bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 646: Targeting MHC class I molecules and immune checkpoints as key immune evasion strategies in EBV-associated nasopharyngeal carcinoma
Authors: Siu Sharie Pui-Kei , To Ka Fai , Chung Grace Tin-Yun , Lui Vivian W.Y. , Li Yvonne Y. , Hammerman Peter S. , Lo Kwok Wai
Published: July 2017

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer that is consistently associated with intensive lymphocytes infiltration and Epstein-Barr virus infection. This study aims at elucidating the mechanism contributing to immune evasion in NPC. During NPC tumorigenesis, multiple intrinsic mechanisms are acquired to escape cellular immune response for the tumor and viral antigens. By genome sequencing, somatic alterations of multiple MHC class I genes including NLRC5, HLA-A, HLA-B, HLA-C and B2M were detected in 30% of primary NPC. Significant correlation of MHC class I gene alterations and poor survival rate in the patients was also shown. The finding suggested that the deficiency of antigen presentation mechanism represents a common strategy for immune evasion in NPC and favors rapid tumor progression. Losses of HLA-A, HLA-B and NLRC5 expression were validated in a panel of NPC tumor lines and selected primary tumors. The role of NLRC5 as key transcription regulator of HLA-A and HLA-B expression was demonstrated in the nasopharyngeal epithelial cells. To further evaluate the mechanisms contributed to immune evasion in NPC, we have elucidated immunosuppressive molecules involved in immune surveillance in Epstein Barr Virus (EBV)-associated NPC and its association with EBV-encoded LMP1 in an independent cohort of primary NPCs. By immunohistochemistry, the expression of EBV-encoded LMP1, MHC class I genes and various immunosuppressive molecules including PDL1, PDL2 and IDO1 was examined in 98 primary tumors. PDL1 and PDL2 were found to be over-expressed in 50% and 33.7% of NPC cases while IDO1 was found to be expressed in 28.8% of cases. Primary NPC cases expressing either PDL1 or PDL2 accounted for 64.3%. A significant association between LMP1 and PDL1 and PDL2 expression was also found. The correlation of PDL1, PDL2 and/or MHC class I molecule expression with the outcome of the patients was determined. The high incidence of MHC class I deficiencies and PDL1/PDL2 overexpression imply that EBV-associated NPC escape from the host immune response by targeting both antigen presentation mechanisms and immune checkpoints. Our findings also suggested that the expression of MHC class I molecules and PDL1/PDL2 is a potential biomarker for immunotherapy in NPC patients.

Citation Format: Sharie Pui-Kei Siu, Ka Fai To, Grace Tin-Yun Chung, Vivian W.Y. Lui, Yvonne Y. Li, Peter S. Hammerman, Kwok Wai Lo. Targeting MHC class I molecules and immune checkpoints as key immune evasion strategies in EBV-associated nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2017-646
Abstract 656: PD-L1 expression in primary lesions vs metastatic sites and by demographics in advanced urothelial carcinoma samples
Authors: Zajac M , Boothman A M. , Ben Y , Gupta A , Jin X , Antal J , Sharpe A , Scott M , Rebelatto M , Walker J
Published: July 2017

Background: Determination of programmed cell death ligand-1 (PD-L1) expression levels in tumors may help physicians understand which patients (pts) are most likely to respond to anti-PD-1/PD-L1 therapies in urothelial carcinoma (UC). Understanding the impact of different sample types and demographics on PD-L1 expression is important to determine suitability of tumor biopsies for testing.

Methods: As of July 24, 2016, 363 pts screened in the UC cohort of Study CD-ON-MEDI4736-1108 (NCT01693562) had tissue available for analysis and 47 pts had provided paired primary and metastatic samples. FFPE samples were tested in a central laboratory with the VENTANA PD-L1 (SP263) Assay using a BenchMark ULTRA instrument. Pts were classified as having either PD-L1 high (PD-L1 expression ≥25% either on tumour cells [TC] or immune cells [IC]) or PD-L1 low/negative (<25% on TC and IC) tumors. PD-L1 high prevalence was reported in primary vs metastatic sites, and by age, sex and race.

