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. 2025 Mar 4;13(1):37.
doi: 10.1186/s40364-025-00752-8.

Folate receptor 1 is a stemness trait-associated diagnostic and prognostic marker for hepatocellular carcinoma

Yuto Shiode #  1   2 , Takahiro Kodama #  1 , Yu Sato  1 , Ryo Takahashi  1 , Takayuki Matsumae  1 , Kumiko Shirai  1 , Akira Doi  1 , Yuki Tahata  1 , Hayato Hikita  1 , Tomohide Tatsumi  1 , Moto Fukai  3 , Akinobu Taketomi  3 , Mathuros Ruchirawat  4   5 , Xin Wei Wang  2   6 , Tetsuo Takehara  7
Affiliations

Folate receptor 1 is a stemness trait-associated diagnostic and prognostic marker for hepatocellular carcinoma

Yuto Shiode et al. Biomark Res. .

Abstract

Background: Hepatocellular carcinoma (HCC) can be classified into several subtypes based on molecular traits, aiding in prognostic stratification. The subtype with a poor prognosis is often associated with stem/progenitor features. This study focused on identifying circulating biomarkers for aggressive HCC.

Methods: We searched for secretory proteins whose expression was positively associated with the stem/progenitor markers KRT19, EPCAM, and PROM1 in 2 independent HCC cohorts. Serum folate receptor 1 (FOLR1) levels were measured in 238 chronic liver disease and 247 HCC patients, evaluating their diagnostic and prognostic capabilities.

Results: FOLR1 was identified as a secretory protein that was positively correlated with all 3 stem/progenitor markers and a poor prognosis in both the discovery and validation cohorts. Higher FOLR1 expression was detected in tumor than nontumor tissues and was associated with aggressive subtypes, and activation of p53, DNA repair, Myc, E2F, and PI3K/AKT/mTOR pathways. Serum FOLR1 levels correlated with tumoral FOLR1 expression in HCC patients and were significantly elevated compared with those in patients with chronic hepatitis or nonliver disease. Serum FOLR1 levels demonstrated diagnostic performance for HCC comparable to that of alpha-fetoprotein (AFP), and their combination increased the diagnostic accuracy. Elevated serum FOLR1 levels were associated with poor prognosis in HCC patients, regardless of treatment, especially in patients with early-stage disease. The multivariate analysis revealed that the serum FOLR1 level and the Gender, Age, AFP-L3, AFP, and Des-gamma-carboxy prothrombin (GALAD) score were independent predictors of a poor prognosis with their combination further stratifying prognosis.

Conclusions: FOLR1 is a stemness-associated biomarker for HCC, with serum levels serving as a diagnostic marker for HCC and a prognostic indicator for early-stage disease.

