Misdiagnosis of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (RCC) represents the most common histological subtype of malignant kidney tumors. Based on symptoms alone, clear cell RCC is indistinguishable from other histological classes of RCC unless the tumor is present in the context of an RCC syndrome. Histopathological examination is, therefore, important to accurately identify clear cell RCC. Clear cell RCCs have characteristic morphological criteria; these tumors can be easily identified upon typical presentation, but diagnosis can be challenging when tumor cell pattern is unusual or when availability of tissue samples is limited. In this Review, the clinical, radiological and pathological characteristics of clear cell RCCs are described, as well as the potential tumors that can be confused with clear cell RCC and need to be considered in the differential diagnoses. Finally, the importance of an accurate diagnosis is highlighted in the context of the increasing use of preoperative tissue sampling and the prevalence of clear cell tumors associated with hereditary syndromes, which could have different therapeutic and prognostic implications for patients and their families.     

Clear cell hepatocellular carcinoma: origin,metabolic traits and fate of glycogenotic clear and ground glass cells

Clear cell hepatocellular carcinoma (CCHCC) has hitherto been considered an uncommon, highly differen-tiated variant of hepatocellular carcinoma (HCC) with a rela-tively favorable prognosis. CCHCC is composed of mixtures of clear and/or acidophilic ground glass hepatocytes with excessive glycogen and/or fat and shares histology, clinical features and etiology with common HCCs. Studies in animal models of chemical, hormonal and viral hepatocarcinogenesis and observations in patients with chronic liver diseases prone to develop HCC have shown that the majority of HCCs are preceded by, or associated with, focal or diffuse excessive stor-age of glycogen (glycogenosis) which later may be replaced by fat (lipidosis/steatosis). In ground glass cells, the glycogenosis is accompanied by proliferation of the smooth endoplasmic reticulum, which is closely related to glycogen particles and frequently harbors the hepatitis B surface antigen (HBsAg). From the findings in animal models a sequence of changes has been established, commencing with preneoplastic glycogenot-ic liver lesions, often containing ground glass cells, and pro-gressing to glycogen-poor neoplasms via various intermediate stages, including glycogenotic/lipidotic clear cell foci, clear cell hepatocellular adenomas (CCHCA) rich in glycogen and/or fat, and CCHCC. A similar process seems to take place in humans, with clear cells frequently persisting in CCHCC and steatohepatitic HCC, which presumably represent intermedi-ate stages in the development rather than particular variants of HCC. During the progression of the preneoplastic lesions, the clear and ground glass cells transform into cells charac-teristic of common HCC. The sequential cellular changes are associated with metabolic aberrations, which start with an activation of the insulin signaling cascade resulting in pre-neoplastic hepatic glycogenosis. The molecular and metabolic changes underlying the glycogenosis/lipidosis are apparently responsible for the dramatic metabolic shift from gluconeo-genesis to the pentose phosphate pathway and Warburg-type glycolysis, which provide precursors and energy for an ever increasing cell proliferation during progression.     

Clear cell hepatocellular carcinoma arising 25 years after the successful treatment of an infantile hepatoblastoma

Primary liver tumors in children are rare with hepatoblastoma (HB) being the most common malignancy. Clear cell carcinoma, a variant of hepatocellular carcinoma (HCC), is another rare tumor of the liver that tends to affect adults. We describe the diagnosis and management of the only known documented case of a primary clear cell HCC arising twenty-five years after the patient was successfully treated with chemotherapy and surgical resection for a malignant HB as an infant. While some evidence has shown a genetic link between HB and various types of HCC, other research has shown distinct chromosomal alterations and molecular mechanisms unique to both. Further knowledge of liver tumorigenesis will help elucidate the complicated genetic, molecular, and environmental factors involved in the development of these two rare hepatic malignancies.     

Genomic alteration is not associated with fatty and clear cell change in hepatocellular carcinomas and its precursor nodular lesions in cirrhotic liver.

