Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation.

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5'-CACGTGT-3') sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole-imidazole (PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5'- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth.

Allogeneic hematopoietic stem cell transplantation against recurrent rhabdomyosarcoma.

The prognosis of high-risk rhabdomyosarcoma (RMS) with metastatic or recurrent disease remains poor. We report a 6-year-old girl who successfully underwent allogeneic hematopoietic stem cell transplantation against recurrent metastatic alveolar RMS. The disease recurred at distant lymph node metastasis with bone marrow involvement. After chemotherapy and radiotherapy for the metastatic site, she underwent allogeneic bone marrow transplantation during complete remission from her 5/8 HLA-matched father. She developed acute graft-versus-host disease after preemptive donor lymphocyte infusion and remains in a disease-free condition for 31 months after transplantation. A graft-versus-tumor effect through allogeneic immune cells might produce a beneficial effect for high-risk RMS.

Clinical assessments in patients ten years after pylorus-preserving gastrectomy with or without preserving both pyloric and hepatic branches of the vagal nerve for early gastric cancer.

BACKGROUND/AIMS: To clarify the differences in the postoperative quality of life (QOL) of patients after pylorus preserving gastrectomy (PPG) between those with preserved pyloric and hepatic branches of the vagal nerve (PHV) and those without PHV, we investigated the postoperative gastrointestinal symptoms at 10 years after PPG patients with or without PHV. METHODS: Twenty eight subjects who underwent PPG with D2 lymphadenectomy without preserving the PHV (group A: 18 male and 10 female subjects aged 38 to 70 years with a mean age of 60.2 years) were interviewed to inquire about gastrointestinal symptoms (appetite, weight loss, epigastric fullness, reflux esophagitis, and early dumping syndrome), and compared with 30 PPG patients with D1 lymphadenectomy with preserving PHV (group B: 20 male and 10 female subjects aged 33 to 72 years with a mean age of 61.3 years). Esophagogastric endoscopy and abdominal ultrasonography were also studied. RESULTS: There were no differences in the postoperative gastrointestinal symptoms, endoscopic reflux esophagitis, and endoscopic gastritis between groups A and B. However, cholecystolithiasis was significantly found in group A but was not found in group B. In addition, there was significant difference between groups A and B (p = 0.0074). CONCLUSIONS: It is important to preserve the PHV to prevent cholecystolithiasis formation in patients after PPG.

Are there any functional differences of the enteric nervous system between jejunum and ileum in normal humans?

BACKGROUND/AIMS: Functional differences of the enteric nervous system (ENS) between the jejunum and ileum in humans are still unknown. To clarify the physiological differences of the ENS in the normal human jejunum and ileum, we investigated the enteric nerve responses on normal jejunum and normal ileum in vitro. METHODOLOGY: Twenty-two preparations of normal jejunum were taken from 22 patients with gastric cancer (14 men and 8 women, aged 43 to 65 years with a mean age of 49.0 years). Twenty preparations of normal ileum were also taken from 20 patients with right sided colonic cancer (15 men and 5 women, aged 40 to 63 years with a mean age of 50.9 years). A mechanographic technique was used to evaluate in vitro muscle responses to electrical stimulation (EFS) of adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers. RESULTS: Responses to EFS before blockade of the adrenergic and cholinergic nerves; Muscle strips in jejunum and ileum demonstrated significant contraction reactions rather than relaxation reactions (p < 0.0001, p = 0.0001, respectively). In addition, jejunal muscle strips showed significant contraction reactions than those in ileal muscle strips (p = 0.0274). Responses to EFS after blockade of the adrenergic and cholinergic nerves; Jejunal muscle strips demonstrated relaxation reactions rather than contraction reactions by EFS (p = 0.0704). Ileal muscle strips significantly demonstrated relaxation reactions rather than contraction reactions by EFS (p < 0.0001). In addition, ileal muscle strips showed significant relaxation reactions more than those in jejunal muscle strips (p = 0.0451). Tetrodotoxin abolished the EFS responses in the muscle strips both jejunum and ileum. CONCLUSIONS: Cholinergic nerves are mainly involved in regulation of enteric nerves in the jejunum rather than the ileum. In contrast, the ileum was more strongly innervated by non-adrenergic noncholinergic inhibitory nerves than the jejunum.

