bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PR12: Mutant p53 drives early events in fallopian tube tumorigenesis through mesenchyme-associated autocrine production of matrix that supports survival and mesothelial intercalation.
Authors: Iwanicki Marcin P. , Chen Hsing-Yu , Zervantonakis Ioannis , Novak Marian , Muranen Taru , Ince Tan A. , Drapkin Ronny , Brugge Joan S.
Published: May 2016

High-grade serous ovarian cancer (HGS-OvCa) harbor p53 mutations and predominantly originates from the secretory epithelial cell compartment of the fallopian tube fimbriae. From this site, tumor cells detach, survive in the peritoneal cavity, and form cellular clusters that clear the mesothelium and expand to form peritoneal masses. To examine the contribution of mutant p53 to any of these phenotypic alterations associated with HGS-OvCA tumor progression, we developed live-cell microscopy assays that recapitulate these phenotypes and examined dynamic processes regulated by depletion of p53 or gain-of-function (GOF) mutations in p53 in cultured secretory fallopian tube epithelial cells. Expression of GOF mutant variants of p53, but not depletion of endogenous wild-type p53, promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of spheroids with mesothelium-clearing capacity. These phenotypes were blocked by mutant p53 reactivation and depended on the expression of the mesenchyme transcription factor TWIST1, inhibition of p63 and de novo formation of a unique cell-matrix adhesion niche supported by autocrine fibronectin production and activation of integrins in detached cells. Inhibition of fibronectin or integrin α5 in suspension blocked cell-to-cell adhesion leading to HGS-OvCa spheroid disintegration and ultimately anoikis. These results indicate the detached FTE cells-expressing GOF mutants of p53 undergo a mesenchymal transition under conditions of matrix detachment leading to autocrine production of matrix and anchorage-independent survival and proliferation, as well as mesothelial clearance activity. These findings provide important new insights into activities of mutant p53 that may play a critical role in early events in tumorigenesis of high-grade ovarian serous tumors and provide a rationale for the development of therapeutics that target ovarian cancer cells transiting through the peritoneal cavity.

This abstract is also presented as Poster B03.

Citation Format: Marcin P. Iwanicki, Hsing-Yu Chen, Ioannis Zervantonakis, Marian Novak, Taru Muranen, Tan A. Ince, Ronny Drapkin, Joan S. Brugge. Mutant p53 drives early events in fallopian tube tumorigenesis through mesenchyme-associated autocrine production of matrix that supports survival and mesothelial intercalation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR12.
Abstract 21: Mutation profiling of patients with multiple primary cancers reveals some common genomic alterations in signaling pathways
Authors: Wang Ruoyu , Zhao Dewei , Wang Zhiqiang , Gu Xuesong , Wen Shu , Ji Xuening , Cao Hong , Yang Bin , Wu Fei , Zhong Jianguo , Li Nina , Liang Shanshan , Li Heming , Zhang Yuewei , Ding Yan
Published: May 2016

Multiple primary cancers in a single individual are rare and usually challenging for effective treatments. Identification common cancer gene driver mutations and targets for patients with multiple primary malignant cancers will offer potential opportunities for effective personalized treatment. We have collected and analyzed tumor tissue samples from patients diagnosed with multiple primary cancers including lung cancer, kidney cancer, prostate cancer, and bone cancer from the Affiliated Zhongshan Hospital of Dalian University. Deep sequencing of the tumor samples was performed using TruSeq Amplicon - Cancer Panel (TSACP) which covers 48 cancer genes with more than 200 mutation hotspots. We have found that TP53 (75%), PIK3CA (50%), KDR(50%), VHL(50%), KRAS (25%), , FGFR(30%), EGFR(25%), HER2(25%) and ATM (25%) mutations are among the most common gene alterations across different types of primary cancers, they share some common cancer signaling pathways. Missense exon variation(4.13%) , non-coding exone variation(2.8%), and frameshift variation (0.41%) are among the most frequent alterations for functional effects. Targeted and chemotherapies have been recommended for personalized cancer treatments and circulating tumor cells (CTC) enumeration was conducted to monitor the prognosis of therapeutic efficacy.

Citation Format: Ruoyu Wang, Dewei Zhao, Zhiqiang Wang, Xuesong Gu, Shu Wen, Xuening Ji, Hong Cao, Bin Yang, Fei Wu, Jianguo Zhong, Nina Li, Shanshan Liang, Heming Li, Yuewei Zhang, Yan Ding. Mutation profiling of patients with multiple primary cancers reveals some common genomic alterations in signaling pathways. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 21.
Abstract 31: A new research solution for circulating cell free DNA with short amplicons allow accurate detection of 70 COSMIC mutations at 1% detection limit
Authors: Mongan Ann , Chien Richard , Martinez-Alcantara Antonio , Hyland Fiona
Published: May 2016

