bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 357: Rapid harvesting of highly concentrated circulating tumor cells from bulk human blood with buoyant immuno-microbubbles
Authors: Wang Guankui , Pingle Sandeep , Kesari Santosh , Liu Yu-Tsueng , Simberg Dmitri
Published: July 2016

Circulating tumor cells (CTCs) are the extremely rare cells shed from primary tumor during metastatic disease. A technology to quickly and inexpensively isolate viable CTCs will enable widespread clinical utilization of CTCs for cancer prognosis and drug testing. The lipid-shelled microbubble is a biocompatible material that has been used as ultrasound contrast media. We have previously shown that MB is a promising vehicle to isolate rare cells from biological fluids by flotation. Here we investigated the role of lipid composition, MB/cell ratio, and type of tumor surface marker [epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erbB-2 or human epidermal growth factor receptor 2 (HER2/neu) and epithelial growth factor receptor (EGFR)] on MB stability and binding efficiency to breast cancer cells. Optimized MBs coated with anti-EpCAM antibody showed 95% binding efficiency to human breast carcinoma SKBR-3 cells. Cells were able to proliferate in culture with MBs attached to them. We further developed an inverted setup in order to harvest (skim) highly concentrated tumor cells in a microliter volume on a nitrocellulose membrane. Using vacuum-assisted harvesting we demonstrated a high efficiency (∼80%) of isolation of rare tumor cells spiked in 7 ml blood by anti-EpCAM MBs. A blinded study using anti-EpCAM MBs detected epithelial CTCs in samples of metastatic breast and ovarian cancers with brain metastases, but not in samples from non-epithelial brain malignancies, including primary glioblastoma multiforme, hemangioblastoma and metastatic melanoma. The simple and robust MB approach can be developed into an inexpensive tool for batch CTC isolation in basic research and clinical applications.

Citation Format: Guankui Wang, Sandeep Pingle, Santosh Kesari, Yu-Tsueng Liu, Dmitri Simberg. Rapid harvesting of highly concentrated circulating tumor cells from bulk human blood with buoyant immuno-microbubbles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 357.
Abstract 3571: Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer
Authors: Pilling Amanda , Hwang Ok , Hwang Clara
Published: July 2016

Prostate cancer is the most common malignancy and the 2nd leading cause of cancer death in men worldwide. First-line treatment includes androgen deprivation therapy in addition to androgen receptor (AR) antagonists that work to block androgen signaling. Although most patients respond initially to hormone deprivation therapy, responses are not durable and the disease progresses to a lethal stage, termed castrate resistant prostate cancer (CRPC). Interestingly, most CRPCs are still dependent on AR signaling for growth and survival, therefore more potent AR-antagonists such as enzalutamide have recently been FDA approved for treatment of CRPC. However, response rates to enzalutamide can be limited by emergence of acquired resistance or intrinsic resistance. A universal mechanism of resistance to cytotoxic therapies is upregulation of anti-apoptotic programs that function to override death signals and permit survival of the tumor cell. In prostate cancer, overexpression of the Inhibitor of Apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP has been demonstrated in progression to CRPC. Additionally, aberrant expression of these proteins is reported to contribute to anti-androgen resistance. Therefore, to achieve more robust responses to AR inhibition with enzalutamide, we targeted IAP proteins in order to amplify the apoptotic signal and increase cell death in prostate cancer cells. Using a small-molecule IAP inhibitor, we evaluated response to enzalutamide upon IAP inhibition in prostate cancer cell lines LNCAP and C4-2. Here we demonstrate greater proliferation inhibition, reduced cell survival and synergistic induction of apoptosis signaling in response to combined AR antagonism and IAP inhibition. Furthermore, we demonstrate depletion of cIAP1 protein expression upon enzalutamide treatment indicating a possible role for AR in regulating anti-apoptotic pathways. Taken together these results demonstrate that IAP proteins may be a critical survival pathway in CRPC and antagonism of these proteins through small-molecule inhibition can amplify apoptosis and increase cell death in response to enzalutamide. Targeting a critical survival pathway can lower the apoptotic threshold providing a possible therapeutic opportunity to treat and prevent resistance to enzalutamide and increase benefit to patients with this lethal form of prostate cancer.

