Optical Imaging with a Cathepsin B Activated Probe for the Enhanced Detection of Esophageal Adenocarcinoma by Dual Channel Fluorescent Upper GI Endoscopy

Despite significant advances in diagnosis and treatment, the prognosis of esophageal adenocarcinoma remains poor highlighting the importance of early detection. Although white light (WL) upper endoscopy can be used for screening of the esophagus, it has limited sensitivity for early stage disease. Thus, development of new imaging technology to improve the diagnostic capabilities of upper GI endoscopy for early detection of esophageal adenocarcinoma is an important unmet need. The goal of this study was to develop a method for the detection of malignant lesions in the esophagus using WL upper endoscopy combined with near infrared (NIR) imaging with a protease activatable probe (Prosense750) selective for cathepsin B (CTSB). An orthotopic murine model for distal esophageal adenocarcinoma was generated through the implantation of OE-33 and OE-19 human esophageal adenocarcinoma lines in immunocompromised mice. The mice were imaged simultaneously for WL and NIR signal using a custom-built dual channel upper GI endoscope. The presence of tumor was confirmed by histology and target to background ratios (TBR) were compared for both WL and NIR imaging. NIR imaging with ProSense750 significantly improved upon the TBRs of esophageal tumor foci, with a TBR of 3.64$\pm$0.14 and 4.50$\pm$0.11 for the OE-33 and OE-19 tumors respectively, compared to 0.88$\pm$0.04 and 0.81$\pm$0.02 TBR for WL imaging. The combination of protease probes with novel imaging devices has the potential to improve esophageal tumor detection by fluorescently highlighting neoplastic regions

Exome and whole genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a five-year survival rate of 15%, identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole exome sequencing of 149 EAC tumors/normal pairs, 15 of which have also been subjected to whole genome sequencing. We identify a mutational signature defined by a high prevalence of A to C transversions at AA dinucleotides. Statistical analysis of exome data identified significantly mutated 26 genes. Of these genes, four (TP53, CDKN2A, SMAD4, and PIK3CA) have been previously implicated in EAC. The novel significantly mutated genes include chromatin modifying factors and candidate contributors: SPG20, TLR4, ELMO1, and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 reveal increased cellular invasion. Therefore, we suggest a new hypothesis about the potential activation of the RAC1 pathway to be a contributor to EAC tumorigenesis

Mutational heterogeneity in cancer and the search for new cancer genes

Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention

Optical Imaging with a Cathepsin B Activated Probe for the Enhanced Detection of Esophageal Adenocarcinoma by Dual Channel Fluorescent Upper GI Endoscopy

<p>Despite significant advances in diagnosis and treatment, the prognosis of esophageal adenocarcinoma remains poor highlighting the importance of early detection. Although white light (WL) upper endoscopy can be used for screening of the esophagus, it has limited sensitivity for early stage disease. Thus, development of new imaging technology to improve the diagnostic capabilities of upper GI endoscopy for early detection of esophageal adenocarcinoma is an important unmet need. The goal of this study was to develop a method for the detection of malignant lesions in the esophagus using WL upper endoscopy combined with near infrared (NIR) imaging with a protease activatable probe (Prosense750) selective for cathepsin B (CTSB). An orthotopic murine model for distal esophageal adenocarcinoma was generated through the implantation of OE-33 and OE-19 human esophageal adenocarcinoma lines in immunocompromised mice. The mice were imaged simultaneously for WL and NIR signal using a custom-built dual channel upper GI endoscope. The presence of tumor was confirmed by histology and target to background ratios (TBR) were compared for both WL and NIR imaging. NIR imaging with ProSense750 significantly improved upon the TBRs of esophageal tumor foci, with a TBR of 3.64&#177;0.14 and 4.50&#177;0.11 for the OE-33 and OE-19 tumors respectively, compared to 0.88&#177;0.04 and 0.81&#177;0.02 TBR for WL imaging. The combination of protease probes with novel imaging devices has the potential to improve esophageal tumor detection by fluorescently highlighting neoplastic regions.</p

BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors

<div><p>BRD4 is a transcriptional co-activator functioning to recruit regulatory complexes to acetylated chromatin. A subset of High-grade Serous Ovarian Cancer (HGSOC) patients are typified by focal, recurrent BRD4 gene amplifications. Despite previously described cancer dependencies, it is unclear whether BRD4 amplification events are oncogenic in HGSOC. We find that physiologically relevant levels of expression of BRD4 isoforms in non-transformed ovarian cells result in cellular transformation. Transcriptional profiling of BRD4-transformed ovarian cells, and BRD4-amplified HGSOC patient samples revealed shared expression patterns, including enriched MYC, and E2F1 gene signatures. Furthermore, we demonstrate that a novel BET inhibitor, AZD5153, is highly active in BRD4-amplified patient derived xenografts and uncover Neuregulin-1 as a novel BRD4 effector. Experiments involving Neuregulin-1 inhibition and exogenous addition, demonstrate Neuregulin-1 as necessary and sufficient for BRD4-mediated transformation. This study demonstrates the oncogenic potential of BRD4 amplification in cancer and establishes BRD4-amplified HGSOC as a potential patient population that could benefit from BET inhibitors.</p></div

NRG1 is a pharmacodynamic marker of BRD4-amplified HGSOC patient-derived xenografts.

<p><b>(A)</b> NRG1 mRNA expression measured using RNA-seq in OV0857F tumors at 4 hr and 24 hr following 10 mg/kg AZD5153 p.o. once (QD) or for four days (QDx4). <b>(B)</b> NRG1 protein levels measured by immunoblot in OV0857F tumors at 4 hr and 24 hr following a single or 4-repeat p.o. dose of 10 mg/kg AZD5153. <b>(C)</b> NRG1 mRNA expression measured using RNA-seq in HOXF062 tumors at 4 hr following 30 mg/kg AZD5153 p.o. (QD). <b>(D)</b> NRG1 protein levels measured by immunoblot in HOXF062 tumors at 4 hr following a single p.o. dose of 30 mg/kg AZD5153. <b>(E)</b> NRG1 mRNA measured using RNA-seq in OV2022F tumors at 4 hr following either 10 mg/kg p.o. AZD5153 QD or 5 mg/kg AZD5153 BIDx3. <b>(F)</b> NRG1 protein levels measured by immunoblot in OV2022F tumors at 4 hr following either 10 mg/kg QD or 5 mg/kg BIDx3 AZD5153.</p

BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells.

<p><b>(A)</b> Analysis of BRD4 copy-number from 1042 cell lines (CCLE). <b>(B)</b> BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan^® Copy-number Assay by qPCR. <b>(C)</b> Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. <b>(D)</b> Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. <b>(E)</b> Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.</p