Aisha Siebert

Expression of the Long Non-Coding RNA HOTAIR Correlates with Disease Progression in Bladder Cancer and Is Contained in Bladder Cancer Patient Urinary Exosomes.

Exosomes are 30-150nM membrane-bound secreted vesicles that are readily isolated from biological fluids such as urine (UEs). Exosomes contain proteins, micro RNA (miRNA), messenger RNA (mRNA), and long non-coding RNA (lncRNA) from their cells of origin. Although miRNA, protein and lncRNA have been isolated from serum as potential biomarkers for benign and malignant disease, it is unknown if lncRNAs in UEs from urothelial bladder cancer (UBC) patients can serve as biomarkers. lncRNAs are > 200 nucleotide long transcripts that do not encode protein and play critical roles in tumor biology. As the number of recognized tumor-associated lncRNAs continues to increase, there is a parallel need to include lncRNAs into biomarker discovery and therapeutic target algorithms. The lncRNA HOX transcript antisense RNA (HOTAIR) has been shown to facilitate tumor initiation and progression and is associated with poor prognosis in several cancers. The importance of HOTAIR in cancer biology has sparked interest in using HOTAIR as a biomarker and potential therapeutic target. Here we show HOTAIR and several tumor-associated lncRNAs are enriched in UEs from UBC patients with high-grade muscle-invasive disease (HGMI pT2-pT4). Knockdown of HOTAIR in UBC cell lines reduces in vitro migration and invasion. Importantly, loss of HOTAIR expression in UBC cell lines alters expression of epithelial-to-mesenchyme transition (EMT) genes including SNAI1, TWIST1, ZEB1, ZO1, MMP1 LAMB3, and LAMC2. Finally, we used RNA-sequencing to identify four additional lncRNAs enriched in UBC patient UEs. These data, suggest that UE-derived lncRNA may potentially serve as biomarkers and therapeutic targets.

Nasonia vitripennis venom causes targeted gene expression changes in its fly host

Parasitoid wasps are diverse and ecologically important insects that use venom to modify their hosts metabolism for the benefit of the parasitoids offspring. Thus, the effects of venom can be considered an ‘extended phenotype’ of the wasp. The model parasitoid wasp Nasonia vitripennis has approximately 100 venom proteins, 23 of which do not have sequence similarity to known proteins. Envenomation by N. vitripennis has previously been shown to induce developmental arrest, selective apoptosis and alterations in lipid metabolism in flesh fly hosts. However, the full effects of Nasonia venom are still largely unknown. In this study, we used high throughput RNA sequencing (RNA‐Seq) to characterize global changes in Sarcophaga bullata (Diptera) gene expression in response to envenomation by N. vitripennis. Surprisingly, we show that Nasonia venom targets a small subset of S. bullata loci, with ~2% genes being differentially expressed in response to envenomation. Strong upregulation of enhancer of split complex genes provides a potential molecular mechanism that could explain the observed neural cell death and developmental arrest in envenomated hosts. Significant increases in antimicrobial peptides and their corresponding regulatory genes provide evidence that venom could be selectively activating certain immune responses of the hosts. Further, we found differential expression of genes in several metabolic pathways, including glycolysis and gluconeogenesis that may be responsible for the decrease in pyruvate levels found in envenomated hosts. The targeting of Nasonia venom effects to a specific and limited set of genes provides insight into the interaction between the ectoparasitoid wasp and its host.

Prenatal PAH Exposure is Associated with Chromosome-specific Aberrations in Cord Blood

Chromosomal aberrations are associated with increased cancer risk in adults. Previously, we demonstrated that stable aberrations involving chromosomes 1-6 in cord blood are associated with prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) measured in air and are disproportionate to genomic content. We now examine whether the association with air PAHs is chromosome-specific and extends to smaller chromosomes. Using whole chromosome paints for chromosomes 1-6, 11, 12, 14 and 19, and a 6q sub-telomere specific probe, we scored 48 cord bloods (1500 metaphases per sample) from newborns monitored prenatally for airborne PAH exposure in the Columbia Center for Childrens Environmental Health cohort. Frequencies of stable aberrations were calculated as incident aberrations per 100 cell equivalents scored, and examined for association with airborne PAHs. Aberrations in chromosome 6 occurred more frequently than predicted by genomic content (p<0.008). Levels of both prenatal airborne PAHs and stable aberration frequency in chromosomes 1-6 decreased to half the levels reported previously in the same cohort (mean PAH decreased from 3.6 to 1.8ng/m(3); mean stable aberration frequency from 0.56 to 0.24, SD=0.19). The mean stable aberration frequency was 0.45 (SD=0.15) in chromosomes 11-19. After adjusting for gender, ethnicity, and household smokers, the mean stable aberration frequency increased with increasing PAH exposure: with a doubling of prenatal PAH exposure, the mean stable aberration frequency for the chromosome1-6 group increased by a factor of 1.49 (95% CI: 0.84, 2.66; p=0.17); for chromosomes 11-19 mean stable aberration frequency increased by 2.00 (95% CI: 1.11, 3.62; p=0.02); for chromosome 6 alone, it increased by 3.16 (95% CI: 0.93, 10.77; p=0.06); there was no increase for chromosomes 1-5 (p>0.8). Aberrations in chromosomes 11, 12, 14, 19 and 6 were associated with prenatal exposure to PAHs in air, even at lower levels of PAH in air. The observed chromosome-specific effects of prenatal airborne PAHs raise concern about potential cancer risk.

