiology of this malignancy.

Methods: Patients diagnosed with duodenal “carcinoid” tumors from 1983-2010 were identified in the SEER tumor registry. Information within the registry was used to classify patients as Stage I, II, III or IV utilizing the East Carolina University modified AJCC stage for D-NET.

Results:A total of 1,258 patients were identified.  The mean age was 64 years. The majority of patients were male and white, 55.6% and 68.8% respectively. Most patients presented with Stage I disease (66.2%), while 10.3% presented with Stage II, 12.6% with Stage III and 11.0% with Stage IV disease. Patients who met inclusion criteria were divided in to two cohorts: group one, those diagnosed from 1983 to 2005 and group two, those diagnosed from 2005-2010. Over the study period there was a clear increase in the incidence rate of D-NET, rising from 0.27 per 100,000 in 1983 to 1.1 per 100,000 in 2010 (Figure). The p-value for this trend was < 0.001. The 5-year disease-specific survival was significantly improved for Group two as compared to Group one, 89.3 vs. 85.2%, p=0.05. However, the disease-specific survival by stage remained similar.  The survival difference between the two groups is likely due to stage migration. When comparing Group two to Group one, patients in the former group were more likely to present with stage I disease (69.9 vs. 57.5 %, p <0.01) and less likely to present stage III (11.8 vs. 14.4%, p 0. 039) and IV disease (19.8 vs. 7.3%, p <0.001).

Conclusion:Prognosis for D-NET, in contrast to other small bowel NET, is excellent.  As with all GEP-NET, the prevalence of D-NETs has steadily increased over the last three decades. This has been coincident with a migration to earlier disease stage and improved disease-specific survival.  These data suggest an increase in the incidental discovery of indolent D-NETs.   

 

3.06 Immunotherapeutic Virus GLV-1h153 Fascilitates 131I Radiotherapy and Imaging in Cholangiocarcinoma

Posted on December 22, 2014

C. Johnsen1, J. W. Ady1, K. Mojica1, A. Pugalenthi1, D. Love1, V. Longo6, P. Zanzonico6, N. G. Chen5, R. J. Aguilar5, Y. A. Yu5, A. A. Szalay5, Y. Fong2  1Memorial Sloan-Kettering Cancer Center,Surgery,New York, NY, USA 2City Of Hope National Medical Center,Surgery,Duarte, CA, USA 3University Of California – San Diego,4Department Of Radiation Medicine And Applied Sciences, Rebecca & John Moores Comprehensive Cancer Center,San Diego, CA, USA 4University Of Würzburg,5Department Of Biochemistry, Rudolph Virchow Center For Experimental Biomedicine, And Institute For Molecular Infection Biology,Würzburg, BAVARIA, Germany 5Genelux,Research And Development,San Diego, CA, USA 6Memorial Sloan-Kettering Cancer Center,Small Animal Imaging Core,New York, NY, USA

Introduction:  Cholangiocarcinoma (CC), a deadly carcinoma of the bile ducts, is clinically silent in the majority of patients until curative surgery is no longer an option. With most patients succumbing to the disease within 6 months of diagnosis, novel treatment options are needed.  Oncolytic viruses are promising cancer therapy agents because they selectively infect, replicate within, and kill cancer cells.  In this study, we assess the ability of the human sodium iodide symporter (hNIS) expressing recombinant oncolytic vaccinia virus GLV-1h153 to kill and image CC when combined with 131I radiotherapy.

Methods:  Three human CC cell lines were assayed for infectivity, cytotoxicity and viral replication in vitro.  hNIS mediated 131I radiouptake was assayed in vitro. Flank CC xenografts were treated with intratumoral GLV-1h153 alone and/or with 131I to assess tumor burden reduction. SPECT/CT was performed to visualize 131I uptake in infected tumor in vivo.

Results: All cell lines demonstrated infectivity and oncolysis in a time and concentration dependent manner. Significant viral replication was supported in all cell lines. Flank xenografts treated with GLV-1h153 combined with 131I demonstrated significant tumor reduction as compared to controls.  131I mediated SPECT/CT demonstrated significant uptake in infected tumors. 

Conclusion: GLV-1h153 efficiently kills human CC in vitro. When combined with 131I, GLV-1h153 allows for SPECT/CT imaging of CC tumors and significantly reduces tumor burden in vivo. These results indicate that GLV-1h153 is a promising novel imaging and therapeutic agent for patients with CC.

