L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

Epigenetic alterations critically affect tumor development. Polycomb-group complexes constitute an evolutionarily conserved epigenetic machinery that regulates stem cell fate and development. They are implicated in tumorigenesis, primarily via histone modification. Polycomb repressive complex 1 (PRC1) complexes 1-6 (PRC1.1-6) mediate the ubiquitination of histone H2A on lysine 119 (H2AK119ub). Here, we studied the functional roles of a PRC1.6 molecule, L3MBTL2, in neuroblastoma (NB) cells. L3MBTL2-knockout and knockdown revealed that L3MBTL2 depletion suppressed NB cell proliferation via cell-cycle arrest and gamma-H2A.X upregulation. L3MBTL2-knockout profoundly suppressed xenograft tumor formation. Transcriptome analysis revealed suppressed cell-cycle-related and activated differentiation-related pathways. Break repair meiotic recombinase recruitment factor 1 (BRME1) and nuclear receptor interacting protein 3 (NRIP3) were notably de-repressed by L3MBTL2-knockout. The deletion of L3MBTL2 reduced enrichment of H2AK119ub and PCGF6 at transcriptional start site proximal regions of the targets. Add-back studies unveiled the importance of L3MBTL2-BRME1 and -NRIP3 axes for NB cell proliferation. We further manifested the association of MYCN with de-repression of NRIP3 in an L3MBTL2-deficient context. Therefore, this study first revealed the significance of L3MBTL2-mediated gene silencing in MYCN-amplified NB cells.

Establishment and characterization of a novel MDM2/MYCN-co-amplified neuroblastoma cell line, NBN-SHIM, established from a late recurrent stage MS tumor.

The biological heterogeneity of neuroblastoma underscores the need for an in vitro model of each molecularly defined subgroup to investigate tumorigenesis and develop targeted therapies. We have established a permanently growing cell line from a 12-year-old girl who developed a late recurrent stage MS, MDM2-amplified neuroblastoma arising in the liver and performed histological, molecular, cytogenetic, exome, and telomere analyses of the recurrent tumor and the cell line. On histology, the recurrent tumor was immunoreactive for TP53, CDKN1A, and MDM2. A molecular cytogenetic study of the recurrent tumor revealed the amplification of MDM2 but no amplification of MYCN. The established cell line, NBM-SHIM, showed amplification of both MDM2 and MYCN on double-minute chromosomes. A copy number evaluation based on exome data confirmed the finding for MYCN and MDM2 and further identified high ploidy on CDK4 and GLI2 loci in the recurrent tumor and the cell line. The telomere maintenance mechanism on the cell line is unusual in terms of the low expression of TERT despite MYCN amplification and alternative lengthening of telomeres suggested by positive value for C-circle assay and telomere contents quantitative assay. The cell line is unique because it was established from a MYCN-nonamplified, MDM2-amplified, late-relapsed stage MS neuroblastoma, and MYCN amplification was acquired during cell culture. Therefore, the cell line is a valuable tool for investigating neuroblastoma tumorigenesis and new molecular targeted therapies for disrupted ARF-TP53-MDM2 pathway and amplification of MDM2 and CDK4.

STAT3 Contributes a Favorable Response to Pembrolizumab Through IFN-γ-induced Apoptosis in Urothelial Cancer.

BACKGROUND/AIM: Pembrolizumab, a second-line therapy for platinum-refractory advanced urothelial carcinoma (UC), is needed to improve objective response rate. Hence, it is crucial to identify optimal predictive biomarkers of responses. This study aimed to clarify the predictive value and role of signal transducer and activator of transcription 3 (STAT3) in selecting patients with advanced UC who might benefit clinically from pembrolizumab therapy. PATIENTS AND METHODS: We retrospectively analyzed 31 patients who received pembrolizumab therapy for UC. STAT3, phosphorylated STAT3 (p-STAT3), and PD-L1 expression were determined using tissue microarrays constructed from patient-derived specimens, and the association of these expression levels with overall survival was analyzed. We assessed the functional role of STAT3 in bladder cancer cell lines in response to interferon-gamma (IFN-γ). RESULTS: Patients with high STAT3 or p-STAT3 expression, and high platelet-to-lymphocyte ratio (PLR) (n=6) had a significantly shorter OS; in the other patients (n=25), high STAT3 or p-STAT3 expression was significantly associated with improved prognosis. IFN-γ-induced apoptosis was partially dependent on STAT3 in T24 cells but not in JMSU1 cells. CONCLUSION: In patients with advanced UC, STAT3 plays a key role in mediating the efficacy of pembrolizumab through apoptosis in response to IFN-γ.

