MYC-family protein overexpression and prominent nucleolar formation represent prognostic indicators and potential therapeutic targets for aggressive high-MKI neuroblastomas: a report from the children's oncology group.

Neuroblastomas with a high mitosis-karyorrhexis index (High-MKI) are often associated with MYCN amplification, MYCN protein overexpression and adverse clinical outcome. However, the prognostic effect of MYC-family protein expression on these neuroblastomas is less understood, especially when MYCN is not amplified. To address this, MYCN and MYC protein expression in High-MKI cases (120 MYCN amplified and 121 non-MYCN amplified) was examined by immunohistochemistry. The majority (101) of MYCN-amplified High-MKI tumors were MYCN(+), leaving one MYC(+), 2 both(+), and 16 both(-)/(+/-), whereas non-MYCN-amplified cases appeared heterogeneous, including 7 MYCN(+), 36 MYC(+), 3 both(+), and 75 both(-)/(+/-) tumors. These MYC-family proteins(+), or MYC-family driven tumors, were most likely to have prominent nucleolar (PN) formation (indicative of augmented rRNA synthesis). High-MKI neuroblastoma patients showed a poor survival irrespective of MYCN amplification. However, patients with MYC-family driven High-MKI neuroblastomas had significantly lower survival than those with non-MYC-family driven tumors. MYCN(+), MYC-family protein(+), PN(+), and clinical stage independently predicted poor survival. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and neuroblastoma growth. Together, MYC-family protein overexpression and PN formation should be included in new neuroblastoma risk stratification and considered for potential therapeutic targets.

Age-dependent prognostic effect by Mitosis-Karyorrhexis Index in neuroblastoma: a report from the Children's Oncology Group.

Prognostic effects of Mitosis-Karyorrhexis Index (MKI) used in the International Neuroblastoma Pathology Classification (INPC) are age-dependent. A total of 4,282 neuroblastomas reviewed at the Children's Oncology Group Neuroblastoma Pathology Reference Laboratory (8/1/2001-3/31/2012) included 2,365 low-MKI (L-MKI), 1,068 intermediate-MKI (I-MKI), and 849 high-MKI (H-MKI) tumors. Cox proportional hazards models were fit to determine age cut-offs at which the relative risk of event/death was maximized in each MKI class. Backward-selected Cox models were fit to determine the prognostic strength of the age cut-offs for survival in the presence of other prognostic factors. The age cut-offs used in the INPC for L-MKI tumors (<60 months, n  =  2,710, 84.0% ± 1.0% event-free survival [EFS], 93.8 ± 0.7% overall survival [OS] vs ≥60 months, n  =  195, 49.8% ± 4.6% EFS, 71.7% ± 4.1% OS; P < 0.0001) and I-MKI tumors (<18 months, n  =  568, 83.8% ± 2% EFS, 93.7% ± 1.3% OS vs ≥18 months, n  =  500, 51.4% ± 2.9% EFS, 66.7% ± 2.7% OS; P < 0.0001) were within the effective range for distinguishing prognostic groups. As for H-MKI tumors (no cut-off age in the INPC, 51.0% ± 2.2% EFS, 64.4% ± 2.1% OS), a new cut-off of 3-4 months was suggested (<4 months, n  =  38, 82.3% ± 8.4% EFS, 81.8% ± 8.5% OS vs ≥4 months, n  =  811, 49.6% ± 2.2% EFS, 63.7% ± 2.1% OS, P  =  0.0034 and 0.0437, respectively). Multivariate analyses revealed that cut-offs of 60 and 18 months for L-MKI and I-MKI tumors, respectively, were independently prognostic. However, the cut-off of 4 months for H-MKI tumors did not reach statistical significance in the presence of other factors. The age cut-offs for MKI classes (60 months for L-MKI, 18 months for I-MKI, no cut-off for H-MKI) in the current INPC are reasonable and effective for distinguishing prognostic groups with increased risk of event/death for older patients.

Identification of a KEAP1 germline mutation in a family with multinodular goitre.

BACKGROUND: The familial clustering of multinodular goitres (MNGs) with a dominant mode of inheritance has been repeatedly reported. Linkage studies have revealed several genetic loci responsible for familial MNG; however, most of the causative variants remain unknown. METHODS AND RESULTS: Through linkage analysis using single-nucleotide polymorphism markers, we identified a new MNG locus on 19p13.2-q12 in a five-generation Japanese MNG family. Subsequent mutation searches focusing on the candidate 25-Mb region of chromosome 19 identified a heterozygous mutation, c.879_880delinsA, p.Asp294Thr, fs*23, in exon 3 of the KEAP1, which plays a central role in the cytoprotection pathway against oxidative stress. Reverse transcriptase-PCR analysis showed low expression of wild type KEAP1 accompanied by no transcription product of mutant allele in the normal and goitre region of thyroid tissues obtained from the proband. In agreement with previous studies showing that KEAP1 negatively regulates NFE2L2, the NFE2L2 target genes GSTA4 and GCLC were up-regulated in the thyroid tissues of the patient. CONCLUSIONS: This study identified the first KEAP1 mutation in MNG. The results provide insights into the pathogenesis of goitre which develops in the organ continuously exposed to oxidative stress during hormone synthesis.

