Integrated molecular characterization of sarcomatoid hepatocellular carcinoma

Article information

Clin Mol Hepatol. 2025;31(2):426-444

Publication date (electronic) : 2024 December 10

doi : https://doi.org/10.3350/cmh.2024.0686

Rong-Qi Sun ¹^,²^,^*, Yu-Hang Ye ¹^,²^,^*, Ye Xu ¹^,²^,^*, Bo Wang ³^,^*, Si-Yuan Pan ¹^,², Ning Li ¹^,², Long Chen ¹^,², Jing-Yue Pan ¹^,², Zhi-Qiang Hu ¹^,², Jia Fan ¹^,², Zheng-Jun Zhou^,¹^,²

, Jian Zhou^,¹^,²

, Cheng-Li Song^,⁴

, Shao-Lai Zhou^,¹^,²

¹Department of Liver Surgery and Transplantation, Zhongshan Hospital, Fudan University, Shanghai, China

²Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China

³Department of General Surgery, Second Affiliated Hospital, Dalian Medical University, Dalian, China

⁴Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China

Corresponding author : Shao-Lai Zhou Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, 1609 Xie Tu Road, Shanghai 200032, China Tel: +86-21-64041990, Fax: +86-21-64037181, E-mail: zhoushaolai99@sina.com

Cheng-Li Song Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning 116044, China Tel: +86-411-86110499, Fax: +86-411-86110499, E-mail: songchengli@dmu.edu.cn

Jian Zhou Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, 1609 Xie Tu Road, Shanghai 200032, China Tel: +86-21-64041990, Fax: +86-21-64037181, E-mail: zhou.jian@zs-hospital.sh.cn

Zheng-Jun Zhou Liver Cancer Institute, Zhongshan Hospital, Fudan University, 1609 Xie Tu Road, Shanghai 200032, China Tel: +86-21-64041990, Fax: +86-21-64037181, E-mail: zhouzhengjun1221@126.com

*These authors contributed equally to this work.

Editor: Ju Hyun Shim, University of Ulsan, Korea

Received 2024 August 21; Revised 2024 November 15; Accepted 2024 December 6.

See the commentary-article "Genetic insights into sarcomatoid hepatocellular carcinoma: Critical role of ARID2 in pathogenesis and immune feature: Editorial on “Integrated molecular characterization of sarcomatoid hepatocellular carcinoma”" on page 635.

Abstract

Background/Aims

Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated.

Methods

In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs.

Results

Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment.

Conclusions

We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

Keywords: Sarcomatoid hepatocellular carcinoma; Multiomics; ARID2; Epithelial–mesenchymal transition

Graphical Abstract

INTRODUCTION

The incidence and mortality rates of hepatocellular carcinoma (HCC), one of the most prevalent types of cancer, have increased in recent years [1,2]. Over the past decade, we have expanded our understanding of HCC pathogenesis at the molecular level. Following technological advances, several studies revealed the genetic landscape of alterations that underlie liver carcinogenesis [3-8]. Previously, we and others delineated the genomic events that characterize Chinese HCCs [9,10].

Sarcomatoid HCC, a rare histological type of HCC characterized by spindle-shaped cells with increased mitotic activity [11], accounts for approximately 1.8% to 3.9% of all HCC tumors [12,13]. In a recent report by Liao et al. [11], the cumulative incidence of sarcomatoid HCC was 0.79% among a total of 5,047 patients with histologically proven HCC. Because of its rarity, there are few reports of sarcomatoid HCC in the English literature, and most of these reports have mainly focused on histopathological characteristics. The prognosis for sarcomatoid HCC remains unsatisfactory because of high rates of recurrence and metastasis compared with non-sarcomatoid HCC [11,14]. Furthermore, the molecular characterization of sarcomatoid HCC is unclear.

In this study, we conducted an integrated multiomics study of whole-exome sequencing (WES), RNA-seq, spatial transcriptome (ST), and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC to ascertain frequently altered genes, track the mutation process, infer the tumor subclonal architectures and delineate the transcriptional characteristics. We also explored the role of ARID2, a recurrently mutated gene in sarcomatoid HCC, in HCC progression and epithelial–mesenchymal transition (EMT).

MATERIALS AND METHODS

Patients and follow-up

From January 2013 to December 2016, a total of 3,417 consecutive patients diagnosed with histologically proven primary HCC who received tumor resection in the Department of Liver Surgery and Transplantation in Zhongshan Hospital, Fudan University, were recruited. 46 patients with sarcomatoid HCC were identified from the pathology database, and their pathological information was retrieved for reconfirmation. Five patients were excluded from the study because the sarcomatoid tumor components and conventional HCC components were not in the same paraffin blocks. For the remaining 41 patients, 1 case was excluded due to palliative surgery, and 9 cases were eliminated because of prior interventions. Finally, 31 cases with both sarcomatoid tumor components and conventional HCC components were identified and included in this study (Supplementary Fig. 1, Supplementary Table 1), and 10 cases were involved in integrated multiomics study. The pathological diagnosis of sarcomatoid HCC was confirmed through histological examination (using hematoxylin and eosin [H&E] staining) and immunohistochemical staining (including Hep, Par-1, arginase-1, cytokeratin [CK], and Vimentin). Hepatocellular markers were typically negative in the malignant spindle-cell component, whereas CK and Vimentin were positive in most cases [11]. The histological tumor differentiation was determined according to the system proposed by Edmondson and Steiner [15]. Liver function was assessed by the Child–Pugh scoring system. Tumor stage was determined using the 2017 International Union Against Cancer tumor-node-metastasis (TNM) classification system [16]. Before surgical operation and tissue sample collection, we obtained oral and written informed consent from each participant, which included information on the use of clinical characteristics and tissue samples for scientific research. For this research, the Research Ethics Committee of Zhongshan Hospital reviewed and approved the study and granted ethical approval for the use of human subjects. We also obtained oral informed consent for inclusion in the study from the participants at the time of follow-up. The Institutional Review Board (IRB) number is IRB00008978.

