Morphologic and Immunophenotypic Spectrum of 11 Gastric Cancers with Elevated Serum Alpha-Fetoprotein: Emphasis on Hepatoid or Enteroblastic Differentiation

Abstract

Aim

A retrospective analysis of 11 gastric cancer patients with elevated serum alpha-fetoprotein levels was conducted to investigate the clinical-pathological characteristics, diagnostic approaches, and surgical experiences associated with this disease, thereby enhancing understanding of its biological behavior.

Methods

A retrospective analysis of clinical data from gastric cancer patients diagnosed at our hospital between 2020 and 2025 presented with elevated serum alpha-fetoprotein levels, combined with a discussion of disease classification based on relevant literature.The exclusion criteria were:such as hepatocellular carcinoma, cirrhosis/active liver disease, and germ cell tumors.

Results

Among the 11 patients, 9 were male, accounting for approximately 81.8%, and 2 were female, accounting for approximately 18.2%. The median age was 68 years.Clinical manifestations included upper abdominal pain, melena, and elevated serum alpha-fetoprotein levels. Eleven patients underwent radical gastrectomy for gastric cancer, including four cases of total gastrectomy. Tumors were located in the cardia in seven patients (63.6%), in the gastric body in three patients (27.3%), and in the gastric antrum in one patient (9.1%).Based on histological type, the cases can be classified as follows: 6 cases of hepatocellular adenocarcinoma and 5 cases of gastric adenocarcinoma with intestinal differentiation.Immunohistochemical staining revealed varying degrees of expression for alpha-fetoprotein, Glypican-3, and SALL-4. Five cases showed positivity for all three markers, eight cases showed positivity for two markers, and eleven cases showed positivity for only one marker.

Conclusion

This type of gastric adenocarcinoma is rare, often detected at an advanced stage with a poor prognosis. In clinical diagnosis, it must be distinguished from conventional gastric adenocarcinoma.

Introduction

Alpha-fetoprotein (AFP) is an embryonic serum glycoprotein first discovered in 1956. It is primarily produced by the yolk sac epithelium and fetal liver, with minor synthesis occurring in the embryonic gastrointestinal epithelium.^1,2 Gastric cancer with elevated serum alpha-fetoprotein is clinically rare. These tumors are highly malignant, prone to metastasis, and carry a poor prognosis.^3,4 The 5th Edition of the WHO Classification of Tumors of the Digestive System indicates that gastric adenocarcinomas with serum AFP are classified into hepatocellular adenocarcinoma, gastric adenocarcinoma with embryonal differentiation, gastric adenocarcinoma with yolk sac differentiation, and mixed types.⁵ Some studies have reported genetic alterations in this type of tumor, such as those involving P53, CCNE1, ERBB2, and EGFR.⁶ Currently, there are limited reports on the histological morphology, immunophenotype, and treatment of this tumor. We hypothesized that gastric cancers with elevated serum AFP in our surgical cohort would exhibit a reproducible morphologic–immunophenotypic spectrum, and that integrated assessment of morphology together with AFP, Glypican-3, SALL4, and lineage-associated markers could provide more practical diagnostic information than serum AFP elevation or single-marker staining alone. Although the prognostic impact of AFP-producing gastric cancer (AFPGC) has gained attention, a comprehensive description of its entire morphological spectrum—ranging from conventional adenocarcinoma to hepatoid and fetal intestinal types—along with their corresponding immunophenotypic variants based on a systematic, single-center pathological review remains lacking. This knowledge gap not only limits diagnostic precision but also poses a substantial challenge in differential diagnosis from metastatic hepatocellular carcinoma (HCC) in routine practice. Given that distinct pathological subtypes may correlate with divergent biological behaviors and therapeutic responses (eg, differential efficacy of anti-angiogenic agents or immune checkpoint inhibitors), accurate subtyping holds potential clinical implications for guiding individualized treatment decisions and refining surveillance protocols.

