Development of a Flow Cytometry Assay to Predict Immune Checkpoint Blockade-Related Complications

Abstract

Treatment of advanced melanoma with combined immune checkpoint inhibitor (ICI) therapy is complicated in up to 50% of cases by immune-related adverse events (irAE) that commonly include hepatitis, colitis and skin reactions. We previously reported that pre-therapy expansion of cytomegalovirus (CMV)-reactive CD4+ effector memory T cells (TEM) predicts ICI-related hepatitis in a subset of patients with Stage IV melanoma given αPD-1 and αCTLA-4. Here, we develop and validate a 10-color flow cytometry panel for reliably quantifying CD4+ TEM cells and other biomarkers of irAE risk in peripheral blood samples. Compared to previous methods, our new panel performs equally well in measuring CD4+ TEM cells (agreement = 98%) and is superior in resolving CD4+ CD197+ CD45RA- central memory T cells (TCM) from CD4+ CD197+ CD45RA+ naive T cells (Tnaive). It also enables us to precisely quantify CD14+ monocytes (CV = 6.6%). Our new “monocyte and T cell” (MoT) assay predicts immune-related hepatitis with a positive predictive value (PPV) of 83% and negative predictive value (NPV) of 80%. Our essential improvements open the possibility of sharing our predictive methods with other clinical centers. Furthermore, condensing measurements of monocyte and memory T cell subsets into a single assay simplifies our workflows and facilitates computational analyses.

Advanced Immune Cell Profiling by Multiparameter Flow Cytometry in Humanized Patient-Derived Tumor Mice

“…Similar to the approach described here, flow cytometric protocols for immune cell profiling were already used to identify checkpoint-related complications in melanoma patients receiving combined PD-1/CTLA-4 blockade [34,90]. In another study, a flow cytometric assay was applied to analyze tumor and peripheral blood samples addressing the cellular "immunome" in patients with melanoma, breast cancer and glioblastoma [91].…”

Applications of Flow Cytometry in Drug Discovery and Translational Research

“…Flow cytometric techniques have been increasingly deployed in early-phase clinical trials but are most often limited to measuring exploratory endpoints in support of POM or in providing mechanistic insights enabling reverse translation. Several studies have leveraged flow cytometry or related methods such as CyTOF to identify biomarkers of response to immune-checkpoint blockade [101,102] or to monitor safety-related safety complications [101]. Clinical trials in asthma patients often rely on indirect measurements of circulating cytokines or clinical endpoints related to lung function.…”

Prediction of immune checkpoint blockade‐related hepatitis in metastatic melanoma patients

“…This approach showed great impact as none of the four patients developed liver inflammation. This treatment concept will now be examined within a clinical trial using a validated flow cytometry assay for immune phenotyping blood samples of melanoma patients [16].…”