All patients with sarcomatoid HCC were monitored after surgery until December 30, 2019 as previously described [17,18]. We diagnosed tumor recurrence on the basis of computed tomography scans, magnetic resonance imaging, digital subtraction angiography, and elevated serum alpha-fetoprotein (AFP) level, with or without histological confirmation [10]. We defined recurrence-free survival (RFS) as the interval between the surgery and any diagnosis of recurrence (intrahepatic or extrahepatic) [19]. We defined overall survival (OS) as the time from the date of surgery until death or the end of follow-up. The surviving patients were censored at the time of the end of follow-up.

Additional materials and methods can be found in the Supplementary Materials and Methods.

RESULTS

Patients with sarcomatoid HCCs showed poor prognosis compared with patients with non-sarcomatoid HCCs

We calculated propensity scores according to sex, age, and TNM stage to match the patients with sarcomatoid HCC at a 1:4 ratio to patients with non-sarcomatoid HCC as a control group from our previous next-generation sequencing study of HCC (n=182) [20]. As a result, a total of 124 matched patients with non-sarcomatoid HCC from the prior study were included as controls (Supplementary Table 1). The median follow-up was 40.7 months and 73.7 months for the sarcomatoid and non-sarcomatoid groups, respectively. The 1-year, 3-year, and 5-year OS rates of the patients with non-sarcomatoid HCC were significantly higher than those of the patients with sarcomatoid HCC (87.1% versus 61.3%, 62.1% versus 28.0%, and 43.9% versus 16.8%, respectively; Supplementary Fig. 2A). Similarly, the patients with sarcomatoid HCC had poorer prognosis at 1, 3, and 5 years, with lower RFS rates than the patients with non-sarcomatoid HCC (40.0% versus 79.7%, 15.9% versus 34.3%, and 0.0% versus 26.0%, respectively; Supplementary Fig. 2B). Univariate and multivariate analyses revealed that the tumor subtype (sarcomatoid HCC vs. non-sarcomatoid HCC) was an independent prognostic factor for both OS (P=0.002; hazard ratio [HR]=2.27) and RFS (P=0.002; HR=2.24; Supplementary Fig. 2).

Overview of genomic alterations in sarcomatoid HCCs

We performed WES of 28 paired sarcomatoid tumor components and conventional HCC components and 10 matched normal liver samples from 10 patients with sarcomatoid HCC. The average sequencing depth was 360.3-fold for the sarcomatoid tumor components, 364.3-fold for the conventional HCC components, and 133.5-fold for normal liver samples (Supplementary Table 2). We mapped the sequence reads to the human reference genome and identified a total of 3,696 somatic non-synonymous mutations in the sarcomatoid tumor components (42–1,160 per tumor) and 2,302 somatic non-synonymous mutations in the conventional HCC components (39–1,222 per tumor; Fig. 1A, Supplementary Table 3). The mean number of somatic non-synonymous mutations per patient in the sarcomatoid tumor components was 220.1±333.9, which was comparable to that in the conventional HCC components (215.7±361.3). Sanger sequencing to validate 320 randomly selected non-synonymous mutations showed a high true-discovery rate (91.6%).

Figure 1.

Genomic landscape of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC. (A) The mutational spectrum of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC identified by whole-exome sequencing. (B) Comparison of the most frequently mutated cancer-related genes between patients with sarcomatoid HCC (n=10) and patients with non-sarcomatoid HCC in cohorts from three previous studies. (C) Heatmap of copy-number variations in sarcomatoid HCCs. The x-axis shows chromosomal coordinates. (D) GISTIC analysis revealed the genome distribution of copy-number alterations in sarcomatoid tumor components (lower panel) and conventional HCC components (upper panel). GISTIC q-values (y-axis) for deletions (blue) and amplifications (red) are plotted across the genome (x-axis). HCC, hepatocellular carcinoma; TNM, tumor-node-metastasis.

We compared the most frequently mutated cancer-related genes identified in the sarcomatoid HCC cohort (n=10) to those identified in other non-sarcomatoid HCC cohorts, including our previous cohort [10], the Gao cohort [21], and a TCGA cohort [4]. We found that ARID2 mutation occurred in 70% (7/10) of the patients with sarcomatoid HCC but only 1–5% of the patients with non-sarcomatoid HCC in the other three cohorts (P<0.001 for each comparison; Fig. 1B}.