Methods

We conducted a retrospective analysis of 1500 gastric cancer patients admitted to Jinhu County People’s Hospital in Jiangsu Province between January 2020 and December 2025. Among them, 11 cases were pathologically diagnosed as alpha-fetoprotein (AFP)-positive gastric cancer. The screening criteria were serum alpha-fetoprotein (AFP) levels above the laboratory’s upper limit of normal (>10 ng/mL), and cases of gastric cancer in which primary hepatocellular carcinoma, liver metastases, and germ cell tumors had been ruled out based on imaging (contrast-enhanced abdominal CT/MRI) and serological tests (including HBsAg, HCV antibodies, and germ cell tumor markers such as β-hCG and CA19-9).This study was approved by the People’s Hospital of Jinhu County, Jiangsu Province and received written consent from the patients or their families. Medical records were documented and telephone follow-ups were conducted with patients through March 2026.Overall survival was used as the prognostic outcome measure.4 μm serial sections were cut from formalin-fixed, paraffin-embedded tumor tissues. The sections were conventional dewaxing to water and performed antigen retrieval using EDTA, after incubated in 3% H2O2 for 10 min, sections were blocked with 1% bovine serum albumin (BSA) for 10 min, and incubated with AFP (EP209,1:200,Fujian Maixin), Hepatocyte (MX119,1:300,Fujian Maixin), CD10 (MX002,1:200,Fujian Maixin), Glypican-3(MAXIM001,1:150,Fujian Maixin), CDX2(MXR024,1:200,Fujian Maixin) and HER2(MXR001,1:200,Fujian Maixin) at 4 °C overnight. The slides were incubated with anti-rabbit/mouse IgG antibody the next day, after incubating with reaction enhancer, the slides were colored with DAB and counterstained with hematoxylin. Negative control was performed by the absence of the primary antibody. AFP, Hepatocyte and CD10 are localized to the cytoplasm. Glypican-3 is localized to the cytoplasm and/or cell membrane. SALL4, CDX2 are localized to the nucleus.HER2 expression is localized to the cell membrane. The results of IHC were interpreted independently by two pathologists who were blinded to all clinical and pathological data. Immunoreactivity was semi-quantitatively scored based on the percentage of positive tumor cells as negative (0), focal (<10%), patchy (10–50%), or diffuse (>50%).

Results

Among the 1500 patients who underwent surgical resection, 11 had elevated AFP levels, accounting for approximately 0.7%, preoperative serum AFP levels exceeded 10 ng/mL. Postoperative serum AFP values decreased by 90%, indicating that gastric cancer cells cause elevated serum AFP levels in patients. The male-to-female ratio in this cohort was 9:2. The age range at diagnosis was 58–77 years, with a median age of 68 years. The median serum AFP level was 632 ng/mL (Table 1). Preoperative abdominal CT scans were performed in 11 patients, including 8 with contrast-enhanced scans and 3 with plain scans. The primary CT findings were gastric mass shadows or ulcerative changes (Figure 1). All 11 patients underwent gastroscopy prior to surgery, with lesions distributed in the cardia, body, and antrum of the stomach. The average tumor diameter ranged from 2.5 to 10 cm, with a median diameter of 4.75 cm. The cardia was the most common site of origin. The majority of tumors presented as ulcerative types. Vascular invasion and lymph node metastasis were observed (Table 2). All 11 patients underwent surgery, and 9 patients received postoperative combination chemotherapy with oxaliplatin and capecitabine.

Table 1.

Clinical Data of 11 Patients

Sex	Age (Years)	Chief Complaint	AFP Value (preoperative,ng/mL)	Surgical Procedure	Postoperative Chemotherapy	Survival Time After Surgery (MONTHS)
Male	58	Elevated AFP for 3 months	986	Total gastrectomy	Yes	13
Female	68	Vomit blood	1256	Proximal gastrectomy	No	10
Male	72	Elevated AFP for 6 months	1325	Proximal gastrectomy	Yes	Survive(50)
Male	58	Abdominal pain for 1 year	698	Total gastrectomy	Yes	Survive(36)
Male	70	Stomach cancer detected on physical examination	212	Distal gastrectomy	No	6
Female	77	Black stool for over a month	412	Proximal gastrectomy	Yes	Survive(24)
Male	71	Acid reflux and heartburn	456	Proximal gastrectomy	Yes	Survive(13)
Male	66	Elevated AFP for 4 months	732	Proximal gastrectomy	Yes	Survive(9)
Male	67	Black stool	96	Total gastrectomy	Yes	Survive(7)
Male	70	Abdominal pain for a month	128	Total gastrectomy	Yes	Survive(5)
Male	70	Hematochezia	653	Proximal gastrectomy	Yes	2

Figure 1.

