Identification of predicted neoantigen vaccine candidates in follicular lymphoma patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 8054-8054
Author(s):  
Cody Ramirez ◽  
Felix Frenkel ◽  
Olga Plotnikova ◽  
Vladislav Belousov ◽  
Alexander Bagaev ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Pilot Trial ◽  
In Silico Analysis ◽  
Clinical Results ◽  
Immune Checkpoint Blockade ◽  
Missense Mutations ◽  
Rna Seq ◽  
High Quality ◽  
Vaccine Candidates

8054 Background: Follicular lymphoma (FL) is incurable with conventional therapies and poorly responsive to immune checkpoint blockade. There is a need for new therapies without long-term complications of chemotherapy and with curative potential. We hypothesize that FL contains tumor-specific mutant antigens (TSMAs) that can be targeted by the immune system by vaccination. Recent reports have highlighted the potential for unique immunoglobulin peptides to elicit immune response in lymphomas. We utilized whole exome sequencing (WES) and RNA sequencing (RNA-Seq) of FL patient samples to infer HLA genotype, and predict TSMAs with the goal of designing a personalized cancer vaccine, supported by recent reports of this approach in solid cancers. Methods: DNA and RNA from 58 patients’ FL biopsies underwent WES and RNA-Seq. pVACtools and MiXCR predicted potential somatic and B-cell clonotype neoantigens, which were filtered to identify high quality TSMAs. B-cell oligoclonality was determined by comparison to B-cell receptor (BCR) repertoire profiling of healthy individual lymph nodes. RNA-seq data allowed us to identify expressed TSMAs. Complementary in silico analysis based on mRNA-based peptide reconstruction and custom HLA affinity binding predictions were performed. Results: An average of 52 somatic mutations per patient (range: 2-172) were identified. At least one high quality TSMA was predicted for 57 of 58 patients. Five or more TSMA candidates were identified for 52 (90%) patients with a mean of 17 predicted peptides per patient (range: 0-45). 81% (813/1,004) of the total predicted TSMA peptides arose from missense mutations, 9% (94/1,004) from indels, and 10% (97/1,004) from BCR. 78% (45/58) of patients have both somatic and BCR vaccine candidates, while 21% (12/58) of patients had only somatic vaccine candidates. Predicted TSMAs were identified in multiple genes recurrently mutated in lymphoma (e.g., BCL2). There was a high prediction concordance with the orthogonal BostonGene Vaccine Module V1 pipeline. These pre-clinical results led to a first-in-human pilot trial of personalized TSMA vaccine combined with anti-PD-1 mAb for rel/ref FL patients (NCT03121677), with one response observed within 4 patients evaluable for response to date. Conclusions: TSMA peptides suitable for cancer vaccines were identified for most FL patients via next-generation sequencing, MiXCR and pVACtools. This pre-clinical study suggests that FL patients will be candidates for TSMA vaccine clinical trials and pilot clinical results provide proof of concept for this approach.

scholarly journals Neoantigen Landscape Supports Feasibility of Personalized Cancer Vaccine for Follicular Lymphoma

2022 ◽  
Author(s):  
Cody A Ramirez ◽  
Felix Frenkel ◽  
Michelle Becker ◽  
Erica K Barnell ◽  
Ethan D McClain ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cancer Vaccine ◽  
Cell Receptor ◽  
Missense Mutations ◽  
Rna Seq ◽  
Vaccine Strategy ◽  
High Quality ◽  
Treatment Paradigm ◽  
Personalized Cancer

Personalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that polyvalent vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole exome sequencing (WES) and RNA sequencing (RNA-Seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-Seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by alignment of B-cell receptor (BCR) CDR3 regions from RNA-Seq data, grouping at the protein level, and comparison to the BCR repertoire of RNA-Seq data from healthy individuals. An average of 52 somatic mutations per patient (range: 2-172) were identified, and two or more (median: 15) high-quality neoantigens were predicted for 56 of 58 samples. The predicted neoantigen peptides were composed of missense mutations (76%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide (SLP) vaccines were successfully synthesized for and administered to all four patients enrolled to date. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field.

