A multimodal atlas of hepatocellular carcinoma reveals convergent evolutionary paths and 'bad apple' effect on clinical trajectory.

BACKGROUND & AIMS: Hepatocellular Carcinoma (HCC) is a highly fatal cancer characterized by high intra-tumor heterogeneity (ITH). A panoramic understanding of its tumor evolution, in relation to its clinical trajectory, may provide novel prognostic and treatment strategies. METHODS: Through the Asia-Pacific Hepatocellular Carcinoma (AHCC) trials group (NCT03267641), we recruited one of the largest prospective cohorts of HCC with over 600 whole genome and transcriptome samples from 123 treatment-naïve patients. RESULTS: Using a multi-region sampling approach, we revealed seven convergent genetic evolutionary paths governed by the early driver mutations, late copy number variations and viral integrations, which stratify patient clinical trajectories after surgical resection. Furthermore, such evolutionary paths shaped the molecular profiles, leading to distinct transcriptomic subtypes. Most significantly, although we found the coexistence of multiple transcriptomic subtypes within certain tumors, patient prognosis was best predicted by the most aggressive cell fraction of the tumor, rather than by overall degree of transcriptomic ITH level - a phenomenon we termed the 'bad apple' effect. Finally, we found that characteristics throughout early and late tumor evolution provide significant and complementary prognostic power in predicting patient survival. CONCLUSIONS: Taken together, our study generated a comprehensive landscape of evolutionary history for HCC and provided a rich multi-omics resource for understanding tumor heterogeneity and clinical trajectories. CLINICAL TRIAL NUMBER: NCT03267641 (Observational cohort) IMPACT AND IMPLICATIONS: This prospective study, utilizing comprehensive multi-sector, multi-omics sequencing and clinical data from surgically resected HCC, reveals critical insights into the role of tumor evolution and intra-tumor heterogeneity (ITH) in determining the prognosis of Hepatocellular Carcinoma (HCC). These findings are invaluable for oncology researchers and clinicians, as they underscore the influence of distinct evolutionary paths and the 'bad apple' effect, where the most aggressive tumor fraction dictates disease progression. These insights not only enhance prognostic accuracy post-surgical resection but also pave the way for developing personalized therapies tailored to specific tumor evolutionary and transcriptomic profiles. The co-existence of multiple sub-types within the same tumor prompts a re-appraisal of the utilities of depending on single samples to represent the entire tumor and suggests the need for clinical molecular imaging. This research thus marks a significant step forward in the clinical understanding and management of HCC, underscoring the importance of integrating tumor evolutionary dynamics and multi-omics biomarkers into therapeutic decision-making.

Multi-region sampling with paired sample sequencing analyses reveals sub-groups of patients with novel patient-specific dysregulation in Hepatocellular Carcinoma.

BACKGROUND: Conventional differential expression (DE) testing compares the grouped mean value of tumour samples to the grouped mean value of the normal samples, and may miss out dysregulated genes in small subgroup of patients. This is especially so for highly heterogeneous cancer like Hepatocellular Carcinoma (HCC). METHODS: Using multi-region sampled RNA-seq data of 90 patients, we performed patient-specific differential expression testing, together with the patients' matched adjacent normal samples. RESULTS: Comparing the results from conventional DE analysis and patient-specific DE analyses, we show that the conventional DE analysis omits some genes due to high inter-individual variability present in both tumour and normal tissues. Dysregulated genes shared in small subgroup of patients were useful in stratifying patients, and presented differential prognosis. We also showed that the target genes of some of the current targeted agents used in HCC exhibited highly individualistic dysregulation pattern, which may explain the poor response rate. DISCUSSION/CONCLUSION: Our results highlight the importance of identifying patient-specific DE genes, with its potential to provide clinically valuable insights into patient subgroups for applications in precision medicine.

A genomic enhancer signature associates with hepatocellular carcinoma prognosis.

Background & Aims: Lifestyle and environmental-related exposures are important risk factors for hepatocellular carcinoma (HCC), suggesting that epigenetic dysregulation significantly underpins HCC. We profiled 30 surgically resected tumours and the matched adjacent normal tissues to understand the aberrant epigenetic events associated with HCC. Methods: We identified tumour differential enhancers and the associated genes by analysing H3K27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C/HiChIP data from the resected tumour samples of 30 patients with early-stage HCC. This epigenome dataset was analysed with previously reported genome and transcriptome data of the overlapping group of patients from the same cohort. We performed patient-specific differential expression testing using multiregion sequencing data to identify genes that undergo both enhancer and gene expression changes. Based on the genes selected, we identified two patient groups and performed a recurrence-free survival analysis. Results: We observed large-scale changes in the enhancer distribution between HCC tumours and the adjacent normal samples. Many of the gain-in-tumour enhancers showed corresponding upregulation of the associated genes and vice versa, but much of the enhancer and gene expression changes were patient-specific. A subset of the upregulated genes was activated in a subgroup of patients' tumours. Recurrence-free survival analysis revealed that the patients with a more robust upregulation of those genes showed a worse prognosis. Conclusions: We report the genomic enhancer signature associated with differential prognosis in HCC. Findings that cohere with oncofoetal reprogramming in HCC were underpinned by genome-wide enhancer rewiring. Our results present the epigenetic changes in HCC that offer the rational selection of epigenetic-driven gene targets for therapeutic intervention or disease prognostication in HCC. Impact and Implications: Lifestyle and environmental-related exposures are the important risk factors of hepatocellular carcinoma (HCC), suggesting that tumour-associated epigenetic dysregulations may significantly underpin HCC. We profiled tumour tissues and their matched normal from 30 patients with early-stage HCC to study the dysregulated epigenetic changes associated with HCC. By also analysing the patients' RNA-seq and clinical data, we found the signature genes - with epigenetic and transcriptomic dysregulation - associated with worse prognosis. Our findings suggest that systemic approaches are needed to consider the surrounding cellular environmental and epigenetic changes in HCC tumours.

Dynamic phenotypic heterogeneity and the evolution of multiple RNA subtypes in hepatocellular carcinoma: the PLANET study.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types.

PD-L1 protein expression and copy number gains in HIV-positive locally advanced cervical cancer.

BACKGROUND: The programmed death-1/programmed death-ligand-1 (PD-1/PD-L1) axis may represent a target for cervical cancer; however, it is poorly understood in human immunodeficiency virus (HIV)-infected patients. METHODS: We evaluated HIV-positive (n = 42) and HIV-negative (n = 110) women with locally advanced cervical cancer regarding their PD-L1 expression, determined by combined positive score (CPS) ⩾ 1 and tumor proportion score (TPS) ⩾ 25%, and PD-L1 copy number alterations, assessed by fluorescence in situ hybridization. RESULTS: Regardless of HIV status, 84.9% and 44.8% of cases were PD-L1-positive according to CPS ⩾ 1 and TPS ⩾ 25%. Per CPS ⩾ 1, PD-L1 positive rate was similar between HIV-positive and HIV-negative women, whereas a significant difference was seen per TPS ⩾ 25%. Tumor size and parametrial invasion were correlated with PD-L1 positivity in HIV-negative women, whereas anti-retroviral therapy (ART) was correlated with TPS < 25%. Low CD4-positive cell counts were associated with CPS < 1 in HIV-positive women. No significant difference was observed in PD-L1 copy number status between HIV-positive and HIV-negative women. PD-L1 amplification and polysomy were independently associated with TPS ⩾ 25%, whereas the presence of parametrial invasion was independently associated with CPS ⩾ 1. Cancer stage and PD-L1 amplification were identified as independent predictors of recurrence-free survival [hazard ratio (HR) = 2.40 (1.32-4.36) and HR = 5.33 (1.94-14.61)] and cancer-specific survival [HR = 13.62 (5.1-36.38) and HR = 3.53 (1.43-8.69)]. PD-L1 polysomy was an independent predictor of locoregional recurrence-free survival [HR = 3.27 (1.27-8.41)]. HIV status and PD-L1 expression (CPS ⩾ 1 or TPS ⩾ 25%) were not associated with poor patient outcomes. CONCLUSION: PD-L1 amplification and polysomy are the strongest drivers of PD-L1 expression in cervical cancer, and could represent prognostic biomarkers for anti-PD-1/PD-L1 therapy. Cervical cancer biology may be modulated by HIV infection, CD4-positive cells, and HIV treatments.

Impact of antiretroviral drugs on PD-L1 expression and copy number gains with clinical outcomes in HIV-positive and -negative locally advanced cervical cancers.

Cervical cancer has become a leading cause of death in both HIV-infected and uninfected women. Previous studies have revealed that antiretroviral therapy (ART) possesses anti-human papillomavirus (HPV) and antitumour properties, potentially serving as an anticancer agent and improving functional immunity in HIV-positive individuals. However, to the best of our knowledge, no studies have examined the association between ART and the clinical outcome of patients with pre-existing invasive cervical cancer. The current study analysed 48 HIV-positive and 123 HIV-negative patients with locally advanced stage IB2-IVA cervical cancer between December 2008 and December 2016. Tumours were categorized based on programmed cell death-ligand 1 (PD-L1) immunoreactivity and copy number alterations in the PD-L1 gene, as determined by fluorescence in situ hybridization. The results revealed that ART-treated patients exhibited a lower prevalence of PD-L1 immunopositivity, PD-L1 amplification and polysomy compared with patients that did not receive ART and those that were HIV-negative. Furthermore, ART-treated patients with PD-L1 immunonegativity exhibited an improved recurrence-free survival (RFS) compared with patients that did not receive ART and HIV-negative individuals with PD-L1 immunopositivity (P=0.041 vs. P=0.030). Additionally, ART-exposed patients with PD-L1 disomy demonstrated improved locoregional recurrence-free survival (LRR) when compared with HIV-negative patients with PD-L1 amplification and polysomy (P=0.039 vs. P=0.007), RFS (P<0.001 vs. P=0.006) and cancer-specific survival (CSS) (P=0.021 vs. P=0.025). ART-exposed patients with PD-L1 disomy also exhibited improved RFS (P<0.001) and CSS (P<0.001) compared with HIV-negative patients with PD-L1 amplification. Improved LRRs were demonstrated in ART-exposed patients with PD-L1 disomy (P=0.028) compared with non-HIV patients with polysomy. Following multivariate analysis, International Federation of Gynaecology and Obstetrics stage and PD-L1 amplification were determined to be predictors of poor a RFS [hazard ratio (HR), 2.43; 95% confidence interval (CI), 1.37-4.30; P=0.002 vs. HR, 7.03; 95% CI, 2.79-17.74; P<0.001) and CSS (HR, 11.47; 95% CI, 4.70-27.99; P<0.001 vs. HR, 4.05; 95% CI, 1.64-9.98; P=0.002). However, only PD-L1 polysomy was determined to be a predictor of poor LRR (HR, 2.50; 95% CI, 1.11-5.63; P=0.027). HIV status was not associated with poor outcomes, as determined using Cox models. The results of the current study indicated that ART may be used for the treatment of cervical cancer in both HIV-infected and uninfected patients. However, additional research is required to further elucidate these results.