Cytokeratin (CK)-18 and CK18 Fragments As Sensitive and Specific Biomarkers of Acute Hepato-Intestinal Gvhd: Results of a Prospective Observational Study
Authors: Sauer Sandra , Radujkovic Aleksandar , Hegenbart Ute , Ho Anthony D , Dreger Peter , Luft Thomas 
Published: October 2019

Abstract
Introduction:
Preliminary data have suggested that CK18, a highly abundant protein in liver and gut epithelial cells, and its apoptotic degradation product CK18 fragments (CK18F) could indicate apoptotic end organ damage induced by acute hepato-intestinal GVHD and, thus, may be used as an acute GVHD biomarker (Luft et al, Blood 2011; 118:1685). Here, we present the first results of a clinical study aiming at prospective validation of the capacity of CK18/ CK18F to predict acute hepato-intestinal GvHD activity and its response to treatment.
Patients and methods:
Main endpoints of the study were (1) prediction of imminent acute hepato-intestinal GvHD by comparing CK18/CK18F serum levels measured 7 to 14 days prior to symptom onset to with baseline levels (pre-conditioning, at transplant and one week after alloSCT) of the same subject; and (2) prediction of response to GVHD treatment by measuring CK18/CK18F kinetics 3, 7 and 14 days after start of immunosuppressive therapy for hepato-intestinal GvHD. Eligible were all adult patients undergoing allogeneic stem cell transplantation (alloSCT) at our institution from December 2008 onwards. Target sample size was 100. Blood samples were collected in the study population prior to the initiation of the conditioning regimen and from the day of transplantation on a weekly basis for one year. The M30-Apoptosense ELISA kit was used for measuring serum levels of CK18 fragments, CK18 was analyzed with the M65 (EpiDeath) ELISA Kit (both provided by PEVIVA, Nacka, Sweden). Serum levels measured at the pre-specified landmarks were compared by Wilcoxon's signed rank test. The study was registered at ClinicalTrials.gov (Identifier: NCT00935324).
Results:
Between December 2008 to February 2011, 109 patients were enrolled. The median age was 53 years (range 19 - 69), 72 were male. The underlying disease was acute myeloid leukemia in 39 patients, myeloproliferative / myelodysplastic disorders in 18 patients, acute lymphoblastic leukemia in 10 patients, and lymphoproliferative malignancies in 42 patients. Donors were matched related (36), matched unrelated (52), or mismatched unrelated (21). Stem cell source was peripheral blood in 100 patients and bone marrow in 9 patients. Reduced intensity conditioning protocols (n = 89) prevailed over myeloablative (n = 7) and aplasia conditioning protocols (n = 13). Immunosuppression was based on a calcineurin inhibitor (CNI) backbone with mycophenolate or short-course methotrexate, plus ATG in mismatched or unrelated transplants. Patients received pravastatin (20 mg daily dose) for routine prophylaxis of CNI-induced vascular complications as per institutional policy as of 2010.
Acute hepato-intestinal GVHD grade II-IV developed in 38 patients (34,9 %) at a median of 53 days post transplant and was steroid-refractory in 12 patients. In patients with biopsy-proven acute hepato-intestinal GVHD, CK18 significantly raised 7 to 14 days before clinical GVHD onset and can therefore serve as an early predictor of impending acute hepato-intestinal GVHD (median at baseline vs preGVHD, 149 ng/ml vs 527 ng/ml; p=0.012), and peaked at symptom onset (median 1281 ng/ml; p=0.003, compared to baseline). CK18F kinetics paralleled that of CK18 but was less pronounced. In contrast, patients with isolated acute skin GVHD, patients without acute GVHD (cut-off day+53), and patients with GVHD-typical symptoms but a negative biopsy did not show any increase CK18 or CK18F in serum (Figure 1). However, CK18/CK18F levels were not correlated with acute GVHD severity. Neither marker predicted refractory GVHD or non-relapse mortality after acute GVHD at any of the time points measured.
Conclusions:
This prospective study validates CK18 and CK18F as specific biomarkers suitable for prediction and diagnosis of suspected imminent and clinically manifest acute hepato-intestinal GVHD. In contrast, neither CK18 nor CK18F can be used to predict the prognosis of an ongoing GVHD episode. Interventional trials employing CK18 as trigger for pre-emptive GVHD therapy seem to be warranted.

Disclosures
Hegenbart: Janssen: Honoraria, Other: travel support. Ho:Genzyme/Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Luft:Immundiagnostik AG: Research Funding.