Soluble BTN2A1 Is a Potential Prognosis Biomarker in Pre-Treated Advanced Renal Cell Carcinoma

The development of immune checkpoint inhibitors (ICI) has dramatically changed the landscape of therapies for metastatic renal cell carcinoma. However, many patients do not benefit from such therapy and prognostic or predictive validated biomarker validated for ICI are still needed to better select and treat patient. Plasmatic soluble immune checkpoints have been described as potential immune biomarkers in hematological malignancies and solids tumors, then, we would like to explore the prognostic value of different soluble immune checkpoints in patients with mRCC treated with nivolumab after TKI. We prospectively collected plasma samples before nivolumab infusion from 38 patients previously treated for mRCC with TKI at Paoli-Calmettes Institute, from the NIVOREN GETUG-AFU 26 study (NCT03013335). Enzyme-linked immunosorbent assays (ELISA) were performed for soluble forms of PD-1, PD-L1, global BTN3, BTLA, BTN3A1 and BTN2A1. Among the different soluble checkpoints analyzed, only high baseline plasmatic level of BTN2A1 was significantly associated with shorter PFS: median PFS was 3.95 months for sBTN2A1high vs 14.30 months for sBTN2A1low (sBTN2A1 cut-off: 6.7ng/mL; HR = 2.26, 95%CI [0.68 – 4.60], p = 0.0307). There was no statistical difference in OS between sBTN2A1high and sBTN2A1low. Our results suggest that the baseline level of plasmatic BTN2A1 could be an independent prognosis factor of PFS after nivolumab for pre-treated patient with mRCC. However, these results need to be validated in a larger prospective cohort and the biological role of BTN subfamily and γδ T cell immunity in mRCC must be elucidated.


INTRODUCTION
The development of immune checkpoint inhibitors (ICI) has dramatically changed the landscape of therapies for metastatic renal cell carcinoma (mRCC), as such in first line associated to tyrosine kinase inhibitors (TKI) (1) or as monotherapy (2). However, many patients do not benefit from such therapy and prognostic or predictive validated biomarker validated for ICI are still needed to better select and treat patient according to the best genomic profiles. The International Metastatic RCC Database Consortium (IMDC) prognostic model (3) is used to predict survival and adapted treatment strategy of untreated mRCC patients and was validated for patients previously treated with TKI, before second line TKI. Identifying new prognostic biomarkers in this population is needed to improve treatment strategy for mRCC patients.
We aimed to explore the prognostic value of different soluble immune checkpoints in mRCC treated with nivolumab after TKI.

sBTN2A1 Is a Prognostic in Patients With Pre-Treated Advanced Renal Cell Carcinoma
To explore the prognostic value of different soluble immune checkpoints, we prospectively collected plasma samples before nivolumab infusion from patients previously treated for mRCC with TKI at Paoli-Calmettes Institute (Nivo-Rein cohort), from the NIVOREN GETUG-AFU 26 study (NCT03013335). Enzyme-linked immunosorbent assays (ELISA) were performed for soluble forms of PD-1, PD-L1, global BTN3, BTLA, BTN3A1 and BTN2A1. All patients provided informed consent prior to enrolment. The receiver operating characteristic (ROC) curves were generated for each marker. The areas under the curves (AUC) were assessed to evaluate each marker performance for discriminating short term responder (patients with PFS > median) from long-term responder (patients with PFS < median). Cut-off was selected to obtain the best sensitivity and specificity for discriminating long-term responders from short term responders. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Multivariate analysis of survival was performed using the Cox-regression model, including all variables associated with progression-free survival (PFS) (p < 0.1).
We included 38 patients between March 2016 and November 2017. Patients' characteristics are summarized in Table 1. With a median follow-up of 19.8 months, 31 (82%) patients had tumor progression and 25 (66%) died. Among the different soluble checkpoints analyzed, only high baseline plasmatic level of BTN2A1 was significantly associated with shorter PFS. Cut-off retained was 6.7ng/mL, that approximate the median value of 6.8ng/mL. There was no significant difference between patients with sBTN2A1 low and sBTN2A1 high level; excepted for age (  Figure 1).

DISCUSSION
To our knowledge, this study is the first that described sBTN2A1 as potential prognostic biomarker in patients with pre-treated mRCC. In the previous NIVOREN GETUG-AFU 26 study, it was reported that tissue PD-1 and AXL expression by immunohistochemistry were associated with PFS and confirmed that PBRM-1 loss was a strong prognostic factor (10). Incorvaia et al. explored the prognostic value of soluble forms of PD-1, PD-L1, global BTN3, BTN3A1 and BTN2A1 in a prospective cohort of metastatic renal cell carcinoma patients (11). They identified sPD-1, sPD-L1 and sBTN3A1 as potential biomarkers. However, our study did not significantly confirm these results, with only a tendency for sPD-1 and sPD-L1 and this difference could be explained by the small number of patients in our cohort. Conversely, sBTN2A1 was not significantly associated with survival in their study but patients with PFS > 18 months seemed to have lower level of sBTN2A1 than patients with PFS < 18 months. The small number of patients in their learning cohort (n=21) could also explain the difference between the two works; and reinforce the necessity to validate these results in a larger prospective cohort.
The BTN sub-family plays an important role in V ϒ 9V d 2 Tcells activation and regulation by direct or indirect presentation of self and nonself phosphoantigens. The cell surface molecule BTN2A1 binds to the gd TCR, in conjunction with BTN3A1, and signals the presence of phosphoantigens to gd T cells (12). Gamma delta T (gdT) lymphocytes are members of the immune system which display both innate and adaptive functions (13). They recognize low molecular mass nonpeptide ligands named "phosphoantigens" that comprise isoprenoid pathway metabolites, as (E)-4-hydroxy-3-methyl-but-2-enyl PPi (HMB-PP) derived from bacteria and protozoa (14) and isopentenyl PPi (IPP) derived from host cells (15). There are evidence of their cytotoxic activity against tumor cells in certain cancers (16), and they are able to recognize tumor cells without the need for major histocompatibility complex (MHC) antigen presentation. However, there also evidence that suggested that gdT lymphocytes may also have a pro-tumorigenic role (17), by impairing the function of other antitumor immune cells or enhancing immunosuppressive cell function such as MDSCs. We can hypothesize that high level of circulating BTN2A1 could lead to Vg9Vd2 T-cells exhaustion that facilitate tumor immune escape mechanisms (18), and/or activate Vg9Vd2 T cells with pro-tumorigenic characteristics, and explain the poorer outcomes of patients with high level of circulating BTN2A1. These Vg9Vd2 T-cells alterations could also impaired anti-PD-1 activities on these cells and then, impaired clinical efficacy of anti-PD-1 agents in patients with mRCC.
Soluble BTN2A1 could be a new approach to predict resistance and avoiding toxicities for mRCC treated by nivolumab. Targeting BTN sub-family molecule could represent a new therapy approach for mRCC patients. Despite the small number of patients in this cohort, our results suggest that the baseline level of plasmatic BTN2A1 could be an independent prognosis factor of PFS after nivolumab for pretreated patient with mRCC. However, these results need to be validated in a larger prospective cohort and the biological role of BTN subfamily and gd T cell immunity in mRCC must be elucidated.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institut Paoli Calmettes. The patients/participants provided their written informed consent to participate in this study.