View papers showing how the growing community of cancer researchers are breaking new ground in this field. Below are links to recently published peer-reviewed articles and their abstracts, as well as Illumina commentary briefly highlighting how our products enabled the research.
Villani AC, Satija R, Reynolds G, et al.
Science
356: eaah4573
2017
Abstract
Dendritic cells (DCs) and monocytes play a central role in pathogen sensing, phagocytosis, and antigen presentation and consist of multiple specialized subtypes. However, their identities and interrelationships are not fully understood. Using unbiased single-cell RNA sequencing (RNA-seq) of ~2400 cells, we identified six human DCs and four monocyte subtypes in human blood. Our study reveals a new DC subset that shares properties with plasmacytoid DCs (pDCs) but potently activates T cells, thus redefining pDCs; a new subdivision within the CD1C+ subset of DCs; the relationship between blastic plasmacytoid DC neoplasia cells and healthy DCs; and circulating progenitor of conventional DCs (cDCs). Our revised taxonomy will enable more accurate functional and developmental analyses as well as immune monitoring in health and disease.
Illumina Commentary
Dendritic cells (DCs) and monocytes are immune cells composed of multiple specialized sub-types. Given their essential role in pathogen sensing and antigen presentation, fully characterizing their identities and interrelationships is critically important. Historically, they have been defined by morphological features, biological features, and restricted sets of surface markers. This approach has led to a biased and incomplete understanding of DC sub-types. In this study, the authors used the HiSeq 2500 System to perform singe-cell sequencing of 2400 individual DCs from blood donors. Their data reveal six human DC sub-types and four monocyte subtypes in human blood. Further, the data reveal a new DC subset that potently activates T cells. The revised taxonomy enabled by this unbiased NGS approach should enable more accurate biological analyses and immune monitoring.
Le DT, Durham JN, Smith KN, et al.
Science
357: 409-413
2017
Abstract
The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers' tissue of origin.
Illumina Commentary
Mismatch-repair (MMR) deficient tumors contain many mutation-associated neoantigens (MANAs). The MANAs might be recognized by the host immune system, suggesting the possibility that MMR deficient cancers may be particularly responsive to PD-1 blockade immunotherapy. In this study, the investigators initiated a phase II clinical trial to test the efficacy of PD-1 blockade therapy in different subtypes of MMR deficient cancers. To identify somatic mutations, the authors performed whole-genome sequencing of tumor/normal tissues using the HiSeq 2000/2500 and MiSeq Systems. The authors found that more than half of patients responded well to PD-1 blockade therapy. Furthermore, the authors used additional genomic approaches to identify neoantigen-specific T cell clones that specifically reacted to mutant neopeptides found in these tumors. These data suggest that MMR deficient cancers, regardless of tissue of origin, may be sensitive to immune checkpoint blockade therapy.
Davoli T, Uno H, Wooten EC and Elledge SJ
Science
355: eaaf8399
2017
Abstract
Immunotherapies based on immune checkpoint blockade are highly effective in a subset of patients. An ongoing challenge is the identification of biomarkers that predict which patients will benefit from these therapies. Aneuploidy, also known as somatic copy number alterations (SCNAs), is widespread in cancer and is posited to drive tumorigenesis. Analyzing 12 human cancer types, we find that, for most, highly aneuploid tumors show reduced expression of markers of cytotoxic infiltrating immune cells, especially CD8+ T cells, and increased expression of cell proliferation markers. Different types of SCNAs predict the proliferation and immune signatures, implying distinct underlying mechanisms. Using published data from two clinical trials of immune checkpoint blockade therapy for metastatic melanoma, we found that tumor aneuploidy inversely correlates with patient survival. Together with other tumor characteristics such as tumor mutational load, aneuploidy may thus help identify patients most likely to respond to immunotherapy.
Illumina Commentary
Somatic copy number alterations (SCNAs) are abundant in cancers and have been proposed to drive cancer formation. Immune evasion is a hallmark of cancer, but it is not known to what extent SCNAs may play a role in this process. The authors analyzed approximately 5000 tumor/normal samples form The Cancer Genome Atlas (TCGA) and assigned a SCNA score to each tumor. They found that SCNA levels positively correlated with the total number of mutations for most tumors. Further, tumors with high levels of SCNAs had enhanced expressions of cell proliferation markers and reduced levels of expression of immune cell infiltrate markers. Finally, the authors found that high SCNA levels correlated with poor survival of melanoma patients. The data suggest that cell proliferation and immune evasion in cancer can be predicted by SCNAs, and that SCNAs might be a useful biomarker for predicting response to immunotherapies.
Shin DS, Zaretsky JM, Escuin-Ordinas H, et al.
Cancer Discov
7: 188-201
2017
Abstract
Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti-PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair-deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti-PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy.
SIGNIFICANCE: A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188-201. ©2016 AACR. See related commentary by Marabelle et al., p. 128 This article is highlighted in the In This Issue feature, p. 115. ©2016 American Association for Cancer Research.
Illumina Commentary
PD-1 blockade immunotherapy can lead to a durable anti-tumor response for several metastatic cancers. These cancer immunotherapies inhibit the binding of PD-1 to its ligand, PD-L1, leading to reactivation of anti-tumor T cells. This T cell anti-tumor activity is mediated by the activation of interferon gamma receptors, which is mediated by the activation of Janus kinases JAK1/2. Further, acquired resistance to PD-1 blockade is associated with loss-of-function mutations in JAK1/2. In this study, the authors used the HiSeq 2000 System to perform whole-exome sequencing of melanoma and colon cancer tumors and cell lines. They found that JAK1/2 mutations were present in tumors which were not responsive to PD-1 blockade, and that JAK1/2 mutations in melanoma cell lines were associated with an inability to respond to interferon gamma. Finally, they analyzed data from The Cancer Genome Atlas (TCGA) and found JAK1/2 loss-of-function mutations were associated with poor outcomes in patients. Their data suggest that tumors harboring JAK1/2 loss-of-function mutations would be unlikely to respond to PD-1 blockade therapy.
Balar AV, Galsky MD, Rosenberg JE, et al.
The Lancet
389: 67-76
2017
Abstract
BACKGROUND: First-line chemotherapy for patients with cisplatin-ineligible locally advanced or metastatic urothelial carcinoma is associated with short response duration, poor survival, and high toxicity. This study assessed atezolizumab (anti-programmed death-ligand 1 [PD-L1]) as treatment for metastatic urothelial cancer in cisplatin-ineligible patients.
