An update on the histological subtypes of hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Microscopically, it is characterized by thickened cell plates, malignant tumor cell cytology, capillarization of sinusoids and evidence of invasion. Histological evaluation of HCC specimens plays a key role in tumor staging, and in distinguishing HCC from its precursor lesions or other liver nodules. With reference to the multi-step process of hepatocarcinogenesis which could also be observed histologically, some classification systems have been proposed for HCC. For instance, the terms “early HCC” and “progressed HCC” have been defined based on the size and differentiation of tumor^[1]. In recent years, further investigations have been carried out on the subtyping of HCC specifically referencing the morphological characteristics of tumor cells. The significance of these subtypes was further substantiated by their clinical relevance and the genetic makeup. In the latest 5th edition of the World Health Organization (WHO) Classification of Digestive System Tumors, several histological subtypes were described^[2]. In this review, we will focus on elaborating the recent understanding on 5 subtypes: macrotrabecular-massive HCC (MTM-HCC), steatohepatitic HCC (SH-HCC), lymphocyte-rich HCC, scirrhous HCC and fibrolamellar carcinoma (FLC). In addition, an update on the entity combined hepatocellular-cholangiocarcinoma (cHCC-CCA) will be discussed.

MTM-HCC

Published in 2018, histological review of clinical samples archive led to the identification of a novel and distinct subtype of HCC defined by the histological features of tumor cells - MTM-HCC. It is defined as the presence of macrotrabeculae of more than 6 cells thick in > 50% tumor, and was identified in 16% on average in 2 large cohorts comprising 237 surgical resection samples and 284 biopsy samples^[3]. And this subtype was statistically associated with aggressive parameters including tumor size, alpha-fetoprotein (AFP) levels, satellite nodules and vascular invasion. Besides, it was an independent prognosticator for early recurrence (within 2 years) and overall recurrence^[3]. The prognostic significance was further validated by other group^[4]. In another study by Jeon et al.^[5], MTM-HCC, as defined by > 30% of macrotrabecular pattern, was associated with large tumor, hepatitis B virus infection, and less frequent cirrhosis. This subtype was also found to be associated with higher tumor grade, tumor stage, higher AFP level, and a worse recurrence-free survival.

In addition, investigations were carried out to identify specific radiological, immunohistochemical, and genetic features of this entity. Radiologically, MTM-HCC was reported to preferentially demonstrate irregular rim-like arterial phase enhancement on gadoxetate-enhanced magnetic resonance imaging^[6]. Extending from their initial observation, Calderaro et al.^[7] attempted to identify potential biomarkers for this entity. On analysis of the TCGA dataset, endothelial-specific molecule 1 (ESM1) was identified and validated as a biomarker for MTM-HCC. In addition, angiotensin 2 and VEGFA overexpression was observed in MTM subtype^[8]. Recent studies also shed light on the genetic composition of MTM-HCC, which was found to be related to cell cycle activation, chromosomal instability, the G3 transcriptomic subgroup^[8] according to Boyault et al.^[9] and TP53 mutation^[8].

SH-HCC

The steatohepatitic subtype was first described by Salomao et al.^[10]. It is characterized by prominent steatotic changes in the tumor cells namely fat accumulation, ballooning degeneration, presence Mallory-Denk bodies and peri-cellular fibrosis. In a study examining HCV-related liver explants, SH-HCC was identified in 35.5% of the cohort^[10]. According to a follow-up study by the same group in 2012, SH-HCC constituted 14% of HCC explants^[11]. A more recent paper reported a diagnosis of SH-HCC in around 20% of 96 HCC cases reviewed, and made an observation that SH-HCC was not associated with cirrhosis^[12]. It was noted that around 60% (14 of 22) SH-HCCs was associated with one more known risk factor for non-alcoholic fatty liver disease (NAFLD)^[10]. The association with and NAFLD and metabolic syndrome was consolidated in other studies^[13,14]. While most studies suggested a link of fatty liver disease with this subtype, in 2015 Yeh et al.^[15] looked at a series of SH-HCC and identified a group of patients without any underlying causes for metabolic disease.