Positive regulatory loop of platelet-derived growth factor DD-induced STAT3 activation is associated with poor prognosis in advanced urothelial carcinoma.

Immune checkpoint inhibitor (ICI) therapy has been established for patients with advanced urothelial cancer (UC). The necessity of overcoming resistance to ICIs and identifying a predictive factor for the same has been highlighted, such as the assessment of combination therapy with other targeted drugs and the characterization of molecular signatures in the tumor microenvironment. Recently, we reported that low hemoglobin (Hb) levels and a high platelet-to-lymphocyte ratio (PLR) were significantly associated with overall survival in patients with UC who did not benefit from pembrolizumab treatment. In the present study, we identified a possible link between these unfavorable prognostic indicators and PDGF-DD-induced STAT3 activation in UC. Overlapping patients between the high STAT3- or phosphorylated STAT3-positive score group (as assessed by immunohistochemistry) and low Hb levels or high PLR group (as assessed by blood tests) showed significantly worse outcomes after pembrolizumab treatment. Additionally, using the bladder cancer JMSU1 cell line, we demonstrated a possible positive regulatory loop between autocrine/paracrine PDGF-DD and STAT3 signaling. Therefore, we suggest that STAT3 inhibition and PDGF-DD detection in the tumor microenvironment might represent a potential therapeutic strategy to overcome resistance to pembrolizumab. Moreover, this can help identify patients with UC who could benefit from combination treatment.

ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors.

BACKGROUND: Genomic alterations, including loss of function in chromosome band 11q22-23, are frequently observed in neuroblastoma, which is the most common extracranial childhood tumour. In neuroblastoma, ATM, a DNA damage response-associated gene located on 11q22-23, has been linked to tumorigenicity. Genetic changes in ATM are heterozygous in most tumours. However, it is unclear how ATM is associated with tumorigenesis and cancer aggressiveness. METHODS: To elucidate its molecular mechanism of action, we established ATM-inactivated NGP and CHP-134 neuroblastoma cell lines using CRISPR/Cas9 genome editing. The knock out cells were rigorously characterized by analyzing proliferation, colony forming abilities and responses to PARP inhibitor (Olaparib). Western blot analyses were performed to detect different protein expression related to DNA repair pathway. ShRNA lentiviral vectors were used to knockdown ATM expression in SK-N-AS and SK-N-SH neuroblastoma cell lines. ATM knock out cells were stably transfected with FANCD2 expression plasmid to over-expressed the FANCD2. Moreover, knock out cells were treated with proteasome inhibitor MG132 to determine the protein stability of FANCD2. FANCD2, RAD51 and γH2AX protein expressions were determined by Immunofluorescence microscopy. RESULTS: Haploinsufficient ATM resulted in increased proliferation (p < 0.01) and cell survival following PARP inhibitor (olaparib) treatment. However, complete ATM knockout decreased proliferation (p < 0.01) and promoted cell susceptibility to olaparib (p < 0.01). Complete loss of ATM suppressed the expression of DNA repair-associated molecules FANCD2 and RAD51 and induced DNA damage in neuroblastoma cells. A marked downregulation of FANCD2 expression was also observed in shRNA-mediated ATM-knockdown neuroblastoma cells. Inhibitor experiments demonstrated that the degradation of FANCD2 was regulated at the protein level through the ubiquitin-proteasome pathway. Reintroduction of FANCD2 expression is sufficient to reverse decreased proliferation mediated by ATM depletion. CONCLUSIONS: Our study revealed the molecular mechanism underlying ATM heterozygosity in neuroblastomas and elucidated that ATM inactivation enhances the susceptibility of neuroblastoma cells to olaparib treatment. These findings might be useful in the treatment of high-risk NB patients showing ATM zygosity and aggressive cancer progression in future.

