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Male germ cell tumors (GCTs) are a model for a curable solid tumor. GCTs can differentiate into mature teratomas. Embryonal carcinomas (ECs) represent the stem cell compartment of GCTs and are the malignant counterpart to embryonic stem (ES) cells. GCTs and EC cells are useful to investigate differentiation therapy and chemotherapy response. This study explored mechanistic interactions between all-trans-retinoic acid (RA), which induces differentiation of EC and ES cells, and the Hedgehog (Hh) pathway, a regulator of self-renewal and proliferation. RA was found to induce mRNA and protein expression of Patched 1 (Ptch1), the Hh ligand receptor and negative regulator of this pathway. PTCH1 is also a target gene of Hh signaling through Smoothened (Smo) activation. Yet, this observed RA-mediated Ptch1 induction was independent of Smo. It occurred despite co-treatment with RA and Smo inhibitors. Retinoid induction of Ptch1 also occurred in other RA-responsive cancer cell lines and in normal ES cells. Notably, this enhanced Ptch1 expression was preceded by induction of the homeobox transcription factor Meis1, a direct RA target. Direct interaction between Meis1 and Ptch1 was confirmed using chromatin immunoprecipitation assays. To establish the translational relevance of this work, Ptch1 expression was shown to be deregulated in human ECs relative to mature teratoma and the normal seminiferous tubule. Taken together, these findings reveal a previously unrecognized mechanism through which RA can inhibit the Hh pathway via Ptch1 induction. Engaging this pathway is a new way to repress the Hh pathway that can be translated into the cancer clinic.

The multicellular tumor spheroid (MCTS) model represents a suitable in vitro model recreating in vivo tumor formation. The aim of this study was to identify differentially expressed genes that could potentially serve as predictive gene markers for MCTS and be involved in the formation of MCTS. Using the suppression subtractive hybridization (SSH) method, we identified ERBB2/HER2-interacting protein (Erbin), Tumor rejection gp96 (Tr-gp96), 12S ribosomal RNA (12S rRNA), ATP synthase, Kruppel-like transcription factor 5 (KLF5), transcription factor-like 5 (TCFL5), and the dual-specificity phosphatase 11 (DUSP11) to be overexpressed in 3-day-old HT-29 colon carcinoma MCTSs compared to HT-29 colon carcinoma cells grown in monolayer. We could also confirm overexpression of these genes in HT-29 MCTSs and in MCTSs formed by the human glioblastoma tumor cell lines U343 MG, U373 MG, and DBTRG 05 MG. Knockdown of KLF5, Erbin, DUSP11, and TCFL5 was effectively achieved after transfection of HT-29 cells with the appropriate short-interfering RNAs (siRNAs), and correlated with a significant inhibition of MCTS formation in the case of KLF5, Erbin, and TCFL5 siRNAs. We suggest that KLF5, Erbin, and TCFL5 are essential for MCTS formation and play a key role in the development of tumor diseases.

The lack of an effective detection method for lung circulating tumor cells (CTCs) presents a substantial challenge to elucidate the value of CTCs as a diagnostic or prognostic indicator in lung cancer, particularly in nonsmall cell lung cancer (NSCLC). In this study, we prepared a capture surface exploiting strong multivalent binding mediated by poly(amidoamine) (PAMAM) dendrimers to capture CTCs originating from lung cancers. Given that 85% of the tumor cells from NSCLC patients overexpress epidermal growth factor receptor (EGFR), anti-EGFR was chosen as a capture agent. Following in vitro confirmation using the murine lung cancer cell lines (ED-1 and ED1-SC), cyclin E-overexpressing (CEO) transgenic mice were employed as an in vivo lung tumor model to assess specificity and sensitivity of the capture surface. The numbers of CTCs in blood from the CEO transgenic mice were significantly higher than those from the healthy controls (on average 75.3 ± 14.9 vs 4.4 ± 1.2 CTCs/100 μL of blood, p < 0.005), indicating the high sensitivity and specificity of our surface. Furthermore, we found that the capture surface also offers a simple, effective method for monitoring treatment responses, as observed by the significant decrease in the CTC numbers from the CEO mice upon a treatment using a novel anti-miR-31 locked nucleic acid (LNA), compared to a vehicle treatment and a control-LNA treatment (p < 0.05). This in vivo evaluation study confirms that our capture surface is highly efficient in detecting in vivo CTCs and thus has translational potential as a diagnostic and prognostic tool for lung cancer.

Catechins have been reported to possess anti-cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti-tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin-3 gallate (EGCG)-treated HT 29 cells have been analysed by high-density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5-50 microm) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3-day-old spheroids relative to normal adherent cells using three replicates. Oncogenes like c-fos and c-jun are significantly up-regulated in spheroids. We identified several signal transduction and proliferation genes which are down-regulated in response to EGCG treatment. Increase in the mRNA expression profile of c-Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG-treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells. Our findings suggest that EGCG may exert its anti-cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell-cell contacts and cell-matrix interactions.

