Analysis of tumor infiltrating CD4+ and CD8+ CDR3 sequences reveals shared features putatively associated to the anti-tumor immune response.

Introduction: Tumor-infiltrating lymphocytes (TILs) have predictive and prognostic value in breast cancer (BC) and exert a protective function against tumor growth, indicating that it is susceptible to treatment using adoptive cell transfer of TILs or T cell receptor (TCR)-based therapies. TCR can be used to identify naturally tumor-reactive T cells, but little is known about the differences in the TCR repertoires of CD4+ and CD8+ TILs. Methods: TCR high-throughput sequencing was performed using TILs derived from the initial cultures of 11 BC biopsies and expanded and sorted CD4+ and CD8+ TILs as well as using PBMCs from healthy donors expanded and sorted using the same methodology. Results: Physicochemical TCR differences between T cell subsets were observed, as CD4+ TILs presented larger N(D)Nnt TRB sequences and with a higher usage of positively charged residues, although only the latest was also observed in peripheral T cells from healthy individuals. Moreover, in CD4+ TILs, a more restricted TCR repertoire with a higher abundance of similar sequences containing certain amino acid motifs was observed. Discussion: Some differences between CD4+ and CD8+ TCRs were intrinsic to T cell subsets as can also be observed in peripheral T cells from healthy individuals, while other were only found in TILs samples and therefore may be tumor-driven. Notably, the higher similarity among CD4+ TCRs suggests a higher TCR promiscuity in this subset.

Epstein-Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report.

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

Application of failure mode and effects analysis to intracranial stereotactic radiation surgery by linear accelerator.

PURPOSE: The aim of this study was to analyze the application of the failure modes and effects analysis (FMEA) to intracranial stereotactic radiation surgery (SRS) by linear accelerator in order to identify the potential failure modes in the process tree and adopt appropriate safety measures to prevent adverse events (AEs) and near-misses, thus improving the process quality. METHODS AND MATERIALS: A working group was set up to perform FMEA for intracranial SRS in the framework of a quality assurance program. FMEA was performed in 4 consecutive tasks: (1) creation of a visual map of the process; (2) identification of possible failure modes; (3) assignment of a risk probability number (RPN) to each failure mode based on tabulated scores of severity, frequency of occurrence and detectability; and (4) identification of preventive measures to minimize the risk of occurrence. RESULTS: The whole SRS procedure was subdivided into 73 single steps; 116 total possible failure modes were identified and a score of severity, occurrence, and detectability was assigned to each. Based on these scores, RPN was calculated for each failure mode thus obtaining values from 1 to 180. In our analysis, 112/116 (96.6%) RPN values were <60, 2 (1.7%) between 60 and 125 (63, 70), and 2 (1.7%) >125 (135, 180). The 2 highest RPN scores were assigned to the risk of using the wrong collimator's size and incorrect coordinates on the laser target localizer frame. CONCLUSION: Failure modes and effects analysis is a simple and practical proactive tool for systematic analysis of risks in radiation therapy. In our experience of SRS, FMEA led to the adoption of major changes in various steps of the SRS procedure.