METHODS: For this single-arm, multicentre, phase 2 study, in 47 academic medical centres and community oncology practices in seven countries in North America and Europe, we recruited previously untreated patients with locally advanced or metastatic urothelial cancer who were cisplatin ineligible. Patients were given 1200 mg intravenous atezolizumab every 21 days until progression. The primary endpoint was independently confirmed objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 (central review), assessed in prespecified subgroups based on PD-L1 expression and in all patients. All participants who received one or more doses of atezolizumab were included in the primary and safety analyses. This study was registered with ClinicalTrials.gov, number NCT02108652.
FINDINGS: Between June 9, 2014, and March 30, 2015, we enrolled 123 patients, of whom 119 received one or more doses of atezolizumab. At 17·2 months' median follow-up, the objective response rate was 23% (95% CI 16 to 31), the complete response rate was 9% (n=11), and 19 of 27 responses were ongoing. Median response duration was not reached. Responses occurred across all PD-L1 and poor prognostic factor subgroups. Median progression-free survival was 2·7 months (2·1 to 4·2). Median overall survival was 15·9 months (10·4 to not estimable). Tumour mutation load was associated with response. Treatment-related adverse events that occurred in 10% or more of patients were fatigue (36 [30%] patients), diarrhoea (14 [12%] patients), and pruritus (13 [11%] patients). One treatment-related death (sepsis) occurred. Nine (8%) patients had an adverse event leading to treatment discontinuation. Immune-mediated events occurred in 14 (12%) patients.
INTERPRETATION: Atezolizumab showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use in untreated metastatic urothelial cancer.
FUNDING: F Hoffmann-La Roche, Genentech.
Illumina Commentary
Cisplatin-based chemotherapy is currently the standard first-line treatment for urothelial cancer and improves overall survival. However, only a minority of patients receive cisplatin due to renal dysfunction or other comorbidities. The authors describe the results of a phase 2 clinical trial that assessed the efficacy of atezolizumab (a PD-L1 blocking antibody) in urothelial carcinoma patients ineligible for cisplatin-based chemotherapy. They found that atezolizumab treatment was well-tolerated in patients with renal insufficiency and significantly improved clinically-defined objective response rates. They also found that that tumors with high mutational load positively correlated with atezolizumab response and overall survival. This study demonstrates the potential of atezolizumab as a first-line treatment option for urothelial cancer patient ineligible for cisplatin-based therapy. The study also highlights the importance of mutational load in urothelial carcinoma in cancer immunotherapy outcomes.
Rosenberg JE, Hoffman-Censits J, Powles T, et al.
The Lancet
387: 1909-1920
2016
Abstract
BACKGROUND: Patients with metastatic urothelial carcinoma have few treatment options after failure of platinum-based chemotherapy. In this trial, we assessed treatment with atezolizumab, an engineered humanised immunoglobulin G1 monoclonal antibody that binds selectively to programmed death ligand 1 (PD-L1), in this patient population.
METHODS: For this multicentre, single-arm, two-cohort, phase 2 trial, patients (aged ≥18 years) with inoperable locally advanced or metastatic urothelial carcinoma whose disease had progressed after previous platinum-based chemotherapy were enrolled from 70 major academic medical centres and community oncology practices in Europe and North America. Key inclusion criteria for enrolment were Eastern Cooperative Oncology Group performance status of 0 or 1, measurable disease defined by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1), adequate haematological and end-organ function, and no autoimmune disease or active infections. Formalin-fixed paraffin-embedded tumour specimens with sufficient viable tumour content were needed from all patients before enrolment. Patients received treatment with intravenous atezolizumab (1200 mg, given every 3 weeks). PD-L1 expression on tumour-infiltrating immune cells (ICs) was assessed prospectively by immunohistochemistry. The co-primary endpoints were the independent review facility-assessed objective response rate according to RECIST v1.1 and the investigator-assessed objective response rate according to immune-modified RECIST, analysed by intention to treat. A hierarchical testing procedure was used to assess whether the objective response rate was significantly higher than the historical control rate of 10% at α level of 0·05. This study is registered with ClinicalTrials.gov, number NCT02108652.
FINDINGS: Between May 13, 2014, and Nov 19, 2014, 486 patients were screened and 315 patients were enrolled into the study. Of these patients, 310 received atezolizumab treatment (five enrolled patients later did not meet eligibility criteria and were not dosed with study drug). The PD-L1 expression status on infiltrating immune cells (ICs) in the tumour microenvironment was defined by the percentage of PD-L1-positive immune cells: IC0 (<1%), IC1 (≥1% but <5%), and IC2/3 (≥5%). The primary analysis (data cutoff May 5, 2015) showed that compared with a historical control overall response rate of 10%, treatment with atezolizumab resulted in a significantly improved RECIST v1.1 objective response rate for each prespecified immune cell group (IC2/3: 27% [95% CI 19-37], p<0·0001; IC1/2/3: 18% [13-24], p=0·0004) and in all patients (15% [11-20], p=0·0058). With longer follow-up (data cutoff Sept 14, 2015), by independent review, objective response rates were 26% (95% CI 18-36) in the IC2/3 group, 18% (13-24) in the IC1/2/3 group, and 15% (11-19) overall in all 310 patients. With a median follow-up of 11·7 months (95% CI 11·4-12·2), ongoing responses were recorded in 38 (84%) of 45 responders. Exploratory analyses showed The Cancer Genome Atlas (TCGA) subtypes and mutation load to be independently predictive for response to atezolizumab. Grade 3-4 treatment-related adverse events, of which fatigue was the most common (five patients [2%]), occurred in 50 (16%) of 310 treated patients. Grade 3-4 immune-mediated adverse events occurred in 15 (5%) of 310 treated patients, with pneumonitis, increased aspartate aminotransferase, increased alanine aminotransferase, rash, and dyspnoea being the most common. No treatment-related deaths occurred during the study.
INTERPRETATION: Atezolizumab showed durable activity and good tolerability in this patient population. Increased levels of PD-L1 expression on immune cells were associated with increased response. This report is the first to show the association of TCGA subtypes with response to immune checkpoint inhibition and to show the importance of mutation load as a biomarker of response to this class of agents in advanced urothelial carcinoma.
FUNDING: F Hoffmann-La Roche Ltd.
Illumina Commentary
The authors describe the results of a phase 2 clinical trial that assessed the efficacy of atezolizumab (a PD-L1 blocking antibody) in urothelial carcinoma patients refractory to platinum-based chemotherapy. They found that atezolizumab treatment significantly improved clinically-defined objective response rates. In additional exploratory studies, they demonstrated that tumors with high mutational load were predictive for response to atezolizumab. Further, PD-L1 expression on immune cells correlated with increased response. This study demonstrates that PD-L1 expression on immune cells is a potential biomarker for the selection of patients for treatment with atezolizumab.
Subudhi SK, Aparicio A, Gao J, et al.