Non-invasive determination of the resistance or recurrence of tumor development will allow for a safer, more cost effective method for cancer research. As circulating tumor DNA makes up only a small fraction of cell-free DNA (cfDNA) recovered from blood plasma, targeted sequencing offers an ideal tool for mutation detection. Furthermore, as cfDNA is more fragmented than genomic DNA, it presents new technical challenges. In particular, reports in the literature have shown that the majority of cfDNA fragment sizes fall between 120-200 bp, with a median length of 170 bp. Consistent with these reports, we observed an amplicon size dependent improvement in coverage with input samples containing high amounts of fragmented DNA (FFPE or cfDNA). To optimize for cfDNA selection, we have released an Ion AmpliSeq™ designer that offers amplicons of 100-140 bp in size. In this study we conducted a head-to-head comparison between short (100-140 bp) and long (135-175 bp) designs for the same set of targets. These two panels evaluate 1000 COSMIC hotspot mutations from 73 frequently mutated genes. We applied a protocol using magnetic beads to isolate cfDNA from one control (Coriel GM23485) and 3 cfDNA samples. We completed the molecular characterization of the isolated cfDNA using the multiplexing capabilities of Ion AmpliSeq™ library preparation and Ion PGM™ sequencer. Our results showed the short amplicon design led to an improvement of library yield that correlated with the amounts of fragmented DNA contained in the cfDNA samples. The shorter amplicon design also offered improved depth coverage and uniformity for fragmented DNA, while maintaining high performance with genomic DNA. Variant analysis of GM23485 with 70 known mutations at 1% allelic frequency showed >95% positive predictive value with no false negative. In short, the bead-based cfDNA isolation protocol and short amplicon designs offer a simple sample preparation workflow to facilitate rapid mutation detection for cfDNA in cancer research.

Citation Format: Ann Mongan, Richard Chien, Antonio Martinez-Alcantara, Fiona Hyland. A new research solution for circulating cell free DNA with short amplicons allow accurate detection of 70 COSMIC mutations at 1% detection limit. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 31.
Abstract 35: Evolution of resistance to glucocorticoid receptor agonists
Authors: Dalin Simona , Zhao Boyang , Hemann Michael T.
Published: May 2016

Chemotherapy resistance is a major obstacle to curing cancer patients. Over 90% of metastatic tumors are resistant to chemotherapy, leading to treatment failure. Although glucocorticoid receptor agonists (GRAs) are a very commonly used class of component drugs in front-line combination chemotherapies for hematopoietic cancer, our understanding of its mechanism(s) of action and resistance are very limited. Here using a preclinical murine model of Burkitt's lymphoma, we investigated the mechanism of resistance to the glucocorticoid receptor agonist dexamethasone. Dose responses of dexamethasone on the parental Eμ-myc; Arf-null cell line revealed a consistently small population of persistent survivors, suggesting a pre-existing subpopulation with innate dexamethasone resistance. To further address the evolution and selection of these survivors in the presence of GRAs, we derived resistant cell lines in vitro by treating the parental cell line with dose escalating concentrations of dexamethasone. These resistant cell lines were expectedly cross-resistant to other GRAs. However, loss of expression of the glucocorticoid receptor (GR) was not observed – ruling out one possible resistance mechanism. To explore alternative or downstream pathways that may be implicated, we subsequently acquired dose responses against a broad set of targeted kinase inhibitors, and observed modest sensitivity to the HSP90 inhibitor 17-AAG. This suggests that the resistance mechanism could be related to HSP90's role of holding ligand-free glucocorticoid receptor (GR) in the cytoplasm. We are now re-evolving the parental populations, and deriving clonal populations to confirm this phenotype. A better understanding of how hematopoietic cancer cells evolve resistance to GCAs will lead to improved design of drug combinations for prolonged survival.

Citation Format: Simona Dalin, Boyang Zhao, Michael T. Hemann. Evolution of resistance to glucocorticoid receptor agonists. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 35.
Abstract 43: HBEGF cooperates with Cdkn2a and Pten loss to form malignant gliomas
Authors: Shin Clifford , Holmen Sheri
Published: May 2016

Malignant gliomas are aggressive brain tumors that carry a grim prognosis to over 10,000 patients a year in the United States. The highest grade tumor, glioblastoma, has an average survival of one year with the current standard of care, which includes surgical resection, radiotherapy, and temozolomide chemotherapy. Heparin-binding EGF-like growth factor (HBEGF), first discovered in macrophages as a mitogen for vascular smooth muscle cells, is a ligand for epidermal growth factor receptor, the most commonly amplified receptor-tyrosine kinase in glioblastoma. About 30% of glioblastomas express HBEGF, and it is also commonly upregulated in tumors that express the common EGFR mutant, EGFRvIII. To date, HBEGF has not been evaluated for its ability to promote the development of malignant gliomas in vivo. Using a method for somatic cell gene transfer, we show that HBEGF cooperates with loss of tumor suppressors Cdkn2a and Pten to form malignant gliomas in vivo. These tumors share many characteristics found in human glioblastoma such as necrosis and nuclear atypia. We also demonstrate using high-throughput sequencing that these tumors share a common gene signature to the human glioblastoma samples from The Cancer Genome Atlas study. No tumors were seen with loss of Cdkn2a and Pten alone, which are the two most common tumor suppressors altered in glioblastoma. Our findings demonstrate that HBEGF can drive the development of high grade gliomas in the context of Cdkn2a and Pten loss and that these tumors closely resemble the human disease.

