Primary liver cancer is currently the 4th most common malignant tumor in China and the 2nd leading cause of cancer-related death, posing a serious threat to the lives and health of the Chinese people.
Primary liver cancer mainly includes three different pathological types: hepatocellular carcinoma (hepatocellular carcinoma, HCC), intrahepatic cholangiocarcinoma (intrahepatic cholangiocarcinoma, ICC), and combined hepatocellular-cholangiocarcinoma (combined hepatocellular-cholangiocarcinoma, cHCC-CCA). The three types differ significantly in terms of pathogenesis, biological behavior, histopathology, treatment methods, and prognosis, with HCC accounting for 75% to 85% and ICC for 10% to 15%. In this article, “liver cancer” refers only to HCC.
Pathological Diagnosis of Liver Cancer
Primary liver cancer: refers to malignant tumors originating from hepatocytes and intrahepatic bile duct epithelial cells, mainly including HCC, ICC, and cHCC-CCA.
HCC: refers to malignant tumors arising from hepatocytes. The pathological diagnostic terms “hepatocellular carcinoma” or “hepatocellular type liver cancer” are not recommended.
ICC: refers to malignant tumors arising from the epithelial cells lining the branches of the intrahepatic bile ducts, with adenocarcinoma being the most common. Histologically, it can be divided into:
① Large duct type: originating from larger bile ducts above the portal area between the liver lobules, with large and irregular glandular duct diameters;
② Small duct type: originating from small bile ducts or fine bile ducts below the bile ducts of the liver lobule septa, with small and relatively regular glandular duct diameters, or can appear as solid, thin strands with closed lumens. Studies have shown that the biological behavior and genetic phenotypic characteristics of the above two subtypes of ICC also differ, with patients of the small duct type having a better clinical prognosis than those of the large duct type.
The clinical and pathological significance of molecular typing of HCC and ICC is still under research and validation, but recent studies have shown that EB virus-associated ICC has unique clinical pathological, immune microenvironment, and molecular characteristics, with better prognosis and greater benefit from immune checkpoint therapy, making it a potential new subtype; while high expression of fructose-1,6-bisphosphate aldolase in ICC tissues is a useful indicator for assessing postoperative recurrence risk. The 2019 edition of the WHO classification of digestive system tumors no longer recommends using the pathological diagnostic term “cholangiocellular carcinoma” or “cholangiolocellular carcinoma” for ICC. The gross sampling and microscopic examination requirements for ICC should mainly refer to HCC.
cHCC-CCA: refers to the simultaneous presence of both HCC and ICC tissue components in the same tumor nodule, excluding collision tumors. Although some scholars suggest using a pathological diagnostic standard of ≥30% for both tumor components as the criterion for cHCC-CCA, there is currently no internationally unified pathological diagnostic standard for the proportion of HCC and ICC tumor components in cHCC-CCA. Therefore, it is recommended to annotate the proportion of the two tumor components when diagnosing cHCC-CCA pathologically for clinical evaluation of tumor biological characteristics and formulation of treatment plans.
Pathological Diagnosis Standards for Liver Cancer
The pathological diagnosis standards for liver cancer consist of specimen processing, specimen sampling, pathological examination, and pathological reporting.
Key Points for Specimen Processing:
① The surgical physician should clearly indicate the site, type, and quantity of the specimen on the pathological examination application form, and mark the surgical margins and important lesions with dye or sutures;
② The tumor specimen should be delivered intact to the pathology department for cutting and fixation within 30 minutes post-excision. Specimen collection for the tissue bank should be conducted under the guidance of the pathology department to ensure accuracy in sampling and should primarily meet the needs of pathological diagnosis;
③ Fixation in 4% neutral formaldehyde (10% neutral formalin) solution for 12 to 24 hours.
Key Points for Specimen Sampling: The periphery of liver cancer is a representative area of the tumor’s biological behavior. Therefore, a 7-point baseline sampling method is required (Figure 1), sampling at a 1:1 ratio at the 12 o’clock, 3 o’clock, 6 o’clock, and 9 o’clock positions at the junction of the cancer and adjacent liver tissue; at least one sample should be taken from within the tumor; and one sample each should be taken from liver tissue within ≤1cm (near the cancer) and >1cm (far from the cancer). For small liver cancers with a maximum diameter of ≤3cm, all should be sampled for examination. The actual sampling sites and quantities should also consider the size and number of tumors (evidence level 2, recommendation A).
