Usefulness and analytical performances of complement multiplex assay for measuring complement biomarkers in plasma.

INTRODUCTION: The complement system is involved in numerous diseases, through diverse mechanisms and degree of activation. With the emergence of complement targeting therapeutic, simple and accessible tools to evaluate the extent of complement activation are strongly needed. METHODS: We evaluated two multiplex panels, measuring complement activation fragments (C4a, C3a, C5a, Bb, Ba, sC5b9) and intact components or regulators (C1q, C2, C3, C4, C5, FD, FP, FH, FI). The specificity of each measurement was assessed by using complement proteins depleted sera and plasma collected from patients with complement deficiencies. Normal values distribution was estimated using 124 plasma samples from healthy donors and complement activation profile was assessed in plasma collected from 31 patients with various complement-mediated disorders. RESULTS: We observed good inter-assay variation. All tested protein deficiencies were accurately detected. We established assay-specific reference values for each analyte. Except for C3, C4 and C4a, the majority of the measurements were in good agreement with references methods or published data. CONCLUSION: Our study substantiates the utility of the Complement Multiplex assay as a tool for measuring complement activation and deficiencies. Quantifying complement cleavage fragments in patients exhibiting classical or alternative pathway activation allowed evaluating the activation state of the whole cascade.

International Validation of the Immunoscore Biopsy in Patients With Rectal Cancer Managed by a Watch-and-Wait Strategy.

PURPOSE: No biomarker capable of improving selection and monitoring of patients with rectal cancer managed by watch-and-wait (W&W) strategy is currently available. Prognostic performance of the Immunoscore biopsy (ISB) was recently suggested in a preliminary study. METHODS: This international validation study included 249 patients with clinical complete response (cCR) managed by W&W strategy. Intratumoral CD3+ and CD8+ T cells were quantified on pretreatment rectal biopsies by digital pathology and converted to ISB. The primary end point was time to recurrence (TTR; the time from the end of neoadjuvant treatment to the date of local regrowth or distant metastasis). Associations between ISB and outcomes were analyzed by stratified Cox regression adjusted for confounders. Immune status of tumor-draining lymph nodes (n = 161) of 17 additional patients treated by neoadjuvant chemoradiotherapy and surgery was investigated by 3'RNA-Seq and immunofluorescence. RESULTS: Recurrence-free rates at 5 years were 91.3% (82.4%-100.0%), 62.5% (53.2%-73.3%), and 53.1% (42.4%-66.5%) with ISB High, ISB Intermediate, and ISB Low, respectively (hazard ratio [HR; Low v High], 6.51; 95% CI, 1.99 to 21.28; log-rank P = .0004). ISB was also significantly associated with disease-free survival (log-rank P = .0002), and predicted both local regrowth and distant metastasis. In multivariate analysis, ISB was independent of patient age, sex, tumor location, cT stage (T, primary tumor; c, clinical), cN stage (N, regional lymph node; c, clinical), and was the strongest predictor for TTR (HR [ISB High v Low], 6.93; 95% CI, 2.08 to 23.15; P = .0017). The addition of ISB to a clinical-based model significantly improved the prediction of recurrence. Finally, B-cell proliferation and memory in draining lymph nodes was evidenced in the draining lymph nodes of patients with cCR. CONCLUSION: The ISB is validated as a biomarker to predict both local regrowth and distant metastasis, with a gradual scaling of the risk of pejorative outcome.