---
title: AI-Derived Macrophage Density Reveals Hot and Cold Fibrosis Endotypes with Distinct Biomarker Profiles in Metabolic Dysfunction-Associated Steatohepatitis
description: Agreement Across 10 Artificial Intelligence Models in Assessing Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Breast Cancer Whole-Slide Images
---

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# [AI-Derived Macrophage Density Reveals Hot and Cold Fibrosis Endotypes with Distinct Biomarker Profiles in Metabolic Dysfunction-Associated Steatohepatitis](https://www.pathai.com/publications/ai-derived-macrophage-density-reveals-hot-and-cold-fibrosis-endotypes-with-distinct-biomarker-profiles-in-metabolic-dysfunction-associated-steatohepatitis)

 Written by [Admin](https://www.pathai.com/publications/author/admin) | May 27, 2026 4:00:00 AM

Background and aims: In metabolic dysfunction-associated steatohepatitis (MASH), the biological activity driving fibrogenesis is heterogenous and not adequately represented by CRN fibrosis stage alone. We evaluated a hot vs cold fibrosis framework, defined by AImeasured macrophage density and linked to non-invasive tests (NITs), to identify biologically distinct endotypes and assess candidate NITs for patient selection in early clinical trials. Method: Liver micro-architecture was quantified in 150 biopsyconfirmed MASH patients from the Stravitz-Sanyal Institute using LiverExplore* (PathAI, Boston, MA, *For Research Use Only. Not for use in diagnostic procedures) and by SHG/TPEF (Histoindex, Singapore) to assess the qFibrosis score. CRN Fibrosis was centrally staged. Hot fibrosis was defined as significant fibrosis by CRN staging (F ≥ 2) with macrophage density ≥ 250 cells/mm2 by LiverExplore, and cold fibrosis as CRN F ≥ 2 with density < 250 cells/mm2 . Clinical variables and NITs were compared, including PRO-C3 (Roche Elecsys PRO-C3), CTX III, CTX III/PRO-C3 ratio, enhanced liver fibrosis (ELF) score, thrombospondin 2 (TSP2), and liver stiffness by vibration-controlled transient elastography (VCTE). Results: The median qFibrosis score was 1.86 (IQR 1.27–3.07). Hot fibrosis accounted for 65% of cases and showed higher AST, ELF, PROC3, and TSP2 compared to cold fibrosis cases, while liver stiffness was similar. CTX III and CTX III/PRO-C3 ratios tended to be higher in cold fibrosis. NAS, steatosis, and ballooning did not differ, but hot fibrosis showed greater lobular inflammation. Within standard Phase 2b/3 MASH criteria (F ≥ 2; NAS ≥ 4 with ≥1 in each domain), tightening the lobular inflammation threshold from ≥ 1 to ≥ 2 substantially reduced the proportion of cold fibrosis cases, from ∼ 50% to ∼ 25%. High PROC3 (≥ 40 ng/mL) further enriched for hot fibrosis (88%; ∼ 10% cold). Low PRO-C3 in cold fibrosis reflects low fibrogenic activity and limited capacity for further PRO-C3 decline. Conclusion: Hot and cold fibrosis represent biologically distinct MASH endotypes. Elevated PRO-C3 enriches for active fibrogenesis, while cold fibrosis reflects a stable, low-fibrogenic state unlikely to respond to anti-fibrogenic therapies. Endotype- and biomarkerbased enrichment may improve alignment between mechanism and therapeutic response in MASH trials.

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