Hepatoblastoma Tumors Research Articles

Effect of neoadjuvant chemotherapy on resectability of stage III and IV hepatoblastoma.

The potential for surgical resection of primary hepatoblastoma tumours was assessed at diagnosis, and after two and four cycles of neoadjuvant chemotherapy. Available radiographic images for patients with stage III and IV hepatoblastoma diagnosed between 1991 and 2008 were reviewed. The extent of disease was determined at diagnosis using the PRETEXT staging system, and after two and four cycles of therapy by POST-TEXT staging. Tumour resectability based on radiographic studies was assessed independently by two surgeons with expertise in hepatic surgery who were blinded to treatment and clinical outcome. Radiographic images from 20 patients with hepatoblastoma were reviewed. Six of 20 tumours were downstaged after two cycles, and three additional tumours were downstaged following four cycles. All PRETEXT stage III and IV tumours were determined to be surgically unresectable at diagnosis. The number of tumours considered unresectable decreased from 16 of 20 at diagnosis to seven of 20 after two cycles, and to four of 20 after four cycles. Five of the seven tumours that were unresectable after two cycles, and all four tumours that were unresectable after four cycles would have qualified for liver transplant based on radiographic studies. The majority of stage III and IV hepatoblastomas achieved radiographic resectability after two cycles of chemotherapy. There may be an opportunity for earlier surgical intervention and potential for a reduction in chemotherapy in a considerable number of patients.

Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex.

Hepatoblastoma (HB) is the most common primary liver tumor in children. Mutations in the β-catenin gene that lead to constitutive activation of the Wnt pathway have been detected in a large proportion of HB tumors. To identify novel mutations in HB, we performed whole-exome sequencing of six paired HB tumors and their corresponding lymphocytes. This identified 24 somatic nonsynonymous mutations in 21 genes, many of which were novel, including three novel mutations targeting the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) genes in the Wnt pathway, and genes previously shown to be involved in the ubiquitin ligase complex (SPOP, KLHL22, TRPC4AP, and RNF169). Functionally, both the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) were observed to be gain-of-functional mutations, and the CAPRIN2 (R968H/S969C) was also shown to activate the Wnt pathway in HB cells. These findings suggested the activation of the Wnt pathway in HB, which was confirmed by immunohistochemical staining of the β-catenin in 42 HB tumors. We further used short hairpin RNA (shRNA)-mediated interference to assess the effect of 21 mutated genes on HB cell survival. The results suggested that one novel oncogene (CAPRIN2) and three tumor suppressors (SPOP, OR5I1, and CDC20B) influence HB cell growth. Moreover, we found that SPOP S119N is a loss-of-function mutation in HB cells. We finally demonstrated that one of the mechanisms by which SPOP inhibits HB cell proliferation is through regulating CDKN2B expression. These results extend the landscape of genetic alterations in HB and highlight the dysregulation of Wnt and ubiquitin pathways in HB tumorigenesis.

Influence and clinical significance of chemotherapy on angiogenesis in pediatric difficult removal solid tumor

Objective To explore the influence and clinical significance of chemotherapy on the expressions of micro-vessel density (MVD) and vascular endothelial growth factor (VEGF) in pediatric difficult removal solid tumor. Methods From Jan.2002 to Jan.2012, totally 80 cases of the hospitalized pediatric patients with solid tumor (Ⅲb-Ⅳb stage) were enrolled in this study in Department of Pediatric Surgery of the Second Hospital of Hebei Medical University.All of the tumors were identified as difficult removal malignant tumor by imageology and physical examinations.After biopsy of the tumors, these patients were divided into neuroblastoma group (25 cases), hepatoblastoma group (25 cases) and Wilms' tumor group (30 cases). All patients in these 3 groups underwent chemotherapy respectively according to their different pathologic findings, thereafter, the tumor resections were performed.The resection rates of tumor in each groups were recorded and the expressions of MVD and VEGF between preoperative biopsy and postoperative specimens were detected and compared by immunohistochemistry in different groups. Results With chemotherapy, the resection rate of neuroblastoma was 80.00%(20/25 cases), hepatoblastoma was 92.00%(23/25 cases)and Wilms' tumor was 96.67%(29/30 cases). There was no significant difference among them (χ2=4.286, P=0.117). Before chemotherapy, the positive expressions of VEGF in neuroblastoma, hepatoblastoma and Wilms' tumor were 80.00%(20/25 cases), 90.00%(18/20 cases) and 83.33%(25/30 cases) respectively, at the same time, the values of MVD were 48.39±15.87, 52.16±16.12 and 46.82±19.07.After chemotherapy, the expressions of VEGF and MVD in neuroblastoma, hepatoblastoma and Wilms' tumor were 52.00%(13/25 cases), 45.00%(9/20 cases), 30.00% (9/30 cases)and 31.31±14.21, 25.19±15.31 and 29.06±13.05.Compared with the expressions of them in each group before chemotherapy, significant differences were found(all P<0.05). Conclusions Chemotherapy can efficiently promote the resection rate for pediatric difficult removal solid tumor.MVD and VEGF can be considered as the markers in evaluating the outcomes of chemotherapy and promoting the resection rate of tumors. Key words: Solid tumor; Chemotherapy; Micro-vessel density; Vascular endothelial growth factor; Child

