Tumour infiltrating B cells discriminate checkpoint blockade-induced responses.

BACKGROUND: Immune cell-driven anti-cancer activity is paramount for effective responses to checkpoint inhibitors (ICB). However, the contribution of the different immune cell subsets in the circulation and within the tumour is poorly understood. MATERIALS AND METHODS: To elucidate the role of the different cell subsets in anti-tumour responses elicited by ICB, we performed single-cell analysis of the transcriptome and surface proteome of paired pre- and early on-treatment metastatic melanoma tumour biopsies and matched peripheral blood mononuclear cell samples. We next compared the survival of metastatic melanoma patients treated with ICB according to the abundance of pre-treatment tumour-infiltrating B cell clonotypes. RESULTS: We identified cell clusters associated with disease control or progression, defined differential expression of biological pathways likely involved in the immune awakening against the tumour and examined how cell-cell communication patterns between the tumour cell subsets change during treatment. Furthermore, we discovered that B cells (immunoglobulin expression and abundance of B cell clonotypes) discriminate the clinical response after ICB and propose that B cells likely contribute to anti-tumour immunity by antigen presentation through major histocompatibility complex molecules. Finally, we demonstrated that the abundance of tumour-infiltrating B cell clonotypes at baseline identifies two distinct risk groups, a finding that we confirmed in an independent cohort. CONCLUSIONS: Our exploratory translational study provides new insights on the mechanistic role of B cells in anti-melanoma immunity during treatment with ICB. Additionally, we support pre-treatment B cell tumour infiltration as a promising prognostic biomarker to be further validated as a tool for clinical risk stratification.

T cell immune awakening in response to immunotherapy is age-dependent.

BACKGROUND: Precision immuno-oncology approaches are needed to improve cancer care. We recently demonstrated that in patients with metastatic melanoma, an increase of clonality or diversity of the T cell receptor (TCR) repertoire of peripheral T cells following one cycle of immunotherapy is coincident with response to immune-checkpoint blockade (ICB). We also identified a subset of peripheral CD8+ immune-effector memory T cells (TIE cells) whose expansion was associated with response to ICB and increased overall survival. To improve our understanding of peripheral T cell dynamics, we examined the clinical correlates associated with these immune signatures. METHODS: Fifty patients with metastatic melanoma treated with first-line anti-PD-1 ICB were included. We analysed TCR repertoire and peripheral TIE cell dynamics by age before treatment (T0) and after the first cycle of treatment at week 3 (W3). RESULTS: We observed a correlation between TIE abundance and age at T0 (r = 0.40), which reduced following treatment at W3 (r = 0.07). However, at W3, we observed two significantly opposing patterns (p = 0.03) of TCR repertoire rearrangement in patients who responded to treatment, with patients ≥70 years of age showing an increase in TCR clonality and patients <70 years of age showing an increase in TCR diversity. CONCLUSIONS: We demonstrate that immunotherapy-induced immune-awakening patterns in patients with melanoma are age-related and may impact patient response to ICB, and thus have implications for biomarker development and planning of personalised therapeutic strategies.

The T cell receptor repertoire of tumor infiltrating T cells is predictive and prognostic for cancer survival.

Tumor infiltration by T cells is paramount for effective anti-cancer immune responses. We hypothesized that the T cell receptor (TCR) repertoire of tumor infiltrating T lymphocytes could therefore be indicative of the functional state of these cells and determine disease course at different stages in cancer progression. Here we show that the diversity of the TCR of tumor infiltrating T cell at baseline is prognostic in various cancers, whereas the TCR clonality of T cell infiltrating metastatic melanoma pre-treatment is predictive for activity and efficacy of PD1 blockade immunotherapy.

Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in cancer.

BACKGROUND: Resistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia (AML) and the drug efflux pump ABCB1 is a critical mediator. Recent studies have identified promoter translocations as common drivers of high ABCB1 expression in recurrent, chemotherapy-treated high-grade serous ovarian cancer (HGSC) and breast cancer. These fusions place ABCB1 under the control of a strong promoter while leaving its open reading frame intact. The mechanisms controlling high ABCB1 expression in AML are largely unknown. We therefore established an experimental system and analysis pipeline to determine whether promoter translocations account for high ABCB1 expression in cases of relapsed human AML. METHODS: The human AML cell line THP-1 was used to create a model of chemotherapy resistance in which ABCB1 expression was driven by a promoter fusion. The THP-1 model was used to establish a targeted nanopore long-read sequencing approach that was then applied to cases of ABCB1high HGSC and AML. H3K27Ac ChIP sequencing was used to assess the activity of native promoters in cases of ABCB1high AML. RESULTS: Prolonged in vitro daunorubicin exposure induced activating ABCB1 promoter translocations in human THP-1 AML cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancers. Targeted nanopore sequencing proved an efficient method for identifying ABCB1 structural variants in THP-1 AML cells and HGSC; the promoter translocations identified in HGSC were both previously described and novel. In contrast, activating ABCB1 promoter translocations were not identified in ABCB1high AML; instead H3K27Ac ChIP sequencing demonstrated active native promoters in all cases studied. CONCLUSIONS: Despite frequent high level expression of ABCB1 in relapsed primary AML we found no evidence of ABCB1 translocations and instead confirmed high-level activity of native ABCB1 promoters, consistent with endogenous regulation.

Immune-awakening revealed by peripheral T cell dynamics after one cycle of immunotherapy.

Our understanding of how checkpoint inhibitors (CPI) affect T cell evolution is incomplete, limiting our ability to achieve full clinical benefit from these drugs. Here we analyzed peripheral T cell populations after one cycle of CPI and identified a dynamic awakening of the immune system revealed by T cell evolution in response to treatment. We sequenced T cell receptors (TCR) in plasma cell-free DNA (cfDNA) and peripheral blood mononuclear cells (PBMC) and performed phenotypic analysis of peripheral T cell subsets from metastatic melanoma patients treated with CPI. We found that early peripheral T cell turnover and TCR repertoire dynamics identified which patients would respond to treatment. Additionally, the expansion of a subset of immune-effector peripheral T cells we call TIE cells correlated with response. These events are prognostic and occur within 3 weeks of starting immunotherapy, raising the potential for monitoring patients responses using minimally invasive liquid biopsies."