scholarly journals The immune landscape and response to immune checkpoint blockade therapy in lymphoma

Blood ◽  
2020 ◽  
Vol 135 (8) ◽  
pp. 523-533 ◽  
Author(s):  
Justin Kline ◽  
James Godfrey ◽  
Stephen M. Ansell
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Immune Checkpoint ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Classical Hodgkin Lymphoma ◽  
Large B Cell

Abstract The clinical development of effective cancer immunotherapies, along with advances in genomic analysis, has led to the identification of tumor environmental features that predict for sensitivity to immune checkpoint blockade therapy (CBT). Early-phase clinical trial results have demonstrated the remarkable effectiveness of CBT in specific lymphoma subtypes, including classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. Conversely, CBT has been relatively disappointing in follicular lymphoma and diffuse large B-cell lymphoma. These clinical observations, coupled with important scientific discoveries, have uncovered salient features of the lymphoma microenvironment that correlate with immunotherapy response in patients. For example, classical Hodgkin lymphoma is characterized by an inflammatory environment, genetic alterations that facilitate escape from immune attack, and sensitivity to PD-1 blockade therapy. On the other hand, for lymphomas in which measures of immune surveillance are lacking, including follicular lymphoma and most diffuse large B-cell lymphomas, anti-PD-1 therapy has been less effective. An improved understanding of the immune landscapes of these lymphomas is needed to define subsets that might benefit from CBT. In this article, we describe the immune environments associated with major B-cell lymphomas with an emphasis on the immune escape pathways orchestrated by these diseases. We also discuss how oncogenic alterations in lymphoma cells may affect the cellular composition of the immune environment and ultimately, vulnerability to CBT. Finally, we highlight key areas for future investigation, including the need for the development of biomarkers that predict for sensitivity to CBT in lymphoma patients.

scholarly journals Systematic Evaluation of Somatic Cis-Regulatory Mutations in Follicular Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-27
Author(s):  
Firat Uyulur ◽  
Findlay Bewicke-Copley ◽  
Chinedu Anthony Anene ◽  
Matthias Schlesner ◽  
Icgc MMML-Seq Project ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Mutation Rate ◽  
Research Funding ◽  
Whole Genome ◽  
Rna Seq ◽  
Regulatory Mutations ◽  
Flanking Sequences ◽  
Bona Fide ◽  
Flanking Regions