Results: PD-L1 status was evaluable for 332/363 (91.5%) pts (175/332 [52.7%] PD-L1 high and 157/332 [47.3%] PD-L1 low/negative) whose UC specimens were tested (intent to diagnose [ITD] population). Overall percentage agreement between paired primary and metastatic samples, based on combined TC/IC scoring ≥25%, was 74.5% (95% CI 59.7 - 86.1%). In the ITD population, using only the samples from which patient PD-L1 expression status was determined, PD-L1 high prevalence in primary and metastatic samples was 57.1% and 50.9% respectively (p=0.343, not significant). The proportion of pts with PD-L1 high status was not enriched in any demographic group (Table).

Conclusions: Initial data from UC pts in Study 1108 showed similar PD-L1 high prevalence in primary and metastatic lesions and good concordance between paired primary and metastatic samples. These results build optimism that samples obtained from either location could be used to determine PD-L1 status. Further data are needed to confirm these findings.

Patients screened for UC cohort with evaluable PD-L1 result - ITD population (n = 332)ParameterPD-L1 high, n (%)P valueAge, years<65 (n=131)73 (55.7%)0.438≥65 (n=201)102 (50.8%)SexMale (n=236)127 (53.8%)0.610Female (n=96)48 (50.0%)RaceAsian (n=52)23 (44.2%)Asian vs White: 0.406Black or African American (n=10)6 (60.0%)White (n=218)113 (51.8%)Other (n=9)4 (44.4%)

Citation Format: M Zajac, A M. Boothman, Y Ben, A Gupta, X Jin, J Antal, A Sharpe, M Scott, M Rebelatto, J Walker. PD-L1 expression in primary lesions vs metastatic sites and by demographics in advanced urothelial carcinoma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2017-656
Abstract 666: CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma
Authors: Xu-Monette Zijun Y. , Young Ken H.
Published: July 2017

PD-1 immune checkpoint blockade reconstituting antitumor immunity has changed the cancer treatment paradigm. PD-1 blockade has been successful in many types of solid tumors and Hodgkin lymphoma, but not yet for diffuse large B cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. We found several biomarkers including loss of CD37 tetraspanin expression in DLBCL correlated with increased PD-1 expression suggesting potential sensitivity of these DLBCL subsets to PD-1 blockade. CD37 (TSPAN26) is a member of the tetraspanin superfamily, widely expressed on normal and malignant mature B-cells and downregulated in plasma cells. It has been documented that CD37 plays important roles in T-cell-B-cell interactions, B-cell humoral response triggered by B-cell receptor cross-linking, and a balance between immune responses and tolerance. Interestingly, in a large cohort of DLBCL patients, we found that loss of CD37 expression in DLBCL predicts strikingly decreased overall and progression-free survival rates, and that PDCD1 gene expression was upregulated in CD37-negative activated B-cell-like (ABC) DLBCL by gene expression profiling, whereas the costimulatory molecule ICOSLG was upregulated in CD37+ germinal center B-cell-like (GCB) DLBCL. Using the new fluorescent multiplex technology, we further measured PD-1 and PD-L1 expression at the protein level on lymphoma cells and immune cells in the tumor microenvironment, and found that PD-1 protein levels were increased on both cytotoxic and helper T-cells infiltrating in CD37-negative DLBCL either of GCB or ABC subtype. These novel discoveries suggest that CD37 is important for sustained antitumor adaptive immunity, that immune dysregulation plays an important role for poor clinical outcomes in DLBCL, and that CD37-negative DLBCL may be sensitive to PD-1 blockade. In summary, loss of a CD37 tetraspanin was found to correlate with PD-1 overexpression in DLBCL clinical samples, and CD37 may serve as a novel biomarker for anti-PD-1 immunotherapy clinical trials in DLBCL.

Note: This abstract was not presented at the meeting.

Citation Format: Zijun Y. Xu-Monette, Ken H. Young. CD37 tetraspanin as a novel biomarker for PD-1 blockade in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2017-666
Abstract 680: Prognostic impact of OPN and DKK1 in patient of hepatocellular carcinoma after hepatectomy
Authors: Seo YunSung , Byeon Hye Rim , Park In Hae , Lee Seung Duk
Published: July 2017