CD20 expression predicts survival in paediatric post‐transplant lymphoproliferative disease (PTLD) following solid organ transplantation

The prognostic role of CD20 expression and Epstein–Barr virus (EBV) positivity in post‐transplant lymphoproliferative disease (PTLD) after solid organ transplantation (SOT) in paediatric patients is poorly understood. We retrospectively examined the relationship of CD20 and EBV with the time interval from SOT to PTLD diagnosis, and PTLD‐related event‐free (EFS) and overall survival (OS) in 45 consecutive PTLD patients (≤25 years) following SOT. These 45 paediatric SOT patients (28 heart, 11 liver, six kidney) were diagnosed with PTLD 45 months (mean; SD 43; range 4–153; median 24·5) after SOT, with PTLD diagnosis at 118 months (mean) (SD 77; range 14–287) of age. Of 40 evaluable tumours (11 monomorphic, 19 polymorphic, five early lesions, five rare subtypes), 32 (80%) had detectable EBV and 28 (70%) were classified as CD20+. Patients whose PTLD expressed CD20 or EBV had shorter intervals between SOT and PTLD onset (28 vs. 64 or 77 months for CD20 and EBV respectively) (P < 0·02), even after adjusting for age at SOT. Patients with CD20+ tumours had higher 5‐year PTLD‐related EFS (83·7% vs. 28·6%, P < 0·001) and OS (95·8% vs. 56·3%, P = 0·01). EBV expression was unrelated to PTLD‐related EFS or OS. CD20 expression is associated with timing of development of PTLD and predicts survival in PTLD diagnosed following paediatric SOT.

A new approach for investigating venom function applied to venom calreticulin in a parasitoid wasp.

A new method is developed to investigate functions of venom components, using venom gene RNA interference knockdown in the venomous animal coupled with RNA sequencing in the envenomated host animal. The vRNAi/eRNA-Seq approach is applied to the venom calreticulin component (v-crc) of the parasitoid wasp Nasonia vitripennis. Parasitoids are common, venomous animals that inject venom proteins into host insects, where they modulate physiology and metabolism to produce a better food resource for the parasitoid larvae. vRNAi/eRNA-Seq indicates that v-crc acts to suppress expression of innate immune cell response, enhance expression of clotting genes in the host, and up-regulate cuticle genes. V-crc KD also results in an increased melanization reaction immediately following envenomation. We propose that v-crc inhibits innate immune response to parasitoid venom and reduces host bleeding during adult and larval parasitoid feeding. Experiments do not support the hypothesis that v-crc is required for the developmental arrest phenotype observed in envenomated hosts. We propose that an important role for some venom components is to reduce (modulate) the exaggerated effects of other venom components on target host gene expression, physiology, and survival, and term this venom mitigation. A model is developed that uses vRNAi/eRNA-Seq to quantify the contribution of individual venom components to total venom phenotypes, and to define different categories of mitigation by individual venoms on host gene expression. Mitigating functions likely contribute to the diversity of venom proteins in parasitoids and other venomous organisms.

Quality Improvement in Cystectomy Care with Enhanced Recovery (QUICCER) study

To determine if patients managed with a cystectomy enhanced recovery pathway (CERP) have improved quality of care after radical cystectomy (RC), as defined by a decrease in length of hospital stay (LOS) without an increase in complications or readmissions compared with those not managed with CERP.

MP51-19 MULTI-COMPONENT VIDEO-BASED FEEDBACK USING ADDITIONAL WEBCAM INPUTS IMPROVES DAVINCI SURGICAL SKILLS SIMULATOR (DVSSS) PERFORMANCE

INTRODUCTION AND OBJECTIVES: The use of cognitive training in the form of mental imagery (MI) to develop surgical skill has been previously demonstrated. However inconsistent results and the limited practical application of current cognitive training methods has limited its uptake. We have developed the first generic cognitive training tool for surgical skills development and validated its use for laparoscopic suturing training. METHODS: The MI training tool was developed in conjunction with cognitive psychologists and expert laparoscopic surgeons. By providing a framework within which trainees develop a personalised MI script, the training tool enables participants to undertake independent cognitive training. Ongoing critical self-evaluation promotes further refinement and improvement in the quality of the MI. The training tool was validated with a randomised controlled trial comparing MI to standard lecture based training for laparoscopic suturing. Participants randomised to cognitive training used the training tool to compose a personalised MI script. All participants then underwent 7 training sessions. Each session was video recorded and technical performance was blindly assessed post-hoc using a validated laparoscopic suturing score. Following training participants’ opinions of the MI training tool were collected. Quality of the MI was measured using the validated MI questionnaire (MIQ). RESULTS: 27 novice participants completed the study. The MI training tool was found to be very effective, with mean ratings of 6.5/7 and 6.29/7 for content and effectiveness respectively compared to scores of 4.9/7 and 3.8/7 for standard training. The quality of MI was also rated highly with a mean MIQ score of 6/7. No significant differences in technical performance were found between the groups either at baseline assessment or during the 1st 4 training sessions. Cognitive training resulted in significantly better performance after the 5th training session with increasing divergence in scores between the two groups (Figure 1). CONCLUSIONS: Our novel and highly adaptable cognitive training tool has been shown to effectively aid laparoscopic suturing training. Further validation of the cognitive training tool in more experienced robotic surgeons is now required to determine the optimal integration of cognitive training into surgical training.