Proc Natl Acad Sci U S A
113: 11919-11924
2016
Abstract
Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2-3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2-3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab.
Illumina Commentary
Immune checkpoint therapies have made a significant impact on cancer treatment, inducing clinical responses in patients with different types of solid tumors. Complications of immunotherapy include immune-mediated adverse events (irAEs). In some cases, these AEs can be severe, and early intervention can attenuate the severity of some AEs. However, there are currently no biomarkers for detecting or managing these immune-related AEs. In this phase II clinical trial, the authors treated 27 patients with metastatic prostate cancer with androgen deprivation therapy plus ipilimumab, a CTLA-4 blocking antibody. They isolated expanded CD8 T cells from these patients and used T cell receptor sequencing to identify more than 50 specific CD8 T cell clones consistently present in patients that experienced grade 2-3 immune-related AEs. These data suggest that CD8 T cell expansion may serve as a biomarker to enable monitoring and early intervention for patients receiving ipilimumab immunotherapy.
Tran E, Robbins PF, Lu YC, et al.
N Engl J Med
375: 2255-2262
2016
Abstract
We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.
Illumina Commentary
Tumor-infiltrating lymphocytes (TILs) can be expanded ex vivo and can block tumor growth for some metastatic cancers, likely through T cell recognition of tumor neoantigens. Mutations in the KRAS oncogene are frequent and drive the formation of many cancer types, yet no currently available drugs target oncogenic KRAS. In this study, the authors present clinical and biological data from a single patient with metastatic colorectal cancer. TILs were isolated from tumor tissues, and tested for reactivity against tumor neoepitopes. CD8+ T cells recognizing the KRAS G12D oncogenic variant were identified. The most KRAS G12D reactive CD8+ T cells were expanded and infused into the patient. This adoptive transfer of T cells specifically targeting mutant KRAS G12D led to the regression of metastatic colon cancer in this patient. The tumor that progressed despite treatment was subjected to whole exome and RNA sequencing using the NextSeq 500 System, and this resistant metastasis contained additional mutations in the HLA locus. The data suggest that TILs targeting oncogenic KRAS neoantigens may be an effective treatment option for some metastatic cancers, but that additional targeting of HLA neoantigens may also be required for full efficacy.
McGranahan N, Furness AJ, Rosenthal R, et al.
Science
351: 1463-9
2016
Abstract
As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens.
Illumina Summary
Coming soon.
Sen DR, Kaminski J, Barnitz RA, et al.
Science
354: 1165-1169
2016
Abstract
Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells.
Illumina Commentary
T-cell exhaustion is an acquired state of T-cell dysfunction that occurs in response to chronic infection or cancer. Immune checkpoint blockade agents (including PD-1 and PD-L1 inhibitors) aim to reverse T-cell exhaustion in cancer and have proven effective for hematologic cancers in clinical trials; however, their broader use in solid tumors has been impeded by development of T-cell exhaustion. In this study, the authors examined the molecular mechanisms differentiating exhausted T-cells from functional memory T-cells. They isolated antigen-specific as well as exhausted CD8+ T-cells from lymphocytic choriomeningitis virus (LCMV)-infected mice and profiled their epigenetic landscapes by performing ATAC-seq using the HiSeq 2500 sequencer. Their data demonstrate that the genetic landscape of regulatory regions are vastly different in exhausted and functional T-cells, suggesting that these cell types use divergent epigenetic mechanisms to regulate gene expression. In particular, they found that PD-1 expression was regulated by an exhaustion-specific enhancer, and they demonstrated that enhancer removal via CRISPR-Cas9-mediated gene editing abrogated PD-1 expression. In addition to identifying molecular mechanisms underlying T-cell exhaustion, this study suggests potential approaches for overcoming exhaustion in chimeric antigen receptor (CAR) T-cell immunotherapies.
Shukla SA, Rooney MS, Rajasagi M, et al.
Nat Biotechnol
33: 1152-8
2016
Abstract
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.
Illumina Commentary
Human leukocyte antigen (HLA) genes are highly polymorphic with thousands of documented alleles. Current strategies for finding somatic HLA mutations are effective when using targeted deep sequencing of HLA regions. However, the process is more challenging when using whole exome sequencing (WES) with standard short-read technology at low- to medium-coverage. In this study, the authors describe Polysolver, a computational pipeline that extrapolates HLA type from WES data. Polysolver first identifies HLA sequences by matching WES reads with a tag library derived from all known HLA alleles and then infers HLA alleles using several factors. The Polysolver HLA alleles can then be used as a reference to identify mutations in the HLA genes. Using Polysolver, they analyzed WES data from approximately 8000 tumor and paired normal samples and identified a number of known as well as novel HLA mutations across 20 tumor types. Their data demonstrate that HLA gene mutations are abundant in some tumor types, but absent from others. Further, they identified 29 recurrent HLA mutation sites that were associated with enhanced effector lymphocyte gene expression, suggesting that altered HLA function may play a role in immune evasion in cancer.
Giannakis M, Mu XJ, Shukla SA, et al.
Cell Rep
17: 1206
2016
Abstract
Large-scale genomic characterization of tumors from prospective cohort studies may yield new insights into cancer pathogenesis. We performed whole-exome sequencing of 619 incident colorectal cancers (CRCs) and integrated the results with tumor immunity, pathology, and survival data. We identified recurrently mutated genes in CRC, such as BCL9L, RBM10, CTCF, and KLF5, that were not previously appreciated in this disease. Furthermore, we investigated the genomic correlates of immune-cell infiltration and found that higher neoantigen load was positively associated with overall lymphocytic infiltration, tumor-infiltrating lymphocytes (TILs), memory T cells, and CRC-specific survival. The association with TILs was evident even within microsatellite-stable tumors. We also found positive selection of mutations in HLA genes and other components of the antigen-processing machinery in TIL-rich tumors. These results may inform immunotherapeutic approaches in CRC. More generally, this study demonstrates a framework for future integrative molecular epidemiology research in colorectal and other malignancies.
Illumina Commentary
High throughput sequencing approaches for cancer has refined not only understanding of the genomic landscape of cancer, but also understanding immune system regulation of tumorigenesis. In this study, the authors developed a new model for molecular epidemiology research by integrating genomic data with clinical, epidemiological and pathological annotation data linked to colorectal cancer (CRC). Whole exome sequencing (WES) was performed on 619 archived tumor-normal colorectal cancer (CRC) tissue pairs obtained from the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS), well-known long-term cancer epidemiology studies. They identified recurrent mutations in 90 genes, including 73 new CRC driver genes. Somatic mutations were used to predict immunogenic peptides, and tumors with high neoantigen load were associated with infiltration of lymphocytes and memory T cells as well as improved CRC-specific survival. These results demonstrate an integrated model for genetic epidemiology in cancer and may inform CRC immunotherapeutic approaches.
