Clinicopathological and Prognostic Characteristics of Hepatoid Adenocarcinoma of the Stomach

Objectives The aim of this study is to analyze the clinical and pathological features of HAS and investigate the factors affecting prognosis. Methods From January 2014 to May 2020, 31 patients were diagnosed as HAS in in the First Aliated Hospital of Zhengzhou University. The clinicopathologic features and the relevant prognosis of these patients were analyzed. Results Of the 31 patients, 23 were male, with a median age of 62 years. And among the 31 patients, 22 patients underwent a radical gastrectomy and a D2 lymph node dissection. During the follow up period, 18 patients died of the disease. The median OS (mOS) was 28 months. The 1-year survival rate was 55%, the 3-year survival rate was 47%, and the 5-year survival rate was 34%. An elevated CA125 level, distant metastasis, and radical surgery were associated with the prognosis of HAS, and distant metastasis is an independent prognostic risk factor for survival. Conclusion HAS had a poor prognosis because of its frequent vascular involvement, and lymph node and liver metastasis. Aggressive multimodal treatment comprising radical gastrectomy and adjuvant chemotherapy is warranted. This article describes the Clinicopathological of Hepatoid Adenocarcinoma of the Stomach and through survival analysis, the factors affecting the patient’s prognosis were studied. fact that more patients undergo radical surgery and adjuvant chemotherapy in our group.Univariate analysis showed that radical surgery is associated with prognosis(P = 0.0001;HR = 0.087,95%CI: 0.025 ~ 0.299), but between receiving adjuvant chemotherapy or not, the difference was not statistically signicant (P = 0.936 (cid:0) HR = 1.068 (cid:0) 95%CI (cid:0) 0.214 ~ 5.319).Multivariate analysis revealed that only distant metastasis is an independent prognostic risk factor for survival (P = 0.038; HR = 27.016, 95%CI: 1.205 ~ 605.664). Due to the sample size is small, risk stratication was not done based on the TNM staging of this group of patients and other risk factors. In addition, the single-center retrospective study may have a selection bias. The benet of radical surgery and adjuvant chemotherapy need to be explored by further large-scale clinical research. And whether targeted drugs such as sorafenib can be used to treat HAS also look forward to additional studies.


Introduction
Hepatoid adenocarcinoma (HAC) is a particular type of extrahepatic adenocarcinoma with morphological characteristics similar to hepatocellular carcinoma (HCC) [1].In 1985, Ishikura et al. reported a case of AFP-producing primary gastric adenocarcinoma and rst described HAC as a type of tumor that produces large amounts of AFP and has morphological and immunohistochemical characteristics of liver differentiation [2].However, in 1997, he reported a case of primary gastric cancer with histological characteristics similar to hepatocellular carcinoma and negative AFP expanding the scope of HAC to have liver differentiation characteristics regardless of whether AFP is positive [3]. Nagai et al. also proposed that HAC should be de ned based on histological features similar to hepatocellular carcinoma, regardless of whether the tumor produces AFP [4].The stomach is the most common site of HAC [5]. In addition, HAC can also be found in the gallbladder, uterus, lung, bladder, esophagus, colon, and ovary [1,6].The incidence of hepatoid adenocarcinoma of stomach (HAS) accounts for only 0.17-1% of all gastric cancers [7][8][9][10]. The clinical symptoms are not speci c compared with the common gastric cancer (CGC), but the prognosis of HAS is worse than CGC because of its frequent vascular involvement, and lymph node and liver metastasis [5,11].In this retrospective study, we analyzed the clinical and pathological features of 31 cases of HAS and described the factors affecting prognosis.

Materials And Methods
Patients and pathological staging criteria Between January 2014 and May 2020 a total of 6,078 cases of gastric cancer were treated at our center,of which 31 (0.51%) were HAS.The enrolled 31 patients were all diagnosed as HAS by our pathology department and the pathological diagnosis information is complete.All tumours were staged according to the American Joint Committee on Cancer (AJCC) TNM Staging Classi cation for Carcinoma of the Stomach, 8th edition.This study was approved by the Ethics Committee of the First A liated Hospital of Zhengzhou University.

Survival analysis and statistical analysis
The overall survival (OS) time was calculated from the date of diagnosis to the last day of follow-up or the date of death. Follow-up visits were conducted using outpatient visits and telephone calls. All patients were regularly followed up from the date of rst hospitalization at our hospital.
The nal follow-up date was June 2020,and no patients were lost to follow-up.
Statistical analysis was performed using IBM SPSS 25.0 software. The survival rate was calculated using the Kaplan-Meier method. Univariate and multivariate survival analysis used the Cox risk proportional regression model, a P value of < 0.10 of univariate analysis as the multifactor analysis inclusion standard, and a P value of < 0.05 was considered signi cant. with anorexia.Among 5 patients with a history of cancer, 3 patients had a family history of gastric cancer, and 2 patients had a family history of esophageal cancer.A total of 27 patients were tested for serum AFP level at initial diagnosis, and serum AFP levels were normal in 5 patients(18.5%), and elevated in 22 patients(81.5%). Among 22 patients whose serum AFP levels were elevated, 7 patients had serum AFP levels above the upper limit of the instrument. The clinical characteristics of patients are shown in Table 1.

