What is GvHD?
Bone marrow transplantation is sometimes the only treatment option for patients with blood cancers like leukemia and lymphoma. In some cases, the donor cells attack a patient’s healthy tissues in a phenomenon known as graft-versus-host disease (GvHD).
In gut GvHD, the donor cells attack the patient’s gut mucosa, causing severe inflammation. More severe cases of gut GvHD have a survival rate of only 25%. The standard therapy for gut GvHD is the administration of glucocorticoids, but a substantial number of patients relapse and their prognosis is extremely poor. Determining who will respond well to such therapy is critical to enabling healthcare providers to design optimal treatment strategies.
In recent years, we have begun to understand the types of immune reactions that take place in the gut, noted Dr. Miyawaki. Various immune cells interact with each other in response to gut microbiota, playing a critical role in gut health. It is still unknown, however, which immune reactions cause the effects of gut GvHD.
Before describing the methods of the study, Dr. Miyawaki explained some of the limitations they faced with their samples. Most gut GvHD samples are obtained via endoscopic biopsy from severely ill patients, making it difficult to obtain a large number of samples. They are also commonly archived as formalin-fixed, paraffin-embedded (FFPE) tissues.
RNA extracted from FFPE is usually severely degraded, making it unsuitable for whole transcriptome analysis. Because tissues are already fixed, they cannot be used to generate single-cell suspensions, which also rules out global immune profiling through single-cell analysis methods like single-cell sequencing or flow cytometry.
With these issues in mind, Dr. Miyawaki’s team instead used two different technologies: probe-based gene expression profiling and the CODEX® system for ultra-high multiplex immunofluorescence, which can be used in both fresh frozen and FFPE tissues.
Using probe-based gene expression profiling in gut GvHD samples, the team observed that mast cell-related genes were upregulated in patients responsive to steroidal therapy, while macrophage-related genes were upregulated in non-responders. “These results motivated us to see whether we could stratify patients by mast cell or macrophage signatures and associate them with prognosis”, said Dr. Miyawaki.
“We performed multiplex imaging and analysis on CODEX…we stained the gut GvHD tissues with 38 antibodies and performed high-plex spatial analysis.”
In order to determine the mechanisms behind these observations, the team decided to perform multiplex imaging analysis with CODEX. To do so, they collaborated with Aaron Mayer at Enable Medicine, a service provider which specializes in high-dimensional spatial analysis methods. They stained gut GvHD samples with 38 antibodies and performed high-plex spatial analysis using the CODEX platform to validate and expand on the gene expression profiling results.