Histopathological Diagnosis of Early HCC through Biopsy: Efficacy of Victoria Blue and Cytokeratin 7 StainingKobayashi S.a · Kim S.R.b · Imoto S.b · Ando K.b · Hirakawa M.b · Saito J.b · Fukuda K.b · Otono Y.b · Sakaki M.b · Tsuchida S.c · Kim S.K.e · Hayashi Y.d · Nakano M.f · Kudo M.g
aDepartment of Hepatology, Osaka City University Graduate School of Medicine, Osaka, bDepartment of Gastroenterology, Kobe Asahi Hospital, cDivision of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, and dDivision for Infectious Disease Pathology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, eDepartment of Gastroenterology, Kyoto University, Kyoto, fDivision of Pathology, Ohfuna Chuo Hospital, Kamakura, and gDepartment of Gastroenterology and Hepatology, Kinki University School of Medicine, Osaka-Sayama, Japan Corresponding Author
Objectives and Methods: Findings of histological analyses of 2 cases of liver biopsy revealing hypovascular nodules are described. Results: Ultrasound examination revealed hypovascular and hypoechoic nodules (8 mm in diameter) in segment 1 (case 1) and (8 mm) in segment 8 (case 2). The nodules were detected by only Gd-EOB-DTPA-enhanced MRI. Hematoxylin and eosin staining of ultrasound-guided biopsy of the nodules revealed slight hypercellularity without the features of early hepatocellular carcinoma (HCC) such as cell atypia, fatty change and pseudoglandular formation. Early HCC was suspected; however, Victoria blue staining disclosed terminal portal tract invasion, the most important finding of early HCC. Also, cytokeratin 7 staining revealed decreased ductular reaction compatible with early HCC. Taken together, these histological analyses confirmed the two nodules to be early HCC. Conclusion: Based on the criteria of the International Consensus Group, the two nodules were diagnosed as early HCC through biopsy.
Copyright © 2012 S. Karger AG, Basel
In 2009, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) published a consensus paper describing stromal invasion as most helpful in differentiating early hepatocellular carcinoma (HCC) from high-grade dysplastic nodules (HGDN) .
Here, we describe 2 cases of early HCC in chronic hepatitis C patients. Ultrasound (US)-guided biopsy of the nodules subjected to hematoxylin and eosin (HE) staining revealed slight hypercellularity without cell atypia, fatty change and pseudoglandular formation. Victoria blue staining and immunohistochemical staining with cytokeratin 7 (CK7) showed a stromal (portal tract) invasion and reduced ductular reaction compatible with early HCC.
Laboratory data on admission disclosed α-fetoprotein (AFP) 4.4 ng/ml (normal 0–20) and protein induced by vitamin K absence II (PIVKA II) 31 mAU/ml (0–40). A routine abdominal ultrasonograph (US) revealed an 8-mm hypoechoic nodule in segment 1 (S1) (fig. 1a). Sonazoid-enhanced US revealed no hypervascular nodules in the early vascular phase and no defect in the Kupffer phase. Contrast-enhanced computed tomography (CT) revealed no enhanced lesion in the arterial phase and no washout lesion in the equilibrium phase. Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) revealed no hypervascular nodule in the arterial phase, but disclosed a defective nodule in the hepatobiliary phase (fig. 1b). Arterioportal angiography revealed no enhanced lesion in CT hepatic arteriography and no perfusion defect in CT arterial portography. Histological analysis of a US-guided biopsy of the nodule revealed slight hypercellularity without cell atypia, fatty change and pseudoglandular formation. Victoria blue staining showed stromal (portal tract) invasion compatible with early HCC (fig. 2a–c). Immunohistochemical staining with CK7 showed no ductular reaction in areas of stromal invasion (fig. 2d–f).