Hepatoblastoma in a 15‐month‐old female with trisomy 13

Trisomy 13 (T13) is a rare autosomal aneuploidy. Greater than 90% of patients die during the first year of life. Malignancies reported in association with T13 include two cases of Wilms tumor and one case of pilocytic astrocytoma. There is no previous report of hepatoblastoma in patients with T13. We report a unique case of hepatoblastoma in a 15-month-old female with constitutional T13. Our patient was born at 38 weeks gestation and was noted to have phenotypic features consistent with T13. Genetic testing confirmed an abnormal karyotype of 47,XX,+13 in all cells. At fifteen months of age she was noted to have a right hepatic lobe mass on a routine follow-up renal ultrasound for hydronephrosis. Serum alpha-fetoprotein level was 55,300 ng/ml. Staging work-up revealed the absence of metastases. She underwent a complete surgical resection with right hepatic lobectomy. Histopathology was consistent with hepatoblastoma, mixed epithelial and mesenchymal type. She had a protracted postoperative course complicated by Enterobacter aerogenes urosepsis, a significant biloma, chronic pancreatitis, and apneic episodes of uncertain etiology. She received four courses of doxorubicin monotherapy without any severe or unexpected toxicity. She continues to be in remission 8 months following diagnosis. This is the first reported case of hepatoblastoma in a child with constitutional T13. This may represent a non-random association, as somatic trisomy of chromosome 13 in hepatoblastoma tumors has been previously described in the literature. Prolonged survival may have allowed for hepatoblastoma to present in our patient.

RASSF1A methylation indicates a poor prognosis in hepatoblastoma patients

The RAS association domain family protein 1 (RASSF1A) is known to be frequently inactivated by promoter hypermethylation in cancers. This study investigated the association of RASSF1A methylation with clinical outcomes in hepatoblastoma patients and whether it is correlated with the histological phenotype of hepatoblastoma tumors. Seventy-four hepatoblastoma tumors were obtained from patients enrolled in the Japanese study group for pediatric liver tumor protocol-2. From nine formalin-fixed, paraffin-embedded specimens, we extracted DNA by dissection under a light microscope. We examined the methylation status of the RASSF1A promoter region by bisulfite pyrosequencing. Twenty-five (33.8 %) hepatoblastoma tumors were classified as having methylated RASSF1A. The RASSF1A methylation was significantly associated with metastatic tumors and a poor prognosis. Despite the complete resection, five pretreatment extent of disease II tumors showed recurrence or distant metastasis postoperatively. Among these cases, four tumors were found to show RASSF1A methylation. When compared to histologically different types of cell, RASSF1A methylation values in samples of the normal liver, fetal type, and embryonal type, were significantly elevated in ascending order. We confirmed that RASSF1A methylation is a significant prognostic indicator in hepatoblastomas, and it may become a promising molecular marker to stratify patients into appropriate risk groups.

Socioeconomic status and incidence of pediatric embryonal tumors in the United States.

10036 Background: Of the 13,000 children diagnosed with cancer each year in the United States (US), the embryonal solid tumors, neuroblastoma (NB), retinoblastoma (RB), Wilms tumors (WT), hepatoblastoma (HB), rhabdomyosarcomas (RMS) and germ cell tumors (GCT), account for over 30% of the cases. Social disparities in cancer are well studied for adults, but few studies have focused on children, mostly for leukemia. The aim of this study is to evaluate the differences in incidence of rare cancers according to socioeconomic status (SES). Methods: Cases aged 0-19 were identified from the Surveillance Epidemiology and End Results (SEER) cancer registries from 1992-2009. Using data from the US 2000 Census, the county of residence of the cases was categorized above or below the national average for SES measures including: % persons with&lt; high school education, % persons below poverty, % persons unemployed and % households with &gt; 1 person/room. Age standardized rates per million (ASR), rate ratios (RR) and 95% confidence intervals (CI) were obtained. The findings were validated using cases from the National Program of Cancer Registries (NPCR) from 1999-2009, analyzed with the same SES variables. Results: Among cases identified in SEER, rates of NB and WT are higher in counties with upper SES measures whereas RB and GCT occurred more frequently in counties with lower SES measures. No association was found between SES and rates of HB and RMS. The results were reproducible with NPCR cases. For instance, ASR of NB is lower (SEER: 5.86; NPCR: 7.48) in counties where &gt;19.6% of the population had not completed high school and higher (SEER: 8.41; NPCR: 8.47) in counties where ≤19.6% had not achieved a high school degree. (SEER: RR=0.69; 95%CI=0.62-0.77; NPCR: RR=0.88; 95%CI=0.84-0.93). Analysis of NB rates according to poverty, unemployment and crowding showed consistent results, with higher rates in counties with higher SES. Conclusions: The findings are suggestive of a relation between SES and cancer susceptibility that may be connected to environment and lifestyle. Understanding the role of contributing causes demands further studies to evaluate why cancer rates vary across cultural and ethnic groups as well as the magnitude of specific SE aspects.

FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo

Hepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas.

Targeting EpCAM (CD326) for immunotherapy in hepatoblastoma

Hepatoblastoma (HB) is the most common liver cancer in children. Recurrence of HB after chemotherapy and surgery is frequent among high-risk patients and is associated with chemoresistance. Immunotherapy may improve poor treatment outcomes in HB patients. Cytotoxic leukocytes of the innate and adaptive immune system including different populations of cytotoxic T cells play a major role in fighting developing tumors. In this setting, monoclonal antibodies may be employed to specifically direct immune responses toward tumor cells. We addressed this issue by using humanized antibodies that recognize the cell surface molecule EpCAM (CD326, overexpressed in hepatic tumor cells) to enhance immune responses against HB. EpCAM was constantly expressed on HB cells and its expression was independent of previous therapy based on the DNA-damaging agent cisplatin. Co-culture assays performed with two well-described HB cell lines and tumor tissue cultures demonstrated that tumor cell lysis by γδ T cells can be dramatically augmented by applying EpCAM-specific monoclonal antibodies. These data emphasize the value of antitumor immune responses and encourage adapting immunotherapeutic regimens to improve the outcome of high risk HB.

Evaluating RASSF1 promoter methylation and the 16-gene signature as a prognostic marker in hepatoblastoma.

63 Background: Hepatoblastoma (HB) is the most common malignant liver tumor in childhood. Although combination of neoadjuvant chemotherapy and surgery has improved the outcome of standard-risk HB-patients up to 94% the outcome of high-risk HB-patients with metastatic tumors or invasion of large hepatic veins is still dismal and only 60% of these patients survive. Therefore, the identification of biomarkers that could help early detection of high-risk HB-patients and lead to risk-adapted therapies is of utmost importance. Methods: Genomic DNA of tumor and normal liver samples from 21 HB patients was extracted with standard procedures and bisulfite-treated using EpiTect Bisulfite Kit (Qiagen). Methylation status of the Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) gene was analyzed by methylation-specific-PCR. cDNA of tumor and normal liver samples was used to determine the 16-gene expression signature according to Cairo et al. (2008, Cancer Cell) using real-time PCR. Association studies of molecular and clinical data were performed with GraphPad Prism Version 3.0 using two-tailed Chi square test. Results: We identified 9/21 HB cases hypermethylated in the RASSF1 promoter region. Interestingly, RASSF1 promoter methylation was significantly associated with metastases (p=0.0166) and occurrence of this epigenetic marker was associated with a reduced overall survival of patients (p=0.0008). Moreover, we discriminated HB tumors in C1 and C2 subclasses by using the 16-gene signature and correlation of these subtypes with the methylation status of RASSF1 showed a significant association (p=0.0037) for aggressive C2 tumors (8/11), but not C1 tumors (1/10). Conclusions: RASSF1 promoter hypermethylation is a strong prognostic marker for metastasis and worse outcome and correlates with an aggressive C2 HB-tumor profile. Since the detection of tumor-derived methylated DNA in the blood stream of cancer patients is nowadays practical, our data advise RASSF1 methylation assays to be used for risk assessment in HB-patients in the future.

