Early-Stage Breast Carcinomas Are Infiltrated By Oligoclonal T Cell Populations Highly Enriched Relative To The Blood

Abstract BACKGROUD: The immunogenicity of some human tumors towards T lymphocytes is well established. Recently, encouraging results have been obtained with immunotherapies inhibiting immune checkpoints in cancers such as melanoma, NSCLC and bladder cancer. Fewer studies explored these treatments in breast cancer (BC) as these tumors are often considered to be poorly immunogenic. METHODS: We analysed the T cell receptor β-chains variable genes (TCRBV) repertoires of tumor-infiltrating T cells in 17 early BC. We looked for clonally amplified T cells as their presence is an expected consequence of tumor immunogenicity. RNA was extracted and reverse-transcribed from formalin-fixed, paraffin-embedded tumor tissues. A short random sequence was added to the cDNA and used as a unique molecular identifier (UMI) for each cDNA molecule. cDNA encoding TCRBV genes was then amplified and sequenced using high throughput sequencing. Usage of UMIs during this procedure strongly improved the accuracy of the analysis by avoiding amplification biases inherent to the construction of the TCRBV library and by allowing an absolute quantification of TCRBV mRNA molecules normalized with the RPP30 housekeeping gene. TCRBV sequences were aligned using IMGT/HighV-QUEST. The Simpson's index was used to evaluate TCRBV repertoires diversity (ranging from 0 = infinite diversity to 1 = no diversity). For 3 patients, the same procedure was applied on blood T cells collected a few days before tumor resection and the analysis was also carried out on 3 normal tissues obtained from breast reduction surgery. RESULTS: T cell infiltration varied strongly from one tumor to another ranging from 5 to 2498 TCRBV/103 RPP30 mRNA molecules. TCRBV repertoires analysis indicated that infiltrated T cells corresponded to oligoclonal populations. We observed 3 clonotypes in the smaller repertoire and 74 in the largest one and the Simpson's index ranged from 0.01 to 0.65. Most tumors (16/17) contained at least one clonotype that made up ≥10% of the infiltrating T cells, with the highest observed proportions reaching 80%. Normal breast samples were infiltrated by a more diverse repertoire: 130 to 368 clonotypes were identified in those tissues and Simpson's index ranged from 0.002 to 0.008. Highest observed frequency among those clonotypes was 2%. For 3 BC patients, the frequencies of the most prevalent clonotypes in the tumor were compared to those of the same clonotypes in blood prior to surgery. These T cell clones were 250 to >34000 times more frequent in the tumor than in the blood. CONCLUSIONS: Some early BC are infiltrated by oligoclonal T cell populations that are highly enriched relative to the blood. Quantitative T cell repertoire analysis allows to distinguish 3 types of BC: (1) tumors without T cell infiltration, (2) tumors with a high T cell infiltration and a small T cell repertoire, and (3) tumors with a high T cell infiltration and a large repertoire. Our observations suggest that anti-tumor T cell responses are ongoing in some early BC and this warrants boosting such responses with immune checkpoint inhibitors in selected patients. T cell repertoire evaluation could be used as a predictive biomarker to identify patients who will benefit from this treatment. Citation Format: Carrasco J, Schröder D, Coulie PG, Godelaine D, Berlière M, Theate I, Delrée P, Vannuffel P, Galant C, Duhoux FP, Machiels J-P, Canon J-L. Early-stage breast carcinomas are infiltrated by oligoclonal T cell populations highly enriched relative to the blood. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-10.