# Whole-Blood Longitudinal Molecular Profiling Maps the Road of Graft Versus Host Disease (GVHD)

## TL;DR

This study uses a minimally invasive blood test to track gene activity in transplant patients, revealing molecular patterns linked to graft versus host disease (GVHD) over time.

## Contribution

A microfluidics-based method enables frequent, low-volume blood testing to map molecular changes associated with GVHD onset and progression.

## Key findings

- Neutrophil activation and interferon signatures mark the start of acute GVHD.
- Erythroid signatures distinguish acute and mild chronic GVHD.
- Protein-synthesis and B-cell-related signatures are linked to late acute/overlap GVHD.

## Abstract

With a method that measures the abundance of 264 genes from a few drops of fingerstick-collected blood, we analyzed blood changes in patients after allogeneic hematopoietic cell transplantation for the first time at high frequency: every week/second week. By correlating the results with patients’ health status, we discovered important biological processes conducive to graft versus host disease (GVHD). Such genes may suggest a way to prevent GVHD.

Background: Graft versus host disease (GVHD) and the graft versus tumor (GVT) effect after allogeneic hematopoietic cell transplantation (allo-HCT) result from complex interactions between the donor immune system and the recipient environment. High-temporal longitudinal monitoring might be necessary to identify triggering events of GVHD and GVT and to intercept these events before their occurrence. But it would require an overall considerable amount of blood by venipuncture, which is unfeasible in such a fragile population. Methods: In this study, we implemented a targeted multiplex microfluidics q-PCR-based transcriptional fingerprint assay (TFA) on 50 µL of blood collected by a simple fingerstick to evaluate post-allo-HCT systemic immune perturbations associated with the development of GVHD. Fluctuations of a panel of 264 genes were measured in 31 allo-HCT patients by frequent (weekly or biweekly) analysis of 50 µL serial blood samples. Cross-sectional and longitudinal analyses correlated with detailed clinical annotations were performed. Results: Signatures of neutrophil activation and interferon (IFN) characterized the onset of acute GVHD, while an ongoing cytotoxic response was modulated in chronic mild GVHD and protein-synthesis and B-cell-related signatures characterized late acute/overlap GVHD. An unexpected erythroid signature distinguished patients with acute and mild chronic GVHD. Conclusions: Our micro-invasive approach unveiled the molecular heterogeneity of GVHD and identified hierarchically important biological processes conducive to different forms of GVHD. These findings increase our understanding of GVHD and reveal potentially targetable alterations. This approach might be implemented clinically to intercept GVHD before its occurrence and to modulate therapeutic interventions accordingly.