Abstract 4337: Effect of sorafenib in combination with cytostatic agents on hepatoblastoma in vitro and in vivo

Abstract Background: Hepatoblastoma (HB) represents the most common malignant liver tumour in childhood. For patients with high risk or recurrent HB treatment results remain still poor due to a developing multidrug resistance during chemotherapy. Therefore new therapy strategies have to be evaluated. The primary objective was to investigate the combined therapy of cytotoxic agents with sorafenib. Sorafenib has been shown to reduce tumour progression and angiogenesis. In order to investigate a treatment close to a clinical setting the effect of the combined administration of cytotoxic agents with sorafenib on HB cell lines and xenotransplanted HB tumours was determined. Methods: Cell viability of two HB cell lines (HUH6 and HepT1) was evaluated in using MTT assays after treatment with sorafenib and different cytostatic agents (cisplatin (CDDP), doxorubicin, irinotecan, topotecan). ERK signalling was investigated by Western blot analysis. NMRI mice (nu/nu) bearing subcutaneous HUH6 derived tumours were treated with Sorafenib orally (30 mg/kg, on days 4-13 and 17-24) alone or in combination with CDDP i.p. (3 mg/kg, on days 1-3 and 14-16). Tumor progression and viability were monitored by tumour volume and AFP levels. Apoptosis was assessed by TUNEL assay. Angiogenesis was determined by CD31 staining and mean vascular density (MVD) was calculated. Results: In vitro the combination of sorafenib with CDDP showed a remarkable decrease in cell viability compared to the other agents tested. However with increasing concentrations of CDDP this additive effect disappeared. CDDP alone showed an enhanced phosphorylation of ERK1/2, which is a common pathway for apoptosis induction through NOXA. CDDP in combination with sorafenib led to an overall reduced phosphorylation level of ERK1/2, which also resulted in a low HB cell viability. Since CDDP and sorafenib have an opposing effect on phosphorylation of ERK1/2, we avoid the concomitant administration of both drugs in vivo. In HB xenografts both sorafenib alone or in alternating combination with CDDP reduced tumour growth compared to the control untreated group (p &amp;lt; 0.05). The combination treatment showed a higher and earlier reduction of tumor volume than sorafenib alone. In addition AFP levels did not significantly change in the treatment groups. Apoptotic areas as determined by TUNEL analysis increased significantly after administration of sorafenib or sorafenib and CDDP (p &amp;lt; 0.08). Furthermore CD31 staining showed a significant inhibition of angiogenesis as revealed by determination of the MVD in both treatment groups (p &amp;lt; 0.02). Discussion: Taken together the combination therapy of Sorafenib and CDDP led to an enhanced decrease in cell viability as well as in tumour progression and angiogenesis than sorafenib alone. Therefore a combined therapy might be a promising option in the treatment of high risk or recurrent hepatoblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4337. doi:1538-7445.AM2012-4337

Abstract 1051: UHRF1 is highly expressed in childhood liver tumors and interacts with DNMT1 and USP7 to guide tumor suppressor gene silencing

Abstract Embryonal tumors are thought to arise from immature tissue during intrauterine or early postnatal development and display apart from specific genetic changes in developmental signaling pathways a relatively normal genomic background. Instead, epigenetic events appear to be involved in key steps of the earliest phases of neoplastic evolution. However, the mechanistic basis responsible for epigenetic alterations in these early-onset tumors and the role of the DNA methylation machinery therein is still not known. In order to define the impact of epigenetic factors in embryonal tumor development we made use of hepatoblastoma (HB), the most common liver tumor of early childhood, as a model for a pristine and genetically “clean” tumor. In this study we identified ubiquitin-like with PHD and ring finger domains 1 (UHRF1) to be highly overexpressed in HB tumors compared to normal liver tissue, a protein known to preferentially bind to hemi-methylated DNA. Using immunoprecipitation, we furthermore show that UHRF1 binds in concert with DNA methyltransferase 1 (DNMT1) and ubiquitin specific peptidase 7 (USP7) as a trimeric complex to promoter regions of tumor suppressor genes relevant in HB, such as hedgehog interacting protein (HHIP), insulin-like growth factor binding protein 3 (IGFBP3), and secreted frizzled-related protein 1 (SFRP1). These genes are epigenetically silenced in HB, as evidenced by heavy DNA methylation and enrichment of the repressive H4K27me3 chromatin mark. Interestingly, knockdown of UHRF1 expression via RNA interference resulted in promoter demethylation and a shift towards the active H3K4me2 chromatin mark of the three tumor suppressor genes. Altogether, these data suggest that aberrant expression of the epigenetic regulator UHRF1 and excessive binding of a trimeric complex consisting of UHRF1/DNMT1/USP7 results in silencing of tumor suppressor genes in HB and may display an important mechanism in the initial phases of embryonal tumorigenesis in general. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1051. doi:1538-7445.AM2012-1051