Pretreatment Hemoglobin Levels and Platelet-to-Lymphocyte Ratio Predict Survival Benefit from Pembrolizumab in Advanced Urothelial Carcinoma.

BACKGROUND/AIM: Several prognostic risk factors have been recognized when using cisplatin-based conventional chemotherapy for the treatment of advanced urothelial carcinoma (UC); these include performance status (PS), liver metastasis, hemoglobin (Hb) levels, time from prior chemotherapy (TFPC), and other systemic inflammation scores including neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). However, the benefit of these indicators for predicting outcome of immune checkpoint inhibitors is not fully understood. Here, we investigated the predictive value of the indicators in patients who received pembrolizumab for the treatment of advanced UC. PATIENTS AND METHODS: Seventy-five patients who received pembrolizumab treatment for advanced UC were included. The Karnofsky PS, liver metastasis, hemoglobin levels, TFPC, NLR, and PLR were analyzed, and their relationship with overall survival (OS) was determined. RESULTS: All factors were highlighted as significant prognostic indicators for OS in the univariate proportional regression analysis (p<0.05 for each). Multivariate analysis revealed that Karnofsky PS and liver metastasis were independent prognostic indicators for OS (p<0.01) but were applicable only for a small number of patients. Notably, the combined analysis with low Hb levels and high PLR was significantly associated with OS in patients who could gain less benefit from pembrolizumab at a median of 6.6 [95% confidence interval (CI)=4.2-9.0] versus 15.1 (95% CI=12.4-17.8) months (p=0.002). CONCLUSION: The combination of Hb levels and PLR may be a broadly applicable indicator for the outcome of pembrolizumab as second-line chemotherapy in patients with advanced UC.

Development of an osteosarcoma model with MYCN amplification and TP53 mutation in hiPS cell-derived neural crest cells.

Mesenchymal stem cell- or osteoblast-derived osteosarcoma is the most common malignant bone tumor. Its highly metastatic malignant phenotypes, which are often associated with a poor prognosis, have been correlated with the modulation of TP53- and cell-cycle-related pathways. MYC, which regulates the transcription of cell-cycle modulating genes, is used as a representative prognostic marker for osteosarcoma. Another member of the MYC oncoprotein family, MYCN, is highly expressed in a subset of osteosarcoma, however its roles in osteosarcoma have not been fully elucidated. Here, we attempted to create an in vitro tumorigenesis model using hiPSC-derived neural crest cells, which are precursors of mesenchymal stem cells, by overexpressing MYCN on a heterozygous TP53 hotspot mutation (c.733G>A; p.G245S) background. MYCN-expressing TP53 mutated transformed clones were isolated by soft agar colony formation, and administered subcutaneously into the periadrenal adipose tissue of immunodeficient mice, resulting in the development of chondroblastic osteosarcoma. MYCN suppression decreased the proliferation of MYCN-induced osteosarcoma cells, suggesting MYCN as a potential target for a subset of osteosarcoma treatment. Further, comprehensive analysis of gene expression and exome sequencing of MYCN-induced clones indicated osteosarcoma-specific molecular features, such as the activation of TGF-ß signaling and DNA copy number amplification of GLI1. The model of MYCN-expressing chondroblastic osteosarcoma was developed from hiPSC-derived neural crest cells, providing a useful tool for the development of new tumor models using hiPSC-derived progenitor cells with gene modifications and in vitro transformation.

Polycomb EZH1 regulates cell cycle/5-fluorouracil sensitivity of neuroblastoma cells in concert with MYCN.

In the present study, we found that EZH1 depletion in MYCN-amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1-depleted cells resulted in downregulation of the cell cycle progression-related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F-mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1-related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5-fluorouracil (5-FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN-amplified neuroblastoma cells to 5-FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

Platelet-to-Lymphocyte Ratio Predicts the Efficacy of Pembrolizumab in Patients With Urothelial Carcinoma.

BACKGROUND/AIM: This study aimed to determine useful predictive factors for selecting patients with advanced urothelial carcinoma (UC) who might benefit clinically from treatment with pembrolizumab. PATIENTS AND METHODS: We retrospectively analyzed 54 patients who underwent pembrolizumab treatment for UC. The hemoglobin, albumin, lymphocyte and platelet (HALP) score, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) were calculated as indices of systemic inflammatory response, and the relationships between these scores and the initial tumor response or overall survival, as well as other clinicopathological factors, were assessed. RESULTS: High NLR and PLR were associated with a poor initial tumor response to pembrolizumab. A HALP score <30.05 and a PLR ≥173.73 were associated with worse overall survival. In the multivariate Cox regression analysis, a high PLR was a significant independent prognostic factor for unfavorable outcomes. CONCLUSION: A high pretreatment PLR may be a valuable indicator for choosing therapy other than pembrolizumab in patients with advanced UC.

Loss of p53 suppresses replication stress-induced DNA damage in ATRX-deficient neuroblastoma.

Genetic aberrations are present in the ATRX gene in older high-risk neuroblastoma (NB) patients with very poor clinical outcomes. Its loss-of-function (LoF) facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells and is strongly linked to replication stress (RS) and DNA damage through G-quadruplex (G4) DNA secondary structures. However, limited information is available on ATRX alteration-related NB tumorigenesis. We herein knocked out (KO) ATRX in MYCN-amplified (NGP) and MYCN single copy (SK-N-AS) NB cells with wild-type (wt) and truncated TP53 at the C terminus, respectively, using CRISPR/Cas9 technologies. The loss of ATRX increased DNA damage and G4 formation related to RS in TP53 wt isogenic ATRX KO NGP cells, but not in SK-N-AS clones. A gene set enrichment analysis (GSEA) showed that the gene sets related to DNA double-strand break repair, negative cell cycle regulation, the G2M checkpoint, and p53 pathway activation were enriched in NGP clones. The accumulation of DNA damage activated the ATM/CHK2/p53 pathway, leading to cell cycle arrest in NGP clones. Interestingly, ATRX loss did not induce RS related to DNA damage response (DDR) in TP53-truncated SK-N-AS cells. p53 inactivation abrogated cell cycle arrest and reduced G4 accumulation in NGP clones. The loss of p53 also induced G4 DNA helicases or Fanconi anemia group D2 protein (FANCD2) with ATRX deficiency, suggesting that ATRX maintained genome integrity and p53 deficiency attenuated RS-induced DNA damage in NB cells featuring inactivated ATRX by regulating DNA repair mechanisms and replication fork stability.

Frequent breakpoints of focal deletion and uniparental disomy in 22q11.1 or 11.2 segmental duplication region reveal distinct tumorigenesis in rhabdoid tumor of the kidney.

SMARCB1 is mutated in most rhabdoid tumors (RTs) developing in the kidney (RTK) and various other organs. Focal deletions found in patients with 22q11.2 deletion syndrome show breakpoints within clusters of segmental duplications (SDs), and those in some RTs show breakpoints in the 22q11-q12 region. SDs are known to cause focal deletion mediated by non-allelic homologous recombination. The present study identified SMARCB1 alterations in all 30 RTKs, using SNP array CGH, MLPA, and sequence analyses. Twenty-eight tumors had a total of 51 breakpoints forming focal 22q deletion and/or uniparental disomy (22qUPD), and the other two had compound mutation with no breakpoints in 22q. Twenty-four (47.1%) of the 51 breakpoints were within SDs, and occurred in 16 (53.3%) of the 30 tumors. The association of breakpoints with SDs was found not only in focal deletion, but also in 22qUPD, indicating that SDs mediate the first and second hits (focal deletion) and the second hit (22qUPD) of SMARCB1 alteration. Of the 51 breakpoints, 14 were recurrent, and 10 of the 14 were within SDs, suggesting the presence of hotspots in the 22q11.2 region. One recurrent breakpoint outside SDs resided in SMARCB1, suggesting inactivation of the gene by out-of-frame fusion. The association between SDs and focal deletion has been reported in two other types of cancer. RTKs may be the third example of SD-associated tumors. Thus, the present study indicated that RTKs exploit genomic instability in the 22q11.1-11.2 SDs region, and 22qUPD caused by mitotic recombination may also be mediated by SDs.

Blastemal predominant type Wilms tumor in Japan: Japan Children's Cancer Group.

BACKGROUND: Persistence of blastemal components after chemotherapy is a marker of poor outcome in Wilms tumor (WT). Recent reports from local Japanese areas have described pre-chemotherapy blastemal predominant type WT to also be a risk factor for relapse. The significance, however, of blastemal predominant WT remains to be evaluated in a larger study. This study retrospectively evaluated the prognostic significance of pre-chemotherapy blastemal predominant type WT in the Japan Wilms tumor Study (JWiTS) trials. METHODS: The JWiTS trial (1996-2013) was a prospective, single-arm study. The outcomes of blastemal predominant type WT were retrospectively evaluated compared with non-blastemal type WT excluding anaplasia between 1996 and 2013. Relapse-free survival (RFS) and overall survival (OS) were estimated. RESULTS: Of 319 primary renal tumors diagnosed by the central pathology review system, advanced stage of pre-chemotherapy blastemal predominant type WT (n = 53; 16.1%) occurred more frequently in older children than non-blastemal type WT (n = 225), and was especially frequent in female patients registered in the JWiTS trials. No significant difference in 10 years RFS and OS (78.8% vs 84.5; P = 0.201) or in 10 years RFS and OS (89.3% vs 93.5; P = 0.45) was seen between pre-chemotherapy blastemal predominant type and non-blastemal type WT. CONCLUSIONS: Relapse-free survival and OS are not significantly different between pre-chemotherapy blastemal predominant type and non-blastemal type WT.

Combined Genetic and Chromosomal Characterization of Wilms Tumors Identifies Chromosome 12 Gain as a Potential New Marker Predicting a Favorable Outcome.

To identify prognostic factors, array CGH (aCGH) patterns and mutations in WT1 and 9 other genes were analyzed in 128 unilateral Wilms tumors (WTs). Twenty patients had no aCGH aberrations, and 31 had WT1 alterations [silent and WT1 types: relapse-free survival (RFS), 95% and 83%, respectively]. Seventy-seven patients had aCGH changes without WT1 alterations (nonsilent/non-WT1 type) and were subtyped into those with or without +12, 11q-, 16q-, or HACE1 loss. RFS was better for those with than those without +12 (P = .010) and worse for those with than those without 11q-, 16q-, or HACE1 loss (P = .001, .025, or 1.2E-04, respectively). Silent and WT1 type and 8 subtype tumors were integrated and classified into 3 risk groups: low risk for the silent type and +12 subgroup; high risk for the no +12 plus 11q-, 16q-, or HACE1 loss subgroup; intermediate risk for the WT1 type and no +12 plus no 11q-, 16q-, or HACE1 loss subgroup. Among the 27 WTs examined, the expression of 146 genes on chromosome 12 was stronger in +12 tumors than in no +12 tumors, while that of 10 genes on 16q was weaker in 16q- tumors than in no 16q- tumors. Overexpression in 75 out of 146 upregulated genes and underexpression in 7 out of 10 downregulated genes correlated with better and worse overall survival, respectively, based on the public database. +12 was identified as a potential new marker predicting a favorable outcome, and chromosome abnormalities may be related to altered gene expression associated with these abnormalities.

Outcome of renal tumors registered in Japan Wilms Tumor Study-2 (JWiTS-2): A report from the Japan Children's Cancer Group (JCCG).

BACKGROUND: Japan Wilms Tumor Study-2 (JWiTS-2) mandated central pathology review for all case registrations. The study aimed to compare the outcomes of patients with unilateral Wilms tumor enrolled on the JWiTS-1 and JWiTS-2 trials. PROCEDURE: The JWiTS-2 trial (2006-2014), a prospective, single-arm study, required compulsory submission of histologic slides to central pathology, while in the JWiTS-1 trial, such submission was not compulsory. Relapse-free survival (RFS) and overall survival (OS) versus cases in the JWiTS-1 trial (1996-2005) were statistically evaluated. RESULTS: Of 277 enrolled patients with primary renal tumors diagnosed by the central pathology review system, 225 patients with unilateral renal tumors were followed up over 9 years. The RFS and OS of Wilms tumor (n = 178) were 90.4% (P = 0.0003) and 96.8% (P = 0.054), respectively, as compared to 74.9% and 89.4% in JWiTS-1. RFS rates of stages I-III Wilms tumor in JWiTS-2 were more than 90%, although the outcome of stage IV Wilms tumor was significantly poorer (RFS: 66.2%) (P = 0.0094). RFS and OS of clear cell sarcoma of the kidney (CCSK; n = 31) were 82.4% (P = 0.30) and 91.3% (P = 0.42), respectively, as compared to 68.8% and 81.3% in JWiTS-1, and those of rhabdoid tumor of the kidney (RTK; n = 16) were 18.8% (P = 0.88) and 25.0% (P = 0.80), respectively, as compared to 23.5% and 23.5% in JWiTS-1. CONCLUSIONS: RFS and OS for stages I-III Wilms tumor were improved in JWiTS-2 compared to JWiTS-1, whereas outcomes for stage IV Wilms tumor, CCSK, and RTK did not improve.

EZH2 regulates neuroblastoma cell differentiation via NTRK1 promoter epigenetic modifications.

The polycomb repressor complex 2 molecule EZH2 is now known to play a role in essential cellular processes, namely, cell fate decisions, cell cycle regulation, senescence, cell differentiation, and cancer development/progression. EZH2 inhibitors have recently been developed; however, their effectiveness and underlying molecular mechanisms in many malignancies have not yet been elucidated in detail. Although the functional role of EZH2 in tumorigenesis in neuroblastoma (NB) has been investigated, mutations of EZH2 have not been reported. A Kaplan-Meier analysis on the event free survival and overall survival of NB patients indicated that the high expression of EZH2 correlated with an unfavorable prognosis. In order to elucidate the functional roles of EZH2 in NB tumorigenesis and its aggressiveness, we knocked down EZH2 in NB cell lines using lentivirus systems. The knockdown of EZH2 significantly induced NB cell differentiation, e.g., neurite extension, and the neuronal differentiation markers, NF68 and GAP43. EZH2 inhibitors also induced NB cell differentiation. We performed a comprehensive transcriptome analysis using Human Gene Expression Microarrays and found that NTRK1 (TrkA) is one of the EZH2-related suppression targets. The depletion of NTRK1 canceled EZH2 knockdown-induced NB cell differentiation. Our integrative methylome, transcriptome, and chromatin immunoprecipitation assays using NB cell lines and clinical samples clarified that the NTRK1 P1 and P2 promoter regions were regulated differently by DNA methylation and EZH2-related histone modifications. The NTRK1 transcript variants 1/2, which were regulated by EZH2-related H3K27me3 modifications at the P1 promoter region, were strongly expressed in favorable, but not unfavorable NB. The depletion and inhibition of EZH2 successfully induced NTRK1 transcripts and functional proteins. Collectively, these results indicate that EZH2 plays important roles in preventing the differentiation of NB cells and also that EZH2-related NTRK1 transcriptional regulation may be the key pathway for NB cell differentiation.

CFC1 is a cancer stemness-regulating factor in neuroblastoma.

BACKGROUND: Despite the use of aggressive therapy, survival rates among high-risk neuroblastoma (NB) patients remain poor. Cancer stem cells (CSCs) are considered to be critically involved in the recurrence and metastasis of NB and are isolated as NB spheres. METHODS: The gene expression profiling of adherent (control) and sphere-forming primary NB cells was conducted using a gene expression microarray. CFC1, which functions in the development of embryos and decides the left-right axis, was strongly expressed in sphere-forming cells only and was related to the unfavorable prognosis of NB patients. The knockdown and overexpression of CFC1 were performed using a lentiviral system in NB cell lines. Sphere formation, cell proliferation, colony formation in soft agar, and xenograft tumor formation were analyzed. RESULTS: The overexpression of CFC1 increased sphere formation, cell growth, and colony formation. These phenotypes, particularly sphere formation, and xenograft tumor formation were significantly suppressed by the knockdown of CFC1. CFC1 inhibited Activin A-induced NB cell differentiation and Smad2 phosphorylation in NB cell lines, indicating its involvement in tumorigenesis related to EGF-CFC co-receptor family molecule pathways. Collectively, these results indicate that CFC1 is a candidate molecule for the development of CSC-targeted therapy for NB.

BRCA1 alterations with additional defects in DNA damage response genes may confer chemoresistance to BRCA-like breast cancers treated with neoadjuvant chemotherapy.

The BRCA-like phenotype is a feature that some sporadic breast cancers share with those occurring in BRCA1 or BRCA2 mutation carriers. As tumors with the phenotype have defects in the DNA damage response pathway, which may increase sensitivity to drugs such as DNA cross-linking agents and PARP inhibitors, a method to identify this phenotype is important. The prediction of chemoresistance, which frequently develops in these tumors, is also crucial for improving therapy. We examined genomic aberrations and BRCA1 promoter methylation in tumors of 73 breast cancer (20 HR-/HER2- and 53 HR+/HER2-) patients, who received neoadjuvant chemotherapy with anthracycline, cyclophosphamide, and taxane, using SNP array CGH and quantitative PCR. The methylation and/or loss or uniparental disomy (UPD) of BRCA1 (BRCA1 alterations) and the loss or UPD of BRCA2 (BRCA2 alterations) were detected in 27 (37%) and 21 (29%), respectively, of the 73 tumors. Tumors with BRCA1 or BRCA2 alterations were associated with a higher number of genomic aberrations (P < 0.001 and P < 0.001) and higher percentage of TP53 alterations (P < 0.001 and P < 0.001) than those without. Overall survival (OS) rates were similar between patients with or without BRCA1 or BRCA2 alterations. However, when 27 patients with BRCA1-altered tumors were classified into those with or without the loss or UPD of PALB2, PAGR1, RAD51B, FANCM, MLL4, or ERCC1/2 in tumors, patients with additional defects in DNA damage response genes had worse OS (P = 0.037, 0.045, 0.038, 0.044, 0.041, or 0.019) than those without. These defects may confer chemoresistance and predict poor outcomes in patients with BRCA1-altered breast cancer.

Anaplastic histology Wilms' tumors registered to the Japan Wilms' Tumor Study Group are less aggressive than that in the National Wilms' Tumor Study 5.

PURPOSE: To evaluate the clinical features and treatment results of anaplastic histology (AH) Wilms' tumor (WT) patients registered in the Japan Wilms' Tumor Study (JWiTS) group to elucidate the clinical characteristics of AH in the Japanese population. PATIENTS AND METHODS: Of 344 WT patients who were enrolled in JWiTS between 1995 and 2013, 17 had AH. Treatment using the JWiTS protocols was similar to the fifth National Wilms' Tumor Study 5 (NWTS-5) protocols. Clinical characteristics and mutation status of TP53 gene were evaluated and compared with those in NWST-5 study. RESULTS: AH incidences in JWiTS were 4.9 %, lower than that in NWTS-5. Seven tumors had focal AH and 10 had diffuse AH. Clinical stages of AH patients were stage I in seven, stage II in three, stage III in five, stage IV in one and unknown in one. Four-year event-free survival and overall survival rates were 90.9 and 86.7 %, respectively. Two patients with diffuse AH and none with focal AH had TP53 mutation. CONCLUSION: Japanese patients presented with higher incidence, earlier stages and may have better outcomes than American patients, indicating a possible biological heterogeneity of AH WT. Further analysis is necessary to elucidate the different characteristic of AH WT between Japanese and American populations.

Clinical prognostic value of DNA methylation in hepatoblastoma: Four novel tumor suppressor candidates.

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.