Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing ...reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these 'hotspot' sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.
Genome sequencing has revealed a large number of shared and personal somatic mutations across human cancers. In principle, any genetic alteration affecting a protein-coding region has the potential ...to generate mutated peptides that are presented by surface HLA class I proteins that might be recognized by cytotoxic T cells. To test this possibility, we implemented a streamlined approach for the prediction and validation of such neoantigens derived from individual tumors and presented by patient-specific HLA alleles. We applied our computational pipeline to 91 chronic lymphocytic leukemias (CLLs) that underwent whole-exome sequencing (WES). We predicted ∼22 mutated HLA-binding peptides per leukemia (derived from ∼16 missense mutations) and experimentally confirmed HLA binding for ∼55% of such peptides. Two CLL patients that achieved long-term remission following allogeneic hematopoietic stem cell transplantation were monitored for CD8+ T-cell responses against predicted or confirmed HLA-binding peptides. Long-lived cytotoxic T-cell responses were detected against peptides generated from personal tumor mutations in ALMS1, C6ORF89, and FNDC3B presented on tumor cells. Finally, we applied our computational pipeline to WES data (N = 2488 samples) across 13 different cancer types and estimated dozens to thousands of predicted neoantigens per individual tumor, suggesting that neoantigens are frequent in most tumors.
•Tumor neoantigens are a promising class of immunogens based on exquisite tumor specificity and the lack of central tolerance against them.•Massively parallel DNA sequencing with class I prediction enables systematic identification of tumor neoepitopes (including from CLL).
Cancer genome sequencing has enabled the rapid identification of the complete repertoire of coding sequence mutations within a patient's tumor and facilitated their use as personalized immunogens. ...Although a variety of techniques are available to assist in the selection of mutation-defined epitopes to be included within the tumor vaccine, the ability of the peptide to bind to patient MHC is a key gateway to peptide presentation. With advances in the accuracy of predictive algorithms for MHC class I binding, choosing epitopes on the basis of predicted affinity provides a rapid and unbiased approach to epitope prioritization. We show herein the retrospective application of a prediction algorithm to a large set of bona fide T cell-defined mutated human tumor antigens that induced immune responses, most of which were associated with tumor regression or long-term disease stability. The results support the application of this approach for epitope selection and reveal informative features of these naturally occurring epitopes to aid in epitope prioritization for use in tumor vaccines.
Regain of immunocompetence after myeloablation and bone marrow cell (BMC) reconstitution essentially depends on T progenitor homing into the thymus and intrathymic T cell maturation. CD44 facilitates ...progenitor homing and settlement in the bone marrow and is known as a T progenitor marker. In search for improving regain of immunocompetence after BMC reconstitution, we explored whether the CD44 standard (CD44 s) and/or variant isoforms CD44v6 and CD44v7 contribute to thymus repopulation and thymocyte maturation. Antibody‐blocking studies and cells/mice with a targeted deletion of CD44v6/7 or CD44v7 revealed that CD44s, but not CD44v6 and CD44v7, has a major impact on progenitor cell homing into the thymus. Instead, CD44v6 strengthens apoptosis resistance and expansion of early thymocytes. CD44v6‐induced apoptosis resistance, most strong in double‐negative (DN) thymocytes, is accompanied by Akt activation. CD44v6‐induced proliferation of DN cells proceeds via activation of the MAPK pathway. At later stages of T cell maturation, CD44 acts as an accessory molecule, initiating and supporting TCR/CD3 complex‐mediated signal transduction in double‐positive and single‐positive thymocytes. Thus, CD44 plays a major role in thymus homing. In addition, CD44v6 is important for survival and expansion of early thymocytes. These findings suggest that strengthening CD44v6 expression on lymphoid progenitors could well contribute to accelerated regain of immunocompetence.
The recent successes of cancer immunotherapies have stimulated interest in the potential widespread application of these approaches; haematological malignancies have provided both initial proofs of ...concept and an informative testing ground for various immune-based therapeutics. The immune-cell origin of many of the blood malignancies provides a unique opportunity both to understand the mechanisms of cancer immune responsiveness and immune evasion, and to exploit these mechanisms for therapeutic purposes.
Analyzing the growth of fibrosarcoma lines derived from IL-1α–, IL-1β–, or IL-1αβ–knockout (−/−) mice in the immunocompetent host revealed that tumor-derived IL-1α and IL-1β exert strong and opposing ...effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1–deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1–deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1α−/−β–competent (comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1βcomp lines, IL-1α strengthens anchorage-independent growth, but both IL-1α and IL-1β support drug resistance. Corresponding to the aggressive growth, IL-1βcomp cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated αv/β3 and matrix metalloproteinase expression. Recruitment of myeloid cells requires IL-1β but is regulated by IL-1α, because inflammatory chemokine and cytokine expression is stronger in IL-1α−/−βcomp than in IL-1wt lines. This regulatory effect of tumorderived IL-1α is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1β. Both sarcoma cell–derived IL-1α and IL-1β promote tumor growth. However, IL-1α exerts regulatory activity on the tumor cell–matrix cross-talk, and only IL-1β initiates systemic inflammation.
In an exploratory analysis, we investigated the association between programmed death ligand 1 (PD-L1), tumor mutational burden (TMB), T-cell-inflamed gene expression profile (TcellinfGEP), and ...stromal signature with outcomes of pembrolizumab in urothelial carcinoma (UC).
Patients with advanced UC received first-line pembrolizumab 200 mg every 3 weeks in the single-arm phase II KEYNOTE-052 trial (NCT02335424) and salvage pembrolizumab 200 mg every 3 weeks or chemotherapy (paclitaxel/docetaxel/vinflunine) in the randomized phase III KEYNOTE-045 trial (NCT02256436). The association of each biomarker (continuous variable) with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) was evaluated using logistic regression (ORR) and Cox PH (PFS, OS), adjusted for ECOG PS; nominal P values were calculated without multiplicity adjustment (one-sided, pembrolizumab; two-sided, chemotherapy). Significance was prespecified at α = 0.05.
In KEYNOTE-052, PD-L1, TMB, and TcellinfGEP were significantly associated with improved outcomes; stromal signature was significantly associated with worse outcomes. In KEYNOTE-045, although findings for TMB and TcellinfGEP with pembrolizumab were consistent with those of KEYNOTE-052, PD-L1 was not significantly associated with improved outcomes, nor was stromal signature associated with worse outcomes with pembrolizumab; chemotherapy was not associated with outcomes in a consistent manner for any of the biomarkers. Hazard ratio (HR) estimates at prespecified cutoffs showed an advantage for pembrolizumab versus chemotherapy regardless of PD-L1 or TMB, with a trend toward lower HRs in the combined positive score ≥10 and the TMB ≥175 mutation/exome subgroup. For TcellinfGEP, PFS and OS HRs were lower in the TcellinfGEP-nonlow subgroup regardless of treatment.
Multiple biomarkers characterizing the tumor microenvironment may help predict response to pembrolizumab monotherapy in UC, and potential clinical utility of these biomarkers may be context-dependent.
The recent successes of cancer immunotherapy have stimulated interest for the potential widespread application of these approaches; hematologic malignancies have provided both initial ...proofs-of-concept and an informative testing ground for a variety of immune-based therapeutics. The immune-cell origin of many of the blood malignancies provides a unique opportunity to both understand the mechanisms of human immune-responsiveness and immune-evasion as well as to exploit the unique therapeutic opportunities they provide.
Tumor neoantigens are a promising class of vaccine immunogens as they arise from gene alterations in tumor cells and are hence exquisitely tumor-specific. We recently reported the development of a ...pipeline that leverages massively parallel sequencing data with HLA-peptide binding predictions to identify candidate neoantigens. By applying this pipeline to cases of chronic lymphocytic leukemia (CLL) with known HLA typing, we described the prediction of personal tumor neoantigens against which long-lived memory T cell responses developed following remission-inducing therapy. Our pipeline thus provides a method for selecting neoantigens for developing future personalized tumor vaccines. In order to extend this approach beyond CLL, we sought to gain estimates of tumor neoantigen loads across cancers. We hypothesized that the numbers of neoantigens within cancers would be proportional to their mutation frequency.
To examine this hypothesis, we turned to the extensive collections of whole-exome sequencing (WES) data that have been generated through recent large-scale cancer sequencing projects. In order to generate accurate estimates of personal tumor neoantigen loads, HLA typing information is required. While in theory this information should be directly extractable from WES, direct inference of HLA type from standard WES reads has not been previously possible due to suboptimal alignments against a standard reference genome arising from the highly polymorphic nature of the HLA region. We therefore developed a strategy to optimize alignment. Based on the IMGT database, we constructed a reference library of all known HLA alleles (6597 unique entries) and aligned WES reads containing one or more short sequence segments corresponding to any HLA allele against this reference using the Novoalign software. HLA alleles were then inferred through a model that enabled calculation of allele probabilities by taking into account the number and quality of reads aligned to each allele. Alleles with the highest probabilities were then identified as winners. We trained the algorithm on 8 CLL cases for which WES data and HLA typing (based on conventional molecular typing) were available, and established a performance accuracy of ∼94% (45 of 48 alleles). This was further validated using a set of 133 Hap Map samples with known HLA typing, in which 94.61% (755 of 798) alleles were identified correctly at protein level resolution.
We applied the HLA typing algorithm together with the neoantigen discovery pipeline across WES from 2488 cases collected from publicly available datasets of 13 diverse cancers. Mutation rates in solid tumor malignancies were consistently higher, in some cases by more than an order of magnitude, than the blood malignancies. For example, the high mutation rate tumor melanoma displayed a median of 300 (range, 34-4276) missense mutations per case, while renal cell carcinoma (RCC) had 41 (range, 10-101) and CLL had 16 (range, 0-75). The number of frame-shifting events (indels and termination read-throughs) was generally 10-fold or more lower in each tumor type than missense mutations and did not correlate with the number of missense mutations. As expected, the rate of predicted HLA binding peptides mirrored the somatic mutation rate per tumor type. The median number of predicted class I HLA-binding neopeptides (with IC50 < 500 nM) per sample generated from missense and frameshift events for melanoma was 488 (range: 18-5811), for RCC, 80 (range: 6-407), and for CLL 24 (range 2-124). Overall, we found an average of 1.5 HLA-binding peptides (i.e. with IC50<500nM) was generated per missense mutation and 4 binding peptides per frameshift mutation.
By predicting tumor neoantigens in a variety of low and high mutation rate cancers, we established that dozens to hundreds of potential neoantigens are present in most tumors. In the process, we developed a highly accurate analytic approach that provides a solution for extracting HLA typing information from WES data but which could, in principle, be applied to other highly polymorphic regions of the genome. Ongoing studies focus on integrating estimates of tumor neoantigen load with understanding of HLA expression in order to optimize selection of antigen targets to build future personalized tumor vaccines.
No relevant conflicts of interest to declare.
A blockade of CD44 can interfere with haematopoietic and leukemic stem cell homing, the latter being considered as a therapeutic option in haematological malignancies. We here aimed to explore the ...molecular mechanism underlying the therapeutic efficacy of anti‐CD44. We noted that in irradiated mice reconstituted with a bone marrow cell transplant, anti‐CD44 exerts a stronger effect on haematopoietic reconstitution than on T lymphoma (EL4) growth. Nonetheless, in the non‐reconstituted mouse anti‐CD44 suffices for a prolonged survival of EL4‐bearing mice, where anti‐CD44‐prohibited homing actively drives EL4 cells into apoptosis. In vitro, a CD44 occupancy results in a 2–4‐fold increase in apoptotic EL4 cells. Death receptor expression (CD95, TRAIL, TNFRI) remains unaltered and CD95 cross‐linking‐mediated apoptosis is not affected. Instead, CD44 ligation promotes mitochondrial depolarization that is accompanied by caspase‐9 cleavage and is inhibited in the presence of a caspase‐9 inhibitor. Apoptosis becomes initiated by activation of CD44‐associated phosphatase 2A (PP2A) and proceeds via ERK1/2 dephosphorylation without ERK1/2 degradation. Accordingly, CD44‐induced apoptosis could be mimicked by ERK1/2 inhibition, that also promotes EL4 cell apoptosis through the mitochondrial pathway. Thus, during haematopoietic stem cell reconstitution care should be taken not to interfere by a blockade of CD44 with haematopoiesis, which could be circumvented by selectively targeting leukemic CD44 isoforms. Beyond homing/settlement in the bone marrow niche, anti‐CD44 drives leukemic T cells into apoptosis via the mitochondrial death pathway by CD44 associating with PP2A. Uncovering this new pathway of CD44‐induced leukemic cell death provides new options of therapeutic interference.
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