Computational drug prediction in hepatoblastoma by integrating pan-cancer transcriptomics with pharmacological response.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the predominant form of pediatric liver cancer, though it remains exceptionally rare. While treatment outcomes for children with HB have improved, patients with advanced tumors face limited therapeutic choices. Additionally, survivors often suffer from long-term adverse effects due to treatment, including ototoxicity, cardiotoxicity, delayed growth, and secondary tumors. Consequently, there is a pressing need to identify new and effective therapeutic strategies for patients with HB. Computational methods to predict drug sensitivity from a tumor's transcriptome have been successfully applied for some common adult malignancies, but specific efforts in pediatric cancers are lacking because of the paucity of data. APPROACH AND RESULTS: In this study, we used DrugSense to assess drug efficacy in patients with HB, particularly those with the aggressive C2 subtype associated with poor clinical outcomes. Our method relied on publicly available collections of pan-cancer transcriptional profiles and drug responses across 36 tumor types and 495 compounds. The drugs predicted to be most effective were experimentally validated using patient-derived xenograft models of HB grown in vitro and in vivo. We thus identified 2 cyclin-dependent kinase 9 inhibitors, alvocidib and dinaciclib as potent HB growth inhibitors for the high-risk C2 molecular subtype. We also found that in a cohort of 46 patients with HB, high cyclin-dependent kinase 9 tumor expression was significantly associated with poor prognosis. CONCLUSIONS: Our work proves the usefulness of computational methods trained on pan-cancer data sets to reposition drugs in rare pediatric cancers such as HB, and to help clinicians in choosing the best treatment options for their patients.

The landscape of genomic alterations across childhood cancers.

Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.

Author Correction: The landscape of genomic alterations across childhood cancers.

In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.

Sarcopenia is a prognostic outcome marker in children with high-risk hepatoblastoma.

BACKGROUND: Children with hepatoblastoma (HB) are at risk of sarcopenia due to immobility, chemotherapy, and malnutrition. We hypothesized that children with HB have a low preoperative total psoas muscle area (tPMA), reflecting sarcopenia, which negatively impacts outcome. PROCEDURE: Retrospective study of children (1-10 years) with hepatoblastoma treated at a large university children's hospital from 2009 to 2018. tPMA was measured as the sum of the right and left psoas muscle area (PMA) at intervertebral disc levels L3-4 and L4-5. z-Scores were calculated using age- and gender-specific reference values and were compared to anthropometric measurements, clinical variables, and outcomes. Sarcopenia was defined as a tPMA z-score below -2. RESULTS: Thirty-three children were included. Mean tPMA z-score was -2.18 ± 1.08, and 52% were sarcopenic. A poor correlation between tPMA and weight was seen (r = 0.35; confidence interval [CI] 0.01, 0.62; P = .045), and most children had weights within the normal range (mean z-score -0.55 ± 1.39). All children categorized as high risk with relapse (n = 5/12) were sarcopenic before surgery. Relapse was significantly higher in the high-risk sarcopenic group compared to the nonsarcopenic group (P = .008). The change in tPMA z-score 1-4 months after surgery did not improve in patients with relapse, but did improve in 75% of children without relapse. CONCLUSIONS: The majority of children with HB were sarcopenic prior to surgery. Especially in children with high-risk hepatoblastoma, sarcopenia is an additional risk factor for relapse. Large multicenter studies are needed to confirm these preliminary results.

Epigenetic footprint enables molecular risk stratification of hepatoblastoma with clinical implications.

BACKGROUND & AIMS: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. METHODS: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. RESULTS: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. CONCLUSIONS: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. LAY SUMMARY: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer.

MiR-492 regulates metastatic properties of hepatoblastoma via CD44.

BACKGROUND & AIMS: MicroRNAs are important genetic regulators of physiological and pathophysiological processes including cancer initiation and progression of hepatoblastoma, the most common liver tumour in childhood. We aimed to identify malignant and metastasis promoting effects of miR-492, a miRNA, previously reported to be overexpressed in metastatic hepatoblastoma. Furthermore, we intended to evaluate its diagnostic and prognostic potential. METHODS: Stable and transient overexpression of miR-492 in two liver tumour cell lines HepT1 and HUH7 was used to analyse features of metastatic tumour progression such as proliferation, anchorage-independent growth, migration and invasion. Via a mass spectrometry based proteomic screen, we investigated miRNA-492-dependent effects on proteome level and explored the underlying biology. One of the predicted target genes, CD44, was experimentally validated via luciferase assays. Diagnostic and prognostic properties of miR-492 were studied in hepatoblastoma tumour samples. RESULTS: We show that miR-492 significantly enhances cell proliferation, anchorage-independent growth, migration and invasion of hepatoblastoma cells. We also identified and validated CD44, a transmembrane adhesion receptor for hyaluronan, as direct and functional target of miR-492. This miRNA has a strong direct impact on two CD44 isoforms (standard and v10). High miR-492 expression correlates with high-risk or aggressive tumours and further bears potential for predicting reduced event-free survival. CONCLUSIONS: We identified miR-492 and its target CD44 as regulators of a number of biological features important for malignancy and metastasis. Furthermore, we demonstrated the diagnostic and prognostic potential of miR-492, a promising novel therapeutic target and biomarker for hepatoblastoma.

Patient-derived mouse xenografts from pediatric liver cancer predict tumor recurrence and advise clinical management.

UNLABELLED: Identification of new treatments for relapsing pediatric cancer is an unmet clinical need and a societal challenge. Liver cancer occurrence in infancy, 1.5 for million children per year, falls far below the threshold of interest for dedicated drug development programs, and this disease is so rare that it is very difficult to gather enough children into a phase II clinical trial. Here, we present the establishment of an unprecedented preclinical platform of 24 pediatric liver cancer patient-derived xenografts (PLC-PDXs) from 20 hepatoblastomas (HBs), 1 transitional liver cell tumor (TCLT), 1 hepatocellular carcinoma, and 2 malignant rhabdoid tumors. Cytogenetic array and mutational analysis of the parental tumors and the corresponding PLC-PDXs show high conservation of the molecular features of the parental tumors. The histology of PLC-PDXs is strikingly similar to that observed in primary tumors and recapitulates the heterogeneity of recurrent disease observed in the clinic. Tumor growth in the mouse is strongly associated with elevated circulating alpha-fetoprotein (AFP), low rate of necrosis/fibrosis after treatment, and gain of chromosome 20, all indicators of resistance to chemotherapy and poor outcome. Accordingly, the ability of a tumor to generate PLC-PDX is predictive of poor prognosis. Exposure of PLC-PDXs to standards of care or therapeutic options already in use for other pediatric malignancies revealed unique response profiles in these models. Among these, the irinotecan/temozolomide combination induced strong tumor regression in the TCLT and in a model derived from an AFP-negative relapsing HB. CONCLUSION: These results provide evidence that PLC-PDX preclinical platform can strongly contribute to accelerate the identification and diversification of anticancer treatment for aggressive subtypes of pediatric liver cancer. (Hepatology 2016;64:1121-1135).

Transcriptional activation of Hedgehog pathway components in aggressive haemangioma.

Infantile hemangioma is a vascular neoplasm and is one of the most common tumors diagnosed in young children. Although most hemangiomas are harmless and involute spontaneously, some show severe progression, leading to serious complications, such as high-output cardiac failure, ulcerations, compression of the trachea or deprivation amblyopia, depending on their size and localization. However, the pathogenesis and cause of hemangioma are largely unknown to date. The goal of this study was to identify markers that could predict hemangiomas with aggressive growth and severe progression that would benefit from early intervention. By using a PCR-based screening approach, we first confirmed that previously known markers of hemangioma, namely FGF2 and GLUT1, are highly expressed in hemangioma. Nevertheless, these genes did not show any differential expression between severely progressing tumors and mild tumors. However, transcriptional upregulation of several Hedgehog signalling components, comprising the ligand Sonic Hedgehog (SHH), the transcription factor GLI2 and its target gene FOXA2 were detected in extremely aggressive hemangioma specimens during the proliferation phase. Notably, GLI2 was even overexpressed in involuting hemangiomas if they showed an aggressive growth pattern. In conclusion, our data suggest that overexpression of the Hedgehog components SHH, GLI2 and FOXA2 might be used as markers of an aggressive hemangioma that would benefit from too early intervention, while FGF2 and GLUT1 are more general markers of hemangiomas.

Childhood cancer predisposition syndromes-A concise review and recommendations by the Cancer Predisposition Working Group of the Society for Pediatric Oncology and Hematology.

Heritable predisposition is an important cause of cancer in children and adolescents. Although a large number of cancer predisposition genes and their associated syndromes and malignancies have already been described, it appears likely that there are more pediatric cancer patients in whom heritable cancer predisposition syndromes have yet to be recognized. In a consensus meeting in the beginning of 2016, we convened experts in Human Genetics and Pediatric Hematology/Oncology to review the available data, to categorize the large amount of information, and to develop recommendations regarding when a cancer predisposition syndrome should be suspected in a young oncology patient. This review summarizes the current knowledge of cancer predisposition syndromes in pediatric oncology and provides essential information on clinical situations in which a childhood cancer predisposition syndrome should be suspected.

Activation of the Wnt/ß-catenin pathway is common in wilms tumor, but rarely through ß-catenin mutation and APC promoter methylation.

PURPOSE: The Wnt/ß-catenin pathway is known to be crucial for the regulation of embryogenesis and cell differentiation, and its constitutive activation is associated with a wide range of malignancies. There are two major principles for an activated Wnt/ß-catenin pathway. The first is caused by the failure of the destruction complex, mainly due to the decreased expression of the tumor suppressor gene adenomatous polyposis coli (APC); the second is the mutation of the ß-catenin (CTNNB1) protein itself. Wilms tumors (WTs) are also thought to be malignancies with a high rate of Wnt/ß-catenin pathway activation. The aim of this study was to analyze a large cohort of WT for activated Wnt/ß-catenin pathway. METHODS: The transcription of axis inhibition protein 2 (AXIN2) and APC was analyzed by real-time PCR. Expression was compared with those in healthy renal tissues as a control. Methylation status of the APC promoter was measured by pyrosequencing and correlated with APC expression. Finally, the mutations of CTNNB1 itself were detected by Sanger sequencing. RESULTS: The analysis was done in a cohort of 103 WTs, treated in our institution. There was a significant overexpression of AXIN2 in WTs (P < 0.0001), with 33 (32 %) tumors showing higher expression (median + 3× SD) than normal kidney tissue. In contrast, the expression of APC as well as its promoter methylation did not differ from control (P = 0.78; P = 0.82). Finally, there were only seven (6.8 %) mutations detectable in CTNNB1, and five out of seven were seen in WTs with AXIN2 overexpression. CONCLUSION: The finding that AXIN2, one of the major Wnt target genes, is overexpressed in our cohort of WTs, is indicative for the activation of the Wnt/ß-catenin pathway. However, neither the alteration of APC nor frequent CTNNB1 mutations were seen in our analyses. Therefore, other mechanisms might be responsible for the common activation of the Wnt/ß-catenin pathway.

Platelet Derived Growth Factor Has a Role in Pressure Induced Bladder Smooth Muscle Cell Hyperplasia and Acts in a Paracrine Way.

PURPOSE: Bladder outlet obstruction is a finding in many urological disorders, leading to bladder wall hyperplasia. We investigated platelet derived growth factor and its receptor in human bladder smooth muscle cells and urothelial cells exposed to hydrostatic pressure or PDGF in vitro. MATERIALS AND METHODS: Bladder smooth muscle cells and urothelial cells were exposed to elevated hydrostatic pressure for 1 hour. The expression of PDGF and PDGFR was evaluated using reverse transcriptase-polymerase chain reaction and Western blot analysis. Pressure or PDGF induced proliferation of bladder smooth muscle cells with or without pretreatment with lovastatin or imatinib was measured by enzyme-linked immunosorbent assay. PDGFRα was knocked down with siRNA. RESULTS: After hydrostatic pressure bladder smooth muscle cells showed increased PDGFRα and ß expression. PDGF was not expressed in bladder smooth muscle cells. Urothelial cells showed no expression of PDGFR but PDGF expression was noted. Western blot analysis of bladder smooth muscle cells revealed a pressure induced increase in PDGFR in the membrane fraction. Phosphorylation of PDGFR occurred with pressure induction. Bladder smooth muscle cell proliferation was increased in pressure and PDGF mediated fashion. Pretreatment with lovastatin or imatinib prevented proliferation. There was no cell proliferation after PDGFRα knockdown. CONCLUSIONS: Increased expression and phosphorylation of PDGFR in bladder smooth muscle cells after hydrostatic pressure suggests a pivotal role of the PDGF pathway in pressure induced hyperplasia of bladder smooth muscle cells. PDGF expressed in urothelial cells may act in a paracrine way. Cholesterol depletion, inhibition of receptor tyrosine kinase activity and knockdown of PDGFRα in bladder smooth muscle cells prevent pressure and PDGF mediated cell proliferation. Targeting PDGFR seems a promising way to influence pressure induced bladder wall hyperplasia.

The genomic landscape of hepatoblastoma and their progenies with HCC-like features.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most common childhood liver cancer and occasionally presents with histological and clinical features reminiscent of hepatocellular carcinoma (HCC). Identification of molecular mechanisms that drive the neoplastic continuation towards more aggressive HCC phenotypes may help to guide the new stage of targeted therapies. METHODS: We performed comprehensive studies on genetic and chromosomal alterations as well as candidate gene function and their clinical relevance. RESULTS: Whole-exome sequencing identified HB as a genetically very simple tumour (2.9 mutations per tumour) with recurrent mutations in ß-catenin (CTNNB1) (12/15 cases) and the transcription factor NFE2L2 (2/15 cases). Their HCC-like progenies share the common CTNNB1 mutation, but additionally exhibit a significantly increased mutation number and chromosomal instability due to deletions of the genome guardians RAD17 and TP53, accompanied by telomerase reverse-transcriptase (TERT) promoter mutations. Targeted genotyping of 33 primary tumours and cell lines revealed CTNNB1, NFE2L2, and TERT mutations in 72.5%, 9.8%, and 5.9% of cases, respectively. All NFE2L2 mutations affected residues of the NFE2L2 protein that are recognized by the KEAP1/CUL3 complex for proteasomal degradation. Consequently, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signalling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumours was significantly associated with metastasis, vascular invasion, the adverse prognostic C2 gene signature, as well as poor outcome. CONCLUSIONS: Our study demonstrates the importance of CTNNB1 mutations and NFE2L2-KEAP1 pathway activation in HB development and defines loss of genomic stability and TERT promoter mutations as prominent characteristics of aggressive HB with HCC features.

Hepatoblastoma cells express truncated neurokinin-1 receptor and can be growth inhibited by aprepitant in vitro and in vivo.

BACKGROUND & AIMS: Multidrug resistance presents a major problem in hepatoblastoma (HB), and new anti-tumor strategies are desperately needed. The substance P (SP)/neurokinin-1 receptor (NK1R) complex has been discovered to be pivotal in the development of a variety of human cancers, and NK1R antagonists, such as the clinical drug aprepitant, are promising future anticancer agents. Yet, the role of the SP/NK1R complex as a potential anticancer target in HB is unknown. METHODS: Human HB cell lines HepT1, HepG2, and HuH6, human tumor samples from 17 children with HB as well as mice xenografted with human HB cell line HuH6 were analyzed regarding the SP/NK1R complex as a potential new anti-tumor target in HB. RESULTS: Therapeutic targeting with the NK1R antagonists aprepitant, L-733,060, and L-732,138 led to growth inhibition and apoptosis in HepT1, HepG2, and HuH6 cells in a dose-dependent manner. Intriguingly, HB cells predominantly expressed the truncated splice variant of NK1R. Human fibroblasts showed only dismal NK1R expression and were significantly more resistant. Stimulation of HB cells with SP, NK1R's natural ligand, caused increased growth rates and abrogated the anti-proliferative effect of NK1R antagonists. Expression analysis of 17 human HB samples confirmed the clinical relevance of NK1R. Most importantly, oral treatment of a HuH6 xenograft mouse model with 80mg/kg/day aprepitant for 24days resulted in a striking reduction of tumor growth, as evidenced by reduced tumor volume and weight, lowered tumor-specific alpha-fetoprotein (AFP) serum levels, and decreased number of Ki-67 positive cells. Furthermore, aprepitant treatment inhibited in vivo angiogenesis. CONCLUSIONS: For the first time, we describe the NK1R in its truncated splice variant as a potent target in human HB and an inhibitory effect in vivo and in vitro by NK1R antagonists. Therefore, NK1R antagonists should be considered promising new candidates for innovative therapeutic strategies against HB.

Intestinal expression of the anti-inflammatory interleukin-1 homologue IL-37 in pediatric inflammatory bowel disease.

OBJECTIVES: The function of interleukin (IL)-37 has not been resolved. We recently showed that IL-37 suppresses colonic inflammation in mice. To gain more insight into its relevance in human disease, we investigated the expression of IL-37 in the intestine of pediatric patients with chronic inflammatory bowel disease (IBD). METHODS: Intestinal biopsies were obtained from children with IBD (18 Crohn disease [CD], 14 ulcerative colitis [UC] and 11 controls) during endoscopy and analyzed for IL-37 expression by immunohistochemistry and real-time polymerase chain reaction. Results were correlated with immunostaining for IL-18 and IL-17, messenger RNA (mRNA) levels of pro- and anti-inflammatory cytokines, and clinical parameters. RESULTS: IL-37 protein was detected in epithelial cells and submucosal lymphoid cells of patients with CD and UC as well as healthy controls. IL-37 protein expression tended to be higher with submucosal lymphoid cell infiltration of patients with CD and UC and correlated with histological severity score of inflammation. IL-18 showed a staining pattern similar to that of IL-37, whereas staining for IL-17 revealed distinct positive cells scattered in the submucosal layer. mRNA expression of IL-8, IL-17, and IL-10 was upregulated in patients with CD and UC. mRNA levels of IL-18 and IL-37 were not significantly elevated compared with controls. Levels of IL-37 and IL-18 mRNA showed a positive correlation in the CD group. CONCLUSIONS: IL-37 protein is expressed in healthy and diseased bowel tissue. IL-37 and IL-18 show a similar expression pattern and correlate at mRNA levels. Future studies are warranted to delineate the specific contribution of IL-37 to modulate chronic bowel inflammation in humans.

Targeting G9a/DNMT1 methyltransferase activity impedes IGF2-mediated survival in hepatoblastoma.

BACKGROUND: As the variable clinical outcome of patients with hepatoblastoma (HB) cannot be explained by genetics alone, the identification of drugs with the potential to effectively reverse epigenetic alterations is a promising approach to overcome poor therapy response. The gene ubiquitin like with PHD and ring finger domains 1 (UHRF1) represents an encouraging epigenetic target due to its regulatory function in both DNA methylation and histone modifications and its clinical relevance in HB. METHODS: Patient-derived xenograft in vitro and in vivo models were used to study drug response. The mechanistic basis of CM-272 treatment was elucidated using RNA sequencing and western blot experiments. RESULTS: We validated in comprehensive data sets that UHRF1 is highly expressed in HB and associated with poor outcomes. The simultaneous pharmacological targeting of UHRF1-dependent DNA methylation and histone H3 methylation by the dual inhibitor CM-272 identified a selective impact on HB patient-derived xenograft cell viability while leaving healthy fibroblasts unaffected. RNA sequencing revealed downregulation of the IGF2-activated survival pathway as the main mode of action of CM-272 treatment, subsequently leading to loss of proliferation, hindered colony formation capability, reduced spheroid growth, decreased migration potential, and ultimately, induction of apoptosis in HB cells. Importantly, drug response depended on the level of IGF2 expression, and combination assays showed a strong synergistic effect of CM-272 with cisplatin. Preclinical testing of CM-272 in a transplanted patient-derived xenograft model proved its efficacy but also uncovered side effects presumably caused by its strong antitumor effect in IGF2-driven tumors. CONCLUSIONS: The inhibition of UHRF1-associated epigenetic traces, such as IGF2-mediated survival, is an attractive approach to treat high-risk HB, especially when combined with the standard-of-care therapeutic cisplatin.

Impact of BCL-2 Expression on Course of Disease in Neuroblastoma.

OBJECTIVE: The antiapoptotic BCL-2 protein has implications for maturation and differentiation of neural tissue and acts as a strong modulator of carcinogenesis in different tumors. Recent research focuses not only on its benefit as a prognostic factor, but also as a potential therapeutic target. The role of BCL-2 in neuroblastoma, the most common extracranial solid tumor in childhood, remains controversial. The aim of our study was to determine the gene expression level of BCL-2 in a large cohort of neuroblastoma patients and its correlation with clinical parameters. METHODS: Tumor samples and clinical data were collected from 100 neuroblastoma patients treated according to the NB2004 protocol of the German Society of Pediatric Oncology and Hematology. BCL-2 gene expression levels were measured by quantitative reverse transcription polymerase chain reaction and correlated with clinical parameters. RESULTS: BCL-2 expression was detected in all tumor samples. Relative BCL-2 expression levels were higher in females versus males (1.839 vs. 1.342; p = 0.0143), in patients with low versus high International Neuroblastoma Staging System stage (2.051 vs. 1.463; p = 0.0206), in nonmetastatic versus metastatic disease (1.801 vs. 1.342; p = 0.0242), as well as in patients without presurgical chemotherapy (2.145 vs. 1.402; p = 0.0016), but was not associated with overall survival and MYCN amplification. CONCLUSION: Our study demonstrates the ubiquitous expression of BCL-2 in neuroblastoma and suggests the possibility for targeted therapy with BCL-2 inhibitors, even in lower-stage neuroblastoma. It also underlines the need for further research on concomitant genetic alterations for a better understanding of the impact of BCL-2 on this pediatric tumor type.

Hepatoblastoma Relapse-Findings from the German HB99 Trial and the German Liver Tumor Registry.

Survival rates for HB patients have improved; however, outcomes for patients who relapse remain poor. A retrospective review of information gathered for the HB99 study and the German Liver Tumor Registry identified 25 relapse patients (6.9%, 25/362). The median time from initial diagnosis to first relapse was 13 months (range: 5-66 months). Two patients relapsed >36 months after initial diagnosis. A total of 68% (17/25) of relapses were metastatic, 24% local, and 8% combined. 67% of local relapses were alive at the last follow-up, in contrast to 53% of metastatic and 0% of combined relapses. At the last follow-up, 73% (8/11) of patients with lung relapses were still alive (0/4 with peritoneal, 1/2 with CNS involvement). A total of 20% of the patients had AFP-negative relapses, 64% of the relapse patients achieved a second complete remission, 69% were still in complete second remission at the last follow-up (median FU of 66 months), and 83% (5/6) of irinotecan-naïve patients who received relapse treatment including irinotecan were in second complete remission at the last follow-up. The 3-year overall survival/event-free survival from relapse was 63%/48% respectively. There is a good chance that HB patients will achieve a second remission despite a first relapse. However, patients who suffer further relapses tend to have a poorer prognosis.

Neutrophils express distinct RNA receptors in a non-canonical way.

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.

Epigallocatechin-3-gallate inhibits hepatoblastoma growth by reactivating the Wnt inhibitor SFRP1.

Activation of Wnt signaling plays a central role in the formation of hepatoblastoma (HB), the most common pediatric liver cancer. Blocking this pathway with specific inhibitors is currently the target of various research endeavours. This study provides evidence that the naturally occurring flavonoid epigallocatechin-3-gallate (EGCG) is highly effective against HB growth through inhibition of Wnt signaling. We demonstrate that EGCG has a strong cytotoxic effect on HB cells in a time- and dose-dependent manner by impinging on cell viability, while leaving normal fibroblasts unaffected. Apoptotic features, including morphological changes, caspase 3 activity, and proteolytic cleavage of poly(ADP-ribose) polymerase, were frequently found in EGCG-treated HB cells, thereby suggesting involvement of the mitochondrial intrinsic apoptotic pathway. We furthermore show that EGCG effectively inhibits Wnt signaling, as evidenced by down-regulation of Wnt-responsive reporter gene activity and expression of the Wnt target genes MYC and CCND1. Interestingly, EGCG induced reexpression of the tumor suppressor gene SFRP1, which is transcriptionally silenced in HB cells and known to down-regulate Wnt signaling. Considering the lack of toxic effects on normal cells, EGCG should be preclinically validated as an adjuvant therapy in vivo with the ultimate goal of determining its efficacy in human trials.

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1.

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo.