A transcriptomic intratumour heterogeneity-free signature overcomes sampling bias in prognostic risk classification for hepatocellular carcinoma

BACKGROUND & AIMS: Intratumour heterogeneity (ITH) fosters the vulnerability of RNA expression-based biomarkers derived from a single biopsy to tumour sampling bias, and is regarded as an unaddressed confounding factor for patient precision stratification using molecular biomarkers. This study aimed to identify an ITH-free predictive biomarker in hepatocellular carcinoma (HCC). METHODS: We interrogated the confounding effect of ITH on performance of molecular biomarkers and quantified transcriptomic heterogeneity utilising three multiregional HCC transcriptome datasets involving 142 tumoural regions from 30 patients. A de novo strategy based on the heterogeneity metrics was devised to develop a surveillant biomarker (a utility gadget using RNA; AUGUR) using three datasets involving 715 liver samples from 509 patients with HCC. The performance of AUGUR was assessed in seven cross-platform HCC cohorts that encompassed 1,206 patients. RESULTS: An average discordance rate of 39.9% at the level of individual patients was observed applying 13 published prognostic signatures to classify tumour regions. We partitioned genes into four heterogeneity quadrants, from which we developed and validated a reproducible robust ITH-free expression signature AUGUR that showed significant positive associations with adverse features of HCC. High AUGUR risk increased the risk of disease progression and mortality independent of established clinicopathological indices, which maintained concordance across seven cohorts. Moreover, AUGUR compared favourably to the discriminative ability, prognostic accuracy, and patient risk concordant rates of 13 published signatures. Finally, a well-calibrated predictive nomogram integrating AUGUR and tumour-node-metastasis (TNM) stage was established, which generated a numerical probability of mortality. CONCLUSIONS: We constructed and validated an ITH-free AUGUR and nomogram that overcame sampling bias and provided reliable prognostic information for patients with HCC. IMPACT AND IMPLICATIONS: Intratumour heterogeneity (ITH) is prevalent in hepatocellular carcinoma (HCC), and is regarded as an unaddressed confounding factor for biomarker design and application. We examined the confounding effect of transcriptomic ITH in patient risk classification, and found existing molecular biomarkers of HCC were vulnerable to tumour sampling bias. We then developed an ITH-free expression biomarker (a utility gadget using RNA; AUGUR) that overcame clinical sampling bias and maintained prognostic reproducibility and generalisability across multiple HCC patient cohorts from different commercial platforms. Furthermore, we established and validated a well-calibrated nomogram based on AUGUR and tumour-node-metastasis (TNM) stage that provided an individualised prognostic information for patients with HCC.