The utility of muscle sparing axillar skin crease incision for pediatric thoracic surgery.

BACKGROUND: Posterolateral or standard axillar incisions for the pediatric thoracic surgery are occasionally associated with poor motor as well as cosmetic results, including chest deformities and large surgical scars. A muscle sparing axillar skin crease incision (MSASCI) was initially proposed by Bianchi et al. (in J Pediatr Surg 33:1798-1800, 1998) followed by Kalman and Verebely (in Eur J Pediatr Surg 12:226-229, 2002) resulting in satisfactory cosmetics. However, they performed operations through the third or fourth intercostals space (ICS), therefore the target organs were restricted in the upper two-thirds of the thoracic cavity. PATIENTS AND METHODS: Thoracic surgeries were performed using MSASCI in 27 patients (1-day to 9-year old). There were ten patients with esophageal atresia, seven with congenital cystic adenomatoid malformation, five with pulmonary sequestration, two with mediastinal neuroblastoma, two with right diaphragmatic hernia, and one with pulmonary hypertension. A thoracotomy was performed through the appropriate ICS (from third to eighth). RESULTS: In all patients, the expected procedures, including pulmonary lower lobectomy, were successfully performed by MSASCI throughout the thoracic cavity. A good operational field was easily obtained in neonates and infants. Most of the patients achieved excellent motor and aesthetic outcomes. CONCLUSIONS: MSASCI may become the standard approach for the thoracic surgery for small children.

Correlation between the number of segmental chromosome aberrations and the age at diagnosis of diploid neuroblastomas without MYCN amplification.

BACKGROUND: In neuroblastomas (NBs) without MYCN amplification, segmental chromosome aberrations SCAs such as 1p loss, 11q loss, and 17q gain have been suggested to be associated with the prognosis of the patients. We assessed the correlation between the number of SCAs and other biological factors in primary NBs samples. METHOD: The status of SCAs in 54 primary NBs samples was analyzed using the single-nucleotide polymorphism (SNP) array (Human CMV370-Duo; Illumina, San Diego, CA). The status of MYCN amplification was determined by an SNP array and the fluorescence in situ hybridization method. The DNA ploidy was determined by flow cytometry. RESULTS: Nine of 54 samples showed MYCN amplification. All 9 samples with MYCN amplification and 20 of 45 samples without MYCN amplification showed diploidy/tetraploidy, and the other 25 samples without MYCN amplification showed aneuploidy. The most frequent SCAs were 17q gain (26/54; 48.1%) and 11q loss (16/54; 29.6%), followed by 1p loss (15/54; 27.8%). The number of SCAs in diploidy/tetraploidy NBs without MYCN amplification (7.00 ± 4.67) was higher than that in NBs with MYCN amplification (4.78 ± 2.82) and in aneuploid NBs (1.64 ± 2.78) (P < .05). In diploid/tetraploid NBs without MYCN amplification, there was a significant difference between an age at diagnosis less than 12 months (n = 7) and over 12 months (n = 13) (4.14 ± 3.63 vs 8.54 ± 4.54; P = .04). Moreover, the number of SCAs correlated with the age at diagnosis in diploid/tetraploid samples without MYCN amplification (r = 0.70, P = .0006). In NBs with MYCN amplification, the number of SCAs did not correlate with the age at diagnosis. CONCLUSION: The number of SCAs significantly increased in proportion to age at diagnosis in diploid/tetraploid NBs without MYCN amplification. The increase in the number of these SCAs may play an important role in the prognosis of patients without MYCN amplification over 12 months of age.

Usefulness of exchanging a tunneled central venous catheter using a subcutaneous fibrous sheath.

OBJECTIVES: The reserve of the venous route to the central veins is important for long-term parenteral nutrition (PN). Frequent catheter-related bloodstream infection (CRBSI) induces occlusion of the venous routes. Therefore, a modified exchange procedure using a tunneled central venous catheter (CVC) with a fibrous sheath was developed to preserve the route to the central veins. METHODS: Seven patients who required long-term PN received the modified exchange procedure and the outcome of exchanged CVC was retrospectively reviewed. RESULTS: The procedure was performed 10 times in seven patients. The venous routes were either the subclavicular or the internal jugular vein in all patients. The exchange of the catheter was due to CRBSI or occlusion in almost all patients. The mean duration of new catheter use was 296.2 days following the exchange. Four catheters continued to be used, and the remaining ones were removed. The reasons for removal were severe CRBSI and occlusion, each of which occurred in two catheterized patients, while the reason for removing the remaining catheters was because the patients no longer needed the catheters. CONCLUSION: The modified catheter exchange using fibrous sheath, even in patients with CRBSI, appears to be an effective procedure for reserving the venous route to the central veins in patients who require either long-term PN or other treatments.