We identified an average of 878.4 MB gain and 534.3 MB loss in the sarcomatoid tumor components, which was comparable to the total length of copy-number variations in the conventional HCC components (668.9 MB gain and 415.2 MB loss; Fig. 1C). GISTIC analysis identified 5 recurrently amplified segments and 12 recurrently lost segments in the sarcomatoid tumor components, and 4 recurrently amplified segments and 11 recurrently lost segments in the conventional HCC components (Fig. 1D, Supplementary Table 4). Furthermore, 8p23.2 was recurrently deleted in the sarcomatoid tumor components but not in the conventional HCC components. qRT-PCR results demonstrated that the expression of EGR3, GATA4, SOX7, and LZTS1, which are common tumor-suppressor genes located on 8p23.2 [22-25], was downregulated in the sarcomatoid tumor components compared with the conventional HCC components (Supplementary Fig. 3A). Immunohistochemical staining results also confirmed that EGR3 and SOX7 expression was lower in the sarcomatoid tumor components compared with the conventional HCC components (Supplementary Fig. 3B). These results suggested a possible role of 8p23.2 deletion in sarcomatoid HCC development and progression.

Genetic phylogeny and clonal evolution of sarcomatoid HCCs

To infer the evolutionary trajectory of sarcomatoid HCCs, we analyzed the subclonal architectures using calculated mutation clusters based on all identified somatic mutations (SNVs+indels). Figure 2 shows the results rendered using MapScap [26], with different colored ovals indicating clones and subclones and diameters representing the corresponding cancer cell fractions (CCFs). There were 4–8 clonal clusters estimated per patient (Fig. 2), suggesting that multiple clones were present in the samples. The phylogenetic trees showed that all of the sarcomatoid tumor components shared a proportion of mutations with their matched conventional HCC components, confirming the existence of common ancestors from which both tumor components were derived in each patient. We also observed branched tumor evolution during sarcomatoid HCC development and progression; however, we did not observe any difference in the number of clones between the sarcomatoid tumor components and the conventional HCC components in each patient.

Figure 2.

Subclonal architectures and clone phylogenies of sarcomatoid HCCs. Each subclonal architecture represents an individual patient. The diameter of each oval with color is proportional to the estimated cancer cell fraction, which reflects the proportion of cells in that sample that contain the somatic mutations. For the clone phylogenies, FFPE samples with hematoxylin and eosin staining are arrayed in the middle. The clone phylogenies inferred from each conventional HCC component or sarcomatoid tumor component are displayed on the left and right side, respectively. Phylogenetic trees constructed from each patient are displayed on the bottom. Line lengths reflect the numbers of clustered somatic mutations attributed to that clone or subclone. Driver mutations are listed on the corresponding clone or subclone in each phylogenetic tree. FFPE, formalin-fixed and paraffin-embedded; HCC, hepatocellular carcinoma.

We next analyzed the distribution of mutations affecting known HCC driver genes across the clonal phylogenies to determine the timing of acquisition and clone specificity of the driver mutations. TP53 mutations occurred in seven of the 10 patients and were always exhibited in a trunk clone (black clone, 6/7, except for P13), supporting a relatively ancestral role for TP53 mutation. ARID2 mutations appeared in trunk clones in only two patients, whereas they appeared in trunk or branch subclones in four patients. These results confirmed that TP53 mutations commonly occurred at tumor initiation during sarcomatoid HCC development, whereas ARID2 mutations often occurred later as a prerequisite for rapid growth and progression.

The mutational spectrum and signatures in sarcomatoid HCCs

We further analyzed the mutational spectrum according to the genetic phylogenies of the sarcomatoid HCCs (i.e., trunk mutations, conventional HCC component mutations, sarcomatoid tumor component mutations). T>A transversion was enriched in trunk mutations, whereas C>T transition was enriched in mutations occurring in either the conventional HCC component or the sarcomatoid tumor component (Supplementary Fig. 4A).

We next analyzed mutational signatures in all 28 paired sarcomatoid tumor components and conventional HCC components using DeconstructSigs, which accurately reconstructs mutational profiles based on a predefined mutational spectrum of 30 COSMIC signatures. One prominent feature revealed by the mutational signature analysis was that the mutational signatures were similar in the sarcomatoid tumor components and the conventional HCC components (Supplementary Fig. 4B), indicating that both types follow similar mutational processes. The other significant feature was that the heterogeneity of mutational signatures across patients was considerably greater than the heterogeneity across different evolutionary stages within any given patient (Supplementary Fig. 4B). That finding suggests that a given sarcomatoid HCC accesses only a subset of the mutational processes that are potentially available to it, but these mutational processes contribute genomic variation on an ongoing basis. Nonetheless, there were some shifts in the relative contributions of the mutational processes over time. For example, signature 22, which corresponds to T>A transversions and is correlated with aristolochic acid exposure [10], was strongly enriched among trunk mutations, especially in patients P13, P14, and P15. Signature 22 is usually associated with a high mutational burden [10], which might account for the relatively high mutational burden in those three patients. Similarly, alcohol-related signature 16 was also enriched among trunk mutations, suggesting that alcohol-related etiology mainly contributed to sarcomatoid HCC formation but not progression. By contrast, signature 1 and signature 3, which are associated with age and homologous recombination deficiency, respectively, were enriched in conventional HCC components and further enriched in sarcomatoid tumor components, suggesting that those mutational processes contributed more to the stage of sarcomatoid HCC progression.