Citation Format: Amanda Pilling, Ok Hwang, Clara Hwang. Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3571.
Abstract 3578: Novel insights into the cellular function and gene regulation of a master mutator, APOBEC3B
Authors: Gara Sudheer Kumar , Liu-Chittenden Yi , Kotian Shweta , Patel Dhaval , Kebebew Electron
Published: July 2016

Unequivocally, cumulative mutations are required for the development of cancer and many sources of mutagenic activity contribute to tumorigenesis. Recent evidence has implicated that a member of cytidine deaminase family, APOBEC3B (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B or A3B) is enriched in the genomes of many human cancers such as cervical, bladder, lung, head and neck and breast cancers (Burns et al., Nature, 2013). APOBEC3B deaminate cytosines in the host genome to generate C-T mutations. Here we report for the first time that APOBEC3B is overexpressed with adrenocortical carcinoma (ACC), a very aggressive and lethal malignancy. We found that APOBEC3B is significantly overexpressed in primary and metastatic ACC as compared to normal adrenal and cortical adenomas by qPCR, immunofluorescence. We observed that γH2AX, a marker of DNA double-strand breaks co-localizes with the expression of APOBEC3B in patients with primary and metastatic ACC. Also ACC tumor samples with high APOBEC3B expression had high chromosomal gain/loss particularly in chromosome 4 and 8 compared to tumor samples with low APOBEC3B expression. To identify the cellular function of APOBEC3B, we performed transient knock-down studies in three ACC cell lines and found that it reduces cellular proliferation and induces cell cycle arrest. Furthermore, we observed that knockdown of APOBEC3B inhibits MAPK signaling by downregulating phospho ERK1/2 levels. Interestingly, we found that tumors with high APOBEC3B expression levels had twice as many TP53 inactivating mutations as compared to tumors with low levels of APOBEC3B. In order to understand the regulation of APOBEC3B expression, we analyzed the methylation status, copy number variation and potential targeting miRNAs of APOBEC3B from genome wide array platform but found no associations in human ACC samples. Therefore, we did a functional, knockdown screen of 90 cancer-associated transcription factors and found that GATA3 showed positive regulation of APOBEC3B. Our data provide novel insights into the function and regulation of APOBEC3B expression beyond its known mutagenic ability.

Citation Format: Sudheer Kumar Gara, Yi Liu-Chittenden, Shweta Kotian, Dhaval Patel, Electron Kebebew. Novel insights into the cellular function and gene regulation of a master mutator, APOBEC3B. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3578.
Abstract 358: In vitro assessment of tumor relapse in a 3D tumor microtissue model
Authors: Dhar Sumeer
Published: July 2016

One of the most pressing problems in the treatment of cancer is tumor relapse. Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. Current in vitro assays based on monolayer cultures do not provide the required time frame to study tumor recurrence.

We developed a new method to study tumor relapse in a 3D in vitro tumor microtissue model. HCT116 colorectal tumor microtissues were produced and dosed with 3 reference compounds, Taxol (cell division inhibitor), Staurosporine (protein kinase inhibitor) and Doxorubicin (DNA synthesis inhibitor). Based on an initial range finding to determine IC20 and IC50 values, tumor growth was monitored via size profiling and viability (intra-tissue ATP content) over 10 days. Three different conditions were tested: (i) continuous treated tumor microtissues, (ii) microtissues which were supplemented with the drug for 5 days and (iii) non-treated tumor microtissues. Growth differences couldn't be observed comparing IC20 concentrations, however growth profiles significantly varied comparing IC50 concentrations. Whereas a 5-day treatment of Taxol was leading to a complete growth inhibition after removing the drug, Staurosporine treated tumor microtissues relapsed even with a similar growth kinetic as the untreated control group. Pulsed treatment of Doxorubicin was leading to a slower growth kinetic as compared to the control but not to a complete reminiscence. Based on the individual growth profiles relative to the continuous treated and non-treated groups we calculated a relapse index (ReI, patent pending) to enable classification of compounds and compound combinations according to their risk for tumor relapse.

Here we present a novel in vitro tumor relapse assay method based on a 3-dimensional tumor microtissue model, to enable compound classification based on their efficiency to prevent tumor relapse.

Citation Format: Sumeer Dhar. In vitro assessment of tumor relapse in a 3D tumor microtissue model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 358.
Abstract 3585: KIF11 silencing or inhibition induces chromosome instability
Authors: Asbaghi Yasamin , Lichtensztejn Zelda , Thompson Laura , McManus Kirk
Published: July 2016