Background: Follicular lymphoma (FL) is an incurable indolent B cell malignancy characterized in the majority of cases by the t(14;18) translocation. While the mutational landscape of the coding genome is nearing completion, less is known about the characteristics of its noncoding genome. Our expectation is that the distribution of noncoding mutations will be non-random, determined by epigenomics features, such as chromatin modification and accessibility. Our strategy is therefore to integrate whole genome epigenomic marks and mutations in order to enrich for variants with regulatory potential and identify unique mutational processes specific to thesecis-regulatory elements (CREs).Allele-specific expression (ASE) patterns allow further refinement to resolve functional CREs and bona fide mutations associated with changes in gene expression. Methods: The H3K27Ac consensus CREs were determined using the ChIP-Seq data of 9 FL patient primary cells (Koues et al., Immunity2015). DNase I-hypersensitive sites (DHSs) and Hi-C of the B-lymphocyte cell line GM12878 was downloaded from ENCODE. Whole genome sequencing and RNA-Seq of 70 FL patients with relative high tumour cellularity (≥30% for DNA tumour purity and ≥25% for RNA B-cell content) were obtained from the International Cancer Genome Consortium project ICGC MMML-Seq. The mutation rate within CREs and DHSs in comparison to their flanking sequences was estimated using a previous pipeline (Sabarinathan et al., Nature2016). We further developed cis-ASE, an integrated analytic pipeline for the identification of recurrent ASE genes, and significantly associated CREs and mutations (Fig1.A). Results: In total 1.04 million noncoding mutations, corresponding to 14.8K mutations per sample with a median of 9,991 noncoding mutations were identified in our series of 70 FL samples. 62K (6.0%) mutations were located within H3K27Ac bound CREs and there was an elevated mutation rate in H3K27Ac CREs compared to the corresponding flanking regions of 1kb up and downstream (χ² test p = 1.95e-19, Fig1.B). For DHSs, we observed the opposite pattern, with a lower mutation rate in DHSs than in the flanking regions (p = 0.04, Fig1.C), most likely reflecting the higher accessibility to global genome repair machinery in DHSs in relation to flanking sequences. Dividing mutations into high (≥0.3) and low (<0.3) adjusted variant allele frequency (VAF) groups (accounting for tumour purity), we observed significantly higher mutation rate in CREs than in the flanking sequences, that was specific for mutations with higher VAFs (p= 6.95e−38), as the difference was much weaker for low VAF mutations (p= 0.02). Mutation signatures 6 and 20, linked to defective DNA mismatch repair, were highly enriched for mutations within H3K27Ac CREs (44%) versus other regions outside (27%) (p< 0.001). ASE was assessed using 45.6k informative SNPs (1.7%) per sample wherein matched genotype data was available from WGS and RNA-Seq profiles. Cis-ASE identified on average 480 ASE genes per sample (binomial test, adjusted p<0.05), corresponding to 1,943 recurrent ASE genes with a minimum threshold of ≥5 samples. These ASE events were not significantly influenced by local copy number changes or promoter methylation. KEGG pathway analysis of recurrent ASE genes identified adherens junction, B cell receptor signaling pathway and Fc gamma R-mediated phagocytosis as the most overrepresented pathways. Cis-ASE further identified 18 ASE-CRE interactions where CRE mutations were significantly correlated with an imbalance in elevated alternative allele ratios. These ASE genes included recognized lymphoma related genes including BCL2, STAT6, MAF, and additional novel targets. The pattern of aberrant somatic hypermutation (aSHM) was assessed for these 18 CREs, and we narrowed down to 15 significant ASE-CRE interactions not strongly affected by aSHM, consisting of 92 bona fide mutation candidates present in 37 FL samples of our cohort (52.9%). Conclusion: Our study identified unique mutation processes operating in H3K27Ac CREs. Using an integrated genomic approach of whole genome mutations, chromatin marks and RNA-Seq, we explored ASE-CRE interactions, and identified 15 H3K27Ac bound CREs enriched for cis-regulatory mutations significantly associated with ASE and total expression of targeted genes, deserving for further exploration to enrich our understanding of FL noncoding genome. Disclosures Okosun: BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Research Funding.

scholarly journals Pipeline for Discovering Neoepitopes Generated By Alternative Splicing in B-ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1342-1342
Author(s):  
Sisi Zheng ◽  
Ammar S. Naqvi ◽  
Elisabeth Bolton-Gillespie ◽  
Mukta Asnani ◽  
Katharina Hayer ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Full Length ◽  
Missense Mutations ◽  
Rna Seq ◽  
New Paradigm ◽  
Junction Site ◽  
Therapeutic Development ◽  
Normal Controls

