Published online Nov 27, 2026. doi: 10.4240/wjgs.124589
Revised: August 5, 2026
Accepted: September 28, 2026
Published online: November 27, 2026
Processing time: 146 Days and 18.8 Hours
This study explored the key points of intraoperative frozen pathological diagnosis and the clinical diagnostic value of primary alpha-fetoprotein (AFP)-positive hepatoid adenocarcinoma of the gallbladder (HAGB).
The clinical data, imaging findings, intraoperative frozen pathology, postope
Intraoperative frozen pathology can rapidly and accurately identify AFP-HAGB. Combined with serum AFP, imaging, and immunohistochemistry, a clear dia
Core Tip: Intraoperative frozen section pathology combined with serum alpha-fetoprotein, imaging, and immunohistochemistry enables rapid and accurate diagnosis of primary alpha-fetoprotein-positive hepatoid adenocarcinoma of the gallbladder, which is crucial for guiding intraoperative surgical decisions and reducing misdiagnosis.
- Citation: Shui XL, Liu YF. Intraoperative frozen section diagnosis of primary alpha-fetoprotein–positive hepatoid adenocarcinoma of the gallbladder: A case report. World J Gastrointest Surg 2026; 18(11): 124589
- URL: https://www.wjgnet.com/1948-9366/full/v18/i11/124589.htm
- DOI: https://dx.doi.org/10.4240/wjgs.124589
Primary alpha-fetoprotein (AFP)-positive hepatoid adenocarcinoma of the gallbladder (AFP-HAGB) is an extremely rare and highly invasive epithelial-derived malignant tumor and a specific subtype of hepatoid adenocarcinoma[1]. The main characteristic of this tumor is that its histological morphology is similar to that of hepatocellular carcinoma, accompanied by abnormally high expression of AFP. Because of its extremely low incidence, clinical manifestations often lack specificity, and preoperative imaging findings are easily confused with those of common gallbladder cancer, hepatocellular carcinoma invading the gallbladder, or metastatic liver cancer, leading to significant challenges in clinical diagnosis[2].
Research suggests that the occurrence of AFP-HAGB may be related to abnormal differentiation of homologous cell lineages in the liver and gallbladder systems during embryonic development, but the exact molecular pathogenesis of this disease has not yet been clarified[3]. Currently, complete surgical resection remains the only possible means of achieving a cure for this disease. However, because of the aggressive biological behavior and rapid progression of this tumor, most patients are in the middle or advanced stages at the time of diagnosis and have lost the optimal opportunity for surgery[4]. Even if surgery can be performed, quickly and accurately determining the status of the surgical margins and lymph node metastasis directly affects the formulation of the surgical plan and assessment of prognosis. Intraoperative frozen pathological examination, as a key technique guiding surgeons in making immediate decisions, plays an indispensable role in the diagnosis of such rare tumors[5]. Existing data indicate that AFP-HAGB has unique characteristics in cell morphology and immunophenotype, and there is relatively limited experience with conventional frozen section diagnosis, which is prone to misdiagnosis or missed diagnosis, thereby affecting the accuracy of subsequent treatment[6].
In recent years, with exploration of the application of immunohistochemistry (IHC) in intraoperative frozen sections, the diagnostic efficacy of hepatocellular markers such as AFP, glypican-3 (GPC3), and Hepatocyte in frozen sections has gradually become a research focus[7]. Given the unique clinical and pathological characteristics of AFP-HAGB and the higher requirements it poses for rapid intraoperative diagnosis, systematically exploring its morphological features and immunophenotypic combinations under frozen pathological conditions is of significant clinical importance. However, there are currently no systematic reports in the domestic or international literature on the intraoperative frozen pa
A 61-year-old woman was admitted to our hospital because of a gallbladder lesion detected during a routine physical examination.
The patient had no symptoms such as abdominal pain, abdominal distension, fever, or jaundice.
The patient had no history of hepatitis or liver cirrhosis and no smoking or drinking habits.
The patient had no family history of tumors.
On admission, the patient was alert and in good general condition. Vital signs were stable: Blood pressure, 128/76 mmHg; heart rate, 78 beats/minute; respiratory rate, 16 breaths/minute; and body temperature, 36.5 °C. Physical examination revealed no jaundice of the skin or sclera. The abdomen was flat and soft, with a palpable, firm, ill-defined mass approximately 4 cm in diameter in the right upper quadrant, without tenderness or rebound tenderness. The liver and spleen were not palpable below the costal margin. Shifting dullness was negative, and no edema was present in the lower extremities.
