Tumor-specific Mutations Research Articles

Abstract 4982: Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade

Abstract Background: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can effect long-term durable regression in patients with metastatic melanoma but has not been widely tested in common epithelial cancers. Similar to metastatic melanoma, recent studies on epithelial GI cancers report that tumor regressions can be mediated by adoptively transferred TIL recognizing non-synonymous somatic mutations. Breast cancer metastatic tumors are infiltrated by TIL and reactivity against autologous somatic mutations can be identified. Methods: TIL were grown from a metastatic soft tissue tumor of a patient with hormone and chemotherapy-refractory ER+, HER2- breast cancer. DNA was extracted from tumor and matched normal peripheral blood samples for whole exome sequencing (WES) and RNAseq. Non-synonymous somatic mutations were identified and tested for potential recognition by autologous TIL using previously described tandem mini-gene and long peptide approaches. Recognition was assessed by IFN-γ release on ELISPOT and/or CD137 (4-1BB) upregulation with appropriate controls. Mutation reactive TIL were rapidly expanded in culture and transferred back to the patient. Adoptive cell transfer was preceded by a lymphodepleting preparative regimen and one dose of pembrolizumab. Cells were supported post-transfer with intravenous IL-2 administration, and pembrolizumab was administered for three additional doses. Deep sequencing of TCR rearrangement sequences was performed on the TIL and the pre- and post-infusion peripheral blood. Results: The resected subcutaneous tumor no longer expressed ER and was negative for PD-L1 staining. WES/RNASeq identified 96 non-synonymous mutations for testing. Multiple CD4+ clones specifically recognizing a mutation expressed in the solute carrier SLC3A2 (p.K94T) and a single CD8+ clone recognizing a mutation in the proteasome-associated protein KIAA0368 (p.S186F) were identified with no recognition of wild-type peptides. These eight T cell clones constituted 23% of the infusion bag TIL. Nine months after transfer of 82x109 cells, the patient has an ongoing partial response, with target lesions down 96% from baseline, including multiple hepatic metastases and disabling brachial plexus adenopathy. Seven of the eight T cell clones with known reactivity persisted in the peripheral blood comprising 2.4% of all CDR3 sequences at 7 months. Only two of these TCR clones were detectable, with frequencies totaling 0.005%, in the pre-treatment circulation. Conclusions: Tumor-infiltrating lymphocytes derived from a patient with metastatic breast cancer recognized tumor-specific non-synonymous somatic mutations, expanded in vitro and were used for adoptive cell transfer. TIL, co-administered with IL-2 and a short course of pembrolizumab, persisted in vivo and mediated regression of soft tissue, nodal and visceral metastases of breast cancer. Citation Format: Nikolaos Zacharakis, Katarzyna Trebska-McGowan, Robert Somerville, Yong-Chen Lu, Anna Pasetto, Mary Black, Harshini Chinnasamy, Hui Xu, Jared J. Gartner, Todd D. Prickett, Paul F. Robbins, Steven A. Rosenberg, Stephanie L. Goff, Steven A. Feldman. Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2017-4982

Abstract 4461: Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells

Abstract Exosomes are small vesicles, ranging from 30 to 100 nm in size, secreted from most types of cells. They contain proteins, lipids, and nucleic acids (e.g. DNA, mRNA, and microRNA) and are thought to play an important role in cell proliferation, invasion, and metastasis in human malignancies. Recently, circulating exosomes have also been of interest as a source for liquid biopsies. In this study, we examined the usefulness of exosomal RNA as a biomarker and attempted to detect the tumor-specific gene mutations from exosomal RNA derived from human oral squamous cell carcinoma (OSCC) cells. We used 3 human OSCC cells (HSC2, KT-T, KT-N). KT-T and KT-N cells were established from the lower gingival tumor and lymph node metastasis, respectively. First, we detected the somatic mutations in genomic DNA extracted from these cells by next-generation sequencing (NGS) with the use of HaloPlex Cancer Research Panel (Agilent Technologies). PIK3CA H1047R mutation was identified in all OSCC cells, and KT-N cells had the additional HRAS Q61R mutation. We confirmed these mutations of PIK3CA and HRAS in OSCC cells by TaqMan® SNP Genotyping Assays (Thermo Fisher Scientific). Subsequently, we evaluated the gene mutations in exosomal RNA from these OSCC cells. Exosomal RNA was isolated from the conditioned medium using exoRNeasy Serum/Plasma Maxi Kit (Qiagen) according to the manufacture’s protocol. In exosomal RNA, we found the PIK3CA H1047R mutation from all OSCC cells and HRAS Q61R mutation from KT-N cells by genotyping assays. Furthermore, we assessed the detection of these mutations in tumor xenograft mouse models. KT-T and KT-N cells (5 x 106) complexed with Matrigel® (BD) in 100-µl aliquots were injected subcutaneously space in the flank of male athymic nude mice. Exosomal RNA was isolated from the serum of tumor-bearing nude mice using exoRNeasy Serum/Plasma Midi Kit (Qiagen). By genotyping assays using serum exosomal RNA, PIK3CA H1047R mutation was observed in both types of xenograft mice and HRAS Q61R mutation was positive in KT-N xenograft mice. Cancer cell-derived exosomal RNA had the same gene mutations in those cells, and could be detected in the serum of tumor-bearing nude mice. These results suggest that exosomal RNA is useful for detecting somatic mutations in OSCC cells. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2017-4461

Abstract 728: Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP’s) and insertion/deletions (indels) mutations can be detected. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression. Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in colon cancer development, early detection, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2017-728

Abstract 5674: Cancer immunogenomics for the development of personalized ovarian cancer vaccine

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer death in the United States with approximately 20,000 women being diagnosed every year. Since OC development is mainly asymptomatic, patients are often diagnosed at late stage and present local and distal metastases. This offers a clinical challenge, as roughly 70% of the patients develop chemoresistant disease. With the advent of cancer immunogenomics it is now possible to identify tumor specific mutations, or neoantigens/neoepitopes, that can be exploited for the development of personalized T cell therapies via DC vaccines or adoptive cell therapy (ACT). We therefore hypothesize that tumor cells isolated from malignant ascites reflect the genomic landscape of the primary tumor, offering a unique opportunity to identify clinically actionable tumor neoantigens. We utilized primary tumor, tumor cells isolated from malignant ascites and metastatic samples to create patient-derived xenografts (PDX) mice and to interrogate the tumor mutational landscape. Somatic mutations were identified by whole exome sequencing (WES) and RNASeq analysis in tumor samples, using PBMCs as germline control. In parallel 20 PDX of ovarian cancer were established in NSG mice by injecting 2×106 cells/mouse subcutaneously (SQ, n=10) and intraperitoneally (IP, n=10). Tumor growth was monitored weekly via caliper measurement for mice injected SQ and via abdominal circumference for mice injected IP. Mutational analysis and prediction of high affinity peptides was done using bioinformatics approach. We have utilized different algorithms that are based on Artificial Neural Networks or published motifs for predicting binding of 8-11 mer peptides to number of Human MHC alleles (HLA) and out of 168 mutated peptides identified, 30 were found to have high affinity for different HLAs compared to wild type peptide. Mutated and wild type peptides were pulsed into dendritic cells and co-cultured with CD8+ T cells for 20 days. Validation of stimulated CD8+ T cells by these mutated peptides using ELISPOT is underway. TCR specific for neoantigen from CD8+T cells will be identified and cloned using retroviral vectors. Transfected CD8+ T cells with neoantigen specific TCR will be confirmed by co-culturing them with dendritic cells pulsed with wild type and mutated peptides. Further engineered CD8+ T cells specific for neoantigens will be tested for adoptive cell therapy in the established PDX models of ovarian cancer. Citation Format: Muzamil Y. Want, Takemasa Tsuji, Richard Koya, Sebastiano Battaglia. Cancer immunogenomics for the development of personalized ovarian cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5674. doi:10.1158/1538-7445.AM2017-5674

Abstract 3804: Comprehensive comparison of bisulfite conversion kits: A guide for optimal sensitivity and specificity of circulating cell-free DNA methylation-based biomarkers

Abstract Analysis of circulating cell-free DNA (cfDNA) is a promising approach for non-invasive detection of human cancer. The approach has wide-spread applications including early detection of disease in asymptomatic individuals, residual disease detection and disease monitoring. A challenge for the clinical utilization of cfDNA is the small fragment size of cfDNA and the limited amount of cfDNA present in blood. For detection of circulating tumor DNA (ctDNA) highly sensitive methods for cfDNA detection e.g. targeted sequencing and digital droplet PCR (ddPCR) target tumor specific mutations or DNA methylation alterations. Detection of methylation-based biomarkers requires that cfDNA is bisulfite converted prior to biomarker detection, which is known to be associated with some loss of cfDNA. Yet, surprisingly few efforts have focused on ensuring high cfDNA bisulfite conversion (BSC) efficiency and -recovery, and few commercial kits are especially designed for this purpose. In this study we aimed to find a BSC method suited for cfDNA methylation biomarker research, with high BS conversion efficiency and minimum loss of cfDNA, in order to increase the amount of cfDNA template available for downstream analysis. We studied 13 different methods for BSC and examined both the recovery and the BSC efficiency using quantitative real-time PCR (qPCR), polyacrylamide gel electrophoresis (PAGE) and ddPCR. A significant variation in DNA recovery was observed between the 13 methods tested using both high and low molecular weight DNA templates. Especially five kits performed consistently well on all DNA templates tested, as evaluated by qPCR and PAGE. As the lengths of cfDNA is 146-167 base pairs, only methods that are able to recover fragments of this lengths is suitable for cfDNA pipelines. Results showed that few methods enabled recovery of DNA fragments ≤ 150 base pairs. However, three kits showed a higher amount of 150 base pair fragments compared to the other methods tested. In a clinical setting it is expected that the target cfDNA compose only a minor fraction of the total cfDNA in plasma. Thus, the five best performing kits were tested using ddPCR on serial dilutions of leucocyte DNA fragmented to ~ 150bp to mimic cfDNA. The best performing kit had a recovery of 69% of the initial input compared to only 13% for the poorest performing kit. Collectively, our comprehensive comparison hereby guide which BS conversion methods are suited for cfDNA methylation-based biomarker pipelines. Citation Format: Sarah Ø. Jensen, Mai-Britt W. Ørntoft, Jesper B. Bramsen, Torben F. Ørntoft, Claus L. Andersen. Comprehensive comparison of bisulfite conversion kits: A guide for optimal sensitivity and specificity of circulating cell-free DNA methylation-based biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2017-3804

Abstract 3751: Allogeneic EGFRvIII chimeric antigen receptor T cells for treatment of glioblastoma

Abstract Glioblastoma multiforme (GBM) is a highly aggressive form of brain tumors with a 5-year survival rate of less than 10%. Standard-of-care combining radiation therapy with temozolomide only yields a median survival of 14.6 months and more effective therapeutic options to extend patient lives are urgently needed. EGFR variant III (EGFRvIII) is a tumor specific mutant of EGFR found in 25-30% of GBM but not expressed in healthy tissues. EGFRvIII is formed by an in-frame deletion of exons 2-7 of the wild-type EGFR which leads to removal of 267 amino acids in the extracellular domain of the receptor. The truncated receptor loses its ability to bind ligands but acquires constitutive kinase activity. The lack of normal tissue expression makes EGFRvIII an ideal target for developing CAR T therapy. Most CAR T therapies in clinical development use the patients’ own T cells for CAR T manufacturing. Our approach is to develop an “off-the-shelf” CAR-T treatment for GBM using healthy donor T cells with the TCR (T-cell receptor) disrupted to prevent graft-versus-host disease. This allogeneic approach could circumvent challenges faced by some patients due to limited availability and quality of their own T cells and rapid progress of their diseases, and has the potential to reduce the high cost associated with autologous CART therapy. Using phage panning and hybridoma approaches, we generated a series of humanized and fully human EGFRvIII antibodies with a wide range of affinities. Recombinant EGFRvIII protein binding by Biacore and FACS assays using EGFRvIII and EGFR wild-type expressing cells demonstrated that these antibodies are highly specific for EGFRvIII. The antibodies were converted to ScFvs and cloned into a CAR vector. Subsequently, EGFRvIII CARs were transduced into primary human T cells for functional studies. We developed a series of assays to evaluate CAR expression, degranulation activity and target dependent cytotoxicity. Our goal is to select CAR candidate that is safe, persistent and has potent target dependent cytotoxicity. Citation Format: Oi Kwan Wong, Mathilde Dusseaux, Jing-Tyan Ma, Melinda Au, Sophie Leduc, Joyce Chou, Jessica M. Yu, Marjorie Bateman, Thomas Pertel, Kevin C. Lindquist, Julianne Smith, Barbra Sasu, Shu-Hui Liu. Allogeneic EGFRvIII chimeric antigen receptor T cells for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3751. doi:10.1158/1538-7445.AM2017-3751

Abstract 2746: Detection of tumor-specific mutations in plasma DNA: A potential esophageal adenocarcinoma biomarker

Abstract BACKGROUND: Recent studies have shown that tumor-specific DNA from multiple types of tumors can be detected circulating in plasma and this has raised the possibility of “liquid biopsies” using mutated tumor DNA as a potential diagnostic and prognostic biomarker. Detection of mutations with allele frequencies below 0.1% remains challenging however given that circulating cell-free DNA is highly degraded and in low abundance. Detection of multiple different mutations in the same sample presents an additional challenge particularly when the mutation panel may change from patient to patient. We have developed a novel approach, called SimSen-Seq, to introduce molecular barcodes into sequencing libraries with DNA inputs as low as 5ng. Barcodes enable differentiation of true mutants from background noise introduced by Taq polymerase errors and permits detection of variant alleles with frequencies below 0.1%. The barcodes are protected from mis-priming using a hairpin structure which permits a high degree of multiplexing and flexibility for detection of multiple mutations from one plasma sample. We are using this technology to test the utility of liquid biopsy as a biomarker for esophageal adenocarcinoma (EAC) diagnosis and disease monitoring. METHODS: Blood samples were obtained at a single time point from patients with various stages of EAC and longitudinal blood samples were also collected from patients undergoing neoadjuvant therapy followed by surgery. Tumor samples were obtained from biopsy or resection specimens. Tumor DNA was sequenced using a targeted EAC panel to identify mutations in each tumor. SimSen-Seq assays were designed to identify these mutations in plasma, and hairpin barcodes were attached. Sequencing libraries were generated from circulating DNA, sequenced and analyzed using the barcodes to reduce background noise. RESULTS: Mutations were identified in tumor samples from 37 patients. To date, 29 patients have had plasma analyzed; 5 stage I, 6 stage II, 13 stage III, and 5 stage IV. Of these 29, the same mutations have been identified in 15 plasma DNA samples (20% stage I, 50% stage II, 54% stage III, 80% stage IV. Six patients demonstrated multiple mutant alleles in plasma DNA. One patient with detectable pre-treatment ctDNA underwent serial blood draws during their treatment course, and post-operative detection of tumor-specific markers preceded physical evidence of disease recurrence. DISCUSSION: SimSen-Seq shows promise as a novel ultra-sensitive, highly multiplexed sequencing method for identifying rare circulating mutations. Possible applications include prognostication in early stage patients and rapid monitoring of therapeutic response and recurrence. Further work is to evaluate this is ongoing. Citation Format: Matthew Egyud, Jennifer Jackson, Emiko Yamada, Anders Ståhlberg, Paul Krzyzanowski, Virginia Litle, Lincoln Stein, Tony Godfrey. Detection of tumor-specific mutations in plasma DNA: A potential esophageal adenocarcinoma biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2017-2746

Abstract 4540: MiVax: an innovative cancer neoantigen prediction system

Abstract T cell epitopes bearing tumor-specific mutations discovered using next generation sequencing stimulate T cell-mediated processes that lead to tumor regression. Although neoantigen prediction using computational methods rapidly identifies epitope candidates in the mutanome, a large proportion prove to be non-immunogenic. Innovative computational tools validated for infectious disease can be applied to enhance design of personalized cancer immunotherapies by classification of predicted epitopes according to potential for mounting a tumor-specific response. We developed the JanusMatrix algorithm that parses query sequences into MHC-facing and T cell receptor (TCR)-facing sequences and screens sequence databases to identify MHC ligands that share TCR faces with host-related proteins. A database of human protein sequences is available to identify tumor-specific epitopes that may reduce anti-tumor activity by sequences that activate regulatory T cells (Tregs) trained in the thymus on self-antigens. Similarly, tumor-specific epitope candidates are screened using databases composed of human commensal- or pathogen-derived sequences to identify epitopes that, respectively, may detrimentally or beneficially cross-react with T cells raised over the course of an individual’s immune history. We conducted retrospective analyses of cancer vaccine efficacy studies performed in mice [Kreiter et al. 2015 Nature 520, 692-696] showing that mutanome-directed vaccines effective at preventing tumor growth contained higher numbers of class I and II MHC neo-epitopes and had lower potential to cross-react with other murine proteins, as identified by the MiVax platform. Likewise, an evaluation of mutanomes derived from non-small cell lung cancer patients [Rizvi et al. 2014 Science 348, 124-128] revealed that improved clinical outcomes were observed in patients with mutanomes enriched in class I MHC neo-epitopes with TCR faces distinct from other self-epitopes. While retrospective in nature, the suite of tools used for these analyses have been extensively validated in prospective vaccine studies for infectious disease. Removal of Treg epitopes identified by JanusMatrix has led to the development of a novel H7N9 influenza vaccine scheduled for Phase I clinical trial. These results highlight the benefits of using in silico tools for the selection of high-value neoantigen candidates and how they can improve the design and efficacy of cancer vaccines. Neoantigens with low Treg activation potential may then be used to support development of personalized therapies including vaccination and in vitro expansion of tumor infiltrating lymphocytes for adoptive cell transfer. Citation Format: Lenny Moise, Guilhem Richard, Frances Terry, William Martin, Ann De Groot. MiVax: an innovative cancer neoantigen prediction system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2017-4540

Abstract 1695: Exploiting the pancreatic cancer mutome for immune interception

Abstract This study aims to translate tumor-specific mutations identified by next-generation sequencing techniques into an effective anti-tumor vaccine for a classically non-immunogenic tumor, pancreatic ductal adenocarcinoma (PDA). Pancreatic ductal adenocarcinomas develop an average of 63 mutations during tumorigenesis, collectively referred to as the “mutome.” Of these, approximately 45 are amino acid point mutations. These mutations are not present in normal cells and provide a source of tumor-specific neoepitopes for targeted immunotherapy. Next-generation sequencing technologies allow for rapid identification of mutations present in patient tumors; however, methods for rapid identification of immunogenic neoepitopes which can be effectively targeted for tumor clearance, and optimized vaccination strategies for targeting them, still need to be developed. In this study, we are using a transplantable murine PDA model, Panc02, to develop vaccination strategies for inducing neoepitope-specific T cell antitumor responses, and to define parameters for selecting appropriate targets. Exome sequencing identified 878 nonsynonymous mutations in the Panc02 cell line. Three immunological epitope prediction servers identified 878 potentially immunogenic peptides in the Panc02 model, 29 of which were strong candidates (scores <50) and another 240 candidate epitopes within the limits of prediction (scores <1000). Panc02 neoepitope peptide vaccinations in mice confirmed 14% of the predicted epitopes to be immunogenic by IFNγ ELISPOT, which may include both CD8+ and CD4+ T cell responses. Tumor transplant experiments demonstrate that therapeutic vaccination against neoepitopes in combination with a human STING-activating cyclic dinucleotide adjuvant is capable of inducing a temporary regression of implanted tumors. After tumor escape and regrowth, flow cytometry reveals a large population of infiltrating lymphocytes, the majority of which are co-expressing the checkpoint molecules PD-1, Tim3, and Lag3, indicating that treatment of vaccinated animals with checkpoint blockade therapy may potentiate infiltrating lymphocytes and ultimately result in permanent tumor regression. Thus, a broad and unbiased approach for screening vaccine targets will allow for the evaluation of predictive algorithms and ultimately the development of more effective vaccination approaches for targeting the tumor mutome. Citation Format: Heather Kinkead, Eric Lutz, Thomas W. Dubensky, Elizabeth Jaffee. Exploiting the pancreatic cancer mutome for immune interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1695. doi:10.1158/1538-7445.AM2017-1695

Synthetic lethality and cancer.

A synthetic lethal interaction occurs between two genes when the perturbation of either gene alone is viable but the perturbation of both genes simultaneously results in the loss of viability. Key to exploiting synthetic lethality in cancer treatment are the identification and the mechanistic characterization of robust synthetic lethal genetic interactions. Advances in next-generation sequencing technologies are enabling the identification of hundreds of tumour-specific mutations and alterations in gene expression that could be targeted by a synthetic lethality approach. The translation of synthetic lethality to therapy will be assisted by the synthesis of genetic interaction data from model organisms, tumour genomes and human cell lines.

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

BackgroundDuring posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR).MethodsTP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.ResultsIn all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2–422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667–156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%.ConclusionsOur results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

The frequency of neoantigens per somatic mutation rather than overall mutational load or number of predicted neoantigens per se is a prognostic factor in ovarian clear cell carcinoma

ABSTRACTNeoantigens derived from tumor-specific somatic mutations are excellent targets for anti-tumor immune responses. In ovarian clear cell carcinoma (OCCC), checkpoint blockade yields durable responses in a subset of patients. To approach the question of why only some patients respond, we first investigated neoantigen loads and immune signatures using exome sequencing and expression array data for 74 OCCC patients treated conventionally. Neither the number of missense mutations nor total predicted neoantigens assessed in the tumor correlated with clinical outcomes. However, the number of neoantigens per missense mutation (“neoAg frequency”) did correlate with clinical outcomes. Cox multivariate regression analysis demonstrated that low neoAg frequencies correlated with increased progression-free survival (PFS) and was an independent predictive factor for PFS in OCCC (p = 0.032), especially at stage I-II (p = 0.0045). Immunity-associated genes including those related to effector memory CD8 T cells were dominantly expressed in tumors with low neoAg frequencies in stage I-II patients, suggesting CD8 T cell-mediated elimination of immunogenic sub-clones expressing neoantigens (immunoediting) had occurred. In contrast, we observed decreased HLA-A, -B, and -C expression (p = 0.036, p = 0.026, and p = 0.030, respectively) as well as increased ratios of CTLA-4, PD-1, Tim-3, and LAG3 to CD8A expression (p = 0.0064, p = 0.017, p = 0.033 and p = 0.0136, respectively) in stage I-II tumors with high neoAg frequencies. Constrained anti-tumor immunity may thus result in limited immunoediting, and poor prognosis. Our results show that neoAg frequency in OCCC is an independent prognostic factor for clinical outcome and may become a potential candidate biomarker for immunomodulatory agent-based treatments.

Synthetic Genomics makes anticancer DNA

Synthetic Genomics, the biotech firm founded by J. Craig Venter, has opened what it calls the first current Good Manufacturing Practice (cGMP) facility for making synthetic DNA. The company is working with Advaxis on an investigational cancer immunotherapy called ADXS-NEO that will enter a Phase I clinical trial this year. The patient-customized therapy is based on tumor-specific mutations that can trigger an immune response from the patient’s T cells. Advaxis uses Synthetic Genomics’ automated genomic workstation to produce DNA based on the mutations. It inserts the DNA sequence into bacteria to produce the protein that the mutation expresses. The proteins are then taken up by a patient’s antigen-presenting cells to activate a tumor-specific T-cell response.

CloudNeo: a cloud pipeline for identifying patient-specific tumor neoantigens.

SummaryWe present CloudNeo, a cloud-based computational workflow for identifying patient-specific tumor neoantigens from next generation sequencing data. Tumor-specific mutant peptides can be detected by the immune system through their interactions with the human leukocyte antigen complex, and neoantigen presence has recently been shown to correlate with anti T-cell immunity and efficacy of checkpoint inhibitor therapy. However computing capabilities to identify neoantigens from genomic sequencing data are a limiting factor for understanding their role. This challenge has grown as cancer datasets become increasingly abundant, making them cumbersome to store and analyze on local servers. Our cloud-based pipeline provides scalable computation capabilities for neoantigen identification while eliminating the need to invest in local infrastructure for data transfer, storage or compute. The pipeline is a Common Workflow Language (CWL) implementation of human leukocyte antigen (HLA) typing using Polysolver or HLAminer combined with custom scripts for mutant peptide identification and NetMHCpan for neoantigen prediction. We have demonstrated the efficacy of these pipelines on Amazon cloud instances through the Seven Bridges Genomics implementation of the NCI Cancer Genomics Cloud, which provides graphical interfaces for running and editing, infrastructure for workflow sharing and version tracking, and access to TCGA data.Availability and implementationThe CWL implementation is at: https://github.com/TheJacksonLaboratory/CloudNeo. For users who have obtained licenses for all internal software, integrated versions in CWL and on the Seven Bridges Cancer Genomics Cloud platform (https://cgc.sbgenomics.com/, recommended version) can be obtained by contacting the authors.Supplementary information Supplementary data are available at Bioinformatics online.

Amplification of N-Myc is associated with a T-cell-poor microenvironment in metastatic neuroblastoma restraining interferon pathway activity and chemokine expression

ABSTRACT Immune checkpoint inhibitors have significantly improved the treatment of several cancers. T-cell infiltration and the number of neoantigens caused by tumor-specific mutations are correlated to favorable responses in cancers with a high mutation load. Accordingly, checkpoint immunotherapy is thought to be less effective in tumors with low mutation frequencies such as neuroblastoma, a neuroendocrine tumor of early childhood with poor outcome of the high-risk disease group. However, spontaneous regressions and paraneoplastic syndromes seen in neuroblastoma patients suggest substantial immunogenicity. Using an integrative transcriptomic approach, we investigated the molecular characteristics of T-cell infiltration in primary neuroblastomas as an indicator of pre-existing immune responses and potential responsiveness to checkpoint inhibition. Here, we report that a T-cell-poor microenvironment in primary metastatic neuroblastomas is associated with genomic amplification of the MYCN (N-Myc) proto-oncogene. These tumors exhibited lower interferon pathway activity and chemokine expression in line with reduced immune cell infiltration. Importantly, we identified a global role for N-Myc in the suppression of interferon and pro-inflammatory pathways in human and murine neuroblastoma cell lines. N-Myc depletion potently enhanced targeted interferon pathway activation by a small molecule agonist of the cGAS-STING innate immune pathway. This promoted chemokine expression including Cxcl10 and T-cell recruitment in microfluidics migration assays. Hence, our data suggest N-Myc inhibition plus targeted IFN activation as adjuvant strategy to enforce cytotoxic T-cell recruitment in MYCN-amplified neuroblastomas.

Patient monitoring through liquid biopsies using circulating tumor DNA.

Tumors release components such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and tumor-derived extracellular vesicles into the circulation. Multiple studies have demonstrated that molecular information about tumors and metastases can be extracted from these factors, which are therefore frequently referred to as "liquid biopsies." Liquid biopsies allow the longitudinal monitoring of tumor genomes non-invasively and may hence ensure that patients receive appropriate treatments that target the molecular features of their disease. Accordingly, the number of studies employing liquid biopsy based assays has been skyrocketing in the last few years. Here, we focus on three important issues, which are of high relevance for monitoring tumor genomes. First, we analyze the relation between the allele frequency of somatic tumor-specific mutations and the tumor fraction within plasma DNA. Second, we ask how well current tumor evolution models correlate with findings in longitudinal liquid biopsy studies. And, finally, as sensitivity is one of the key challenges of mutation detection, we address the challenge of detecting mutations occurring at very low allele frequencies in plasma DNA.

Prognostic value of various subtypes of circulating DNA in ovarian cancer patients.

e17092 Background: Patients with ovarian cancer represent a heterogeneous population with a variable prognosis and response to chemotherapy. Circulating tumor DNA has been shown to have a prognostic value in different types of cancer including ovarian carcinoma. Whether total circulating DNA, which can be assessed much easier without knowing the tumor-specific mutations, has similar informative value is currently unknown. The aim of this study was to evaluate the prognostic value of circulating DNA in advanced ovarian cancer. Methods: This prospective study included 67 patients (pts) with ovarian cancer treated with 1st line paclitaxel and carboplatin (25 pts) and bevacizumab (42 pts). Thirty-five patients had optimal debulking before chemotherapy. Circulating plasma DNA was quantified using real time PCR before administration of chemotherapy (67 pts) and after 6 cycles of chemotherapy (44 pts). Results: Total extracellular DNA (ecDNA), as well as extracellular DNA of nuclear (nDNA) and mitochondrial origin (mtDNA) significantly (p < 0.05) decreased after 6 cycles of chemotherapy (by 54%, 63% and 52%, respectively. Patients with stage I disease had significantly lower mtDNA compared to patients with stage II-IV (8604 vs. 16984 ge/mL, p = 0.03). Patients with lower baseline nDNA had superior progression-free (HR=0.35 (0.14-0.86)) and overall survival (HR=0.18 (0.04-0.77). The prognostic value of nDNA was confirmed independent of tumor stage and confirmed in multivariate analysis. Conclusions: Our data suggest that ecDNA of both, nuclear and mitochondrial origin, could be added to prognostic markers in ovarian cancer. This is of importance as this analysis does not require the knowledge of tumor-specific mutations in contrast to the quantification of tumor-derived ecDNA. Analysis of the dynamics and cell type-specific source of the ecDNA could shed light on its biology in cancer and might help to direct the treatment of ovarian cancer.

Detection of tumor-derived DNA mutations in cerebrospinal fluid of patients with primary or metastatic brain tumors.

2070 Background: Because detecting tumor-derived cell free DNA (cfDNA) in the blood of patients with primary or metastatic brain tumors is challenging, here we studied whether cerebrospinal fluid (CSF) could be serve as an alternative “liquid biopsy” by enabling measurement of circulating DNA within CSF to characterize tumor specific mutations. Methods: The paired cfDNA in CSF and plasma were collected from 20 patients with brain tumors and was subjected to enrichment for a 1.15M size panel cover exon regions from 1,086 genes. Followed by next generation sequencing on an Illumina X10 platform, the captured sequencing data was further processed using bioinformatics analysis to identify somatic mutations, including single nucleotide variants (SNV) and short insertions/deletions (indels). Results: The mutation profiles of 48 tumor associated genes in cfDNA were compared between the CSF and plasma. Our results showed that both average somatic mutation number and frequency identified in the cerebrospinal fluid was much higher than that in the corresponding plasma samples (25 vs. 18 & 1.39% vs. 0.55%). Among the twenty cases, one more potential actionable mutation, EGFR exon 19 deletion mutation with a 25.38% allele frequency variation, was only detected in the CSF cfDNA of a patient with brain metastasis lung cancer. Conclusions: Tumor mutations were detectable in CSF cfDNA of patients with different primary and metastatic brain tumors. Thus cerebrospinal fluid cell free DNA analysis could be a potential alternative analysis for patients with primary or metastatic brain tumors.

Somatic Tumor Mutations Detected by Targeted Next Generation Sequencing in Minute Amounts of Serum-Derived Cell-Free DNA

The use of blood-circulating cell-free DNA (cfDNA) as ‘liquid-biopsy’ is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients.

Mutational profiling of acral melanomas in Korean populations.

The proportion of acral melanoma (AM) is much higher in Asian populations than in Caucasian populations. Although mutational profiles associated with AM have been discovered in Caucasian populations, knowledge of its genetic alterations in Asian populations is limited. To describe the molecular nature of AM in Korean patients, we performed mutational profiling of AM and matched normal tissues in patients. Fifty-one formalin-fixed paraffin-embedded AM samples and 32 matched pairs from patients' saliva DNA were analysed by next-generation sequencing. Only mutations confirmed via digital droplet PCR or in BRAF, KIT and NRAS, the most frequently altered cancer genes in cutaneous melanoma, were considered as positive. The relationship between mutational status and clinicopathological features were examined. Of the 47 AM patients screened, alteration of BRAF, NRAS and KIT genes was observed in 6.4%, 4.3% and 8.5%, respectively. We also tested matched normal tissues of patients to identify tumor-specific mutations. Examination of the mutational profile in a cohort of 28 primary melanomas and matched normal controls found BRAF mutations in two cases (7.1%), KIT mutations in three cases (10.7%) and CTNNB1 mutations in one case (3.6%). The BRAF, NRAS and KIT mutation status did not correlate with clinicopathological characteristics. Our results show that KIT, NRAS and BRAF hotspot mutations occur at a low frequency in Korean populations. We also observed a case with the CTNNB1 mutation, which raises the possibility that other pathways are associated with AM development.