Mesenchymal hamartoma of the liver originating in the caudate lobe with t(11;19)(q13;q13.4): report of a case.

We herein report the case of a 35-month-old female child presenting with mesenchymal hamartoma of the liver (MHL), with t(11;19)(q13;q13.4) originating in the caudate lobe. This case is the eighth known description of a cytogenetic abnormality in mesenchymal hamartoma of the liver. It is similar to the seven cases previously reported, in that one of the breakpoints involves the chromosome band 19q13.3 or 19q13.4, but it is the first report of an abnormality originating in the caudate lobe.

Usefulness of power Doppler ultrasonography and superparamagnetic iron oxide enhanced magnetic resonance imaging for diagnosis of focal nodular hyperplasia of the liver after treatment of neuroblastoma.

Focal nodular hyperplasia (FNH) of the liver is rare in children, and it is usually diagnosed through a biopsy of the liver or hepatectomy. The authors report a case of a 10-year-old girl with multiple focal nodular hyperplasia lesions of the liver after the completion of tumor therapy for advanced neuroblastoma, and review the usefulness of the combination of power Doppler ultrasonography (US) and superparamagnetic iron oxide (SPIO) enhanced magnetic resonance imaging (MRI) for the diagnosis of FNH without a biopsy of the liver or hepatectomy.

Studies on pudendal nerve terminal motor latency in patients after ileal J-pouch-anal anastomosis for ulcerative colitis and adenomatous coli in childhood.

BACKGROUND/AIMS: To clarify the neurological function with respect to external anal sphincter (EAS) muscles in child patients with or without soiling after ileal J pouch anal anastomosis (IPAA), we examined the terminal motor latency in the pudendal motor nerves (PNTML). METHODOLOGY: A total of nine patients after IPAA for UC (7 cases) and AC (2 cases) were studied (6 males and 3 females, 10 to 15 with a mean age of 13.8 years). Patients one year after IPAA with soiling were also subdivided by the see page of mucous and/or stool group A (5 cases); rare soiling with loose stool, group B (4 cases); occasional soiling (1 time per 2 or 3 days). However, all patients showed continence 2 years after IPAA (Group C). Group D served as controls without gastrointestinal symptoms and digestive diseases and consisted of 12 subjects (8 males and 4 females aged 12 to 16 years old with a mean age of 14.8 years). Examinations were performed 1 and 2 years after ileostomy closure. Bilateral (left-sided and right-sided) PNTML tests were performed on all patients in order to measure the latency of the response in the bilateral EAS muscle following digitally directed transrectal pudendal nerve stimulation. RESULTS: 1) Values of the PNTML at the right-sided of the PN: The conduction delay in group B was the longest, followed by groups A, C, and D. Moreover, significant differences in PNTML were noted between groups A and B, between groups A and D, between groups B and C, and between groups B and D (p < 0.0001, p = 0.0316, p < 0.0001, p < 0.0001 respectively). There were no significance differences between group A and C or between groups C and D (p = 0.1733, p = 0.2957, respectively). 2) Values of the PNTML at the left-sided of the PN: The conduction delay in group B was the longest, followed by groups A, C, and D. Moreover, significant differences in PNTML were noted between groups A and B, between groups A and D, between groups B and C, and between groups B and D (p < 0.0001, p = 0.0584, p < 0.0001, p < 0.0001 respectively). There were no significance differences between groups A and C or between groups C and D (p = 0.3042, p = 0.2553, respectively). CONCLUSIONS: These findings support the hypothesis that child patients' soiling after IPAA may be caused by damage to the bilateral pudendal motor nerves.

Effects of mosapride citrate on patients after vagal nerve, lower esophageal sphincter, and pyloric sphincter-preserving nearly total gastrectomy reconstructed by jejunal J pouch interposition, and postoperative quality of life.