Citation Format: Clifford Shin, Sheri Holmen. HBEGF cooperates with Cdkn2a and Pten loss to form malignant gliomas. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 43.
Abstract 46: Functional analysis of long noncoding RNAs in Bcr-Abl-mediated tumorigenesis
Authors: Guo Guijie , Wang Xuefei , Chen Jilong
Published: May 2016

Human genome is pervasively transcribed and the majority does not have apparent protein coding potential, which is defined as noncoding RNA (ncRNA). Long noncoding RNAs (lncRNAs) are greater than 200 nucleotides in length, transcribed by RNA polymerase II or III, and poorly conserved. Aberrant expression of numerous functional lncRNAs is associated with various human cancers. Chronic myeloid leukemia (CML) is a hematologic malignancy associated with a translocation between chromosome 9 and 22 that results in the formation of bcr-abl hybrid gene which occurs in over 90% of CML cases. However, the role of lncRNAs in Bcr-Abl-mediated chronic myeloid leukemia (CML) is not fully understood. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using a microarray of cDNAs encoding lncRNAs. The microarray analysis revealed 338 upregulated lncRNAs and 108 downregulated lncRNAs after silencing Bcr-Abl in human CML cells. To identify functional lncRNAs that are important in Bcr-Abl-mediated transformation, we selected the most significantly affected lncRNAs for further studies. These lncRNAs were examined by ORF finder from NCBI and protein-coding potential was determined. We identified several lncRNAs that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Expression of these lncRNAs was further examined in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Experiments including ectopic overexpression or knockdown of these lncRNAs were performed to address the role and mechanism of how these lncRNAs function in the leukemic cells. Together, these results reveal that several lncRNAs are critically involved in Bcr-Abl-mediated cellular transformation and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia.

Note: This abstract was not presented at the conference.

Citation Format: Guijie Guo, Xuefei Wang, Jilong Chen. Functional analysis of long noncoding RNAs in Bcr-Abl-mediated tumorigenesis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 46.
Abstract 49: Genomic analysis and simulation for understanding heterogenous cancer evolution
Authors: Niida Atsushi , Uchi Ryutaro , Takahashi Yusuke , Mimori Koshi , Miyano Satoru
Published: May 2016

Cancer results from accumulation of mutations in the genome and evolutionary selection for growth advantage. In the evolutionary process, it is assumed that multiple clones appear, and this phenomenon called intratumor heterogeneity could cause therapeutic failures by fostering evolutionary adaptation. To study intratumor heterogeneity in colorectal cancer, we performed whole exome sequencing of samples from multiple regions within a single tumor. our genomic analysis revealed high intratumor heterogeneity; however, principles underlying it remained unclear. To explore the principles , we built a cellular automaton model, termed the BEP model, which can reproduce the branching cancer evolution in silico. We then extensively searched for conditions leading to high intratumor heterogeneity as observed in the experimental data, by performing simulations with various parameter settings on a supercomputer. The simulation result suggests that a high mutation rate leads to high intratumor heterogeneity, which is generated by neutral evolution. Collectively, this study provides provide novel insights into heterogenous cancer evolution.

Citation Format: Atsushi Niida, Ryutaro Uchi, Yusuke Takahashi, Koshi Mimori, Satoru Miyano. Genomic analysis and simulation for understanding heterogenous cancer evolution. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 49.
Abstract 51: Molecular profiling of relapsed childhood cancer
Authors: Wadham Carol , Saletta Federica , Byrne Jennifer A. , Trahair Toby , Ziegler David S. , McCowage Geoffrey , Marshall Glenn , Haber Michelle , Norris Murray
Published: May 2016

Background: Despite the overall success achieved in treating childhood cancers, a major challenge is to find alternative treatment options for those children who suffer relapse. This requires more detailed understanding of the molecular pathways responsible for maintaining and promoting an aggressive drug resistant phenotype, as well as the successful targeting of these pathways.

Materials and methods: We are implementing a molecular profiling approach to individual tumours, which includes Fluidigm Biomark qRT-PCR analysis (a panel of 96 marker genes), next-generation sequencing (Ion Proton custom AmpliSeq panel of 480 genes) and automated immunohistochemistry (IHC, 30 antibodies to 25 proteins/ phosphoproteins).