(A) CT scan shows irregular thickening from the cardia to the gastric body. (B) CT contrast-enhanced imaging demonstrates mild to moderate irregular enhancement from the cardia to the gastric body.

Table 2.

Pathological Examination results of 11 Patients

Tumour Site	Broad Type	Maximum Diameter of Tumor	Infiltration Depth	Histological Type	Intravascular Cancerous Embolus	Lymph Node Metastasis	TMN Staging
Gastric body	Protruded	3.2cm	Subserosal	Enteroblastic differentiation	Yes	Yes	pT3N2M0
Cardia	Ulceration	3.5cm	Subserosal	Hepatoid differentiation	Yes	Yes	pT3N1M0
Cardia	Ulceration	3.0cm	Serosa	Hepatoid differentiation	Yes	Yes	pT4N3M0
Gastric Body	Ulceration	3.0cm	Subserosal	Hepatoid differentiation	No	No	pT3N0M0
Gastric Sinus	Protruded	2.5cm	Subserosal	Enteroblastic differentiation	Yes	Yes	pT3N3M0
Cardia	Protruded	5.0cm	Serosa	Hepatoid differentiation	Yes	Yes	pT4N1M0
Cardia	Ulceration	2.0cm	Serosa	Enteroblastic differentiation	Yes	Yes	pT4N2M0
Gastric Body	Protruded	6.0cm	Serosa	Hepatoid differentiation	Yes	Yes	pT4N2M0
Cardia	Protruded	4.5cm	SUBSEROSAL	Hepatoid differentiation	Yes	Yes	pT3N3M0
Cardia	Ulceration	10cm	Subserosal	Enteroblastic differentiation	Yes	Yes	pT3N1M0
Cardia	Ulceration	9.5cm	Subserosal	Enteroblastic differentiation	Yes	Yes	pT3N2M0

Based on histological features, it can be classified into two subtypes, including 6 cases (54.5%) of hepatocellular adenocarcinoma. The tumor consists of tubular or papillary adenocarcinoma areas and hepatocellular differentiation areas, with transitional zones between histologically distinct regions. Hepatocyte-like differentiated cells are arranged in trabecular and nests (Figure 2A and B). They are separated by sinusoids. Tumor cells exhibit abundant eosinophilic cytoplasm, large irregular nuclei with coarse chromatin, and readily visible mitotic figures. Five cases (45.5%) of gastric adenocarcinoma with accompanying intestinal metaplasia showed cancer cells forming tubular, papillary, or cystic structures with clear or vacuolated cytoplasm, resembling primitive intestinal epithelium during embryonic development (Figure 3A and B). Homogeneous red-stained round bodies or red-stained material within the glandular lumens were observed. Among the 11 patients in this group, the depth of cancer cell invasion (T stage) revealed that 7 cases showed invasion to the subserosal layer (T3), while 4 cases demonstrated invasion to the serosal layer (T4).Immunohistochemical staining revealed varying degrees of expression for Hepatocyte (Figure 2C), SALL-4 (Figure 2D), Glypican-3, and AFP (Figure 2E) (Figure 3C–E). The intestinal progenitor differentiation zone shows focal expression of CD10 and CDX2 (Figure 3F and G). 11/11 at least one of AFP/GPC3/SALL4 positive, 8/11 at least two positive, 5/11 triple positive, enteroblastic areas showed focal CD10/CDX2, HER2 equivocal in 6 but no FISH amplification (Figure 2F) and (Figure 3H).