Transcriptome analyses revealed EMT and hypoxic phenotypes in sarcomatoid HCCs

To comprehensively understand the transcriptional characteristics of sarcomatoid HCCs, we selected one tissue specimen (P03) from among 10 patients with sarcomatoid HCC and applied ST sequencing using the 10×Genomics Visium platform. After clustering, four spatially sequential sections were obtained (Fig. 3A, Supplementary Fig. 5A). Based on marker gene expression and histological characteristics, we identified the four sections: peritumor liver cluster, conventional HCC cluster, sarcomatoid tumor cluster, and margin cluster (Fig. 3A, Supplementary Fig. 5A, 5B). Moreover, we compared the H&E staining images with the ST data regarding the marker gene expression and verified that the transcriptional features were consistent with the histological information (Fig. 3B). Gene Ontology (GO) results showed that different clusters diversified pathway activities within certain tissue components. The activities of cell cycle and DNA repair pathways, which were reported to promote HCC occurrence and progression in previous studies, were higher in conventional HCC components than in peritumor liver components [9,10]. Furthermore, some other pathways, such as cell junction, cell adhesion, and cell migration, were enriched in sarcomatoid tumor components. Importantly, we found that EMT and hypoxic signaling exhibited high enrichment in both conventional HCC and sarcomatoid tumor components, and this enrichment was highest in the sarcomatoid tumor components (Fig. 3C).

Figure 3.

Transcriptome analyses of sarcomatoid HCCs. (A) Spatial transcriptome (ST) sequencing for patient P03. (B) Marker genes of the four clusters in ST sequencing. (C) Gene Ontology (GO) results of conventional HCC cluster compared with peritumor liver cluster, or sarcomatoid tumor cluster compared with conventional HCC cluster in ST sequencing. (D) The heatmap of RNA-seq for 35 samples from 9 patients with sarcomatoid HCCs. (E) GO results of conventional HCC compared with peritumor liver tissues, or sarcomatoid tumor components compared with conventional HCC components in RNA-seq. (F) Gene Set Enrichment Analysis (GSEA) results of EMT and hypoxic pathways in conventional HCC tissues or in sarcomatoid HCCs. (G) TPM of E-cadherin and Vimentin in peritumor liver tissues, conventional HCC components, and sarcomatoid tumor components from 9 patients involved in RNA-seq. EMT, epithelial–mesenchymal transition; ES, enrichment score; HCC, hepatocellular carcinoma; NES, normalized enrichment score; TPM, transcripts per million.

Next, we collected 35 samples (including 12 conventional HCC components, 14 sarcomatoid tumor components, and 9 matched peritumor liver tissues) from the remaining 9 patients with sarcomatoid HCC and performed bulk RNA-seq to validate and supplement the transcriptional results obtained by ST sequencing. The results showed that 103 genes were upregulated both in conventional HCC components (compared with peritumor liver samples) and in sarcomatoid tumor components (compared with conventional HCC components), while 76 genes were similarly downregulated (Fig. 3D, Supplementary Fig. 5C). GO analysis also demonstrated that the activities of the cell cycle, DNA repair, response to hypoxia, and regulation of EMT pathways were enriched in conventional HCC components compared with peritumor liver tissues. Furthermore, when compared with conventional HCC components, sarcomatoid tumor components displayed further enrichment of cell migration, cell adhesion, regulation of EMT, and response to hypoxia (Fig. 3E). Gene Set Enrichment Analysis (GSEA) also indicated that the gene sets of EMT and hypoxic signaling were enriched in conventional HCC components, compared with peritumor liver tissues, and further enriched in sarcomatoid tumor components (Fig. 3F). In addition, we observed that compared with peritumor liver tissues, conventional HCC components had downregulated expression of E-cadherin, and upregulated expression of Vimentin, indicating an EMT phenotype, and these changes in EMT marker expression were even more pronounced in the sarcomatoid tumor components (Fig. 3G).

Immunohistochemical analyses confirmed the EMT phenotype and hypoxic microenvironment in sarcomatoid HCCs

To verify the EMT phenotype of sarcomatoid HCCs revealed by transcriptome analyses, we investigated the expression of the EMT markers E-cadherin, Vimentin, and N-cadherin by immunohistochemical staining in 31 sarcomatoid HCCs with both sarcomatoid tumor components and conventional HCC components. Representative cases of immunohistochemical staining are shown in Figure 4. Compared with peritumor liver tissues, both the conventional HCC components and the sarcomatoid tumor components exhibited a typical EMT phenotype characterized by downregulated E-cadherin and upregulated Vimentin and N-cadherin; moreover, these changes in EMT marker expression were more pronounced in the sarcomatoid tumor components than in the conventional HCC components (Fig. 4). In addition, immunohistochemistry results confirmed a hypoxic microenvironment in these sarcomatoid tumor components, which exhibited dense staining of HIF-1α and CAIX (Fig. 4).