Hundal J, Carreno BM, Petti AA, et al.
Genome Med
8: 11
2016
Abstract
Cancer immunotherapy has gained significant momentum from recent clinical successes of checkpoint blockade inhibition. Massively parallel sequence analysis suggests a connection between mutational load and response to this class of therapy. Methods to identify which tumor-specific mutant peptides (neoantigens) can elicit anti-tumor T cell immunity are needed to improve predictions of checkpoint therapy response and to identify targets for vaccines and adoptive T cell therapies. Here, we present a flexible, streamlined computational workflow for identification of personalized Variant Antigens by Cancer Sequencing (pVAC-Seq) that integrates tumor mutation and expression data (DNA- and RNA-Seq). pVAC-Seq is available at https://github.com/griffithlab/pVAC-Seq.
Illumina Commentary
Cancer vaccine immunotherapy is facilitated by the discovery of patient-specific epitopes arising from cancer-specific somatic mutations. These neoepitopes, or neoantigens, can be targeted by CD8+ T-cells and can enhance immune system anti-tumor activity. Approaches for identifying good quality neoantigens are currently fragmented and suboptimal. In this study, the authors developed pVAC-Seq, an in silico automated pipeline for predicting good quality cancer neoepitopes. The pipeline integrates patient-specific HLA alleles with patient-specific DNA-Seq and RNA-Seq data, allowing somatic mutations to be qualified by tumor-specific gene expression data. The pipeline was validated in a phase I vaccine clinical trial for metastatic melanoma, where it identified a restricted set of good quality immunogenic neoepitopes that were verified experimentally. The pVAC-Seq computational workflow is publicly available, flexible, and automated; thus, increasing its potential utility for clinical use.
Hackl H, Charoentong P, Finotello F, and Trajanoski Z.
Nat Rev Genet
17: 441-58
2016
Abstract
Recent breakthroughs in cancer immunotherapy and decreasing costs of high-throughput technologies have sparked intensive research into tumour-immune cell interactions using genomic tools. The wealth of the generated data and the added complexity pose considerable challenges and require computational tools to process, to analyse and to visualize the data. Recently, various tools have been developed and used to mine tumour immunologic and genomic data effectively and to provide novel mechanistic insights. Here, we review computational genomics tools for cancer immunology and provide information on the requirements and functionality in order to assist in the selection of tools and assembly of analytical pipelines.
Illumina Summary
Coming soon.
Tirosh I, Izar B, Prakadan SM, et al.
Science
352: 189-96
2016
Abstract
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.
Illumina Commentary
Single-cell sequencing has the potential to inform treatment response and drug resistance by assessing malignant, microenvironmental, and immunologic states within tumors. In this study, the authors applied scRNA-Seq to 4645 single cells (malignant, stromal, immune, and endothelial) isolated from 19 patients with metastatic melanoma. They found that malignant cells within the same tumor displayed transcriptional heterogeneity associated with cell cycle, spatial context, and drug resistance. The same tumor had cells with high expression levels of microphthalmia-associated transcription factor (MITF), as well as cells with low MITF levels and elevated levels of AXL kinase (cells prone to early drug resistance). Infiltrating T-cell analysis revealed exhaustion programs, connection to T-cell activation/expansion, and patient variability. This study demonstrates how single-cell genomics can unravel the cellular ecosystem of tumors, with implications for targeted and immune therapies.
Anagnostou V, Smith KN, Forde PM, et al.
Cancer Discov
33: 1152-8
2016
Abstract
Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance.
Illumina Commentary
Tumor cells contain somatic mutations that alter the amino acid sequence of expressed proteins. These alterations in coding sequences lead to expression of tumor-specific neoantigens that can induce a host immune response against the tumor. Some cancers, especially those expressing PD-L1, respond well to PD-1 blockade therapy. However, cancers can develop resistance to PD-1 blockade. Upregulation of alternate immune checkpoints or somatic mutations have been hypothesized to underlie resistance mechanisms, but the specific mechanisms have not been identified. In this study, the authors used the HiSeq 2000/2500 System to perform whole exome sequencing of non-small cell lung cancer (NSCLC) tumors that developed resistance to PD-1 blockade. Their work suggests that the acquisition of PD-1 resistance in NSCLC is associated with loss of somatic mutations encoding NSCLC-specific neoantigens. These data suggest that genomic analysis of acquired resistance may be useful in patient-specific immunotherapy approaches.
Abstract
Tumor-derived exosomes (TEX) are harbingers of tumor-induced immune suppression: they carry immunosuppressive molecules and factors known to interfere with immune cell functions. By delivering suppressive cargos consisting of proteins similar to those in parent tumor cells to immune cells, TEX directly or indirectly influence the development, maturation, and antitumor activities of immune cells. TEX also deliver genomic DNA, mRNA, and microRNAs to immune cells, thereby reprogramming functions of responder cells to promote tumor progression. TEX carrying tumor-associated antigens can interfere with antitumor immunotherapies. TEX also have the potential to serve as noninvasive biomarkers of tumor progression. In the tumor microenvironment, TEX may be involved in operating numerous signaling pathways responsible for the downregulation of antitumor immunity.
Illumina Summary
Coming soon.
Nat Med
22: 8-10
2016
Abstract
Coming soon.
Illumina Summary
Coming soon.
Sotillo E, Barrett DM, Black KL, et al.
Cancer Discov
5: 1282-95
2015
Abstract
The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.
SIGNIFICANCE: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
Illumina Commentary
CART-19 targets the CD19 antigen, expressed in most cases of pre-B cell acute lymphoblastic leukemia (B-ALL). Treatment with CART-19 antigen yields 70% response in patients of B-ALL, but it also can lead to escape variants that result in relapse with epitope loss. The aim of this study was to identify the underlying mechanism of this resistance. To do this, the authors analyzed CD19-positive leukemia cells pre- and post-CART-19 therapy, as well as relapsed cells. They detected hemizygous deletions in spanning the CD19 locus, de novo mutations in exon 2 of the gene, and alternatively spliced CD19 mRNA isoforms lacking exon 2. They identified SRSF3 as splicing factor involved in the retention of exon 2. With the use of genome-editing techniques, they provided evidence suggesting a mechanism of resistance based on a combination of deleterious mutations and selecting for alternatively spliced RNA isoforms.
Carreno BM, Magrini V, Becker-Hapak M, et al.
Science
348: 803-8
2015
Abstract
BACKGROUND: First-line chemotherapy for patients with cisplatin-ineligible locally advanced or metastatic urothelial carcinoma is associated with short response duration, poor survival, and high toxicity. This study assessed atezolizumab (anti-programmed death-ligand 1 [PD-L1]) as treatment for metastatic urothelial cancer in cisplatin-ineligible patients.