Phenylarsonic acid compounds with broad-spectrum and potent cytotoxic activity against human cancer cells.

The in vitro cytotoxic activity profile of nine novel phenylarsonic acid (CAS 98-05-5, PAA) compounds against 17 human cancer cell lines including (a) ovarian cancer cell lines ES-2, PA-1, CAOV-3, OVCAR-3, (b) testicular cancer cell lines Ntera-2, Tera-2, N2NICP, 833K, and 64CP, (c) multiple myeloma cell lines ARH77, HS-Sultan, RPMI-8226, and U266, and (d) acute lymphoblastic leukemia (ALL) cell lines NALM-6, MOLT-3, ALL-1, and RS4; 11, was determined by the MTT assay. The lead compounds, 2-methylthio-4-[(4'-aminophenylazo)-phenylarsonic acid] pyrimidine (PHI-370) and 2-methylthio-4-(4'-phenylarsonic acid)-aminopyrimidine (PHI-380) caused apoptotic death in all 17 cancer cell lines at low micromolar concentrations, as documented by TUNEL assays and confocal laser scanning microscopy. PHI-380 was also tested and found to be very active against primary tumor cells isolated from surgical biopsy specimens of 14 patients with therapy-refractory non-small cell lung cancer, breast cancer, colon cancer, lymphoma, hepatoblastoma, or Wilm's tumor as well. Because of their broad-spectrum and potent anticancer activity and ability to induce apoptosis in primary tumor cells from therapy-refractory cancer patients, PAA compounds such as PHI-370 and PHI-380 may provide the basis for effective salvage regimens for patients with recurrent cancer.

Treatment effects of the multikinase inhibitor sorafenib on hepatoblastoma cell lines and xenografts in NMRI‐Foxn1nu mice

Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours. Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence. Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment. Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.

HGF/c-Met related activation of β-catenin in hepatoblastoma

BackgroundActivation of beta-catenin is a hallmark of hepatoblastoma (HB) and appears to play a crucial role in its pathogenesis. While aberrant accumulation of the beta-catenin is a common event in HB, mutations or deletions in CTNNB1 (beta-catenin gene) do not always account for the high frequency of protein expression. In this study we have investigated alternative activation of beta-catenin by HGF/c-Met signaling in a large cohort of 98 HB patients enrolled in the SIOPEL-3 clinical trial.MethodsWe performed immunohistochemistry, using antibodies to total beta-catenin and tyrosine654-phosphorylated beta-catenin, which is a good surrogate marker of HGF/c-Met activation. CTNNB1 mutation analysis was also carried out on all samples. We also investigated beta-catenin pathway activation in two liver cancer cell lines, HuH-6 and HuH-7.ResultsAberrant beta-catenin expression was seen in the cytoplasm and/or nucleus of 87% of tumour samples. Our results also revealed a large subset of HB, 83%, with cytoplasmic expression of tyrosine654-phosphorylated beta-catenin and 30% showing additional nuclear accumulation. Sequence analysis revealed mutations in 15% of our cohort. Statistical analysis showed an association between nuclear expression of c-Met-activated beta-catenin and wild type CTNNB1 (P-value = 0.015). Analysis of total beta-catenin and Y654-beta-catenin in response to HGF activation in the cell lines, mirrors that observed in our HB tumour cohort.ResultsWe identified a significant subset of hepatoblastoma patients for whom targeting of the c-Met pathway may be a treatment option and also demonstrate distinct mechanisms of beta-catenin activation in HB.