Figure 4.

Phenotypic and microenvironment features in sarcomatoid HCCs. Immunohistochemical staining showed the expression of E-cadherin, Vimentin, N-cadherin, HIF-1α, and CAIX in 31 sarcomatoid HCCs with both sarcomatoid tumor components and conventional HCC components; scale bars=100 μm. HCC, hepatocellular carcinoma.

ARID2 somatic mutations resulted in lower ARID2 expression and were associated with poor outcomes

A total of 25 ARID2 somatic mutations (10 different mutations) were identified in 7 conventional HCC components and 10 sarcomatoid tumor components from 7 patients (Fig. 5A). We used Sanger sequencing to validate these results, except for c.1201-1202del in ST16 because of insufficient DNA (Supplementary Fig. 6). The 10 different ARID2 somatic mutations included 4 frame-shift indels, 2 in-frame indels, 3 nonsense SNVs, and 1 missense SNV. Polyphen-2 analysis revealed that the missense SNV was predicted to adversely affect the function of the ARID2 protein.

Figure 5.

Clinical significance of ARID2 alteration in patients with sarcomatoid HCC and patients with non-sarcomatoid HCC. (A) Distribution of the somatic mutations in ARID2 identified in this study. (B) Representative ARID2 staining in peritumor tissues and tumor tissues from non-sarcomatoid HCCs and in conventional components and sarcomatoid components from ARID2-mutated sarcomatoid HCCs; scale bars=100 μm. (C, D) The statistics of ARID2 staining density in different groups, ^*P<0.05, ^**P<0.01, ^***P<0.001. (E) Kaplan-Meier survival analysis showing OS and RFS rates based on ARID2 expression in 124 matched patients with non-sarcomatoid HCC. Multivariate analysis shows the ability of ARID2 expression to predict OS and RFS compared with that of other clinical parameters, ^*P<0.05, ^**P<0.01. CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; MT, mutant; OS, overall survival; RFS, recurrence-free survival; WT, wild-type.

We next evaluated ARID2 expression by immunohistochemistry in 10 sarcomatoid HCCs and 124 non-sarcomatoid HCCs from matched patients (Fig. 5B). The results showed that ARID2 expression was downregulated in the tumors overall compared with that in adjacent peritumor liver samples (Fig. 5C). Furthermore, ARID2 expression was more downregulated in sarcomatoid HCCs than in non-sarcomatoid HCCs, even those carrying ARID2 somatic mutations (Fig. 5D, Supplementary Fig. 7).

We correlated ARID2 expression with clinical characteristics and outcomes in 124 patients with non-sarcomatoid HCCs. Low ARID2 expression was correlated with increased tumor size and vascular invasion and poor tumor differentiation (Supplementary Table 5). Kaplan–Meier survival analysis revealed significantly shorter OS and RFS among patients with low ARID2 expression (Fig. 5E). Univariate and multivariate analyses confirmed that low ARID2 expression was an independent prognostic factor for OS and RFS (Fig. 5E).

ARID2 functions as a tumor-suppressor gene in HCC

Our results suggested a possible tumor-suppressor role for ARID2 in HCC. To test that hypothesis, we analyzed the genotype and expression of ARID2 in six HCC cell lines. All six HCC cell lines were validated as wild-type (WT) by Sanger sequencing, using the same filter criteria used for WES. Western blots confirmed that ARID2 protein levels in the high metastatic potential HCC cells (MHCC97L, MHCC97H, and HCCLM3) were lower than that in the low metastatic potential HCC cells (HepG2, PLC/PRF/5, and Hep3B) (Fig. 6A) [2,17].

Figure 6.

Identification of ARID2 as a tumor-suppressor gene in HCC. (A) ARID2 expression examined by qRT-PCR and western blot in six HCC cell lines (HepG2, PLC/PRF/5, Hep3B, MHCC97L, MHCC97H, and HCCLM3) and in stably transfected cells. (B) Proliferation of HepG2 cells after ARID2 knockdown and of HCCLM3 cells expressing wild-type or mutant ARID2 compared with that of controls. (C) Colony formation activity of HepG2 cells after ARID2 knockdown and of HCCLM3 cells expressing wild-type or mutant ARID2 compared with that of controls. Bar graphs illustrate quantification of the colony formation assay, ^*P<0.05, ^**P<0.01, ^***P<0.001. (D) Invasion of HepG2 cells after ARID2 knockdown and of HCCLM3 cells expressing wild-type or mutant ARID2 compared with that of controls. The graphs depict the number of invasive cells after 48 hr, ^*P<0.05, ^**P<0.01, ^***P<0.001. (E) Representative bioluminescence images of mouse liver tumors and pulmonary metastasis. The color scale bar depicts the photon flux emitted from the mice, ^*P<0.05, ^**P<0.01, ^***P<0.001. HCC, hepatocellular carcinoma.

Next, we knocked down ARID2 in HepG2 cells and conducted biofunctional investigations to determine the phenotypic effects. We found that the knockdown of ARID2 resulted in increased HCC cell proliferation and colony formation activity and led to enhanced invasive ability (Fig. 6B–6D). In vivo HCC mouse models showed that ARID2 knockdown accelerated tumor growth and pulmonary metastasis (Fig. 6E).