METHODS: For this single-arm, multicentre, phase 2 study, in 47 academic medical centres and community oncology practices in seven countries in North America and Europe, we recruited previously untreated patients with locally advanced or metastatic urothelial cancer who were cisplatin ineligible. Patients were given 1200 mg intravenous atezolizumab every 21 days until progression. The primary endpoint was independently confirmed objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 (central review), assessed in prespecified subgroups based on PD-L1 expression and in all patients. All participants who received one or more doses of atezolizumab were included in the primary and safety analyses. This study was registered with ClinicalTrials.gov, number NCT02108652.
FINDINGS: Between June 9, 2014, and March 30, 2015, we enrolled 123 patients, of whom 119 received one or more doses of atezolizumab. At 17·2 months' median follow-up, the objective response rate was 23% (95% CI 16 to 31), the complete response rate was 9% (n=11), and 19 of 27 responses were ongoing. Median response duration was not reached. Responses occurred across all PD-L1 and poor prognostic factor subgroups. Median progression-free survival was 2·7 months (2·1 to 4·2). Median overall survival was 15·9 months (10·4 to not estimable). Tumour mutation load was associated with response. Treatment-related adverse events that occurred in 10% or more of patients were fatigue (36 [30%] patients), diarrhoea (14 [12%] patients), and pruritus (13 [11%] patients). One treatment-related death (sepsis) occurred. Nine (8%) patients had an adverse event leading to treatment discontinuation. Immune-mediated events occurred in 14 (12%) patients.
INTERPRETATION: Atezolizumab showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use in untreated metastatic urothelial cancer.
FUNDING: F Hoffmann-La Roche, Genentech.
Illumina Commentary
The genome of melanoma cells, as for any cancerous cell, displays several somatic mutations. Amino acid substitutions (AASs) are the result of missense mutations, and they can provide patient-specific antigens that trigger immune response and tumor-specific T cell immunity. The aim of this study was to evaluate these putative neoantigens as targets of anti-tumor activity and to verify whether a vaccination could augment such immune response. To do so, the authors performed exome sequencing on excised tumors from 3 patients with stage III resected cutaneous melanoma to identify AASs. They then enrolled the 3 patients in a phase 1 clinical trial, administering a dendritic cell vaccine, directed at tumor-specific amino acids. They found that the vaccine increased the naturally occurring neoantigen-specific immunity and revealed previously undetected human leukocyte antigen (HLA) class I-restricted neoantigens. Vaccination also promoted a diverse neoantigen-specific T cell receptor (TCR) repertoire in terms of both TCR-ß and clonal composition.
Rizvi NA, Hellmann MD, Snyder A, et al.
Science
348: 124-8
2015
Abstract
Immune checkpoint inhibitors, which unleash a patient's own T cells to kill tumors, are revolutionizing cancer treatment. To unravel the genomic determinants of response to this therapy, we used whole-exome sequencing of non-small cell lung cancers treated with pembrolizumab, an antibody targeting programmed cell death-1 (PD-1). In two independent cohorts, higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival. Efficacy also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations; each factor was also associated with mutation burden. In one responder, neoantigen-specific CD8+ T cell responses paralleled tumor regression, suggesting that anti-PD-1 therapy enhances neoantigen-specific T cell reactivity. Our results suggest that the genomic landscape of lung cancers shapes response to anti-PD-1 therapy.
Illumina Commentary
Immune checkpoint inhibitors use a patient’s own T cells to kill tumors. The aim of this study was to characterize the genomic determinants of response to therapy with antibody targeting programmed cell death protein (PD-1). To do so, they used exome sequencing data from two cohorts of non-small-cell lung cancers and the matched normal DNA (n = 16 and n = 18). In both cohorts, higher nonsynonymous mutation burden in tumors was associated with improved response, durable clinical benefit, and progression-free survival. These results are consistent with a genomic-shaped response to anti-PD-1 therapy.
Sivan A, Corrales L., Hubert N, et al.
Science
350: 1084-9
2015
Abstract
T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the mechanisms underlying variable immune responses between individuals are not well understood. One possible modulator could be the intestinal microbiota. We compared melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or after fecal transfer. Sequencing of the 16S ribosomal RNA identified Bifidobacterium as associated with the antitumor effects. Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein 1 ligand 1 (PD-L1)-specific antibody therapy (checkpoint blockade), and combination treatment nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced CD8(+) T cell priming and accumulation in the tumor microenvironment mediated the effect. Our data suggest that manipulating the microbiota may modulate cancer immunotherapy.
Illumina Commentary
The mechanism underlying variable immune response to cancer is not well understood. In this study, the authors aimed at assessing the role of the intestinal microbiota in modulating this response. To do so, they compared subcutaneous melanoma growth in 2 lines of C57BL mice, which are genetically similar but differ in their commensal microbes. They observed significant differences in spontaneous anti-tumor immunity and melanoma growth rate. These differences were eliminated after cohousing or fecal transfer. They sequenced the 16S ribosomal RNA of mice subjected to administration of fecal permutations and identified Bifidobacterium as significantly associated with anti-tumor immune-mediated response. They then administered Bifidobacterium alone to treated mice, improving tumor control at the same extent as programmed cell death protein 1 ligand (PD-L1)-specific antibody therapy. A combination of both Bifidobacterium and anti-PD-L1 treatment nearly abolished tumor outgrowth. These results highlight an important role for the gut microbiome in anti-tumor immunity.
Linnemann C, van Buuren MM, Bies L, et al.
Nature Medicine
21: 81–5
2015
Abstract
Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen-specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen-specific CD4+ T-cell reactivity is a common property in human melanoma.
Illumina Summary
Coming soon.
Diken M, Kreiter S, Kloke B, et al.
Prog Tumor Res
42: 44-54
2015
Abstract
Developments in sequencing technologies have not only led to a rapid generation of genomic and transcriptional data from cancer patients, but also revealed the vast diversity of cancer-specific changes in patient tumors. Among these, mutation changes in the protein sequence can result in novel epitopes recognized by the immune system and, therefore, can be employed in the development of personalized vaccines. Thanks to its easy design and scalable GMP production, vaccines based on mRNAs coding for mutated epitopes have emerged as a reliable strategy for the exploitation of the potential of patient-specific genomic data. In this review, we provide an overview of recent developments in actively personalized vaccinations, with a special focus on the promise of mRNA vaccines.