The new biomarkers are essential for improving the survival and prognosis of hepatocellular carcinoma(HCC) patients. Alpha-fetoprotein(AFP) is the most widely used biomarker. But the low sensitivity and specificity limits its clinical application. Recent study validated the diagnostic capability of osteopontin(OPN) and dickkopf-1(DKK1) and assessed the combination of AFP, DKK1, and OPN as a panel for the diagnosis of HCC. Based on these previous studies, we hypothesized that combination of OPN and DKK1 can be used to as a marker for prognosis to patient of HCC after hepatectomy. From January 2006 to December 2008, patients undergoing hepatectomy for hepatocellular carcinoma were screened serum that has been stored in the Bank of tumor. To target the selected patient, it was confirmed that the remaining tissue specimen is stored after diagnosis. Serum of the patients was used to investigate the OPN and DKK1 by ELISA. In the paraffin block were prepared unstained slide and OPN and DKK1 level checked by IHC. It examined the correlation between prognosis and biomarkers through statistical analysis. AFP, OPN(serum level) and DKK1(serum level) are an independent prognostic factor for overall survival(OS) in HCC after hepatectomy(n=60, P=0.0204, 0.0167 and 0.0455 respectively). New biomarkers combinations based on the AFP existing biomarker are showed a falling curve of the overall survival(OS) and disease-free survival(DFS) in Kaplan Meier curve. In conclusion, combination of OPN, DKK1 and AFP as a biomarker could support the correct diagnosis for HCC after hepatectomy.

Note: This abstract was not presented at the meeting.

Citation Format: YunSung Seo, Hye Rim Byeon, In Hae Park, Seung Duk Lee. Prognostic impact of OPN and DKK1 in patient of hepatocellular carcinoma after hepatectomy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2017-680
Abstract 686: onTARGET: a new computational strategy to predict in vivo relevant targets against neuroblastoma
Authors: Almstedt Elin , Lönnstedt Ingrid , Krona Cecilia , Nelander Sven
Published: July 2017

One of the main bottlenecks of anticancer drug development is the assessment of the in vivo relevance of emerging therapies. Previously, drugs that suppress the in vivo progression of neuroblastoma have been hard to identify. To address this problem, we propose a new computational technique, onTARGET, that enables researchers to select, with good accuracy, compounds that are likely to induce changes in cellular pathways that are consistent with specific clinical outcomes and subgroups.

onTARGET prediction is based on an integration of massive expression and other data resources, including the NIH LINCS dataset (with 1,300,000 expression profiles), the NIH TARGET and R2-AMC neuroblastoma cohorts, and our own in-house databases. In a first step, the datasets are preprocessed by factor analysis to remove systematic bias in these large data sets. In a second step, Bayesian statistics are applied to prioritize compounds whose cellular responses match clinically relevant differences between patient subsets, such as MYCN status, survival hazard ratio, or histopathological assessment.

To evaluate the method, we used onTARGET prediction to identify compounds and gene targets relevant for clinical outcomes in the NIH-TARGET (n=249) and R2-AMC (n=98) neuroblastoma cohorts. The algorithm confirmed existing targets and made interesting predictions. Of 50 the top-ranking compounds associated with a survival outcome, agents targeting the mTOR/PI3K axis are highly prevalent. The algorithm correctly predicts targeting of the MYC pathway, and predicts possible modulators of MYC signaling, such as TRRAP, AURKA and BMP2. In addition to nominating agents with a favorable outcome, the algorithm can also flag compounds that might worsen prognosis, further facilitating the prioritization of targets for evaluation.

In summary, onTARGET may guide target selection for neuroblastoma studies and can be adapted for other cancers as well. The many targets that were identified immediately suggest an opportunity for continued evaluation in cells and in vivo models. This work has been initiated in collaboration with researchers at Lund University.

Citation Format: Elin Almstedt, Ingrid Lönnstedt, Cecilia Krona, Sven Nelander. onTARGET: a new computational strategy to predict in vivo relevant targets against neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 686. doi:10.1158/1538-7445.AM2017-686
Abstract 70: Elucidating the roles of antibody pharmacokinetics and maleimide stability in the toxicology of antibody-drug conjugates
Authors: Neff-LaFord Haley , Zapata Franciso , Schultz Wendi , Balasubramanian Cindy , Pittmen Paul , Hengel Shawna , Sanderson Russell , Chemuturi Nagendra , Setter Jocelyn , Lyon Robert P.
Published: July 2017