We next generated lentiviral constructs to re-express WT or mutant (MT) ARID2 in HCCLM3 cells. We found that overexpression of WT ARID2 substantially suppressed HCC cell proliferation, colony formation, and invasion ability. By contrast, all MT ARID2 variants failed to cause these effects in whole or in part (Fig. 6B–6D). In agreement with the in vitro studies, analysis of an in vivo HCC mouse model showed that WT ARID2 significantly suppressed tumor growth and pulmonary metastasis, whereas MT ARID2 yielded a larger tumor volume and increased pulmonary metastasis compared with WT ARID2 (Fig. 6E). These results support the notion that ARID2 is a tumor-suppressor gene in HCC and that certain somatic mutations abolish its function and its tumor-inhibitory effect.

Hypoxia facilitated EMT of HCC cells carrying inactivated ARID2

Considering the high frequency of ARID2 mutations in sarcomatoid HCCs, which exhibited an EMT phenotype and hypoxic microenvironment, especially in sarcomatoid tumor components, we speculated that HCC cells carrying ARID2 mutation may undergo EMT under hypoxic conditions, contributing to sarcomatoid HCC formation and development. We observed that the HepG2 cells with ARID2 knockdown took on a spindle-like fibroblastic morphology, whereas the HCCLM3 cells overexpressing WT ARID2 presented a cobblestone-like appearance of epithelium (Supplementary Fig. 8). Next, we screened for epithelial and mesenchymal markers in HCC cells. The results showed that knockdown of ARID2 in HepG2 cells resulted in downregulation of E-cadherin and upregulation of Vimentin and N-cadherin (Fig. 7C, 7D). In HCCLM3 cells, reexpression of WT ARID2 had an inverse effect, including upregulation of E-cadherin and downregulation of Vimentin and N-cadherin, whereas expression of MT ARID2 failed to cause these effects in whole or in part (Fig. 7A, 7B). Furthermore, these HCC cells displayed consistent snail expression with phenotypes typical of cells undergoing EMT, but not consistent expression of other transcriptional factors such as slug, twist, E47, ZEB1, or ZEB2 (data not shown). We validated the in vitro results with immunohistochemical staining of tumor sections from a mouse model (Fig. 7G). These results suggested that ARID2 inactivation leads to EMT of HCC cells. More importantly, we found that in HCCLM3 and HepG2-shARID2 cells—two cell lines with ARID2 deficiency—hypoxia further induced phenotypes typical of cells undergoing EMT, including further downregulation of E-cadherin and further upregulation of Vimentin and N-cadherin (Fig. 7C–7F). These results demonstrated that hypoxia facilitated EMT of HCC cells carrying inactivated ARID2, which may contribute to sarcomatoid HCC formation and development.

Figure 7.

Hypoxia facilitated EMT of HCC cells carrying inactivated ARID2. (A) Results of qRT-PCR and (B) western blot analysis showed changes in EMT marker (E-cadherin, N-cadherin, Vimentin, and snail) expression in HCCLM3 cells expressing wild-type or mutant ARID2 compared with that of controls. (C) Results of qRT-PCR and (D) western blot analysis showed changes in EMT marker expression in HepG2 cells after ARID2 knockdown compared with that of controls and under hypoxic conditions. (E) Results of qRT-PCR and (F) western blot analysis showed changes in EMT marker expression in HCCLM3 cells under hypoxic conditions compared with that of controls. (G) Representative images of serial tumor sections from xenograft tumor models. Scale bars=100 μm. EMT, epithelial–mesenchymal transition; HCC, hepatocellular carcinoma.

DISCUSSION

Sarcomatoid HCC is a rare histological subtype of HCC with a highly aggressive nature, which is characterized by combined features of epithelial (HCC) and mesenchymal (sarcoma) [11]. Patients with sarcomatoid HCCs are frequently diagnosed at an advanced stage with a larger tumor size or lymph node metastasis. All these characteristics lead to the extremely poor prognosis with a high risk of recurrence and metastasis [11,14]. Our cohort study featuring a comparison of outcomes between sarcomatoid and non-sarcomatoid HCCs revealed a consistent conclusion: that the sarcomatoid subtype is the most sensitive predictor of both OS and RFS.

Until now, no study has explored the genomic alterations in sarcomatoid HCC. Using laser capture microdissection, we isolated and sequenced paired sarcomatoid tumor components and conventional HCC components. The results showed no significant difference in driver gene mutation between the sarcomatoid components and the conventional HCC components, suggesting that the two components shared the same genetic background. By comparing mutation profiles in cancer-related genes between patients with sarcomatoid HCC and patients with non-sarcomatoid HCC, we found that ARID2 mutation was enriched in patients with sarcomatoid HCC, which suggests a potentially crucial role of ARID2 mutation in sarcomatoid HCC pathogenesis. Therefore, HCC patients harboring ARID2 mutations should be followed closely after surgery, because their tumors may contain sarcomatoid tumor components, leading to extremely poor prognosis [11].

