Histopathology of Hepatocellular Carcinoma MED3205

Histopathology of Hepatocellular Carcinoma

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, accounting for approximately 90% of primary liver cancers. It is the sixth leading cause of cancer in the world and the fourth leading cause of death from cancer. The geography of distribution of HCC shows distinctness, with prevalence highest in Eastern Asia and sub-Saharan Africa. HCC is a multifactorial aetiology with chronic liver disease as the predominant predisposing condition. The most significant risk factor worldwide is chronic HBV infection, and HCV is a substantial contributor in Western countries and Japan. Other significant risk factors include alcoholic liver disease, type 2 diabetes, type 2 diabetes with NAFLD, NASH, aflatoxin B1 exposure, hereditary haemochromatosis, and alpha-1-antitrypsin deficiency (Renne et al., 2021). Histopathological diagnosis is the cornerstone of HCC management in defining HCC as malignancy, which helps to define treatment. By integrating histopathological examination, biochemical markers, and advanced imaging technologies, the diagnostic approach to liver cancer is early detection, accurate staging, and choice of treatment.

Aim long

The objectives of this essay are:

• To provide a comprehensive overview of the laboratory tests utilised in the diagnosis of hepatocellular carcinoma, with particular emphasis on histopathological techniques, biochemical markers, and imaging modalities.


• To elucidate the diagnostic criteria, methodological approaches, and interpretative challenges associated with each diagnostic modality.


• To evaluate the integration of these complementary approaches in establishing an accurate diagnosis, determining prognosis, and guiding therapeutic interventions.


• To identify the limitations of current diagnostic methods and explore emerging technologies that may enhance diagnostic accuracy and efficiency in the future.

Methods

Histopathological Techniques

Histopathological examination remains the gold standard for definitive diagnosis of HCC. The process begins with tissue acquisition, which can be performed through various approaches. The most commonly used method is percutaneous needle biopsy, usually performed under ultrasound or CT guidance to help ensure the targeting of suspicious lesions. For patients with ascites or coagulation disorders, an alternative approach is transjugular liver biopsy. Detailed histopathological assessment is offered with surgical specimens, including reservation specimens and explanted livers.references

Once obtained, tissue specimens undergo a standardised processing protocol. Cellular structures are stabilised by fixation in 10% neutral buffered formalin, and autolysis is prevented. After fixation, tissues are dehydrated through ascending concentrations of alcohol, cleared with xylene, and embedded in paraffin wax. Sections of the paraffin blocks, 35 ?m thick, are cut on a microtome and mounted on glass slides. Morphological assessment is based upon standard hematoxylin and eosin (H&E) staining in which nuclei are stained blue by hematoxylin and cytoplasm and extracellular matrix components are in varying shades of pink by eosin. references

Special stains complement routine H&E evaluation, providing additional diagnostic information. Reticulin stains (silver impregnation) assess the integrity of the reticulin framework; usually, the reticulin framework is reduced or lost in HCC. Glycogen accumulation is diagnosed by periodic acid-Schiff (PAS) with diastase, and fibrosis surrounding the liver parenchyma is demonstrated by Masson's trichrome and Sirius red. In cases with hemochromatosis, a stain for Prussian blue may detect excessive iron deposition. references

Immunohistochemistry (IHC) plays a crucial role in HCC diagnosis, particularly in challenging cases. Hepatocyte-specific markers include Hepatocyte Paraffin 1 (HepPar1), which shows mitochondrial reactivity, which is granular cytoplasmic staining, and Arginase 1, which has superior sensitivity and specificity. High specificity of glypican-3 for malignant hepatocellular lesions (Renne et al., 2021). In HCC, polyclonal CEA and CD10 have a typical canalicular appearance. Additional markings include AFP, which is positive in about 30% of cases, and glutamine synthetase positive in a diffuse cytoplasmic distribution. references