Results: Preliminary testing of a subset of these genes has been conducted on 12 cell lines representing the major childhood cancer subtypes, as well as 8 patient-derived primary cultures including 4 inflammatory myofibroblastic tumours, 2 diffuse intrinsic pontine gliomas and 2 acute lymphoblastic leukemia (ALL). The potential for molecular profiling to assist in drug selection was subsequently tested in high throughput in vitro drug sensitivity assays using a drug library based on the profiling data. Gene expression has also been analysed in forty five cell lines from mice bearing patient-derived ALL xenografts. The individualised mouse xenografts will allow in vivo testing of the efficacy of molecularly targeted drugs. We are now undertaking a retrospective study of diagnostic frozen and formalin fixed paraffin embedded samples from 55 paediatric cancer patients who subsequently experienced relapse.

Conclusion: We have implemented a screening approach to identify molecular targets in paediatric cancer patients who have failed the best current treatments available, and for whom treatment options following relapse are extremely limited. The ultimate goal is to undertake a multicentre prospective clinical trial in paediatric patients with recurrent or refractory solid tumours or leukemias for whom targeted drug therapy will be identified using molecular profiling.

Citation Format: Carol Wadham, Federica Saletta, Jennifer A. Byrne, Toby Trahair, David S. Ziegler, Geoffrey McCowage, Glenn Marshall, Michelle Haber, Murray Norris. Molecular profiling of relapsed childhood cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 51.
Abstract IA03: The evolutionary landscape of CLL: Therapeutic implications
Authors: Wu Catherine J.
Published: May 2016

Clonal evolution is a key feature of cancer progression and relapse. Recent studies across cancers have demonstrated the extensive degree of intratumoral heterogeneity present within individual cancers. We hypothesized that evolutionary dynamics contribute to the variations in disease tempo and response to therapy that are highly characteristic of chronic lymphocytic leukemia (CLL). We have recently investigated this phenomenon by developing a pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequence (WES) and local copy number data (Landau et al., Cell 2013). By applying this analysis approach to 149 CLL cases, we discovered earlier and later cancer drivers, uncovered patterns of clonal evolution in CLL and linked the presence of subclones harboring driver mutations with adverse clinical outcome. Thus, our study, generated from a heterogeneous sample cohort, strongly supports the concept that CLL clonal evolution arises from mass extinction and therapeutic bottlenecks which lead to the emergence of highly fit (and treatment resistant) subclones. We further hypothesized that epigenetic heterogeneity also shapes CLL clonal evolution through interrelation with genetic heterogeneity. Indeed, in recent work, we have uncovered stochastic methylation disorder as the primary cause of methylation changes in CLL and cancer in general, and that this phenomena impacts gene transcription, genetic evolution and clinical outcome. Thus, integrated studies of genetic and epigenetic heterogeneity in CLL have revealed the complex and diverse evolutionary trajectories of these cancer cells.

Immunotherapy is exquisitely suited for specifically and simultaneously targeting multiple lesions. We have developed an approach that leverages whole-exome sequencing to systematically identify personal tumor mutations with immunogenic potential, which can be incorporated as antigen targets in multi-epitope personalized therapeutic vaccines. We are pioneering this approach in an ongoing trial in melanoma and will now expand this concept to address diverse malignancies. Our expectation is that the choice of tumor neoantigens for a vaccine bypasses thymic tolerance and thus generates highly specific and potent high-affinity T cell responses to eliminate tumors in any cancer, including both trunk and branch lesions.

Citation Format: Catherine J. Wu. The evolutionary landscape of CLL: Therapeutic implications. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA03.
Abstract IA07: The application of integrative sequencing for precision oncology
Authors: Chinnaiyan Arul
Published: May 2016

Studies of the genomic landscape of human cancers have demonstrated that most solid cancers have a multitude of mutations, a subset of which may be “actionable” with current drugs. Thus, the personalization of therapy for cancer will require molecular characterization of unique and shared genetic aberrations. Patients who have advanced/refractory cancer and are candidates for clinical trials could potentially benefit by identifying eligibility for “targeted” drugs based on the “actionable” genes in their specific tumor. Growing technological advances in genomic sequencing has now made it possible to consider the use of sequence data in a clinical setting. MI-ONCOSEQ initiative brings together expertise at the University of Michigan including clinical oncology, cancer genetics, bioinformatics, clinical pathology, social and behavioral sciences, and bioethics in order to implement clinical cancer sequencing. Our study identifies patients with advanced or refractory cancers that are eligible for clinical trials, consent them to the study, obtain biospecimens (tumor tissue, germline tissue) and store clinical data. The clinical sequencing team processes biospecimens, performs comprehensive sequencing and analysis of tumors to identify point mutations, copy number changes, rearrangements/gene fusions, and aberrant gene expression. Finally, a multi-disciplinary Precision Medicine Tumor Board deliberates on return of actionable or incidental genomic results.