Antibody-drug conjugates (ADCs) continue to emerge as effective therapeutics in a variety of oncology indications, with two agents currently approved and many more in late-stage clinical trials. These ADCs employ drug-linkers that were developed many years ago, and are now recognized to have properties that may adversely impact the activity and toxicology of the ADCs prepared with them. Two such properties that are now well appreciated are the reversibility of maleimide-based drug conjugation, and the impact of drug conjugation on the pharmacokinetics of the ADC. We recently reported advances in drug-linker design that independently address both of these properties, resulting in the irreversible conjugation of drugs which have minimal impact on antibody pharmacokinetics, even at high levels of drug loading (Nature Biotechnology 32, 1059-1062 (2014), Nature Biotechnology 33, 733-735 (2015)). We have now prepared drug-linkers of monomethylauristatin E (MMAE) that orthogonally employ these features to enable a systematic evaluation of the relative contributions of maleimide instability and accelerated plasma clearance on the in vivo behavior of MMAE ADCs. Biodistribution studies with these molecules have revealed that the concentration of released MMAE in normal tissues is greatly impacted by the rate of ADC clearance (fast clearance results in greater Cmax of free drug), while stabilization of the maleimide has a relatively small effect. These differences in observed free drug concentrations were paralleled in tolerability studies, with ADC clearance rates exerting a greater impact on hematology parameters than maleimide stability. Collectively, these results suggest that ADC pharmacokinetics dominate the biodistribution and toxicology profiles for a given drug payload, with conjugate stability playing a relatively minor role.

Citation Format: Haley Neff-LaFord, Franciso Zapata, Wendi Schultz, Cindy Balasubramanian, Paul Pittmen, Shawna Hengel, Russell Sanderson, Nagendra Chemuturi, Jocelyn Setter, Robert P. Lyon. Elucidating the roles of antibody pharmacokinetics and maleimide stability in the toxicology of antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 70. doi:10.1158/1538-7445.AM2017-70
Abstract 710: Identification of autoantibody to ECH1 & HNRNPA2B1 as potential biomarkers in the early detection of lung cancer
Authors: Dai Liping , Li Jitian , Tsay Jun-Chieh J. , Wang Xiao , Munger John S. , Pass Harvey , Rom William N. , Tan Eng M. , Zhang Jian-Ying
Published: July 2017

Background: Identification of biomarkers for early detection of lung cancer (LC) may lead to more effective treatment and reduction of mortality.

Methods: Serological proteome analysis (SERPA) was used to identify proteins around 34 kDa, which had been previously recognized by autoantibody in sera from LC patients. We have validated autoantibody response in sera from 90 LC patients, 89 normal controls by using immunoassay. Another independent cohort of 25 LC patients with 219 serial serum samples and 56 matched normal controls were examined to evaluate whether the autoantibody can be detected in the preclinical stage.

Results: The proteins with molecular weight of 34 kDa were identified as ECH1, GAPDH and HNRNPA2B1. In the validation study, autoantibody to ECH1 achieved an area under the curve (AUC) of 0.799 with sensitivity of 62.2% and specificity of 95.5% in discriminating LC from normal individuals, and showed negative correlation with tumor size (rs=0.-256, p=0.023). Autoantibody to HNRNPA2B1 performed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%, and showed negative correlation with lymph node metastasis (rs=0.-279, P=0.012). By using longitudinal preclinical samples, autoantibody to ECH1 showed an AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3% in distinguishing LC with matched normal controls, and elevated autoantibody levels could be detected greater than two years prior to LC diagnosis.

Conclusions: ECH1 and HNRNPA2B1 are autoantigens that elicit autoimmune responses in LC and can be used as potential biomarkers for the early detection of LC.

Funding support: This work was supported by the National Natural Science Foundation of China (81672917, 81372371) and the National Institutes of Health (SC1CA166016 and U01CA086137).

Citation Format: Liping Dai, Jitian Li, Jun-Chieh J. Tsay, Xiao Wang, John S. Munger, Harvey Pass, William N. Rom, Eng M. Tan, Jian-Ying Zhang. Identification of autoantibody to ECH1 & HNRNPA2B1 as potential biomarkers in the early detection of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2017-710
Abstract 722: Circulating EBV microRNA BART2-5p helps to diagnose nasopharyngeal carcinoma < screen for high risk individuals precisely
Authors: Jiang Chen , Li Lei , Jiangchao Li , Xie Shang-hang , Cao Su-mei , Guan Xin-yuan
Published: July 2017

Nasopharyngeal carcinoma is an EB-virus associated malignancy which is highly prevalent in Southeast Asia. If diagnosed early, the 5 year disease-free survival rate is about 90%. However, most patients have already developed local or regional invasion when diagnosed, which results in poor prognosis. So it is essential to develop biomarkers to screen high risk individuals or diagnose the disease at early stages. As nasopharyngeal carcinoma is closely related to EBV infection and microRNAs encoded by BART region (BamHI A Rightward Transcripts) is abundant in NPC, we propose to identify biomarkers from those BART microRNAs. To quantify the copy number of microRNAs in serum samples, we used Taqman Probe-based qPCR. After adding Spiked-in Control (Mixture of Cel-39,Cel-54, Cel-238) to each 100ul serum sample, we extracted total RNA and reverse transcribed microRNA into cDNA with corresponding Taqman primers. Meanwhile, microRNA mimics with known copy number were serial diluted and reverse transcribed along with the samples. qPCR was performed to quantify the level of microRNAs in these samples with diluted cDNA and Taqman Probe. All data was normalized by Spiked-in controls and exact copy number of each microRNA was calculated through the standard curve generated by the serial diluted microRNA mimics. Here, we screened 17 BART microRNAs in discovery stage and found BART 2-5p as a candidate biomarker. In training cohort consisting of 266 patients with nasopharyngeal carcinoma and controls from Hong Kong, the sensitivity, specificity and AUC is 93.2%, 89.8%,0.976 respectively. The results are similar in validation cohort 1 constituted by 376 patients and controls from Guang Zhou. To evaluate the ability of BART 2-5p to distinguish preclinical NPC patients from healthy high risk individuals, we established a nested case-control study with serum samples prospectively collected from 10 NPC patients at least one year prior to their clinical diagnosis and 92 matched healthy high risk controls from a screening scheme conducted in South China. The sensitivity and specificity is 90.0% and 31.5%. Taken together, EBV microRNA BART2-5p is a promising biomarker to improve NPC diagnosis and target preclinical patients more precisely than serologic test.

Citation Format: Chen Jiang, Lei Li, Li Jiangchao, Shang-hang Xie, Su-mei Cao, Xin-yuan Guan. Circulating EBV microRNA BART2-5p helps to diagnose nasopharyngeal carcinoma < screen for high risk individuals precisely [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 722. doi:10.1158/1538-7445.AM2017-722
Abstract 728: Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay
Authors: Powell Michael J. , Peletskaya Elena , Vallerga Anne , Sun Qing , Pastor Larry , Zhang Aiguo
Published: July 2017

Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated.

Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP’s) and insertion/deletions (indels) mutations can be detected. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression.

Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in colon cancer development, early detection, disease monitoring and therapeutic interventions.

Citation Format: Michael J. Powell, Elena Peletskaya, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2017-728
Abstract 738: Highly multiplexed and precisely calibrated reference materials for copy number variation detection
Authors: Huang Catherine , Konigshofer Yves , Dickens Jessica , Anekella Bharathi
Published: July 2017

Introduction: Genomic instability is a hallmark of cancer, and copy number variation (CNV), including gene duplication and deletion, can be a key driver of oncogenesis. Detection of CNV is needed for personalized treatment, and may involve microarray analysis or Next Generation Sequencing. Often, DNA from patient-derived cell lines is used as positive control material in these assays. However, these materials are often poorly characterized, may change over time, and have additional confounding genetic alterations. Here we describe the development of new reference materials that are highly characterized by digital PCR and can aid in the validation and calibration of new diagnostic assays for CNV.

Methods: The GM24385 reference cell line has been extensively characterized by the Genome in a Bottle project1 and originates from a participant in the Personal Genome Project. DNA from this cell line was used to make a library of full-length gene inserts. The library had >12X coverage of the GM24385 genome and an average insert size of ~135 Kb. PCR based screening was used to identify and isolate full length MYC, FGFR3 and ERBB2 genes. Next Generation Sequencing, using Illumina Nextera library preparation kits and a MiSeq instrument, was used to verify the gene of interest. The genomic DNA and gene inserts were then formulated into reference materials, in purified DNA format to amplify the copy numbers of these key genes. Digital PCR was used to control the mixing and verify the number of copies of genes and/or mutations within the reference material.

Results: The sequence confirmed genes were formulated into GM24385 genomic DNA at levels consistent with 5X and 10X amplification. Testing on the ArcherDx VariantPlex™ Solid Tumor Kit indicated that each amplification could be detected at the expected level.

Conclusions: Highly characterized and consistently manufactured reference materials for copy number detection are currently lacking. The described Seraseq CNV Reference Materials provide proof of concept for a biosynthetic, engineered material that uses digital PCR as an orthologous reference method and provides a “ground truth” copy number level for key oncogenes.

Citation Format: Catherine Huang, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Highly multiplexed and precisely calibrated reference materials for copy number variation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 738. doi:10.1158/1538-7445.AM2017-738
Abstract 739: Orthogonal validation of oncomine cfDNA panel data with digital PCR using TaqMan Rare Mutation Assays
Authors: Venkatesh Vidya , Laig Marion , Varma Kamini , Li Yanchun , Bramlett Kelli , Dhingra Dalia , Chien Richard
Published: July 2017

The discovery of circulating tumor DNA (ctDNA) in the blood, urine and other bodily fluids of cancer patients has led to a new type of non-invasive method of characterizing cancer-causing mutations, the liquid biopsy. With NGS technologies becoming increasingly sensitive, down to a 0.1% Limit of Detection (LOD), they are rapidly gaining traction as a valid assay for cancer genotyping and have potential to direct cancer treatment plans. The wide-angle view provided by NGS panels, combined with dPCR’s zoomed-in precision detection of DNA provide a comprehensive picture of a cancer’s genetic makeup. By applying these complementary techniques at the appropriate time based on the disease type and stage, cancer treatment will become quicker, more precise and more cost-effective in the future. NGS and digital PCR together provide a complete picture of the cancer genome. As part of our research, we wet-lab tested a subset of Rare Mutation Assays corresponding to the Oncomine cfDNA panel for next generation sequencing. Synthetic plasmid (GeneArt) carrying the mutation was spiked into wild-type genomic DNA to reflect a mutation rate of 0.1%. Wild-type genomic DNA was used as negative control. Thermal cycling was performed according to protocol for digital PCR using the QuantStudio 3D. In this study, we tested a pilot set of samples using AcroMetrix Oncology Hotspot Control and Horizon cfDNA Reference Standard with both NGS using Oncomine cfDNA panel and digital PCR with Rare Mutation Assays. Comparison of NGS and digital PCR results for the same sample showed excellent correlation in the low mutation range around 0.1%. This study confirms that digital PCR using QuantStudio 3D and Rare Mutation Assays is effective as a method for orthogonal validation of NGS data using the Oncomine cfDNA panel. Additionally, TaqMan Rare Mutation Assays offer a sensitive and precise solution for downstream mutation tracking over a time course. For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Vidya Venkatesh, Marion Laig, Kamini Varma, Yanchun Li, Kelli Bramlett, Dalia Dhingra, Richard Chien. Orthogonal validation of oncomine cfDNA panel data with digital PCR using TaqMan Rare Mutation Assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 739. doi:10.1158/1538-7445.AM2017-739
Abstract 740: Rapid detection of IDH1/2 mutations using RNaseH2 dependent, multiplex quantitative PCR Assays
Authors: Bao Yun , Baig Aymen , Wang Yu , Iafrate A. John , Chen Caifu , Borger Darrell R.
Published: July 2017

Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are enzymes that normally catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate during glucose metabolism. Heterozygous mutations in the IDH1 and IDH2 genes have been identified in a specific subset of cancers. Mutations typically affect the enzyme active sites at codon R132 in the IDH1 gene, and R140 or R172 in the IDH2 gene. This confers neomorphic enzyme activity that produces the oncometabolite 2-hydroxyglutarate that is involved in cancer initiation and progression. The detection of IDH1/IDH2 mutations has important diagnostic, prognostic, and therapeutic implications and testing for these variants is being incorporated in clinical settings. In fact, the identification of IDH1 or IDH2 gene mutations is now required for the classification of tumors of the central nervous system under the 2016 WHO guidelines. Therefore, there is a significant need for an IDH1 + IDH2 test that can be performed in a rapid manner to support clinical diagnosis and treatment decisions. A strategy utilizing allele-specific quantitative PCR (qPCR) is well suited for evaluating a small number of hotspot variants in IDH1 and IDH2 from clinical tissue samples in a matter of few hours. Using RNase H2-dependent PCR (rhPCR) and a universal reporter system, we have developed new multiplex qPCR assays that effectively discriminate between closely related sequences that differ by only a single nucleotide base at the same position or adjacent position in a single tube reaction. In addition, the rhPCR mechanism of the assays also greatly reduces the impact of primer-primer interactions, further improving the multiplex capability of the assays. During initial testing, specificity, accuracy, and sensitivity were evaluated. Mutations present in patient samples at frequencies as low as 2% were identified with high reproducibility. The assays have been tested across 49 patient samples that included FFPE tissue, blood, and bone marrow aspirate with results that are concordant to those found using an orthogonal method of next generation sequencing. Our study showed that this assay provides a rapid method for detection of multiple IDH1 or IDH2 mutations in single rhPCR reactions with high accuracy, specificity, and sensitivity.

Citation Format: Yun Bao, Aymen Baig, Yu Wang, A. John Iafrate, Caifu Chen, Darrell R. Borger. Rapid detection of IDH1/2 mutations using RNaseH2 dependent, multiplex quantitative PCR Assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2017-740
Abstract 741: Rapid detection of necrosis in breast cancer with ex vivo and in situ mass spectrometry analysis methods
Authors: Zarrine-Afsar Arash , Shrestha Bindesh , Tata Alessandra , Woolman Michael , Ventura Manuela , Bernards Nicholas , Ganguly Milan , Ginsberg Howard , Zheng Jinzi , Bluemke Emma
Published: July 2017

Necrosis is a form of cell death that is often associated with highly aggressive forms of cancer, is of prognostic value in treatment planning. Mass Spectrometry (MS) is a highly sensitive analytic platform capable of providing a molecular profile of cancer on the basis of mass to charge (m/z) ratio of tissue constituent molecules. MS analysis of ex vivo tissue slices from metastatic murine xenograft tumors from LM2-4 cell line with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) allowed direct comparisons with histology images to determine the molecular profile of necrotic tissues. The necrotic tissue is characterized by the presence of a ceramide absent from the viable cancer regions. The spatial distribution of this ion fully correlated to necrotic areas from pathology in additional independent tumor samples examined. The same ion was detected from in situ necrotic tissue using tissue aerosols generated by hand-held ablation probes coupled to evaporative ionization interface in only a few seconds of sampling. These developments further establish MS as a novel tool for rapid pathology that is highly complementary to current histology based methods widely used in characterization of cancer in both imaging mode (to provide spatial information on cancer border) and profiling mode (to provide information on cancer type and subtype); all based on unique molecular profile associated with each cancer type and subtype. Current efforts in creating cancer molecular profile libraries will facilitate translation.

Citation Format: Arash Zarrine-Afsar, Bindesh Shrestha, Alessandra Tata, Michael Woolman, Manuela Ventura, Nicholas Bernards, Milan Ganguly, Howard Ginsberg, Jinzi Zheng, Emma Bluemke. Rapid detection of necrosis in breast cancer with ex vivo and in situ mass spectrometry analysis methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 741. doi:10.1158/1538-7445.AM2017-741
Abstract 767: Breast cancer in sub-Saharan Africa: a histopathologic review from a tertiary hospital in Northern Nigeria
Authors: Ahmed Saad A. , Kabir Balarabe , Abubakar Nasiru
Published: July 2017

Introduction: Breast cancer is the commonest cancer in women world-wide, however, there are discrepancies in the incidence among different geographical regions. The highest incidence rate is in North American, Northern and Western European, while the lowest rate is found among Asian and sub-Sahara African women. Breast cancer is undoubtedly heterogeneous in terms of its presentation, morphology and molecular profiles. Studies from the developing countries suggested that there are differences between the biology of BC in the developing and developed countries.

Methods: This is a retrospective histopathologic review of breast cancer cases between 1st January 2003 and 31st December 2015 from the Ahmadu Bello University Teaching Hospital Zaria - Nigeria.

Results: A total of 784 breast cancer cases were seen during the study period and majority of the cases (99.4%) were females. The prevalence of breast cancer was found to be 12.6%. Triple-negative breast cancers (TNBC) constitute 67.7% of cases. The mean age was 45.8 ±12.0 years and the youngest patient was 18 years while the oldest was 90 years. Out of the 784 cases, 208 were mastectomies while the remaining were incisional, trucut needle and excisional biopsies. Infiltrating ductal carcinoma NST was the most common histologic variant seen (94.7%) and most of these cases (59.1%) were Grade II tumors. The Nottingham Prognostic Index (NPI) shows that 91.4% of the cases have NPI Score of ≥3.5 with attendant poor 5-year survival rate.

Conclusion: This study found that breast cancer is common and affects mostly women in fifth decade of life. There is a high TNBC among the population studied and therefore androgen receptor profile and genomic studies may be useful to determine the course of therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Saad A. Ahmed, Balarabe Kabir, Nasiru Abubakar. Breast cancer in sub-Saharan Africa: a histopathologic review from a tertiary hospital in Northern Nigeria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 767. doi:10.1158/1538-7445.AM2017-767
Abstract 790: Regulation of tumor angiogenesis by low dose Aspirin
Authors: Chakraborty Jinia , Maity Gargi , Banerjee Snigdha , Banerjee Sushanta K.
Published: July 2017

Background and Objective: Tumor angiogenesis is a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs for the nourishment of cancer cells and metastatic growth. Thereby, targeting tumor angiogenesis is an important area of research for cancer therapy. Abnormal structure of blood vessels (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA) has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis.

Methods: To do so, we determined the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA-treated (2.5mM) conditioned media (231-CMASA) or vehicle-treated conditioned media (231-CMVT) of MDA-MB-231 cells on different endothelial cell physiology. These include endothelial cells’ migration towards serum using 3D modified Boyden chamber assay, in vitro capillary-like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes-endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on in vivo tumor angiogenesis using in vivo Angiogenesis Assay (Trevigen)

Results and Conclusions: We found that 231-CMASA significantly blocks in vitro migration, the formation of in vitro capillary-like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231-CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF-A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis.

Citation Format: Jinia Chakraborty, Gargi Maity, Snigdha Banerjee, Sushanta K. Banerjee. Regulation of tumor angiogenesis by low dose Aspirin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2017-790
Abstract 797: Glioblastoma animal model using CRISPR-Cas9 technology
Authors: Jeong Da Eun , Lee Kee Hang , Kim Sung Soo , Bae Yoon Kyung , Nam Hyun , Hwang Ji Yoon , Pyeon Hee Jang , Song Hye Jin , Joo Kyeung Min
Published: July 2017

Current in vivo model system poses limitation on fully recapitulating genomic characteristics of a tumor due to high complexity and poor understanding of the heterogeneous microenvironment conditions in cancer pathogenesis. In an effort to address such issues, strategic models are required. In present study, we propose that the most representative cancer models have consistent tumor microenvironments and genomic mutations. The Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a powerful genome editing tool for efficient and precise genome engineering. Here, we employed CRISPR-Cas9 system in vivo to generate Cre-dependent Cas9 knock-in mouse (B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J, Jackson lab.). The Cre-dependent Cas9 mouse models harbor combinations of genomic alterations including well-established oncogenes such as EGFRviii, c-MET, PDGFRa, IDH1 R132H and KRAS, EGFR, ALK, BRAF in Brain and Lung cancer models, respectively. While, they also consist of tumor suppressor genes including PTEN, NF1, Ink4a/ARF, Rb, TP53 and TP53, PTEN, NKx-1, APC in both Brain and Lung models, respectively. Cre-dependent model allows us to study in-depth into the tumor initiation and progression, while able to follow up in the role of tumor microenvironment in cancer maintenance. A better understanding of cancer models for preclinical research including their uses, as well as their limitations, may aid future potential studies regarding the development and implementation of new immune targeted therapies and potential validation of novel therapeutic biomarkers.

Citation Format: Da Eun Jeong, Kee Hang Lee, Sung Soo Kim, Yoon Kyung Bae, Hyun Nam, Ji Yoon Hwang, Hee Jang Pyeon, Hye Jin Song, Kyeung Min Joo. Glioblastoma animal model using CRISPR-Cas9 technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 797. doi:10.1158/1538-7445.AM2017-797
Abstract 803: Targeting β-catenin/CBP signaling in OSCC
Authors: Alamoud Khalid , Sadykov Khikmet , Kartha Vinay , Monti Stefano , Belkina Anna , Snyder-Cappione Jennifer , Pai Sara , Kukuruzinska Maria
Published: July 2017

Objectives: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype.

Methods: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24hiCD29hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24lowCD29low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice.

Results: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24hiCD2hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24lowCD29low. Treatment of CD44+CD24hiCD29hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy.

Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000.

Citation Format: Khalid Alamoud, Khikmet Sadykov, Vinay Kartha, Stefano Monti, Anna Belkina, Jennifer Snyder-Cappione, Sara Pai, Maria Kukuruzinska. Targeting β-catenin/CBP signaling in OSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2017-803