The critical determinants of effective antitumor immune responses, whether pre-existing or induced by therapy, remain poorly understood due to the complexity and plasticity of the immune system. To better profile and track these responses, we have performed single cell RNA sequencing for paired gene expression and immune repertoire analysis on tumor fine needle aspirates and peripheral blood of lymphoma patients undergoing immunotherapy on one of two clinical trials (NCT02927964, NCT03410901).

In these in situ vaccination studies, patients with low-grade lymphoma receive local low-dose radiation and a TLR9 agonist intratumorally to one site of disease, along with either ibrutinib or an OX40 agonist. Tumor fine needle aspirates (FNAs) and peripheral blood samples are obtained prior to treatment, and at one week and six weeks after treatment start. We performed single cell RNA-sequencing to an average targeted depth of 50,000 reads/cell for gene expression libraries and 5000 reads/cell for TCR sequencing. Identification of variable genes, principal component and/or canonical correlation analysis, graph-based clustering and differential expression analysis was performed using the Seurat algorithm. Single-cell TCR repertoires were analyzed using TCR-specific analysis software. Sequencing libraries have been prepared from 67 longitudinal samples from 9 patients thus far.

We have successfully generated single cell gene expression and TCR libraries from 3,000-10,000 cells per sample from tumor fine needle aspirates and peripheral blood, with excellent sequencing quality metrics obtained. Analyzing 23 samples sequenced thus far, we consistently identify many B and T cell subsets of interest within tumors and reliably distinguish tumor B cells from normal B cells. In two responding patients, we confirm shrinkage of the tumor B cell compartment and find specific increases in CD8 and CD4 effector cells with decreases in T follicular helper cells and/or regulatory T cells at the treated site, results that are corroborated by multiparameter flow cytometry performed in parallel. Comparing pre-and post-treatment tumor FNAs from the same sites, we find evidence of increased cytotoxicity and interferon response following treatment. We also find distinct transcriptional shifts in tumor B cells both at the treated and at the distant untreated sites. Sample collection, sequencing, and analysis are ongoing.

Deep profiling of serial biopsies during immunotherapy using single cell RNA sequencing promises to illuminate underlying cellular dynamics, and paired with clinical outcome data, determinants of response. Ultimately, this may provide a roadmap for successful translation of single-cell genomics into the clinic for treatment monitoring and response prediction.

Citation Format: Tanaya Shree, Anuja Sathe, Hanlee Ji, Ronald Levy. Single cell RNA sequencing of serial tumor and blood biopsies from lymphoma patients undergoing in situ vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4045.