Chromosome Instability (CIN) is defined as an increase in the rate at which whole chromosomes or large parts are gained or lost. CIN is not only associated with virtually all tumor types, but it is associated with aggressive tumors, the acquisition of multi-drug resistance and consequently poor patient prognosis. Despite these associations, the genes and molecular defects that contribute to CIN are only poorly understood. Recently, we performed a high content screen that identified KIF11, a microtubule associated motor protein, as a candidate CIN gene. Here, we couple RNAi-based gene silencing with biochemistry and cell biology to show that diminished KIF11 expression and/or function induce CIN. HCT116 cells were employed, as they are a karyotypically stable colorectal cancer cell line of epithelial origin that has been used for similar CIN studies. KIF11 was either silenced (both individual and pooled siRNA duplexes) or inhibited (Monostrol) and expression levels were determined by Western blots. To determine whether KIF11 silencing or inhibition affects DNA content, two phenotypes frequently associated with CIN, namely increases in nuclear area and micronucleus formation were evaluated. Fluorescence microscopy was employed on DAPI-counterstained samples and revealed statistically significant increases both nuclear area and micronucleus formation following KIF11 silencing and inhibition relative to controls. Next, flow cytometry was performed on propidium iodide labeled samples to assess whether increases in DNA content were associated with the changes in nuclear area. As predicted, increases in the proportion of cells with >G2/M DNA content occurred within the KIF11 silenced populations. Finally, mitotic chromosome spreads were generated and chromosomes were manually enumerated from 100 spreads per condition/control. Subsequent Kolmogorov-Smirnov tests identified statistically significant increases in the cumulative distribution frequencies of mitotic chromosome numbers within the spreads generated from the KIF11 silenced cells relative to controls. To extend our findings beyond the colorectal cancer cell context employed above, analogous studies were performed in hTERT cells (karyotypically stable fibroblast cell line) with very similar results. Collectively, these data indicate that KIF11 expression and function are normally required to maintain genome integrity. They further suggest that the loss of KIF11 expression and/or function may be a contributing factor in the etiology of tumorigenesis in colorectal cancer and perhaps other tumor types as well.

Citation Format: Yasamin Asbaghi, Zelda Lichtensztejn, Laura Thompson, Kirk McManus. KIF11 silencing or inhibition induces chromosome instability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3585.
Abstract 3586: Overexpression of Emi1 causes chromosomal instability and cancer
Authors: Vaidyanathan Srividya , Cato Kathleen , Pavey Sandra , Haass Nikolas K. , Gabrielli Brian G. , Duijf. Pascal HG
Published: July 2016

Chromosomal Instability (CIN) is a common occurrence in solid cancer associated with poor survival in patients. A significant percentage of these tumors show abnormal chromosomes with near-triploid or tetraploid numbers exhibiting numerical CIN (nCIN). nCIN is caused mainly due to deregulation of cell cycle proteins. Early Mitotic Inhibitor 1 (Emi1) is a cell cycle protein that regulates the activity of E3 ubiquitin ligase, Anaphase Promoting Complex/Cyclosome (APC/C), which targets many cell cycle proteins for degradation. Emi1 is overexpressed in many solid tumors but not blood cancers.

Unpublished data from our lab has shown that Emi1 overexpression in mouse models causes more penetrant and metastatic tumor than control tumors. Breast cancer tissues show significantly high expression of Emi1 at protein level as compared to control breast tissue samples, in agreement with the microarray data from The Cancer Genome Atlas (TCGA). In addition, Emi1 expression from breast cancer TCGA data strongly correlated with the chromosomal instability signature than many of the well-established genes known to promote CIN phenotype. To understand the mechanisms of CIN due to Emi1 overexpression, we overexpressed Emi1 in HeLa cells expressing GFP-tagged histone protein to monitor the cells during mitosis by live cell imaging.

HeLa cells overexpressing Emi1 show CIN following delays in both chromosome alignment at the metaphase plate and, subsequently, anaphase onset (p = 0.0008 and p = 0.0028). There is a significant increase in CIN phenotypes and abnormal mitoses such as anaphase bridges/lagging chromosomes, mitotic arrest and cell death. These results indicate that Emi1 overexpression actively drives tumorigenesis.

Citation Format: Srividya Vaidyanathan, Kathleen Cato, Sandra Pavey, Nikolas K. Haass, Brian G. Gabrielli, Pascal HG Duijf. Overexpression of Emi1 causes chromosomal instability and cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3586.
Abstract 3589: Nucleotide excision repair is elevated in stage IV commonly used breast cancer cell lines as compared to stage I breast tumor explants
Authors: As Sobeai Homood , Johnson Jennifer M. , Lalanne Nancy , Ibrahim Omar , Grant Stephen G. , Wenger Sharon L. , Latimer Jean J.
Published: July 2016