Figure 2.

(A and B) Hematoxylin and eosin(HE) staining(A×100,B×400).Hepatocyte-like differentiated cells are arranged in trabecular and nests. (C) Tumor cells express Hepatocyte (×200). (D) Tumor cells express SALL-4 (×200). (E) Tumor cells focally express AFP (×200). (F) HER2 expression is approximately 1+ (×200).

Figure 3.

(A and B) Hematoxylin and eosin(HE) staining(A×100,B×400).The tumor cells exhibit a papillary structure, with cells resembling intestinal blast cells. (C) Tumor cells focally express Hepatocyte (×200). (D) Tumor cells express Glypican-3 (×200). (E) Tumor cells express SALL-4. (F and G) Tumor cells focally express CDX2, CD10 (×200). (H) HER2 expression is approximately 2+ (×200).

Discussion

Japanese scholars Ishikura et al reported in 1985 a case of gastric adenocarcinoma with elevated serum AFP.⁷ Its histomorphology and immunohistochemistry exhibited unique differentiation characteristics, leading to its designation as gastric carcinoma with elevated serum AFP. This represents a rare, distinct pathological subtype of gastric cancer, accounting for 0.3% to 1.0% of all gastric cancer cases. It is predominantly observed in Asian regions, particularly Japan and China.^8,9 This tumor predominantly affects middle-aged and elderly males, often accompanied by elevated serum AFP levels. Due to its high malignancy, propensity for liver and lymph node metastasis, and poor response to chemotherapy, it has garnered significant attention. This study enrolled 11 patients with a median age of 68 years, 81.8% of whom were male—consistent with published literature. He primary contribution of this study lies not in reaffirming the well-established fact that AFP-associated gastric cancer is generally associated with poor prognosis, but rather in systematically documenting, through a single-center cohort of resected cases, the morphological heterogeneity and actual immunophenotypic patterns of AFP-elevated gastric adenocarcinomas, particularly hepatoid and enteroblastic differentiation.

Currently, the pathogenesis of this disease remains unclear. The prevailing view suggests that the stomach and liver share a common origin in the embryonic endoderm and foregut, which may represent a potential underlying mechanism for disease onset. Furthermore, genetic evolution or mutation is believed to accompany the progression of the disease.¹⁰ Numerous molecular studies have been conducted on this disease. Liu et al found that dysfunction of the p53 protein family may serve as an initiator of hepatocellular differentiation. Hepatocellular adenocarcinoma exhibits a highly heterogeneous and active tumor microenvironment, characterized by high tumor cell stemness and robust methionine metabolism activity.¹¹ The clinical features of gastric cancer patients with elevated serum AFP are nonspecific. Clinical manifestations observed in our study population included epigastric pain, melena, and hematemesis. The most prominent finding may be elevated serum AFP. Studies have reported a positive rate of 73.7% in patients with hepatocellular adenocarcinoma, with 49.5% exhibiting concentrations exceeding 500 ng/mL. All 11 patients in our study exhibited elevated serum AFP levels, ranging from a minimum of 96 ng/mL to a maximum of 1325 ng/mL. Abdominal CT findings showed little difference from typical gastric cancer, presenting as gastric wall thickening, ulceration, and localized masses.

