Letter | Published:

Immunogenic neoantigens derived from gene fusions stimulate T cell responses

Abstract

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.

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Whole-exome and RNA-sequencing data have been deposited in SRA and are available under project number PRJNA527992.

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Acknowledgements

The authors wish to acknowledge our patients and their families who selflessly contributed time and samples to support this research, the patients and investigators who contributed to the TCGA studies analyzed here, McKenzy and Beth Hupke, members of the Timothy Chan Laboratory for insightful discussions, members of the MSKCC Immunogenomics and Precision Oncology Platform and the Molecular Cytology Core Facility of MSKCC. This work was supported by the NIH/NCI Cancer Center Support Grant no. P30 CA008748 (to MSKCC), Cycle for Survival (R.J.W., L.G.T.M. and T.A.C.), the Frederick Adler Chair at MSKCC, the Jayme Flowers Fund, the Sebastian Nativo Fund, the Damon Runyon Cancer Research Foundation, NIH grant nos. K08 DE024774 and NIH R01 DE027738 (L.G.T.M.), the Adenoid Cystic Carcinoma Cancer Research Foundation, (T.A.C., A.L.H. and L.G.T.M.), The Geoffrey Beene Junior Faculty Chair (A.L.H.), NIH grant nos. R01 CA205426 and NIH R35 CA232097, the Pershing Square Sohn Cancer Research Alliance, the STARR Cancer Consortium and the PaineWebber Chair at MSKCC (T.A.C.).

Author information

W.Y., R. M. Srivastava, K.-W.L., T.A.C. and L.G.T.M. contributed to the study conception and design. L.W., M.A.C., J.T., N.S., A.L.H. and L.G.T.M. contributed to clinical treatment and clinical research coordination. W.Y., R. M. Srivastava, M.G.D., Z.N., J.T. and L.G.T.M. contributed to biospecimen processing. R.G. and N.K. contributed to pathologic analysis. The New York Genome Center (S.K.T., N.R., K.A., H.G. and P.A.) and MSKCC IGO core facility (K.H., N.B. and K.V.) contributed to DNA and RNA sequencing and analyses. V.M., F.K., C.K., D.H., D.C., J.S.S. and L.G.T.M. contributed to the bioinformatics, computational and statistical analyses. W.Y., R. M. Srivastava, K.-W.L. and L.G.T.M. contributed to the experimental design and execution. M.G.D., J.J.H., R.M., R. M. Samstein, N.R, T.A.C., I.G., A.L.H., R.J.W. and L.G.T.M. contributed to the interpretation of data. W.Y., K.-W.L., T.A.C. and L.G.T.M. wrote the manuscript.

Competing interests

K.-W.L. and J.S.S. are now full-time employees of Regeneron Pharmaceuticals. R. M. Srivastava received speaker fees and travel reimbursement from Innovent Biologics, Inc. A.L.H. receives research funding from Eli Lilly, Genentech, AstraZeneca, Bayer, Kura Oncology, Kotan Pharmaceuticals, Eisai, Bristol-Myers Squibb, Astellas Pharma, Novartis, Merck, Pfizer, Ayla Pharmaceuticals, Allos Therapuetics, Daiichi Sankyo, consulting fees from Bristol-Myers Squibb, Eisai, Genzyme, Merck, Novartis, Sun Pharma, Regeneron, TRM Oncology, Ayala Pharmaceuticals, AstraZeneca, Sanofi Aventis, and travel reimbursement from Janssen Oncology, Merck, Kura Oncology, Ignyta, Ayala Pharmacueticals. J.J.H’s spouse is a full-time employee of Regeneron Pharmaceuticals. R. M. Samstein, T.A.C. and L.G.T.M. are inventors on a provisional patent application (62/569,053) filed by Memorial Sloan Kettering (MSK) relating to the use of TMB in cancer immunotherapy. D.C. and T.A.C. are inventors on a PCT patent application (PCT/US2015/062208) filed by MSK relating to the use of TMB in cancer immunotherapy. MSK and the inventors may receive a share of commercialization revenue from license agreements relating to these patent applications. T.A.C. is a co-founder of Gritstone Oncology and holds equity. He acknowledges grant funding from Bristol-Myers Squibb, AstraZeneca, Illumina, Pfizer, An2H, and Eisai, and has served as an advisor for Bristol-Myers Squibb, Illumina, Eisai and An2H. L.G.T.M. received consulting fees from Rakuten Aspyrian and speaker fees from Physician Educational Resources.