Genomic instability is a hallmark of human carcinogenesis. We have previously published that sporadic stage I breast tumors exhibit deficient Nucleotide Excision Repair (NER) capacity relative to non-diseased breast reduction primary cultures (Latimer et al., 2010). In the present work, we hypothesized that this feature of early human breast cancer would not be maintained in cell lines established from late stage tumors. Our objective was to determine functional NER capacity and relative NER gene expression in a series of commonly used stage IV breast cancer-derived cell lines relative to stage I breast cancer explants and cell lines. NER capacity was determined using the functional unscheduled DNA synthesis (UDS) assay in six established breast cancer cell lines (MCF-7, MCF-7LY2, MDA-MB-231, SK-BR-3, CAMA-1, BT-20). JL BTL-12 (Jean Latimer Breast Tumor Line-12), derived from a stage III breast tumor, was included to represent an advanced stage tumor cell line established using our culture system. The NER capacity of the cell lines was compared to that of 19 stage I breast tumor primary cultures. NER gene expression was determined by expression microarray using the Affymetrix HG U-133 plus 2.0 chip in five of these established cell lines (MCF-7, MDA-MB-231, SK-BR-3, CAMA-1, BT-20), JL BTL-12 and a representative stage I tumor-derived cell line (JL BTL-8, Breast Tumor Line-8). The stage IV cell lines and JL BTL-12 all manifested significantly higher NER capacity than explants of stage I breast cancer. Unsupervised as well as supervised analyses, based on expression of the canonical 20 NER genes, placed JL BTL-12 and the five stage IV commercial cell lines in one cluster, while JL BTL-8 clustered separately. Four NER genes were significantly upregulated in JL BTL-12 and all commercial cell lines relative to JL BTL-8. In conclusion, based on NER these five cell lines commonly used in breast cancer research are more representative of late stage disease than early stage breast cancer, regardless of the type of breast cancer they represent. The most commonly diagnosed stage of breast cancer in the U.S. is now stage I, and it is now possible to study stage I cell lines or explants.

Citation Format: Homood As Sobeai, Jennifer M. Johnson, Nancy Lalanne, Omar Ibrahim, Stephen G. Grant, Sharon L. Wenger, Jean J. Latimer. Nucleotide excision repair is elevated in stage IV commonly used breast cancer cell lines as compared to stage I breast tumor explants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3589.
Abstract 3591: Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells
Authors: Gomes Cecil J. , Harman Michael , Centuori Sara , Wolgemuth Charles , Martinez Jesse
Published: July 2016

In normal lung tissue the expression levels of 14-3-3 gamma are very low and tightly regulated, however, in cancer it is evident that 14-3-3 gamma's expression patterns become deregulated and significantly elevated. In patients with advanced non-small cell lung cancer (NSCLC), increased expression of this isoform is associated with poorer survival and significantly correlates with lymph node and distant metastasis. These data prompted us to analyze TCGA's NSCLC gene expression dataset, and it is clear that 14-3-3 gamma's expression continues to increase in the progression from early to late stage cancers. Taken together, these data suggest that overexpression of 14-3-3 gamma is correlated with a more aggressive tumor phenotype, an observation also seen with breast and hepatocellular carcinoma. The focus of this study is to elucidate the mechanism(s) causing these more aggressive cancer phenotypes. We have previously shown that overexpressing 14-3-3 gamma in human lung cancer cells harboring very low levels of endogenous 14-3-3 gamma and no wildtype p53 results in a stable subpopulation of cells with polyploid DNA content. Interestingly, approximately 40% of lung adenocarcinomas present with hyper-triploid karyotypes, and even a higher percentage, 40-60%, have inactivation of p53. It is well established that polyploid tumors have the capacity to increase tumorigenicity by allowing resistance to conventional therapies and also permitting elevated tolerance to chromosomal instability (CIN). This leads us to hypothesize that in the absence of p53, NSCLC tumors overexpressing 14-3-3 gamma result in a polyploid population of cells that may be influencing the aggressiveness of the tumor. Further in vitro analysis of these 14-3-3 gamma induced tetraploid cells show that they have a prolonged mitosis with significantly more lagging chromosomes in anaphase than their diploid counterparts, indicating an increase in chromosomal instability (CIN). After isolating isogenic tetraploid clones with or without 14-3-3 gamma expression, the stability of the tetraploid cell state was assessed. Clones not expressing 14-3-3 gamma quickly reverted back to a pseudo-diploid state, whereas overexpression of 14-3-3 gamma significantly prolonged the tolerance of tetraploidy and eventually resulted in an aneuploid cell state. Our data suggests that overexpression of 14-3-3 gamma increases tumorigenicity through the stabilization of a polyploid cell state.

Citation Format: Cecil J. Gomes, Michael Harman, Sara Centuori, Charles Wolgemuth, Jesse Martinez. Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3591.
Abstract 3592: BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells
Authors: Thaiparambil Jose T. , El-Zein Randa
Published: July 2016

Lung cancer remains the leading cause of cancer related deaths in the United States. Cancer results from an accumulation of multiple genetic events leading to genomic instability that is a crucial early event in carcinogenesis. Aneuploidy is the most frequently identified genomic abnormality in cancer. We have previously reported a significantly higher level of aneuploidy in smokers who developed lung cancer as compared to smoker controls. Benzo(a)pyrene [B(a)P], a polycyclic aromatic hydrocarbon, is a pulmonary carcinogen and one of 60 different carcinogens found in tobacco smoke. In this study, we hypothesize that B(a)P disrupts mitotic spindle assembly that impairs the fidelity of mitosis leading to chromosome missegregation and aneuploidy.