|Fig. 1. B-mode US imaging; 8-mmhypoechoic nodule in S1 (a). Gd-EOB- DTPA-enhanced MRI revealed a defect in the hepatobiliary phase (b).|
|Fig. 2. Histological findings of the US-guided biopsy specimens. HE staining. Slight hypercellularity without cell atypia, fatty change and pseudoglandular formation (a). Victoria blue staining. Low magnification (b) and high magnification of oval enclosure: stromal invasion is observed (arrows) (c). Immunohistochemical staining with CK7. Low magnification (d), high magnification of tumor lesion (e), and high magnification of non-tumor lesion (f). Ductular reaction, absent in areas of stromal invasion (e) is florid around the cirrhotic nodules (non-tumor lesion) (f).|
Laboratory data on admission disclosed AFP 2.3 ng/ml and PIVKA II 158 mAU/ml. A routine abdominal US examination revealed an 8-mm hypoechoic nodule in S8 (fig. 3a). Sonazoid-enhanced US revealed no hypervascular nodule in the arterial phase and no defect in the Kupffer phase. Contrast-enhanced CT revealed no enhanced lesion in the early arterial phase and no washout lesion in the portal phase. Gd-EOB-DTPA-enhanced MRI revealed no enhanced nodule in the arterial phase, no washout lesion in the late phase, but revealed a defect in the hepatobiliary phase (fig. 3b). CT hepatic arteriography revealed no enhanced lesion, and CT arterial portography revealed no perfusion defect. Histological analysis of a US-guided biopsy revealed slight hypercellularity without cell atypia, fatty change and pseudoglandular formation (fig. 4a). Victoria blue staining showed stromal invasion compatible with early HCC (fig. 4b). Immunohistochemical staining with CK7 showed a scanty ductular reaction between the lesion and the adjacent liver tissue (fig. 4c).
|Fig. 3. B-mode US imaging. 8-mm hypoechoic nodule in S8 (a). Gd-EOB- DTPA-enhanced MRI revealed a defect in the hepatobiliary phase (b).|
|Fig. 4. Histological findings of US-guided biopsy. HE staining. Slight hypercellularity without cell atypia, fatty change and pseudoglandular formation are observed (a). Victoria blue staining. Stromal invasion is observed (b). Immunohistochemical staining with CK7. Ductular reaction is scanty between the lesion and the adjacent liver tissue (c).|
The status of imaging studies in the diagnosis of HCC <2 cm has changed with the introduction of new contrast agents used in US and MRI. First, Sonazoid was exclusively approved in Japan in 2007 as a second-generation US contrast agent; second, Gd-EOB-DTPA, a new liver-specific contrast agent used in MRI [2,3], was approved in 2008. Mita et al.  have compared the diagnostic sensitivity of contrast-enhanced CT, Sonazoid-enhanced US, Gd-EOB-DTPA-enhanced MRI, and CT arteriportal angiography in diagnosing HCC in nodules <2 cm. In the study of 34 nodules, 24 were moderately-differentiated and 10 well-differentiated HCC. Overall, the sensitivity was 53.9% by contrast-enhanced CT, 67.6% by Sonazoid-enhanced US, 76.5% by Gd-EOB-DTPA-enhanced MRI, and 88.2% by CT arteriportal angiography. A significant difference was observed in the sensitivity between contrast-enhanced CT and CT arteriportal angiography, but no difference among Sonazoid-enhanced US, Gd-EOB-DTPA-enhanced MRI and CT arteriportal angiography. The authors concluded that changing the main diagnostic modality for HCC <2 cm from CT arteriportal angiography to Sonazoid-enhanced US and Gd-EOB-DTPA-enhanced MRI was recommended.
Advances in these imaging techniques and the establishment of surveillance protocols for high-risk populations have led to the detection of small hepatic nodules in patients with chronic liver diseases, particularly those with cirrhosis or chronic hepatitis caused by B or C virus. Such nodules, comprising a broad range of diagnostic entities (some benign and some with malignant potential) are currently defined histologically, and their clinical management often depends on the ability to make a reliable histological diagnosis.