BACKGROUND: The aim of this study was to investigate whether fatty and clear cell areas in large regenerative nodules (LRN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC) show higher degree of genomic mutation compared to non-fatty/clear cell area in the same nodule or non-lesional tissue. METHODS: We examined 22 nodular lesions (9 HCC, 5 DN and 8 LRN) from seven cirrhotic livers removed at transplantation. Frozen sections were used for manual microdissection of areas with fatty/clear cell change. DNA from microdissected tissue was amplified using arbitrarily primed polymerase chain reaction (AP-PCR), and PCR products were run on polyacrilamide gel generating a "fingerprint" band pattern. Autoradiographs were analysed using Adobe Photoshop version 6.0. Fingerprints from lesional tissue were compared to reference tissue and the total number of bands in excess or defect was calculated and divided by the total number of bands identified, obtaining the genomic damage fraction (GDF). RESULTS: Increasing GDF average values were seen from cirrhotic liver (0.13+/-0.04), to LRN (0.16+/-0.1), DN (0.28+/-0.08) and HCC (0.30+/-0.07). A statistically significant difference in GDF values was documented between cirrhotic liver and DN (p=0.008) and HCC (p=0.005) and between HCC and LRN (p=0.02). No significant difference was documented between DN and HCC, and between LRN and cirrhotic liver. Eleven nodules containing fat/clear cell areas were compared to the other 11 nodules without fat/clear cell areas. The GDF was not different between the two groups: 0.29+/-0.11 versus 0.25+/-0.12; p=0.5. The average value of genomic damage fraction between fat/clear cell areas (0.29+/-0.11) and no fat/clear cell areas (0.25+/-0.1) within the same nodules were not significantly different (p=0.11). CONCLUSION: Fatty and clear cell change in nodular lesions in cirrhotic liver may be an epigenetic phenotypic modification caused by microenvironmental factors such as ischaemia rather than indicating areas of increased malignant potential per se.     

Telomere shortening and inactivation of cell cycle checkpoints characterize human hepatocarcinogenesis

Telomere shortening and inactivation of cell cycle checkpoints characterize carcinogenesis. Whether these molecular features coincide at specific stages of human hepatocarcinogenesis is unknown. The preneoplasia-carcinoma sequence of human HCC is not well defined. Small cell changes (SCC) and large cell changes (LCC) are potential precursor lesions. We analyzed hepatocellular telomere length, the prevalence of DNA damage, and the expression of p21 and p16 in biopsy specimens of patients with chronic liver disease (n = 27) that showed different precursor lesions and/or HCC: liver cirrhosis (n = 25), LCC (n = 26), SCC (n = 13), and HCC (n = 13). The study shows a decrease in telomere length in nondysplastic cirrhotic liver compared with normal liver and a further significant shortening of telomeres in LCC, SCC, and HCC. HCC had the shortest telomeres, followed by SCC and LCC. Hepatocytes showed an increased p21 labeling index (p21-LI) at the cirrhosis stage, which remained elevated in most LCC. In contrast, most SCC and HCC showed a strongly reduced p21-LI. Similarly, p16 was strongly expressed in LCC but reduced in SCC and not detectable in HCC. gammaH2AX-DNA-damage-foci were not detected in LCC but were present in SCC and more frequently in HCC. These data indicate that LCC and SCC represent clonal expansions of hepatocytes with shortened telomeres. CONCLUSION: The inactivation of cell cycle checkpoints coincides with further telomere shortening and an accumulation of DNA damage in SCC and HCC, suggesting that SCC represent more advanced precursor lesions compared with LCC.     

