Identifying Biomarkers for Acute GVHD.

Acute graft versus host disease (aGVHD) is a complication of allogeneic hematopoietic cell transplantation (HCT). Current prevention and treatment approaches are effective in many but not all cases. Acute GVHD after a myeloablative HCT has a median onset of ~21 days, with a few cases occurring as late as 3 months post-HCT. We hypothesized that global gene expression profiling of RNA from white blood cells (WBC) could identify biomarkers associated with onset of aGVHD and potentially provide insight into the mechanisms responsible for causing clinically significant aGVHD. Patients were enrolled prospectively and blood samples collected weekly until onset of aGVHD or day 90. RNA samples adequate for hybridization were collected between day 19 and 24 from 23 patients who developed aGVHD (GVHD+) and compared with RNA from 13 patients who remained free of aGVHD through day 90 (GVHD−). All blood samples from GVHD+ patients were obtained prior to initiation of steroid therapy.

Biotin-labeled cRNA was hybridized on Affymetrix HG-U133A. Realizing quantitative complexities underlying gene expression assessment and different assumptions required by different algorithms, we used 5 different summary algorithms (Mas5, PLIER, RMA, gcRMA, Dchip) to compute gene expression levels. Subsequently, we carried out analyses and identified genes that were consistently identified in more than one analyses; replications imply robustness of discoveries, even though discovery efficiency is compromised to some degree. A total of 141 genes showing differential expression were discovered.

Thirteen functional classes were identified for 101 genes. The remaining 40 genes had unknown function.

Sixteen transcription factors were identified, 12 of them showing increased expression such as the early response genes EGR1, EGR2, FOS and JUN. Among 12 immune response genes identified increased expression was observed for CD83 and IL-18, associated with antigen presentation and adaptive immunity; and NRS2, MMP9 and CD157, associated with innate immune response. Decreased expression was observed for CD52 and PTPN11.

Among 7cell cycle progression and proliferation genes, we observed increased expression for CSF2, VEGF, PTN and TGFa. These data are consistent with the hypothesis that the development of acute GVHD is associated with acute inflammation. Ongoing validation studies using Taqman have confirmed up regulation of CD83 and EGR2 and down regulation of CD52 in GVHD+ patients.

To further understand the functional implications of the 141 genes identified, probe sets were also input into Ingenuity Pathway Analysis software to identify possible signaling and regulatory pathways. The Ingenuity defined pathways most densely populated by the 141 genes included pathways controlling cytokine production and inflammation. Network analysis showed upregulation of transcriptional pathways responsible for cell proliferation, growth and activation: IL18, CSF2, TGFa and the transcription factors EGR1, EGR2, FOS and JUN were linked to networks that appear to have important role in regulation of expression of VEGF suggesting that it may be involved in sustaining inflammatory response in GVHD.

This study demonstrates that whole genome transcriptional analysis of WBC RNA can be informative for detecting biomarkers associated with aGVHD. Furthermore these findings suggest that these biomarkers can be used to better define cellular pathways associated with clinical onset and/or severity of acute GVHD and thereby provide potential targets for therapy.