Clinicopathological ndings
In this group of patients, 20 (64.5%) had tumors located in the upper third of the stomach, 1 (3.2%) had tumors in the middle third, 9 (29.1%) had tumors in the lower third, and 1 (3.2%) had tumors occupying the whole stomach. All 31 patients presented with advanced gastric cancer, according to the Borrmann gross type classi cation, Borrmann type I, II,III and IV were found in 5 (16.1%), 2 (6.5%) ,17(54.8% and 1 (3.2%) patients. In addition, the Borrmann type of 6 patients were unknown. Of the 31 tumors, 20 (64.5%) were pathologically con rmed as poorly differentiated, 9 (29.0%) were moderately to poorly differentiated and 2 (6.5%) were moderately differentiated, and there was no welldifferentiated tumors. The pathological ndings are shown in Table 1.
Among the 31 patients, TNM classi cation revealed the presence of 1 patient 3.2%) with stage I, 6 patients (19.4%) with stage II, 15 patients (48.4%) with stage III, and 9 patients (29.0%) with stage IV cancer. At the rst visit, 25 patients (80.6%) had regional lymph node metastasis and 9 (29.0%) had distant metastasis. Of the 9 cases, liver metastasis was found in 8 cases, retroperitoneal lymph node metastasis was found in 4 cases, and left supraclavicular lymph node metastasis was found in 1 case.
Immunohistochemical staining of AFP was performed in tumor specimen for each patient, of these, 24 patients (77.4%) showed positive or focal positive. And among the 24 patients, 1 patient with normal serum AFP level. Immunohistochemical staining of CEA were used in 17 patients, 11 (64.7%) were positive or focal positive. And immunohistochemical staining of Glypican-3 was performed in 18 patients, 14 (77.8%) were positive or focal positive. For Ki67, they presented an expression positive at 100%, and among them, 22(71.0%) had positive area more than 50%.Immunohistochemical ndings are shown in Table 2.

Treatment
Among the 31 patients, 22 patients underwent a radical gastrectomy and a D2 lymph node dissection, 1 patient underwent palliative surgery, and 8 patients only received palliative chemotherapy. In 22 patients who who underwent a radical operation, 16 patients had received postoperative adjuvant chemotherapy, the chemotherapy regimen is mostly 5-uorouracil (5fu) plus platinum.

Outcomes of HAS patients
The median follow-up time for the 31 patients was 14 months (range 0.5-63 months).During the follow up period, 18 patients died of the disease.
The median OS was 28 months. The 1-year survival rate was 55%, the 3-year survival rate was 47%, and the 5-year survival rate was 34% (Fig. 1).
Univariate Cox regression analysis revealed that an elevated CA125 level, distant metastasis, and radical surgery were associated with prognosis (P<0.05).A multivariate analysis further revealed that distant metastasis remained an independent prognostic risk factor for survival (P=0.038; HR=27.016, 95%CI: 1.205 ~ 605.664). The survival analysis results are presented in Table 3.

Discussion
HAC is a rare special type of extrahepatic adenocarcinoma, the stomach is the most common site of HAC. The mechanism of HAS is not fully understood. Ishikura et al. believed that HAS originated from endoderm stem cells, which can differentiate into hepatocyte cell lines and/or intestinal cell lines; HAS can also be caused by the transdifferentiation of cells from adenoid to hepatoid [12].Research by Akiyama et al. also showed that hepatoid adenocarcinoma has the same origin as coexisting tubular adenocarcinoma [11].Gastric cancer is closely related to liver cancer. During the embryonic development, the stomach and liver are both developed from the foregut. Therefore, some primary gastric cancers have a differentiation disorder, which eventually leads to their differentiation into hepatocytes and the formation of hepatoid adenocarcinoma [5].
Histopathological features are the gold standard for the diagnosis of HAS. As long as hepatocellular differentiation areas appear in the primary focus of gastric cancer, regardless of whether serum AFP is elevated or immunohistochemical staining is positive for AFP, it can be diagnosed as HAS [3,5]. The typical histology of HAS is usually the coexistence of adenocarcinoma area and hepatocellular differentiation area, the two can migrate between each other, and a few are all hepatocellular differentiation areas. The adenocarcinoma area is often located in the gastric HAS has a lower incidence rate, previous studies have shown that it accounts for only 0.17-1% of all gastric cancers. The difference in proportion is not only related to race and region, but also closely related to the level of diagnosis and treatment. In our study, HAS accounted for 0.51% of all gastric cancers during the same period, which was consistent with previous studies. Compared with CGC, the clinical manifestations of HAS are not speci c [13]. Our analysis shows that the clinical manifestations of HAS include upper abdominal pain, upper abdominal discomfort, meconium, nausea, and anorexia. Wang et al. found that HAS is more common in the elderly, with a higher proportion of males and a higher frequency of intestinal type and liver metastases [14]. Qu et al. studied 95 HAS patients from China, and the results showed that HAS mainly occurs in the elderly, males are more common than females, mainly in the gastric antrum, usually ulcerative, and the degree of differentiation is mostly poorly differentiated [15]. Lin et al. reported that the most common type in HAS is Borrmann type III, and the presence of recent bleeding ulcers can often be seen. In the present study, most of the patients were elderly men with a median age of 62 years. 54.8% (17/31) were Borrmann III type, 64.5% (20/31) were poorly differentiated type, consistent with previous studies, but the tumor sites were more common in the upper third of the stomach (20/31). And 22.6% (7/31) of patients presented with black stool as the initial symptom, which is consistent with the recent bleeding ulcers often seen in tumor lesions in previous studies [10].
Elevated serum AFP levels are an important feature of HAS, but not all HAS patients have elevated serum AFP levels. Su et al. reported an elevated serum AFP level in 84.8% of patients with HAS, ranging from 1.0 to 475,000 ng/mL [1]. Another study reported that about 80.6% of patients with HAS had elevated serum AFP levels [16]. Of the 31 patients in this study, 27 had serum AFP levels detected at the time of diagnosis, and 81.5% (22/27) patients had elevated serum AFP levels, of which 26.0% (7/27) patients had higher serum AFP levels than the instrument measurable upper limit. AFP is a glycoprotein produced and secreted by the fetal liver, yolk sac, and gastrointestinal tract, and serum AFP levels drop rapidly after birth. The increase in AFP levels is mainly related to the occurrence of hepatocellular carcinoma (HCC). In addition, many other tumors, such as nonseminoblastic germ cell tumors and endodermal sinus tumors, can also cause AFP levels to increase. Serum AFP level has the signi cance of disease monitoring, the level generally begin to decrease 1 to 2 weeks after tumor resection, return to normal 2 months after surgery, and increase again when distant metastasis or recurrence occur [17]. Ishikura et al. believe that AFP has an immunosuppressive effect, and its production will affect the prognosis of HAS [12].Wang et al. believe that preoperative serum AFP levels ≥ 500 ng/ml are related to OS (P = 0.007) and DFS (P = 0.05), and preoperative serum AFP level is a sensitive prognostic biomarker for DFS and OS [14]. For patients with HAS, elevated serum AFP level is an important feature, which is great helpful to con rm the diagnosis. It is recommended as a routine test item for patients with gastric cancer to reduce the missed diagnosis rate of HAS.
Immunohistochemical detection is also of great signi cance for the diagnosis of HAS, but there is no very clear marker. AFP is often diffusely positive in the hepatocellular differentiation area, while it is weakly positive or focally positive in the adenocarcinoma area [2]. The adenocarcinoma area is composed of well-differentiated intestinal epithelial cells and usually contains CEA [5]. The positive rate of Glypican-3 in HAS is higher than that of CGC, and it can be used for the diagnosis of HAS [1]. SALL4 is not expressed in adult normal gastric tissues and HCC, but is partially expressed in normal gastric cancer. In addition, it is diffusely expressed in HAS [18]. Akiyama et al. found that the hepatocellular differentiation area contains AFP positive cells [11]. Kumashiro et al. found that 83% of patients showed AFP expression [19]. Osada et al. reported that the positive rate of AFP staining in HAS patients was 80%, and the positive rate of Glypican 3 staining was 56% [20]. Another study reported that AFP staining was found to be positive in 91.6% of patients, and CEA staining was found to be positive in 78.7% of patients [1]. In our study, 77.4% (24/31) patients were positive for AFP staining, 64.7% (11/17) patients were positive for CEA staining, and 77.8% (14/18) patients were positive for Glypican-3 staining. We also found that Ki67 positive rate was 100%, of which 71.0% (22/31) positive area was > 50%. Previous studies also reported that the expression of Ki-67 in the tumor cell nucleus was moderately increased [21]. These results indicate that HAS has a high malignant potential, such as high proliferative activity, weak apoptosis and abundant neovascularization [22], which may be the reason for its poor prognosis.
Compared with CGC, HAS is more common in lymph node metastasis, liver metastasis, and extensive vascular invasion, with a worse prognosis.
Lin et al. found that 90% of patients had lymph node metastasis and/or distant metastasis at the time of diagnosis [10]. Another study found that the total incidence of liver metastases in the HAS group was 75.6%, while the total incidence of liver metastases in the CGC group was 11.5% (P < 0.01) [13]. Osada et al. found that the incidence of vascular in ltration (P = 0.005) and distant metastases (P = 0.0458) was higher in hepatoid adenocarcinoma compared with non-hepatoid adenocarcinoma [20]. In the present study, 80.6% (25/31) of patients had regional lymph node metastasis at rst visit, and 29.0% (9/31) of patients had distant metastasis at rst visit. 25.8% (8/31) of the patients had liver metastasis at the time of rst diagnosis, and 4 patients had liver metastasis during follow-up. That is, the incidence of liver metastasis was 38.7% (12/31), including 25.8% (8/31) simultaneous liver metastasis and 12.9% (4/31) metachronous liver metastasis. 66.7% (14/21) patients had vascular tumor thrombus, and 68.8% (11/16) patients had nerve invasion. This aggressive biological behavior of HAS leads to a poor prognosis. According to Liu et al., the one-year, three-year and ve-year survival rates of HAS are 30%, 13% and 9%, respectively, while the CGC group is 95%, 57% and 38% (P < 0.01) [13]. Qu et al. reported that the 3-year survival rate of HAS was 7.4%, and the median OS was 10 months. Compared with patients with metastasis, the survival time of patients without metastasis was longer (P = 0.001), and the survival of patients receiving surgical treatment longer than patients treated with non-surgical methods (P = 0.046). Survival analysis showed that survival time was associated with metastasis (P = 0.002) and liver metastasis (P = 0.036) [15]. Another study found that the interval between gastrectomy and liver metastases in patients with HAS is shorter than CGC, resulting in a poorer prognosis [8]. Baek et al. reported that the median OS of patients with stage I-III and stage IV HAS was 28.0 and 8.2 months, respectively [6]. Zeng et al. found that vascular in ltration, pTNM stage and adjuvant therapy are independent risk factors for prognosis [16]. The median OS of the patients in this study was 28 months. The 1-year survival rate was 56%, the 3-year survival rate was 47%, and the 5-year survival rate was 40%. The survival rate in this study is higher than previous studies, probably because the data was collected retrospectively in a single center, more patients underwent radical surgery and adjuvant chemotherapy, and fewer patients in stage IV, resulting in selection bias. Univariate Cox regression analysis revealed that an elevated CA125 level, distant metastasis, and radical surgery were associated with prognosis (P < 0.05). A multivariate analysis further revealed that distant metastasis remained an independent prognostic risk factor for survival (P = 0.038; HR = 27.016, 95%CI: 1.205 ~ 605.664).
The prognosis of HAS is poor, but due to its low incidence, there is currently no standard treatment plan, and clinical treatment of HAS is similar to the treatment of CGC. Radical surgery, including the resection of liver metastases and adjuvant therapy, is considered the best treatment option.
However, compared with CGC, the radical resection of HAS is more challenging because of its higher incidence of vascular invasion, lymph node metastasis and distant metastasis before surgery. Radical surgery, early pTNM and adjuvant therapy can signi cantly improve the prognosis of HAS [16].

Conclusions
In conclusion, HAS is a rare special type of extrahepatic adenocarcinoma. The clinical symptoms are not speci c compared with the CGC, but the prognosis of HAS is worse than CGC because of its frequent vascular involvement, and lymph node and liver metastasis. An elevated CA125 level, distant metastasis, and radical surgery were associated with the prognosis of HAS, and distant metastasis is an independent prognostic risk factor for survival. Due to the low incidence rate of HAS, the understanding of HAS is still insu cient from a clinical point of view, and missed diagnosis and misdiagnosis easily occur under these circumstances. Early and accurate diagnosis is required for a relatively long survival time.
And aggressive multimodal treatment comprising radical gastrectomy and adjuvant chemotherapy is warranted. In addition, molecular targeted therapy also can be tried.

Declarations
Ethics approval and consent to participate Not applicable.

Patient consent for publication
Not applicable

Avaliability of date and materials
The datasets used and/or analyzed during the current study areavailable from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by National Natural Science Foundation of China (Grant no.81872264).
Authors' contributions YRQ and ZWC designed the study; CXL was the major contributor in writing the manuscript; CL, LD and WJW collected the patient data. All authors read and approved the nal manuscript.
23. Xie Y, Zhao Z, Li P, Wang Y, Guo C, Wang X, Tang W, Liu Q, Lu N, Xue L et al: Hepatoid adenocarcinoma of the stomach is a special and easily misdiagnosed or missed diagnosed subtype of gastric cancer with poor prognosis but curative for patients of pN0