There has been considerable confusion regarding the nomenclature of and diagnostic approaches to these hepatic nodules. To clarify these issues, the International Working Party (IWP) of the World Congresses of Gastroenterology proposed a consensus – now widely adopted – and diagnostic criteria for hepatocellular nodular lesions in 1995 . The IWP classified nodular lesions found in chronic liver disease into large regenerative nodules, low-grade dysplastic nodules, HGDN, and HCC. In addition, the IWP introduced the concept of dysplastic focus as a cluster of hepatocytes with features of early neoplasia (in particular small cell change of iron-free foci in a siderotic background) measuring <0.1 cm, and defined small HCC as a tumor measuring <2 cm.
More recent studies support the division of small HCC into two clinicopathological groups termed early HCC and progressive HCC . Early HCC has a vaguely nodular appearance and is well differentiated. Progressive HCC has a distinctly nodular pattern and is mostly moderately differentiated, often with evidence of microvascular invasion. Early HCC takes longer to recur and has a higher 5-year survival rate compared with progressive HCC.
Small lesions with malignant potential are subtly different from the surrounding parenchyma, making them difficult to assess reproducibly. The ICGHN was convened to refine and make current the international consensus on the histopathological diagnosis of nodular lesions, such as dysplastic nodules and early HCC. Early HCC tumors are vaguely nodular and characterized by various combinations of the following major histologic features [7,8,9]: (1) increased cell density (more than twice that of the surrounding tissue) with an increased nuclear/cytoplasm ratio and an irregular thin trabecular pattern; (2) varying numbers of portal tracts within the nodule (intratumoral portal tract); (3) a pseudoglandular pattern; (4) diffuse fatty change, and (5) varying numbers of unpaired arteries. Nonetheless, all of these features may also be found in HGDN. Stromal invasion, described by ICGHN as helpful in differentiating early HCC from HGDN , is invasion into portal tracts of septal stroma within a hepatocellular nodule and is confirmed by Victoria blue staining.
Stromal invasion, which may be difficult to identify in early HCC because tumor cells show little or no cytologic atypia, is often focal; its identification is still more difficult when dealing with biopsied liver tissue in which only limited portions of nodules are sampled. Recently, Park et al.  have reported that CK7 immunostaining is useful in identifying stromal invasion. Ductular reaction, confirmed by CK7 staining, is frequently found in non-cancerous hepatocellular nodular lesions, whereas it is less frequently found in HCCs with true stromal invasion.
Here, the nodules of the 2 cases of early HCC were found hypovascular and were detected by only Gd-EOB-DTPA-enhanced MRI. HE staining of US-guided biopsy of the nodules revealed slight hypercellularity without the features of early HCC such as cell atypia, fatty change and pseudoglandular formation. Early HCC was suspected, however additional Victoria blue staining disclosed terminal portal tract invasion, which is the most important finding of early HCC. Also, CK7 staining revealed reduced ductular reaction compatible with early HCC. Thus, these histological analyses confirmed that these two nodules were early HCC. Victoria blue and CK7 staining are very useful in the diagnosis of early HCC through biopsy tissue. Further study is needed, however, to obtain consensus among pathologists regarding the efficacy of these two techniques in the diagnosis of early HCC.
The authors declare that no financial or other conflict of interest exists in relation to the content of the article.
Soo Ryang Kim, MD
Department of Gastroenterology, Kobe Asahi Hospital
3-5-25 Bououji-cho, Nagata-ku
Kobe 653-0801 (Japan)
Published online: December 13, 2012
Number of Print Pages : 6
Number of Figures : 4, Number of Tables : 0, Number of References : 10
Digestive Diseases (Clinical Reviews)
Vol. 30, No. 6, Year 2012 (Cover Date: December 2012)
Journal Editor: Malfertheiner P. (Magdeburg)
ISSN: 0257-2753 (Print), eISSN: 1421-9875 (Online)
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