Illumina Commentary
Sequencing technologies are enabling the rapid characterization of genomic and transcriptional data from cancer patients, revealing the high degree of cancer-specific variability. This is a breakthrough in personalized medicine and cancer diagnostics, which sets great expectations in personalized therapies. The several mutations in protein sequences from cancerous cells can provide a valuable source of antigens that can be recognized by the immune system and used in the development of personalized vaccines. Of the various forms of possible cancer vaccines, mRNA-based vaccine encoding for the mutated epitopes has given promising results and been proven safe in preclinical and clinical settings. After the preclinical proof of concept in mice, actively personalized mRNA cancer vaccination was first introduced to the clinic in 2013, in a phase I study. The approach consists in, first, the use of next-generation sequencing (NGS) to characterize a cancer’s mutanome, immunome, and transcriptome by comparing healthy and cancer tissues from each patient. Then, patients receive 2 polypeptide-encoding RNA molecules, which are patient specific. This approach is aimed at targeting multiple epitopes in a specific patient’s tumor to address cancer heterogeneity, and is therefore, applicable to any cancer that carries more than 1 mutation.
Nat Biotechnol
33: 571-2
2015
Abstract
T cell immunity directed against tumor-encoded amino acid substitutions occurs in some melanoma patients. This implicates missense mutations as a source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as targets of antitumor immunity is lacking. Moreover, it remains unknown whether vaccination can augment such responses. We found that a dendritic cell vaccine led to an increase in naturally occurring neoantigen-specific immunity and revealed previously undetected human leukocyte antigen (HLA) class I-restricted neoantigens in patients with advanced melanoma. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor (TCR) repertoire in terms of both TCR-ß usage and clonal composition. Our results demonstrate that vaccination directed at tumor-encoded amino acid substitutions broadens the antigenic breadth and clonal diversity of antitumor immunity.
Illumina Commentary
Chimeric antigen receptor (CAR) T cell (CAR-T) treatments are next-generation therapies that belong to the adoptive cell therapy class of treatments. These treatments comprise the ex vivo modification of T cells to direct a response to a tumor antigen. Recently, more biopharma companies have been licensing deals to access CAR-based cellular immunotherapies, following the example initially set by Novartis in 2012, when it acquired the exclusive rights to a CAR-T program developed at the University of Pennsylvania by Dr Carl June. Although this technology still presents challenges, these come with high-improvement opportunities. Examples are the ability to regulate T cell gene expression and response after they have been infused back into the patient using small molecules as activators, and the simplification of the procedure itself.
Rosenberg SA, Restifo NP.
Science
348: 62-8
2015
Abstract
Adoptive cell therapy (ACT) is a highly personalized cancer therapy that involves administration to the cancer-bearing host of immune cells with direct anticancer activity. ACT using naturally occurring tumor-reactive lymphocytes has mediated durable, complete regressions in patients with melanoma, probably by targeting somatic mutations exclusive to each cancer. These results have expanded the reach of ACT to the treatment of common epithelial cancers. In addition, the ability to genetically engineer lymphocytes to express conventional T cell receptors or chimeric antigen receptors has further extended the successful application of ACT for cancer treatment.
Illumina Commentary
Adoptive cell transfer (ACT) consists of the administration of cancer-bearing host immune cells. While other therapies rely on sufficient expansion of anti-tumor cell types in vivo, in ACT, lymphocytes are grown in vitro, activated, and selected on the basis of their anti-tumor activity before injection into patient. Once injected, they can expand more than thousandfold. Lymphocytes infiltrating the stroma of tumors are capable of recognizing cancer cells. It was demonstrated in the 1980s that adoptive transfer of such tumor infiltrating lymphocytes (TILs) can mediate regression in certain tumors. Such populations of TILs are usually CD8+ and CD4+ T cells. In early studies, the effect of the transferred cells was often of short duration, and cells were rarely found in circulation a few days after treatment. An important observation in 2002 demonstrated that lymphodepletion using a nonmyeloablative chemotherapy regimen administered prior to TIL transfer led to an improvement in cancer regression, as well as oligoclonal population of the host. TILs can be grown from several tumors; however, melanoma is the 1 cancer that has given the most results. In fact, ACT using TILs is the most effective way to treat metastatic melanoma. Thanks to exome studies on tumor vs control screening to identify patients’ mutanomes, it is now known that TILs recognize and target products of cancer mutations. This is true for melanoma, but recent studies have demonstrated the existence of TILs from other epithelial cancers that can recognize cancer mutations. Also, lately, the ability to engineer lymphocytes to express conventional T cell receptors or chimeric antigens has further advanced the applications of ACT in cancer treatment.
Abstract
Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances and provided a new weapon against cancer. This therapy has elicited durable clinical responses and, in a fraction of patients, long-term remissions where patients exhibit no clinical signs of cancer for many years. The way forward for this class of novel agents lies in our ability to understand human immune responses in the tumor microenvironment. This will provide valuable information regarding the dynamic nature of the immune response and regulation of additional pathways that will need to be targeted through combination therapies to provide survival benefit for greater numbers of patients.
Illumina Commentary
Immune checkpoint therapy targets specific regulatory pathways in T cells, enhancing immune response. This approach has provided significant and durable benefit to a subset of patients and has led to the development and release of 2 new cancer treatments: ipilimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody, approved in 2011 by the US Food and Drug Administration (FDA), and pembrolizumab and nivolumab, antibodies against programmed cell death protein (PD-1) that were approved by the FDA in 2014. Although in some cases, these treatments have led to long-term remission with no clinical signs of cancer for several years, cancer and patient characteristics that will lead to successful use of immune checkpoint therapy still need to be defined. Recent studies are proving that mutational load is likely associated to treatment outcome for certain cancers. The identification of prognostic biomarkers, as well as the identification of new pathways and development of new therapies, will lie in our ability to understand the immune system and its responses in the tumor microenvironment. This will also be useful in understanding what therapies, or combination of therapies, will be appropriate for which patients.
Sharma P, Allison JP.
Cell
161: 205-14
2015
Abstract
Research in two fronts has enabled the development of therapies that provide significant benefit to cancer patients. One area stems from a detailed knowledge of mutations that activate or inactivate signaling pathways that drive cancer development. This work triggered the development of targeted therapies that lead to clinical responses in the majority of patients bearing the targeted mutation, although responses are often of limited duration. In the second front are the advances in molecular immunology that unveiled the complexity of the mechanisms regulating cellular immune responses. These developments led to the successful targeting of immune checkpoints to unleash anti-tumor T cell responses, resulting in durable long-lasting responses but only in a fraction of patients. In this Review, we discuss the evolution of research in these two areas and propose that intercrossing them and increasing funding to guide research of combination of agents represent a path forward for the development of curative therapies for the majority of cancer patients.