Regarding correlation with other histological tumor parameters, there was a lack of microsatellite nodules or microvascular invasion in SH-HCC^[8]. On the immunohistochemical phenotype, C reactive protein (CRP) expression was frequent^[8]. Immunohistochemical expression of serum amyloid A and CRP was significantly higher in this subtype than conventional HCC as revealed by another study^[16]. It was also found that the cancer-associated fibroblasts in SH-HCC more frequently expressed senescence-associated secretory phenotype by immunohistochemical staining^[14]. On genetic and genomic levels, SH-HCC was shown to associate with IL6/JAK/STAT pathway activation, as well as wild type CTNNB1^[8,17] and TP53^[8]. By multivariate modeling, it was shown to be related to the S1 subclass^[12] according to Hoshida et al.^[18].

Lymphocyte-rich HCC

Previously known as lymphoepithelioma-like HCC, lymphocyte-rich HCC is characterized by an immune-rich stroma^[19,20]. Wada et al.^[21] defined this subtype by the presence of more than 100 tumor-infiltrating lymphocytes in 10 high-power fields. Despite a difference in immune cells infiltration, immunohistochemically the tumor cells express epithelial markers and HepPar-1^[22-27]. The tumor-infiltrating lymphocytes were largely composed of CD3+ T cells^[19-29]. In contrast to lymphoepithelioma-like carcinoma originating in the nasopharynx, vast majority of lymphocyte-rich HCC were EBER negative^{[19,22,23,26,28,30]}. In 2017, Labgaa et al.^[31] published a comprehensive review on a total of 66 lymphocyte-rich HCC cases. In this report, 64% patients were male and liver cirrhosis was present in 46%. While a few studies demonstrated a trend of better survival with this subtype^[19-21,28], the prognostic significance of this subtype remains to be clarified due to its rarity. The genomic landscape of 12 lymphocyte-rich HCC was determined by whole-exome sequencing in a recent report^[32]. Mutations of CTNNB1, AXIN1, APC, NOTCH1 and NOTCH2 were less frequently observed in lymphocyte-rich HCC than conventional HCC. Since activation of Wnt/beta-catenin pathway was correlated with poorer clinical response to immune checkpoint inhibitors^[33], lymphocyte-rich HCC is possibly more susceptible to immunotherapies. The potential significance in terms of treatment response was in line with in a recent study examining the immunohistochemical expression in 217 HCCs, that a high programmed death-ligand 1 expression was correlated with the lymphocyte-rich subtype^[34].

Scirrhous HCC

Scirrhous HCC shows peculiar histology with small oval cells arranged in nests or trabecular among an abundant fibrous stroma^[35]. It comprises 0.19% of all HCC from the National Cancer Database from 2004-2015^[36]. The survival outcome for this subtype remains to be further delineated. Overall survival of patients was found comparable with non-scirrhous HCC in some studies^[36,37], while both better^[38-40] and worse^[35] survival outcomes were also reported. Furthermore, scirrhous HCC was associated with less frequent HBV infection, lower serum AFP level and less liver cirrhosis when compared with conventional HCC^[37]. Radiologically, scirrhous HCC was reported to show distinct computed tomography (CT) scan features including presence of washout areas^[41]. Immunohistochemical analyses revealed expression of stem/progenitor markers in scirrhous HCC; and gene expression profiling highlighted a TGF-β signature^[35].

FLC

FLC was first introduced in 1956^[42] illustrating a primary liver cancer displaying characteristic large eosinophilic tumor cells with prominent nucleoli and pale bodies, and the prominent fibrotic bands traversing the tumor cells in lamellae. The latter feature led to the coining of its nomenclature^[43]. FLC occurs more often in young adults with a mean age of diagnosis at 25 years^[43-45]. FLC express CK7 and HepPar-1 immunohistochemical staining^[46]. From a nationwide study published in 2014 using the SEER data base, the incidence of FLC was 1% among 7225 patients^[47]. In the same study, it was reported that patients tend to be younger, female, and associated with longer overall survival on univariate analysis. In 2014, it was reported that a chimeric transcript was identified, which was further found to be due to a deletion in chromosome 19 detected by whole genome sequencing, which in turn leads to the generation of the DNAJB1-PRKACA chimeric protein, with the kinase activity is retained in the latter component^[48]. This discovery is significant since it provides a pathognomonic genetic feature for this subtype. The tumorigenicity of the fusion transcript was validated by in vivo mouse model with hydrodynamic tail vein injection of Crispr/cas9 generated DNAJB1-PRKACA vector^[49]. Subsequent study revealed an interaction between the fusion kinase and β-catenin^[50], suggesting a contributory role of the fusion protein and β-catenin in the pathogenesis of FLC. In addition, analysis of clinical samples suggested the recruitment of heat shock protein 70 by the fusion enzyme and further in phosphoproteomic profiling using cell line models highlights the activation of ERK signaling in DNAJ-PKAC cells^[51].

cHCC-CCA

cHCC-CCA is defined as a primary liver cancer showing unequivocal presence of both hepatocytic and cholangiocytic differentiation in the same tumor^[2,52]. The 2 components histologically can either be juxtaposed with or intermingled with each other. There is no definite cutoff value as to the minimal proportion of each component present in a tumor to render a diagnosis of cHCC-CCA. In this type of liver cancer, small uniform epithelial cells with scanty cytoplasm and showing CK19, EpCAM, CD56, CD117 or CD133 expression has been observed^[2]. The radiological feature with CT scan/magnetic resonance imaging was reviewed by a French group^[53]. In the study, a mixed pattern comprising HCC, intrahepatic cholangiocarcinoma and atypical radiological pattern was observed in cHCC-CCA; and this mixed pattern showed a sensitivity of 48% and a specificity of 81%. Protein expression for of diagnostic purpose of cHCC-CCA has been investigated, and malic enzyme 1 (ME1) was proposed as a potential immunohistochemical marker for cHCC-CCA, in which 77% express ME1^[54].

Previous study demonstrated an intermediate clinical outcome of cHCC-CCA between HCC and intrahepatic cholangiocarcinoma (iCCA), when overall survival after resection, disease-free survival after resection, and overall survival after liver transplantation were considered^[55]. A more recent study comprising 250 cHCC-CCA in the training cohort and 99 cases in the validation cohort demonstrated that the 1-, 2 and 3-year overall survival was 67.7%, 46.8% and 37.9% respectively; and the 1-, 2 and 3-year cancer-specific survival was 73.1%, 52.0% and 43.0%, respectively^[56]. At times of recurrence or metastasis, as reported by He et al.^[56], the heterogeneity tends to be retained rendering the clinical behavior of cHCC-CCA recurrence is largely unpredictable^[57].

Despite the deviation in clinical outcome, a study on 20 cHCC-CCA samples by capture-based next-generation sequencing revealed similar genomic profiles to conventional HCC. Recurrent alterations in TERT, TP53, cell cycle genes, receptor tyrosine kinase/Ras/PI3K pathway genes, chromatin regulators, etc., were identified in cHCC-CCA, while IDH1, IDH2, FGFR2 or BAP1 mutations were absent^[58]. On a side note, genomic and genetic profiling of cholangiolocellular carcinoma, which was previously classified as a subtype of cHCC-CCA in the 4th edition of WHO Classification of Digestive Tumors^[59], showed that this entity was likely biliary tract origin featuring NCAM expression, chromosomal stability and TGF-β activation^[60]. Consistent findings were reported by Balitzer et al.^[61]. By comparing immunohistochemical expressions, mutational profiles and copy number variation patterns, cholangiolocellular carcinoma was shown to display a highly similar pattern with iCCA, suggesting that the former should instead be classified as a form of well differentiated iCCA.

Future perspectives

In this review, latest understanding on 5 HCC subtypes and the distinct entity cHCC-CCA were discussed. These entities in common demonstrate peculiar pathognomonic histological features. Among these entities, MTM-HCC, lymphocyte-rich HCC and cHCC-CCA are known carry potential prognostic significance. In addition, lymphocyte-rich HCC may represent a subtype showing relatively favorable response to immunotherapy. SH-HCC may represent a spectrum of HCC arising from specific etiology. Further delineation of the genetic and genomic signatures of FLC and cHCC-CCA may provide insights on the cell of origin and pathogenesis of primary liver cancer.

Apart from defining specific subtypes, some histological features in HCC were found to be closely related to certain genetic alterations. For instance, well differentiated tumors with pseudoacinar pattern, tumor cell cholestasis and lack of immune cell infiltration were associated with CTNNB1 mutations^[8,62]. Calderaro et al.^[63] summarized a histological-molecular correlation of liver cancer. In this review, the molecular subclasses^[9,18] and genetic alterations of histological subtypes including MTM-HCC, SH-HCC, scirrhous HCC, lymphocyte-rich HCC were discussed. Besides, the immune microenvironment of 158 HCC cases was recently characterized by Kurebayashi et al.^[64] using multiplex immunohistochemistry. The accumulating body of information, together with integrated analyses of the expression profiles of HCC at transcriptomic, genomic and proteomic levels may facilitate formulating a classification system of clinical relevance.