Serum alpha-fetoprotein (AFP)-positive gastric cancer (AFP-GC) is a rare subtype characterized by highly aggressive biological behavior and a high propensity for liver metastasis. Although there is ample evidence that elevated AFP levels (>10 ng/mL) are associated with poor prognosis and hepatocellular differentiation, diagnostic strategies for this disease remain unclear in routine clinical practice, particularly regarding the optimal threshold for biopsy selection and the interpretation of heterogeneous histological patterns. Against this backdrop, our study of 11 cases provides a detailed description of the clinical and pathological data. Histopathology remains the gold standard for diagnosing this disease. Hepatocellular adenocarcinoma is characterized histologically by polygonal, eosinophilic hepatocellular-like cells arranged in solid nests or trabecular patterns. Areas of hepatocellular differentiation typically coexist with those of conventional adenocarcinoma, with transitional features between the two.^12,13 Traditional adenocarcinoma typically exhibits tubular, sieve-like, or glandular structures, with some areas containing papillary components. It is commonly located on the mucosal surface, separated from normal gastric mucosa by a transition zone. Gastric adenocarcinoma with intestinal blastocyst differentiation exhibits columnar tumor cells with well-defined borders and characteristically abundant, clear cytoplasm. This transparent cytoplasm may form vacuoles above or below the nucleus, resembling the secretory phase of the endometrium. This study found that all six cases of hepatocellular adenocarcinoma expressed AFP-positive, with positive localization in the cytoplasm. This may be because AFP is a secreted protein synthesized within tumor cells and subsequently released into the bloodstream. GPC3, as a cell membrane surface protein, is widely expressed in embryonic liver and HCC. Clinical trials for GPC3-targeted drug therapies are currently underway, such as the antibody drugs GC33 and ERY974.¹³ All five gastric adenocarcinoma regions with enteroblast differentiation in our study expressed the intestinal epithelial marker CDX-2. Additionally, all five cases expressed CD10. CDX-2 and CD10 are markers of primitive intestinal epithelium, reflecting their primitive intestinal epithelial differentiation characteristics. Our findings suggest that no single marker should be considered mandatory for recognition of this spectrum. Instead, an integrated panel is more informative: AFP, Glypican-3, and SALL4 supported AFP-related differentiation across cases, while CD10/CDX2 helped highlight enteroblastic differentiation in selected areas. This is particularly relevant in routine practice, where AFP staining may be focal and morphology may overlap with conventional gastric adenocarcinoma or metastatic hepatocellular carcinoma.

As previously described, p53 mutations occur at a higher rate in gastric adenocarcinomas with enteroblast differentiation. P53 gene mutations may occur at a higher rate in gastric adenocarcinomas with enteroblast differentiation and play a significant role in their development. Therefore, combining the histological and cytological features with the immunophenotype of gastric adenocarcinomas with enteroblast differentiation enables accurate diagnosis of this subtype. According to the guidelines for human epidermal growth factor receptor 2 (HER-2) testing in gastric cancer,¹⁴ HER-2-positive patients may be resistant to HER-2 therapy. Literature reports indicate that gastric cancers with elevated serum AFP levels exhibit a higher HER-2 positivity rate of approximately 2 positive cases may be eligible for anti-HER-2 therapy. Literature reports indicate a higher HER-2 positivity rate (approximately 22%) in gastric cancers with elevated serum AFP, significantly exceeding that of conventional gastric adenocarcinoma (12%).¹⁵ In this study, immunohistochemical detection of HER-2 protein expression revealed only 6 cases as HER-2(2+), with all others testing negative. FISH testing was subsequently performed on the 6 HER-2(2+) cases, revealing no HER-2 gene amplification. Although six tumors showed equivocal HER2 (2+) immunostaining, none demonstrated HER2 amplification by FISH. In our view, this finding underscores the need for confirmatory molecular testing before inferring targetability in AFP-elevated gastric cancers.

Conclusion

In summary, gastric cancers with elevated serum AFP represent a diagnostically challenging and morphologically heterogeneous subgroup, in which integrated morphologic and immunohistochemical evaluation is critical for accurate recognition and subclassification. Larger-scale, multicenter studies incorporating standardized pathological evaluation, molecular profiling, and long-term clinical follow-up are needed to validate these diagnostic findings. The limitations of this study include the retrospective single-center design, the absence of a comparator group, the absence of survival analyses, and the lack of molecular characterization, all of which limit the generalizability of the conclusions. The proposed diagnostic framework is still in its preliminary stages and needs to be validated through larger-scale, multicenter cohort studies.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics Approval

All studies conducted in this research were in accordance with the regulations and guidelines of Jinhu County People’s Hospital were carried out in compliance with the Helsinki guidelines. Informed consent was obtained from all individual participants who were included in the study.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no competing interests.