Illumina Commentary
Somatic mutation events in oncogenesis inactivate genes normally involved in regulation of cell division and programmed cell death, as well as DNA repair. The identification of these mutations has triggered the study and development of therapies that can lead to clinical response but not provide durability in time. Memory, as well as specificity, are the characteristics that make the use of the immune system particularly advantageous in cancer treatment. Thanks to advances in DNA sequencing and genomic technologies, several tumor antigens defined by tumor-specific cells have been identified in mice and humans. However, the activation of T cells requires antigen presentation by antigenpresenting cells (APCs), which provide the co-stimulatory molecule B7 that is normally lacking in tumors. B7 molecules will engage their ligand CD28 expressed on the T cell to activate it. This priming elicits both a program to activate the response and a program that will eventually inactivate it. This inhibitory program is mediated by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which is homologous to CD28 but much more affine to the B7 activator molecules. Expression of CTLA-4 is fundamental in healthy tissues, but its accumulation in the immunological synapse in the context of tumor response will eventually attenuate it. CTLA-4 blockade is a promising therapy for certain cancers, and it was approved as a treatment for melanoma in 2011. This success has opened a new field called “immune checkpoint therapy” that has identified other pathways such as programmed cell death protein (PD-1) and its ligand PD-L1, also expressed in activated T cells only. Anti-PD-L1 antibodies have shown promising results in multiple cancers. As any cancer treatment, checkpoint inhibitors have side effects, mainly related to immune-related adverse events. Many other pathways are showing promising results, and it is likely that in the future, combination treatments—as in chemotherapy—will hold the key to better results in cancer treatments.
Le DT, Uram JN, Wang H, et al.
N Engl J Med
372: 2509-20
2015
Abstract
BACKGROUND: Somatic mutations have the potential to encode "non-self" immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to mismatch-repair defects may be susceptible to immune checkpoint blockade.
METHODS: We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti-programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair-deficient colorectal cancers, patients with mismatch repair-proficient colorectal cancers, and patients with mismatch repair-deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate.
RESULTS: The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair-deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair-proficient colorectal cancer (hazard ratio for disease progression or death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P=0.05]). Patients with mismatch repair-deficient noncolorectal cancer had responses similar to those of patients with mismatch repair-deficient colorectal cancer (immune-related objective response rate, 71% [5 of 7 patients]; immune-related progression-free survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of 1782 somatic mutations per tumor in mismatch repair-deficient tumors, as compared with 73 in mismatch repair-proficient tumors (P=0.007), and high somatic mutation loads were associated with prolonged progression-free survival (P=0.02).
CONCLUSIONS: This study showed that mismatch-repair status predicted clinical benefit of immune checkpoint blockade with pembrolizumab. (Funded by Johns Hopkins University and others; ClinicalTrials.gov number, NCT01876511.).
Illumina Commentary
Antibodies to the programmed cell death protein (PD-1) pathway lead to a remarkable response in certain cancers, such as melanomas, non-small-cell lung cancer, renal cell carcinoma, bladder cancer, and Hodgkin’s lymphoma. The authors of this study hypothesized that this treatment is particularly effective in patients whose tumors have large numbers of somatic mutations. To prove their hypothesis, they conducted a phase 2 study in 41 patients with progressive metastatic carcinoma, either repair-deficient or repair-proficient. To estimate the number of mutation-associated antigens in each tumor, they performed exome sequencing and human leukocyte antigen (HLA) haplotyping from a primary tumor sample and matched peripheral blood, and used the data for an epitope prediction algorithm. They observed that in repair-deficient cancer patients, the immune-related objective response rate and immune-related progression-free survival rate were 40% (4 out of 10 patients) and 78% (7 out of 9 patients). On the other side, these percentages were as low as 0% and 11% (2 out of 18 patients) in mismatch repair-proficient patients. Furthermore, mutation loads were significantly associated with prolonged progression-free survival (p = 0.02).
Van Allen EM, Miao D, Schilling B, et al.
Science
350: 207-11
2015
Abstract
Monoclonal antibodies directed against cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), such as ipilimumab, yield considerable clinical benefit for patients with metastatic melanoma by inhibiting immune checkpoint activity, but clinical predictors of response to these therapies remain incompletely characterized. To investigate the roles of tumor-specific neoantigens and alterations in the tumor microenvironment in the response to ipilimumab, we analyzed whole exomes from pretreatment melanoma tumor biopsies and matching germline tissue samples from 110 patients. For 40 of these patients, we also obtained and analyzed transcriptome data from the pretreatment tumor samples. Overall mutational load, neoantigen load, and expression of cytolytic markers in the immune microenvironment were significantly associated with clinical benefit. However, no recurrent neoantigen peptide sequences predicted responder patient populations. Thus, detailed integrated molecular characterization of large patient cohorts may be needed to identify robust determinants of response and resistance to immune checkpoint inhibitors.
Illumina Commentary
Inhibition of immune checkpoints through monoclonal antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a promising therapy for metastatic melanoma. The aim of this study was to characterize the role of tumor-specific neoantigens and alterations of the tumor microenvironment in response to anti-CTLA-4 antibodies. To do so, the authors sequenced exomes from pre-treatment melanoma biopsies and matched normal DNA from 110 patients. They found that overall mutation load, neoantigen load, and expression of cytolytic markers in the immune microenvironment were significantly associated with clinical benefit. However, no recurrent neoantigen peptide was able to predict response in the population.
Vetizou M, Pitt JM, Daillere R, et al.
Science
350: 1079-84
2015
Abstract
Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy. We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species. In mice and patients, T cell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Tumors in antibiotic-treated or germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage with B. fragilis, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis-specific T cells. Fecal microbial transplantation from humans to mice confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties. This study reveals a key role for Bacteroidales in the immunostimulatory effects of CTLA-4 blockade.
Illumina Commentary
The use of antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a promising treatment in cancer immunotherapy. The aim of this study was to address the role of the gut microbiota in the immunomodulatory effects of CTLA-4 blockade. To do so, the authors compared the therapeutic effects on sarcoma model mice housed in a pathogen-free vs germ-free environment, and demonstrated that the effect of CTLA-4 blockade was effective in mice that were hosted in a pathogen-free environment but not in a germ-free environment. By sequencing of the 16S ribosomal RNA subunit, they then identified T cell responses specific to Bacteroides thetaiotaomicron and Bacteroides fragilis as associated with the response to against CTLA-4 treatment. Furthermore, antibiotic administration also inhibited the effect of therapy. This inhibition was overcome by gavage administration of B. thetaiotaomicron, immunization with B. fragilis polysaccharides, or adoptive cell transfer of B. fragilis-specific T cells. These results were confirmed by fecal transplantation from human to mice, as melanoma patients with anti-CTLA-4 antibodies favored the outgrowth of B. fragilis having anticancer properties. These results demonstrated a key role for Bacteroides in the immunostimulatory effects of CTLA-4 blockade.