Correspondence to Timothy A. Chan or Luc G. T. Morris.

Extended data

  1. Extended Data Fig. 1 Visualization of DEK–AFF2 and AFF2–DEK gene fusions.

    a,b, Visualization of the DEK–AFF2 (a) and AFF2–DEK (b) gene fusions in the primary tumor of patient MSK-HN1 shown on IGV plots of RNA-seq data.

  2. Extended Data Fig. 2 Screen of SNV-derived and alternative splicing-derived nonamer peptides.

    a, A screen for binding of SNV-derived nonamer peptides (10 µM) to HLA-A*02:01 on T2 cells reveals no peptides with significant binding affinity. The MFI values are normalized to DMSO. NY-ESO-1 was used as a positive control. b, IFN-γ ELISpot assay of patient MSK-HN1 T cells after 18 h co-culture with autologous PBMCs (n = 3) pulsed with 10 µM of indicated peptides derived from mutations. Due to limited numbers of autologous PBMCs, in several samples (<1>, <2>, <3>, <4>, <5> and <6>), multiple mutant-derived peptides corresponding to a single wild-type peptide were pulsed together. The grouped peptides are indicated. c, IFN-γ ELISpot assay of patient MSK-HN1 T cells after 18 h co-culture with autologous PBMCs (n = 3) pulsed with 10 µM of indicated peptides derived from potential alternative splicing events. Means ± s.e.m. are plotted, with sample n representing the number of independently treated samples.

  3. Extended Data Fig. 3 DEK–AFF2 generates an immunostimulatory peptide recognized by autologous T cells.

    a, Flow cytometry analysis of CD137 expression on CD8+ T cells after 18 h co-culture with patient MSK-HN1 PBMCs pulsed with indicated peptides. Data are representative of two independent experiments. b, IFN-γ ELISpot assay of patient MSK-HN1’s T cells after 18 h co-culture with autologous PBMCs (n = 3) which have been pulsed with DMSO or DEKSEEEVS peptide, co-treated with either immunoglobulin G (IgG) control or anti-MHC-I antibody overnight (two-tailed t-tests, 95% CI = −2.187 to +19.85, effect size η2 = 0.685, P = 0.042). c, IFN-γ ELISpot assay of patient MSK-HN1’s T cells after 18 h co-culture with SCC-9 expressing DEK-N-term or DEK–AFF2 fusion (n = 3). Cells are treated with either IgG control or anti-MHC-I antibody (two-tailed t-tests, 95% CI = −278.3 to −147.7, η2 = 0.954, P = 0.0008). d, IFN-γ ELISpot assay of patient MSK-HN1’s T cells after 18 h co-culture with COS-7 cells (n = 3) co-transfected with HLA-C*04:01 plasmid and pLVX–DEK-N-term or pLVX–DEK–AFF2. T cells and COS-7 cells were used at a 6:1 ratio (two-tailed t-test, 95% CI = 48.73–571.9, η2 = 0.731, P = 0.0301). e, Active caspase-3 staining of SCC-9 target cells (n = 2) expressing either DEK-N-term or DEK–AFF2 fusion after 3 h incubation with MSK-HN1 CD8 + TEM cells (CCR7-CD45RA-) at the indicated ratios. Means ± s.e.m. are plotted, with sample n representing the number of independently treated samples.

  4. Extended Data Fig. 4 Screen of HLA binding by fusion peptides predicted to bind to members of HLA-A2 alleles.

    a,b, The graphs show stabilization of HLA-A*02:01 on the surface of T2 cells by MYB–NFIB and MYBL1–NFIB peptides (a) and NFIB–MYB peptides (b). The peptides in which we further tested are indicated by arrows. Data are representative of two independent experiments.

  5. Extended Data Fig. 5 Validation of MYB–NFIB and NFIB–MYB fusions.

    Validation of MYB–NFIB and NFIB–MYB fusions expressed in patient ACC_M9 by visualization on IGV plots of RNA-seq data.

  6. Extended Data Fig. 6 Schematic of ACC_M9-, ACC_M1-, ACC_P11-, ACC_P14-derived MYB–NFIB fusion constructs that were cloned into pcRNA6SL.

