Longitudinal Assessment of Tumor-Infiltrating Lymphocytes in Primary Breast Cancer Following Neoadjuvant Radiation Therapy.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have prognostic significance in several cancers, including breast cancer. Despite interest in combining radiation therapy with immunotherapy, little is known about the effect of radiation therapy itself on the tumor-immune microenvironment, including TILs. Here, we interrogated longitudinal dynamics of TILs and systemic lymphocytes in patient samples taken before, during, and after neoadjuvant radiation therapy (NART) from PRADA and Neo-RT breast clinical trials. METHODS AND MATERIALS: We manually scored stromal TILs (sTILs) from longitudinal tumor samples using standardized guidelines as well as deep learning-based scores at cell-level (cTIL) and cell- and tissue-level combination analyses (SuperTIL). In parallel, we interrogated absolute lymphocyte counts from routine blood tests at corresponding time points during treatment. Exploratory analyses studied the relationship between TILs and pathologic complete response (pCR) and long-term outcomes. RESULTS: Patients receiving NART experienced a significant and uniform decrease in sTILs that did not recover at the time of surgery (P < .0001). This lymphodepletive effect was also mirrored in peripheral blood. Our SuperTIL deep learning score showed good concordance with manual sTILs and importantly performed comparably to manual scores in predicting pCR from diagnostic biopsies. The analysis suggested an association between baseline sTILs and pCR, as well as sTILs at surgery and relapse, in patients receiving NART. CONCLUSIONS: This study provides novel insights into TIL dynamics in the context of NART in breast cancer and demonstrates the potential for artificial intelligence to assist routine pathology. We have identified trends that warrant further interrogation and have a bearing on future radioimmunotherapy trials.

Genetic and immune landscape evolution in MMR-deficient colorectal cancer.

Mismatch repair-deficient (MMRd) colorectal cancers (CRCs) have high mutation burdens, which make these tumours immunogenic and many respond to immune checkpoint inhibitors. The MMRd hypermutator phenotype may also promote intratumour heterogeneity (ITH) and cancer evolution. We applied multiregion sequencing and CD8 and programmed death ligand 1 (PD-L1) immunostaining to systematically investigate ITH and how genetic and immune landscapes coevolve. All cases had high truncal mutation burdens. Despite pervasive ITH, driver aberrations showed a clear hierarchy. Those in WNT/ß-catenin, mitogen-activated protein kinase, and TGF-ß receptor family genes were almost always truncal. Immune evasion (IE) drivers, such as inactivation of genes involved in antigen presentation or IFN-γ signalling, were predominantly subclonal and showed parallel evolution. These IE drivers have been implicated in immune checkpoint inhibitor resistance or sensitivity. Clonality assessments are therefore important for the development of predictive immunotherapy biomarkers in MMRd CRCs. Phylogenetic analysis identified three distinct patterns of IE driver evolution: pan-tumour evolution, subclonal evolution, and evolutionary stasis. These, but neither mutation burdens nor heterogeneity metrics, significantly correlated with T-cell densities, which were used as a surrogate marker of tumour immunogenicity. Furthermore, this revealed that genetic and T-cell infiltrates coevolve in MMRd CRCs. Low T-cell densities in the subgroup without any known IE drivers may indicate an, as yet unknown, IE mechanism. PD-L1 was expressed in the tumour microenvironment in most samples and correlated with T-cell densities. However, PD-L1 expression in cancer cells was independent of T-cell densities but strongly associated with loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and may contribute to a higher recurrence risk of MMRd CRCs with impaired CDX2 expression. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Determinants of anti-PD-1 response and resistance in clear cell renal cell carcinoma.

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action.

The T cell differentiation landscape is shaped by tumour mutations in lung cancer.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

Geospatial immune variability illuminates differential evolution of lung adenocarcinoma.

Remarkable progress in molecular analyses has improved our understanding of the evolution of cancer cells toward immune escape1-5. However, the spatial configurations of immune and stromal cells, which may shed light on the evolution of immune escape across tumor geographical locations, remain unaddressed. We integrated multiregion exome and RNA-sequencing (RNA-seq) data with spatial histology mapped by deep learning in 100 patients with non-small cell lung cancer from the TRACERx cohort6. Cancer subclones derived from immune cold regions were more closely related in mutation space, diversifying more recently than subclones from immune hot regions. In TRACERx and in an independent multisample cohort of 970 patients with lung adenocarcinoma, tumors with more than one immune cold region had a higher risk of relapse, independently of tumor size, stage and number of samples per patient. In lung adenocarcinoma, but not lung squamous cell carcinoma, geometrical irregularity and complexity of the cancer-stromal cell interface significantly increased in tumor regions without disruption of antigen presentation. Decreased lymphocyte accumulation in adjacent stroma was observed in tumors with low clonal neoantigen burden. Collectively, immune geospatial variability elucidates tumor ecological constraints that may shape the emergence of immune-evading subclones and aggressive clinical phenotypes.

Neoantigen-directed immune escape in lung cancer evolution.

The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.

Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.

Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8(+) T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8(+) T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK's immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8(+) T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

Global outcome, productivity and epilepsy 3--8 years after severe head injury. The impact of injury severity.

OBJECTIVE: To assess long-term outcome in survivors after severe head injury and relate outcome to injury severity. DESIGN: Follow-up 3-8 years post injury in a retrospectively collected sample. SETTING: A neurosurgical department in a regional trauma centre. SUBJECTS: Of 146 individuals admitted, 135 were actively treated. Twenty-four per cent of these died within six months. Ninety-three of the 94 (aged 1-88 years) who survived more than three years were included in the follow-up. They were separated into groups based on their level of consciousness at four weeks post injury: oriented (n = 39), confused (n = 22) or in a minimally conscious/vegetative state (MCS/VS) (n = 26) and not possible to assess (n = 6). OUTCOME MEASURES: Glasgow Outcome Scale Extended (GOSE), participation in work/education (productivity) and posttraumatic epilepsy. RESULTS: GOSE scores were: vegetative state: 3%, severe disability: 28% (22% lower level, 6% upper level), moderate disability: 39% (22% lower level, 17% upper level) and good recovery: 27% (10% lower level, 17% upper level). Productivity was 34% (aged 7-64 years). The three severity groups had different GOSE scores (P<0.001) and different proportion of productive individuals (P<0.001). Twenty-three per cent experienced posttraumatic epilepsy and this was significantly associated with the highest injury severity (P<0.001) and intracranial surgery (P = 0.01). CONCLUSIONS: Being independent in daily life but unable to work was the typical long-term outcome. Stratifying the patients based on consciousness at four weeks we found different outcomes. Among oriented patients, almost all regained independency, whereas in the most severe group, poor outcomes and posttraumatic epilepsy was common.