Elafin Is a Biomarker of Graft Versus Host Disease of the Skin

There are no plasma biomarkers that are specific to any of the three target organs of acute graft versus host disease (GVHD): skin, GI tract and liver. We sought to identify a biomarker that is specific for GVHD of the skin in an initial discovery step using an intact proteomic analysis system. We compared plasma pooled from ten patients with GVHD only of the skin (sGVHD) to plasma pooled from ten patients with no GVHD to plasma pooled from ten patients with GVHD only of the GI tract. Of four candidate proteins that were both significantly elevated only in the plasma of sGVHD patients and that could be measured by ELISA, we selected elafin, an epidermal proteinase inhibitor that is induced by TNF-α and found in inflamed epidermis in diseases such as psoriasis. We therefore measured levels of elafin (expressed hereafter as mean ± SEM pg/ml) in individual samples of the discovery set, confirming that they were significantly higher in plasma from patients with sGVHD compared to patients presenting only with GI GVHD or without GVHD, respectively (13,312 ± 2456 vs. 3968 ± 537 vs. 3257 ± 332, p < 0.0001). We next analyzed a validation set of 429 plasma samples from allogeneic BMT patients transplanted at the University of Michigan from 2000–2008. We obtained samples at regular intervals from all patients until day 100 after BMT and at the first clinical signs of sGVHD. 275 (64%) patients had no GVHD and 154 (36%) had sGVHD. There were no statistically significant differences between patients with sGVHD and without GVHD with respect to age, underlying hematological malignancies and conditioning intensity. Recipients of grafts from donors who were not family members or who were less than 8/8 HLA identical matches were overrepresented in the sGVHD group. The median post-transplant day for sample collection was day 31 in GVHD-group and day 30 in sGVHD group. Elafin levels were two fold higher in plasma from patients with sGVHD as compared to plasma from patients without GVHD (7532 ± 488 vs. 3925 ± 112, p < 0.0001). We next analyzed whether elafin levels provided prognostic information regarding

• eventual maximum stage of GVHD skin,

• transplant-related mortality and

• overall survival.

We divided the patients into 2 groups using a threshold level of elafin that provided 85% specificity (5500 pg/ml). The group with the high levels of elafin comprised 29% of the total patient population and developed more severe skin GVHD (maximum stage) than the group with low levels (p < 0.0001, Table 1). Patients with high elafin levels also experienced greater transplant-related mortality (TRM) at 1 year: 18% (95% CI, 11–25%) vs. 7% (95% CI, 4–11%) (p = 0.002). This difference remained significant when adjusted for age, family member donor, conditioning intensity and HLA mismatch (p = 0.01). Patients with high elafin levels also experienced lower overall survival (OS) at 1 year: 61% (95% CI, 52–71%) vs. 76% (95% CI, 71–82%) (p < 0.0001, adjusted for all the factors above) (Figure 1). Within the population of patients with sGVHD (n =154), patients with high elafin had a mortality-hazard ratio of 1.98 compared to patients with low elafin levels (p = 0.003). This hazard ratio of 1.98 remained significant (p = 0.017) after adjusting for stage of skin disease at onset, demonstrating that the association of mortality risk with elafin levels was independent of the GVHD clinical stage. In additional studies, we determined whether elafin levels measured 7 days prior to clinical onset of skin disease predicted a subsequent occurrence of sGVHD. In samples available from 133 patients, 7 days prior to sGVHD onset, elafin levels were 1.26 fold higher than in patients who did not later develop GVHD (4279 ± 222 vs. 3448 ± 108, p = 0.001). We conclude that elafin is a novel biomarker for sGVHD that can provide important diagnostic and prognostic information, including long term survival.

Table 1: Correlating low/high elafin with maximum skin stage Maximum skin stage

.	Maximum skin stage .		.
.	0–1 .	2–4 .	p-value .
Low elafin	83%	17%	<0.0001
High elafin	38%	62%

.	Maximum skin stage .		.
.	0–1 .	2–4 .	p-value .
Low elafin	83%	17%	<0.0001
High elafin	38%	62%

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