To test our hypothesis, we use confocal imaging in combination with traditional molecular biology techniques to dissect the precise mechanism by which B[a]P induces chromosome missegregation and aneuploidy. Mitotic spindle defects in the form of chromosome lags and bridges, monopolar and multipolar spindles, were assessed in normal lung epithelial cells after treatment with 10μM B[a]P for 6 hrs. Spindle defects were assessed at 24, 48, 72 and 96 hours after treatment. Immunofluorescence revealed abnormal mitosis in the prometaphase through the telophase stage of mitosis. Our result show that B[a]P exposure induced abnormal spindles in the form of monopolar (14%) and multipolar (4%) spindles. High resolution confocal images further revealed that B[a]P exposure led to misoriented mitotic spindles with an abnormal spindle angle [-380] relative to normal plane [180]. Chromosome lagging at both metaphase (17%) and anaphase (9%) has also been recorded in response to B[a]P exposure, observations that result from defects in the mitotic apparatus and kinetochores. Furthermore, we assessed the extent of B[a]P induced genetic instability [in the form of double strand DNA breaks] using gama-H2AX in order to elucidate the underlying mechanism associated with accumulated genetic damage and chromosome missegregation. Our data indicate that B[a]P results in accumulation of mitotic abnormalities and gama-H2AX foci (22%) peaks at 48 hr compared to other time points.

Taken together, our data show that B[a]P potently induces mitotic abnormalities by disrupting kinetochore microtubules. The induction of mitotic abnormalities by B[a]P leading to chromosome missegregation and aneuploidy, would unravel a novel targetable molecular mechanism of cigarette smoke induced lung tumorigenesis.

Citation Format: Jose T. Thaiparambil, Randa El-Zein. BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3592.
Abstract 3595: Improved therapeutics for hepatocellular carcinoma
Authors: Zavadil Jessica A. , Walter Christi A.
Published: July 2016

Eighty percent of hepatocellular carcinoma (HCC) within the USA is diagnosed late when chemotherapy provides only 2 months increased survival. We fortuitously found that treatment with an alkylating agent, methylnitrosurea (MNU), prior to tumorigenesis reduced tumor prevalence later in life by ∼50% in C3HeB/FeJ male mice, a model of spontaneous HCC. A similar effect was observed with temozolomide (TMZ), a glioblastoma therapy that induces the same DNA damage at similar proportions to MNU. O6-methylguanine-DNA methyltransferase (MGMT) repairs the highly mutagenic and cytotoxic O6-methylguanine lesions induced by TMZ, and MGMT abundance influences therapy response in glioblastoma. MGMT is reduced in 1/3 of human HCC, leading us to hypothesize that TMZ could be effective in treating HCC. We proposed to combine TMZ with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, to maximize efficacy, reasoning that SAHA would allow TMZ greater access to DNA to induce greater levels of damage. We tested this drug combination in two human HCC cell lines, Huh7, with reduced MGMT expression, and SNU398, with higher expression. TMZ and SAHA synergistically reduced cell viability in both cell lines with multiple dose combinations. Based on this outcome, we tested the drugs in tumor-bearing C3HeB/FeJ mice. Surprisingly, DMSO treated solvent control mice showed reduced tumor burden. The DMSO treatment was repeated adding a sham control group and confirmed that DMSO significantly reduces tumor burden. The TMZ cohort also displayed a significantly lower tumor burden compared to the sham cohort, but no significant difference was found with SAHA alone or SAHA+TMZ. We previously showed that a hMGMT transgene protects from hepatocarcinogenesis in C3HeB/FeJ mice, but surprisingly, there is not additional protection when transgenic mice are treated with MNU. These results suggest that the more prevalent cytotoxic lesions induced by MNU and TMZ, e.g. N7-methylguanine, may be more important in HCC than O6-methylguanine. These lesions are recognized by methylpurine-DNA glycosylase (MPG) and repaired through base excision repair. Therefore, we directly tested the role of MPG in TMZ therapy through altered expression of MPG in Huh7 and SNU398 cell lines and showed that it does affect sensitivity to TMZ.

Citation Format: Jessica A. Zavadil, Christi A. Walter. Improved therapeutics for hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3595.
Abstract 3598: Laser micro-irradiations of large amounts of cells for high content applications
Authors: Mistrik Martin , Vesela Eva , Furst Tomas , Frydrych Ivo , Gursky Jan , Bartek Jiri
Published: July 2016

A new methodical approach for the evaluation of photo-manipulated samples is presented and tested in various biological setups. The method is based on an atypical setting of a standard laser-scanning microscope (LSM) and image-processing software solution. Newly, a whole population of several dozen of cells is simultaneously micro-irradiated by a laser beam in a precisely defined pattern of collinear rays. Such laser induced striation pattern is automatically analyzed by specialized software routine providing quantitative assessment of various photo-manipulations, such as DNA-damage induced protein translocations or modifications, in both live and fixed cells. Automation of the process reduces workload and opens new possibilities for various dynamic studies and/or high content experiments. Results obtained during the testing of the method are presented and offer an insight into several DNA damage recognition and response pathways.