Citation Format: Arul Chinnaiyan. The application of integrative sequencing for precision oncology. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA07.
Abstract IA11: Clinical application of next-generation sequencing as a guide to treatment selection
Authors: Solit David B.
Published: May 2016

Profound and durable responses are often observed in early stage clinical trials of novel cancer agents in only a small minority of patients. It has long been postulated that these responses have a definable genetic basis but until now it was not feasible to perform a comprehensive genomic analysis of such patients. Rather, at best a few candidate genes were examined. Technical feasibility thus ensured that oncology trials were designed to identify agents that have a statistically significant benefit in a genetically unselected population, a paradigm that has led to the development of many agents that have modest or no benefit in the vast majority of patients. Agents with profound activity in only a small number of patients were on the other had deemed inactive and abandoned. Our preliminary experience shows that next generation sequencing methodology can now be feasibly applied in the clinical setting to identify previously unrecognized biomarkers of response for agents that show profound responses in only a minority of patients. As an example, we explored the molecular basis of an outlier phenotype, an apparent disease cure of a 51-year old woman with recurrent metastatic small cell carcinoma. The patient was treated on a phase 1 clinical trial that combined topoisomerase I inhibition with an ATP-competitive inhibitor of checkpoint kinase 1 (Chk1). Whole genome sequencing revealed a complex but highly clonal tumor genome with a somatic, missense mutation in the Mre11 complex gene RAD50, which functions to initiate double stranded break (DSB) repair by homologous recombination or non- homologous end joining. Through modeling in yeast, we confirmed that this heterozygous RAD50 mutation, which affects a highly conserved residue and whose wild type copy was focally deleted, conferred sensitivity to topoisomerase I inhibition. Drug sensitivity was markedly enhanced upon genetic ablation of the DNA damage checkpoint pathway, suggesting a synthetic lethal interaction between checkpoint kinase inhibition and clastogenic chemotherapy. These results and additional examples to be presented demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs. The use of novel clinical trial designs to confirm genotype-phenotype associations will also be discussed.

Citation Format: David B. Solit. Clinical application of next-generation sequencing as a guide to treatment selection. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA11.
Abstract IA20: Moving CAR T cell therapy forward: CARs and armored CARs
Authors: Brentjens Renier J.
Published: May 2016

T cells may be genetically modified to express chimeric antigen receptors (CAR) targeted to antigens expressed by tumor cells. We have developed this technology in the laboratory and translated this adoptive T cell approach in the clinic. Treatment of patients with CD19 targeted CAR T cells has resulted remarkable remission rates in relapsed B cell acute lymphoblastic leukemia (B-ALL). Specifically, based on currently updated clinical outcomes, we have achieved >85% CR rates in adult patients with relapsed B-ALL treated with CD19 targeted CAR T cells which far exceed historical expectations. Further, by deep sequencing analysis, most treated patients were MRD- after CAR T cell therapy. Significantly, remissions were observed in both patients with overt morphological residual disease at the time of therapy as well as in patients with only residual MRD+ disease. Clinical trials form other centers which confirm the presented clinical trial outcomes from our center will be reviewed. While overall generally well tolerated, we found that patients treated with CAR T cells in the setting of overt disease experienced a cytokine release syndrome (CRS) with associated fevers, hypotension, and mental status changes including overt seizures. In contrast, far more modest responses were seen in patients with relapsed chronic lymphocytic leukemia (CLL). The discordant clinical outcomes between B-ALL and CLL patients treated with CD19 targeted CAR T cells remains a subject of conjecture although the tumor microenvironment is a strong focus of further investigation. To this end, we will present novel data on a next generation of CAR T cells, termed “armored CARs” further genetically designed to overcome an immune suppressive tumor microenvironment through further genetic modification of CAR T cells. Promising preclinical studies utilizing these “armored CAR” T cell approaches and their role in future clinical trials will be discussed.

Citation Format: Renier J. Brentjens. Moving CAR T cell therapy forward: CARs and armored CARs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA20.
Abstract A004: Upregulation of soluble programmed death ligands in patients with colorectal cancer
Authors: Du Jipei , Cheng Yao , Mao Zhigang , Liao Shiping , Zheng Yanjiang , Zhang Ji , Wang Yufang
Published: May 2016

Objective: The programmed death-1/programmed death-1 ligands (PD-1/PD-Ls) pathway is critical for attenuating autoimmunity and maintaining T cell homeostasis. It has been suggested that PD-1/PD-Ls plays an important role in tumor evasion from host immunity. Although strong evidence supports the concept of immunosurveillance and immunoediting in colorectal cancer, the immune regulatory role of PD-1/PD-Ls pathway is not clear in colorectal cancer (CRC) patients. Here we investigated the expression of PD-1 and the soluble ligands (sPD-Ls) in blood plasma of patients with inflammatory Bowel Disease (IBD) and CRC.

Methods: Age-matched 107 health controls, 109 patients with colorectal cancer and 109 patients with IBD from West China Hospital (Chengdu, Sichuan, China.) were enrolled in the present study. The levels of PD-1 and sPD-Ls in blood plasma were analyzed by enzyme linked immunosorbent assay (ELISA). Expression of PD-L1 and PD-L2 were investigated in colorectal tissue by immunohistochemical (IHC) staining.

Results: The level of PD-1 in the plasma of the CRC patients was significantly up-regulated compared with healthy people (P=0.004), but there was no significant difference between IBD patient and healthy people. The levels of PD-1 were higher in CRC patients than in IBD patients. The levels of PD-L1 and soluble PD-L2 in peripheral blood were significantly increased in CRC patients comparing to health patients (P=0.0028 and p=0.0264, respectively). sPD-Ls were correlated with circulatory PD-1 expression in the CRC patients. These results indicated that the PD-1/PD-Ls pathway is activated in the CRC patients. However, we didn't observe significant differences either between IBD patients and healthy people or between CRC patients and IBD patients. We further confirmed that PD-L1 and PD-L2 were localized in colorectal cancer cells by IHC. In 67 CRC patients, the positive rates of PD-L1 and PD-L2 were 35.3% and 65%, respectively. In addition, expression of PD-1, PD-L1 and PD-L2 were higher in the patients with metastatic CRC compared to the patients with primary CRC.

Conclusion: The higher levels of plasma sPD-Ls may be associated with CRC and may predict CRC metastatic progression. The existence of immunosuppressive PD-1 and sPD-Ls in the circulation of CRC patients may indicate that PD-Ls suppress immune responses against CRC not only locally, but also systemically. This information will provide a novel insight into the role of PD-1/PDLs in CRC-mediated immunosuppression.

Citation Format: Jipei Du, Yao Cheng, Zhigang Mao, Shiping Liao, Yanjiang Zheng, Ji Zhang, Yufang Wang. Upregulation of soluble programmed death ligands in patients with colorectal cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A004.
Abstract A036: Anti-PD1 therapy and CD8 T cell invigoration in metastatic melanoma
Authors: Huang Alexander , Xu Wei , Harmon Shannon , Quagliarello Felix , Herati Ramin , Pauken Kristen , Bengsch Bertram , Schuchter Lynn , Amaravadi Ravi , McGettigan Suzanne , Gangadhar Tara , Wherry John
Published: May 2016

Antibodies targeting the PD-1 pathway can reverse T cell exhaustion resulting in re-invigoration of immune responses. Anti-PD1 therapies such as pembrolizumab have been shown to be efficacious across a broad range of human cancers including melanoma with clinical responses correlating with immune changes in the tumor microenvironment. However, our understanding of the immune mechanism of anti-PD1 therapy in humans remains incomplete. Specifically, there is relatively little information about changes in the differentiation, activation and reversal of exhaustion in peripheral blood CD8 T cells and correlation with clinical outcome. We collected peripheral blood at serial time points before and after pembrolizumab therapy on 39 consecutive patients with Stage IV melanoma and analyzed changes in T cell subsets and differentiation using 16 parameter flow cytometry. Here we show that pembrolizumab treatment results in increases in the peripheral blood CD8/Treg ratio, as well as invigoration of CD8 T cells. Focusing on key CD8 T cell subsets expressing combinations of T-bet, Eomes, PD-1 and other inhibitory receptors has allowed greater focus on populations responding to re-invigoration by pembrolizumab treatment. This invigoration can be demonstrated by upregulation of granzyme B (GzmB) and Ki67 in key CD8 T cell subsets. An early increase in GzmB+Ki67+ cells translates into a later increase in GzmB+ cells suggesting an early wave of proliferation giving rise to a pool of re-invigorated CD8 T cells. T cell subsets that express markers of exhaustion including T-bet, Eomes, and inhibitory receptors may represent populations that are invigorated by anti-PD1 therapy. These changes may also correlate with clinical outcomes and therefore may be useful as a biomarker of response. These results show that T cell responses to pembrolizumab treatment can be tracked in an easily accessible peripheral blood compartment during therapy. The kinetics of immune response in key CD8 T cell subsets may inform us of optimal treatment duration as well as the nature and timing of cancer response to therapy.

Citation Format: Alexander Huang, Wei Xu, Shannon Harmon, Felix Quagliarello, Ramin Herati, Kristen Pauken, Bertram Bengsch, Lynn Schuchter, Ravi Amaravadi, Suzanne McGettigan, Tara Gangadhar, John Wherry. Anti-PD1 therapy and CD8 T cell invigoration in metastatic melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A036.
Abstract A037: IMCgp100 ImmTAC: A new immunotherapeutic reagent for the treatment of malignant melanoma
Authors: Pentier Johanne , Connolly Mary , Canestraro Martina , Ryan Ruth , Weigand Luise , Hassan Namir , Vuidepot Annelise , Jakobsen Bent
Published: May 2016

For years cancer therapies were mainly concentrated around surgery, chemotherapy and radiotherapy. Recently, a new domain has emerged. Immunotherapies aim to exploit, enhance and optimise the patient's immune system to be more potent at eradicating tumours. T-cell immunotherapies, however, often encounter challenges with low cell surface expression of tumour-associated-antigens (TAAs), down-regulation of class I MHC molecules and low T-cell avidity for TAAs, all contributing to tumour escape.

To address these challenges, Immunocore Ltd. has developed ImmTAC (Immune Mobilising mTCR Against Cancer) technology that aims to recruit and redirect circulating T-cells to target and kill cancer cells with high specificity. ImmTACs are composed of an affinity enhanced monoclonal T-cell receptor (mTCR) combined with an anti-CD3 specific antibody fragment (CD3-scFv). The mTCR targets cancer cells that express TAAs displayed on class I MHC molecules, with high affinity. The CD3-scFv effector domain then drives the recruitment and activation of T-cells. ImmTACs enable an immune synapse to form, leading to the destruction of cancer cells by T-cells.

TAA candidates are selected according to their level of gene expression by measuring their mRNA levels by RT-PCR in healthy and cancer cells, in order to reduce off-target toxicity, as well as by measuring their cell surface expression on class I MHCs by mass spectrometry, to verify target presentation.

We present here our current clinical ImmTAC candidate, IMCgp100, which targets the melanoma-associated gp100 antigen presented on HLA-A2. IMCgp100 has been extensively studied in vitro and is undergoing a phase IIa clinical trial in advanced melanoma patients in the UK and US. The maximum tolerated dose has been established and a dose expansion phase is currently underway. T-cell mobilisation, relevant cytokine release as well as tumour shrinkage have been monitored. The very encouraging results observed so far in our clinical trials show ImmTAC technology to be a promising efficient immunotherapy for cancer.

Citation Format: Johanne Pentier, Mary Connolly, Martina Canestraro, Ruth Ryan, Luise Weigand, Namir Hassan, Annelise Vuidepot, Bent Jakobsen. IMCgp100 ImmTAC: A new immunotherapeutic reagent for the treatment of malignant melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A037.
Abstract A039: Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells
Authors: Moserova Irena Kusova , Truxova Iva , Garg Abhishek G, , Agostinis Patrizia , Cartron Piere Francois , Vosahlikova Sarka , Spisek Radek , Fucikova Jitka
Published: May 2016

Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy and high hydrostatic pressure (HHP). HHP induces immunogenic cell death of cancer cell is currently tested in running II. and III. phase of clinical trial for preparation of active cellular cancer immunotherapy (DCVAC). Previous studies have shown that the immunogenicity of ICD mainly relies on surface exposed calreticulin (ecto-CRT); CRT is normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the cell surface within hours after treatment. Although ICD can be triggers by HHP, underlying danger signaling pathways remain unclear. Therefore ovarian (OV90) cancer cell line was treated by HHP, UV-B and anthracycline (Idarubicin). The kinetics of ICD markers and key components of ER stress-mediated apoptotic pathway were analyzed by flow cytometry, western blotting and confocal microscopy.

In this work we described the ability of HHP triggers oxidative ER-stress (the production of reactive oxygen species (ROS) together with ER stress response) which results in phosphorylation of PERK and eIF2α. We also observed a rapid caspase-8 activation subsequently followed by BAP31 cleavage, Bid activation and caspase-3 activation. While caspase-8 activation was necessary for cell surface exposure of calreticulin (ecto-CRT) after HHP treatment, proapoptotic proteins Bax and Bak were dispensable for this process. Moreover, we identified that HHP treatment leads to caspase-2 activation. Whereas Idarubicin treatment has no effect on ecto-CRT exposure in Caspase-2 knockdown tumor cells, ecto-CRT was significantly decreased in these cells after HHP treatment. In addition, phagocytosis of HHP-treated Caspase-2 knockdown tumor cells was compromised in contrast to Idarubicin-treated knockout tumor cells.

In conclusion, we characterized the molecular mechanism of ecto-CRT exposure pathway induced by HHP which is analogous to that induced by anthracycline treatment. For the first time we provide evidence that caspase-2 might play an important role in the HHP-mediated ecto-CRT exposure.

Citation Format: Irena Kusova Moserova, Iva Truxova, Abhishek G, Garg, Patrizia Agostinis, Piere Francois Cartron, Sarka Vosahlikova, Radek Spisek, Jitka Fucikova. Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A039.
Abstract A045: Dendritic cell vaccination with partial Treg depletion in relapsed glioblastoma – results from the pilot phase of the HIT-HGG Rez Immunvac study
Authors: Eyrich Matthias , Krauss Jürgen , Löhr Mario , Technau Antje , Rachor Johannes , Monoranu Camelia , Warmuth-Metz Monika , Wölfl Matthias , Kramm Christof , Schlegel Paul Gerhardt
Published: May 2016