 

3.07 A Flavonoid and Midkine. Novel Targeted Therapeutic Approach for Hepatocellular Carcinoma

Posted on December 22, 2014

K. M. Sokolowski1, S. Kunnimalaiyaan1, M. Balamurugan1, S. T. Koprowski1, T. C. Gamblin1, M. Kunnimalaiyaan1  1Medical College Of Wisconsin,Surgical Oncology/Surgery/Medical College Of Wisconsin,Milwaukee, WI, USA

Introduction: Despite improvement in therapeutic strategies, median survival in advanced hepatocellular carcinoma (HCC) remains less than one year. Therefore, molecularly targeted compounds with less toxic profiles are needed. Xanthohumol (XN), a prenylated chalcone flavonoid has been shown to have anti-proliferative effects in various cancers types in vitro. XN treatment in healthy mice and humans yielded favorable pharmacokinetics and bioavailability. Although a report providing therapeutic potential of XN in prostate cancer in transgenic mice, the effects of XN on HCC proliferation is unknown. A potential molecular target, midkine (MK- heparin-binding growth factor) is negatively correlated with HCC prognosis and readily detected in serum. Therefore, investigating the effects of XN on HCC cellular proliferation and the evaluation of midkine as a possible therapeutic biomarker is integral.

Methods: The effects of XN on a panel of HCC cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colometric and colonogenic assay. Cell lysates were analyzed via Western blotting for pro-apoptotic (c-PARP and cleaved caspase-3) and anti-apoptotic proteins (Bcl2, Survivin, and Mcl-1). Mechanism of XN on HCC cellular proliferation was also examined. Midkine expression was evaluated following XN treatment of conditioned HCC media.

Results: Up to 2 µM concentration of XN resulted in cell viabilities exceeding 90% in HCC cell lines. However, 5 µM and above of XN significantly reduced cell viability in a dose-dependent manner. Colonogenic ability also decreased with increasing concentrations of XN (Figure). Additionally, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced anti-apoptotic proteins. Importantly, MK secretion into media was significantly reduced following XN treatment. Furthermore, XN inhibited Notch1 signaling.

Conclusion: Xanthohumol effectively inhibits HCC growth in cell culture and reduces MK secretion. This may provide XN as a novel therapeutic agent and midkine as a potential biomarker. We report for the first time that XN targets Notch1 signaling in HCC cells. The favorable cytotoxic profile based on both healthy mice and human studies along with these findings warrant further preclinical in vivo analysis. Evaluation of Notch1 reduction and its effect on HCC cells following XN treatment is innovative. In addition, confirmation of MK as a therapeutic marker for HCC to XN treatment will be novel. To our knowledge, this is the first characterization of MK and Notch1 inhibition in HCC in response to XN treatment.

 

3.08 Bidirectional TSTA of SHIP-DTA Enhances the Effect of Gene Therapy for Insulinoma

Posted on December 22, 2014

S. Liu1, J. Wu1, G. Zhou1, J. Yu1, R. Sanchez1, F. Brunicardi1  1University Of California – Los Angeles,Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA

Introduction: Two limitations of gene therapy are tissue specificity and tissue expression of the transgene. We hypothesize that synthetic promoters have greatly enhanced activities over endogenous promoters to drive gene expression and can be used to enhance the effect of gene therapy without loss of target specificity. We have demonstrated that the synthetic human insulin promoter (SHIP) has far greater activity than HIP or rat insulin promoter (RIP) in driving gene expression with subsequent enhanced effect of gene therapy in neuroendocrine tumors which overexpress pancreatic and duodenal homeobox1 (PDX1) transcription factor. Bidirectional two-step transcriptional amplification (TSTA) has been shown to enhance gene expression. In this study we sought to determine that TSTA enhances the expression and therapeutic effect of SHIP-diphtheria toxin A (SHIPTSTA-DTA) in PDX1 expressing cells, such as insulinoma.

Methods: SHIP-eGFP vs SHIPTSTA-eGFP, SHIP- firefly luciferase (FLuc) vs SHIPTSTA-FLuc and SHIP-DTA vs SHIPTSTA-DTA vectors were constructed using subcloning techniques. Activity and specificity of vectors were determined by bioluminescence and eGFP assays in mouse insulinoma (βTC6) cells, human primary pancreatic (HPP) cells, pancreatic ductal epithelial (HPDE) cells and PDX1-HPDE cells, since PDX1 is the primary activator of SHIP, with or without PDX-1 shRNA co-transfection. Cytotoxicity was determined by MTS assay and glucose-stimulated insulin secretion (GSIS) was determined by ELISA. Statistical analysis was performed via paired T test; p<0.05 = significant.

Results: SHIPTSTA-FLuc activity in βTC6 cells was 8 fold higher than that of SHIP-FLuc (p<0.05)(Fig. A). SHIP-FLuc was equal to CMV-driven expression, however was specific for cells expressing PDX1. PDX-1 shRNA co-transfection resulted in decreased SHIPTSTA-FLuc activity.  Similarly, increased cell numbers of eGFP and expression intensity of eGFP were observed in SHIPTSTA-eGFP transfected βTC6 cells vs controls. No FLuc activity was detected in both SHIPTSTA-FLuc and SHIP-FLuc transfected HPP and HPDE cells. SHIPTSTA-DTA resulted in enhanced cytotoxicity and enhanced suppression of proliferation and GSIS from βTC6 cells compared to SHIP-DTA (p<0.05)(Fig. B).

Conclusions:  SHIPTSTA markedly enhanced SHIP activity without loss of tissue specificity; SHIPTSTA-DTA in PDX1+ insulinoma cells enhanced cytotoxicity and suppressed proliferation and insulin secretion. The data support the hypothesis that synthetic promoters of the target gene have greatly enhanced expression efficiency over endogenous promoters and can be used to enhance the effect of gene therapy without loss of target specificity. 
 

3.17 Changes in Liver Lobe Function After Portal Vein Ligation Determined by Selective Biliary Drainage

Posted on December 22, 2014

A. Szijarto1, A. Fulop1, A. Budai1, G. Lotz2, A. Kiss2, L. Harsanyi1  1Semmelweis University,1st Department Of Surgery,Budapest, BUDAPEST, Hungary 2Semmelweis University,2nd Department Of Pathology,Budapest, BUDAPEST, Hungary

Introduction: Portal vein ligation (PVL) and embolization (PVE) are techniques used before extended hepatic resections to prevent posthepatectomy liver failure. These therapies redirect portal blood to liver lobes that will remain after surgery resulting in hypertrophy, while the portal deprived lobes undergo atrophy. Although, the effect of PVL on liver volume is well-documented, the parallel alterations in lobar liver function are still the subject of controversy. Therefore, the aim of the present study was to evaluate the morphological, hemodynamic and functional alterations caused by the selective occlusion of the portal vein in a well-established rat model.

 

Methods:  Male Wistar rats (n=84) underwent PVL by the ligation of the portal veins feeding the median, left lateral and caudate lobes (approximately 80% of total liver mass). Before PVL, as well as 24-, 48-, 72-, 120-, 168 hours after PVL, liver morphology (liver weight; mitotic activity; necrotic-, apoptotic cell death and lobular area), hepatic microcirculation (laser Doppler flowmetry), global liver function (laboratory blood test; total hepatic bile flow; plasma disappearance rate of indocyanine-green (PDR); the percentage of biliary ICG excretion to the administered ICG during the first 20 minutes (ICG%20min)) as well as hepatic lobar function (lobar bile flow and lobar biliary ICG excretion) were examined.

Results: PVL induced atrophy of ligated lobes (from 0.35±0.037 to 0.09±0.018g/BWkg) and hypertrophy of non-ligated lobes (from 0.1±0.102 to 0.31±0.019g/BWkg), while the total liver weight remained unchanged. The microcirculation of ligated lobes impaired, while the microcirculatory blood flow of non-ligated lobes significantly increased with the peak response at 48th postoperative hours. Serum albumin-, total bilirubin levels and total hepatic bile flow did not changed significantly throughout the entire experiment. PDR and ICG%20min significantly decreased after PVL, with the lowest value at postoperative 48th hours and returned near to the baseline at 168 hours after the operation. The bile flow and biliary ICG excretion of ligated lobes decreased significantly after PVL (from 81.99±8.6 to 17.62±3.9g/BWkg and from 1.16±0.03 to 0.1±0.03g/BWkg, respectively), while bile flow and biliary ICG excretion of non-ligated lobes showed a significant increase (from 23.65±2.75 to 88.8±4.67g/BWkg and from 0.34±0.03 to 1.4±0.03g/BWkg, respectively).

 

Conclusion: PVL induced a temporary impairment in global liver function, followed by a rapid recovery mainly caused by the increase in the function of non-ligated liver lobes. In the non-ligated lobes, the functional increase was more pronounced than suggested by the degree of hypertrophy. Consequently, the functional capacity of the liver was shifted towards the regenerating lobes in a greater extent than would be expected according to the volumetric alterations.

3.20 Self-assembly Nanoparticles of PLGA-polyethylenimine (PLGA-PEI) Copolymer for Gene Delivery

Posted on December 22, 2014

J. LU1, Z. Liang1, Q. Yao1, C. Chen1  1Baylor College Of Medicine,Surgical Research/Surgery,Houston, TX, USA

Introduction: Although gene therapy holds great promise, the progress has been slow because the current gene delivery systems are less successful for clinical applications due to their low efficiency and high toxicity. The objective of this study was to develop a better gene delivery system from two biocompatible polymers, poly(lactic-co-glycolic acid) (PLGA) and polyethylenimine (PEI), potentially for clinical applications.

Methods: The PLGA-PEI copolymer was prepared by directly mixing PLGA and PEI in organic solvent. Self-assembly nanoparticles (NPs) of PLGA-PEI copolymer and DNA were prepared by adding the DNA solution to the water solution of PLGA-PEI copolymer. The size and morphology of PLGA-PEI/DNA NPs were determined with dynamic light scattering and scanning electronic microscopy. The cytotoxicity of PLGA-PEI/DNA NPs at different copolymer to DNA ratios was performed in pancreatic cancer cell line (PANC-1) with an MTT assay. For the gene transfection assay in PANC-1 cells, plasmid DNA containing a green or red fluorescence protein (GFP or RFP) gene was used. In vivo toxicity and transfection efficiency of PLGA-PEI PLGA-PEI/DNA NPs were carried in mouse models with the tail vein administration.

Results: PLGA-PEI copolymer was produced in one-step by mixing PEI and PLGA in the tetrahydrofuran solution and PLGA to PEI ratio (0.5:1). Through the analysis of primary amines of PEI before and after its chemical reaction with PLGA, the chemical structure of the PLGA-PEI copolymer was demonstrated.  PLGA was broken down to LGA single units, which were covalently linked to the primary amine groups of PEI; while PEI was intact. PLGA-PEI copolymers spontaneously formed NPs (~100 nm in diameter) with plasmid DNA at the 1.5:1 ratio due to change of the surface charge, achieving 100% DNA loading. The particle size can be controlled by justifying copolymer and DNA ratios.  PLGA-PEI significantly reduced the toxicity of PEI in both PANC-1 cells and mouse models. PLGA-PEI copolymer more efficiently delivered GFP or RFP plasmid into PANC-1 cells compared with commercially available transfection reagents with additional advantages of less toxicity, serum independency and long duration of transgene expression. More importantly, PLGA-PEI/DNA NPs were tested in the mouse model and showed an effective gene delivery to liver, spleen, and pancreas. Direct intratumor administration of PLGA-PEI/DNA NPs also showed a high transfection rate in the nude mouse model.

Conclusions: PLGA-PEI copolymer is a new gene delivery material, which has high DNA loading capacity and low toxicity in vitro and in vivo; and it condenses DNA into small sized NPs. The PLGA-PEI/DNA NPs have a high transfection efficiency in cell cultures and mouse models. The current study demostrates a better gene delivery system, which may have board clinical applications.

30.01 Profile of hepatocellular carcinoma in surgical area: about 100 cases.

Posted on December 22, 2014

P. S. Diop1, I. Ka1, M. Faye1, J. M. Ndoye1, B. Fall1 1Department of General Surgery , General Hospital of Grand Yoff , Dakar, Senegal

The aim of our study was to determine the characteristics of hepatocellular carcinoma (HCC) in surgery and consider therapeutic strategies

PATIENTS AND METHODS:
This was a retrospective analysis of 100 records of patients referred for treatment of HCC from January 2006 to March 2013 .

Results:
The study population involved 72 men and 28 women, mean age 58,49ans with extremes of 18 and 78. The underlying cirrhosis was present in 93 % of cases; due to hepatitis B ( 91 % ) and hepatitis C ( 2 % ) . The topography was as follows 66 % left , and right 23 % 11 % of bilateral tumors . The evaluation criteria resectability showed: a mean tumor size of 12 ± 4.7 cm , 87% of score CHILD A, 13% of score CHILD B and 12% of venous invasion . The mean follow-up for the whole population was 10.2 ± 9mois .

Surgical resection was effective in 31% of patients. Rates without recurrence in1 and 2 years survival rates were 30.43% and 21.73 % for patients resected .

Conclusion:Hepatic resection is possible in our context in selected patients with acceptable immediate results. The major obstacles to improving disease-free survival are delayed consultation and inaccessibility of adjuvant therapies for HCC.
 

30.02 Occult Metastases in Node-negative Breast Cancer: A SEER-based Analysis

Posted on December 22, 2014

C. W. Kimbrough1, K. M. McMasters1, A. R. Quillo1, N. Ajkay1  1University Of Louisville,Hiram C. Polk, Jr. Department Of Surgery,Louisville, KY, USA

Introduction: Although multiple retrospective studies suggest that occult metastases are a significant prognostic factor in breast cancer, the results of two prospective randomized trials have questioned the role of immunohistochemistry (IHC) in detecting occult disease for patients initially found to be node-negative. In this study, we sought to evaluate factors associated with overall survival in node-negative breast cancer patients staged by immunohistochemistry using the Surveillance, Epidemiology, and End Results (SEER) database.

Methods: The SEER database was queried for all patients between 2004 and 2011 with invasive lobular or ductal carcinoma and no evidence of distant metastases. Only patients with regional lymph nodes coded as negative on hematoxylin and eosin (H&E) stains that underwent additional studies using IHC were included for analysis. Patients were stratified by nodal involvement and overall survival was compared using Kaplan-Meier analysis with a log-rank test. Multivariate analysis controlling for patient and tumor characteristics was performed using a Cox-proportional hazards regression model.

Results: Overall, 93,070 patients were identified. Of these, 11,377 patients (12.2%) had occult metastases; 4657 with isolated tumor cells (N0(i+)) and 6720 with micrometastases (N1mi). On Kaplan-Meier analysis, occult metastases were associated with a small but significant decrease in overall survival (p<0.001). The 5-year survival approached 92.2% in patients without occult disease, while 5-year survival for occult metastases was 89.6%. Once further stratified by N-stage, there was no difference in overall survival observed between N0(i-) and N0(i+) patients (p<0.449), although N1mi patients demonstrated worse survival compared to both N0(i-) and N0(i+) groups (p<0.001). On multivariate analysis, micrometastasis remained an independent predictor for decreased survival compared to IHC-negative patients (HR 1.40, 95% CI 1.28–1.53), while isolated tumor cells were not a significant predictor (HR 1.05, 95% CI 0.92-1.20). Other negative prognostic factors included male sex, age at diagnosis, African-American ethnicity, increasing tumor grade, increasing T-stage, and negative hormone receptor status.

Conclusions: Patients with occult metastases found via IHC demonstrated a significant, but relatively small 2.6% overall survival difference at 5-years compared to patients with no evidence of nodal disease. Most of this survival difference is attributable to micrometastases, as isolated tumor cells have no prognostic significance in this study.  Discontinuing the classification of isolated tumors cells as a separate subgroup of N0 disease warrants further consideration.

30.03 The Impact of Malignant Pleural Mesothelioma Histologic Subtype on Outcomes in the SEER Database

Posted on December 22, 2014

C. J. Yang1, P. Speicher1, B. Gulack1, R. R. Meyerhoff1, M. Hartwig1, T. D’Amico1, D. Harpole1, M. Berry1,2  2Stanford,Cardiothoracic Surgery,Palo Alto, CA, USA 1Duke University Medical Center,Department Of Surgery,Durham, NC, USA

Introduction: Histologic subtype for malignant pleural mesothelioma (MPM) is known to be an important determinant of both treatment and survival.  This study was conducted to specifically quantify the impact of MPM histology on the use of surgery and survival in a population-based analysis.

 

Methods: Overall survival (OS) of patients with stage I-III epithelioid, sarcomatoid (including spindled and desmoplastic mesothelioma and fibrous mesothelioma not otherwise specified), and biphasic MPM in the Surveillance, Epidemiology, and End Results database from 2004 to 2010 was evaluated using multivariable Cox proportional hazard models.

 

Results: Of 1,199 patients who met inclusion criteria, the histologic subtype was epithelioid in 823 patients (69%), biphasic in 149 patients (12%), and sarcomatoid in 227 patients (19%).  Median survival was 14 months in the epithelioid group, 10 months in the biphasic group and 4 months in the sarcomatoid group (p<0.0001) (figure).  Cancer-directed surgery was utilized more often in epithelioid (37%, n=303) and biphasic patients (44%, n=65) compared to sarcomatoid patients (26%, n=58) (p=0.004).  Among patients who underwent surgery, median survival was 19 months in the epithelioid group, 12 months in the biphasic group and 4 months in the sarcomatoid group (p<0.0001).  In multivariable analysis, surgery was associated with improved survival in the epithelioid group (hazard ratio [HR] 0.70; p<0.001) but not in the biphasic (HR 0.68; p=0.10) or sarcomatoid (HR 0.82; p=0.27) groups. 

 

Conclusions: Cancer-directed surgery is associated with improved survival for MPM patients with epithelioid histology, but patients with sarcomatoid and biphasic histologies have poor prognoses that may not be improved by operative treatment.  The specific histology of patients with MPM should be identified prior to treatment whenever possible, so that patients with non-epithelioid histologies and particularly sarcomatoid MPM are not exposed to the risks of surgery without any likely benefit.

 

 

30.04 Trends in Radiation Therapy for Elderly Women with Early Stage Breast Cancer: A Report from the NCDB

Posted on December 22, 2014

O. Kantor1, E. Leiderbach2, C. Wang3, D. J. Winchester2, C. E. Pesce2, K. Yao2  1University Of Chicago,Department Of Surgery,Chicago, IL, USA 2NorthShore University Health System,Department Of Surgery,Evanston, IL, USA 3Northshore University Health Systems,Center For Biomedical Research Informatics,Evanstol, IL, USA

Introduction:
Several randomized controlled trials in 2004 have examined the efficacy of radiation in elderly women with early stage breast cancer without demonstrating a survival benefit to radiation. Omission of radiation for this cohort has been incorporated into National Comprehensive Cancer Network guidelines for patients meeting criteria. We examined trends in radiation utilization for elderly patients since publication of these trials.

Methods:
Using the National Cancer Data Base (NCDB), radiation therapy utilization was determined for patients with early stage, estrogen receptor positive (ER+), clinically node negative invasive cancer treated with breast conserving surgery and hormone therapy from 2004 to 2011. Chi square tests and logistic regression models were used for analysis.

Results:
Adjuvant radiation therapy after breast conserving surgery decreased from 83.8% to 76.0% among those ≥70yo with ER+ tumors ≤2cm. For patients who did not receive hormonal therapy, radiation utilization significantly decreased from 60.7% to 52.4% over the study period.  Among those patients who did not undergo radiation therapy, the rate of hormone therapy significantly increased from 36.9% to 52.7%. There is variation in the use of radiation according to patient and tumor factors; 95.3% of 60-69 year olds, 81.6% of 70-79 year olds, and only 59.1% of 80-90 year olds received radiation therapy in 2011. 83.2% of grade 3 tumors received radiation in 2011, compared to 73.3% of grade 1 tumors. Patients without lymph node staging were much less likely to receive radiation therapy compared to those that did (45.7% versus 77.8%).

The rate of external beam radiation has significantly decreased from 69.9% to 46.2%, while alternate forms of radiation have increased twofold from 14.2% to 29.8% (Figure 1). Brachytherapy has increased from 5% in 2004 to 11.7% in 2011. Independent predictors of receiving alternate forms of radiation include being treated at a community cancer center (OR 1.43, CI: 1.32-1.54), living >50 miles from the hospital (OR 1.50, CI:1.28-1.76), and living in the Mid-Atlantic (OR 2.42, CI: 2.16-2.71) or South Atlantic (OR 2.14, CI: 1.91-2.39) regions. Caucasian women, those with private insurance, grade 1 tumors, T1a tumors, and PR positive tumors were also more likely to receive alternate forms of radiation.

Conclusion:
Time trends in the NCDB reflect a gradual acceptance of evidence from randomized clinical trials supporting the omission of radiation therapy for women 70 and older with ER+ early stage breast cancer.  However, the majority still receive radiation, influenced by tumor size and grade, and many are receiving alternate forms of radiation, particularly brachytherapy.
 

3.03 Oncolytic Recombinant Vaccinia Virus GLV-2b372 Efficiently Kills Hepatocellular Carcinoma

Posted on December 22, 2014

J. W. Ady1, C. Johnsen1, K. Mojica1, J. Heffner1, D. Love1, A. Pugalenthi1, J. Belin1, J. R. Aguilar5, N. Chen5, Y. A. Yu5, A. Szalay5, Y. Fong4  1Memorial Sloan-Kettering Cancer Center,Surgery,New York, NY, USA 2University Of California – San Diego,3Department Of Radiation Medicine And Applied Sciences, Rebecca & John Moores Comprehensive Cancer Center,San Diego, CA, USA 3University Of Würzburg,4Department Of Biochemistry, Rudolph Virchow Center For Experimental Biomedicine, And Institute For Molecular Infection Biology,Würzburg, BAVARIA, Germany 4City Of Hope National Medical Center,Surgery,Duarte, CA, USA 5Genelux,Research And Development,San Diego, CALIFORNIA, USA

Introduction:  Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with limited treatment options and poor prognosis. Therefore, there is a great need to develop novel therapies for HCC.   Oncolytic viruses that specifically infect, replicate within, and kill cancer cells represent an emerging and promising platform for cancer therapy.   In this study we assess at the ability of GLV-2b372, a novel nonattenuated vaccinia virus derived from the Lister 1.1.1 strain, to kill HCC.

Methods:  The infectivity, viral replication and cytotoxicity of GLV-2b372 was assayed in 4 human HCC cell lines.  Huh-7 flank xenografts were generated in athymic nude mice.  Mice were treated with intratumoral injections of GLV-2b372.  Biodistribution of viral presence in animals treated with GLV-2b372 was assessed.

Results: Infectivity increased in a time and concentration dependent manner in all cell lines. All cell lines supported efficient replication of virus. Flank xenografts treated with GLV-2b372 demonstrated complete erradication of tumor in 75% of animals with significant tumor reduction as compared to controls (p < 0.05).  Biodistribution confirmed sustained intratumoral presence of virus at 2 weeks post infection, with rapid clearance of tumor from all other tissues. 

Conclusion: Our results demonstrate that the novel oncolytic vaccinia virus GLV-2b372 selectively infects and replicates in HCC tissue.  We demonstrate infectivity and killing of HCC both in vitro and in vivo.  These findings indicate that GLV-2b372 is a potent oncolytic vector that could serve as a promising platform for oncolytic cancer immunotherapy.  

 

3.05 Controlled Release of Nitric Oxide Enhances Gemcitabine Cytotoxicity in Pancreatic Adenocarcinoma

Posted on December 22, 2014

J. Fernandez-Moure1,2, D. Kirui1, J. Van Eps1,2, N. Dhanani1,4, F. Cabrera1, M. Ferrari1,3, E. Tasciotti1  1Houston Methodist Research Institute,Nanomedicine,Houston, TX, USA 2Houston Methodist Hospital,Surgery,Houston, TX, USA 3University Of Texas Health Science Center At Houston,Houston, TX, USA 4Texas A & M Health Science Center College Of Medicine,Bryan, TX, USA

Introduction:

Gemcitabine (GEM) is the first-line treatment for pancreatic adenocarcinoma (PAC). Despite such broad use, intrinsic and acquired chemoresistance is common. Loss of the tumor suppressor SMAD4 is present in more than 50% of PAC and is known to play a role in chemoresistant mechanisms. Nitric oxide (NO) is the predominant species responsible for the cytotoxic action of macrophages against cancer cells yet localized delivery is difficult given the short half-life and volatility. Silica (Si) nanoparticle mediated local delivery has been shown to be an effective strategy for NO delivery and controlled release. We sought to study the effects of locally delivered NO on GEM mediated PAC cytotoxicity and the potential role of SMAD4 in this effect. We hypothesized that NO would enhance the cytotoxicity of GEM in a SMAD4 independent manner.

 

Methods:

NO-Silica nanoparticles (NO-Si) were synthesized via a co-condensation of tetraethoxysilane with aminoalkoxysilane under high pressure nitrous oxide for 3 or 4 days. Particle characterization included scanning electron microscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. NO release was measured using a chemiluminescence nitric oxide analyzer. A SMAD4 negative PAC cell line (SMAD4-) was made using lentiviral knockdown of Panc1 PAC cells and confirmed by western blot. Panc1 and SMAD4- cells were then treated with gemcitabine (100nm to 30μm), 30 mg NOSi particles (NOSihi or NOSilo), or both for 72 hours. Cell viability was then quantified by MTS cell proliferation assay.

 

Results:

Half-life of NOSi NO release is 3.5±0.6 hours. NOSilo maximum concentration of NO release was 750ppb/mg and NOSihi was 2250ppb/mg. NOSilo alone reduced cell viability by 30% and 35% in SMAD4 and Panc1 respectively. When combined with GEM, NOSilo led to a significant reduction in cell viability of both cell lines at all concentrations used. The greatest effect was seen at 1μm where cytotoxicity was increased by 50% in Panc1 and 70% in SMAD4-. NOSihi led to >90% reductions in cell viability in Panc1 and no difference was seen when combined with GEM. NOSihi alone led to only 70% reduction in SMAD4- cells. When combined with GEM, cytotoxicity was improved to 90% at GEM concentrations as low as 5μm.

 

Conclusion:

We have demonstrated the in vitro dose dependent cytotoxic effects of Si nanoparticle NO controlled release on PAC. When combined with GEM there is a synergistic effect resulting in improved cytotoxicity seen in both Panc1 and SMAD4- PAC cells with a differential pattern of cell death seen at high concentrations of NO. These findings suggest not only that NOSi is useful chemosensitizing agent but that SMAD4 may play a role in its synergism with GEM. Creation of a novel class of therapeutics where local controlled release of NO is used as a method of chemosensitization may lead to a paradigm shift in not only treating PAC but all solid tumors where chemoresistance is common.

 

27.01 Electrophysiological Characterisation Of Human Visceral Afferent Nerves: First In Man

Posted on December 22, 2014

K. S. Ng1,2, N. A. Montes-Adrian2, D. A. Mahns3,M. A. Gladman1,2 1Academic Colorectal Unit, Sydney Medical School – Concord Hospital Campus, University of Sydney, NSW, Australia. 2Enteric Neuroscience & Gastrointestinal Research Group, ANZAC Research Institute, University of Sydney, Australia 3Department of Integrative Physiology, School of Medicine, University of Western Sydney, NSW, Australia

Introduction:  During the last decade, abnormal afferent activity gained recognition as being important in the development of functional gastrointestinal disorders. Since it is not possible to directly measure visceral afferent activity ‘in vivo’ in humans, in this study we aimed to make direct electrophysiological recordings (in vitro) from extrinsic afferents supplying the human colon and rectum. 

Methods:  SSections of normal rectum and colon were procured from anterior resection and right hemicolectomy specimens, respectively. Sections were pinned flat and mesenteric nerves dissected. Extracellular visceral afferent nerve activity was recorded. Neuronal responses to chemical (capsaicin and ‘inflammatory solution’ [IS]1) and mechanical (Von Frey probing) stimulation were recorded and quantified by determining peak firing rates [range] in onesecond intervals. 

Results: 21 nerves were studied from six rectums. Of these, spontaneous afferent activity was recorded in 18 nerves. Peak discharge rates increased significantly following capsaicin (7 [4-25] spikes/sec vs. 3 [2-6], P=0.001) and IS (5 [3-18] spikes/sec vs. 4 [3-12], P=0.003) application. Punctate mechanosensitive ‘hot-spots’ were identified in 11 nerves (threshold 2.0g [1.4–4.0g]). In six of these, the threshold decreased following IS (1.0g [0.4–1.4g]). By comparison, no ‘hot spots’ were identified and spontaneous activity in only one of 18 nerves studied from five
colons. 

Conclusion:  This is the first study to record from extrinsic rectal afferent nerves and to confirm their chemoand mechanosensitivity. Colonic afferents appear less responsive to mechanical stimulation, suggesting differences in electrophysiological characteristics. This technique offers the opportunity to measure electrophysiological properties of extrinsic nerves in disease states. 

References:  

  1. 10μM each of histamine, serotonin, bradykinin, and prostaglandin E2, as in Feng B, Gebhart GF. Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum. Am J Physiol Gastrointest Liver Physiol. 2011; 300(1): G170-80.

 

 

27.02 BET Inhibitor Blocks Neurosphere Formation And Promotes Differentiation In Neuroblastomas

Posted on December 22, 2014

E. J. Rellinger1, S. Lee1, J. Qiao1, B. T. Craig1, K. Kim1, C. V. Romain1, D. H. Chung1  1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA

Introduction:  High-risk group neuroblastoma (NB) patients have a poor prognosis with ~50% overall survival. Nearly half of high-risk NBs demonstrate MYCN amplification; however, conventional drug discovery strategies have had limited success in targeting this transcription factor. The bromodomain and extraterminal (BET) family of proteins coactivates transcription by interacting with histones. BET inhibitors, such as JQ1, have recently been shown to block NB proliferation and induce apoptosis in vitro and halt tumor formation in in vivo murine models by inhibiting MYCN signaling. Our laboratory has characterized the role of gastrin-releasing peptide (GRP) signaling in NB tumor initiation, progression, and metastasis. Its cognate receptor, GRP-R, modulates expression of N-myc, along with PI3K/AKT and ERK. As such, we set out to delineate the effects of JQ1 treatment on GRPR signaling and assess the phenotypic effects of this epigenetic regulator in NBs.

Methods:  Cell viability was determined using CCK-8 assay. Immunoblotting was performed to assess for N-myc, GRP-R, p-AKT, p-ERK expression after JQ1 treatment in NB cells. Tumor sphere formation in serum-free media was used to select for stem-like cancer cells. Tumor sphere formation and expression of neural and stem cell markers served as measures of JQ1 efficacy. We used a subcutaneous murine xenograft model to determine the effects of intraperitoneal JQ1 administration on tumor progression. At harvest, tumor size and immunohistochemistry were our primary measures of tumor progression and differentiation.

Results: JQ1 inhibited cell growth in both MYCN-amplified and MYCN-nonamplified NBs; n-myc expression was decreased in MYCN-amplified cell lines (Fig. A; *p < 0.05 vs. control). Notably, JQ1 blocked the expression of GRP-R and its downstream pathways, PI3K/AKT and ERK, highlighting that BET inhibitors act on both MYCN-dependent and MYCN-independent signaling pathways (Fig. B). We next selected for purported tumor-initiating cells utilizing an in vitro sphere assay and found that JQ1 diminished sphere formation, decreased expression of stem cell markers, and induced neural differentiation. JQ1 was shown to decrease tumor size and induced neural differentiation in our xenograft model.

Conclusion: BET inhibitor downregulates NB cell proliferation, induces apoptosis, and promotes neural differentiation irrespective of MYCN copies, further implicating its therapeutic potential in the treatment of high-risk group of NBs.

 

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