Citation Format: Martin Mistrik, Eva Vesela, Tomas Furst, Ivo Frydrych, Jan Gursky, Jiri Bartek. Laser micro-irradiations of large amounts of cells for high content applications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3598.
Abstract 3604: Validated structural variant detection with prioritisation of known cancer related changes
Authors: Ahdesmäki Miika , Chapman Brad , Luke Sally , Carr Hedley , Stetson Daniel , Hofmann Oliver , Johnson Justin
Published: July 2016

Reliable detection of structural variation (SV) is playing an increasingly important role in cancer diagnostics and treatments. We introduce a best practice integrative workflow for detecting large structural variations (SVs) such as deletions, duplications, inversions, fusions and translocations from standard DNA re-sequencing techniques, annotating and prioritising for clinically actionable events.

While established RNA-Seq fusion workflows exist to detect and filter fusion events in tumors detecting large structural changes from exome- and WGS data remains challenging. Current DNA-based SV callers predict a large percentage of false positive events, and the resulting event lists are not prioritised for validation. To streamline follow-up studies we automate running multiple structural variant callers (Manta, WHAM, Lumpy, MetaSV), identifying potentially disruptive large scale events. We evaluate the integrated workflow sensitivity and specificity against the ICGC-TCGA DREAM Mutation Calling Challenge data set, optimise filtering criteria based on quality and supporting information, and annotate the filtered calls using known cancer related alterations from CIViC and publicly available gene lists from AstraZeneca. This enables us to prioritise known and potentially interesting events over intergenic events or non-whole exon deletions.

We show validation results from test data and clinically actionable inversions, duplications and fusion events from cancer cell lines. Our approach is based on a two tier approach of first focusing on known events that yield fusion transcripts such as the TACC3-FGFR3 tandem duplication and ALK-EML4 inversion, and secondarily on events in oncogenes and tumor suppressors of interest. While whole genome sequencing is the preferred approach to detecting these events, we also show how by design or by the nature of the breakpoint being close enough to exons, hybrid capture data can also be surprisingly useful for SV inference.

Citation Format: Miika Ahdesmäki, Brad Chapman, Sally Luke, Hedley Carr, Daniel Stetson, Oliver Hofmann, Justin Johnson. Validated structural variant detection with prioritisation of known cancer related changes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3604.
Abstract 3608: Detection of somatic structural variants using nanopore sequencing
Authors: Norris Alexis L. , Workman Rachael E. , Fan Yunfan , Eshleman James R. , Timp Winston
Published: July 2016

Structural variants (SVs) are a hallmark of human cancer, but remain difficult to reliably and accurately detect with next generation sequencing (NGS). This is in part due to the difficult in mapping NGS's short reads (<300bp) to repetitive elements, which often flank the genomic rearrangements of SVs. Nanopore sequencing, a 3rd generation sequencing technology, offers long sequencing reads (up to 20kb), that should allow for more reliable mapping of SVs. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Here, we have tested the ability of nanopore sequencing to detect a series of well-characterized SVs (including large deletions, inversions, and translocations), that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we show that nanopore sequencing on the MinION platform can detect large deletions, translocations, and inversions at dilutions as low as 1:100. Given the speed, small footprint (USB size), and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of somatic SVs, with clinical applications of molecular relapse, early detection, or therapeutic monitoring.

Citation Format: Alexis L. Norris, Rachael E. Workman, Yunfan Fan, James R. Eshleman, Winston Timp. Detection of somatic structural variants using nanopore sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3608.
Abstract 3618: NGS-based detection of FLT3-ITDs with Anchored Multiplex PCR
Authors: Bessette Marc , Van Deusen Benjamin , Banos Michael , Johnson Laura , Berlin Aaron , Reckase Erik , Stahl Joshua , Licon Abel , Kudlow Brian
Published: July 2016

FLT3 internal tandem duplications (ITDs) are found in > 20% of pediatric and adult acute myeloid leukemia (AML) cases and are generally associated with a poor prognosis. Nevertheless, detection of FLT3-ITDs presents a challenge to NGS-based approaches, as many variant callers fail to identify the highly variable repeated sequences associated with FLT3-ITDs.

We have developed a novel targeted assay, the Archer™ VariantPlex™ Core AML Panel, that utilizes Anchored Multiplex PCR (AMP™) with probes in multiple locations proximal to exons 13, 14, and 15 of FLT3. These exons encompass the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. This panel yields high-complexity, dual-strand coverage of known FLT3-ITD locations. Because AMP probes, unlike traditional PCR probes, function independently of each other, we are able to produce multiple overlapping “snapshots” of the region of interest, thereby enhancing the ability to confidently identify complex mutation types. The sequenced reads are assembled using a novel de novo assembly algorithm in the Archer Analysis bioinformatics pipeline, in which the resulting consensus is annotated and events that involve FLT3 exons 13 14 or 15 are marked as FLT3 abnormalities.

In order to assess Archer Analysis and the VariantPlex Core AML panel, we tested it on >20 patient-derived samples with known FLT3-ITDs. In addition, we generated over two thousand in silico datasets, representing the spectrum of known ITDs, to further test our panel design and analysis algorithm. Each in silico dataset was constructed to simulate reads originating from probes in VariantPlex Core AML Panel. This methodology permitted rigorous optimization of both the VariantPlex Core AML Panel and the analysis algorithm.

The VariantPlex Core AML Panel, in conjunction with our novel detection algorithm, showed both exceptional sensitivity and specificity in the detection of FLT3-ITDs. FLT3-ITDs were successfully identified in all patient samples tested, and no false positives were detected. Our in silico datasets showed similarly high sensitivity and specificity.

These data indicate that AMP libraries targeting the FLT3-ITD region are ideal for the detection of this complex mutation type, largely because of the overlapping and anchored read structure. Therefore, the Archer VariantPlex Core AML panel, in conjunction with Archer Analysis represents a reliable platform for the detection of FLT3-ITDs, in addition to other mutations commonly found in AML.

Citation Format: Marc Bessette, Benjamin Van Deusen, Michael Banos, Laura Johnson, Aaron Berlin, Erik Reckase, Joshua Stahl, Abel Licon, Brian Kudlow. NGS-based detection of FLT3-ITDs with Anchored Multiplex PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3618.
Abstract 3623: Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay
Authors: Choi Jonathan
Published: July 2016

Circulating tumor DNA can be found in the plasma of cancer patients, and can be utilized for non-invasive cancer detection and genotyping through Next Generation Sequencing. However, unique characteristics of ctDNA pose technical challenges that limit technical sensitivities and robustness. ctDNA are fragmented, short, rare in abundance, and found in high background of normal DNA from leukocytes. Cancer variants range as low as 0.1-1% minor allele fractions, within the noise of NGS methods. Furthermore, clinical interest includes large number of genes and mutation types including single nucleotide variants, copy number amplification, and fusions. We compared two popular target enrichment categories, digital PCR and a hybridization-based capture panel for target enrichment. We assessed the assay performance and detection limitations, using a diverse sample set to test the range of input material that reflects ctDNA found in plasma. We titrated important cancer mutations (BRAF, EGFR, KRAS, PI3KCA, etc.) testing minor allele fractions ranging from 33% down to 0.1%, MET amplification ranges of 14x to 5x, and CCDC6-RET fusions of 50% to 2%. We also assessed input DNA ranges of 1ng and 10ng, well below manufacturer recommend input of library prep methods. Testing the performance of a 29kb amplicon panel and a 114kb hybridization-probes panel, we found superior on-target rates with digital PCR compared to the hybridization capture method across all samples. However, despite higher depths of coverage with droplet PCR, hybridization based methods had superior read mapping, uniformity (1.4x better), error rate (0.16% vs 0.02%), and greater linearity of SNV variants. Hybridization methods had greater accuracy of variant detection and were able to accurately identify the RET fusion. We demonstrated the feasibility of utilizing target enrichment methods for next generation sequencing for ctDNA variant detection.

Citation Format: Jonathan Choi. Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3623.
Abstract 3628: Improving sequencing quality of libraries prepared from FFPE DNA
Authors: liu pingfang , Chen Lixin , Ettwiller Laurence , Sumner Christine , Stewart Fiona J. , Dimalanta Eileen T. , Davis Theodore B. , Thomas Evans C.
Published: July 2016

Targeted cancer therapy based on genomic alterations can be remarkably effective, and has made significant strides with the recent advances in next-generation sequencing (NGS) technology. Although samples of blood and other bodily fluids are being actively explored for early disease diagnosis and treatment monitoring, DNA isolated from FFPE samples is currently the main source for NGS-based cancer profiling in clinical settings. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. Artifacts associated with FFPE DNA have limited the mutation detection sensitivity to ≥ 5% mutant allele frequency (Frampton et al, Nature Biotechnology 2013), which would unfortunately leave many low-abundance genetic variants of clinical significance undetected. For example, clinical resistance-causing KIT and EGFR mutations can be present in tumors at levels << 1% (Milbury et al, Clin. Chem. 2012).

