bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1354: Developing 384-well and 1536-well cell growth inhibition assay workflow for screening drug-drug combination in tumor cell lines
Authors: Li Yvonne , Ma Mike , Chan Julie , Tian Yang , Pagratis Nikos , Stonich Derek , Chen Victor , Zhang Louis , Kenney Mark
Published: July 2016

Drug combination has been widely used in treating the most debilitating diseases such as cancer. The ideal drug-drug combination will broaden and/or deepen therapeutic efficacy while overcoming resistance and unwanted off-target effects. We have developed a 384-well combination compound plating method in 8×7 format (8 dilutions of Drug A and 7 dilutions of Drug B), and screened a number of compound pairs that showed synergistic effect in inhibiting tumor cell growth in either suspension or solid tumor lines. Compound vehicle DMSO was used as negative control and puromycin treatment as positive control for calculating% inhibition, and both HSA and Bliss independence synergy models were applied for calculating synergy scores. As 384-well 8×7 format contains one compound pair per plate, we aimed to increase the throughput by developing 384-well 5×5 format (5 dilutions of Drug A and 5 dilutions of Drug B) which contains 3 drug pairs per plate, and 1536-well 8×7 and 5×5 format which contains 4 and 12 drug pairs per plate, respectively. We compared EC50s and synergy scores generated from 384-well and 1536-well with both 8×7 and 5×5 formats, single agent EC50s within combination pairs were generally within 3 fold difference and synergy scores are largely consistent. Therefore, we have validated and enabled higher throughput drug-drug combination screen by using 384-well 5×5, 1536-well 8×7, or 5×5 format. The high-throughput method presented here can be readily adopted for combination studies in other disease areas.

Citation Format: Yvonne Li, Mike Ma, Julie Chan, Yang Tian, Nikos Pagratis, Derek Stonich, Victor Chen, Louis Zhang, Mark Kenney. Developing 384-well and 1536-well cell growth inhibition assay workflow for screening drug-drug combination in tumor cell lines. [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 1354.
Abstract 1356: Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity
Authors: Bilsland Alan , Liu Yu , Burns Sharon , Jenkinson David , Roffey Jon , Keith W. Nicol
Published: July 2016

Cellular immortality is a near-universal phenotype of cancer cells with the central role played by the multicomponent telomerase complex firmly established. The reactivation of telomerase is found in approximately 85% of all cancers. Furthermore, frequent activating non-coding mutations in the hTERT promoter region have been identified in multiple tumour types. A considerable body of evidence supports the concept of targeting the signal transduction pathways that contribute to telomerase overexpression in cancer cells. We have exploited our knowledge in this area to develop cell-based luciferase reporter gene assays to measure hTERT and hTR gene promoter activity. To this end we undertook high throughput screening in the A2780 human ovarian cancer cell line against both hTR and hTERT promoters. Secondary specificity and cytotoxicity assays reduced the initial hits to a single chemical series of dual inhibitors which strongly suppress both hTR and hTERT promoter activity without primary cytotoxicity, as predicted of telomerase inhibitors. Through a process of Hit to Lead we have further developed the chemical series. Lead compounds are active across a panel of cell lines from distinct histological origins, reduce endogenous hTERT mRNA levels and decrease telomerase catalytic activity at nM concentrations. Furthermore, these compounds repress both wild type and mutant C228T and C250T hTERT promoter activity. In specificity assays no off-target activity has been identified in an expanded promoter screen against a panel of 15 off-target promoter reporters.

Citation Format: Alan Bilsland, Yu Liu, Sharon Burns, David Jenkinson, Jon Roffey, W. Nicol Keith. Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity. [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 1356.
Abstract 1359: Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor cells
Authors: Gangjee Aleem , Ravindra Manasa P. , Wilson Mike R. , Povrik Adrianne W. , Hou Lisa P. , O’Connor Carrie , Hou Zhanjun , Matherly Larry H.
Published: July 2016

Folates are important single carbon transferring cofactors required for the biosynthesis of purine and pyrimidine nitrogen bases and DNA. Antifolates inhibit synthesis of these nitrogenous bases and function as antineopastic agents. The use of currently marketed antifolates such as methotrexate (MTX and pemetrexed (PMX) is limited by dose-limiting toxicity due to non-selective transport by the ubiquitously expressed reduced folate carrier (RFC). We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT expressing tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and circumvent the important limitation of currently marketed clinical antifolates that lack selective uptake into tumors. As an extension of the SAR, we now report on the role of fluorine atom(s) in improving selectivity as well potency. Fluorine was incorporated into our 6-substituted pyrrolo[2,3-d]pyrimidine antifolates to improve pharmacodynamics by improving binding affinity to target proteins via a number of well documented electrostatic and hydrophobic interactions of fluorine. In addition, fluorines were strategically placed on the side chain of our targeted antifolates for improved potency and selectivity through induced conformational restriction due to a plausible intramolecular fluorine-hydrogen bonding interaction. The fluorinated analogs had IC50s in the subnanomolar range (IC50 = 0.33 to 0.29 nM) for KB tumor cells and nanomolar (IC50 = 2.77 to 3.47 nM) for IGROV-1 cells. In addition, these analogs showed 600- to 1000-fold selectivity for FRα expressing cells over RFC. Protection studies suggest that glycinamide ribonucleotide formyl transferase (GARFTase) is the intracellular targets. In vivo activity was established toward the SKOV3 tumor xenograft model. These preclinical studies suggest that these fluorinated analogs are candidates for possible clinical trial as antitumor agents.

