Dissecting Liver Tumor Heterogeneity to Improve Health Equity

Abstract

Liver cancer burden varies greatly among populations. This disparity stems from the presence of complex etiology, sex, race/ethnicity, and extensive tumor molecular heterogeneity. Our ability to dissect tumor heterogeneity and to identify shared common features of liver cancer in diverse populations may improve precision medicine, thereby reducing disease burden.

Liver cancer disparity

Liver cancer has been among the top five deadliest cancers in the world for decades. Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are two major clinical subtypes, accounting for about 90% and 10% of liver cancer, respectively. A huge disparity in HCC and iCCA incidence, prevalence, mortality and morbidity is evident among different populations characterized by age, gender, race/ethnicity and geographic location [1] (Figures 1A and 1B). For example, men are more likely to develop HCC and iCCA than women. In addition, liver cancer is more common in Asia and Africa than other regions. These variations may reflect the interplay of many factors including potential genetic predispositions and varying etiological factors. Chronic infection with hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) or certain parasites, alcohol abuse, diabetes, dietary-related obesity and/or chemical carcinogens are major risk factors responsible for HCC and iCCA [2]. Noticeably, these risk factors are closely related to geographic locations, with high prevalence of HBV in Asia and Africa and increased HCV infection in Europe and Americas (Figure 1C). The prevalence of HDV is extremely high in Mongolia, a country with the highest incidence of liver cancer in the world. In northeastern Thailand, iCCA has about five times higher incidence than HCC, which is strikingly different from the global burden of HCC and iCCA, where HCC dominates (Figure 1B). This difference is potentially due to the unique etiology of parasitic biliary infections with hepatobiliary flukes in the area. Consequently, different etiological exposures may independently contribute to hepatocarcinogenesis and shape tumor biology, while leaving unique molecular fingerprints among different tumors, including varying mutational signatures, copy number alterations, transcriptomic landscapes, and methylation profiles. However, the molecular features that underlie liver cancer disparity remain partially understood. There is a crucial need to understand etiology and tumor biology using well-defined and diverse patient populations to delineate subgroup-related molecular features to improve precision medicine for the purpose of advancing health equity, which is defined by the World Health Organizationas “the absence of unfair and avoidable or remediable differences in health among population groups defined socially, economically, demographically or geographically ^I.”

Figure 1. Liver cancer disparity.

(A) Global age-standardized incidence and mortality rates (per 100,000 persons) of liver cancer in 2020. LAC, Latin America and the Caribbean. Data source: GLOBOCAN 2020. (B) Age-standardized incidence rates (per 100,000 persons) of HCC and iCCA by sex. The size of each circle represents the incidence rate. (C) Risk factors for liver cancer in different geographical locations.

Molecular heterogeneity

The molecular landscape of liver cancer is highly heterogeneous, with extensive inter- and intra-tumor heterogeneity in genomic, methylomic, transcriptomic, proteomic and metabolomic profiles [3, 4]. TP53, CTNNB1 and ALB are the most frequently mutated genes in HCC [5]. However, the presence of various driver and passenger mutations makes the mutational landscape of liver cancer complex. The vast molecular heterogeneity in liver cancer can be attributed to both genetic and non-genetic factors. As a major source of molecular diversity, genetic instability provides diverse genetic materials for tumor evolution by inducing large-scale chromosomal copy number alterations, gene rearrangements and mutations. In addition, the tumor microenvironment (TME) may impel the generation of a heterogeneous tumor cell population that can evade the immune system and reprogram itself for tumor cell survival. Resources created by the international efforts such as The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC) and the Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC) consortium studies are key to combat disease burden among affected populations. A central question is how to effectively decode the complex and heterogeneous molecular landscape of liver cancer to identify therapeutic vulnerabilities and design interventions that are effective for all.