Pediatric B-cell acute lymphoblastic leukemias (B-ALL) are striking for their low mutational burdens. Ostensibly, this paucity of mutation-associated neoantigens could limit the development of novel immunotherapies. However, our lab has previously shown that globally aberrant splicing is a hallmark of B-ALL, as compared to normal pro-B cells. Here we hypothesize that alternative splicing represents a new source of targetable neo-epitopes, distinct from missense mutations. To test this hypothesis, we constructed a pipeline for neo-epitope discovery, validation, and therapeutic development. First, we obtained a very large RNA-seq dataset covering over 400 pediatric B-ALL samples from St. Jude Children's Research Hospital. For normal controls, RNA-seq was performed on 4 pro-B cell fractions from bone marrow donors. We then applied a bioinformatic splicing analysis, MAJIQ 2.0, to deeply mine the dataset for local splicing variants (LSVs) unique to B-ALL. To bypass MHC presentation barriers, we focused on LSVs mapping to exons encoding extracellular protein domains (ectoLSVs). This filtering yielded a list of 914 ectoLSVs in 430 genes. These ectoLSVs were further filtered for those that preserved opening reading frames, occurred with high prevalence, and demonstrated a large differential expression between B-ALL and normal controls. One such prominent event was the increased skipping in B-ALL of CD22 of exon 5-6 (Δex5-6). We validated this event at the RNA level with RT-PCR in 18 out of 18 primary B-ALL samples from CHOP Biobank. Using Nanopore-based long read RNA sequencing, we confirmed the Δex5-6 event exists within a larger translatable transcript. BALB/c mice were then immunized against a peptide containing the junction site of exon 4 and 7 to generate hybridomas; several monoclonal antibody (mAb) against CD22 Δex5-6 were successfully obtained. To validate the specificity of our mAbs, we used CRISPR to delete endogenous CD22 in OCI-Ly10 cells and reconstituted the cells with either CD22 Δex5-6 or full length CD22. The mAb demonstrated remarkable specificity for CD22 Δex5-6 and did not bind to the full-length isoform. We also demonstrated that at least one of these mAbs (clone 3A3) specifically binds to endogenously expressed CD22 Δex5-6 in multiple B-ALL cell lines by immunoprecipitation and Western blot analysis. Our next steps are to develop 3A3-based antibody-drug conjugates and chimeric antigen receptors for further therapeutic testing. In summary, our results validate that 1) our current discovery pipeline is able to identify targetable splicing-derived neo-epitopes, and that 2) antibodies with impressive specificity can be generated against such neo-epitopes. Furthermore, this new paradigm has the promise of increasing the repertoire of highly specific immunotherapy targets in B-ALL, despite its low mutation burden. Of note, this strategy could also be carried forward into therapeutic development for many other cancers beyond B-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Massively Parallel Single Cell RNA-Seq of Primary Lymphomas Reveals Distinct Cellular Lineages and Diverse, Intratumoral Transcriptional States

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1090-1090
Author(s):  
Noemi Andor ◽  
Erin Simonds ◽  
Jiamin Chen ◽  
Susan Grimes ◽  
Christina Wood ◽  
...  
Keyword(s):  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
Mhc Class Ii ◽  
Research Funding ◽  
Immune Cell ◽  
Single Cells ◽  
Transcriptional Networks ◽  
Rna Seq

Abstract Follicular lymphoma (FL) is a generally incurable B-cell malignancy which has the potential to transform into highly aggressive lymphomas. Genomic studies indicate it is often a small subpopulation rather than the dominant population in the FL that gives rise to the more aggressive subtype.To resolve the underlying transcriptional networks of follicular B-cell lymphomas at single molecule and cell resolution, we leveraged droplet-based barcoding technology for highly parallel single cell RNA-Seq. We analyzed the transcriptomes from tens of thousands of cells derived from five primary FL tumors (average > 5,000 cells/sample). Simultaneously, we conducted multi-dimensional flow cell sorting to validate our characterizing of cellular lineages and critical expressed proteins. For each tumor, we identified multiple cellular subpopulations, matching known hematopoietic lineages. Despite some common features, such as MYC and BCLoverexpression, distinct transcriptional patterns and regulatory programs were evident among the different tumors. Within each tumor, subpopulations of malignant cell transcripts were compared to matched normal B-cells (Figure 1). Transcriptome analysis of >13,500 B-cells across 5 samples reveals sample-specific and joint clusters of B-cells. Each dot represents the t-SNE projection of a single cell's transcriptome profile. Single cells are colored according to sample origin (control PBMC samples 1 & 2; follicular lymphoma samples 1, 2 & 3). Normal B-cells within follicular lymphoma samples cluster together with PBMC derived B-cells. Malignant B-cells cluster according to sample origin, except for the cycling tumor cells (upper left cell cluster). Malignant B-cells were characterized by expression of restricted immunoglobulin light chain type (either kappa or lambda), BCL2 expression, and CD20 expression. We show evidence for the coexistence of small, malignant subpopulations alongside the dominant FL population in the majority of tumors. These smaller subpopulations harbored several transcriptional changes that were absent from the dominant population, including upregulation of MHC class II expression, downregulation of β-2-microglobulin, and exclusive expression of chemokines (CCL22 and CCL17), that would have resulted in alteration in antigen presentation and of the T cell immune system. In addition, we characterize the transcriptomes of the infiltrating immune cell populations that are characteristic of this disease. Our findings provide an unprecedented resolution of distinct immune lineages as seen by transcriptionally characterized cellular diversity. Figure 1 Assignment of 13.5K B-cells to normal and malignant phenotypes. Figure 1. Assignment of 13.5K B-cells to normal and malignant phenotypes. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

scholarly journals Immune checkpoint blockade impairs immunosuppressive mechanisms of regulatory T cells in B-cell lymphoma

2021 ◽  
Vol 14 (9) ◽  
pp. 101170
Author(s):  
Vera Bauer ◽  
Fatima Ahmetlić ◽  
Nadine Hömberg ◽  
Albert Geishauser ◽  
Martin Röcken ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
B Cell ◽  
Immune Checkpoint ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade

scholarly journals Clinicopathologic features of TDO2 overexpression in renal cell carcinoma

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Quoc Thang Pham ◽  
Daiki Taniyama ◽  
Yohei Sekino ◽  
Shintaro Akabane ◽  
Takashi Babasaki ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
In Silico Analysis ◽  
Immunohistochemical Analysis ◽  
Rna Seq ◽  
Anoikis Resistance ◽  
Silico Analysis ◽  
Proliferation And Invasion

Abstract Background Tryptophan 2,3-dioxygenase (TDO2) is the primary enzyme catabolizing tryptophan. Several lines of evidence revealed that overexpression of TDO2 is involved in anoikis resistance, spheroid formation, proliferation, and invasion and correlates with poor prognosis in some cancers. The aim of this research was to uncover the expression and biofunction of TDO2 in renal cell carcinoma (RCC). Methods To show the expression of TDO2 in RCC, we performed qRT-PCR and immunohistochemistry in integration with TCGA data analysis. The interaction of TDO2 with PD-L1, CD44, PTEN, and TDO2 expression was evaluated. We explored proliferation, colony formation, and invasion in RCC cells line affected by knockdown of TDO2. Results RNA-Seq and immunohistochemical analysis showed that TDO2 expression was upregulated in RCC tissues and was associated with advanced disease and poor survival of RCC patients. Furthermore, TDO2 was co-expressed with PD-L1 and CD44. In silico analysis and in vitro knockout of PTEN in RCC cell lines revealed the ability of PTEN to regulate the expression of TDO2. Knockdown of TDO2 suppressed the proliferation and invasion of RCC cells. Conclusion Our results suggest that TDO2 might have an important role in disease progression and could be a promising marker for targeted therapy in RCC. (199 words)

scholarly journals Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia

2021 ◽  
Vol 22 (5) ◽  
pp. 2683
Author(s):  
Princess D. Rodriguez ◽  
Hana Paculova ◽  
Sophie Kogut ◽  
Jessica Heath ◽  
Hilde Schjerven ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Technological Developments ◽  
Cell Acute Lymphoblastic Leukemia

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

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