Biology Reference
In-Depth Information
represented at equal levels, an inherent limitation of this approach is intro-
duced by the availability and selection of antibodies. To overcome these
limitations and create a broader, less-biased approach, we utilized state-of-
the-art proteomics technology that is straightforward, gel-free, and based
on high-resolution MS: the IPAS that was described in detail above. It may be
difficult to find proteins in the blood that are expressed in tissue, but since
tissue proteins can leak into the bloodstream, this approach might be rea-
sonable. To assess the validity of this approach, we compared plasma pooled
from 10 patients with skin-specific GVHD to that from 10 patients without
GVHD, in one IPAS experiment, and plasma pooled from 10 patients with
GI tract-specific GVHD to 10 controls in a second IPAS experiment. Of thou-
sands of spectra analyzed and hundreds of proteins identified and quanti-
fied, elafin emerged as the lead biomarker candidate for skin GVHD at the
time of clinical diagnosis. We showed that plasma elafin concentrations in
samples from 492 patients had significant diagnostic and prognostic value,
including long-term survival, as a biomarker for skin GVHD
[23]
. Compared
to the four previously reported systemic diagnostic biomarkers of aGVHD,
elafin was shown to be the single best discriminator for the diagnosis of
GVHD in patients with a rash; the combination of all five biomarkers further
improves the specificity and sensitivity of the test. These data also provide
a proof-of-principle demonstration that biomarkers of disease-related, tis-
sue-specific changes can be detected in patient plasma. Using the same pro-
teomics strategy, we discovered 74 proteins with increased ratios (i.e., heavy
to light) in patients with GI GVHD, and 5 were of GI origin. Regenerating-
islet-derived-3α (REG3α) was the lead candidate and was validated as a bio-
marker of lower GI GVHD using ELISA. We subsequently validated REG3α
in two independent sets totaling 1014 patients from three different centers.
This marker provides important prognostic information, including response
to GVHD treatment and survival
[22]
. In a follow-up study, REG3α was com-
pared to KRT18 and HGF, previously identified as GI GVHD markers. REG3α
showed that its diagnostic precision for lower GI GVHD was higher than
those of the other two GI GVHD markers
[71]
.
467
There are also shortcomings in the prediction of the response to GVHD
therapy. Luft et al.
[68]
showed that KRT18 and markers of endothelial dys-
function are elevated in steroid-refractory GVHD patients. We measured
our six previously validated diagnostic biomarkers of GVHD from samples
prospectively obtained at the initiation of treatment, day 14, and day 28, in
a multicenter, randomized, four-arm phase II clinical trial for newly diag-
nosed aGVHD. We found that at each of three time points, GVHD onset, 2
weeks into treatment, and 4 weeks into treatment, a panel of six biomark-
ers predicted the important clinical outcomes of day 28 post-therapy non-
response and mortality at day 180 from onset. GVHD biomarker panels
can be used for the early identification of patients at high or low risk for
treatment nonresponsiveness or death and may provide opportunities for
early intervention and improved survival following HSCT. In addition, we
further analyzed biomarkers of resistance to GVHD therapy with an IPAS
experiment and found that they differed from the biomarkers discovered
at GVHD onset. We subsequently identified novel pathways, such as those
for drug resistance, glucocorticoid receptor signaling, glycolysis, cata-
bolic processes, the oxidative stress response, the IL-1 family, and T helper
Search WWH ::
Custom Search