The preoperative AFP level was 1039 ng/mL (normal reference value: 0.0-13.6 ng/mL), carcinoembryonic antigen (CEA) level was 117 ng/mL (normal reference value: 0.0-6.5 ng/mL), carbohydrate antigen 19-9 level was 13.8 U/mL (normal reference value: 0-39 U/mL), carbohydrate antigen 125 level was 91.2 U/mL (normal reference value: 0-38 U/mL), and carbohydrate antigen 72-4 level was 18.7 IU/mL (normal reference value: 0.0-6.9 IU/mL). There were no abnormalities in liver function, routine blood tests, coagulation function, hepatitis B antigen and antibody tests, or hepatitis C antibody tests. Three days after the operation, re-examination revealed that the patient’s AFP and CEA levels had decreased to 475 ng/mL and 68.0 ng/mL, respectively. This study complied with the Declaration of Helsinki. The patient provided informed consent, and this study was approved by the Medical Ethics Review Committee of Affiliated Central People’s Hospital, China Three Gorges University.
Positron emission tomography/computed tomography multimodal imaging showed a soft tissue mass at the bottom of the patient’s gallbladder, with unclear boundaries and abnormally increased metabolism (Figure 1A). Multiple metabolically abnormal enlarged lymph nodes were seen in the retroperitoneum (Figure 1B), and high-uptake lesions were distributed in the gallbladder fossa and retroperitoneal regions (Figure 1C). The lesion showed uneven and significant enhancement in the arterial phase and continuous enhancement in the portal venous phase, with unclear boundaries from the gallbladder wall. In addition, multiple cysts were visible in the liver, but no solid mass was found, and no signs of liver cirrhosis were observed.
Gross features: The gallbladder specimen measured 8.5 cm × 4.0 cm × 3.0 cm. On one side was a piece of liver tissue measuring 8 cm × 6 cm × 3 cm. When the bottom of the gallbladder was dissected, a grayish-white and grayish-red mass measuring 4.3 cm × 2.8 cm × 2.0 cm was observed. The mass was attached by a stalk and had a brittle surface. It invaded the entire thickness of the gallbladder wall (Figure 2A). Additionally, 13 sets of lymph nodes were submitted for examination. One grayish-red fused nodular mass measured 4.3 cm × 3.0 cm × 1.7 cm and had a grayish-white, brittle surface (Figure 2B).
Pathological results of intraoperative frozen hematoxylin and eosin (HE) staining: In the frozen section, the hepatoid adenocarcinoma tumor tissue presented as large solid, sheet-like, and ribbon-like structures. The cancer cells were cuboidal or polygonal, with abundant eosinophilic cytoplasm, vesicular nuclei, and prominent eosinophilic nucleoli, showing a typical “liver-like” morphology; focal areas of glandular differentiation were also observed. Metastatic tumor foci were detected within the submitted lymph nodes. The intraoperative frozen diagnosis, which was based solely on rapid HE staining, was adenocarcinoma, with a high possibility of hepatoid adenocarcinoma.
Routine pathological and IHC results: The routine pathological sections showed that the hepatoid adenocarcinoma exhibited diverse morphologies. Approximately 70% of the area showed solid, sheet-like, and cord-like hepatoid differentiation (Figure 3A); approximately 15% of the area showed a poorly cohesive, ring-like cancer morphology (Figure 3B); approximately 10% of the area showed moderately differentiated glandular and papillary structures (Figure 3C); and in a few localized areas (approximately 5%), a small number of cancer cells had slightly translucent cytoplasm, with the various components intermixed. The tumor invaded the entire thickness of the gallbladder wall, accompanied by perineural invasion and microvascular invasion (Figure 3D).
Postoperative permanent-section IHC staining was performed on formalin-fixed, paraffin-embedded tissue using the standard EnVision 2-step method. In the hepatoid differentiation zone, cancer cells diffusely expressed spalt-like transcription factor 4 (SALL4) (Figure 4A), GPC3 (Figure 4B), and AFP (Figure 4C). Some showed weak positive expression of arginase-1 (Arg-1) (Figure 4D), focal weak positive expression of cytokeratin 7 (CK7) (Figure 4E), partial positive expression of cytokeratin 19 (CK19), and positive expression of cytokeratin 20 (CK20) in individual cells. Hepatocyte and caudal type homeobox transcription factor 2 (CDX2) were negative. The immunophenotype therefore supported both hepatoid differentiation and biliary epithelial differentiation in this case. The Ki-67 proliferation index ranged from 20% to 80%.
The final pathological diagnosis was primary AFP-HAGB. Histologically, the tumor (4.3 cm × 2.8 cm × 2.0 cm) showed 70% solid/cord-like hepatoid differentiation, 15% poorly cohesive ring-like morphology, 10% moderately differentiated glandular and papillary structures, and 5% clear cell features. The tumor invaded the full thickness of the gallbladder wall with perineural invasion and microvascular invasion. Metastasis was confirmed in the submitted lymph nodes. IHC revealed diffuse positivity for SALL4, GPC3, and AFP; negative staining for Hepatocyte; focal positivity for CK7 and CK19; and negative staining for CDX2. The Ki-67 proliferation index ranged from 20% to 80%.
Under general anesthesia, the patient underwent open radical cholecystectomy with partial resection of the adjacent liver bed and regional lymph node dissection. Intraoperative frozen section examination of the gallbladder mass and submitted lymph nodes was performed. The frozen diagnosis of adenocarcinoma with a high possibility of hepatoid adenocarcinoma, together with metastatic tumor foci in the submitted lymph nodes, was communicated to the surgical team, confirming the necessity of radical resection. Intraoperative assessment of the surgical margins was negative. Postoperatively, the patient received routine supportive care, including prophylactic antibiotics, hepatoprotective therapy, and nutritional support. No adjuvant chemotherapy or radiotherapy was administered.
The patient recovered smoothly after radical cholecystectomy without serious perioperative complications such as biliary leakage, abdominal hemorrhage, or infection. Her serum AFP and CEA levels decreased markedly to 475 ng/mL and 68.0 ng/mL, respectively, 3 days postoperatively, reflecting effective radical resection of the primary tumor. The patient was scheduled for regular follow-up every 3 months after discharge, including serum tumor marker testing (AFP, CEA, and carbohydrate antigen 19-9), contrast-enhanced abdominal computed tomography, and routine blood and liver function tests to monitor tumor recurrence and metastasis. At the last follow-up on March 20, 2025, 16 months after surgery, the patient remained asymptomatic. Contrast-enhanced CT showed no newly developed abnormal hypermetabolic lesions in the gallbladder fossa, liver parenchyma, or retroperitoneal lymph nodes; serum AFP maintained a continuous declining trend without secondary elevation.
Primary AFP-HAGB is a rare, highly malignant tumor of the gallbladder[8]. The cause remains unclear, and the clinical misdiagnosis rate is high. Previously, it was believed that its occurrence might be related to chronic cholecystitis and long-term stimulation by gallbladder stones, causing abnormal hyperplasia of the gallbladder mucosal epithelium[9]. However, most patients have no clear history of underlying gallbladder disease. Current studies suggest that AFP-HAGB originates from gallbladder mucosal epithelial stem cells. Under the influence of various pathogenic factors, these cells undergo abnormal differentiation and acquire a hepatocyte-like differentiation phenotype, thereby secreting AFP and forming a malignant tumor with characteristics of both gallbladder adenocarcinoma and hepatocellular carcinoma[10]. Its core pathological feature is a hepatocyte-like arrangement of tumor cells accompanied by positive AFP IHC, whereas the clinical manifestations lack specificity, making it easily confused with primary liver cancer and common gallbladder adenocarcinoma[10]. Intraoperative frozen pathological diagnosis is an important means of rapidly determining the nature of lesions during surgery. Through rapid frozen sectioning and staining, diagnosis can be completed within 30 minutes, allowing differentiation between benign and malignant lesions, clarification of the extent of tumor infiltration, real-time adjustment of the surgical plan, and avoidance of secondary surgery[11,12]. However, because of the short specimen-processing time, limited section quality, and urgent diagnostic timeframe, its accuracy may be affected[13]. Intraoperative frozen pathological diagnosis has been widely applied to lesions of various organs, but systematic research on intraoperative frozen pathological diagnosis of this rare and morphologically distinctive disease remains lacking.
In this case, the patient’s gallbladder specimen was submitted for intraoperative examination. The intraoperative pathological diagnosis was conducted using rapid frozen sectioning and HE staining only; no rapid IHC staining was performed during surgery because of technical limitations at the research center. The initial diagnosis was therefore based entirely on morphological observation. The patient was diagnosed with AFP-HAGB, and the tumor was found to have infiltrated the entire thickness of the gallbladder wall with lymph node metastasis. The surgeon performed a radical cholecystectomy based on this diagnosis. Postoperative routine pathological and IHC examinations further confirmed the diagnosis. The patient recovered well after the operation, with no early recurrence. This might be because rapid HE staining of the intraoperative frozen sections clearly showed that the tumor cells were arranged like hepatocytes, with abundant eosinophilic cytoplasm, large and deeply stained nuclei, prominent nucleoli, and visible pathological mitotic figures, allowing the pathologist to strongly suspect hepatoid adenocarcinoma. Although intraoperative IHC was not available, the characteristic morphology, combined with the patient’s markedly elevated serum AFP and imaging findings, was sufficient to suggest the diagnosis of AFP-HAGB intraoperatively[14]. At the same time, assessment of the extent of tumor infiltration showed full-thickness invasion of the gallbladder wall without gross invasion of adjacent organs; submitted lymph nodes contained metastatic tumor foci, providing a precise basis for radical resection[15]. Postoperative routine pathological examination was supplemented by IHC detection (AFP, CK19, Hepatocyte, GPC3, etc.), among which AFP(+), Hepatocyte(−), and CK19(+) further confirmed that the tumor had both hepatocyte-like and biliary epithelial differentiation characteristics, meeting the pathological diagnostic criteria for AFP-HAGB[16]. GPC3(+) indicated a high degree of malignancy of the tumor, providing an important reference for postoperative adjuvant therapy and prognosis assessment[17]. The intraoperative frozen pathological diagnosis, though based solely on HE staining, was consistent with the postoperative routine pathological diagnosis, demonstrating the reliability of intraoperative frozen pathological diagnosis of AFP-HAGB, even in the absence of rapid IHC support, and its ability to provide timely and accurate guidance for surgical treatment.
AFP-HAGB is a rare malignant tumor of the gallbladder. The accuracy of intraoperative frozen pathological diagnosis and the key points for differential diagnosis of this tumor have gradually drawn attention. This study conducted a literature search using Chinese and English keywords such as “primary AFP-positive hepatoid adenocarcinoma of the gallbladder,” “intraoperative frozen pathological examination,” and “diagnosis” in databases including China National Knowledge Infrastructure, Wanfang, VIP, PubMed, EMBASE, and MEDLINE, up to February 2026. Nine studies describing 13 previously reported patients with AFP-HAGB were included in the analysis. Together with the present case, 14 patients were available for summary in Table 1[18-25]. As shown in Table 1, 9 reports comprising 13 previously reported patients were retained for the frozen-section summary; together with the present case, 14 patients underwent intraoperative frozen pathological diagnosis. There were 9 males and 5 females, with an age range of 38-70 years. The most common underlying disease was gallbladder stones (64.29%, 9/14), followed by chronic cholecystitis (2/14) and no clear underlying disease (3/14).Regarding the diagnostic accuracy of AFP-HAGB, 13 cases (92.86%) were correctly diagnosed as hepatoid adenocarcinoma, whereas 1 case reported by Zhang et al[23] was not correctly diagnosed intraoperatively. Rapid intraoperative IHC was used in 2 accurately diagnosed cases reported by Li et al[8]. Postoperative permanent pathology confirmed AFP-HAGB in all 14 cases, with IHC showing AFP positivity in all cases and variable expression of Hepatocyte, GPC3, CK19, SALL4, and/or Arg-1. Although chronic inflammation and stone-related mucosal injury have been implicated in gallbladder carcinogenesis, their specific role in AFP-HAGB remains unclear. Therefore, in this selected frozen-section series, intraoperative frozen diagnosis showed high consistency with permanent pathology; however, because the available literature remains limited to case reports and small case series, these findings should be interpreted cautiously. Chronic inflammation, immune imbalance, and metabolic abnormalities may jointly participate in the occurrence and progression of the tumor. Currently, the intraoperative frozen pathological diagnosis of AFP-HAGB mainly relies on morphological observation using HE staining, with some advanced centers also employing rapid intraoperative IHC detection. Common IHC indicators include AFP, Hepatocyte, CK19, and GPC3. Among them, positive AFP is a key diagnostic indicator, positive Hepatocyte indicates hepatocyte-like differentiation, and positive CK19 indicates differentiation of the biliary epithelium. The combined detection of these 3 indicators can significantly improve diagnostic accuracy[26]. The main reasons for misdiagnosis were uneven tumor cell differentiation, atypical hepatocyte-like morphology, and lack of support from rapid intraoperative IHC detection. These results indicate that intraoperative frozen pathological diagnosis of AFP-HAGB has a high accuracy rate. Although rapid intraoperative IHC can further reduce the misdiagnosis rate when available, our case demonstrates that a confident intraoperative diagnosis can still be achieved based on careful morphological evaluation, particularly when integrated with clinical data such as markedly elevated serum AFP and imaging features. In addition, this technique can quickly determine the extent of tumor infiltration and lymph node metastasis, which is of great value for evaluating the extent of surgical resection and prognosis. It is worth noting that the literature on intraoperative frozen pathological diagnosis of AFP-HAGB in China consists mostly of case reports, with a lack of large-sample studies and systematic comparisons between intraoperative frozen pathological diagnosis and postoperative routine pathological diagnosis, as well as systematic summaries of key points for differential diagnosis. The pathological morphology of AFP-HAGB is complex and prone to confusion with other lesions. The accuracy of intraoperative diagnosis requires verification in additional cases, and the key points for differential diagnosis also need to be further summarized. Based on this case and the domestic literature, this article systematically analyzed the intraoperative frozen pathological morphological characteristics, key diagnostic points, and differential diagnosis methods of AFP-HAGB. It has innovative and exploratory value in diagnostic strategy and aims to provide new ideas and references for the precise intraoperative diagnosis of this rare gallbladder malignant tumor.
| Reference | Cases | Age (years) | Sex (male/female) | Underlying disease | Frozen diagnosis (correct/total) | Rapid IHC used | Final pathology confirmation |
| Sakamoto et al[18], 2004 | 1 | 58 | Male | Gallbladder stones | 1/1 | No | AFP(+), Hepatocyte(+) |
| Lin et al[19], 2008 | 1 | 62 | Female | Chronic cholecystitis | 1/1 | No | AFP(+), CK19(+) |
| Shi et al[20], 2016 | 1 | 55 | Male | Gallbladder stones | 1/1 | No | AFP(+), GPC3(+) |
| Karayiannakis et al[21], 2012 | 1 | 64 | Male | Gallbladder stones | 1/1 | No | AFP(+), Hepatocyte(+) |
| Li et al[8], 2023 | 4 | 38-68 | 3/1 | Gallbladder stones (3), none (1) | 4/4 | Yes (2 cases) | AFP(+), CK19(+), GPC3(+) |
| Shu et al[22], 2024 | 1 | 70 | Male | Gallbladder stones | 1/1 | No | AFP(+), Hepatocyte(+) |
| Zhang et al[23], 2023 | 2 | 45, 60 | 1/1 | Gallbladder stones (1), none (1) | 1/2 | No | AFP(+), CK19(+) |
| Yang et al[24], 2014 | 1 | 59 | Male | Chronic cholecystitis | 1/1 | No | AFP(+), GPC3(+) |
| Si et al[25], 2017 | 1 | 47 | Female | Gallbladder stones | 1/1 | No | AFP(+), Hepatocyte(+) |
| Present case | 1 | 61 | Female | None | 1/1 | No | AFP(+), GPC3(+), SALL4(+), Arg-1(+) |
| Total | 14 | 38-70 | 9/5 | Gallbladder stones (19), cholecystitis (2), none (3) | 13/14 (92.86%) | Yes in 2 cases | AFP(+) in all cases |
In this case, the intraoperative frozen pathological diagnosis, although based on HE staining alone, successfully identified the characteristic hepatoid morphology and, in conjunction with elevated serum AFP and imaging findings, accurately suggested AFP-HAGB. This provided a key basis for the surgeon to choose radical cholecystectomy, effectively avoiding a second operation and promoting the patient’s postoperative recovery. Based on the analysis of domestic and international literature, intraoperative frozen pathological diagnosis has a relatively high accuracy rate for AFP-HAGB. The combination of morphological observation using HE staining with clinical data (serum AFP and imaging) and, when available, detection of AFP, Hepatocyte, CK19, and other IHC indicators can effectively improve diagnostic accuracy and reduce the misdiagnosis rate. Intraoperative frozen pathological diagnosis is convenient, rapid, and efficient and can provide real-time guidance for surgical treatment, thus having important clinical application value. However, current clinical research on intraoperative frozen pathological diagnosis of AFP-HAGB consists mainly of case reports and small case series. Large-sample, multicenter studies are still lacking to further verify its diagnostic accuracy and influencing factors. The consistency with postoperative routine pathological diagnosis and systematic summary of differential diagnostic points still need to be improved. In the future, larger-scale clinical studies should be conducted to further analyze the intraoperative frozen pathological morphological characteristics of AFP-HAGB, optimize the diagnostic process, improve differential diagnosis strategies, and strengthen the professional training of pathologists to improve the accuracy of intraoperative frozen pathological diagnosis of AFP-HAGB, thereby providing strong support for the precise treatment of this rare malignant tumor.
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