Biochemical and Molecular Tests

Serum biomarkers enable non-invasive assessment and longitudinal monitoring. Alpha-fetoprotein (AFP) is the most established biomarker; abnormally elevated AFP above 400 ng/mL is very suspicious for HCC. Its sensitivity and specificity, on the other hand, are not ideal. In particular, total AFP has been superseded in specificity by AFP-L3, a fucosylated variant of AFP. references

Des-gamma-carboxy prothrombin (DCP), also known as a protein induced by vitamin K absence or antagonist-II (PIVKA-II), serves as a complementary marker. Glypican-3 (GPC3), detectable in serum, offers particular utility in AFP-negative cases. Novel biomarkers include osteopontin, Golgi protein-73 (GP73), and various microRNAs. Combinatorial approaches, such as the GALAD model, demonstrate enhanced diagnostic performance. references

Molecular testing offers more diagnostic and prognostic information. The chromosomal aberrations and viral integration in HBV-related cases can be detected by fluorescence in situ hybridisation (FISH) or by polymerase chain reaction (PCR). Next-generation sequencing (NGS) provides deep genomic sequencing in order to detect mutations in various genes, including TERT promoter, ARID1A, TP53, and CTNNB1 (? catenin). references

Imaging Methods

Imaging modalities offer non-invasive assessment of liver lesions. Ultrasonography (US) serves as the primary screening tool, detecting nodules as small as 1 cm. Contrast-enhanced ultrasonography (CEUS) improves characterisation by assessing vascular patterns. Computed tomography (CT) with multiphasic technique is a cornerstone in the diagnosis of HCC (Miranda et al., 2023). Typically, the protocol consists of unenhanced, arterial, portal venous, and delayed phases. references

Magnetic resonance imaging (MRI) offers superior soft tissue contrast and multiparametric capabilities. Conventional sequences include T1-weighted, T2-weighted and diffusion-weighted imaging (DWI). Vascular patterns seen on dynamic contrast-enhanced MRI are similar to CT. The hepatobiliary phase helps provide extra information as HCC is typically seen as hypointense compared to the background liver using hepatobiliary contrast agents. The sensitivity of 18F-fluorodeoxyglucose (FDG) PET is limited for the detection of conventional HCC. Newer tracers, 11C-acetate and 18F-fluorocholine, are also known to be more sensitive. references

Results

Histological Findings

Histopathological examination reveals diverse morphological patterns reflecting varying degrees of differentiation. Well-differentiated HCC closely resembles normal hepatocytes but demonstrates increased cell density, nuclear atypia, and altered trabecular architecture. The classic trabecular pattern is sinusoidal spaces separating thickened hepatocyte plates. Under the pseudo glandular pattern, gland-like structures are formed; in the compact pattern, the cell growth is dense.image references Although less common, scirrhous, clear cell, fatty change, and sarcomatoid patterns are also seen (Gisder et al., 2022). Cytological findings usually include increased nuclear to-cytoplasmic ratio, hyperchromatic nuclei, prominent nucleoli, and intranuclear inclusions. Intracytoplasmic fat and bile production represent signs of hepatocellular differentiation. references

Architectural features aiding diagnosis include loss of reticulin framework, absence of portal tracts within the lesion, and invasion of surrounding liver parenchyma. The prognostic significance of microvascular invasion is high. The immunohistochemical profile is usually favourable for HepPar-1, Arginase-1, Glypican-3, a canalicular pattern with polyclonal CEA and CD10. These challenges in diagnosis include distinguishing the well-differentiated HCC from the high-grade dysplastic nodules as well as identifying the poorly differentiated HCC from metastatic carcinomas. references

Integration of Biochemical and Imaging Data

According to guidelines from major liver societies, non-invasive diagnosis of HCC can be established in cirrhotic patients when lesions ?1 cm demonstrate the characteristic vascular pattern on dynamic CT or MRI. In this approach, the high specificity of imaging findings is recognised when cirrhosis is present. There is a correlation between serum AFP levels, tumour bulk, and differentiation. AFP, AFP-L3%, and DCP constitute a superior diagnostic combination than the individual markers. references

Correlation between imaging and histopathology reveals that arterial hyperenhancement corresponds to increased arterial vascularisation, while washout reflects reduced portal venous supply and early venous drainage. Imaging correlates with histopathology and shows a mismatch between arterial hyperenhancement and increased arterial vascularisation and for washout versus decreased portal venous supply and early venous drainage. Fibrous pseudocapsule formation correlates to capsular enhancement (Dong et al., 2022). The Liver Imaging Reporting and Data System (LI-RADS) standardises liver characteristic interpretation and reporting in high-risk patients with primary and ancillary features to derive category codes from LRI (definitely benign) to LR5 (definitely HCC). references

Discussion

Critical Evaluation of Methods and Limitations

Histopathological assessment faces challenges related to sampling error, particularly in percutaneous biopsies where tumour heterogeneity may lead to non-representative sampling. Needle track seeding is an unquestionable risk. Even experienced pathologists may find it challenging to distinguish well-differentiated HCC from high-grade dysplastic nodules. Immunohistochemical markers are not sensitive or specific. Poorly differentiated HCC have reduced expression of HepPar-1, whereas the expression of Glypican-3 is regular or absent in well-differentiated tumours. No single marker is perfect at the diagnostic level, so panel-based approaches are needed. References small discussion

Serum biomarkers exhibit limitations in sensitivity and specificity. AFP presents suboptimal performance for early HCC detection, with over 40% of HCCs. Environmental factors, viral hepatitis activity, and pregnancy commonly cause false-positive elevations. Imaging techniques demonstrate modality-specific limitations. However, ultrasonography is operator-dependent and less sensitive in challenging body habitus. Iodinated contrast and ionising radiation are exposed to patients during CT (Bello et al., 2022). MRI contraindications exist in patients with specific implants and take longer to acquire. Non-invasive diagnostic criteria are applicationally dependent on the presence of underlying cirrhosis and are less specific in non-cirrhotic livers. Approximately 40% of small HCCs have atypical imaging features, which represent the reasons for biopsy to make a diagnosis. references

Suggestions for New Diagnostic Tests

Artificial intelligence (AI) and machine learning algorithms applied to radiological images demonstrate potential for improved lesion detection, characterisation, and prognostication. Medical images can be used in radionics, and the quantitative features extracted from them may capture tumour heterogeneity and provide a prediction of biological behaviour. Additionally, molecular profiling by liquid biopsy strategies, including circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), and extracellular vesicles, offer non-invasive approaches. Methylation patterns in cell-free DNA show promise as early detection biomarkers. MicroRNAs and long non-coding RNAs are potential complementary biomarkers as circulating RNAs. references

In addition, spatial transcriptomics and mass spectrometry imaging enable extensive molecular profiling with the spatial context intact. Simultaneous assessment of multiple markers at single-cell resolution can be done in the same cell using multiplex immunofluorescence or imaging mass cytometry. Additionally, metabolomic approaches may provide signals of HCC specific to metabolic changes in the milieu. Hyperpolarised 13C-pyruvate MRI is an emerging technique for metabolic imaging that allows for imaging of tumour metabolism non-invasively. Finally, comprehensive systems biology approaches combined with the integration of multiomics data provide opportunities for precision diagnostics. references

Conclusion

The diagnosis of hepatocellular carcinoma requires a multimodal approach integrating histopathological examination with biochemical markers and advanced imaging techniques. Histopathological assessment is still the gold standard for definitive diagnosis and also for important prognostic information. Immunohistochemical markers help in making the diagnosis accurate, especially in complex cases. Assessment and longitudinal monitoring are made using biochemical markers that are non-invasive but with limited sensitivity and specificity; hence, they should be interpreted with caution. Dynamic imaging techniques exploiting the characteristic vascular profile of HCC are used to make non-invasive diagnoses in settings provided with appropriate clinical contexts. However, the integration of complementary approaches in diagnostics has contributed to the early detection and characterisation of HCC. references