Citation Format: Aleem Gangjee, Manasa P. Ravindra, Mike R. Wilson, Adrianne W. Povrik, Lisa P. Hou, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor 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 1359.
Abstract 1365: Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC
Authors: Gangjee Aleem , Xiang Weiguo , Dehkne Aamod , Wilson Mike R. , Wallace-Povrik Adrianne , O’Connor Carrie , Hou Zhanjun , Matherly Larry H.
Published: July 2016

Drugs usually bind to different targets in different conformations. As antitumor agents, antifolates first need to be selectively transported by tumor cells via the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) or folate receptors (FRs). The second step is the binding of antifolates with the intracellular targets. We are systematically developing novel folate analogs with selective membrane transport for FRs and PCFT over the ubiquitously expressed RFC. These 6-substituted pyrrolo[2,3-d]pyrimidine antifolates are principally inhibitors of de novo purine biosynthesis at the steps catalyzed by glycinamide ribonucleotide formyl transferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). We previously demonstrated that classical antifolates bind to FRα and GARFTase in different conformations. An amide in the bridge region of classical antifolates has two interchangeable lowest energy conformers, pseudo-cis and pseudo-trans. We reported that a novel antifolate (AGF238) with an amide in the bridge region binds to FRα in a pseudo-trans conformation and binds to GARFTase in a pseudo-cis conformation. As part of our continued efforts to pursue targeted antifolates as antitumor agents, we designed an expand series of amide-bridged classical antifolates with different energy barriers between the pseudo-cis and pseudo-trans conformers. Among these, AGF266 was exclusively transported by Chinese hamster ovary (CHO) cells engineered to express human FRα (IC50 = 0.42 nM) or FRβ (IC50 = 0.88 nM) over CHO expressing human RFC (IC50 > 1000 nM). AGF266 was also found to exhibit highly potent antitumor activity (IC50 = 0.79 nM in KB human tumor cells). AGF266 inhibited GARFTase as its principal cellular target, based on patterns of protection from growth inhibition by adenosine and AICA. Our in vitro findings of antitumor activity associated with FR selectivity suggest that further preclinical evaluation of AGF266 as a potential antitumor agent is warranted.

Citation Format: Aleem Gangjee, Weiguo Xiang, Aamod Dehkne, Mike R. Wilson, Adrianne Wallace-Povrik, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC. [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 1365.
Abstract 1375: An ultra-sensitive cell free DNA liquid biopsy assay for cancer treatment monitoring
Authors: Zhao Grace , Weng Li , Tang Paul , Sun Johnny , Huang Yi , Guo Lingchen , Wang Hongyan , Kang Xiaozheng , Shen Wei , Ying Kang , Lin Shengrong
Published: July 2016

In order to take advantage of the narrow time window for optimal treatment efficacy, highly sensitive disease monitoring is critical in the successful management of cancer. Currently, treatment efficacy is assessed by using a combination of protein cancer biomarkers and imaging. However, both methods present limitations with regard to specificity or sensitivity due to their dependency on tumor size. Recently, a number of studies have suggested that monitoring cell free DNA (cfDNA) may provide a more specific alternative for tracking cancer treatment with greatly improved sensitivity. Here we introduce a novel next-generation sequencing based mutation detection system aimed at improving the sensitivity, reliability, and clinical utility of cancer treatment monitoring. Our system, comprised of Nebula, a whole genome amplification technology that is capable of amplifying nanogram quantities of cfDNA >1000-fold, and Firefly, a proprietary technology combining molecular biology and computational algorithm for error-suppression, has reduced the rate of random sequencing errors to 10-6. As a result, we are able to detect 1.5 variant copies from 10ng of input cfDNA with a detection rate of 46%. We have validated the Nebula-Firefly assay on a patient cohort with either colorectal (CRC) or lung cancer. Here we report the successful detection of drug resistant mutations and various genomic alterations associated with minimal residual detection (MRD) in sample cfDNA. These initial findings have led to the exploration of Nebula-Firefly as the technological backbone for a noninvasive, scalable approach for the early detection, treatment, and monitoring of cancer.

Citation Format: Grace Zhao, Li Weng, Paul Tang, Johnny Sun, Yi Huang, Lingchen Guo, Hongyan Wang, Xiaozheng Kang, Wei Shen, Kang Ying, Shengrong Lin. An ultra-sensitive cell free DNA liquid biopsy assay for cancer treatment monitoring. [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 1375.
Abstract 1378: Generating quality control reference standards to evaluate ultrasensitive variant detection
Authors: Debeljak Marija , Haley Lisa , Anderson Derek A. , Adams Emily M. , Suenaga Masaya , Beierl Katie , Lin Ming-Tseh , Goggins Michael , Gocke Christopher D. , Eshleman James R.
Published: July 2016

Introduction: Ultrasensitive point mutation detection can facilitate minimal residual disease detection, early detection of cancer, and therapeutic monitoring of cancer patients. However, nearly identical mutant and wildtype molecules exhibit crosstalk with most technologies, thus producing high background levels. To evaluate technologies against each other for accurate ultrasensitive point mutation detection it is crucial to supply laboratories with reference samples of various dilutions.