A rare case of clear cell adenocarcinoma of the bladder with unique pathological features

Clear cell adenocarcinomas of the urinary bladder are rare tumors with an unknown histogenesis. Since these tumors appear histologically similar to clear cell tumors of the female genital tract, a mullerian histogenesis has been proposed. Several publications have examined the immunohistochemical properties of clear cell adenocarcinomas to improve understanding of the cause and pathogenesis of this tumor. While specific criteria for a diagnosis of clear cell adenocarcinoma have not been defined, there are consistent staining patterns suggested for characterization. We present an important case of clear cell adenocarcinoma of the bladder with a unique staining pattern. We review the literature and discuss the differential diagnosis and various theories concerning the origin of this rare tumor.     

Metastatic clear cell variant of hepatocellular carcinoma with an occult hepatic primary

Metastatic clear cell carcinomas are commonly seen in the kidney and lung. Clear cell variant of hepatocellular carcinoma is an uncommon tumour. Diagnosis is usually made by correlation of histopathology, tumour markers and immunohistochemistry, especially HepPar 1. In this unusual case of metastatic clear cell carcinoma presenting as Sister Mary Joseph's nodule, no primary evidence was observed radiologically in the liver, but the level of alfa fetoprotein was markedly elevated. Metastatic clear cell carcinoma of the liver with an occult hepatic primary was diagnosed by immunohistochemical profile of the tumour.     

不同转移潜能人肝癌细胞系转录因子活性差异分析

目的分析不同转移潜能人肝癌细胞系细胞核内转录因子活性的差异，筛选与肝癌转移相关的转录因子。方法应用转录因子活性芯片技术，在功能水平分析三种不同转移潜能人肝癌细胞系（Hep3B、MHCC97L和MHCC97H）细胞核内转录因子活性的差异，并用电泳迁移率变动分析和蛋白免疫印迹实验验证芯片结果。结果在345个候选的转录因子中，筛选出7个活性差异转录因子。随人肝癌细胞转移潜能的增高（Hep3B〈MHCC97L〈MHCC97H），活性上调的转录因子有5个，包括p53、缺氧诱导因子-1α（HIF-1α）、核因子κb、信号传导及转录活化因子3（Stat3）和Sp1；活性下调的转录因子有2个，包括Rb和Smad3。结论转录因子活性异常与肝癌转移密切相关，本实验筛选出的转录因子可能有助于揭示肝癌转移的分子机制，并寻找新的预测指标及干预治疗的靶点。     

Down-regulation of PTEN expression due to loss of promoter activity in human hepatocellular carcinoma cell lines

AIM: To investigate the regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression in human hepatocellular carcinoma (HCC) cell lines. METHODS: The mRNA and protein levels of PTEN were detected by Northern blot and Western blot in HCC cell lines, respectively. Plasmids containing different fragments of PTEN promoter with Luciferase reporter were constructed and transiently transfected into HCC cell lines to study the promoter activity. DNA analysis and RT-PCR were performed to detect the mutation of PTEN promoter and PTEN cDNA. RESULTS: Either protein or mRNA levels of PTEN in L02 cells (as a control) were significantly higher than that in HCC cell lines. The profile of PTEN promoter activity in 8 cell lines was closely correlated with levels of PTEN mRNA and PTEN protein. Furthermore, the sequence analysis of 8 cells lines showed no mutation in the region of PTEN promoter and PTEN cDNA. CONCLUSION: PTEN expression is down-regulated in HCC cell lines probably due to loss of activity of PTEN promoter.     

Pathomorphologic study on early hepatocellular carcinoma (HCC)--a study of cell density in well-differentiated HCC of the early stage

In order to clarify the histologic characteristics of well-differentiated hepatocellular carcinoma (HCC), a comparative morphometric study on cell density was performed in 15 HCCs smaller than 2 cm in diameter, 6 HCCs with marked fatty and/or clear cell change, 7 hyperplastic nodules, 5 hyperplastic nodules containing foci of HCC, and non-cancerous areas of the livers bearing small HCC. In well-differentiated HCC, marked increase of cell density accompanying by decrease of cell size and increase of nuclear cytoplasm ratio were prominent, and the cell density was approximately two times larger than that of the non-cancerous area in most cases. In HCCs with marked fatty and/or clear cell change, as an increase of cell density was not evident because of swelling of the cytoplasm due to fat and/or glycogen accumulation, it should be careful to differentiate them from non-cancerous nodular lesions including hyperplastic nodule with marked fatty change. Hyperplastic nodules could be divided into two groups; those with marked increase of cell density, and those without increase of cell density. In the former group, 5 of the 7 nodules contained cancerous foci.     

Proliferating cell nuclear antigen and Ki-67 labeling in hepatocellular nodules: a comparative study

Abstract: Aims/Background: The morphologic differential diagnosis of hepatocellular nodules (HCN) is frequently difficult and objective criteria would be useful in the categorization of such lesions. This study evaluated the proliferative activity of HCN, including regenerative, macroregenerative (MRN), cirrhotic, dysplastic, and hepatocellular carcinoma (HCC), as well as intranodular cytologic changes such as bile-stained hepatocytes, eosinophilia, clear, large cell (LCC) and small cell (SCC) change, by comparing the cellular density (CD), labeling indices (LI) and density (DP) of two proliferation markers. Methods: Routinely processed tissue sections from 45 HCN from 17 adult liver explants were studied by immunohistochemistry for PCNA and Ki-67 (MIB-1). Results: A progressive increase in LI from regenerative to dysplastic nodules to HCC was observed with both proliferation markers. The values of the two markers were significantly correlated (p < 0.001). CD, PCNA and MIB-1 LI and DP values were significantly lower in regenerative compared to dysplastic nodules or HCC. MRNs had lower PCNA and MIB-1 LI and DP than regenerative nodules, but similar CD. There were no statistically significant differences in CD, PCNA, and MIB-1 LI and DP between dysplastic nodules and HCC, comparing high versus low grade dysplasia, or HCC smaller than 2 cm with those larger than 2 cm. The CD and proliferation indices LI and DP were higher in HCC than in the surrounding non-neoplastic parenchyma. Lesions with clear cell, eosinophilic and large cell change had CD, PCNA and MIB-1 indices similar to those of regenerative nodules, while these were lower in bile-stained hepatocellular lesions (p < 0.01). SCC showed CD, PCNA and MIB-1LI and DP similar to HCC and higher than surrounding regenerative lesions (p < 0.003). Conclusions: Our results suggest that PCNA and MIB-1 values are closely correlated in HCN. Regenerative nodules are characterized by low cellular proliferation, while dysplastic nodules are usually highly proliferative lesions and may represent an early stage in hepatocarcinogenesis. Hepatocellular lesions characterized by bile stained hepatocytes, eosinophilic, clear and large cell change have low proliferation rates and may not be significant for the development of malignancy.     

肝癌细胞对5-氮杂-2'-脱氧胞苷的敏感性与细胞总DNA甲基化水平无关

目的 比较不同肝癌细胞株对5-氮杂-2'-脱氧胞苷(5-aza-dC)的敏感性,探讨肝癌细胞对5-aza-dC的敏感性是否与细胞总DNA甲基化水平有关.方法 用不同剂量(0.5、5.0、10.0μmol/L)的5-aza-dC处理肝癌细胞株(HepG2、QGY7701和HepG2.2.15细胞)及正常肝细胞株L02,比较不同浓度处理前后的细胞增殖抑制率,比较10 μmol/L 5-aza-dC处理前后的Caspase-3活性及细胞DNA片段化水平(5-溴脱氧尿嘧啶核苷掺入率),比较不同细胞总DNA甲基化水平.组间检测结果比较采用t检验.结果 5-aza-dC对HepG2、QGY7701、HepG2.2.15、L02细胞的半数抑制浓度分别为0.5、0.5、4.5、11.4μmol/L,与HepG2细胞和QGY7701细胞相比,HepG2.2.15绌胞和L02细胞对5-aza-dC不敏感.HepG2和QGY7701细胞中Caspase-3的活性升高较L02和HepG2.2.15细胞明显(P值均＜0.05),QGY7701细胞中5-溴脱氧尿嘧啶核苷掺入率升高较L02细胞明显(P＜0.05).L02、HepG2、QGY7701和HepG 2.2.15细胞的DNA总甲基化水平分别为11.7%±0.9%、10.9%±1.3%、11.7%±1.7%和12.2%±1.0%,差异无统计学意义(P值均＞0.05).结论 细胞对5-aza-dC的敏感性与细胞总DNA甲基化水平无关.     

DNA content of hepatocytes in various stages of liver cirrhosis

In order to search for some parameters that would make it possible to predict a potentially high risk of evolution from liver cirrhosis to hepatocellular carcinoma (HCC), the DNA content of hepatocytes in patients with liver cirrhosis with or without development to HCC was investigated by means of microspectrophotometry in Feulgen-stained specimens. In patients without development of HCC in more than 5 years of follow-up, 90% or more of tested hepatocytes were diploid, whereas in patients with liver cirrhosis who developed HCC within 3 years the frequency histogram of nuclear DNA content was widely spread from diploid to hyperpolyploid with a small peak of triploid. Furthermore, in the latter group of patients, many of the binucleate cells had different DNA contents in each paired nucleus. In non-cancerous portions of HCC, the ploidy histogram of nuclear DNA content was widely spread from diploid to polyploid with different DNA contents of each paired nucleus in binucleate cells, without the small peak of triploid. These results suggest that the deranged cell kinetics of hepatocytes in cirrhosis may be considered to be a state of potentially high risk for the evolution of HCC, and that a patient with liver cirrhosis with such an abnormal hepatocyte DNA content should be followed up carefully for the early diagnosis of HCC.     

DNA损伤与肝癌发生

肝癌是最常见的恶性肿瘤之一,其发生是一个复杂的生物学过程,具体机制尚未完全阐明.研究表明,乙、丙型肝炎病毒感染,黄曲霉毒素污染,饮酒,电离辐射以及具有有遗传毒性的人体代谢产物等能够诱导肝癌的发生,他们大都直接作用于肝细胞的遗传物质,引起DNA的损伤,这是发生肝癌的重要分子基础.DNA损伤后引起细胞一系列的反应,包括损伤信号的传导,损伤修复,诱导细胞死亡.这些诱因也能作用于损伤修复系统中的某个环节,使DNA损伤不能修复或不能正确修复,细胞发生恶性转化.因此,损伤DNA的累积就成为肝癌发生的重要分子机制,对其深入研究将会为肝癌的治疗奠定基础.     

Immunohistochemical detection of proliferating cell nuclear antigen in hepatocellular carcinoma: Relationship to histological grade

Proliferating cell nuclear antigen (PCNA), also known as cyclin, is an auxiliary protein of DNA polymerase-δ and is found only in the nuclei of proliferating cells in the late G₁ and S phases. The proliferation of hepatocellular carcinoma (HCC) by immunohistochemical staining for PCNA using paraffin sections of 20 surgically resected HCC specimens was analysed. The mean percentage of PCNA-positive nuclei in the HCC tissue was 10.3% in grade I of Edmondson and Steiner's classification, 25.5% in grade II, 28.4% in grade III and 41.5% in grade IV. In early HCC, we observed only a few PCNA-positive tumour cells. However, PCNA-positive nuclei were numerous in the tumour thrombi found in portal vein branches, in regions of extracapsular tumour growth, and in the inner nodules of tumours with a nodule-in-nodule formation. Proliferating cell nuclear antigen positivity was correlated with an increase of the nucleocytoplasmic ratio of tumour cells as determined by image analysis. Our findings showed that PCNA positivity was correlated with the histological grade and invasiveness of HCC, suggesting that this antigen may be used as an indicator to predict tumour invasion in patients with HCC.     

