Characterization of an Immunogenic Mutation in a Patient with Metastatic Triple-Negative Breast Cancer

Abstract

Purpose: The administration of autologous tumor-infiltrating lymphocytes (TILs) can mediate durable tumor regressions in patients with melanoma likely based on the recognition of immunogenic somatic mutations expressed by the cancer. There are limited data regarding the immunogenicity of mutations in breast cancer. We sought to identify immunogenic nonsynonymous mutations in a patient with triple-negative breast cancer (TNBC) to identify and isolate mutation-reactive TILs for possible use in adoptive cell transfer.Experimental Design: A TNBC metastasis was resected for TIL generation and whole-exome sequencing. Tandem minigenes or long 25-mer peptides encoding selected mutations were electroporated or pulsed onto autologous antigen-presenting cells, and reactivity of TIL was screened by upregulation of CD137 and IFNγ ELISPOT. The nature of the T-cell response against a unique nonsynonymous mutation was characterized.Results: We identified 72 nonsynonymous mutations from the tumor of a patient with TNBC. CD4⁺ and HLA-DRB1*1501-restricted TILs isolated from this tumor recognized a single mutation in RBPJ (recombination signal binding protein for immunoglobulin kappa J region). Analysis of 16 metastatic sites revealed that the mutation was ubiquitously present in all samples.Conclusions: Breast cancers can express naturally processed and presented unique nonsynonymous mutations that are recognized by a patient's immune system. TILs recognizing these immunogenic mutations can be isolated from a patient's tumor, suggesting that adoptive cell transfer of mutation-reactive TILs could be a viable treatment option for patients with breast cancer. Clin Cancer Res; 23(15); 4347-53. ©2017 AACR.

Figure 1:

Identification of an immunogenic mutation recognized by patient 4062-TIL. 4062-TILs were co-cultured with B cells pulsed with the indicated peptide pool (○, irrelevant peptide; □, pool 1; △, pool 2, ▼ pool 3, ◇; pool 4; ●; pool 5; ⊙, pool 6; X, OKT3). A and B, Twenty-four individual TIL fragments were screened by IFNγ ELISPOT assay (A) or upregulation of 4–1BB (B) as measured by FACS of cells gated on live CD3⁺ (box, positive fragment). C and D, 4062-TILs (F9) were screened against individual peptides comprising peptide pool 3 showing specific recognition of mutated RBPJ by IFNγ ELISPOT (C) and specific upregulation of 4–1BB and OX40 in relation to CD4 expression by FACS (D). Data are gated on live CD3⁺ cells.

Figure 2.

Specific recognition of RBPJ^{c.A611T/p.H204L} by mutation-specific CD4⁺ T cells. A, Autologous B cells pulsed with 25-mer peptides encoding mutated or wild-type RBPJ were titrated against 4062-TILs (F9). Reactivity against mutated RBPJ using serial dilutions of HPLC-purified mutated and wild-type RBPJ 25-mer peptides was assessed by IFNγ ELISPOT. B, Intracellular cytokine staining of mutation-specific RBPJ CD4⁺ T cells. 4062-TIL (F9) cells were cocultured with autologous B cells pulsed with mutated RBPJ or irrelevant mutated 25-mer peptide identified from another patient with breast cancer (4051, QWKDRAETVIIGDGCVGVSLAYHLA) and stained for CD3⁺/CD4⁺/CD8⁺ then fixed and permeabilized prior to staining for IFNγ, IL2, and TNFα. All data are representative of at least two independent experiments. Error bars, SEM.

Figure 3.

Characterization of RBPJ^{c.A611T/p.H204L} mutation–reactive T-cell response. 4062-TILs were cocultured with RBPJ^{c.A611T/p. H204L} 25-mer peptide-pulsed autologous B cells that were preincubated with vehicle or the indicated HLA-blocking antibody. A, HLA-blocking assay demonstrating MHC class II and HLA-DR restriction. B, The HLA-DR restriction element was identified by coculturing 4062-TILs with autologous EBV-B cells or allogeneic EBV-B cells partially matched at theHLA-DR locus. The 4062-TILs CD4⁺ T-cell response was shown to be HLADRB1*15:01 restricted. C, 4062-TILs were cocultured with autologous B cells pulsed with RBPJ^{c.A611T/p.H204L} 25-mer peptide or the indicated truncated peptides. *, location of mutated amino acid at position 13, and the predicted peptide binding core sequence is underlined. All data are based on IFNγ ELISPOT and representative of at least three independent experiments. Error bars, SEM.

Figure 4.

Identification of RBPJ mutation-reactive TCR. A, 4062-TILs were cocultured with autologous APCs presenting RBPJ^A611T peptide and sorted on the basis of expression of CD4⁺/4–1BB⁺ by FACS. Data are gated on live CD3⁺ cells. B and C, TCR analysis revealed an oligoclonal population of TCR-α (B) and TCR-β (C) chains. Of note, 42.9% of the TCR-α chains were unresolved. On the basis of the frequency, the two most frequent TCR-α and TCR-β chains were used to synthesize the four possible TCR combinations. PBL transduced with TCRAV05 (the second most frequent alpha chain) and TCRBV07 (the most frequent beta chain) recognized APCs specifically presenting mutRBPJ based on IFNγ ELISPOT (D) and upregulation of 4–1BB (E) in the CD4⁺ T-cell population (▲, mutRBPJ; △, wtRBPJ; ○, irrelevant peptide). Data are representative of three separate patient TCR-transduced PBL.