Abstract 5354: Characterisation of the cell line HC-AFW1 derived from a paediatric hepatocellular carcinoma

Abstract Paediatric hepatocellular carcinoma (HCC) has an incidence of 1.5 cases per million children and is linked to viral hepatitis in endemic regions. In Europe childhood HCC mostly arises in otherwise healthy liver and has an unknown ethiology. Current treatment results of HCC are dissatisfactory due to advanced disease at diagnosis and lacking response to chemotherapy. To improve the research of childhood HCC and to address specific therapeutic approaches a preclinical model for childhood HCC is needed. So far, there has been one existing cell line, HepG2, which has lately been discussed controversially as being hepatoblastoma. We established a cell line HC-AFW1 from the tumor of a 5 year old boy. The original tumor histology was characteristic as transitional liver cell tumor including hepatoblastoma and HCC morphology. These histological findings were confirmed through several reference pathology laboratories worldwide. The cell line HC-AFW1 was established by xenotransplantation of original tumor into immuno-deficient mice and re-culturing in vitro. The cell line is in sable culture now for over 8 months with a doubling time of 40 h. Cultured cells present epithelial characteristics and express hepatoma proteins like AFP, glypican 3, E-cadherin, CD10, CD326, HepPar1, Vimentin and Desmin. Catenin beta shows a deletion of 49 amino acids in the exon 3 involving the phosphorylation sites of GSK3 and was detected in cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. This involves chromosomal aberrations found in hepatoblastoma as well as in adult HCC. Several alterations of gene copy numbers were detected by genome wide SNP array. Among different tested drugs cisplatin and irinotecan showed an effective inhibition of tumor cell growth in an MTT assay at concentration below 5 µg/ml. Transplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumors with high levels of serum AFP. This xenotransplants as well as stable cultured cells solely consist of HCC components suggesting a selection of HCC and degeneration of formally hepatoblastoma tumor parts. Therefore, HC-AFW1 is the first paediatric HCC-derived cell line and represents a valuable tool to investigate the biology and therapeutic strategies in childhood HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5354. doi:10.1158/1538-7445.AM2011-5354

MicroRNA-492 Is Processed From the Keratin 19 Gene and Up-Regulated in Metastatic Hepatoblastoma σ

MicroRNAs (miRNAs) are well-known regulators of proliferation, apoptosis, and differentiation and are recognized to play an important role in the development of cancers. Here we aimed to identify the functional contribution of miRNAs to the biology of hepatoblastoma (HB), the most common malignant liver tumor in childhood. As overexpression of the oncogene PLAG1 (pleomorphic adenoma gene 1) is a characteristic phenomenon in HB, we used RNA interference and subsequent miRNA array analysis to identify miR-492 as most strongly influenced by PLAG1. We provide novel experimental evidence that miR-492 can originate from the coding sequence of the HB marker gene keratin 19 (KRT19). In agreement with these in vitro observations, significantly elevated levels of coexpressed KRT19 and miR-492 were particularly found in metastatic HB tumor samples. Stable overexpression of miR-492 in HB cell clones served to identify a broad range of differentially expressed transcripts, including several candidate targets of miR-492 predicted by computational algorithms. Among those the liver enzyme BAAT showed significant association with miR-492 expression in HB tumor samples. A close functional relationship between KRT19 and miR-492 was identified that may play an important role in the progression of malignant embryonal liver tumors. Additionally, miR-492 and its associated targets might serve as new HB biomarkers of clinical utility and could assist to explore targeted therapies, especially in metastatic HB with a poor prognosis.

Genome‐wide analysis of allelic imbalances reveals 4q deletions as a poor prognostic factor and MDM4 amplification at 1q32.1 in hepatoblastoma

In a single-nucleotide polymorphism array-based analysis of 56 hepatoblastoma (HB) tumors, allelic imbalances were detected in 37 tumors (66%). Chromosome gains were found in 1q (28 tumors), 2q (24), 6p (8), 8q (8), 17q (6), and 20pq (10), and losses in 1p (6), 4q (9), and 16q (4). Fine mapping delineated the shortest overlapping region (SOR) of gains at 1q32.1 (1.3 Mb) and 2q24.2-q24.3 (4.8 Mb), and losses at 4q34.3-q35.2 (8.7 Mb) and 4q32.3 (1.6 Mb). Uniparental disomy of 11pter-11p15.4 (IGF2) and loss of 11pter-p14.1 were found in 11 and 2 tumors, respectively. Expression of HTATIP2 (11p15.1) was absent in 9 of 20 tumors. Amplification was identified in four tumors at 1q32.1, where the candidate oncogene MDM4 is located. In the 4q32.3-SRO, ANXA10S, a variant of the candidate tumor suppressor ANXA10, showed no expression in 19 of 24 tumors. Sequence analysis of ANXA10S identified a missense mutation (E36K, c.106G>A) in a HB cell line. Multivariate analysis revealed that both 4q deletion and RASSF1A methylation (relative risks: 4.21 and 7.55, respectively) are independent prognostic factors. Our results indicate that allelic imbalances and gene expression patterns provide possible diagnostic and prognostic markers, as well as therapeutic targets in a subset of HB.

Developmental anomalies of permanent teeth identified in children who received chemotherapy: Report of three cases

Childhood cancer, such as that manifested by neuroblastoma and hepatoblastoma tumors, is one of the leading causes of death for young children. Chemotherapy is one of the most important approaches available, though it is considered to cause tooth developmental disturbances when patients receive treatment during that period of development. Microdontia, disturbed root development, hypodontia and enamel hypoplasia are reported to be possible late adverse effects of chemotherapy. We analyzed 3 children, for whom chemotherapy had been given for treatment of cancer from the ages of 1 to 2.5 years old. Intraoral examinations revealed microdont of the bilateral maxillary lateral incisors and canines in 1 case, whereas that of the bilateral mandibular first premolars was shown in another. Orthopantomograph examinations revealed congenital absence of the first and second premolars in all 3 cases. The developmental period of the affected teeth in these cases appeared to correspond to the chronological age when chemotherapy was administered.

Emerging gene-directed anti-tumor strategies against human hepatoblastoma

Background: Despite impressive improvements of treatment results in children suffering from hepatoblastoma (HB), advanced tumor stages still produce unsolved problems for treating physicians. HB tumors are commonly treated following trial regimens, which have been set up by multicenter study groups based on an ongoing treatment evaluation. Recently, treatment outcomes have reached a phase of stagnation and currently alternative treatment options are under investigation. Gene-directed anti-HB treatment approaches have been newly identified as promising option in this regard. Objective: To optimize and extend gene directed treatment strategies in order to improve treatment results of advanced stage HB. Methods: A review of the literature on genetic alterations in HB and of the current state of experimental gene-directed treatment approaches against HB. Results/conclusion: HB seems to be a suitable target for gene-directed treatment approaches. Further development of gene transfer systems and HB-specific strategies will lead to new alternatives for the treatment of this malignancy.

Establishment and characterization of a cancer cell line derived from an aggressive childhood liver tumor

Hepatoblastoma is a rare malignancy of childhood. The scarcity of adequate cell models has limited our understanding of this tumor. Here we describe and characterize a new human liver tumor cell line, Hep293TT, derived from an aggressive childhood hepatoblastoma. Hep293TT cells were established using primary tumor tissues from a 5-year-old Caucasian female child. This cell line has been maintained for more than 34 months and over 20 subcultures, and was characterized by histopathology, ELISA, genotype, cytogenetics, CGH array, immunohistochemistry, and molecular sequence analyses. Cells were confirmed to originate from parental tumor cells, secrete alpha-fetoprotein, and express hepatic markers and beta-catenin. Hep293TT cells were able to form colonies in soft agar. Tumorigenicity was demonstrated by induction of solid tumors after subrenal capsule injection in immunodeficient mice. Hep293TT cells demonstrated a highly aneuploid karyotype, and a whole genome CGH analysis revealed chromosomal imbalances in every chromosome. Allelotype analysis demonstrated loss of alleles at distal 11p15.5 as is typical of embryonal tumors. Both Hep293TT cells and the primary tumor contain a deletion of 351 nucleotides in beta-catenin, as has been seen in other hepatoblastoma tumors. The cell line expressed beta-catenin protein in both full-length and partially deleted forms, and expressed NOTCH2 protein characteristic of hepatoblasts. No mutation was detected in the APC, MYH, MLH1, or MSH2 genes. This cell line, Hep293TT, is a valuable resource for the study of childhood liver cancer and may potentially provide a tool in the development of new agents.