Clinicopathological and prognostic features of primary clear cell carcinoma of the liver.

Aim: Primary clear cell carcinoma of the liver (PCCCL) is a subgroup of primary hepatocellular carcinoma (HCC), pathologically characterized by diffuse clear cells of the tumor, showing a clear cytoplasm that does not stain with hematoxylin-eosin. At present, its clinicopathological and prognostic features are not fully clarified. This study aims to clarify the clinicopathological and prognostic features of PCCCL. Methods: The clinicopathological data of 43 patients with PCCCL treated with hepatectomy in our hospital from January 1999 to December 2003 were retrospectively analyzed. Results: The chi(2)-test showed a positive rate of hepatitis C virus (HCV) infection and capsule formation in the PCCCL group, which was markedly higher than in the common type HCC (CHCC) group (P = 0.000 and P = 0.005). Meanwhile, the vascular invasion rate was notably lower in the PCCCL group, but there were no significant differences between the twogroups (P = 0.129). The Kaplan-Meier method showed that the 1-, 3-, and 5-year survival rates were significantly higher in the PCCCL group than in the CHCC group (P = 0.021). The prognosis of patients in the PCCCL group was related to capsule formation, vascular invasion, liver cirrhosis, and clear cell ratio. The 1-, 3-, and 5-year survival rates were markedly higher in the group with a higher clear cell ratio than in the group with a lower clear cell ratio (P = 0.011). Conclusion: The notable clinicopathological features of the patients in the PCCCL group included a higher rate of HCV infection, capsule formation, and a lower rate of vascular invasion. The prognosis was better than that of the patients in the CHCC group and related to the ratio of clear cells.     

DNA methylation,microRNAs,and their crosstalk as potential biomarkers in hepatocellular carcinoma

Epigenetic alterations have been identified as a major characteristic in human cancers.Advances in the field of epigenetics have contributed significantly in refining our knowledge of molecular mechanisms underlying malignant transformation.DNA methylation and microRNA expression are epigenetic mechanisms that are widely altered in human cancers including hepatocellular carcinoma(HCC),the third leading cause of cancer related mortality worldwide.Both DNA methylation and microRNA expression patterns are regulated in developmental stage specific-,cell type specific-and tissue-specific manner.The aberrations are inferred in the maintenance of cancer stem cells and in clonal cell evolution during carcinogenesis.The availability of genome-wide technologies for DNA methylation and microRNA profiling has revolutionized the field of epigenetics and led to the discovery of a number of epigenetically silenced microRNAs in cancerous cells and primary tissues.Dysregulation of these microRNAs affects several key signalling pathways in hepatocarcinogenesis suggesting that modulation of DNA methylation and/or microRNA expression can serve as new therapeutic targets for HCC.Accumulative evidence shows that aberrant DNA methylation of certain microRNA genes is an event specifically found in HCC which correlates with unfavorable outcomes.Therefore,it can potentially serve as a biomarker for detection as well as for prognosis,monitoring and predicting therapeutic responses in HCC.     

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

AIM To screen clinically relevant micro RNAs (mi RNAs) silenced by DNA methylation in human hepatocellular carcinoma(HCC).METHODS Knockdown of DNA methyltransferases (DNMTs) using si RNAs and mi RNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated mi RNA downregulation. Confirmation using individual quantitative real-time PCR (qR T-PCR) assays was thenperformed followed by DNA methylation quantification at the promoter of the mi RNA genes. Quantification of DNA methylation and mi RNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTS mi RNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of mi R-23, mi R-25 and mi R-183. After q RT-PCR confirmation and Cp G island methylation quantification of these miR NAs in cell lines, further analysis in primary HCC specimens showed that hsa-mi R-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of mi R-183. In HCC patients, hypermethylation at hsami R-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSION Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.