Efficacy of therapeutic dendritic cell vaccines (DCV) can be limited by immunosuppressive mechanisms such as regulatory T cells (Treg) overrepesented in the micromilieu of the tumor. Here, we investigated whether a reduction of Treg with metronomic cyclophosphamide (metrCyc) might be a feasible option to improve vaccine efficacy. 7 patients with replased glioblastoma were treated in the pilot phase of the HIT-HGG Rez Immunovac study (5 pediatric, 2 adult patients). After reoperation and monocyte-apheresis, patients received 4 weekly doses of autologous, TNFa/IL-1ß matured DCs pulsed with tumor lysate. Thereafter 4 monthy, and subsequently threemonthly boosts with tumor lysate were given. The intradermal injection site was prepared with topical imiquimod. Additionally, patients were pretreated with oral metrCyc 2-4 weeks before the first vaccination. MetrCyc was well tolerated with one mild and transient leukopenia in a patient, who received a parallel re-irradiation boost. All patients received at least 7 vaccines (4xDCs, 3xlysate boosts). Treg frequency decreased by 32%, but returned to normal levels after cessation of metrCyc. Importantly, 6/6 analyzed patients showed a positive (>1.5fold increase) IFNg-response of varying magnitude and duration towards the autologous tumor lysate in a IFNg-PCR assay. Furthermore, we observed an increase in VLA4+ T-cells induced by the vaccine, both in the CD4 as well as the CD8 compartment. So far, all patients relapsed after a mean of 4.8 months, however, overall survival is currently 15.2 months with 4/7 patients being still alive after a median follow-up of 21 months (range 12-27 months). One patient is disease-free after reoperation and continuation of vaccination for 26 months now. 6-month overall survival was 86%. We conclude that DC vaccination in combination with partial Treg depletion with metrCyc is feasible, safe, and possibly related with a higher than expected frequency of positive IFNg-responses towards tumor tissue. These pilot data warrant verification in the full HIT-HGG Rez Immunovac trial (Eudra-CT 2013-000419-26), which will start to recruit patients soon.

Citation Format: Matthias Eyrich, Jürgen Krauss, Mario Löhr, Antje Technau, Johannes Rachor, Camelia Monoranu, Monika Warmuth-Metz, Matthias Wölfl, Christof Kramm, Paul Gerhardt Schlegel. Dendritic cell vaccination with partial Treg depletion in relapsed glioblastoma – results from the pilot phase of the HIT-HGG Rez Immunvac study. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A045.
Abstract A048: Phase 1 clinical trial of a therapeutic prostate cancer vaccine containing PSA/IL-2/GM-CSF in PSA defined biochemical recurrent prostate cancer patients
Authors: Head Jonathan F. , Daniels Gregory A. , McKinney Michelle , Ongkeko Weg , Wang-Rodriguez Jessica , Sakamoto Kyoko , Elliott Robert L.
Published: May 2016

Immunotherapy for cancer has had two main approaches that have lead to clinical applications. The first is stimulating immune responses to tumor cells with cytokines or cellular immunotherapy and the second is blocking tumor immune evasion and the associated inhibition of T-cell activation with antibodies to the CTLA-4 receptor, PD-1 receptor or PD-L1. We have taken a different approach and have developed therapeutic cancer vaccines that are a combination of tumor antigens (whole cells or proteins) with biological adjuvants (the cytokines IL-2 and GM-CSF). This study is a Phase 1a/1b clinical trial of a PSA/IL-2/GM-CSF vaccine in recurrent prostate cancer in hormone-naïve and hormone-independent patients. Major inclusion criteria include adenocarcinoma of the prostate, rising serum PSA and no measurable disease. Phase 1a examines the rate of dose limiting adverse events (DLAEs) in an initial course of 6 vaccinations (“induction vaccination”). The Phase 1b examines the rate of DLAEs with a continued course of an additional 6 vaccinations (“maintenance vaccine”). All patients will receive intradermal injections of the PSA/IL-2/GM-CSF vaccine at weeks 1, 2, 3, 7, 11, and 15. In an additional 28 patients the six maintenance vaccines will alternate IL-2 and the complete vaccine (PSA/IL-2/GM-CSF) at weeks 23, 27, 31, 35, 39 and 43. To date, twelve of twenty patients in the Phase 1a portion of the trial have received at least one vaccine injection and ten patients have received all 6 vaccines. Seven of the ten patients that have received 3 vaccines had increased responses to PSA in a lymphocyte blastogenesis assay and five of the nine patients had an increase in their response after 6 vaccines. None of the patients vaccinated in the Phase 1a portion have had a DLAE and enrollment continues in the Phase 1a.

Citation Format: Jonathan F. Head, Gregory A. Daniels, Michelle McKinney, Weg Ongkeko, Jessica Wang-Rodriguez, Kyoko Sakamoto, Robert L. Elliott. Phase 1 clinical trial of a therapeutic prostate cancer vaccine containing PSA/IL-2/GM-CSF in PSA defined biochemical recurrent prostate cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A048.