Molecular subtyping of hepatocellular carcinoma: A step toward precision medicine

1 INTRODUCTION

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer (approximately 90%). Globally, it is the eighth most prevalent and the third leading cause of cancer [1, 2]. China has the highest morbidity and mortality burden of HCC, accounting for over half of both new cases and deaths associated with the disease [3-5]. In China, HCC ranks first both in incidence and mortality in males under 60 years old [6]. Several risk factors, such as cirrhosis, hepatitis virus infection, alcohol abuse, metabolic syndrome, and aflatoxin B1 exposure, have been identified for HCC [7-9]. Evidence from epidemiological studies demonstrates that non-alcoholic fatty liver disease [10, 11], diabetes [12, 13], and tobacco usage [14, 15] increase the risk of HCC. In China, among all cancers, HCC has the lowest survival rate, with the 5-year overall survival rate below 10% [6]. This has been ascribed to several challenges, among which the late diagnosis resulting in the disease mainly detected at advanced stages. In addition to this, high malignancy is another factor attributed to the low overall survival rate of HCC patients, shown to limit the efficacy of therapeutics [16].

The current systems used for HCC subtyping are largely based on tumor burden, which provide a moderate classification of tumors [17]. Meanwhile, the Barcelona Clinic Liver Cancer (BCLC) system is so far the most commonly used model for predicting the prognosis and guiding the selection of therapeutic interventions for HCC. According to this system, treatment options, such as ablation, resection, and transplantation, among others, are effective interventions for early HCC [18], however, they are less effective for advanced stage HCC [19]. Notably, the BCLC staging system is not sufficiently sensitive and specific to delineate the biological and molecular characteristics that influence prognosis and response to therapy, particular among tumors of the same stage [20]. Intriguingly, Nault et al. [21] showed that tumors at the same stage may display diverse genetic blueprints. Thus, it is imperative to develop more precise subtyping methods. Here, we summarize the current research progresses in precision therapy on the backbone of molecular subtypes of HCC.

2 GENOMIC HETEROGENEITY AND PRECISION THERAPY

HCC is a highly heterogeneous disease both at the pathological and molecular levels, stratified into intra- and inter-tumor heterogeneity [22-24]. These heterogeneities may be related to etiological diversity, morphological diversity, genetic predisposition, mutational alterations, dysregulation of signaling pathways, and micro-environmental disturbances [25-28]. Although the advances in next-generation sequencing technology have revealed new insights into the genome-wide landscape of HCC [29-32], molecular features and pathogenesis of HCC are not fully known. Precision medicine approach provides more accurate classification and diagnosis of cancer at the molecular level based on the patient's internal biological information and clinical characteristics [33-35]. On the backbone of large genetic information and clinical data, precision medicine is a key component of effective treatment [36, 37]. Given the highly heterogeneous genomic aberrations and microenvironment in HCC, it is challenging to explicitly simulate how target treatments kill a single cell. Hence, grouping cancer patients into relative homogeneous subtypes based on critical features, such as human epidermal growth factor receptor-2 (HER2)-positive gastric cancer [38] and estrogen receptor-positive breast cancer [39], may offer a great clinical significance. In the following sections, we describe the current progress in molecular subtyping of HCC and how it has revolutionized precision medicine.

3 MOLECULAR ALTERATIONS IN HCC

HCC results from the accumulation of multiple genomic and epigenomic alterations. Even though approximately 40-60 somatic alterations have been detected in exons of the HCC genome [9], only a few of them alter key signaling pathways that drive hepatocarcinogenesis. In fact, most frequently altered genes in HCC, such as telomerase reverse transcriptase (TERT), tumor protein p53 (TP53), and catenin beta-1 (CTNNB1), remain as unactionable targets [32]. Epigenetic dysregulation also plays a crucial role in hepatocarcinogenesis by altering gene expression through various mechanisms, including induction of DNA methylation, modification of histones, chromatin remodeling, and altering the expression of non-coding RNAs (microRNAs and long intergenic non-coding RNAs) [23]. Next-generation sequencing technologies have revealed several signaling pathways and oncogenic alterations associated with HCC, such as telomere maintenance, cell cycle control, epigenetic modifiers, WNT/β-catenin pathway, mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) kinase pathway [7, 40].

4 TARGETED THERAPY BASED ON MOLECULAR FEATURES OF HCC

Sorafenib was the first drug for targeted therapy approved by the U.S. Food and Drug Administration (USFDA) for advanced HCC. It delays hepatocarcinogenesis by blocking tumoral angiogenesis and cell proliferation [41]. Hitherto, four molecular targeted agents, sorafenib [42, 43] and lenvatinib [44] for first-line together with regorafenib [45] and cabozantinib [46] for second-line treatment, have displayed promising benefits in phase III clinical trials for advanced HCC. However, they are associated with adverse reactions, suboptimal efficacy, and frequent resistance [47].

Future trials are therefore needed to test various combinations of multi-kinase inhibitors to provide alternative drugs for patients who fail or do not tolerate first- and second-line therapies. By June 2016, the USFDA had approved 72 targeted anti-tumor drugs, including rapamycin and everolimus (mTOR inhibitors), perifosine and deguelin (AKT kinase inhibitors), tipifarnib (a RAS signaling inhibitor), and wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor). As an example, an HCC patient presented with distant metastases 11 years after liver transplantation. Immunohistochemistry tests performed on tumor cells revealed that this patient was positive for nuclear β-catenin (the WNT pathway) and pS6 (the mTOR pathway). The patient failed to respond to sorafenib alone and was therefore put on everolimus (an mTOR inhibitor). Three months later, the tumor had diminished by nearly half [48]. This case reveals that targeting relevant molecular pathways is effective in treating recurrent HCC. However, there was no evidence to suggest that a combination therapy of everolimus and sorafenib was superior to sorafenib therapy alone in patients with advanced HCC [49]. Overall, the current targeted drugs have only been shown to be effective only in a small portion of patients, hence are not ideal for precision medicine [50]. Studies conducted in the past decade have shed light on the molecular pathogenesis of HCC. Despite this, current therapeutic options are not sufficiently effective, and no biomarker for any approved targeted drug has been defined [51].

Immunotherapy, which remotivates intrinsic immune response to attack tumors by targeting relevant regulatory pathways against immunocytes, has shown remarkable efficacy on different solid tumors [52-54]. Immune checkpoint inhibitors, such as anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody have been approved for the treatment of several tumors [55]. For instance, phase II studies on nivolumab and pembrolizumab (anti-PD-1) for second-line treatment of advanced HCC revealed that the objective response rate and prognosis are promising [56, 57]. However, the heterogeneous immune profile of HCC coupled with high immunotolerant environment limit the efficacy of immunotherapies [58, 59]. Consequently, further researches are required to identify patients likely to benefit from immunotherapy.

5 SUBTYPING OF HCC BASED ON MOLECULAR FEATURES

Several multi-omics technologies have been used to characterize the mutational, transcriptomic, proteomic, metabolic, and immunological alterations in HCC. This has promoted the application of Big Data in the molecular subtyping of HCC [60]. Here, certain molecular signatures reflect specific tumor phenotypes, such as microvascular invasiveness and aggressiveness [61]. This underlines the invaluable benefits of molecular characterization and classification of tumors in the application of precision medicine. To meet these clinical objectives, multiple schemes of HCC sub-grouping have been proposed. The framework of these systems is based on different levels last decades. In the first foundation stage, the aim is establishing single-omic unsupervised molecular subtypes based on apparent biological characteristics and molecular features. The second phase involves molecular subtyping based on multi-omics spectral profiling. Together, precise subtyping with clinical significance is achieved. The following subsections summarize the major recent advances in molecular subtyping of HCC and probable future applications of the molecular feature-based precision medicine (Figure 1).

6 ADVANTAGES AND LIMITATIONS OF SUBTYPING METHODS

The molecular subtyping of HCC based on the aforementioned studies highlights the value of large-scale “-omics” in classification of tumors. Therefore, omics may help the design of potential treatment opportunities that are customized to a homogeneous subgroup of cancer. Molecular subtyping may likely become a common tool for HCC diagnosis and characterization during clinical decision making. However, differences in technological platforms, patient population, preparation and processing of samples make it difficult to develop a common method for classification of HCC tumors. Notably, some subtypes have been repeatedly discovered in independent studies, meaning that different HCC subtypes derived from different subtyping methods may share common molecular features (Figure 2). Given that similarities among independent subtypes still remains superficial, more studies should be conducted to create consensus among different subtyping methods.

7 MOLECULAR SUBTYPE-GUIDED PRECISE TREATMENT

Currently, little is known about the molecular subtypes of HCC to allow effective clinical management of HCC. Integrative experimental and genomic analyses have revealed clinically relevant HCC subtypes and characteristic genomic alterations associated with the subtypes. It has been found that different subtypes of HCC cell lines showed distinct responses to molecular targeted agents. By integrating gene expression pattern and analyzing cellular origins of tumors, Lee et al. [83] identified a subtype of HCC arising from hepatic progenitor cells called “Hepatoblast subtype”, another subtype arising from other hepatic cells named “Hepatocyte subtype”. A Src/Abl small-molecule kinase inhibitor, dasatinib, exhibited preferential killing of the hepatoblast subtype cell lines [84]. The Hoshida's S2 subtype may also be an important biomarker reflecting response to target therapy. Activation of MYC pathway S2 subtype cell lines increased the sensitivity to a small-molecule BET bromodomain inhibitor, (+)-JQ1, which suppressed the activity of MYC. Inhibitors of the fibroblast growth factor receptors (FGFRs) blunted MAPK signaling in S2 cell lines [85].

The results from analysis of genomic data have started to impact clinical decision making and the development of more precise therapies. Many clinical studies have attempted to stratify patients based on biomarkers for precision treatment. In a retrospective analysis of phase II study, patients with advanced HCC harboring RAS mutations exhibited the best clinical responses to a combination of refametinib (MEK inhibitor) and sorafenib [86]. Subsequent studies obtained similar results, but further studies are needed since those studies were performed on few cases [87]. Recently, Sun et al. [88] identified Fms-like tyrosine kinase 3 (FLT3) as a predictive biomarker of sorafenib in advanced HCC. It was found that sorafenib may delay tumor progression in patients with FLT3-high HCC. Tuberous sclerosis complex 1 and 2 (TSC1/2) are mTOR pathway-related genes that are frequently mutated. Mutations of TSC1/2 decreased the activity of mTOR pathway and promoted malignant progression of HCC [89]. Ho et al. [90] identified a novel molecular subgroup of HCCs with aggressive behaviors, harboring TSC1/2 mutations and showing hypersensitivity to rapamycin treatment. However, many targeted drugs lack biomarkers to demonstrate association of genomic subtypes with treatment response.

Although most of these findings are encouraging, there are substantial gaps in translating molecular subtypes to clinical practice [91]. Patient-derived xenograft model is one of the most preferred preclinical models that best recapitulates hepatocarcinogenesis and histologic aggressiveness of different subtypes of HCC [92]. Preclinical models provide a framework that guides preclinical trials of novel drugs, thereby bridging the link between molecular subtypes and precision therapy.

8 PERSPECTIVE

Identification of HCC subtypes have generated new ideas for patient stratification and revealed opportunities for developing therapeutic agents for personalized curative-intent treatment (Figure 3). Molecular subtyping-based analyses of HCC samples have expanded our understanding of the pathogenesis of HCC and provided a basis for clinical subtyping of tumors which have improved treatment of HCC patients. This review reveals that the design of targeted therapies may be visible if sufficient information on the clinicopathological features [93] and the concept of molecular subtyping are optimally utilized in clinical settings. In the foreseeable future, more researches and collaborations are required to apply multi-omics tools to identify the mechanisms of HCC and to evaluate efficacy of novel drugs targeted at molecular pathways in specific subgroups of HCCs patients [94]. Relatively aggressive subtypes of tumors can be converted into another less aggressive subtype through subtype-targeted strategies, e.g., targeting oncogenic pathway. Further advanced molecular monitoring and examination of tumor microenvironment may identify more accurate predictive and prognostic biomarkers of HCC. To produce clinically translatable findings, animal models which reproduce human HCC's specific molecular traits and tumor phenotypes are needed. Finally, prospective clinical trials organized by multi-centers should be conducted to assess subtype-specific responses to therapy.

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