Ludwig Institute Research Articles

The Hallmarks of Cancer in 2023

CURRENT cancer hallmarks, their capabilities, and potential targeted therapies were the focus of the presentation given by Douglas Hanahan, Ludwig Institute for Cancer Research, Lausanne, Switzerland, at the European Society for Medical Oncology (ESMO) Congress 2023, held in Madrid, Spain. Hanahan began by defining hallmarks as acquired functional capabilities that contribute to the manifestation of the disease, which are a “conceptual framework for rationalising the daunting complexity of cancer.”

Heterologous prime-boost viral vector cancer vaccines have protective effects against a syngeneic mouse model of glioblastoma

Abstract Glioblastomas are immunologically “cold” tumors with intense immunosuppressive myeloid cell- and sparse cytotoxic T-cell infiltration. Therefore, viral vector vaccines may be a promising approach to boost the induction of glioblastoma-targeted T cells. It has been previously shown that heterologous prime-boost vaccination with chimpanzee-derived adenovirus ChAdOx1 and modified vaccinia Ankara (MVA) vectors can induce a high magnitude of CD8 +T cells specific for cancer-associated antigens and have therapeutic effects against mouse models of cancer. Therefore, we aimed to evaluate whether using ChAdOx1 and MVA vaccines targeting model antigen P1A, a mouse equivalent to human tumor-associated Melanoma Antigen GenE (MAGE)-type antigens, could have protective effects against a P1A-transfected BGL-1 glioblastoma model. Mice were vaccinated with ChAdOx1/MVA-P1A vaccines, then challenged subcutaneously with P1A-expressing BGL-1 cells. Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impacted the magnitude of antigen-specific CD8 +T-cell responses and protective efficacy. Future studies will investigate the protective effects of vaccination against intracranial P1A-expressing BGl-1 tumors. Supported by grants from the Ludwig Institute for Cancer Research, University of Oxford; the NIH Intramural Program (ZIA BC011877), the NIH Oxford-Cambridge Scholars Program, and the Marshall Scholarship Program.

Top 100 most-cited articles on intraoral squamous cell carcinoma and its risk factors: a bibliometric study.

This study aimed to analyze the 100 most-cited articles on intraoral squamous cell carcinoma and its risk factors. A literature search was conducted on November 12, 2020 using the Web of Science database. Bibliometric data were collected after study selection. Bibliometric maps were generated using VOSviewer software. Articles were ranked based on the number of citations, ranging from 108 to 1513 per study. Eleven studies presented at least 400 citations. Articles were published between 1950 and 2017 and were mostly case-control studies (n = 39) and narrative reviews (n = 29). The author and institution in most articles were S. Franceschi (n = 10) and the International Agency for Research on Cancer (n = 10), respectively. The most prominent countries were the United States (n = 35), the United Kingdom (n = 16), and France (n = 9). Among the 100 most-cited articles, only one article was from Brazil, and the authors were affiliated with the Ludwig Institute for Cancer Research. Bibliometric maps showed strong associations between the terms alcohol, smoked/smokeless tobacco, and human papillomavirus. Emerging terms, such as areca nut and betel quid, were cited in recent articles. In conclusion, alcohol, tobacco, and human papillomavirus were the most prominent risk factors. Case-control design was the most common study design, and the majority of studies were conducted in the United States by the International Agency for Research on Cancer.

Research progress of CTCF in mediating 3D genome formation and regulating gene expression.

In interphase eukaryotic nuclei, chromatin is folded to form a higher-order topological structure. The spatial organization of such chromatin domain has an important impact on the regulation of gene expression. As a key architectural structural protein, CTCF (CCCTC-binding factor) plays an important role in the formation of chromatin three-dimensional chromatin structure. CTCF can also bind to many insulator elements in the genome and insulate enhancers from activating target genes via modulating remote chromatin interactions. A recent study by Dr. Chunliang Li and his team at St. Jude Children's Research Hospital in the United States showed that when CTCF was acutely degraded, significant changes were found in the three-dimensional structure of chromatin. The mechanism by which CTCF binding sites function as insulator elements was investigated by Prof. Qiang Wu's team at Institute of Systems Biomedicine and Shanghai Jiao Tong University in China and Prof. Bing Ren's team at Ludwig Institute for Cancer Research in the United States. Here we mainly review and discuss some of these latest progresses.

Enhancing preclinical predictivity: what is going wrong between preclinical in vitro/in vivo and clinical in vivo settings?

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Christian Schmees copy.jpg" data-image="1"></div><div class="author-det"> <b>Christian Schmees</b> is heading the Tumor Biology group at the Natural and Medical Sciences Institute (NMI) at the University of Tuebingen, Germany. He received his undergraduate degree in biochemistry from Tuebingen University in 2002. Since 2006 he holds a PhD (with highest honors) in cancer immunology from the Technical University of Munich, Germany. His thesis resulted in the identification of gamma-glutamyl-transpeptidase as the major factor for T cell specific immune evasion of the tumorigenic bacterium Helicobacter pylori. As a post-doc he joined the laboratories of Carl-Henrik Heldin at the Ludwig Institute for Cancer Research (LICR) in Uppsala, Sweden (2006-2010) and Philippe Bastiaens at the Max-Planck Institute of Molecular Physiology in Dortmund, Germany (2010-2011). He received fellowships from the German Research Foundation and the LICR to support his research on differential regulation of intracellular PDGF α- and β-receptor trafficking in cancer cells. In 2011 he joined the Natural and Medical Sciences Institute at the University of Tuebingen (NMI) as a senior scientist in tumor biology and was appointed Head of Tumor Biology in 2014. Ongoing projects in his group are focusing on the development of clinically relevant cellular model systems for drug development and target validation in translational oncology. These approaches are combined with gene editing and silencing technologies as well as protein profiling and immunophenotyping platforms for mode-of-action analyses of different compound classes. Dr. Schmees is member of the Association for Cancer Immunotherapy (CIMT). </div></div></div>

MAGE-A3 Recombinant Protein (recMAGE-A3) Immunotherapy and Autologous Peripheral Blood Lymphocyte (PBL) Infusion Is Safe and Induces Robust Humoral Immune Responses In Multiple Myeloma (MM) Patients Undergoing Autologous Stem Cell Transplantation (autoSCT)

▪ BackgroundMAGE-A3 is a cancer-testis antigen expressed in various human cancers, including MM, but not in normal non-gonadal tissues. MAGE-A3 expression inhibits apoptosis in MM cells, and has been associated with a more aggressive clinical course, making it a rational target for immunotherapy. Pre-autoSCT myeloablative conditioning provides tumor antigen release and a lymphopenic environment that may be ideal for expansion of primed PBL and generation of anti-myeloma immune responses. Patients and methodsEligibility for this pilot study required symptomatic MM within 1 year of diagnosis; MAGE-A+ MM cells by immunohistochemistry (IHC); at least VGPR following induction therapy; and meeting institutional criteria for autoSCT. The immunotherapy consists of a recombinant protein containing full-length MAGE-A3 fused to part of H. influenzae Protein D antigen, mixed with the AS15 immunostimulant (containing MPL, QS21, and CpG7909). Six weeks prior to SCT, patients received their 1st injection (300 µg recMAGE-A3 intramuscularly), followed 3 weeks later by steady-state leukopheresis to collect primed PBL. They then underwent stem cell mobilization and a melphalan 200 mg/m2-conditioned autoSCT, followed by re-infusion of unmanipulated PBL on day +3 after SCT, and 7 post-SCT injections (days +10, 31, 52, 73, 94, 180, and 270). Lenalidomide maintenance was permitted. The primary objectives were safety and tolerability. The secondary objectives were cellular and humoral immune responses and clinical outcomes. Humoral immunity was assessed by ELISA using baculovirus-generated recombinant MAGE-A3 protein. ResultsOf 93 pre-screened patients, 30 (32%) were MAGE-A+ by IHC. Of these, 13 (43%) enrolled. Reasons for non-enrollment were: did not achieve VGPR (n=6), declined/received other therapy (n=7), other (n=4). For enrolled patients, median age was 56; ISS stages I/II/III were 58%/25%/17%; 45% had high-risk cytogenetics at diagnosis (n=11 tested). Responses at enrollment were 12 VGPR and 1 CR; most (83%) had received VRD-based induction. All patients had adequate PBL collections (median 1.4 x 108 CD3+ cells/kg, range 1.0-2.6), and all collected stem cells successfully (median 11.4 x 106 CD34+ cells/kg, range 8.3 – 37.2). Autologous PBL infusions were well-tolerated, with no engraftment-like syndromes. Time to neutrophil recovery (median 11 days) and SCT-related toxicities were typical. The immunotherapy schedule was feasible with only 1 missed dose to date. Potential immunotherapy-related AE's (all grade 1 or 2) include injection site pain/reaction (n=6), myalgia/flu-like symptoms (n=5), fever/chills (n=2), fatigue (n=1), and rash (n=1). Three SAE's were observed: 1 immunotherapy-related (grade 2 myalgia requiring a 24-hour hospitalization), and 2 unrelated (grade 3 diarrhea, grade 3 atrial fibrillation with pulmonary edema). There have been no autoimmune toxicities and no deaths on study. Four patients initiated lenalidomide maintenance with no unexpected toxicities. Regarding immune responses, 12 of 12 (100%) evaluable patients have developed high-titer (>104) IgG antibodies against MAGE-A3 (Fig.). Responses were seen in all but 2 patients by day +31 post-SCT, and in all patients by day +75, persisting at 1 year post-SCT in both patients reaching this timepoint so far. All patients also developed strong IgG responses against Protein D. Clinically, at 3 months post-SCT, 50% of patients had achieved CR (33% stringent CR). With a median follow-up of 10 months post-SCT (range 1-20), 92% are progression-free, with follow-up ongoing. ConclusionsMAGE-A3 protein immunotherapy plus autologous PBL transfer is feasible, has an acceptable safety profile, and is strongly immunogenic, capable of overcoming the profound immune suppression typically seen in MM patients post-autoSCT. The presence of IgG-type anti-MAGE antibodies implies the generation of a CD4 T cell response to induce Ig class-switching, though formal assessment of T cell responses against MAGE-A3 and other antigens are needed to confirm this, and are ongoing. Importantly, the use of an “off-the-shelf” immunotherapy product and unmanipulated autologous PBL transfer allows the treatment to be given at any transplant center. Further study of this approach as a strategy to consolidate post-autoSCT responses and potentially eliminate minimal residual disease is warranted. [Display omitted] Disclosures: Cohen:Bristol Meyers Squibb: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria; Millennium: Honoraria. Bertolini:Ludwig Institute for Cancer Research: Employment. Pan:Ludwig Institute for Cancer Research: Employment. Venhaus:Ludwig Institute for Cancer Research: Employment. Fellague-Chebra:GlaxoSmithKline Vaccines: Employment. Gruselle:GlaxoSmithKline Vaccines: Employment.

Clonal Evolution in Newly Diagnosed Multiple Myeloma Patients: A Follow-up Study from the Mmrf Commpass Genomics Project

Multiple Myeloma (MM) is an incurable malignancy of bone marrow plasma cells characterized by wide molecular diversity and complex clonal and subclonal architecture. Intra-tumor heterogeneity reflects the evolution of the disease. Clonal structure may affect response to treatment, which, in turn, may shape tumor evolution and, consequently, contribute to drug resistance. Thus, analysis of tumor clonality has profound implications for personalized medicine.Using our recently developed network model of newly diagnosed MM from the MMRF CoMMpass study (Lagana et al, Leukemia 2017) , MMNet, we have identified patterns of gene co-expression significantly associated with tumor clonality and showed that clonality is correlated with mutational burden and relapse.In order to characterize the evolution of clonal structure from diagnosis to relapse, we analyzed Whole-Exome data (WES) from 19 patients enrolled in CoMMpass for whom sequencing data for multiple time points was available. The cohort included both male (12; 63%) and female (7; 37%) patients of white/caucasian (14; 73%) and black/african-american (5; 27%) origin. Patients were distributed among five of the ten classes defined by MMNet: CCND1 (4; 21%), MAF (1; 5%), MMSET (2; 10%), HY/NRAS (4; 21%), and IMM (4; 21%) (4 were unassigned). Also, six patients had the t(11;14) and two had the t(4;14) chromosomal translocations. All patients had received induction therapy, consisting of combinations of proteasome inhibitors (PI) (Bortezomib: 19, 100%; Carfilzomib: 3, 16%), immunomodulatory drugs (IMiDs) (Lenalidomide: 13, 68%; Pomalidomide: 2, 10%; Thalidomide: 1, 5%), steroids (18, 95%) and monoclonal antibodies (Daratumumab: 1, 5%). Best responses to therapy were CR/sCR (3; 16%), VGPR (7; 37%) and PR (9; 47%). Fourteen (68%) patients had SD or PD at the second sequencing time point, with a median PFS of 456 days.Our analysis inferred subclonal tumor composition and evolution based on somatic mutations and copy number alterations (CNA) using PhyloWGS (Deshwar et al, Genome Biol 2015). Patients had one (14; 74%), two (4; 21%) or three (1; 5%) founding clones at baseline, and numerous subclones. Trajectories of cancer cell populations revealed dramatic changes in most patients in terms of clonal and subclonal cell fractions between baseline and relapse. In most patients we observed emergence of new competing clones at relapse, where one or more subclones decreasing in size were replaced by other subclones, indicating selective pressure introduced by therapy (Fig. 1).Subclones were characterized in terms of mutations and CNA and were labeled as stable/resistant or sensitive based on their trajectories from baseline to relapse. Our analysis revealed significant inter-patient heterogeneity in terms of mutations and clonal composition, with few overlaps. We identified subclonal drivers by screening the observed mutations and CNA against a database of known cancer drivers and analysis of co-occurrence and mutual exclusivity. Our analysis revealed that stable/resistant clones were characterized by concurrent deletion of 17p and 13q, and/or mutations in NRAS. In particular, 7 out of 19 patients had at least one mutation in NRAS, observed at baseline in 5 cases, and with two patients carrying two different mutations in two different clones. Our findings support earlier adoption of targeted therapy against RAS (e.g. Trametinib). Other drivers found exclusively in resistant clones included DIS3, FAM46C, ROBO1 and CCND1.Overall, our analysis provides genomics characterization of relapsed patients in CoMMpass following induction therapy, reveals heterogeneous clonal and subclonal composition and trajectories from baseline to relapse, and defines specific somatic mutations (e.g. NRAS) and CNA as drivers of resistance to induction therapy. [Display omitted] DisclosuresMadduri:Foundation Medicine, Inc.: Consultancy. Chari:Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Amgen: Honoraria, Research Funding; Celgene Corporation: Consultancy, Research Funding. Cho:Bristol Myers-Squibb: Other: advisory board, Research Funding; Agenus, Inc.: Research Funding; Genentech: Other: advisory board, Research Funding; Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding. Barlogie:Millenium Pharmaceuticals: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding. Jagannath:Celgene: Consultancy; Bristol-Meyers Squibb: Consultancy; Merck: Consultancy; Medicom: Speakers Bureau; MMRF: Speakers Bureau; Novartis: Consultancy. Dudley:GlaxoSmithKline: Consultancy; Janssen Pharmaceuticals, Inc.: Consultancy; Ayasdi, Inc.: Equity Ownership; Ecoeos, Inc.: Equity Ownership; NuMedii, Inc.: Equity Ownership, Patents & Royalties; Ontomics, Inc.: Equity Ownership; Personalis: Patents & Royalties; AstraZeneca: Consultancy.

Adar-Mediated Aberrant a-to-I RNA Editing Is Driven By 1q Amplification and Contributes to Proteasome Inhibitor Resistance in Multiple Myeloma

Multiple Myeloma (MM) is an incurable malignancy of plasma cells characterized by wide and remarkable genetic heterogeneity. The amplification of 1q identifies a class of patients characterized by a more aggressive disease course and poor prognosis. While previous research has identified several oncogenes with prognostic relevance located in 1q, the biological mechanisms underlying the poor clinical outcome associated with this alteration is still unclear.ADAR (Adenosine Deaminase Acting on RNA) is an enzyme responsible for A-to-I editing, a post-transcriptional process which contributes to transcriptome and proteome diversity by conversion of specific adenosines into inosines in both coding and non-coding transcripts. Such changes may affect RNA stability and regulation and introduce mutations in the corresponding proteins.Recent research has reported dysregulation of RNA editing in cancer, revealing its oncogenicity and clinical relevance. Since ADAR is located in 1q21, the critical amplified region in MM, we sought to investigate the implications of ADAR activity in MM.We have previously shown increased ADAR expression and A-to-I RNA editing in a small group of newly diagnosed patients with 1q21 amplification enrolled in the MMRF CoMMpass study (Lagana et al, ASH 2016), and that ADAR expression could differentiate prognosis. To further investigate and characterize the role of ADAR and RNA Editing in MM, we have analyzed 590 RNAseq samples from newly diagnosed patients in CoMMpass. ADAR copy number gain was observed in 111 samples (19%).ADAR expression was significantly higher in patients with 1q21 gain (p < 0.0001), supporting the role of copy number gain as a driver of ADAR expression. Moreover, patients with high expression of ADAR and no CN alterations, showed significant activation of Interferon pathways (Fig. 1A). This can be explained by the fact that one isoform of ADAR (p150) is regulated by an interferon-inducible promoter. Multivariate regression of ADAR expression against ADAR CN and STAT1 expression, as a measure of interferon signaling activation, showed that both ADAR CN and STAT1 expression were significant independent predictors of ADAR expression.To investigate the functional impact of ADAR activity, we implemented a computational pipeline to calculate sample-wise Alu Editing Index (AEI). A-to-I RNA editing targets predominantly Alu elements, repetitive sequences abundantly interspersed throughout the human genome, mostly within introns and untranslated regions (UTRs). AEI has been developed and validated as a global measure of RNA editing activity at the sample level (Bazak et al, Genome Res 2014). Our analysis in MM revealed that AEI could discriminate between good and poor prognosis in terms of both PFS and OS (p < 0.0001). Moreover, AEI could critically stratify patients with 1q21 gain in terms of PFS (p < 0.01) (Fig. 1B). This finding suggests that RNA editing can be an adverse prognostic factor independently of 1q21 gain.Patients with high AEI were characterized by down-regulation of immunoglobulin transcripts and of the Unfolded Protein Response (UPR) pathway. In particular, patients with high AEI exhibited down-regulation of key activators of UPR such as NFYC, ATF4 and XBP1, and of transcriptional targets of XBP1s, the active spliced form of XBP1. Decreased UPR and low XBP1s expression in pre-plasmablasts have been shown to induce resistance to proteasome inhibitors (PI) (e.g. Bortezomib) through de-differentiation of plasma cells (Leung-Hagesteijn et al, Cancer Cell 2013). Among patients who received Bortezomib-based first line therapy, we found that AEI was significantly higher (p<0.01) in patients whose best response was stable disease (SD) or progressive disease (PD) (23; 4.5%) compared to patients who had complete (CR) or stringent complete response (sCR) (118; 23%) (Fig. 1C). siRNA meditated knockdown of ADAR increased sensitivity to Bortezomib in MM cell lines with 1q amplification and high ADAR expression (OPM2), consistently with our in-silico findings.In conclusion, we propose a model of MM where ADAR-mediated RNA editing is driven by gain of 1q21 and interferon signaling and contributes to disease aggressiveness and resistance to PI-based therapy through down-regulation of UPR (Fig. 1D). [Display omitted] DisclosuresMadduri:Foundation Medicine, Inc.: Consultancy. Chari:Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Cho:Agenus, Inc.: Research Funding; Genentech: Other: advisory board, Research Funding; Ludwig Institute for Cancer Research: Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Multiple Myeloma Research Foundation: Research Funding. Barlogie:Millenium Pharmaceuticals: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding. Jagannath:Merck: Consultancy; Medicom: Speakers Bureau; Celgene: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy; MMRF: Speakers Bureau. Dudley:Ecoeos, Inc.: Equity Ownership; Ayasdi, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; Personalis: Patents & Royalties; AstraZeneca: Consultancy; NuMedii, Inc.: Equity Ownership, Patents & Royalties; Janssen Pharmaceuticals, Inc.: Consultancy; GlaxoSmithKline: Consultancy.

Single-Cell RNA Sequencing Reveals Distinct Transcriptomic Profiles of Multiple Myeloma with Implications for Personalized Medicine

Multiple Myeloma (MM) is a heterogeneous malignancy with genomic translocations and mutations conferring varying levels of risk of recurrence and confounding treatment options across patients. Intra-tumor clonal heterogeneity has also been observed which further complicates treatment. We hypothesize that the use of Single-Cell RNA sequencing (scRNA-seq) can elucidate the complex clonal transcriptomic structure of individual patients' disease, and can be used in concert with other genomic techniques to better characterize and treat patients on a personalized level.Methods:Individual CD138+ tumor cells were isolated and barcoded from bone marrow aspirates from eight relapsed myeloma patients using the 10x Genomics Chromium microfluidics platform and V2 chemistry for deep sequencing using an Illumina HiSeq 2500 machine. The 10x Genomics Chromium platform allows for large-scale simultaneous cellular sequencing which facilitates a more robust analysis of cellular populations than previous technologies. The number of cells analyzed ranged from 393 to 3,359 per sample, depending on sample cell suspension density. We used 10x Genomics Loupe software to visualize scRNA-seq data via TSNE dimensional reduction plots. Cells were then clustered via Graph-Based, K-Means, or custom gating methods.Results:Analysis of bulk CD138+ whole-exome sequencing data has identified previously known mutational drivers and allowed estimation of cellular sub-populations (Fig. 1a) using copy-number alteration and mutational data via Phylo-WGS (Deshwar et al, Genome Biol 2015). Integrative analysis of bulk RNA-seq data revealed concordant activation of relevant pathways (e.g. MAP kinase) in patients with corresponding somatic mutations. On an individual patient level, we were able to process scRNA-seq data to visualize clusters of single cells with transcriptional similarity using t-distributed stochastic neighbor embedding (t-SNE) plots. We identified pathway activation in individual scRNA-seq clones via differential expression analysis and Signaling Pathway Impact Analysis (SPIA), in concordance with known somatic mutations (Fig. 1b). Genomic drivers and stages of differentiation in cell subsets (plasma cell vs immature B cell-like plasma cells) within a given patient were also revealed by distinct expression profiles (Fig. 1c). We utilized open-source drug repurposing tools with the RNA signature of individual clones to interrogate transcriptional signatures of drugs in public databases such as LINCS. The use of such drug repurposing methods will be effective in generating more targeted personalized therapies, utilizing cluster specific expression profiles. This is especially useful for cellular populations which do not contain a known target based on DNA mutational analysis. In a pooled analysis of all samples, each patient sample remarkably formed its own spatially distinct cluster of cells (Fig. 1d). These data suggest that relapsed myeloma patients form transcriptionally distinct clones, and myeloma therapy may need to be highly individualized to overcome clonal heterogeneity. scRNA-seq is a powerful tool which can be utilized alone, or in harmony with other sequencing technologies and genomic techniques, to improve characterization and treatment of myeloma patients in a clone specific manner. [Display omitted] DisclosuresMadduri:Foundation Medicine, Inc.: Consultancy. Chari:Novartis: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Amgen: Honoraria, Research Funding. Cho:Genentech: Other: advisory board, Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Agenus, Inc.: Research Funding. Barlogie:Celgene Corporation: Consultancy, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding. Jagannath:MMRF: Speakers Bureau; Medicom: Speakers Bureau; Merck: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol-Meyers Squibb: Consultancy. Dudley:NuMedii, Inc.: Equity Ownership, Patents & Royalties; Janssen Pharmaceuticals, Inc.: Consultancy; Ayasdi, Inc.: Equity Ownership; Ecoeos, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; Personalis: Patents & Royalties; AstraZeneca: Consultancy; GlaxoSmithKline: Consultancy.

Neoantigen Synthetic Peptide Vaccine for Multiple Myeloma Elicits T Cell Immunity in a Pre-Clinical Model

Cancer immunotherapies such as immune checkpoint inhibitors (CI) are approved for a broad range of solid tumors and selected hematologic malignancies. Clinical response to CIs has been shown to be associated with mutation burden and is predicated on the presence of T cells specific to tumor-associated antigens. Therefore, increasing the frequency of such T cell responses should increase the efficacy of anti-tumor immunity. Individualized neoantigen peptide vaccines are a promising strategy to activate tumor-specific T cell immunity. Next generation sequencing of tumor specimens is now widely available and novel computational algorithms provide the means for the prediction and selection of neoantigens.We are conducting a preclinical study of a personalized vaccine in an animal model of multiple myeloma (MM). MM is an incurable cancer of plasma cells that resides in the bone marrow (BM) and causes lytic bone disease and other end-organ damage. MM is characterized by an intermediate level of somatic mutation that may yield mutational neoantigens for vaccine design. This investigation pursued two goals: first, to determine if neoantigens are immunogenic, and second, if they induce protective immunity against tumors. To address the immunogenicity of neoantigens, we used the MOPC.315.BM myeloma cell line in the immunocompetent BALB/c mouse background. MOPC.315.BM cells mimic human disease by homing to the BM and causing osteolytic lesions. We performed whole exome sequencing and RNAseq on MOPC.315.BM cells to identify expressed somatic mutations. Neoantigens were then selected based on predicted MHC class I and II binding and expression levels, and corresponding 27-mer peptides were synthesized and tested in vivo for their immunogenicity. Mice were immunized 3 times at day 0, 14 & 21 with pools of peptides in the presence of poly (I:C) adjuvant, and splenocytes were harvested at day 28 to measure peptide-specific T cell activation by IFNg ELISpot. Of the selected peptides, 25 of 66 induce high IFNg T cell responses in the spleen. Interestingly, 88% of these responses were CD4 T cells and 12% were CD8 T cells, consistent with other preclinical and clinical studies where neoantigens predicted to bind MHC-I molecules induced mainly CD4 T cell activation. Humoral immune response specific to the peptides were analyzed by ELISA and showed that 8 of the 25 peptides induce peptide-specific IgG antibodies in the serum.To determine whether neoantigens generate tumor protective immunity, we challenged vaccinated mice with the MOPC.315.BM cell line. We used a well-characterized antigen lambda 2315 idiotype (Id) produced by MOPC cells and known to activate CD4 T cells. Vaccines using only the Id were found to be weakly immunogenic and failed to elicit protective responses. We developed a new strategy of vaccination by adding a proprietary amino acid sequence comprising a high affinity HSP70 binding motif to the idiotype peptide. Mice vaccinated at days 0, 14 & 21 with the so-modified peptide and poly-(I:C) adjuvant had 4 times higher Id-specific IFNg CD4 T cell response in the spleen compared to mice vaccinated with the unmodified Id-peptide. When challenged with MOPC.315.BM cells after vaccination with the modified Id peptide, 60% of these mice maintained non-detectable Id level in their blood and resisted tumor growth for more than 70 days while unvaccinated mice had very high levels of circulating Id and died around 30 days after tumor injection. Id-mediated cytotoxicity is being investigated in vitro to complement these results. We are conducting tumor protection experiments with the 25 immunogenic peptides identified in the vaccination experiments described in the previous section.In summary, we showed that neoantigens derived from bioinformatic analysis of tumor genome sequencing are immunogenic and that they mainly activate CD4 T cells in this setting. Additionally, our vaccination strategy using the idiotype peptide showed that CD4 T cell immunity can be protective against tumor growth. We are currently investigating the potential of the neoantigens identified in the immunogenicity study to protect mice against tumor growth and eradicate pre-existing tumors. Our findings indicate that neoantigen vaccination is very promising and that future investigations combining this strategy with CIs and/or immunomodulatory agents may lead to the efficient generation of protective anti-tumor immunity. DisclosuresBekri:Agenus Inc: Research Funding. Cho:Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Genentech: Other: advisory board, Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Agenus, Inc.: Research Funding.

Phase 1 Study of Elotuzumab and Peripheral Blood Mononuclear Cell Reconstitution in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Phase 1 Study of Elotuzumab and Peripheral Blood Mononuclear Cell Reconstitution in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

Hypoxic and Pharmacological Activation of HIF Inhibits SARS-CoV-2 Infection of Lung Epithelial Cells

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 1 million fatalities to date. Understanding how host factors modify the viral life cycle could inform susceptibility to viral infection and the design of new therapies. Viral replication is shaped by the cellular microenvironment and one important factor is local oxygen tension, where hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via a HIF-1α dependent signalling pathway. Further, hypoxia and Roxadustat inhibit viral replication in SARS-CoV-2 infected cells, showing that post-entry steps in the viral life cycle are oxygen-sensitive. This study highlights the importance of hypoxia and HIF signalling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention and/or treatment of COVID-19.Funding: The McKeating laboratory is funded by a Wellcome Investigator Award (IA) 200838/Z/16/Z, UK Medical Research Council (MRC) project grant MR/R022011/1 and Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002). The Ratcliffe laboratory is funded by the Oxford Branch of the Ludwig Institute for Cancer Research; Wellcome IA 106241/Z/14/Z; the Francis Crick Institute, which receives core funding from Cancer Research UK (FC001501), UK MRC (FC001501) and Wellcome (FC001501); the Paradifference Foundation. PJR, EJH and TB are additionally funded by the COVID-19 Research Response Fund, University of Oxford. SK is funded by the Clarendon Scholarships Fund and the Christopher Welch Trust. The Davis laboratory is funded by Wellcome IA 209412/Z/17/Z and Wellcome Strategic Awards 091911/B/10/Z and 107457/Z/15/Z. JYL is funded by the Medial Sciences Graduate Studentship, University of Oxford. The Hinks laboratory is funded by grants from the Wellcome (104553/z/14/z, 211050/Z/18/z) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre; the views expressed are those of the authors and not those of the NHS or NIHR. Conflict of Interest: EJH is employed under the Cambridge Experimental Medicine Initiative, which is partly funded by AstraZeneca although they have not been involved in this project. The other authors declare no financial interests.Ethical Approval: The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361).

Abstract IA06: The Long-Term Ovarian Cancer Survivor Project: A Department of Defense initiative

Abstract In 2019 there will be 22,500 new cases of ovarian cancer, resulting in approximately 14,000 deaths. Ovarian cancer remains the highest case fatality rate of any gynecologic cancer. Despite significant improvement in the surgical and chemotherapeutic managements of ovarian cancer patients, the overall survival has not changed in 30 years. However, these new therapies have resulted in a significant improvement in median survival, resulting in increasing numbers of patients surviving greater than 10 years with disease. The clinical and molecular features of these unique patients remain uncharacterized. The Department of Defense Ovarian Cancer Research Program (DOD OCRP) funded the creation of our interdisciplinary Long-term Survivor Consortium (LTSC), composed of 6 research sites including MDACC, UAB, IU, UCI, Ludwig Cancer Research, and University Hospitals of Canton Vaud (CHUV) and INOVA, a bioinformatic center at DFCI, and administrative centers at UCI and UAB. The purpose of the consortium is to identify characteristics and predictors of long-term survival in advanced ovarian cancer patients. The consortium is overseen by both a scientific and advocacy advisory board to guide direction and efforts. 285 specimens of advanced-stage high-grade serous ovarian cancers with full clinical annotation including greater than 12 years of follow-up were analyzed for this project. In addition, 340 early-stage high-grade ovarian cancers were available for one of the AIMs of the project. These latter samples were obtained through the creation of an international consortium funded by the DOD OCRP. All specimens were first reviewed for tumor context by H&amp;E staining to determine percentage of tumor. The scientific platforms include whole transcriptome, microRNAs, global proteomics, immune profiling, and methylation. All specimens were analyzed on each individual platform and the results will be integrated in a global process. In parallel with these efforts, a systematic analysis of quality of life of patients at the time of diagnosis and later during the course of their disease based upon patient-reported outcome surveys has been undertaken. These data will also be integrated with genomic results to produce signatures that can further characterize long-term survivors of ovarian cancer. Citation Format: Michael J. Birrer, Lari Wenzel, Austin Miller, Heather Lankes, Nilsa Ramirez, Giovanni Parmigiani, Samuel Mok, Kenneth Nephew, Thomas Conrads, George Coukos, George L. Maxwell, Giulia Fulci, Mary Scroggins. The Long-Term Ovarian Cancer Survivor Project: A Department of Defense initiative [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA06.

The Role of Asunercept as a Selective CD95L Inhibitor in Cutaneous Melanoma: Rationale and Results from an Enhanced TiRP Model

Abstract Background CD95/CD95L signalling plays an important role in cancer cells resistant to CD95-mediated apoptosis and CD95L inhibition can overcome this mechanism. Several mechanisms related to apoptosis induction or dysfunction of effector T-cells via CD95L in the tumour microenvironment (TME) have also been investigated, with supporting evidence from preclinical melanoma models. Methods An autochthonous mouse model of melanoma (TiRP), genetically engineered to express P1A (defined MAGE-type antigen) mimics the T-cell suppressive effects of the TME through CD95L, which normal transplantation models cannot replicate. An advanced version of this murine melanoma TiRP model has been recently developed. This model can generate synchronized tumours in multiple mice in which treatments can be compared simultaneously while maintaining the advantages of the previous model regarding the role of CD95L in the TME interaction. This enhanced TiRP model was used to verify the effect of CD95L inhibition on apoptosis of tumour specific TILs using asunercept. Results In this novel synchronized in vivo melanoma model, asunercept decreased intra-tumour apoptosis among tumour-specific T cells by 67% vs controls (9% vs 28%, p = 0.04). This validates the immune-suppressive interaction between the TME and infiltrating tumour-specific T cells in this model. Furthermore, cross-species specific asunercept prevents T cell apoptosis triggered by the tumour microenvironment, highlighting a rationale for potential application of CD95L inhibition in immuno-oncology for other solid tumours. Conclusion Selective CD95L inhibition by asunercept might have potential clinical implications in different tumours beyond recurrent glioblastoma, including cutaneous melanoma. To date, asunercept has demonstrated clinical efficacy and safety in patients with recurrent glioblastoma and myelodysplastic syndromesand may warrant further investigation in clinical trials. Legal entity responsible for the study The authors. Funding Apogenix AG. Disclosure A. Krendyukov: Full / Part-time employment: Apogenix AG. N. Kneisel: Full / Part-time employment: Apogenix AG. J. Zhu: Research grant / Funding (self), Full / Part-time employment: Ludwig Institute for Cancer Research. C. Merz: Full / Part-time employment: Apogenix AG. D. Richards: Full / Part-time employment: Apogenix AG. C. Gieffers: Full / Part-time employment: Apogenix AG. B. van den Eynde: Research grant / Funding (self), Full / Part-time employment: Ludwig Institute for Cancer Research; Advisory / Consultancy, Shareholder / Stockholder / Stock options, Officer / Board of Directors: iTeos Therapeutics.

1876PD - Immunogenicity of BRCA1-deficient ovarian cancers is driven through DNA sensing and is augmented by PARP inhibition

BackgroundBRCA1 is an essential gene of the homologous recombination repair pathway. Ovarian cancers with BRCA1 mutations represent about 20% of HGSOC and are characterized by loss-of-function TP53 mutations, copy number alterations, chromosomal instability, high neoantigen loads and increased infiltration of intraepithelial CD8 tumor-infiltrating lymphocytes. MethodsTo address this hypothesis, we investigated the effects of BRCA1 loss on tumor immunogenicity in human and mouse ovarian cancer BRCA1 isogenic cell lines. We further studied tumor immunogenicity and immune recognition in situ in human ovarian carcinomas and in vivo using syngeneic transplantable Tp53-/-BrcaWT and Tp53-/-Brca1-/- mouse models. ResultsWe propose that DNA damage induced by BRCA1 loss could be a tumor-autonomous inflammatory mechanism. Our hypothesis was corroborated by studies in human and mouse isogenic ovarian cancer cell lines which revealed that BRCA1 deficiency leads to increased cytoplasmic translocation of nuclear DNA, increased DNA sensing, induction of proinflammatory cytokines and T cell recruiting chemokines and increased tumor CD8 T cell infiltration. PARP inhibition exacerbated type I IFN responses in BRCA1-deficient ovarian cancer cell lines and simultaneously increased surface expression of PDL1. Increased DNA damage, as measured by γH2AX tumor staining, was also detected in situ in human ovarian cancers with BRCA1 mutations. Importantly, we detected tumor expression of pSTAT1 confirming a type I IFN activation in tumors with DNA damage. Both DNA damage and pSTAT1 activation correlated with higher TIL infiltration and better overall survival. Our results translated in mouse models of ovarian cancer where Trp53-/-Brca1-/- but not Trp53-/-Brca1WT tumors presented with biomarkers of DNA damage, type I IFN pathway activation and responded to a therapeutic combination of PARP inhibitor Olaparib with dual checkpoint blockade antibodies. ConclusionsOur results provide a mechanistic link between loss of BRCA1 and induction of tumor-driven inflammatory and immunogenic responses in ovarian cancer that was mediated by tumor-cell autonomous type I IFN signaling, which translated to increased immune surveillance by CD8 T cells. Legal entity responsible for the studyGeorge Coukos. FundingThis study was supported by the Ludwig Institute for Cancer Research and grants P50 CA083638 SPORE in Ovarian Cancer, the Emma Mouschamp Foundation, the Porphyrogenis Fundation and by a Stand Up To Cancer-Ovarian Cancer Research Fund Alliance-National Ovarian Cancer Coalition Dream Team Translational Cancer Research Grant (SU2C-AACRDT16- 15, to EMS). Stand Up To Cancer is a division of the Entertainment 2 Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. DisclosureAll authors have declared no conflicts of interest.

High levels of serum hyaluronan is an early predictor of dengue warning signs and perturbs vascular integrity.

BackgroundA main pathological feature of severe dengue virus infection is endothelial hyper-permeability. The dengue virus nonstructural protein 1 (NS1) has been implicated in the vascular leakage that characterizes severe dengue virus infection, however, the molecular mechanisms involved are not known.MethodsA cohort of 250 dengue patients has been followed from the onset of symptoms to the recovery phase. Serum hyaluronan levels and several other clinical parameters were recorded. The effect of NS1 treatment of cultured fibroblasts and endothelial cells on the expressions of hyaluronan synthetic and catabolic enzymes and the hyaluronan receptor CD44, were determined, as have the effects on the formation of hyaluronan-rich matrices and endothelial permeability.FindingsElevated serum hyaluronan levels (≥70 ng/ml) during early infection was found to be an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells cultured in vitro recruited CD44-expressing macrophage-like cells, suggesting a mechanism for enhancement of inflammation. In cultured endothelial cells, perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity.InterpretationPharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during moderate-to-severe dengue virus infection.FundThis work was supported in part by grants from the Swedish Cancer Society (2018/337; 2016/445), the Swedish Research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106–2314-B-037-088- and 106–2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3 T05) and Academy of Finland. The funders played no role in the design, interpretation or writing of the manuscript.

High Levels of Serum Hyaluronan Predicts Severe Dengue Infection and Perturbs Vascular Integrity

A main pathological feature of severe dengue virus infection is endothelial hyperpermeability. We demonstrate that elevated serum hyaluronan levels (≥70 ng/ml)during early infection is an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlate with high concentration of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells recruited CD44-expressing macrophages, thus perpetuating inflammation. Perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VEcadherin and cytoskeleton re-organization, and exacerbated the NS1 induced disruption of endothelial integrity. Thus, pharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during severe dengue virus infection. Funding Statement: This work was supported in part by grants from the Swedish Cancer Society (2015- 0778), the Swedish research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106- 2314-B-037-088- and 106-2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3T05) and Academy of Finland. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: The human study protocol was approved by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (IRB Number: KMUH-IRB-20110451). Informed consent was obtained from all subjects.