    The amino acid sequences surrounding the junctions are shown. Predicted HLA-A2-binding peptides derived from each fusion are indicated.

  7. Extended Data Fig. 7 MYB–NFIB generates an immunostimulatory peptide recognized by patient ACC_M9’s T cells.

    a, IFN-γ ELISpot assay of patient ACC_M9’s T cells after 18 h co-culture with healthy donor HD1 dendritic cells electroporated with 2 µg of in vitro transcribed mRNA as described in the methods. b, IFN-γ ELISpot assay of patientACC_M9’s T cells after 18 h co-culture with T2 cells pulsed with 10 µM of indicated peptides. Data are representative of two independent experiments.

  8. Extended Data Fig. 8 PD-1, CD40L and CD137 expression on patient ACC_M9’s T cells after 18 h co-culture with autologous PBMCs pulsed with the indicated peptides.

    Flow cytometry analysis of patient ACC_M9’s PBMCs after 18 h pulse with the indicated peptides (n = 3). The immunogenic peptide QFIDSSWYL led to an increased fraction of CD8+ T cells that are CD137+, CD40L+ or PD-1+. Data are representative of three independent experiments.

  9. Extended Data Fig. 9 Patient ACC_M9’s CD8+ T cells that specifically bind to HLA-A*02:01-presented QFIDSSWYL peptide proliferate over 21 days during co-culture with irradiated peptide-pulsed T2 cells.

    Patient ACC_M9’s T cells were expanded on irradiated T2 pulsed with 10 µM of indicated peptides over 21 days and stained with QFIDSSWYL–dextramer–PE. A population of QFIDSSWYL-specific T cells is selectively expanded. Data are representative of two independent experiments.

  10. Extended Data Fig. 10 Healthy donor T cells are stimulated by MYB-NFIB-derived and NFIB-MYB-derived peptides.

    IFN-γ ELISpot assay of healthy donors HD2 and HD3 T cells after 18 h co-culture with T2 cells pulsed with 10 µM of indicated peptides. Data are representative of two independent experiments.

Supplementary Information

  1. Supplementary Information

    Supplementary Figs. 1–8, Supplementary Tables 1–5, 7–9

  2. Reporting Summary

  3. Supplementary Tables

    Supplementary Tables 6, 10–12

Source data

  1. Source Data Fig. 2

  2. Source Data Fig. 3

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DOI

https://doi.org/10.1038/s41591-019-0434-2

Fig. 1: Immunogenomic characterization of patient MSK-HN1, an exceptional responder to anti-PD-1 immunotherapy.
Fig. 2: DEKAFF2 generates an immunostimulatory peptide recognized by autologous T cells.
Fig. 3: MYBNFIB generates an immunostimulatory peptide recognized by autologous T cells.
Fig. 4: Donor T cells are stimulated by MYBNFIB-derived and NFIBMYB-derived peptides.
Extended Data Fig. 1: Visualization of DEK–AFF2 and AFF2–DEK gene fusions.
Extended Data Fig. 2: Screen of SNV-derived and alternative splicing-derived nonamer peptides.
Extended Data Fig. 3: DEK–AFF2 generates an immunostimulatory peptide recognized by autologous T cells.
Extended Data Fig. 4: Screen of HLA binding by fusion peptides predicted to bind to members of HLA-A2 alleles.
Extended Data Fig. 5: Validation of MYB–NFIB and NFIB–MYB fusions.
Extended Data Fig. 6: Schematic of ACC_M9-, ACC_M1-, ACC_P11-, ACC_P14-derived MYB–NFIB fusion constructs that were cloned into pcRNA6SL.
Extended Data Fig. 7: MYB–NFIB generates an immunostimulatory peptide recognized by patient ACC_M9’s T cells.
Extended Data Fig. 8: PD-1, CD40L and CD137 expression on patient ACC_M9’s T cells after 18 h co-culture with autologous PBMCs pulsed with the indicated peptides.
Extended Data Fig. 9: Patient ACC_M9’s CD8+ T cells that specifically bind to HLA-A*02:01-presented QFIDSSWYL peptide proliferate over 21 days during co-culture with irradiated peptide-pulsed T2 cells.
Extended Data Fig. 10: Healthy donor T cells are stimulated by MYB-NFIB-derived and NFIB-MYB-derived peptides.