Tumeh PC, Harview CL, Yearley JH, et al.
Nature
515: 568-71
2014
Abstract
Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.
Illumina Commentary
The process of expression of PD-L1 in response to cytokines has been termed adaptive immune resistance6 and represents a mechanism by which cancer cells attempt to protect themselves from immune-cell-mediated killing. This study sought to determine whether pre-existing tumour associated CD8(+) T cells are a factor in determining clinical response to PD-1 blocking therapy. Using Illumina HiSeq sequencing, the authors characterized and quantified the T-cell receptor clonality in serially sampled tumours. They found that patients responding to treatment showed proliferation of intratumoral CD8(+) T cells, directly correlating with radiographic reduction in tumour size.
Snyder A, Makarov V, Merghoub T, et al.
N Engl J Med
371: 2189-99
2014
Abstract
Abstract
BACKGROUND: Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells.
METHODS: We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients.
RESULTS: Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab.
CONCLUSIONS: These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).
Illumina Commentary
Ipilimumab and tremelimumab are monoclonal antibodies that block cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), resulting in T cell activation. Treatment of melanoma patients with such antibodies enables T cells to kill cancer cells and prolongs overall survival. The authors of this study sequenced exomes of 64 patients treated with CTLA-4; the discovery set consisted of 25 patients treated with ipilimumab, and the validation set consisted of 39 patients treated with ipilimumab, except for 5 treated with tremelimumab. In the discovery set, mutational load was associated with clinical benefit, but it was not sufficient to predict benefit alone. The authors identified tumor neoantigens for each patient using patient-specific human leukocyte antigen (HLA) typing and genome-wide somatic neoepitope analysis. By doing so, they elucidated a neoantigen landscape that was specific to tumors that responded to treatment. They then validated such signature in the validation set, where predicted neoantigens activated T cells from the patients treated with ipilimumab. These results prove and define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for screening exomes from patients who are being considered for CTLA-4 treatment.
Yadav M, Jhunjhunwala S, Phung Q, et al.
Nature
515: 572-6
2014
Abstract
Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
Illumina Commentary
AlthoughCD8 T cells can recognize tumour cells andmediate tumour regression following immunotherapy10, the antigens driving effective anti-tumour CD8 T-cell responses remain largely unknown. This study used a combination of Illumina whole-exome and transcriptome sequencing with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. More than 1,300 amino acid changes were identified using this approach; 13% of those were predicted to bind MHCI and a small fraction of which were confirmed by mass spectrometry. The authors demonstrated how vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses.
Grupp SA, Kalos M, Barrett D, et al.
N Engl J Med
368: 1509-18
2013
Abstract
Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.
Illumina Commentary
Engineered T cells expressing CARs with specificity for CD19 represent a promising therapy for chemotherapy-resistant lymphocytic leukemia (CLL). To verify whether chimeric antigen receptor T cell (CAR-T) treatments have clinical activity in acute lymphoblastic leukemia (ALL) as well, the authors treated 2 children with relapsed and refractory pre-B cell ALL (B-ALL) with infusions of T cells transduced with an anti-CD19 antibody and a T cell-signaling molecule. In both of the patients, infused cells expanded thousandfold and were found in bone marrow and cerebrospinal fluid for up to 6 months after infusion. Although both patients presented cytokine release syndrome as a side effect, cytokine blockade treatment was effective without inhibiting neither the proliferation of CAR-T nor the anti-cancerous effect of the therapy. Both patients had complete remission from the disease. For 1 of the 2 patients, remission lasted at least 9 months, which was confirmed at the molecular level by DNA sequencing. The second patient had a relapse after 2 months, where blast cells no longer expressed CD19. DNA sequencing allowed the identification of the malignant clone in peripheral blood and bone marrow on day 23, hence assessing the relapse earlier than the clinical presentation of symptoms as well as before blast cells were identifiable in the circulation through flow cytometry.
Robbins PF, Lu YC, El-Gamil M, et al.
Nat Med
19: 747-752
2013
Abstract
Significant tumor regressions have been observed in up to 70% of patients receiving adoptively transferred autologous melanoma-reactive tumor infiltrating lymphocytes (TIL) and in pilot trials, 40% of treated patients experienced complete regressions of all measurable lesions for at least five years following treatment. To evaluate the potential association between the ability of TIL to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, a novel screening approach was developed that involved mining whole exome sequence data to identify the mutated proteins that were expressed in patient tumors. Candidate mutated T cell epitopes that were identified using an MHC binding algorithm were then synthesized and evaluated for recognition by TIL. Using this approach, mutated antigens expressed on autologous tumor cells were identified as targets of three TIL that were associated with objective tumor regressions following adoptive transfer. This simplified approach, which avoids the need to generate and laboriously screen cDNA libraries from tumors, may represent a generally applicable method for identifying mutated T cell antigens expressed in melanoma as well as other tumor types.
Illumina Commentary
The positive outcome for up to 70% of melanoma patients receiving tumor-infiltrating lymphocytes (TILs) encourages the use of TILs to mediate durable regressions. To identify the ability of TILs to recognize potent antigens of mutated gene products, this study examined cancer genomes using Illumina HiSeq2000 whole-exome sequencing data to identify mutated proteins expressed in patient tumors. T-cell epitopes were selected using an algorithm evaluating the recognition by TILs. The authors successfully identified mutated antigens expressed on tumor cells that were recognized by three bulk TIL lines from three melanoma patients.
Kalos M, Levine BL, Porter DL, et al.
Sci Transl Med
3: 95ra73
2011
Abstract
Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non–cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR+ T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex–independent approach for the effective treatment of B cell malignancies.
Illumina Commentary
Tumor immunotherapy using T lymphocytes can recognize and destroy malignant cells. T cells can be engineered to express chimeric antigen receptors (CARs), which are composed by an antibody-binding domain and domains that activate the T cell. The expression of CAR domains could overcome tolerance by allowing T cells to respond to cell surface antigens. To test CD19 as a potential tumor target in B cell malignancies, the authors treated 3 patients with chemotherapy-resistant lymphocytic leukemia (CLL) with autologous T cells expressing an anti-CD19 CAR domain that included both the CD3-zeta and the 4-1BB costimulatory domain. They reported that such engineered cells expanded more than thousandfold in vivo, trafficked to bone marrow; showed potent non-cross-resistant clinical activity; and kept expressing CARs at high levels for 6 months. Furthermore, immune profiling revealed a CD19-specific immune response accompanied by complete remission in 2 of 3 patients. A portion of these cells persisted as memory cells and retained anti-CD19 effector functionality.