In this study, we investigated the effects of DNA repair and different sample handling workflows on sequencing quality of libraries prepared from FFPE samples. Careful analysis of sequencing data showed that base calling qualities for all 4 bases are improved, and aberrant G:C to A:T mutations were significantly reduced upon DNA repair. Because the large majority of mutations encountered in human tumors are G:C to A:T mutations (Greenman, C. Nature 2007), we expect that lowering the damage induced background noise of FFPE DNA would allow more reliable detection of clinically important, actionable mutations at lower abundance. In addition, we observed specific sequencing artifacts associated with the method of handling FFPE samples and have since identified effective measurements to avoid such artifacts. We expect that these improvements in sequencing quality of FFPE samples would ultimately enable more sensitive and robust detection of many low level genetic variations in clinically and biologically relevant cancer genes.

Citation Format: pingfang liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona J. Stewart, Eileen T. Dimalanta, Theodore B. Davis, Evans C. Thomas. Improving sequencing quality of libraries prepared from FFPE DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3628.
Abstract 3629: Gene fusion detection in sarcoma using TRAC technology
Authors: Mattinen Laura , Rautio Jari , Salmivaara Jani , Halleen jussi , Sirkiä Marko
Published: July 2016

Genomic rearrangements are a defining feature of cancer, and can lead to gene fusions and chimeric transcripts with respective fusion proteins that have oncogenic properties. The fusion transcripts can have diagnostic, prognostic and therapeutic implications. However, the fusion partners and the exact breakpoints vary from patient to patient and from cancer type to another. This can result in significant sequence variation, and together with the large number of potential genes able to participate in fusion events makes reliable detection of fusion events challenging for many traditional methods. The TRAC (Transcript Analysis with aid of Affinity Capture) method solves a number of these issues by a combination of specific capture and detection probes. By capturing all known 5’ fusion partners and detecting potential 3’ partners for a given cancer type, only a very limited set of positive signals are detected from an individual sample, enabling high-level of multiplexing. In practice, dozens of different fusion events can be detected per sample by using an easily modifiable pool of capture/detection probes. This effectively enables detection of all known fusion types in sarcomas from one sample without the prerequisite of knowing the exact cancer type. TRAC is a direct hybridization based assay that enables rapid quantification of multiple mRNA targets simultaneously from large number of samples. Transcript levels can be measured directly from cell or tissue lysates without the need for RNA extraction, cDNA conversion or PCR-amplification. The TRAC protocol is straightforward and enables sample analysis in a high-throughput format with little hands-on time. In this analysis, TRAC was used to detect the fusion transcripts COL1A1-PDGFB, FUS-DDIT3, and SS18-SSX1/2 from three types of sarcoma tissue biopsies. All fusion types were detected for the respective targets. In addition, twenty Ewing sarcoma cell lines were analyzed for the EWSR1-FLI1 fusion, identifying 12 cell lines positive for the chimeric transcript. Sarcoma tissue lysate was found applicable for direct TRAC analysis. These results demonstrate that the TRAC Fusion Transcript Assay is functional and capable of discriminating sarcomas based on fusion transcript expression. The assay is easy to set up and it can be run using standard laboratory equipment with little hands-on time.

Citation Format: Laura Mattinen, Jari Rautio, Jani Salmivaara, jussi Halleen, Marko Sirkiä. Gene fusion detection in sarcoma using TRAC technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3629.
Abstract 3633: OncoGxOneTM - an advanced genomic test for precision cancer treatment
Authors: Han Yang , Yu Pengfei , Song Qingxuan , Wei Min , Hu Guanghui
Published: July 2016

The fundamental cause of cancer is the aberration of genes that control cell proliferation and apoptosis. With the rapid advancement of genomic technologies and increased arsenal of targeted therapies, genomic profiling has been largely accepted by physicians to assist cancer diagnosis and treatment. Developed by Admera Health, OncoGxOneTM is a CLIA certified and CAP accredited genomic profiling panel detecting all actionable mutations in 64 cancer genes. All the FDA approved targeted therapies and registered clinical trials are covered by this carefully designed panel. Utilizing cutting edge Next Generation Sequencing technology combined with accurate analysis and data interpretation, OncoGxOneTM detects all 4 types of genomic alterations including single nucleotide mutation, insertion/deletion, copy number variation and gene rearrangement. Using fresh frozen tissue or routine FFPE samples, OncoGxOne TM offers both high sensitivity and specificity greater than 99% from samples with as low as 10% tumor cell content.

Citation Format: Yang Han, Pengfei Yu, Qingxuan Song, Min Wei, Guanghui Hu. OncoGxOneTM - an advanced genomic test for precision cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3633.