Activation of a cGAS-STING-mediated immune response predicts response to neoadjuvant chemotherapy in early breast cancer.

BACKGROUND: The DNA-damage immune-response (DDIR) signature is an immune-driven gene expression signature retrospectively validated as predicting response to anthracycline-based therapy. This feasibility study prospectively evaluates the use of this assay to predict neoadjuvant chemotherapy response in early breast cancer. METHODS: This feasibility study assessed the integration of a novel biomarker into clinical workflows. Tumour samples were collected from patients receiving standard of care neoadjuvant chemotherapy (FEC + /-taxane and anti-HER2 therapy as appropriate) at baseline, mid- and post-chemotherapy. Baseline DDIR signature scores were correlated with pathological treatment response. RNA sequencing was used to assess chemotherapy/response-related changes in biologically linked gene signatures. RESULTS: DDIR signature reports were available within 14 days for 97.8% of 46 patients (13 TNBC, 16 HER2 + ve, 27 ER + HER2-ve). Positive scores predicted response to treatment (odds ratio 4.67 for RCB 0-1 disease (95% CI 1.13-15.09, P = 0.032)). DDIR positivity correlated with immune infiltration and upregulated immune-checkpoint gene expression. CONCLUSIONS: This study validates the DDIR signature as predictive of response to neoadjuvant chemotherapy which can be integrated into clinical workflows, potentially identifying a subgroup with high sensitivity to anthracycline chemotherapy. Transcriptomic data suggest induction with anthracycline-containing regimens in immune restricted, "cold" tumours may be effective for immune priming. TRIAL REGISTRATION: Not applicable (non-interventional study). CRUK Internal Database Number 14232

PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics

Multiplex immunofluorescence (mIF) and digital image analysis (DIA) have transformed the ability to analyse multiple biomarkers. We aimed to validate a clinical workflow for quantifying PD-L1 in non-small cell lung cancer (NSCLC). NSCLC samples were stained with a validated mIF panel. Immunohistochemistry (IHC) was conducted and mIF slides were scanned on an Akoya Vectra Polaris. Scans underwent DIA using QuPath. Single channel immunofluorescence was concordant with single-plex IHC. DIA facilitated quantification of cell types expressing single or multiple phenotypic markers. Considerations for analysis included classifier accuracy, macrophage infiltration, spurious staining, threshold sensitivity by DIA, sensitivity of cell identification in the mIF. Alternative sequential detection of biomarkers by DIA potentially impacted final score. Strong concordance was observed between 3,3'-Diaminobenzidine (DAB) IHC slides and mIF slides (R 2 = 0.7323). Comparatively, DIA on DAB IHC was seen to overestimate the PD-L1 score more frequently than on mIF slides. Overall, concordance between DIA on DAB IHC slides and mIF slides was 95%. DIA of mIF slides is rapid, highly comparable to DIA on DAB IHC slides, and enables comprehensive extraction of phenotypic data and specific microenvironmental detail intrinsic to the sample. Exploration of the clinical relevance of mIF in the context of immunotherapy treated cases is warranted

Improving the Diagnostic Accuracy of the PD-L1 Test with Image Analysis and Multiplex Hybridization

Targeting of the programmed cell death protein (PD-1)/programmed death-ligand 1 (PD-L1) axis with checkpoint inhibitors has changed clinical practice in non-small cell lung cancer (NSCLC). However, clinical assessment remains complex and ambiguous. We aim to assess whether digital image analysis (DIA) and multiplex immunofluorescence can improve the accuracy of PD-L1 diagnostic testing. A clinical cohort of routine NSCLC patients reflex tested for PD-L1 (SP263) immunohistochemistry (IHC), was assessed using DIA. Samples of varying assessment difficulty were assessed by multiplex immunofluorescence. Sensitivity, specificity, and concordance was evaluated between manual diagnostic evaluation and DIA for chromogenic and multiplex IHC. PD-L1 expression by DIA showed significant concordance (R² = 0.8248) to manual assessment. Sensitivity and specificity was 86.8% and 91.4%, respectively. Evaluation of DIA scores revealed 96.8% concordance to manual assessment. Multiplexing enabled PD-L1+/CD68+ macrophages to be readily identified within PD-L1+/cytokeratin+ or PD-L1-/cytokeratin+ tumor nests. Assessment of multiplex vs. chromogenic IHC had a sensitivity and specificity of 97.8% and 91.8%, respectively. Deployment of DIA for PD-L1 diagnostic assessment is an accurate process of case triage. Multiplex immunofluorescence provided higher confidence in PD-L1 assessment and could be offered for challenging cases by centers with appropriate expertise and specialist equipment