BACKGROUND/AIMS: Vagal nerve and pylorus-preserving nearly total gastrectomy reconstructed by interposition of a jejunal J pouch (hereinafter called NTGP) is a function-preserving operation for early gastric cancer. However, some patients after NTGP have suffered from postprandial food stasis in the substitute stomach, and postprandial stasis leads to abdominal symptoms. To clarify the clinical effect of mosapride citrate (hereinafter called MS) for prevention of food stasis in the substitute stomach for patients after NTGP, we studied the clinical effects of MS before and after administration of MS. METHODOLOGY: In a total of 24 patients (18 males, 6 females; aged 44-70 years, average 58.1 years) during 5 years after NTGP for early gastric cancer (D1 lymph node dissection, curability A), the relationship between their postoperative quality of life (QOL) and emptying function of the substitute stomach (hereinafter called EFS) was compared using a radioisotope method before MS therapy and after MS therapy at an oral dose of 15mg/day for 3 months. RESULTS: The interviews showd that after MS therapy, patients had more evident appetite and ate more food with a slight increase in body weight (0.52Kg) compared with patients before MS therapy. Before and after MS therapy, patients had no early dumping symtoms, while patients after MS therapy clearly had fewer symptoms such as reflux esophagitis, nausea, and abdominal pain compared with before MS therapy. After MS therapy, patients also had significantly decreased abdominal fullness compared with before MS therapy (p = 0.0046). Endoscopically, we found reflux esophagitis in 4 patients before MS therapy but in no patients after MS therapy. All patients before MS therapy showed residual contents in the substitute stomach, but only 10 patients after MS therapy showed residual contents in the substitute stomach. There was a significant difference between before and after MS therapy (p = 0.0016). Regarding EFS, the time to 50% residual rate before MS therapy (98.7 +/- 13.0 min) was significantly slower than that after MS therapy (83.2 +/- 13.8 min) (p = 0.0134). After MS therapy (37.0 +/- 4.9%), the residual rates at 120 minutes were significantly decreased compared with patients before MS therapy (44.8 +/- 5.3%) (p = 0.0028). Patients after MS therapy clearly had improved stasis of substitute stomach compared with before MS therapy. CONCLUSIONS: It was considered that MS therapy subsequently improves abdominal fullness due to the postprandial food stasis in the substitute stomach, contributing to the improvement of QOL of patients after NTGP.

Are there any functional differences of enteric nervous system between the proximal and distal parts from the dentate line in the normal human internal anal sphincter?

BACKGROUND/AIMS: To clarify the functional differences of the enteric nervous system in the human internal anal sphincter (IAS) between the proximal and distal parts from the dentate line, we investigated the enteric nerve responses of normal proximal and distal IAS in vitro. METHODOLOGY: Normal IAS specimens derived from 20 patients with lower rectal cancer (14 men and 6 women aged from 48 to 77 years, average 66.5 years) were used. These IAS muscles were divided into 2 parts [oral site IAS from dentate line; proximal part (PIAS; n=20), anal site IAS from dentate line; distal part (DIAS; n=20)]. A mechanographic technique was used to evaluate in vitro muscle strip responses to electrical field stimulation (EFS) before and after treatment with various autonomic nerve blockers. RESULTS: 1) Response to EFS before blockade of the adrenergic and cholinergic nerves: In PIAS, the incidence of relaxation reactions was greater than that of contraction reactions (p=0.2059). In DIAS, the incidence of contraction reactions was significantly greater than that of relaxation reactions (p=0.0001). The percentage of relaxation responses in the PIAS was significantly greater than that in the DIAS (p=0.0098). 2) Response to EFS after blockade of the adrenergic and cholinergic nerves: In PIAS, the incidence of relaxation reactions via NANC inhibitory nerve was significantly greater than that of contraction reactions via NANC excitatory nerves (p< 0.0001). In DIAS, the incidence of relaxation reactions via NANC inhibitory nerve was greater than that of contraction reactions via NANC excitatory nerves (p=0.2059). The percentage of relaxation responses via NANC inhibitory nerves in the PIAS was significantly greater than that in the DIAS (p=0.00284). 3) EFS responses in the PIAS and DIAS were blocked by tetrodotoxin. CONCLUSIONS: There are functional differences in the regulation of the enteric nervous system between the PIAS and DIAS. Contraction reaction via excitatory nerves, especially cholinergic nerves, was mainly involved in the regulation of enteric nerve responses to EFS in the DIAS. Relaxation reaction via inhibitory nerves, especially NANC inhibitory nerves, was mainly involved in the regulation of enteric nerve responses to EFS in the PIAS.

A case series of children with high-risk metastatic neuroblastoma treated with a novel treatment strategy consisting of postponed primary surgery until the end of systemic chemotherapy including high-dose chemotherapy.

The aim of this study was to clarify the feasibility of a novel treatment strategy consisting of postponed primary surgery till the end of systemic chemotherapy including HDC without interruption by local therapy for neuroblastoma patients at a high risk for relapse. After induction chemotherapy, patients received double conditioning HDC consisting of thiotepa and melphalan. Radical surgery was applied to local lesions. Irradiation was not applied to any lesions. Eleven consecutive pediatric neuroblastoma patients were treated according to this strategy. Seven of 11 patients remained in complete remission for 21-171 months. This treatment strategy seems feasible and a further study is warranted.

A rare case of multiple duodenal perforations in early infancy.

Duodenal perforation in early infancy is an uncommon condition. We describe a case of duodenal perforation from suspected ulcer. A premature boy was born at the gestational age of 26 weeks with a birth weight of 764 g. The Apgar score at 1 min was 3 and at 5 min had decreased to 2. He was given intermittent mandatory ventilation for one month after the birth. Ninety-eight days after birth, the infant's abdomen became distended. A supine and cross-table lateral radiograph of the abdomen revealed massive pneumoperitoneum. An exploratory laparotomy was performed, which revealed two perforations in the anterior wall of the first portion of the duodenum. The operation procedure was direct closure and intra-abdominal drainage. On the postoperative first day, he had central urorrhagia from hematencephalon. The patient's growth after surgery has been normal, with no recurrence of duodenal ulcer.

Silencing of MYCN by RNA interference induces growth inhibition, apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification.

Although it has been suggested that the MYCN oncoprotein functions may influence tumorigenesis and patient survival in neuroblastoma, the mechanism of these functions remains unclear. To elucidate such molecular and biological mechanisms, we performed knock-down of MYCN expression using RNA interference (RNAi) method. MYCN-siRNAs (MYCN-siRNA) were transfected into the MYCN-amplified cell line NB-1. To verify the sequence specificity of the siRNA, we prepared three control groups (siRNA control group: siRNAs with no significant homology to any known sequences in human genome, mock control group: reagent and PBS, and the untransfected control group). The cells were analyzed by real-time RT-PCR, Western blotting, immunocytochemistry for gene expression. Cell proliferation activity was measured by WST-1 assay. TUNEL staining was performed to evaluate apoptosis. After the MYCN-siRNA transfection, the expression level of the MYCN mRNA was significantly reduced to 30% of those of the three control groups (p<0.05). Western blotting revealed an obvious reduction in MYCN protein level in the MYCN-siRNA group. On immunocytochemistry, intensity of nuclear staining of MYCN was weaker in the MYCN-siRNA group than in the three control groups. On WST-1 viability assay, cell proliferation after the MYCN-siRNA transfection was significantly suppressed compared to the three control groups (p<0.05). The TUNEL positive cells were frequently observed in the MYCN-siRNA group. Additionally, after the MYCN-siRNA transfection, the morphologic change which was suggestive of neuronal cell differentiation was observed and TrkA and TrkC expressions were also significantly up-regulated. Using RNAi method, the knock-down of MYCN expression induced growth-inhibition, apoptotic activity and cell differentiation in MYCN-amplified NB-1 cell line.

In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors.

Mutations of beta-catenin have been identified in the majority of pediatric hepatic malignancies, including hepatoblastoma (HB) and hepatocellular carcinoma (HCC), suggesting its important contribution in hepatic tumorigenesis in this age group. However, the role of beta-catenin/canonical Wnt signaling pathway in the neoplastic growth of cancer cells has not been directly studied. To address beta-catenin's capability in maintaining the malignant phenotype in established pediatric HB and HCC cell lines, HuH-6 and HepG2, harboring mutated and overexpressed beta-catenin, we carried out a series of in vitro analyses through a transfection of short interfering RNAs (siRNAs) to generate a loss-of-function model. HuH-7, another HB cell line derived from a pediatric patient without a stabilizing mutation was used for comparison. RNA interference successfully manipulated the degradation of overexpressed beta-catenin. In all cell lines, beta-catenin mRNA was suppressed by 80-90% after 48 h of transfection, and a reduction of its protein expression was demonstrated. In HuH-6 and HepG2, the pre-existing beta-catenin nuclear accumulation disappeared and reductions of beta-catenin downstream target genes, c-myc and cyclinD1, were also evidenced after the treatment. The in vitro proliferation of both cell lines was transiently inhibited. In contrast, the suppression of beta-catenin in HuH-7 did not lead to a significant change in the expression of target genes or cellular proliferation. Our data indicate that beta-catenin can be considered a specific target for gene therapy in pediatric hepatic tumors with mutations and overexpression of this gene.

Artificially accumulated beta-catenin inhibits proliferation and induces neurite extension of neuroblastoma cell line NB-1 via up-regulation of trkA.

Recent evidence suggests an association between up-regulation of beta-catenin/Wnt signaling pathway and neuronal differentiation of neuroblastoma. We overexpressed beta-catenin into a human neuroblastoma cell line NB-1 and observed its effect on cellular morphology, growth potential and alteration in a known differentiation related gene, trkA. Expression plasmids containing wild-type and mutated forms of beta-catenin gene were transfected into NB-1 cells, using liposome-based transfection method. The mutated forms were a deletion of three nucleotides of codon 45 and a large deletion involving the whole exon 3. In the transient transfection model, cell viability assay demonstrated significant negative effect of mutated beta-catenin transfection, but not wild-type, on the cell proliferation. To investigate impacts of beta-catenin overexpression in detail, a stable transfection model was established. Clones with comparable expression of beta-catenin at the mRNA level were selected. Only the selected clones with mutated form of beta-catenin exhibited neurite extension pattern and stunned cell proliferation, in association with higher accumulation of total cellular beta-catenin protein as evidenced by Western blot and immunocytochemistry. Cell cycle progression demonstrated significantly higher G0-G1 fraction in each stable cell clone with beta-catenin expression plasmid. In addition, retarded G1/S transition was observed exclusively in the cell clones with mutated form. Concomitantly with overexpressed beta-catenin, up-regulations of trkA and Ha-ras were also identified. Our study suggests a potential availability of beta-catenin/Wnt signaling pathway as a target of molecular manipulation for treatment of high-risk neuroblastoma and a potential association between the pathway and the trkA/neurotrophin cascades.

Non-random p53 mutations in pediatric undifferentiated (embryonal) sarcoma of the liver.

Undifferentiated (embryonal) sarcoma of the liver (USL) is a rare hepatic tumor in children. Its pathogenesis is largely unknown, but lines of evidence suggest common links to that of mesenchymal hamartoma of the liver (MH). Previously, we found a p53 mutation in a case of pediatric USL. Recently, there was another published report, demonstrating further evidence of p53 alterations in the adult cases. In this study, we analyzed in three cases of pediatric USL and two cases of MH by using PCR-SSCP and direct sequencing technique. The study identified missense mutations in all three cases of USL, but none of MH. The mutations were found specifically in tumor tissue and not detected in the surrounding normal hepatic tissue. Mutation points were localized in exon 7 (Gly245Ser), exon 6 (Arg196Pro), and exon 8 (Arg273Pro), respectively. Immunohistochemical study of p53 protein expression revealed strong immunoreactivity in cases of USL and negative staining in MH. In summary, this study provided a novel data suggesting that mutations of p53 in pediatric USL are not random genetic events and highly possible to be involved in its tumorigenesis.

Staged approach to the urachal cyst with infected omphalitis.

Embryonal urachus exists as a cord-like structure between the urinary bladder and the umbilicus. In some cases of urachal cysts at the level of the navel, no special symptoms are detected during childhood, but spontaneous drainage at the navel may occur after adolescence, which is called an infected urachal cyst. Especially in cases accompanied by infected omphalitis, no constant opinion has been established to choose either initially curative resection or staged incision. In this study, we evaluated the characteristics of patients with urachal cysts who underwent the staged approach. Twenty patients (14 men and 6 women) with urachal cysts complicated by infected omphalitis were treated in our hospital. Staged surgery was performed for 18 patients. Neither recurrent omphalitis nor subsequent urachal carcinoma was observed. It is desirable that urachal cyst accompanied by intractable omphalitis should be treated by conservative therapy, conducted image diagnosis, and chosen staged surgery.

Epigenetic differences between Wilms' tumours in white and east-Asian children.

BACKGROUND: Variations in the international incidence of Wilms' tumour might be due to genetic factors. The maternal insulin-like growth factor 2 gene (IGF2) is imprinted in normal tissues, whereas in some Wilms' tumours and other tumour types the imprint is lost, leading to biallelic transcription of IGF2. We investigated whether the difference in incidence of Wilms' tumour between children of east-Asian descent and white children is due to variations in proportion of tumours with loss of IGF2 imprinting (IGF2 LOI). METHODS: We assessed IGF2 LOI by use of an ApaI polymorphism in IGF2 exon 9 or quantitative PCR measuring DNA methylation of the H19 and KvDMR1 alleles. The frequencies of perilobar nephrogenic rests associated with Wilms' tumour were assessed histologically in Japanese children and children of white and east-Asian descent. FINDINGS: IGF2 LOI was present in Wilms' tumours from predominantly white children from New Zealand (13 of 41 tumours) but absent in tumours from Japanese children (0 of 21 tumours; difference in proportions 0.32, 95% CI 0.07-0.52). Frequency of perilobar nephrogenic rests accompanying tumours from white American children (1192 of 5002, 24%) was significantly higher than in Japanese (one of 56, 1%, difference in proportions 0.22, 95% CI 0.14-0.25) and east-Asian American children (seven of 92, 8%, 0.16, 0.09-0.21). INTERPRETATION: Wilms' tumours in the east-Asian population rarely arise from the IGF2 LOI mechanism frequently noted in white patients. Our findings that IGF2 LOI and perilobar nephrogenic rests associated with this mechanism arise at low frequency in Japanese and east-Asian American children lend support to this conclusion. Variation in frequency of this epigenetic mechanism provides one explanation for the difference in incidence of Wilms' tumour between populations.

Detection of a novel alteration of the Axin gene in various pediatric neoplasms.

The Wnt signaling pathway is conserved in various species from worms to mammals, and plays critical roles in development, cellular proliferation and differentiation. As part of Wnt signal transduction, the function of the Axin complex is inhibited, leading to the accumulation of beta-catenin protein. Axin gene mutations have been detected in several kinds of human cancers. In this study, we investigated Axin gene alterations in a series of 58 pediatric neoplasms including neuroblastomas, teratomas, rhabdomyosarcomas. Forty-eight non-tumor tissues were used as a control series to compare gene alterations and their frequencies between tumors and normal tissues. The whole coding region of the Axin gene was examined by PCR-SSCP method using 24 sets of the primers. Samples revealing aberrant band patterns were subjected to direct sequencing analysis. In total, we identified six variants in the exons and four intronic nucleotide substitutions in the tumor series. Similar sequence variants except a rare sequence variant at codon 98 (CCG right curved arrow CTG) were observed in the control series and these were regarded as non-pathogenetic polymorphisms. Our results indicated that a tumor-associated mutation of the Axin gene is generally a rare event in our series of pediatric neoplasms.

Hotspot mutations of BRAF gene are not associated with pediatric solid neoplasms.

BRAF (v-raf murine sarcoma viral oncogene homolog B1) activating mutations in a high proportion of melanomas and in a small fraction of other cancers have been recently reported. All the presented mutations of BRAF are located in exons 11 and 15, and the hotspot mutation at codon 599 accounts for 87% of BRAF mutations. Because the mutational status is unclear in pediatric solid neoplasms, we screened BRAF mutations comprehensively in our tumor series presented in childhood. Two pairs of primers were designed to amplify exons 11 and exon 15, respectively, and 181 tumor samples (65 neuroblastomas, 23 Wilms tumors, 19 hepatoblastomas, 16 teratomas, 17 rhabdomyosarcomas, 13 ganglioneuromas, etc.) were investigated by PCR-SSCP method. On agarose gel electrophoresis, amplified PCR fragments showed no size-altered changes in exons 11 and 15, and SSCP analysis revealed uniform band patterns in both exons. Subsequent direct sequencing of selected 10 samples confirmed only normal sequences without any nucleotide substitutions. The current study represents the first genetic analysis of the BRAF gene in pediatric solid tumors. Our data suggest that mutations of BRAF gene as a mechanism of tumorigenesis is unlikely to be associated with most childhood neoplasms.