Methods: We tested a series of sample mixes with digital-droplet PCR and next generation sequencing. Using linearity and accuracy as the gold standard, we empirically introduced two corrections to generate improved quality control reagents. Baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation of variant, while haplotype counting was used to correct technical errors (cell counting and pipetting).

Results: Correlation between the level of dilution and the measured VAF was relatively good (R2 = 0.80) when measured using ddPCR. However, correcting for the parental VAFs substantially improved the correlation (R2 = 0.97). In contrast, correlation of the cell count dilution and the measured VAF, as determined by next generation sequencing (AmpliSeq), was initially relatively poor (R2 = 0.63). However, substantial improvement in correlation was achieved by correcting for the parental VAFs (R2 = 0.94). In addition, correcting by haplotype counting alone only slightly improved accuracy in both technologies (ddPCR, from R2 = 0.80 to R2 = 0.81; AmpliSeq, from R2 = 0.63 to R2 = 0.64). Furthermore, correcting for both factors resulted in the best correlation for ddPCR-generated data (R2 = 0.99); however, introducing both corrections for AmpliSeq data did not prove more beneficial than correcting for parental VAFs alone (both corrections, R2 = 0.94 compared to parental VAF correction only, R2 = 0.94).

Conclusions: Validation of ultrasensitive variant detection requires standardized reference samples. These will also permit assessing various ultrasensitive detection technologies against one another.

Citation Format: Marija Debeljak, Lisa Haley, Derek A. Anderson, Emily M. Adams, Masaya Suenaga, Katie Beierl, Ming-Tseh Lin, Michael Goggins, Christopher D. Gocke, James R. Eshleman. Generating quality control reference standards to evaluate ultrasensitive variant detection. [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 1378.
Abstract 1398: RNA DIRECT: A novel strategy for targeted RNA enrichment of transcripts associated with solid tumors
Authors: Patel Kruti M. , Bowman Sarah K. , Davis Theodore B. , Russello Salvatore , Kraushaar Daniel C. , Emerman Amy B. , Henig Noa , Hendrickson Cynthia L.
Published: July 2016

RNA sequencing (RNA-seq) has become a powerful platform for profiling transcriptomes that enables gene expression analysis and identification of nucleotide or structural variations. RNA-seq, however, can be cost prohibitive due to the size and complexity of most transcriptomes, which require deep sequencing coverage to achieve the necessary sensitivity for a reliable analysis. By combining targeted enrichment strategies with next-generation sequencing (NGS), a subset of transcript regions or sequences can be enriched for and analyzed with the resolution required for both research and clinical applications.

The RNA DIRECT targeted enrichment method utilizes probe-based hybridization to capture only relevant cDNA sequences. Targeted sequences are then ligated to NGS platform specific adaptors and amplified by PCR. The RNA DIRECT strategy provides a robust and cost effective method for the enrichment of transcript sequences for the purpose of identification and detection of known or novel variants and gene fusions. To demonstrate the high specificity, sensitivity and uniformity of this method, we have applied it to the capture of targets commonly associated with solid tumors.

Citation Format: Kruti M. Patel, Sarah K. Bowman, Theodore B. Davis, Salvatore Russello, Daniel C. Kraushaar, Amy B. Emerman, Noa Henig, Cynthia L. Hendrickson. RNA DIRECT: A novel strategy for targeted RNA enrichment of transcripts associated with solid tumors. [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 1398.
Abstract 1404: Pemetrexed decreases circulating S100A9-myeloid derived suppressor cells in patients with non-small cell lung cancer
Authors: Feng Po-Hao , Lee Kang-Yun
Published: July 2016

Background: We previously reported that S100A9-myeloid derived suppressor cells (MDSCs) are negatively associated with chemotherapy effect in non-small cell lung cancer (NSCLC) patients. Pemetrexed-cisplatin chemotherapy is a standard treatment for advanced lung adenocarcinoma. The effect of chemotherapy on MDSCs is still not clear.

Methods: We prospectively enrolled nineteen stage 4 lung adenocarcinoma patients without known driving mutation before chemotherapy. Fourteen patients received pemetrexed-cisplatin and five with docetaxel-cisplatin as first line chemotherapy. Peripheral blood MDSCs percentage was evaluate by flow cytometry before first cycle and second cycle of chemotherapy.

In vitro migration assay with transwell MDSC/A549 cell co-culture system was used to test the effect of chemotherapy on chemotactic ability of A549 cell. Chemotherapy pretreated-A549 cells were cultured in the bottom chamber, and MDSCs, isolated from PBMC with CD14+ magnetic bead, were cultured in the upper chamber.

