Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

Abstract

Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets.

The end of omics? High dimensional single cell analysis in precision medicine

“…One of the most prominent application of HDcyto technology is the investigation of human pathologies, as it allows a deep understanding of inter‐cellular perturbations and the identification of disease‐specific signatures that may serve as diagnostic biomarkers or, eventually, therapeutic targets. HDcyto has already proved to be a powerful platform for the investigation of idiopathic syndromes and pathologies with incomplete understanding of disease‐driving mechanisms .…”

Risk stratification of allogeneic stem cell recipients with respect to the potential for development of GVHD via their pre-transplant plasma lipid and metabolic signature

“…The identification of biomarkers that better enable the risk stratification of SCT patients with respect to the potential for development of GVHD has significant clinical utility in the execution of BMT protocols. Such a capability enables the development of new treatments and disease detection and enable evidence based treatments 32,33 . The strategy used in our study to enable this goal investigated the plasma metabolome and lipidome to profile SCT patients.…”

The search for drug-targetable diagnostic, prognostic and predictive biomarkers in chronic graft-versus-host disease

“…In one of the studies, CXCR3 + CD56 bright NK cells inversely correlated with CXCL10 as a significant biomarker panel for the diagnosis of cGVHD [41]. Four other studies confirmed the role of natural killer cells in cGVHD where patient biopsies revealed the presence of natural killer cells among the lymphocytes that infiltrated into the skin, liver, and the intestine in those GVHD target tissues [72,73,75,76],.…”