Through ST sequencing and RNA-seq, we revealed different transcriptional characteristics in sarcomatoid HCCs; the enrichment of several signaling, including cell adhesion, cell migration, especially the EMT pathway, was increased in sarcomatoid tumor components. H&E staining confirmed that sarcomatoid components of sarcomatoid HCCs consist of spindle-shaped cells that exhibit oval and elongated nuclei with conspicuous nucleoli and spindle-shaped eosinophilic cytoplasm [12]. The spindle cells form a focal storiform pattern and often present with mitotic figures [14]. Immunohistochemical staining also verified the EMT phenotype in sarcomatoid HCCs. The EMT state indicated a higher invasive and metastatic potential, which might account for the poor prognosis of sarcomatoid HCC.

Several hypotheses have been proposed to explain the formation of sarcomatoid tumor components in sarcomatoid HCC. Some authors assumed that these biphasic tumors arise from totipotent stem cells that are able to differentiate into both epithelial cells and mesenchymal cells [27,28]. Others hypothesized that the conventional HCC components were likely to transform into sarcomatoid components through a metaplastic process, as suggested by the transitional areas presenting between conventional HCC components and sarcomatoid tumor components [29,30]. Some authors hypothesized that cancer cells from conventional HCCs were capable of transforming into multipotent immature cells, which, in turn, re-differentiated into sarcomatoid components [31,32]. In addition, World Health Organization tumor classifications suggested that the sarcomatoid component is a carcinoma that has undergone sarcomalike differentiation. The sarcomatoid component represents clonal evolution from the differentiated conventional HCC component [33,34]. Our results suggest that the sarcomatoid tumor components and the conventional HCC components are derived from common ancestors, mostly accessing similar mutational processes. The clonal phylogenies of conventional HCC components and sarcomatoid tumor components demonstrated branched tumor evolution during sarcomatoid HCC development and progression, supporting the clonal evolution hypothesis. This prompted us to examine the possible role of the EMT process in the formation of the sarcomatoid component based on genomic characterization and microenvironment features.

ARID2 is one subunit of the chromatin remodeling complex and is involved in various biological processes, including transcriptional regulation [35], cell cycle modulation [36], embryonic development [37], and DNA damage repair [38]. Although ARID2 mutation has been detected in most cancers, its frequency was low in non-sarcomatoid HCC cohorts [4-7,10,21]. Our results revealed a very high frequency of ARID2 mutations in sarcomatoid HCCs, most of which were truncating mutations that led to partial or complete inactivation of the ARID2 protein. We demonstrated through gain-of-function and loss-of-function studies that ARID2 plays a tumor-suppressor role in HCC, especially during EMT, which might involve regulation of the transcription factor Snail. We further showed that ARID2 expression is a predictor of patient prognosis in non-sarcomatoid HCCs. These features suggest that ARID2 acts as a tumor suppressor in HCC.

To verify the hypoxic phenotype revealed by transcriptome analyses, we stained for HIF-1α and CAIX expression, which demonstrated a hypoxic microenvironment in the sarcomatoid tumor components. Hence, we inferred that the microenvironment may participate in the formation of the sarcomatoid tumor component. Therefore, we hypothesized that HCC cells carrying ARID2 mutations undergo EMT under hypoxia. We subsequently validated this hypothesis through in vitro and in vivo experiments. Our results suggest that genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment) leads to EMT of HCC cells, which may contribute to the formation of the sarcomatoid tumor component, leading to sarcomatoid HCC development and progression. These results indicated that overcoming the tumor hypoxic microenvironment and targeting EMT progress might be effective therapeutic strategies for sarcomatoid HCC patients [39,40].

Taken together, we portrayed the integrated molecular characterization of sarcomatoid HCC through multiomics study, and identified genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), which may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

Notes

Authors’ contribution

RQS, YHY, and YX performed the experiments; SLZ, RQS, and BW analyzed the data; SYP, NL, LC, JYP, ZQH, and ZJZ provided the samples; SLZ, RQS, and JZ wrote the paper; JF and JZ commented on the study and revised the paper; ZJZ, CLS, and SLZ obtained funding and designed the research.

Acknowledgements

This study was jointly supported by the National Natural Science Foundation of China (No. 82372985, No. 82373418, No. 82273247, No. 82173260, No. 82072681, No. 82003082) and Shanghai Medical Innovation Research Project (22Y11907300).

Conflicts of Interest

The authors have no conflicts to disclose.

Abbreviations

AFP

alpha-fetoprotein

CCF

cancer cell fraction

EMT

epithelial–mesenchymal transition

FFPE

formalin-fixed and paraffin-embedded

HCC

hepatocellular carcinoma

overall survival

RFS

recurrence-free survival

spatial transcriptome

TNM

tumor-node-metastasis

WES

whole-exome sequencing

wild-type

SUPPLEMENTAL MATERIAL

Supplementary material is available at Clinical and Molecular Hepatology website (http://www.e-cmh.org).

Supplementary Methods.cmh-2024-0686-Supplementary-Methods.pdf

Supplementary Figure 1.

The patient selection flow chart. The selection flow chart of 31 patients with both sarcomatoid tumor components and conventional hepatocellular carcinoma (HCC) components in this study.

cmh-2024-0686-Supplementary-Figure-1.pdf

Supplementary Figure 2.