Results: MDSCs percentage decreased after patients received first cycle pemetrexed-cisplatin treatment (mean ±SD MDSC% in PBMC: 25.9±13.9% vs 16.3±11.3%, p<0.05, n = 5). In the subgroup patients that had partial response to pemetrexed and cisplatin, the MDSCs percentage decreased most significantly. (25.0±9.1 to 7.1±5.2, p<0.05, n = 5) In vitro study confirmed that pemetrexed reduced numbers of CD14+ MDSC (pemetrexed vs control: 15.0±6.6 vs 107.8± 56.8, p0.05) or cisplatin (96.7±98.2 vs 107.8 ±56.8, p>0.05)

Conclusion: Pemetrexed-cisplatin, but not docetaxel-cisplatin, decreased S100A9-MDSC in patients with lung adenocarcinoma, an effect might be due to a reduction in chemotactic ability of tumors by pemetrexed. In addition to cytotoxic effect, clinical benefit of pemetrexed might be also due to the effect on microenvironment.

Citation Format: Po-Hao Feng, Kang-Yun Lee. Pemetrexed decreases circulating S100A9-myeloid derived suppressor cells in patients with non-small cell lung 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 1404.
Abstract 1406: Analysis of immune responses as a consequence of androgen deprivation therapy in patients with biochemical progression of prostate cancer
Authors: Jochems Caroline , Madan Ravi A. , Fernando Romaine I. , Gulley James L. , Tsang Kwong Y. , Schlom Jeffrey
Published: July 2016

We evaluated the immune responses in 28 patients enrolled in a phase II trial at the National Cancer Institute (NCI). The aim was to investigate the effects of 3 months of androgen deprivation therapy (ADT) with goserelin on the immune responses in patients with biochemical progression of prostate cancer following local definitive therapy. PBMC and sera were collected before treatment and after 3 months. We performed flow cytometry to evaluate the frequencies of subsets of CD4+ and CD8+ T-cells, regulatory T-cells (CD4+ CD25hi CD127− FoxP3+), MDSCs and CD4 and CD8 recent thymic emigrants. CD4 T-cell proliferation and NK-cell lytic activity were evaluated. Serum samples were analyzed for cytokines, sCD27 and sCD40L.

The baseline characteristics were: median age 64 years, testosterone 361 ng/dl, PSA 2.2 ng/ml, PSA-doubling time 7.0 months, and Gleason score 7. After 3 months of ADT we found no significant changes in NK-cell function or frequencies of NK subsets. CD4+ T-cell proliferation decreased slightly. The number of CD4+ effector memory T-cells decreased, but other CD4 subsets were unchanged. The frequencies of CD4 and CD8 recent thymic emigrants did not change. Regulatory T-cells (including the CTLA4+ subset), and the effector:Treg ratio did not change. In contrast, the frequency of granulocytic MDSC decreased (P<0.01). There was an increase in the sCD27:sCD40L ratio (P = 0.012), due to decreased sCD40L. There was a decrease in the serum levels of IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, GM-CSF and IFNγ. There were no changes in the serum levels of TNFα or CXCL2 after therapy.

In conclusion, three months of ADT with goserelin did not alter the immune response in a way that would decrease the likelihood of successful immunotherapy after this treatment. The decreased MDSC and the increased sCD27:sCD40L ratio could indicate a more favorable immune environment.

Citation Format: Caroline Jochems, Ravi A. Madan, Romaine I. Fernando, James L. Gulley, Kwong Y. Tsang, Jeffrey Schlom. Analysis of immune responses as a consequence of androgen deprivation therapy in patients with biochemical progression of 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 1406.
Abstract 1409: Is it what's on the inside that counts? Melanoma mutation profiling and outcomes with immunotherapy
Authors: Freeman Morganna L. , McLeod. Howard
Published: July 2016

Extraordinary advances have been made in the treatment of melanoma over the last few years; immune checkpoint blockade has provided durable responses and meaningful survival outcomes for patients with metastatic disease, yet predicting such responses remains a nascent science. More importantly, the toxicity and financial burden of receiving such therapy warrants a personalized, or precision medicine, approach to appropriate selection of treatment, thus molecular predictors of response may become as important as biomarkers such as PDL1 expression. Recently the high mutational burden of malignant melanoma was shown to be related to the degree of response to the anti-CTLA4 antibody ipilimumab, but mutational burden alone was not sufficient to predict benefit1. Another study has shown NRAS-mutant had a trend to superior outcomes compared with other cohorts in terms of response to therapy with anti-PD1 antibodies and progression-free survival2. Moreover, insight into the molecular evolution of melanoma was recently established3, wherein somatic mutations in oncogenes such as BRAF, NRAS, GNAQ, or GNA11 occur early during progression and additional mutations of TERT, CDKN2A, TP53, and PTEN are seen in late-stage disease. To better evaluate the relationship of these mutations to immunotherapy response, our institution has profiled 95 patients with melanoma with next-generation sequencing (NGS). Of the 95 patients profiled, the most common mutations identified were BRAF (38%), NRAS (21%), TP53 (18%), PTEN (14%), TERT (9%), and CDKN2A (8%). We are presently conducting a retrospective analysis of these selected oncogenes as they relate to clinical responses, and intend to report disease outcomes (duration of response, progression free survival, and overall survival) as they relate to checkpoint inhibition.

Citation Format: Morganna L. Freeman, Howard McLeod. Is it what's on the inside that counts? Melanoma mutation profiling and outcomes with immunotherapy. [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 1409.
Abstract 1424: Predicting response to immune checkpoint therapy using an mutation burden threshold
Authors: Panda Anshuman , Betigeri Anil , Subramanian Kalyanasundaram , Hirshfield Kim , Rodriguez Lorna , Ganesan Shridar , Bhanot Gyan
Published: July 2016

Treatment with antibodies to PD-1 or CTLA-4 leads to prolonged response in some cancer patients. Patients whose tumors have a high mutational burden have a better response to anti-CTLA-4 treatment with ipilimumab in melanoma5, and to anti-PD-1 treatment with pembrolizumab in non-small cell lung6 and colorectal cancer7. These results suggest that a sufficiently high non-synonymous somatic mutation burden in the tumor may lead to protein alterations that serve as neo-antigens to stimulate CD8 T-cell immune response. This immune response may be blocked by the tumor by engaging the immune checkpoint pathway, making such tumors especially vulnerable to immune checkpoint disruption. However, the specific criteria to identify patients likely to respond to immune checkpoint therapy have remained elusive so far.

Using somatic mutation and RNA-Seq data from TCGA, we propose that there exists a mutational threshold, which we call the “Activated Immune Mutational” (AIM) Threshold, which can identify patients likely to respond to immune checkpoint therapy. Compared to AIM- patients (potential non-responders), AIM+ patients (potential responders) have tumors that meet four criteria: (1) a high non-synonymous mutation burden; (2) a high level of immune infiltration in the tumor; (3) a high CD8 T cell fraction in the leukocyte component of the immune infiltrate plus high expression of the T cell marker CD8A; and (4) a high expression of immune checkpoint genes PD-1, PD-L1, PD-L2, CTLA-4.

We find that in melanoma, endometrial, colon, cervical and breast cancer, a clear AIM threshold satisfying all four criteria exists. The distribution of mutations in AIM- versus AIM+ tumors for these cancers is very different. In the former, there are high frequency somatic mutations in only a few genes, while in the latter, high frequency somatic mutations are found in many genes distributed over the whole genome.

Pathological analysis of high-resolution images from 150 TCGA tumors (30 from each of the five tumor types) validated the presence of significantly higher lymphocytic infiltration in the AIM+ tumors compared to AIM- tumors. We show that AIM+ tumors can be identified using sequencing assays which are currently in clinical use. Finally, survival analysis in melanoma patients from TCGA treated with immunotherapy will be presented.

Citation Format: Anshuman Panda, Anil Betigeri, Kalyanasundaram Subramanian, Kim Hirshfield, Lorna Rodriguez, Shridar Ganesan, Gyan Bhanot. Predicting response to immune checkpoint therapy using an mutation burden threshold. [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 1424.
Abstract 143: Mutational landscape of colorectal adenomas reveals potential signatures for progression
Authors: Lin Shu-Hong , Raju Gottumukkala S. , Huff Chad , Gu Jian , Chen Jiun-Shen , Kopetz Scott , Menter David G. , Hawk Ernest , Mishra Lopa , Ye Yuanquing , Wu Xifeng
Published: July 2016

Introduction: Colorectal cancer (CRC) continues to rank as one of the most common cancers in the U.S. and around the world. Colorectal adenoma is the well-established precursor of CRC, yet only a very small proportion of adenomas progress to CRC. Therefore, identification of biomarker associated with adenoma progression is vital for risk stratification and development of intervention strategies. We hypothesize that genomic analysis of adenoma will lead to better understanding of tumorigenesis and identification of novel biomarkers.

Methods: We carried out whole exome sequencing (WES) on tissues and paired blood in 14 sessile serrated adenoma (SSA) and 36 tubulovillous adenoma (TVA) patients. Frequently mutated genes from TVA along with significantly mutated genes of TCGA colorectal adenocarcinoma were further investigated in an additional 100 pairs of TVA by targeted sequencing.

Results: 1) WES revealed similar somatic mutation frequency but distinctive mutated genes in TVA and SSA. 2) All adenomas included in WES were non-hypermutators with mutation frequency ranged from 0.55 to 3.71 (average: 1.49) non-silent somatic mutations per million base, which is significantly lower than that of TCGA non-hypermutators that ranged from 0.48 to 10.11 (average 4.47, p < 2.2×10⁁-16). 3) MutSigCV v1.4 discovered shared and unique significantly-mutated genes (SMG) in TVA and SSA reflecting difference in biology. 4) Targeted sequencing confirmed the findings in WES with a better estimate of population frequency for genes of interest. 5) Unsupervised clustering achieved high discrimination between adenoma and adenocarcinoma. 6) A subset of mutations that best discriminate adenoma and adenocarcinoma were identified by random Forest.

