HOXA13 Overexpression Is Associated With Poor Prognosis In Colorectal Liver Metastases
*David G. Su M.D.1, *Vadim Kurbatov MD1, *Xujun Wang 2, *Zhaoshi zeng 3, *Philip Paty 3, *Caroline Johnson 4, *Jun Lu 5, Sajid A. Khan MD1
1General Surgery, Yale New Haven Hospital, Hamden, CT; 2SJTU-Yale Joint Center for Biostatistics, Shanghai Jiao Tong University, Shanghai, China; 33Department of Surgery, Memorial Sloan Kettering Cancer Center, New York City, NY; 4Environmental Health Sciences, Yale School of Public Health, New Haven, CT; 5Genetics, Yale School of Medicine, New Haven, CT
Background
Curative-intent hepatectomy is dictated by clinical criteria for colorectal cancer liver metastasis (CRCLM); however, genomic studies suggest that molecular biomarkers may be superior. The homeobox (HOX) gene family is dysregulated in multiple cancers and has not been extensively explored in metastatic colorectal cancer. We hypothesized that the transcriptional expression of HOX genes is a prognostic biomarker for biological aggressiveness in CRCLM.
Methods
RNA was extracted from frozen metastatic colorectal liver tumors of patients (N=39) and prepared for paired-end RNA sequencing. We applied unsupervised hierarchical clustering to identify differentially expressed genes between patients. The differential expression of HOX genes between patients with low and high clinical risks was studied using supervised hierarchical clustering. For 28 patients who underwent surgery, we analyzed post liver-resection metastasis recurrence using the Kaplan-Meier method. Genes that were significantly correlated with recurrence-free survival were validated in six external public datasets from the Gene Expression Omnibus database.
Results
We identified 667 differentially expressed genes (p <0.05). Unsupervised hierarchical clustering revealed unique genomic patterns in the patients with similar clinical risk features (p <0.05). Clinical risk-based supervised clustering revealed differential expression of multiple HOXA and HOXD family genes (p<0.01; q<0.01). We also found differential expression in three long non-coding RNA species (lncRNAs): HOTTIP, HOXA-AS5, and HOXD-AS2 (p<0.01; q<0.01). Kaplan-Meier analysis indicated a higher likelihood of metastatic recurrence in patients overexpressing HOXA13 and HOTTIP (log-rank p = 0.004). Moreover, a composite HOX score was found to be predictive of both overall and recurrence-free survival (log-rank p = 0.0269 and 0.0273, respectively). The utility of HOXA13, HOXD4, and HOXD8 as biomarkers of tumor aggressiveness was investigated in six independent cohorts.
Conclusion
In summary, HOX genes and their associated lncRNAs exhibit prognostic associations in patients with CRCLM and may act as biomarkers to refine clinical decision-making. Functional studies will determine whether the HOX pathway is a mechanistic driver of the development of CRCLM.
Higher mRNA expressions of HOXA13 and HOTTIP portend a worse overall survival and recurrence free survival. Survival curves correlating mRNA FPKM expression with OS/RFS on univariate Cox proportional hazards analysis: A. OS by HOXA13 B. RFS by HOXA13 C. OS by HOTTIP D. RFS by HOTTIP. Kaplan-Meier plots with corresponding HRs and log-rank p-values, based on dichotomized (by median) expression. CI, confidence interval; HR, hazard ratio.
Prognostication of OS and RFS utilizing HOX Score. A. OS by HOX Score B. RFS by HOX Score. HOX score was calculated from the log2 of the summative FPKM expression of HOXA13, HOTTIP, and HOXA-AS5 divided by the summative FPKM expression of HOXD4, HOXD8, and HOXD-AS2. Kaplan-Meier plots displaying OS/RFS curves with corresponding HRs from univariate Cox proportional hazards analysis and log-rank p-values, based on dichotomized (by median) expression. CI, confidence interval; HR, hazard ratio.
HOX genes are differentially expressed between primary colon cancer tissue and metastatic colon cancer in GSE131418 dataset (n = 600; 545 CR primary tumors; 55 CRCLM; non-matched). Expression levels between primary tumors and metastases in HOXD4, HOXA13, and HOXD8 genes evaluated using Mann-Whitney tests. ns = non-significant. **** p < 0.0001.
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