Keywords: Biomarker; Diagnosis; FOLR1; HCC; Prognosis.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: This study was conducted in accordance with the Declaration of Helsinki. The validation cohort data were derived from the Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC) consortium, which included patients diagnosed with hepatocellular carcinoma (HCC). Institutional Review Board (IRB) approval was obtained from each participating center in Thailand, namely: Maharaj Nakorn Chiang Mai Hospital, Roi Et Hospital, Chulabhorn Hospital-Bangkok, National Cancer Institute of Thailand and Srinagarind Hospital. All participants provided written informed consent. Additionally, this study included a retrospective cohort of patients admitted to Osaka University Hospital (2014–2018) and Hokkaido University Hospital (2007–2018). The study protocol was approved by the Institutional Review Board Committee of Osaka University Hospital (IRB No. 17097). Informed consent was obtained from all patients prior to their participation. The study adhered to ethical guidelines consistent with the principles of the Declaration of Helsinki. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
FOLR1 is a tumor-derived and stemness trait-associated prognostic marker for HCC. (A) Schematic of the workflow used to identify candidate biomarkers. Genes upregulated in hepatocellular carcinoma (HCC) and correlated with cancer stemness markers (KRT19, PROM1 and EPCAM) were compared to identify common genes. These genes were further filtered to identify those encoding secretory proteins that were associated with overall survival (OS) (created with Biorender). (B) Scatter plot showing the correlations between KRT19 and EPCAM (a), KRT19 and PROM1 (b), and EPCAM and PROM1 (c) mRNA levels in TCGA-LIHC cohort. (C) (a) Venn diagram showing the overlap among genes correlated with KRT19 (1870 genes), PROM1 (1469 genes), EPCAM (1417 genes) and upregulated genes (186 genes). (b) Venn diagram showing the overlap among common genes (873 genes) and predicted secretory proteins (186 genes). (D) Quantitative PCR analysis showing the expression levels of the folate receptor 1 (FOLR1) mRNA in control and HCC samples from TCGA-LIHC cohort. **p < 0.01. (E) Kaplan‒Meier survival curves for TCGA-LIHC cohort stratified by high (red line) and low (black line) FOLR1 mRNA expression levels. (F-G) Scatter plot showing the correlation between FOLR1 mRNA expression and KRT19 (left panel), EPCAM (middle panel), and PROM1 (right panel) mRNA expression levels in TCGA-LIHC cohort (F) and the Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC) cohort (G). (H) Kaplan‒Meier survival curves for the TIGER-LC cohort stratified by high (red line) and low (black line) FOLR1 mRNA expression levels
Fig. 2
Fig. 2
Tumoral FOLR1 expression represents a poor prognostic molecular subtype of HCC with aggressive biological features. A Heatmap showing gene expression, clinical features, molecular subclasses, and gene set enrichment scores of hallmark gene sets evaluated by single-set gene set enrichment analysis (ssGSEA). B FOLR1 mRNA levels in each molecular class. **p < 0.01. ****p < 0.0001. C Gene set enrichment analysis (GSEA) comparing the FOLR1 high- and low-expression groups. Enrichment plot from GSEA showing the distribution of the enrichment scores for the FOLR1-H and FOLR1-L expression groups. D Gene set enrichment scores of HCC patients with either high or low FOLR1 mRNA levels in The Cancer Genome Atlas (TCGA)-LIHC cohort. ****p < 0.0001
Fig. 3
Fig. 3
The serum FOLR1 level is a diagnostic biomarker of HCC, especially in combination with AFP. A Scatter plot showing the correlation between FOLR1 mRNA expression and serum FOLR1 levels in the surgically resected HCC cohort. B Comparison of serum FOLR1 levels among patients with colorectal polyps, patients with chronic hepatitis C (CHC), and hepatocellular carcinoma (HCC) patients. *p < 0.05. ****p < 0.0001. C-D Receiver operating characteristic (ROC) curves representing the diagnostic performance of FOLR1 (C) and alpha-fetoprotein (AFP) (D) for HCC. (E) ROC curve illustrating the combined diagnostic performance of FOLR1 and AFP for HCC. The black line represents the FOLR/AFP combination, and the blue line represents AFP
Fig. 4
Fig. 4
The serum FOLR1 level is a prognostic biomarker of early HCC, especially in combination with the GALAD score. (A) Kaplan‒Meier survival curves for HCC patients stratified by high (red line) and low (black line) serum FOLR1 levels. (B) Comparison of serum FOLR1 levels among patients treated with radiofrequency ablation (RFA), transarterial chemoembolization (TACE), and operation (OP). (C) Subgroup analysis of survival, showing survival curves for patients with high and low serum FOLR1 levels and stratified by treatment with RFA (FOLR1 RFA), TACE (FOLR1 TACE) and OP (FOLR1 OP). (D) Serum FOLR1 levels across different stages of HCC (stage 1 to stage 4). (E) Subgroup analysis of survival, showing survival curves for patients with high and low serum FOLR1 levels and stratified by HCC stage ((a) FOLR1 stage 1, (b) FOLR1 stage 2 and (c) FOLR1 stage 3). (F) Survival curves of HCC patients stratified by the GALAD score. (G) Survival curves of patients stratified by serum FOLR1 levels and GALAD score into the following groups: low/low (L/L), low/high or high/low (L/H or H/L), and high/high (H/H). *p < 0.05. **p < 0.01. ****p < 0.0001
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