Molecular subtypes and shared tumor biology

Given different etiologies in inducing genetic alterations, the vast amount of genetic material in each cell, and tumor evolution, the probability of creating a uniform tumor cell population is almost zero, both within a patient and among patients. However, hepatocarcinogenesis in different patient populations still generates tumors with common features, known as the hallmarks of cancer [6]. Moreover, recurrent chromosome 1q and chromosome 8q gain are relatively stable among HCC and iCCA, where some of these clinically different tumors also share similar molecular subtypes and driver genes among Asian populations [7]. Therefore, despite tumor heterogeneity, shared common features can be traced to reflect tumor biology in diverse liver cancer patient populations.

Molecular subtypes with different survival outcomes have been identified from multiple liver cancer patient cohorts using bulk transcriptome data [5, 7]. Tumors within a molecular subtype tend to share similar malignant features, such as proliferation markers, driver mutations or signaling pathways. Patients of the same race or ethnicity are more likely to have common features linked to a particular etiological exposure or potential genetic predisposition. TCGA study of 363 North America HCC cases indicates that the tumor profiles of patients of Asian ethnicity and high HBV infection is enriched for a molecular subtype characterized by overexpression of proliferation-related marker genes such as MYBL2, PLK1 and MKI67 as well as genetic alterations including lower frequency of CDKN2A silencing, CTNNB1 mutation and TERT promoter mutation [5]. Noticeably, this molecular subtype is associated with poor patient survival, which was validated using three other HCC cohorts of Asian and Caucasian populations. Asian-related prognostic HCC molecular subtypes in North America also share molecular features with Chinese and Thai HCC but not Caucasian HCC, suggesting potential contributions of race and geographic locations to the unique tumor biology [7]. Several additional studies have also been carried out to address population-based molecular subtypes in HCC. For example, in a study of 76 HCC patients from Mongolia, unique driver mutations such as GTF2IRD2B, PNRC2 and SPTA1 are linked to high HDV infection in the region [8]. Genome-wide trans-ancestry analysis revealed evidence of mutational signatures unique to Asian cases of HCC, suggesting ancestry-associated mutational processes in HCC development [9]. Moreover, unique molecular subtypes could be identified in HCC from patients with indigenous American ancestry [10]. These results suggest that patients of the same race, ethnicity or geographic location with similar etiologies could share common molecular features regardless of the extensive intertumoral heterogeneity within a population. These features may represent unique tumor biology specific to a patient population. The successful identification of these features may help to develop corresponding treatment strategies, especially tailored for the underserved populations, to improve liver cancer patient care and health equity (Figure 2). In addition, a better understanding of tumor etiology in contributing to tumor heterogeneity may help develop strategies to reduce potentially modifiable risk factors for liver cancer, which may reduce disparities in liver cancer burden.

Figure 2. Dissecting tumor heterogeneity in diverse liver cancer patient populations with the aim to reduce health disparity and improve health equity.

Future research directions include understanding tumor heterogeneity in the context of TME, cells of origin, tumor evolution and molecular subtypes, to identify shared features. Different populations are shown in dashed circles. The size of the dashed circle represents liver cancer incidence. Light and dark human icons represent healthy individuals and individuals with liver cancer, respectively.

Single-cell analyses of 19 HCC and iCCA reveal two groups of tumors with distinct levels of tumor heterogeneity, where the group of high-diversity tumors share features including activated VEGF as well as TME reprogramming that are linked to poor patient outcome [3]. Single-cell analyses of tumor biopsies before and after treatment with immune checkpoint inhibitors identified three tumor branches in a hierarchical tree with osteopontin as a potential driver of tumor cell evolution and response to immunotherapy in the branches with aggressive tumor features [4]. These studies should be expanded to include diverse patient populations, especially those with high disease burden. Taken together, subgroup-related common features are evident although they may be masked by the appearance of molecular heterogeneity. The identification of these features at the single-cell level may allow for a better understanding of tumor biology of different populations to achieve precision medicine. The application of single-cell technologies to underserved patient populations may help to identify targetable molecular drivers and therapeutic vulnerabilities to advance health equity.

Concluding remarks

The heterogeneous molecular landscape of HCC and iCCA and the associated complex etiological landscape pose great challenges for effective liver cancer interventions. Liver cancer disparity adds another hurdle to the treatment of this complex disease. Currently, no prognostic markers have been incorporated into clinical management for HCC and iCCA. Moreover, treatment options are limited and less effective for advanced liver cancer. Recent clinical trials of a combination of drugs to deal with liver tumor heterogeneity have been tested. The successful IMbrave150 study demonstrated that in patients with unresectable HCC, anti-PD-L1 (atezolizumab) combined with anti-VEGF (bevacizumab) resulted in a median progression-free survival of 6.8 months, which is significantly increased from 4.3 months with sorafenib, a tyrosine kinase inhibitor as the standard of care for frontline therapy for liver cancer [11]. Future treatment decisions will depend on our knowledge of liver cancer heterogeneity and our ability to identify population group-related shared features in diverse liver cancer patient populations with varying etiological backgrounds. Further explorations on the role of the TME, tumor cells of origin, and tumor evolution will help to identify shared factors regulating tumor heterogeneity and disease outcome among different patient populations.

The TME of liver cancer has been recognized as a key player in promoting tumor heterogeneity. TMEs comprise blood vessels, stromal cells, infiltrating immune cells, extracellular matrix, signaling molecules, metabolites, and nutrients. Within a tumor ecosystem, tumor cells continuously communicate with the TME through cell adhesion, signaling molecules and small vesicles. Some noncancerous cells play an anti-tumor role while others have been programmed by tumor cells to promote continuous survival and dissemination of the tumor. Considering the contribution of different etiological factors, carcinogen exposures, and unhealthy lifestyles in hepatocarcinogenesis, distinct patient populations may display different interactions within the TME. Because tissue biopsies are not routinely collected in clinical management of liver cancer, it is difficult to address the roles of race-related TME in hepatocarcinogenesis. However, a recent study has revealed a role for the necroptotic microenvironment in driving lineage commitment in liver cancer [12]. Thus, dissecting the communication between tumor cells and TME in diverse liver cancer patient populations may identify unique vulnerabilities (Figure 2).

In the development of liver cancer, hepatocytes, cholangiocytes, and progenitor cells may serve as the target cell(s) of malignant transformation, i.e., tumor initiating cells (TICs). Hepatic progenitor cells and mature hepatocytes may give rise to HCC while all three cell types (i.e., hepatocytes, cholangiocytes, and progenitor cells) may be sources of iCCA [12, 13]. Noticeably, the three cell types are at different stages of the cellular hierarchy, with stem-cell like features in progenitor cells and differentiated features in mature hepatocytes, suggesting varying degrees of oncogenicity of these TICs. It is plausible that the presence of TICs with similar capability may lead to the formation of tumors with comparable molecular features. In other words, TICs may account for distinct molecular subtypes in liver cancer. Thus, identification of TICs for each tumor and defining features of different groups of TICs may help to identify biomarkers useful for liver cancer surveillance and treatment (Figure 2).

Most studies of tumor heterogeneity only capture a snapshot of the tumor in its long evolutionary course because of the difficulty in obtaining longitudinal clinical tumor specimens. However, tumor evolution is a dynamic process with continuous changes of tumor cells and the interactions with the TME [14]. Animal modeling could serve as an alternative approach to study tumor evolution. The development of mouse models closely resembling of human diseases represents a key step towards this goal and such an effort is being carried out [15]. Future efforts on the analysis of more longitudinal tumor samples may shed light on varying patient responses to treatments and help to identify novel biomarkers for improving precision medicine (Figure 2). The implementation of routine liver biopsy in the clinical management of liver cancer, especially in underserved populations with high disease burden, may help to understand tumor evolution and to identify molecular features that are responsible for tumor heterogeneity to improve liver cancer treatment.

Collectively, a better understanding of tumor heterogeneity from the perspectives of TME, cell of origin, and tumor evolution may identify functional drivers that contribute to tumor heterogeneity among distinct populations. The incorporation of identified molecular features into clinical management of liver cancer could lead to effective strategies to target therapeutic vulnerabilities present in different populations, which ultimately improves patient outcomes for all. A collaborative international effort on studying well-defined and diverse liver cancer patient populations to minimize confounding factors is key to improving our understanding of etiology-related molecular heterogeneity and disease burdens in underserved populations.