Serum MMP-7 as an early and specific biomarker for hepatic graft-versus-host disease

Abstract

Abstract [Introduction] The liver is a major target organ of acute graft-versus-host disease (aGVHD), along with the skin and gastrointestinal tract. Hepatic aGVHD occurs in approximately 10% of patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) and is associated with poor prognosis due to its treatment-resistant nature. Although we previously showed that bile duct injury occurs earlier, the diagnosis of hepatic aGVHD still relies on elevated serum total bilirubin (T-bil), which emerges only after bile duct injury has progressed (Hasegawa Y: Blood 2024). This diagnostic delay may contribute to the poor prognosis of hepatic aGVHD, highlighting the need for the early diagnostic biomarkers to detect bile injury before T-bil elevation. We previously demonstrated that matrix metalloproteinase-7 (MMP-7) expression is elevated in bile duct epithelial cells (BECs) in mouse aGVHD (Hasegawa Y: Blood 2024). Importantly, MMP-7 is also secreted by injured biliary epithelial cells and detectable in serum following BEC damage (Lertudomphonwanit C: Sci Transl Med. 2017). Based on these findings, we evaluated the role of serum MMP-7 as a diagnostic or predictive biomarker of hepatic aGVHD using both murine models and clinical serum samples from patients who underwent allo-HCT. [Methods] We utilized a well-established aGVHD murine HCT model in which BDF1 (H-2b/d) recipients were transplanted with bone marrow cells and splenocytes from allogeneic B6 donors (H-2b/b; Allo) or syngeneic BDF1 (Syn). Liver tissues were collected on day +14 and +28 post-transplant for histological assessment, immunofluorescence staining, and qPCR targeting Mmp7. Serum samples were obtained for T-bil and ELISA-based quantification of MMP-7.
In the clinical study, serum samples were collected prior to allo-HCT and then weekly until day +28. [Results] In the mouse model,livers from Allo group showed marked T-cell infiltration in portal areas and around bile ducts by day +14 (p<0.0001), and apoptosis and exfoliation of BECs were evident by day +28. qPCR revealed a 10-fold increase in Mmp7 mRNA in the Allo group compared to Syn controls on both day +14 and +28 (p<0.001), and immunofluorescence analysis showed that MMP-7 expression was clearly localized to BECs in Allo recipients. Serum MMP-7 levels were significantly elevated in the Allo group on day +14 and +28 (p<0.01), whereas T-bil levels showed only minimal elevation at day +14 and a delayed increase on day +28 (p=0.004).
These findings suggest that serum MMP-7 reflects bile duct injury caused by hepatic GVHD and that its elevation precedes the rise in T-bil. Administration of CCl4 (5 mmol/kg, i.p.), which induces oxidative injury of hepatocytes without affecting bile ducts, did not alter Mmp7 expression at day +6 post-injection, confirming that MMP-7 is a specific biomarker for BEC injury.
In the clinical study, serum samples were collected from 21 allo-HCT recipients and 7 healthy controls. Samples from the allo-HCT recipients were categorized into three groups: hepatic aGVHD (n=8), non-hepatic aGVHD (skin and/or gut aGVHD without liver involvement; n=6), and no aGVHD (n=7). All samples in the hepatic aGVHD group were collected before liver GVHD was diagnosed (T-bil ≥2.0 mg/dL). Serum MMP-7 levels were significantly higher in the hepatic aGVHD group compared to non-hepatic aGVHD, no aGVHD and healthy control groups (mean: 9.9 ng/mL vs. 5.1, 4.4 and ≤ 4.6 ng/mL, respectively; p<0.001). Using a cutoff of 5.9 ng/mL, all patients with elevated serum MMP-7 subsequently developed clinical hepatic aGVHD, with a median interval of 14 days between MMP-7-elevation and T-bil-elevation (range: 6–40 days). Importantly, sequential analysis of serum samples showed that only hepatic aGVHD patients showed progressive increases in serum MMP-7, while non-hepatic aGVHD cases did not. ROC analysis of serum MMP-7 levels on day +28 yielded an AUC of 0.96, indicating excellent predictive performance of this biomarker. [Conclusion] Serum MMP-7 is a promising, sensitive, and BEC injury-specific predictive biomarker for hepatic aGVHD. Its elevation precedes the rise in conventional markers such as T-bil. Incorporating MMP-7 monitoring into clinical practice may facilitate earlier diagnosis and improve treatment outcomes for hepatic aGVHD. Future prospective studies are warranted to validate its clinical utility in larger patient cohorts.