HOXD9 prioritization within developmental regulator-related genes suggests stage-adjusted prognostic relevance in...

Prognostic stratification in hepatocellular carcinoma (HCC) remains difficult because standard staging systems do not fully capture the biological heterogeneity of the disease. We hypothesized that developmental regulators could provide a biologically relevant framework for prioritizing prognostic markers.

RNA sequencing (RNA-seq) and clinical data from The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) cohort were analyzed. Tumor vs. normal differential expression analysis was performed using differential expression analysis for sequence count data 2 (DESeq2). Developmental regulator-related candidate genes were prioritized through a stepwise framework integrating differential expression, clinicopathological association, Kaplan-Meier survival analysis, and Cox regression. A main prognostic model was constructed from the final independent factors and evaluated using a nomogram, calibration analysis, and time-dependent receiver operating characteristic (ROC) analysis. The incremental prognostic value of HOXD9 beyond pathologic stage was assessed using the likelihood ratio test, Harrell's concordance index (C-index), bootstrap-based delta C-index analysis, and time-dependent area under the curve (AUC) comparison. External validation was performed in the LIRI-JP (Liver Cancer-RIKEN, Japan) cohort.

Among the screened developmental regulator-related genes, HOXD9 and MESP2 showed the most consistent signals across differential expression, clinicopathological association, and survival analyses. In multivariable Cox regression in the TCGA-LIHC cohort, only pathologic stage [hazard ratio (HR) =1.565, 95% confidence interval (CI): 1.266-1.935; P<0.001] and HOXD9 expression [HR =1.165, 95% CI: 1.035-1.312; P=0.01] remained independently associated with overall survival (OS). The main prognostic model integrating pathologic stage and HOXD9 yielded AUC values of 0.707 at 1 year and 0.701 at 3 years. Compared with the stage-only model, the stage plus HOXD9 model showed improved model fit (P=0.004), a higher C-index (0.651 vs. 0.601; P=0.004), and a higher 1-year AUC (0.707 vs. 0.656; P=0.02). These findings indicated incremental prognostic value of HOXD9 in the TCGA-LIHC training cohort. In the LIRI-JP cohort, HOXD9 significantly stratified OS (P=0.01), and the main external validation model yielded AUC values of 0.819 at 1 year and 0.739 at 3 years. However, HOXD9 was not independently associated with OS after adjustment for tumor-node-metastasis (TNM) stage in the external multivariable model (HR =1.027, 95% CI: 0.763-1.383; P=0.86).

HOXD9 emerged as a key prognostic candidate and showed incremental prognostic value beyond pathologic stage in the TCGA-LIHC training cohort. However, this stage-adjusted independent association was not reproduced in the LIRI-JP multivariable validation model. HOXD9 should therefore be considered a candidate prognostic marker requiring further validation.