The prognosis of patients with sarcomatoid HCCs. (A) Kaplan–Meier survival analysis showing OS rates based on tumor subtype (sarcomatoid HCC vs. non-sarcomatoid HCC). The ability of tumor subtype to predict OS is compared with that of other clinical parameters by multivariate analyses, ^*P<0.05, ^**P<0.01. (B) Kaplan–Meier survival analysis showing RFS rates based on tumor subtype (sarcomatoid HCC vs. non-sarcomatoid HCC). The ability of tumor subtype to predict RFS is compared with that of other clinical parameters by multivariate analyses. CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; OS, overall survival; RFS, recurrence-free survival, ^**P<0.01.

cmh-2024-0686-Supplementary-Figure-2.pdf

Supplementary Figure 3.

Gene expression located on 8p23.2. (A) Results of qRT-PCR showed changes in EGR3, GATA4, SOX7, and LZTS1 expression in sarcomatoid tumor components and conventional HCC components. (B) Immunohistochemical staining showed the expression of EGR3 and SOX7 in the 10 cases included in multiomics study; scale bars=100 μm. HCC, hepatocellular carcinoma.

cmh-2024-0686-Supplementary-Figure-3.pdf

Supplementary Figure 4.

Mutational spectrum and signatures in sarcomatoid tumor components and conventional HCC components. (A) Dot plots showing six substitution patterns and the distribution of mutational signatures for each patient in the trunk, conventional HCC component, and sarcomatoid tumor component based on phylogenetic trees (left). Pie charts indicating major signature distributions (right). (B) Pie charts indicating mutational signature evolution during sarcomatoid HCC formation and progression based on phylogenetic trees in 10 patients. HCC, hepatocellular carcinoma.

cmh-2024-0686-Supplementary-Figure-4.pdf

Supplementary Figure 5.

Spatial transcriptome and bulk RNA-seq of sarcomatoid HCCs. (A) Uniform Manifold Approximation and Proriptome (ST) sequencing. (B) Heatmap of 4 clusters with unique signature genes by ST sequencing. (C) The expressions of genes were upregulated or downregulated both in conventional HCC components (compared with peritumor liver tissues) and in sarcomatoid tumor components (compared with conventional HCC components) by bulk jection (UMAP) plot displayed 4 clusters identified by spatial transc RNA-seq. HCC, hepatocellular carcinoma.

cmh-2024-0686-Supplementary-Figure-5.pdf

Supplementary Figure 6.

The Sanger sequencing trace files of ARID2 mutations in tumors. ARID2 mutations detected by whole-exome sequencing in patients with sarcomatoid HCCs. The mutant sites highlighted by red arrows. HCC, hepatocellular carcinoma.

cmh-2024-0686-Supplementary-Figure-6.pdf

Supplementary Figure 7.

The expression of ARID2 in sarcomatoid HCCs. (A) Representative ARID2 staining in conventional HCC components and sarcomatoid tumor components from ARID2 wild-type sarcomatoid HCCs; scale bars=100 μm. (B) The statistics of ARID2 staining density in different groups, ^*P<0.05. HCC, hepatocellular carcinoma; MT, mutant; WT, wild-type.

cmh-2024-0686-Supplementary-Figure-7.pdf

Supplementary Figure 8.

The cellular morphology changes of HCC cells with ARID2 knockdown or overexpression. The cellular morphology of the HepG2 cells with ARID2 knockdown (left) and the HCCLM3 cells overexpressing WT ARID2 (right); scale bars=100 μm. HCC, hepatocellular carcinoma; WT, wild-type.

cmh-2024-0686-Supplementary-Figure-8.pdf

Supplementary Table 1.

Clinicopathologic characteristics of 31 sarcomatoid HCCs and 124 matched non-sarcomatoid HCCs

cmh-2024-0686-Supplementary-Table-1.pdf

Supplementary Table 2.

The average depth of WES in 28 paired sarcomatoid tumor components and conventional HCC components and 10 matched normal liver samples from 10 patients with sarcomatoid HCC

cmh-2024-0686-Supplementary-Table-2.pdf

Supplementary Table 3.

The list of non-synonymous somatic mutations identified in 28 paired sarcomatoid tumor components and conventional hepatocellular carcinoma (HCC) components from 10 patients with sarcomatoid HCC

cmh-2024-0686-Supplementary-Table-3.xlsx

Supplementary Table 4.

Significant CNV regions and affected genes identified in 28 paired sarcomatoid tumor components and conventional hepatocellular carcinoma (HCC) components from 10 patients with sarcomatoid HCC

cmh-2024-0686-Supplementary-Table-4.pdf

Supplementary Table 5.

Correlation between ARID2 expression and clinicopathologic characteristics in 124 non-sarcomatoid HCCs

cmh-2024-0686-Supplementary-Table-5.pdf

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Notes

Study Highlights

• Whole-exome sequencing analyses indicate the sarcomatoid tumor components and the conventional HCC components are derived from common ancestors.

• Transcriptome analyses reveal the EMT and hypoxic phenotype in sarcomatoid tumor components.

• ARID2 mutations in 70% of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC.

• The inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment.