Loss of heterozygosity of chromosome 8p and 11p in the dysplastic nodule and hepatocellular carcinoma

BACKGROUND AND AIM: In hepatocarcinogenesis, both de novo and multistep pathways have been suggested, and in the latter a dysplastic nodule is the proposed precancerous lesion. But genetic changes involved in the dysplastic nodule are not well understood. In this study, we tried to determine whether allelic loss of the chromosome 8p and/or 11p could be involved in the development of the dysplastic nodule and/or hepatocellular carcinoma. Platelet-derived growth factor-receptor beta-like tumor suppressor gene (PRLTS) and deletion in liver cancer-1 tumor suppressor gene are located at 8p21.3-p22. The hepatitis B virus integration site and WT1 tumor suppressor gene are located at 11p13. METHODS: We therefore studied loss of heterozygosity (LOH) of chromosome 8p21.3-p22 and 11p13 in 22 dysplastic nodules and 21 hepatocellular carcinomas. The samples, microdissected from paraffin-embedded tissues, were examined using a polymerase chain reaction-based LOH assay using microsatellite markers. RESULTS: Loss of heterozygosity was detected for chromosome 8p21.3-p22 in nine (40.9%) of 22 dysplastic nodules and in eight (42.1%) of 19 hepatocellular carcinomas. D8S261, located adjacent to PRLTS, showed most frequent LOH: 28.6% in dysplastic nodule and 40.0% in hepatocellular carcinoma. Loss of heterozygosity on chromosome 11p13 was found in three (15.8%) of 19 dysplastic nodules and in six (31.6%) of 19 hepatocellular carcinomas. Loss of heterozygosity of D11S995 and D11S907 was found in 33.3% and 7.1% of dysplastic nodules, and 8.3% and 44.4% of hepatocellular carcinomas, respectively. CONCLUSION: These results suggest that at least one putative tumor suppressor gene involved in the development and progression of hepatocellular carcinoma may be located on 8p21.3-p22 and 11p13. Particularly, PRLTS might be related to an early genetic event of hepatocarcinogenesis.     

Premalignant lesions of hepatocellular carcinoma: pathologic viewpoint

The significance of large cell dysplasia and small cell dysplasia as premalignant lesions has been extensively discussed. At present the majority of researchers consider that the former seems to be a secondary change rather than a premalignant lesion, and the latter is strongly suggested to be a premalignant lesion. In the past decade, however, adenomatous hyperplasia (dysplastic nodules), the nodular lesions seen in the cirrhotic liver, has attracted researchers' interest. The significance of adenomatous hyperplasia as a premalignant lesion has been supported by its frequent occurrence in the vicinity of hepatocellular carcinomas (HCC), the presence of adenomatous hyperplasia containing distinct cancerous foci, frequent malignant transformation seen in follow-up studies, and so forth. For histological diagnosis of adenomatous hyperplasia, a thorough understanding of the pathology of well-differentiated HCC at an early stage is essential. Reports from Europe and the United States on dysplastic nodules often include well differentiated HCC at the early stage, possibly because of the lack of opportunity for Western pathologists to see minute early-stage well differentiated HCC. In addition, Western pathologists usually have no concept of "carcinoma in situ" in which no obvious invasive growth is present, and many cases of gastric cancer in the mucosa are diagnosed as dysplasia in Europe and the United State.     

Diagnostic discrepancy of early hepatocellular carcinoma between Japan and West

The diagnoses for a range of hepatocellular neoplastic lesions amongst Japanese and Western pathologists show a striking lack of consistency, especially in distinguishing dysplastic nodules (DN) from very well-differentiated hepatocellular carcinomas (HCC). Namely, very well-differentiated HCC showing a vaguely nodular appearance diagnosed by Japanese pathologists tends to be diagnosed as high-grade DN by Western pathologists. Both Japanese and Western pathologists agreed that "stromal invasion", which is tumor cell invasion into the intra-tumoral portal tracts, is considered the most helpful morphologic clue to distinguish high-grade DN from well-differentiated HCC.     

Recent advances in the diagnosis of hepatocellular carcinoma

In the last decade, new imaging techniques have become available, offering the possibility of investigating contrast perfusion of liver nodules in cirrhosis. It is now accepted that a non-invasive diagnosis of hepatocellular carcinoma (HCC) can be established based on the vascular pattern, obtained with pure blood pool contrast agents. The diagnostic pattern includes: hypervascularity in the arterial phase (15–35 s after contrast injection), consisting in a contrast signal in the nodule greater than in the surrounding parenchyma, followed by contrast wash out, which leads the nodule to show the same, or, more specifically, a lower contrast signal, than the surrounding parenchyma in the portal and late phases (>40 s after injection). Such a pattern can be obtained not only by computed tomography or magnetic resonance imaging, but also by contrast-enhanced ultrasonography, most simply with real-time low mechanical index harmonic imaging ultrasound equipment with second-generation ultrasound contrast agents. The risk of false-positive diagnosis of malignancy isnearly abolished when the functional vascular pattern is not the only feature, but is superimposed on a nodule visible also without contrast. One single contrast imaging technique may suffice to make a diagnosis of HCC if the nodule is >1 cm in diameter and has developed during a surveillance program. Other types of contrast agents, such as those taken up by the reticular-endothelial system cells, may offer additional diagnostic clues, but definitive evidence of their efficacy is still to be produced. In conclusion, contrast-enhanced imaging techniques now offer the possibility of a non-invasive diagnosis of HCC in a large number of cases, reducing the need of invasive investigations, such as ultrasound-guided biopsy or angiography.     

Hypoxia and hepatocellular carcinoma: The therapeutic target for hepatocellular carcinoma

Hypoxia enhances proliferation, angiogenesis, metastasis, chemoresistance, and radioresistance of hepatocellular carcinoma (HCC); suppresses differentiation and apoptosis of HCC; and consequently leads to resistance of transarterial embolization (with or without chemotherapy). Because transarterial embolization contributes to angiogenesis via inducing hypoxia, therapy combined with transarterial embolization and antiangiogenic therapy provides a new strategy for the treatment of HCC. Unfortunately, hypoxia leads to the escape of HCC cells from transarterial embolization and antiangiogenic therapy. Thus combined therapy that induces and targets hypoxia may be of benefit to HCC patients. Because angiogenesis plays an important role in recurrence of HCC after resection, antiangiogenic therapy is beneficial to HCC patients following surgical resection of the tumor.     

肝硬化结节及小肝癌患者3.0T MRI检查结果分析

目的评价3.0 T MRI检查在肝硬化再生性结节（RN）、异型增生性结节（DN）和小肝癌（SHCC）诊断和鉴别诊断中价值。方法回顾性分析经病理检查确诊的69例RN、DN及SHCC患者的3.0 T MRI平扫及容积采集技术（LAVA）三期动态增强扫描特点。结果 RN主要表现为T2WI低信号,动态增强方式呈＂缓慢上升型＂;DN主要表现为T2WI高、低信号,信号较均匀结节的动态方式以＂速升缓降型＂为主,可有＂结中结＂（特征表现）;SHCC主要表现为T1WI低信号、T2WI高信号,动态增强方式为＂速升速降型＂。结论肝硬化结节及小肝癌在3.0 T MRI上各有较为典型的表现,临床可据此进行诊断与鉴别诊断;对其他强化类型的肝脏病灶应结合肿瘤血管的扭曲增粗及包膜等表现排除SHCC。     

Novel markers of cell kinetics to evaluate progression from cirrhosis to hepatocellular carcinoma.

BACKGROUND: We investigated cell cycle kinetics of nodular lesions in cirrhosis to differentiate hepatocellular carcinoma (HCC) from its precursor lesions. METHODS: Twelve small HCC, 10 regenerative (RN), six large regenerative (LRN), and five dysplastic nodules (DN), identified in explant cirrhotic livers of five consecutive patients transplanted at Royal Free Hospital in 2002. Immunoperoxidase for MCM2, geminin and Ki67 was performed and the percentage of positive cells counted. RESULTS: The proportion of cells expressing MCM2 was more than those expressing Ki67, which in turn was more than those expressing geminin (overall median=16%, 2% and 0.5%, respectively, P<0.001). There was a statistically significant trend of increasing Ki67 expression (P=0.006), from RN to HCC; this trend was not statistically significant for geminin (P=0.18) or MCM2 (P=0.51). The median percentage of cells expressing Ki67 was 1% in RN, 0.5% in LRN, 2.2% in DN and 5.4% in HCC. The combination of these markers identified four different cell kinetics patterns: 'resting' (G0 cells: MCM2 -ve, Ki67 -ve, geminin -ve); 'licensed' (MCM2 +ve, Ki67 -ve, geminin -ve); 'slowly growing' (G1 phase arrest, MCM2 +ve, Ki67 +ve, low (0.4%) geminin) and expanding (MCM2 +ve, Ki67 +ve, geminin +ve) nodules. CONCLUSIONS: The combination of MCM2, geminin and Ki67 could represent a valuable tool in the understanding of HCC progression in cirrhosis.     

Genetic and epigenetic alterations of the KLF6 gene in hepatocellular carcinoma

BACKGROUND AND AIM: Kruppel-like factor 6 (KLF6) is a zinc finger tumor suppressor gene that is frequently mutated in several human cancers and is broadly involved in differentiation and development, growth-related signal transduction, cell proliferation, apoptosis, and angiogenesis. The aim of this study was to elucidate the potential etiological role of KLF6 in the development of hepatocellular carcinoma (HCC) in Korea. METHODS: The gene mutation, allelic loss, and methylation status of the KLF6 gene was analyzed in a series of 85 Korean patients: 21 with dysplastic nodules and 85 with HCC. RESULTS: No somatic mutations were observed in the patients with dysplastic nodules or with HCC. Allelic loss was found in five (6.8%) of 73 informative HCC tissues. Three of the five patients with allelic loss had HCC with hepatitis B virus infection and cirrhosis, and the remaining two had no viral infection and a non-specific background. In methylation analysis, unmethylated and methylated DNAs of the KLF6 gene were amplified in all corresponding non-neoplastic liver tissues. Only one HCC tissue showed methylated DNA without unmethylated DNA. CONCLUSIONS: The results suggest that genetic and epigenetic alteration of KLF6 may play a minor role in the development of HCC.     

Computed tomography angiographic findings in hepatocellular carcinoma less than 2 cm detected during follow-up in 29 patients

AIMS: The early stage of hepatocarcinogenesis is not well understood pathologically and clinically. The present study was designed to define small (early) hepatocellular carcinoma (HCC) angiographically using the angio-helical computed tomography (CT) system. METHODS: Arterial portography CT and hepatic arteriography CT were carried out in 29 patients in whom small HCC < or = 20 mm was detected during follow-up. RESULTS: There were 17 males and 12 females, aged 47 to 85 years. The offending virus was hepatitis B in four, hepatitis C in 24 and no virus marker in one case. The follow-up period varied from less than a year to 17 years, averaging 6.4 years. The underlying disease was cirrhosis in 12 and chronic hepatitis in 17 cases. The mass was solitary in 16 and multiple in 13 cases, while the size of the mass ranged from 8-20 mm. All lesions were low in attenuation on arterial portography CT, and in 23 of 30 lesions hepatic arteriography CT showed high attenuation, suggesting arterial blood supply. In the remaining 7 cases, lesions were perhaps in the transition from portal to arterial. CONCLUSIONS: It was concluded that HCC develops frequently in a liver with chronic hepatitis, often muticentrically, and that early HCC lesions are more often overt HCC already with arterial blood supply, rather than extremely well-differentiated supplied by the portal vein as generally believed.     

Multistep hepatocarcinogenesis from a dysplastic nodule to well-differentiated hepatocellular carcinoma in a patient with alcohol-related liver cirrhosis

We describe a rare case of the transformation of a dysplastic nodule into well-differentiated hepato- cellular carcinoma (HCC) in a 56-year-old man with alcoholrelated liver cirrhosis. Ultrasound (US) disclosed a 10 mm hypoechoic nodule and contrast enhanced US revealed a hypovascular nodule, both in segment seven. US-guided biopsy revealed a high-grade dysplastic nodule characterized by enhanced cellularity with a high N/C ratio, increased cytoplasmic eosinophilia, and slight cell atypia. One year later, the US pattern of the nodule changed from hypoechoic to hyperechoic without any change in size or hypovascularity. US-guided biopsy revealed well-differentiated HCC of the same features as shown in the first biopsy, but with additional pseudoglandular formation and moderate cell atypia. Moreover, immunohistochemical staining of cyclase- associated protein 2, a new molecular marker of well- differentiated HCC, turned positive. This is the first case of multistep hepatocarcinogenesis from a dysplastic nodule to well-differentiated HCC within one year in alcohol-related liver cirrhosis.     

Implications of hyperechoic lesions in small hepatocellular carcinoma

Of 34 solitary small hepatocellular carcinomas (HCC) 2 cm in diameter or less, 13 with hyperechoic lesions were observed serially by sonography, and 11 of these were examined histologically. Serial examination showed that hypoechoic areas appeared at the periphery of or within, the hyperechoic tumor, and that these areas expanded more with tumor growth than the hyperechoic areas as if compressing or displacing the existing hyperechoic areas. Histologically, the hyperechoic lesions were composed mostly of well-differentiated cancer cells containing fat droplets, whereas the hypoechoic lesions were composed of cancer cells without fat droplets. In the two tumors that were formed almost completely of cancer cells showing fatty metamorphosis, cancer cells without fat droplets proliferated mainly in the periphery of the tumor. These findings suggest that, in hyperechoic HCC, cancer cells with fat droplets apear in the early stage of HCC, and probably change into concer cells without fat droplets by the time that a certain tumor size is reached, with gradual displacement by the latter type of cell during tumor growth.     

Imaging of multistep human hepatocarcinogenesis

In Japan, there are approximately 32 000 deaths (∼30 deaths per 100 000) per year due to hepatocellular carcinoma (HCC), and it is the third most common cancer in men and fifth in women. Approximately 90% of them are associated with chronic liver diseases due to hepatitis C or B virus infection. Therefore, it has become possible to detect small early stage HCC by the periodic screening in these high-risk patients group. During the screening imaging diagnosis of HCC, various kinds of hepatocellular nodules are also frequentlydetected. To characterize them is very important for the early diagnosis and treatment of HCC. For this purpose, it is necessary to understand the concept of multistep hepatocarcinogenesis and the sequential changes of imaging findings.     

International consensus on histologic diagnosis of early hepatocellular neoplasia

1. The precursor lesions for the development of hepatocellular carcinoma are believed to be high-grade dysplastic nodules. These lesions have atypical and proliferative features that distinguish them from normal or cirrhotic liver but are not sufficient for the diagnosis of carcinoma.
2. Individual HGDN are often heterogeneous and complete sampling may reveal regions of carcinoma within these otherwise benign lesions.
3. Invasion of stroma is considered a definitive feature of HCC. However, this feature is not always present in early HCC and is seldom found in needle biopsies.
4. Accurate diagnosis of dysplastic nodules and well-differentiated HCC requires skill and experience. However, accurate diagnosis with needle biopsies may be impossible if the highest grade of atypia is not sampled. Fine needle aspiration is not appropriate for small lesions that are expected to be early hepatic neoplasia. This technique should be reserved for suspected moderate- or poorly differentiated HCC.     

Expression of the transcription factor Klf6 in cirrhosis, macronodules, and hepatocellular carcinoma

Background and Aims: Macronodules (MN) occurring in cirrhosis are considered to be precursor lesions for hepatocellular carcinoma (HCC). However, early molecular events in hepatocellular carcinogenesis are poorly understood. The aim of this study was to compare gene expression profiling between cirrhotic tissues, MN, and HCC, to identify genes early involved in liver carcinogenesis. Methods: Tissues were obtained from explanted livers: nine cirrhosis, 10 MN, and seven HCC. Total RNAs were extracted by RNeasy and reverse transcribed with labelled [³³P]‐αATP. Hybridations were performed on Atlas Human Cancer 1.2 membranes (1176 genes). Results: A two‐way hierarchical clustering algorithm successfully isolated specific gene expression profiles when comparing MN, cirrhosis, and HCC. A total of 16 and 14 genes were up‐ and down‐expressed, respectively, in HCC as compared to cirrhotic tissues. The molecular signature of MN was characterized by the down‐expression of 23 and 42 genes as compared to cirrhosis and HCC, respectively. Among them, Klf6 was down‐expressed in all MN samples whereas it was over‐expressed in cirrhosis and HCC. This result was confirmed at RNA level by quantitative real time–polymerase chain reaction and at protein level by Western blotting. However, no mutation in the exon 2 of Klf6 was detected. Conclusion: We identified a molecular signature of MN characterized by a down‐expression of several genes. One of them, Klf6 was found to be down‐expressed in all MN without evidence of somatic mutations in the exon 2. This gene could be involved at an early stage of hepatocarcinogenesis.     

Extrahepatic hepatocellular carcinoma metastasis demonstrated by technetium-99 HIDA scan

Summary A patient presented with a 10-cm mass in the left lobe of the liver. Liver biopsy revealed hepatocellular carcinoma with abundant bile staining. A chest roentgenogram showed a 13-mm nodule in the right mid-lung field.A⁹⁹Tc HIDA scan was ordered for the evaluation of the pulmonary nodule. Intense uptake of the radionuclide was noted in the location of the nodule.The findings in this case suggest that the⁹⁹Tc HIDA scan may be a useful staging agent after the diagnosis of hepatocellular carcinoma has been documented.     

肝细胞癌癌前病变富血供转化的风险分析

目的探讨慢性肝病患者钆塞酸二钠增强磁共振成像(MRI)诊断高度异型增生结节(HGDN)转化成富血供肝细胞肝癌(HCC)的危险因素。方法回顾性分析2012年1月至2014年12月期间行钆塞酸二钠增强MRI的患者2037例,筛选出51例有慢性肝病背景的HGDN,行至少2次随访钆塞酸二钠增强MRI扫描,并且在首次MRI前后1个月内行CT增强扫描,研究终点为HGDN转化成富血供HCC,截止时间为2019年4月。根据有无富血供转化分为转化组(A组)和未转化组(B组)。线性回归分析HGDN向富血供HCC转化可能的危险因素。结果A组36个结节,B组79个结节,富血供转化率31.3%(36/115)。单变量分析结节长径>10.2mm(P=0.034)、年增长率>2%(P<0.001)、含脂质成分(P=0.007),与其发生富血供转化具有相关性,多变量分析结节年增长率是发生富血供转化的独立危险因素(P<0.0001)。结论慢性肝病患者钆塞酸二钠增强MRI诊断HGDN年增长率可以作为向富血供转化的潜在预测因子。     

Latest developments in precancerous lesions of hepatocellular carcinoma

Hepatocarcinogenesis in human chronic liver diseases is a multi-step process in which hepatic precancerous lesions progress into early hepatocellular carcinoma(HCC) and progressed HCC, and the close surveillance and treatment of these lesions will help improve the survival rates of patients with HCC. The rapid development and extensive application of imaging technology have facilitated the discovery of nodular lesions of ambiguous significance, such as dysplastic nodules. Further investigations showed that these nodules may be hepatic precancerous lesions, and they often appear in patients with liver cirrhosis. Although the morphology of these nodules is not sufficient to support a diagnosis of malignant tumor, these nodules are closely correlated with the occurrence of HCC, as indicated by long-term follow-up studies. In recent years, the rapid development and wide application of pathology, molecular genetics and imaging technology have elucidated the characteristics of precancerous lesions. Based on our extensive review of the relevant literature, this article focuses on evidence indicating that high-grade dysplastic nodules are more likely to transform into HCC than low-grade dysplastic nodules based on clinical, pathological, molecular genetic and radiological assessments. In addition, evidence supporting the precancerous nature of large cell change in hepatitis B virus-related HCC is discussed.     

Three cases of small hepatocellular carcinoma presenting as obstructive jaundice

Background

Despite improved diagnostic tools, it is often difficult to make a correct diagnosis of small hepatocellular carcinoma (HCC) in patients with obstructive jaundice.

Case outlines

Three cases of small HCC (<2 cm diameter) presenting as obstructive jaundice are reported. All tumours were initially diagnosed as hilar cholangiocarcinoma based on ultrasono-graphy, computed tomography, cholangiography and angiogra-phy. Because of insufficient hepatic function, none of the patients underwent hepatic resection. One patient died 8 months after first admission to our hospital, another died of disseminated intravascular coagulation I month after admission, and the third was treated with hepatic arterial infusion chemotherapy and survived >36 months.

Conclusion

It is important to consider HCC in the diagnosis of obstructive jaundice in patients who are predisposed to HCC because of liver cirrhosis and/or chronic viral hepatitis, and have elevated serum alpha-fetoprotein.     

肝细胞癌癌前结节影像学特征的研究进展

肝细胞癌(HCC)发生是一个多步骤过程,在其发展中检测出HCC癌前病变和进展期HCC,对预测肿瘤行为、判断病变程度、采用最佳治疗策略、改善患者生存至关重要。肝脏成像技术的快速进展和广泛应用,尤其是肝细胞特异性对比剂钆塞酸二钠MRI(Gd-EOB-DTPA MRI)可提供肝结节血管变化、肝细胞功能信息,能够精确区分肝硬化再生结节、低度异型增生结节、高度异型增生结节、早期HCC(early HCC)和HCC,从而进行恶性进展的风险度分层。现综述Gd-EOB-DTPA MRI在HCC早期诊断中的价值,分析HCC多步发展过程中的关键概念,以及癌前病变最终可能转化成典型HCC的影像学表现。