Activation of the epidermal growth factor receptor (EGFR) pathway has been implicated in tumorigenesis in non-small cell lung cancer (NSCLC), the most common type of lung cancer. As a result, EGFR has become a key focus for the development of personalized therapy, with several molecular biomarkers having been investigated as potential predictors of response with EGFR tyrosine kinase inhibitors (TKIs) in NSCLC (e.g., EGFR expression, EGFR gene copy gain, and EGFR mutations). Of these, activating mutations in EGFR have thus far given the most consistent results based on the available evidence from preclinical studies and clinical trials. In an attempt to identify patients who are most likely to benefit from treatment with EGFR TKIs, EGFR mutation testing is being increasingly utilized in clinical practice. Currently in the United States, no EGFR TKI or accompanying mutational test is approved for the identification and first-line treatment of patients with advanced NSCLC. However, the first-generation EGFR TKIs, erlotinib and gefitinib, as well as investigational ErbB family TKIs and EGFR mutation testing methods are being evaluated in this setting. This review will discuss EGFR mutation testing as a biomarker of response to EGFR TKIs and the evolution of EGFR mutational analysis in NSCLC. Completed and ongoing clinical trials evaluating currently available or investigational EGFR TKIs as first-line therapy in molecularly and clinically selected patients with NSCLC, with a focus on trials in patients whose tumors have EGFR mutations, will also be reviewed.

Background: Patients undergoing bariatric surgery are at increased risk for symptomatic venous thromboembolism (VTE). Although the incidence of clinical VTE is lower than in other general and orthopedic surgical procedures, pulmonary embolism is an independent predictor of death after gastric bypass surgery. The optimal strategy for postoperative thrombophylaxis in bariatric surgery has yet to be elucidated. In 2010 our institution proposed a guideline for postoperative thrombophylaxis for bariatric surgery based on risk stratification. Patients were considered high risk if they had history of VTE or a BMI >/= 60 kg/m2 or if they had two or more of the following: age > 50, BMI >/= 50 kg/m2, male sex, recent history of smoking, obstructive sleep apnea, varicose veins, or hormone therapy within 30 days. All patients undergoing bariatric surgery received enoxaparin 40 mg SC twice daily in addition to mechanical thrombophylaxis including venodynes and compression stockings during the hospital stay. Prophylactic IVC filter insertion was not recommended. High-risk patients received an extended course of anticoagulation for 10 days after discharge. Methods: We retrospectively reviewed the medical charts of 692 patients who underwent bariatric surgery at Dartmouth-Hitchcock Medical Center from 2009 to 2014. We analyzed the incidence of VTE and bleeding complications under two different institutional thrombophylaxis protocols: an earlier protocol of VTE prophylaxis based on surgeon's preference (n = 62) and our subsequent protocol based on risk stratification (n = 630). Results: Prior to implementing our protocol the incidence of VTE and bleeding complications in patients who underwent bariatric surgery was 1.6% (1 in 62) for each. After 2010, with the implementation of extended VTE prophylaxis for high-risk patients, the incidence of postoperative VTE was 0% (0 in 630) and the incidence of postoperative bleeding was 2.7% (17 in 630). Of 17 patients who developed postoperative bleeding, 14 (82%) developed bleeding before postoperative day 3 during the hospitalization and 3 (18%) experienced delayed bleeding after hospital discharge. Of 3 patients with delayed bleeding, only 1 was on the extended thrombophylaxis protocol. Severe bleeding, defined as the need for packed red blood cell transfusions or re-operation, occurred in 1.6% (10 in 630). Conclusion: Our study demonstrates that a protocol based on risk stratification is effective at reducing the risk of symptomatic VTE in patients undergoing bariatric surgery. Although the incidence of all postoperative bleeding appears to have increased slightly after implementation of the protocol, the incidence of severe bleeding appears unchanged. As most of the post-operative bleeding events occurred during the hospitalization period, extending the length of pharmacologic thromboprophylaxis was not associated with an increase in bleeding. This study is limited by its retrospective design and the small number of patients studied prior to implementing the extended VTE prophylaxis protocol. Nevertheless, the findings are promising with respect to VTE risk reduction after bariatric surgery and suggest that prospective evaluation of the thromboprophylaxis protocol is in order. Disclosures No relevant conflicts of interest to declare.

Catechins have been reported to possess anti-cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti-tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin-3 gallate (EGCG)-treated HT 29 cells have been analysed by high-density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5-50 microm) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3-day-old spheroids relative to normal adherent cells using three replicates. Oncogenes like c-fos and c-jun are significantly up-regulated in spheroids. We identified several signal transduction and proliferation genes which are down-regulated in response to EGCG treatment. Increase in the mRNA expression profile of c-Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG-treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells. Our findings suggest that EGCG may exert its anti-cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell-cell contacts and cell-matrix interactions.

e13567 Background: MicroRNAs (miRs) have emerged as powerful regulators of cellular processes and are frequently deregulated in cancer. Our previous work showed that miR-31 was overexpressed in human and murine lung cancers and repression of miR-31 inhibits lung cancer proliferation and tumorigenesis. Novel miR-31-targeting compounds were developed using a locked nucleic acid (LNA) drug platform. Compounds were tested in cell-based assays to assess levels of miR-31, miR-31-target genes and cell proliferation. A lead compound was chosen and we are currently examining its in vivo activity in human lung cancer cells and in murine transgenic lung tumor models. Methods: We previously developed a murine model of lung cancer by specifically overexpressing human cyclin E in the lung (CEO mice). The ED1 cell line was derived from a CEO mouse lung tumor. ED1 cells were transfected with the miR-31-LNA-targeting compounds and miR-31 levels and target genes quantified versus controls using RT-PCR assays. Cell proliferation was assessed using the CellTiter-Glo assay. Microarray analysis was performed to further investigate novel miR-31 target genes that were validated using independent RT-PCR assays. A pharmacodynamic study (5 daily tail vein injections) was performed in CEO mice that develop lung tumors spontaneously. Lung tissues were harvested 24 hours after the final dose and analyzed for immunohistochemical biomarkers of drug response (Ki67 and cyclin E). Results: Novel LNA anti-miR-31 compounds decreased miR-31 levels and proliferation rates in murine and human lung cancer cells. The compound, as expected, augmented expression of a known miR-31 target, LATS2. The microarray analysis identified over 100 genes with 2-fold elevated expression in anti-miR-31 treated cells. Validated novel miR-31 targets include GADD45a, Trp53inp1, Txnip and Slc40a1. Preclinical studies in mouse models are ongoing. Conclusions: Targeting miR-31 using LNA–based therapy inhibits cell growth in lung cancer cells and is a promising therapeutic approach for the treatment of lung cancer. Successful preclinical activity of these compounds will warrant translation into the clinic in proof of principle trials to assess pharmacodynamic and pharmacokinetic outcomes.

Abstract 3143 Background: AHA is a rare bleeding disorder caused by autoantibodies (inhibitors) to coagulation factor VIII (FVIII). Acquired FVIII inhibitors are associated with a high rate of complications, with major bleeding reported in up to 90% of affected individuals and mortality rates as high as 22%. There are currently no formal guidelines for the treatment of AHA, due to the paucity of data from randomized clinical trials and limited number of cases. However, there is general agreement that treatment should consist of a two-pronged approach that includes 1) achieving hemostasis in bleeding patients by administering FVIII bypassing agents, and 2) reducing or eradicating the inhibitor with immunosuppressive treatments such as rituximab (R), cyclophosphamide (C), prednisone (P) or a combination. C and P have been used as first-line treatment for AHA until the late 1990s, when R began to be used off-label for inhibitor eradication. Data from case reports and series suggest that R is effective as both first and second-line therapy for AHA. We hypothesized that the use of R rather than C and P may be cost-effective by reducing the overall use of expensive hemostatic agents. The purpose of this study was to compare clinical course, use of hemostatic agents and costs in patients who received R to historical patients who did not receive R for treatment of AHA. Methods: We performed a retrospective study of patients with AHA, defined by prolonged aPTT (>37 sec), FVIII level 0.40 Bethesda Unit (BU)) at Dartmouth-Hitchcock Medical Center (DHMC) using the hospital's electronic medical record system, CIS, to collect pertinent demographic and clinical information. The data collection variables included: age, sex, co-morbidities, FVIII levels, FVIII antibody titers, type of bleeding, amount and total acquisition cost of hemostatic products administered, immunosuppressive regimens administered, time to complete remission (CR) (defined as FVIII level > 30%), and relapses. Results: We identified 16 patients with AHA treated at DHMC from 4/1997 to 3/2010. The median age at diagnosis was 78 (range 66 to 93), and 56% were women. Co-morbidities included current or prior malignancy (62%) and autoimmune disease (12%). Idiopathic cases accounted for 32%. One patient had both a malignancy and autoimmune disease. The median aPTT at diagnosis was 80 sec (range 53 to >160 sec); FVIII level, 3% (range Conclusions: Our findings suggest that R is an effective first-line immunosuppressive treatment for AHA and appears to shorten the time to CR substantially compared to traditional immunosuppression. The cost for hemostatic agents in our population was similar for both R-treated and non-R-treated patients, suggesting that despite the decreased time to CR, the use of R may not result in decreased use of hemostatic products. A hemostatic agent-sparing effect attributable to R may have been missed due to our small AHA population size, however, and is worthy of further investigation. Disclosures: Off Label Use: Rituximab for treatment of acquired hemophilia A.