Metabolic reprogramming of the stromal epigenome in ovarian cancer metastasis

High grade serous ovarian cancer (HGSOC) is characterized by early metastasis throughout the peritoneal cavity and a poor clinical outcome. During the metastatic process, ovarian cancer cells interact with diverse cell populations, including mesothelial cells, adipocytes, and cancer‐associated fibroblasts (CAFs). Ovarian cancer CAFs have well‐described roles in promoting the growth and progression of ovarian cancer by secreting pro‐proliferative cytokines and oncogenic extracellular matrix components (ECM). To better understand the proteomic changes that occur during metastasis in both tumor and stromal compartments, laser‐capture microdissection was coupled to label‐free, quantitative proteomics to independently characterize the proteomes of both tumor and stromal compartments in a cohort of HGSOC patients. We identified a conserved stromal proteomic signature associated with metastasis which included high expression of nicotinamide N‐methyltransferase (NNMT) in the metastatic stroma. NNMT is a metabolic enzyme that catalyzes the methylation of nicotinamide and consumes S‐adenosyl methionine (SAM), thus depleting cellular methylation potential and reducing methylation of DNA and proteins. NNMT regulated the expression of thousands of genes and was necessary and sufficient for the reprogramming of normal fibroblasts to CAFs. Stromal NNMT expression altered multiple metabolic pathways and led to a significant reduction in cellular SAM pools. Attenuated DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) were associated with global changes in gene expression. Using genetic and small‐molecule inhibitor strategies, we found that stromal NNMT activity promoted the progression and metastasis of ovarian cancer. This study provides support to the idea that stromal methyltransferase activities can be targeted to normalize the metastatic stroma and should be further explored as treatment targets for cancer.Support or Funding InformationThis work was supported by a Marsha Rivkin Foundation award (MAE), National Cancer Institute (NCI) grants CA111882 and CA211916 (EL), the Harris Family Foundation (MAE and SDY), the Ludwig Institute for Cancer Research (EL), NIH grant CA175399 and DP2GM128199 (REM), V Foundation for Cancer Research (V2016‐020 to REM), the Körber Foundation/Körber European Science Prize (MM), the Max‐Planck Society for the Advancement of Science (MM), the Novo Nordisk Foundation (Grant agreement NNF14CC0001 and NNF15CC0001; FC and MM), and University of Chicago Cancer Center Support Grant P30CA014599.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cancer and Cytometry.

What would cancer research, diagnosis, and monitoring be without cytometry technologies? Indeed, the first commercial instrument, the Phywe ICP, was designed for quantifying DNA content in cancer cells by Wolfgang Göhde from Münster, Germany more than 50 years ago 1. Since then, the use of cytometry in clinical diagnosis and research of malignancies has diversified and developed, including now such diverse quantitative single cell analysis techniques as mass cytometry 2, image cytometry 3, or label free 4 technologies and various methods to characterize and count circulating tumor cells in the peripheral blood 5. In this issue, I have compiled a small collection of publications around cancer. The first is a multinational study on an anti-CD38 monoclonal antibody for myeloma treatment by Adams II and colleagues (this issue, pp. 279–289). This article is Editor's choice of the month. The authors use Mass cytometry and complex immunophenotyping and present evidence of immunomodulation induced by the therapy. Noteworthy, the manuscript describes in detail quality control and provides publicly available original measurements in the FlowRepository to support reproducible science. In a manuscript by Chumakova and colleagues (this issue, pp. 290–301), the authors investigate cancer stem cell dynamics by image cytometry. To this end, they developed a pipeline consisting of the full process from cell staining to single cell-derived data termed HASCIA. Among other findings, they report the influence of local cell density and SOX2 expression for maintaining stem cell-like phenotype. Finally, Wei and colleagues (this issue, pp. 302–308) demonstrate label free discrimination of three different cancer cell lines by individual light scattering patterns. Whereas conventional light scattering obtained by flow cytometry could not distinguish the different cell lines, individual light scattering patterns in combination with machine learning clearly improved classification. Thus, cytometry has an essential role in cancer therapy evaluation and detection. Consequently, we are appointing Dr. Yolanda Mahnke as a new associate editor on the board of Cytometry Part A this month. She is the first new AE since last November 6. Now, let me briefly introduce Yolanda Mahnke to you. Yolanda Mahnke has been a reviewer of Cytometry Part A articles for many years, and recently joined the journal's editorial board. Since early during her graduate work on T-cell memory, flow cytometry became a central technology in her research. In fact, flow cytometry quickly became more than just a tool, with her research bringing forth seminal publications introducing the concept of primary, secondary, and tertiary antigens for panel development 7, and the spillover-spreading matrix 8. In 2010, with her colleagues Mario Roederer and Pratip Chattopadhyay, she initiated the OMIP publication platform 9 which has become a valuable asset to the flow cytometry community. Overall, Dr. Mahnke published so far seven out of now 53 published OMIPs (OMIP-53, pp. 264–267) Dr. Mahnke is an internationally renowned flow cytometry expert with extensive experience in tumor immunology and infection immunology. Her work at the German Cancer Research Center (DKFZ), the Lausanne Branch of the Ludwig Institute for Cancer Research, NIH's Vaccine Research Center, and Prof. Carl June's CAR T-cell program at the University of Pennsylvania centered on the evolution of T-cell responses in the context of tumors, infections, vaccinations, or cellular therapies, with a particular focus on longitudinal changes in their activation and differentiation status, as well as in their functionality. The development of flow cytometry-based assays and protocols formed an integral part of her correlative studies to elucidate useful biomarkers as well as factors relevant for treatment success. Dr. Mahnke is now president of FlowKnowHow LLC, a flow cytometry consulting service providing education, as well as assistance with experimental design, panel development, troubleshooting, data analyses and interpretation, and reporting. She was an ISAC Marylou Ingram Scholar (2014–2018) and has been active in ISAC, serving on the Scientific Communications Committee, the ISAC Associated Societies Committee, as well as the Programming Committee for several CYTO conferences. As an AE, Dr. Mahnke will be responsible for the field of Cancer Immunology and OMIPs. Yolanda Mahnke, PhD