Figure 1. Diagram of the baseline sampling locations for liver tumor specimens
Key Points for Pathological Examination of Liver Cancer
Gross Specimen Observation and Description: A comprehensive observation should be made of all surgical specimens submitted, focusing on describing the size, number, color, texture, relationship with blood vessels and bile ducts, capsule condition, surrounding liver tissue lesions, type of liver cirrhosis, distance from the tumor to the surgical margin, and status of the surgical margin.
Microscopic Observation and Description: All sampled tissues should be observed comprehensively, and the pathological diagnosis of liver cancer can refer to the 2019 edition of the WHO classification of digestive system tumors, focusing on the following content. The degree of differentiation of liver cancer: the internationally used Edmondson-Steiner four-grade (I–IV) grading method or WHO recommended high, medium, and low differentiation can be employed.
The histological morphology of liver cancer: common types include trabecular, massive, pseudoglandular, and compact types; special subtypes of liver cancer: such as fibrolamellar, sclerosing, clear cell, steatotic, giant cell, chromophobe, neutrophil-rich, lymphocyte-rich, and undifferentiated types; the extent and degree of tumor necrosis (such as after transarterial chemoembolization treatment), lymphocyte infiltration, and stromal fibrosis; the growth patterns of liver cancer: including peritumoral infiltration, capsule invasion or breach, microvascular invasion (MVI), and satellite nodules; assessment of chronic liver disease: liver cancer is often accompanied by varying degrees of chronic viral hepatitis or liver cirrhosis, the relatively simple Scheuer scoring system and the Chinese chronic viral hepatitis histological grading and staging standards are recommended.
MVI Diagnosis: MVI refers to the presence of cancer cell nests within the lumen of blood vessels lined by endothelial cells under the microscope, with portal vein branch invasion (including intraparenchymal vessels) being the most common in liver cancer, while lymphatic invasion can occur in ICC. The pathological grading method is as follows: M0: no MVI detected; M1 (low-risk group): ≤5 MVI, all occurring in adjacent liver tissue (≤1cm); M2 (high-risk group): >5 MVI, or MVI occurring in distant liver tissue (>1 cm). MVI and satellite lesions can be seen as different stages of the intrahepatic metastasis process of liver cancer, and when satellite nodules or lesions in adjacent liver tissue are difficult to distinguish from MVI, they can be included in the MVI pathological grading. MVI is an important reference for assessing the risk of liver cancer recurrence and selecting treatment options and should be included as an indicator in routine histopathological examinations (evidence level 2, recommendation A).
Immunohistochemical Examination
The main purposes of immunohistochemical examination for liver cancer are:
① Differentiation between benign and malignant hepatocellular tumors;
② Differentiation between HCC and ICC as well as other special types of liver tumors;
③ Differentiation between primary liver cancer and metastatic liver cancer. Due to the high heterogeneity of liver cancer histological types, existing liver cancer cell protein markers have certain limitations in specificity and sensitivity for diagnosis, often requiring reasonable combinations, objective assessments, and sometimes the need to be used in conjunction with markers from other systemic tumors.
HCC:
The following markers are positive for hepatocyte labeling and help indicate tumors of hepatocyte origin, but cannot be used as the basis for distinguishing between benign and malignant hepatocellular tumors.
① Arginase-1: positive staining in hepatocyte cytoplasm/nucleus.
② Hepatocyte antigen: positive staining in hepatocyte cytoplasm.
③ Antibodies specific for bile canalicular membrane proteins: such as CD10, polyclonal carcinoembryonic antigen, and bile salt export pump protein, can show specific staining at the bile canalicular surface of hepatocytes, helping to confirm hepatocellular tumors.
The following markers help distinguish between benign and malignant hepatocellular tumors.
① Phosphatidylinositol protein-3: positive staining in hepatocellular carcinoma cytoplasm and cell membrane.
② CD34: Although CD34 immunohistochemical staining does not directly label hepatocytes, it can show the microvascular density and distribution pattern characteristics of different types of liver tumors: for example, hepatocellular carcinoma is diffuse, cholangiocarcinoma is sparse, hepatocellular adenoma is patchy, and focal nodular hyperplasia is cord-like, which, combined with tumor histological morphology, helps in differential diagnosis.
③ Heat shock protein 70: positive staining in hepatocellular carcinoma cytoplasm or nucleus.
④ Glutamine synthetase: often shows diffuse strong positivity in hepatocellular carcinoma; some hepatocellular adenomas, especially β-catenin mutation-activated hepatocellular adenomas, can also show diffuse positivity; in HGDN, it shows moderate intensity focal staining, with positive cells <50%; in focal nodular hyperplasia, it shows characteristic irregular map-like staining; in normal liver tissue, only hepatocytes around the central vein are stained, these characteristics help in differential diagnosis.
ICC:
① Epithelial cell surface glycoprotein (MOC31): positive staining in cholangiocarcinoma cell membranes.
② Cytokeratin (CK) 7/CK19: positive staining in cholangiocarcinoma cytoplasm.
③ Mucin-1 (muc-1): positive staining in cholangiocarcinoma cell membranes.
Although the positive expression of the above markers can suggest tumors of biliary epithelial origin, they can also be positively expressed in non-tumorous biliary epithelium and require careful differentiation.
cHCC-CCA:
Both HCC and ICC components express their respective tumor markers. In addition, positive expression of markers such as CD56, CD117, and epithelial cell adhesion molecule (EpCAM) may suggest that the tumor has stem cell differentiation characteristics, indicating stronger invasiveness.
Pathological Assessment of Resection Specimens After Conversion/Neoadjuvant Therapy
Specimen Sampling: For clinical cases labeled as having undergone conversion/neoadjuvant therapy prior to liver cancer resection, the following process can be followed: cut open at the maximum diameter of the tumor bed (the original location of the tumor before treatment) and measure the three-dimensional size. For small liver cancers ≤3cm, all should be sampled; for tumors >3cm, they should be cut open at intervals of 0.5 to 1 cm at the maximum diameter, choosing the most representative sections of tumor necrosis and residual tumor, while also keeping samples of the tumor bed and surrounding liver tissue for comparison, and can also take photographs of the gross specimens for histological observation comparison.
Microscopic Assessment: The main assessment is the proportion of the three components in the tumor bed of the liver cancer resection specimen:
① Necrotic tumor;
② Viable tumor;
③ Tumor stroma (fibrous tissue and inflammation). The sum of these three areas in the tumor bed equals 100%. The number of samples taken should be noted in the pathological report, and the average percentage of the three components should be determined based on the assessment of each slice to determine the total percentage of residual tumor.
Complete Pathological Response and Significant Pathological Response Assessment: are important pathological indicators for evaluating the efficacy of preoperative treatment and exploring the best timing for surgery.
Complete Pathological Response (CPR): refers to the absence of viable tumor cells in the histological assessment of the tumor bed specimen after preoperative treatment.
Significant Pathological Response (MPR): refers to the reduction of viable tumor to below the threshold that can affect clinical prognosis after preoperative treatment. In lung cancer studies, MPR is often defined as residual tumor cells in the tumor bed reduced to ≤10%, which is consistent with the correlation between tumor necrosis degree and prognosis in liver cancer after TACE treatment. The specific threshold for MPR needs further clinical research confirmation. It is recommended to expand the sampling range for tumors initially diagnosed as MPR for clarification.
Histological Assessment Methods for Tumor Necrosis Degree in Liver Cancer Specimens After Immune Checkpoint Inhibitor Treatment can refer to some tumor types with more related research, deepening the understanding of histological characteristics of liver cancer in practice, while also observing whether there are immune-related liver injuries in the surrounding liver tissue, including hepatocyte injury, lobular hepatitis, and cholangitis.
Liver Cancer Pathological Diagnosis Report
It mainly consists of gross specimen description, microscopic description, immunohistochemical examination, and pathological diagnosis name, and can include explanations and suggestions to the clinic if necessary (Table 1). Additionally, molecular pathological examination results related to the clonal origin detection of liver cancer, drug target detection, biological behavior assessment, and prognosis judgment can be attached to provide clinical reference.
Summary of Key Points
1. The standardized handling and timely submission of liver cancer resection specimens are crucial for maintaining tissue and cell integrity and achieving accurate pathological diagnosis.
2. Sampling for liver cancer specimens should follow the “seven-point baseline sampling” standard, which is beneficial for obtaining representative pathological biological characteristic information of liver cancer.
3. The content of the liver cancer pathological diagnosis report should be standardized and comprehensive, with particular emphasis on the diagnosis and pathological grading assessment of MVI, which significantly affects the prognosis of liver cancer.