Conclusions: We discovered a set of genes with distinctive mutation pattern between adenoma and adenocarcinoma. We are conducting functional analysis to explore the potential of these genes as progression driver and risk predictors. We predict that the findings of this project will help lay the foundation to inform development of surveillance program and preventive intervention in CRC.

Citation Format: Shu-Hong Lin, Gottumukkala S. Raju, Chad Huff, Jian Gu, Jiun-Shen Chen, Scott Kopetz, David G. Menter, Ernest Hawk, Lopa Mishra, Yuanquing Ye, Xifeng Wu. Mutational landscape of colorectal adenomas reveals potential signatures for progression. [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 143.
Abstract 1443: IL-18 is associated with the onset and progression of gastric cancer
Authors: Nguyen Paul , Busuttil Rita , Mielke Lisa , Belz Gabrielle , Boussioutas Alex , Ernst Matthias , Putoczki Tracy
Published: July 2016

INTRODUCTION: Gastric cancer (GC) is the fourth most prevalent, and the third most common cause of cancer-related death worldwide. The disease is generally asymptomatic, and consequently is often diagnosed at an advanced stage when metastasis is present, and limited treatment options are available. Chronic inflammation is recognized as an integral component in the development and progression of GC, and is associated with increased infiltration of immune cells into the tumor microenvironment. The production of pro-inflammatory cytokines by these cells may contribute to tumor progression through activation of pathways promoting tumor cell survival and proliferation. Previous work from our lab has shown that therapeutic inhibition of the inflammatory cytokine interleukin (IL)-11 is effective in ameliorating disease progression in gastrointestinal cancer, however, it is unclear what role other pro-inflammatory cytokines, such as IL-18, might have in disease progression.

METHODS: To characterize the role of different pro-inflammatory cytokines in GC, we first analyzed microarray and qRT-PCR data of from human GC specimens and adjacent non-tumor tissue. Following the generation of a candidate list of cytokines deregulated in human GC, the role of individual cytokines in disease progression was examined using a validated mouse model of intestinal-type GC, referred to as gp130Y757F, by crossing into cytokine knock-out strains and monitoring tumor burden and the expression of genes and proteins classically associated with tumorigenesis.

RESULTS: We found that the expression pro-inflammatory cytokines including IL-1β and IL-18 were significantly elevated in the tumors of human GC patients compared to non-tumor tissue. In gp130Y757F mice, genetic ablation of IL-18, but not of IL-1β significantly reduced gastric tumor burden, which was associated with a reduction in the number of intratumoral macrophages, but not lymphocytes. This observation correlated with decreased expression of pro-inflammatory (Ifng, Tgfb1), antimicrobial (Reg3b, Reg3g), and tissue remodeling (Mmp9) genes, and a reduction in apoptosis in the gastric lesions in these mice.

CONCLUSION: Our results demonstrate that IL-18 has an important role in GC disease progression, and may serve as a potential therapeutic target.

Citation Format: Paul Nguyen, Rita Busuttil, Lisa Mielke, Gabrielle Belz, Alex Boussioutas, Matthias Ernst, Tracy Putoczki. IL-18 is associated with the onset and progression of gastric 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 1443.
Abstract 1446: Discovery of a distinct human lung tissue microbiome and its epidemiologic and clinical features
Authors: Yu Guoqin
Published: July 2016

The human lung tissue microbiota remains largely uncharacterized although a number of studies based on airway samples suggest the existence of a viable human lung microbiota. Here we characterized the microbiota taxonomic and derived functional profiles in 165 non-malignant lung tissue samples from cancer patients. We show that the lung microbiota is unique, differing from the microbiota of oral, nasal, stool, skin, and vagina samples. Microbiota diversity increases with environmental exposures, such as air particulates, residence in low to high population density areas, and pack-years of tobacco smoking. Moreover, the non-malignant lung tissue microbiota is altered in subjects with chronic bronchitis or advanced stage of lung cancer, and differs significantly from the microbiota in lung tumor samples. These findings provide insights on the human lung microbiota composition and function, and their link to human life style and clinical outcomes.

Citation Format: Guoqin Yu. Discovery of a distinct human lung tissue microbiome and its epidemiologic and clinical features. [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 1446.
Abstract 1449: In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity
Authors: Zilio Serena , Vella Jennifer , De La Fuente Adriana , Bronte Vincenzo , Mazza Emilia , Bicciato Silvio , Serafini Paolo
Published: July 2016

Introduction: Depending on their polarization, myeloid cells can either promote or restrain tumor progression. By secreting myelopoietic factors tumours promote myeloid cells polarization toward a phenotype that provides immune protection and that stimulate neoplastic cells growth, invasiveness, and metastasis. Chemokines and their cognate receptors are thought to play a key role in myeloid cell trafficking, however, their study in vivo is hindered by their signal redundancy and integration.

Methods: By taking advantage of a newly developed nanoplatform that allows the in vivo preferential transfection of tumor infiltrating myeloid cells (TIMC, aka Myeloid derived suppressor cells, MDSC, and tumor associated macrophages, TAM), we evaluated the effect of CCR-1, CCR-2, CCR-4, CCR-5 and/or CCR-7 silencing on TAMC trafficking, phenotype, and function.

Results: While CCR-4 and -7 targeted silencing did not affect tumor progression, simultaneous silencing of CCR-1 and CCR-5 significantly restrained tumor growth and greatly modifies tumor micro-environment. Mechanistic in vivo and in vitro analysis surprisingly indicate that myeloid cell trafficking was not altered whereas myeloid cell polarization was. In particular, CCR-1 and CCR-5 blockade restrained MDSC differentiation and promote the accumulation of retinoblastoma positive, neutrophil-like cells with a strong tumoricidal activity.

Discussion: Our data indicate that CCR-1 and CCR-5 engagement (most likely via CCL-3 and CCL-4) is necessary for the pro-tumoral polarization of myeloid cells in cancer. Our findings not only improve our understanding of myeloid cells differentiation in cancer but, also, strongly suggest that targeted inhibition of CCR1 and CCR5 on TIMC can be a new and effective anti-tumor immune-therapeutic strategy that convert pro-tumoral MDSCs into tumoricidal neutrophils.

Citation Format: Serena Zilio, Jennifer Vella, Adriana De La Fuente, Vincenzo Bronte, Emilia Mazza, Silvio Bicciato, Paolo Serafini. In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity. [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 1449.
Abstract 1452: IL-15 overexpression protects cerulein-induced fibrosis in the mouse model of chronic pancreatitis
Authors: MANOHAR MURLI , UPPARAHALLI VENKATESHAIAH SATHISHA , PUTANAPURA MAHADEVAPPA CHANDRASHEKARA , KUMAR VERMA ALOK , MISHRA. ANIL
Published: July 2016

Introduction: Earlier, interleukin (IL)-6, IL-8, IL-1β and IL-10 are implicated in the pathogenesis of pancreatitis. Recently, we observed reduced level of IL-15 in cerulein- induced acute and chronic mouse model of pancreatitis. IL-15 is a pleiotropic cytokine that has an important role in innate immunity. Therefore, we tested the hypothesis that IL-15 overexpression improves the pathogenesis of cerulein-induced pancreatitis.

Experimental procedure: Accordingly, we tested our hypothesis by inducing chronic pancreatitis in rIL-15 pretreated Balb/c mice. Cerulein was given by repetitive intraperitoneal (i.p.) injections as reported earlier (50 μg/kg, 6 hourly injections/day, 3 days/week) along with the rIL-15 (5 μg in 100μl saline/ two-times/week/mice) for up to 4 weeks; the control mice received 100μl saline or saline with 5 μg IL-15. Mice were sacrificed 3 days after the last cerulein injection. Histopathological evaluation of H&E stained tissue sections was performed including tissue remodeling and the accumulation of collagen by Masson's trichrome stain in the tissue sections. Additionally, pro-inflammatory and pro-fibrotic gene i.e. TGF-β1, IL-6, IL-8, Collagen 1 and α-SMA levels were measured by ELISA, immunofluorescence, qPCR and immunoblot analysis.

Summary: We report acinar cells atrophy, induced inflammatiory cells and cytokines in the pancreas of cerulein treated mice along with perivascular collagen deposition compare to saline treated mice. Interestingly, rIL-15 treated and cerulein given mice showed significantly improved morphology of acinar cells and reduced perivascular collagen. Further, we also report that protein levels of TGF-β1 and α-SMA is also reduced in the pancreas of cerulein injected and IL-15 treated mice compare to cerulein treated mice by performing western blot and immunohistochemistry.

Conclusion: Our data show that IL-15 overexpression protects mice from cerulein-induced pancreatic pathogenesis including fibrosis. Taken together, rIL-15 therapy may be a novel strategy to treat pancreatic fibrosis, and may also helpful to treat pancreatic cancer patients by down-regulating tissue fibrogenesis.

Funding: NIH R01 DK067255 (AM), NIH R01 AI080581 (AM),

Citation Format: MURLI MANOHAR, SATHISHA UPPARAHALLI VENKATESHAIAH, CHANDRASHEKARA PUTANAPURA MAHADEVAPPA, ALOK KUMAR VERMA, ANIL MISHRA. IL-15 overexpression protects cerulein-induced fibrosis in the mouse model of chronic pancreatitis. [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 1452.
Abstract 1454: IL-4 modulate the tumor microenvironment and response to cancer therapies
Authors: Ito Shuku-ei A. , Shirota Hidekazu , Ishioka Chikashi
Published: July 2016

recent findings show that immune cells constitute a large fraction of the tumor microenvironment and modulate tumor progression. Clinical data indicate that chronic inflammation is present at tumor sites and that IL-4 in particular is up-regulated. Thus, we tested whether depletion of IL-4 affected in tumor immunity. Current results demonstrate that administration of neutralizing antibody against IL-4 enhances anti-tumor immunity and improve the response to Taxol-based chemotherapy and CpG ODN immunotherapy. Depletion of IL-4 also alter the tumor microenvironment, reducing the generation of immunosuppressive M2 macrophages and myeloid derived suppressor cells and enhancing tumor specific CTLs. These findings suggest that IL-4 impact anti-tumor immunity and constitute an attractive therapeutic target to reduce immune suppression in the tumor microenvironment and thus enhance the efficacy of cancer therapy.

Citation Format: Shuku-ei A. Ito, Hidekazu Shirota, Chikashi Ishioka. IL-4 modulate the tumor microenvironment and response to cancer therapies. [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 1454.
Abstract 146: Genomic analysis of endometrial stromal sarcoma of uterus
Authors: Choi Youn Jin , Hur Soo Young , Park Jong Sup , Lee Keun Ho , Lee Yoon Kyung
Published: July 2016

Although recurrent gene fusions such as JAZF1-JJAZ1 are considered driver events for endometrial stromal sarcoma (ESS) development, other genomic alterations remain largely unknown. In this study, we performed whole-exome sequencing, transcriptome sequencing and copy number profiling for five ESSs (three low-grade ESS (LG-ESS) and two undifferentiated uterine sarcomas (UUSs)). All three LG-ESSs exhibited either one of JAZF1-SUZ12, JAZF1-PHF1 and MEAF6-PHF1 fusions, whereas the two UUSs did not. All ESSs except one LG-ESS exhibited copy number alterations (CNAs), many of which encompassed cancer-related genes. In UUSs, five CNAs encompassing cancer-related genes (EZR, CDH1, RB1, TP53 and PRKAR1A) accompanied their expressional changes, suggesting that they might stimulate UUS development. We found 81 non-silent mutations (35 from LG-ESSs and 46 from UUSs) that included 15 putative cancer genes catalogued in cancer-related databases, including PPARG and IRF4 mutations. However, they were non-recurrent and did not include any well-known mutations, indicating that point mutations may not be a major driver for ESS development. Our data show that gene fusions and CNAs are the principal drivers for LG-ESS and USS, respectively, but both may require additional genomic alterations including point mutations. These differences may explain the different biologic behaviors between LG-ESS and UUS. Our findings suggest that ESS development requires point mutations and CNAs as well as the gene fusions. Clinical and histologic characteristics of five endometrial stromal sarcomasAgeHistopathology gradeDiagnosisSpecimen statusTNMCase 132Low-gradeLG-ESSPrimaryT1bN0M0Case 234Low-gradeLG-ESSPrimaryT1bN1M0Case 357Low-gradeLG-ESSPrimaryTxNxM1Case 465High-gradeUUSMetastaticT2aN0M0Case 557High-gradeUUSMetastaticTxNxM1

Citation Format: Youn Jin Choi, Soo Young Hur, Jong Sup Park, Keun Ho Lee, Yoon Kyung Lee. Genomic analysis of endometrial stromal sarcoma of uterus. [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 146.
Abstract 1461: MHCII-expressing breast cancer cells can induce anti-tumor immune response
Authors: Li Mei , McCaw Tyler R. , Meza-Perez Selene , Randall Troy D. , Weinmann Amy , Buchsbaum Donald J. , Forero Andres , LoBuglio Albert F.
Published: July 2016

We recently reported that the mRNA expression of genes in the Major Histocompatibility Complex class II (MHCII) antigen processing and presentation pathway in breast tumor tissues was strongly prognostic of good clinical outcome in triple negative breast cancer (TNBC) patients (J Clin Oncol 33, 2015 suppl; abstr 1066). Although MHCII expression is most often observed on antigen-presenting cells, such as dendritic cells, macrophages and B cells, we observed MHCII protein expression on TNBC tumor cells. These observations suggested that TNBC cells expressing MHCII may have the ability to directly stimulate CD4 T cells and promote anti-tumor immune responses. To test this hypothesis, we transfected murine TS/A tumor cells (syngeneic, non-immunogenic, and metastatic breast cancer model) with a vector encoding the human MHCII Transcriptional Activator (hCIITA) under the control of a doxycycline (Dox)-inducible promoter. We found that TS/A-hCIITA cells did not express MHCII or CD74 until exposed to Dox, whereupon they expressed MHCII and CD74 mRNA and protein in a dose-dependent fashion. We also found that TS/A-hCIITA cells injected into the mammary fat pad expressed MHCII genes and cell surface MHCII proteins on tumor cells if mice received Dox in their drinking water. Importantly, tumor-bearing, Dox-exposed mice exhibited significantly impaired tumor growth compared to mice that were not exposed to Dox. We also found that tumor-bearing, Dox-exposed mice had a significantly enhanced tumor-specific CD8 T cell response in both the tumor-draining lymph node and tumor bed. Thus, breast cancer tumor cells expressing MHCII proteins promote anti-tumor immune responses, which likely contribute to the control of tumor growth in vivo.

Citation Format: Mei Li, Tyler R. McCaw, Selene Meza-Perez, Troy D. Randall, Amy Weinmann, Donald J. Buchsbaum, Andres Forero, Albert F. LoBuglio. MHCII-expressing breast cancer cells can induce anti-tumor immune response. [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 1461.