Abstract B31: Bladder cancer patient urinary exosomes and tumors contain long noncoding RNA that may serve as therapeutic targets and biomarkers

While long non-coding RNA (lncRNA) play important roles in tumor biology including bladder cancer (BC), no previous studies have shown that lncRNAs in BC patient urinary exosomes (UEs) can function as biomarkers of disease. Exosomes are 30-150nm secreted membrane-bound vesicles that contain active biomolecules like: messenger(m)RNA, micro(mi)RNA, lncRNA and proteins generated from their cells of origin. In this study, we identified a panel of lncRNAs and mRNAs enriched in the UEs and tumors of BC patients, including the well-characterized lncRNA, HOTAIR. In order to elucidate if HOTAIR has a functional role in BC, we knocked it down in BC cell lines. Loss of HOTAIR resulted in reduced expression of known HOTAIR epithelial-to-mesenchyme transition (EMT) target genes, as well as reduced migration, and invasion using trans-well and 3-D culture assays. These data suggest that HOTAIR may serve as both a biomarker in UEs and as a potential therapeutic target. Critically, we RNA-Sequenced primary tumors, distal normal tissue (DNT) and UEs of BC patients and UEs from healthy volunteers (HV) to identify two novel lncRNAs, LINC00477 and LINC00940 enriched in BC patients9 UEs and tumors. These candidate lncRNAs were confirmed with quantitative real time (qRT-PCR). Taken together, UE lncRNA content reflect the lncRNA content of BC tumors and may serve as biomarkers of disease. Further investigation is needed to validate the use of the lncRNAs presented in this study as biomarkers and putative therapeutic targets in relevant patient populations. Citation Format: Claudia Berrondo, Jonathan Flax, Aisha Siebert, Victor Kucherov, Alex Rosenberg, Christopher Fucile, Carla Beckham. Bladder cancer patient urinary exosomes and tumors contain long noncoding RNA that may serve as therapeutic targets and biomarkers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B31.

Abstract LB-402: Chromosomal aberrations in 5-year olds and urinary PAH metabolites

Chromosomal aberrations (CAs) measured in cord blood are associated with maternal exposure to air Polycyclic Aromatic Hydrocarbons (PAH) in newborns from the Columbia Center for Children9s Environmental Health (CCCEH). We hypothesize that naphthalene, a PAH not measureable in air levels and an IARC classified potential carcinogen, might be associated with chromosomal aberrations. Now in 90 five yr olds from this cohort we examined the association between stable CAs (including translocations) scored using whole chromosome painting Fluorescent In Situ Hybridization in chromosomes 1-6, 11,12,14,19 in peripheral blood, and specific gravity adjusted 1OH and 2OH naphthalene (1OHnaph and 2OHnaph) metabolites measured in simultaneously collected urine, using an automated liquid-liquid extraction and quantified by gas chromatography/isotope dilution high-resolution mass spectrometry. Dominican American (DA) 5 yr olds (n=50) had higher specific 2OHnaph levels (mean: 12535.9 ng/m 3 ; SD: 22227.6) than African American (AA) 5 yr olds (n=40)(mean: 4239.2 SD: 3397.0),(Wilcoxon test, p=0.02) consistent with differences previously seen in the entire CCCEH cohort. Bivariate correlations between frequencies of CAs and 1OHnaph and 2OHnaph stratified by sex or ethnic group suggested an ethnic disparity in the association between CAs and naphthalenes. Negative binomial models were used to examine whether this association was dependent on ethnicity, controlling for sex. 1 OHnaph increased total stable CAs frequency in chromosomes 1-6 (r=0.24, p= 0.03), though this increase was only in DA (r=0.47, p=0.0006), not AA (r=0.09). Controlling for sex, the ethnic disparity in the association differs significantly (p=0.006). Frequency of translocations (a subgroup of stable CAs) also increased with increasing levels of 1OHNaph (r=0.20;p=0.07), particularly in DA(r=0.36; p=0.01), not in AA (r=0.07; p=0.0.6);controlling for sex, the ethnic disparity in association was significant (p 1 OHnaph and 2OHnaph did not effect CAs in chromosomes 11,12,14,19. Metabolites of naphthalene are associated with stable CAs including translocations in chromosomes 1-6. Sources of naphthalene may vary with cultural practices and off-label uses can contribute to elevated indoor air levels, surpassing those from outdoor pollutants. Our data suggest there are ethnic differences in both exposure to naphthalenes and response to this exposure, and have potential implications for prevention. The cohort is being followed to determine persistence of CAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-402.