Table 1. Recommended Liver Cancer Pathological Diagnosis Report and Main Description Indicators
Liver Cancer Clinical Diagnosis Standards and Flowchart
Combining the high-risk factors for liver cancer, imaging characteristics, and serological molecular markers, clinical diagnosis of liver cancer can be made according to the steps in the flowchart (Figure 2).
Figure 2. Liver Cancer Diagnosis Flowchart
1.For individuals with HBV or HCV infection, or any cause of liver cirrhosis, at least once every 6 months, perform ultrasound examination and serum AFP testing. If a liver nodule with a diameter ≤ 2cm is found, and at least 2 of the four examinations (multiparametric MRI, dynamic contrast-enhanced CT, ultrasound contrast, or hepatocyte-specific contrast agent Gd-EOB-DTPA-enhanced MRI) show significant enhancement during the arterial phase and lower enhancement in the portal and/or delayed phases compared to liver parenchyma, which are typical features of liver cancer, a clinical diagnosis of liver cancer can be made; for liver nodules with a diameter >2cm, a clinical diagnosis of liver cancer can be made as long as one of the four imaging examinations shows typical liver cancer features.
2.For individuals with HBV or HCV infection, or any cause of liver cirrhosis, if follow-up shows a liver nodule with a diameter ≤ 2cm, and if none or only one of the four imaging examinations shows typical liver cancer features, liver lesion biopsy or follow-up imaging examinations every 2-3 months combined with serum AFP levels can be performed to clarify the diagnosis. For liver nodules with a diameter > 2cm, if none of the four imaging examinations show typical liver cancer features, liver lesion biopsy or follow-up imaging examinations every 2-3 months combined with serum AFP levels can be performed to clarify the diagnosis.
3.For individuals with HBV or HCV infection, or any cause of liver cirrhosis, if serum AFP levels are elevated, especially if persistently elevated, imaging examinations should be performed to clarify the diagnosis of liver cancer; if any of the four imaging examinations show typical liver cancer features, a clinical diagnosis of liver cancer can be made; if no liver nodules are found, and after excluding pregnancy, chronic or active liver disease, germ cell tumors, and gastrointestinal tumors, serum AFP changes should be closely monitored, and imaging re-examinations should be performed every 2-3 months.
Staging of Liver Cancer
The staging of liver cancer is crucial for selecting treatment plans and assessing prognosis. There are various staging systems abroad, such as BCLC, TNM, JSH, and APASL. Combining China’s specific national conditions and practical accumulation, a staging system for liver cancer in China (China liver cancer staging, CNLC) has been established based on the patient’s performance status (PS), liver tumor, and liver function, including: CNLC Stage Ia, Ib, IIa, IIb, IIIa, IIIb, and IV. The specific staging scheme is described in Figure 3.
Figure 3. Clinical Staging and Treatment Flowchart for Liver Cancer in China
CNLC Stage Ia: PS 0-2, liver function Child-Pugh A/B, single tumor, diameter ≤5cm, no imaging-visible vascular cancer thrombus and no extrahepatic metastasis;
CNLC Stage Ib: PS 0-2, liver function Child-Pugh A/B, single tumor, diameter >5cm, or 2-3 tumors, maximum diameter ≤3cm, no imaging-visible vascular cancer thrombus and no extrahepatic metastasis;
CNLC Stage IIa: PS 0-2, liver function Child-Pugh A/B, 2-3 tumors, maximum diameter >3cm, no imaging-visible vascular cancer thrombus and no extrahepatic metastasis;
CNLC Stage IIb: PS 0-2, liver function Child-Pugh A/B, ≥4 tumors, diameter of tumors irrelevant, no imaging-visible vascular cancer thrombus and no extrahepatic metastasis;
CNLC Stage IIIa: PS 0-2, liver function Child-Pugh A/B, tumor status irrelevant, with imaging-visible vascular cancer thrombus and no extrahepatic metastasis;
CNLC Stage IIIb: PS 0-2, liver function Child-Pugh A/B, tumor status irrelevant, with or without imaging-visible vascular cancer thrombus, with extrahepatic metastasis;
CNLC Stage IV: PS 3-4, or liver function Child-Pugh C, tumor status irrelevant, with or without imaging-visible vascular cancer thrombus, with or without extrahepatic metastasis.
References:
National Health Commission Office “Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022)”
* This article is for providing scientific information to medical professionals only and does not represent the views of this platform.
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