Hepatoid adenocarcinoma of the stomach: a unique subgroup with distinct clinicopathological and molecular features

Since Ishikura et al. first proposed the new entity HAS in 1985 [18], sporadic cases of this cancer have been reported [19]. HAS is characterized by HCC-like differentiation and is associated with a high incidence of liver metastasis and a poor prognosis.

Similar to the results of previous research [3], this study showed that HAS was characterized by older patient age, male patient sex, intestinal type, and high liver metastasis frequency.

A few studies have examined the histogenesis or development of this cancer and found that the hepatoid cell component was observed only in invasive lesions, indicating that HAS developed from CGC in the mucosa and differentiated into HAS during the process of tumour invasion and proliferation, acquiring the ability to produce AFP [7]. Our result was consistent with this hypothesis, by revealing that the higher the HAS cell component percentage in a tumour, the more is the AFP secreted by the tumour.

Recent attempts have been made to apply NGS technologies to clinical practice, developing innovative precision treatment for different subtypes of gastric cancer. Several comprehensive datasets have revealed many genetic characteristics of gastric cancer [10, 20, 21]. The assemblers of one of these datasets, the TCGA Research Network, defined four major genomic subtypes of gastric cancer: EBV-infected tumours, MSI tumours, genomically stable tumours, and chromosomally unstable tumours. However, retrospective analysis of the TCGA dataset did not provide any information about genetic alterations in HAS, due to its rarity and geographical distribution [11]. The vast majority of HAS case reports are from Asian regions, especially Japan and China. A previous report by Akazawa et al. revealed that HAS may be subcategorized as a solid type of gastric adenocarcinoma with enteroblastic differentiation (GAED) using NGS for 24 patients with GAED, including 3 HAS cases. The most obvious molecular feature of GAED has been reported to be high-frequency TP53 mutations and CNG of ERBB2 [16]. However, our results identified a TP53 mutation rate of 30% in HAS, which is significantly lower than that in GAED (79.2%) and conventional GC (48.0% in the TCGA database) [10, 16], but similar to that in another report by Akiyama in which 33% (5/15) of patients with HAS had a TP53 mutation [7]. In addition, CNG of ERBB2 was not frequently detected in our study. All these differences indicate that HAS may be a genetically distinct subgroup compared with GAED, although many overlapping clinicopathological features exist between the two subcategories of gastric cancer, including a solid pattern and AFP expression.

Considering the existing literature contains very limited information on the molecular features of HAS, we performed NGS with a 483-gene panel in 24 HAS cases and found that DNA CNG at 20q11.21-13.12, which was not detected in CGC, was clearly the most frequent genetic alteration in our HAS cohort. Previous research suggested that 20q amplification might serve as a cancer-initiating event in the development of many cancers [22, 23]. In GC, CNGs at 20q11-13 were detected in 20–30% of cases [24]; moreover, CNVs tended to be more common in intestinal-type cancers than in diffuse-type cancers [25]. In our HAS cohort, the rate of CNG at 20q12.21-13.12 was as high as 50%. More importantly, with respect to associations between clinicopathological characteristics and genetic alterations, CNGs at 20q 11.21-13.12 were associated with more adverse tumour biobehaviour. Therefore, we hypothesize that potential driver genes located at 20q11.21-13.12 may contribute greatly to the carcinogenesis and development of HAS. Among these amplified genes, TOP1 was found to have the highest amplification frequency. In colorectal cancer, the rate of TOP1 CNG has been reported to range from 53 to 84% [26, 27]; moreover, in a metastatic setting, a borderline significant association (p = 0.007) between an increase in the TOP1 CN and an objective response to second-line treatment with irinotecan monotherapy has been reported [28]. Although no relevant reports have been presented in previous GC studies, in our study the high frequency of TOP1 CNG in HAS, but not in CGC, suggested that this alteration may become a useful predictive biomarker for TOP1-targeting therapy.

Another interesting observation to note is that SALL4, a novel stem cell gene, is located at 20q13.2. A member of the spalt-like (SALL) gene family (SALL1 to SALL4), SALL4, is a key factor in the maintenance of embryonic stem cell pluripotency and self-renewal [29, 30]. An oncofoetal protein similar to AFP, SALL4 is highly expressed in both the murine and human foetal liver, and its expression declines gradually during development and is silenced in adulthood. SALL4 re-expression is recognized in various cancers and is considered an adverse prognostic factor in HCC, breast cancer, and lung cancer [31, 32]. In gastric cancer, SALL4 was reported to play oncogenic roles through the modulation of epithelial–mesenchymal transition (EMT) and cell stemness [33]. In our study, SALL4 expression was detected in 94.7% and 10.5% of HAS and CGC samples, respectively, similar to the 89.0% vs 15.0% noted in a previous report [5]. In addition, pathway enrichment analysis showed that signalling pathways regulating the pluripotency of stem cells were specifically enriched in HAS. Combining all these results, we speculate that SALL4 may play an essential role in HAS carcinogenesis and that it can be considered a novel target for HAS diagnosis and therapy. Given the oncogenic role of SALL4 and its specific expression in a subset of cancers, its usefulness as a therapeutic target has been explored. In HCC cell lines, the SALL4 expression status was associated with histone deacetylase activity, and treatment with a histone deacetylase (HDAC) inhibitor successfully suppressed the proliferation of SALL4-positive HCC cells [34]. In addition, SALL4-expressing lung cancers are sensitive to treatment with the HDAC inhibitor entinostat [35]. Since there is no relevant report on HAS, further investigations on whether SALL4 targeting can treat HAS should be performed.

Overall, our NGS data showed that HAS is highly genetically distinct, as reflected by the frequent CNGs at 20q12.21-13.12. In addition, the mechanism of genomic instability in HAS may be associated with the overexpression of the stem cell marker SALL4. The specific relation between these characteristics needs further research in the future. To conclude, by comprehensively investigating the molecular features of HAS using NGS, several markers may be considered as therapeutic targets in the future.

As a rare, unique type of gastric cancer, HAS has been reported by many authors to exhibit more aggressive biobehaviour and poorer prognosis than CGC. However, the link between AFP and survival has historically been controversial. In H. Katai’s study, for example, the 5-year survival rate was 34%, and survival after surgery was found to not be linked to the preoperative serum AFP level [36]. However, other studies indicated that a high level of serum AFP is an independent prognostic factor in gastric cancer [37]. The 5-year survival rates for patients with gastric cancer with AFP ≤ 20 ng/ml, 20 < AFP ≤ 300 ng/ml, and AFP > 300 ng/ml were 45.8, 17.8, and 0%, respectively [38].

In the present study, the prognosis of HAS patients who received radical surgery was not as poor as previously thought, with a 5-year survival rate of 41.1%. However, the evaluation of more cases and the use of a longer follow-up period are required to confirm these findings and draw accurate conclusions. However, interestingly, a markedly elevated preoperative serum AFP level (≥ 500 ng/ml), but not a higher HAS cell component percentage was significantly associated with poorer DFS and OS in HAS. Indeed, serum AFP, as the most representative biomarker, has been found to play a critical role in the initiation of HCC progenitor/stem cells [39]. Additionally, among GCs, AFP-producing GCs have higher malignant potential (high proliferative activity, weak apoptosis, and rich neovascularization) than AFP-negative GCs; furthermore, interfering with AFP expression reduced cell invasion and metastasis by enhancing anoikis sensitivity [40]. Therefore, we hypothesize that serum AFP not only is involved in the initiation of HAS, but also plays important roles in tumour progression and invasion.

In summary, this study showed that HAS is genetically characterized by CNGs at 20q11.21-13.12. Investigating and targeting potential driver genes at this locus may provide novel personalized therapies for this rare subtype of GC. The serum AFP level is a prognostic biomarker in HAS, which may also be therapeutically exploitable.