Identification of TCTE3 as a gene responsible for congenital diaphragmatic hernia using a high-resolution single-nucleotide polymorphism array.

PURPOSE: Congenital diaphragmatic hernia (CDH) is a birth defect of the diaphragm associated with pulmonary hypoplasia. Although genetic factors have been suggested to play a role, the etiology of CDH is still largely unknown. In this study, we analyzed copy number variants (CNVs) using a single-nucleotide polymorphism (SNP) array to examine whether microdeletions contribute to the pathogenesis of this disease. METHODS: A total of 28 CDH patients, including 24 isolated and 4 non-isolated cases, were available. We performed CNV analysis using high-resolution SNP arrays (370K, 550K, 660K; Illumina Inc.) and CNstream software. Deletions in loci that have been suggested in previous studies to contain candidate genes affecting CDH were analyzed. RESULTS: We detected 335, 6 and 133 deletions specific for patients in 14 (350K array), 3 (550K) and 11 (660K) cases, respectively. Among these deletions, no segments included the previously suggested candidate genes with the exception of an 18-kb deletion observed in the candidate locus 6q27 in two non-isolated patients. This deleted region contains exon 4 of the t-complex-associated-testis-expressed 3 (TCTE3) gene. CONCLUSION: Because TCTE3 encodes a putative light chain of the outer dynein arm of cilia and human diseases caused by ciliary dysfunction show various phenotypes including skeletal defect, TCTE3 may be a genetic candidate influencing CDH.

Effect of insulin-like growth factors on lung development in a nitrofen-induced CDH rat model.

PURPOSE: Both the mortality and morbidity associated with congenital diaphragmatic hernia (CDH) are mainly caused by pulmonary hypoplasia and persistent pulmonary hypertension. A previous study revealed that insulin-like growth factors (IGFs) play important roles in fetal lung development. The aim of this study was to investigate the effect of IGF-1 and IGF-2 on tissue cultures of fetal hypoplastic lungs obtained from nitrofen-induced CDH model rats. METHODS: Pregnant rats were exposed to nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18 by caesarian section. Lung specimens of the CDH (+) fetus were divided into three groups; control, IGF-1, and IGF-2. The specimens from the control group were cultured in culture medium without IGFs. The IGF-1 group specimens were cultured with IGF-1 (500 ng/ml), and those in the IGF-2 group were cultured with IGF-2 (500 ng/ml). The mRNA expression of TTF-1, T1α and α-SMA were analyzed in each group using real-time RT-PCR after 24 and 48 h of incubation. Immunohistochemical staining of these markers was also assessed for each of the cultured specimens. RESULTS: There was a significant increase in the expression of both TTF-1 and T1α mRNA in the IGF-2 group, in comparison to the control group after 48 h of culture. Immunohistochemical staining revealed that the cell morphology was changed from cuboidal to squamous type in the IGF-2 group. CONCLUSIONS: An increased mRNA expression of the markers related to type 1 and 2 alveolar epithelial cells, and morphological changes in the epithelial cells were observed in the IGF-2 group. The administration of IGF-2 to nitrofen-induced hypoplastic lungs might lead to alveolar maturation, which thus results in their improved development.

The genetic and clinical significance of MYCN gain as detected by FISH in neuroblastoma.

PURPOSE: MYCN amplification (MYCN-A) is a strong prognostic factor in neuroblastoma (NB). MYCN gain which is a low level of MYCN-A as determined by FISH. It is unclear whether the MYCN gain is the pre-status of MYCN-A. This study assessed the status of MYCN gene and chromosome 2p of MYCN-A, MYCN gain and no MYCN amplification using a single nucleotide polymorphism (SNP) array, and the clinical implication of MYCN gain in NB. METHODS: The status of the MYCN gene was determined by FISH in 47 primary NB samples and the status of chromosome 2p in all cases was analyzed using an SNP array. RESULTS: 8 of the 47 cases analyzed using FISH showed MYCN-A, 7 cases showed MYCN gain and 32 cases showed no MYCN amplification. An SNP array analysis showed that only 2 of 8 cases with MYCN-A by FISH had both amplification of MYCN region and distal 2p gain and other 6 cases had amplification of the MYCN region without distal 2p gain. All 7 cases with MYCN gain by FISH had distal 2p gain without amplification of the MYCN region, and all 32 cases with no MYCN amplification by FISH demonstrated neither the amplification of the MYCN region nor the 2p gain. 5-year overall survival rate of patients with MYCN gain (n = 7, 71.4%) was not significant different from that of patients with no MYCN amplification (n = 32, 90.6%) by FISH (p = 0.11). CONCLUSIONS: These results suggested that the MYCN gain detected by FISH represents the 2p gain, and the MYCN gain is not considered to represent the pre-status of MYCN amplification.

Connexin43 plays an important role in lung development.

OBJECTIVES: Connexin43 (Cx43) is one of the proteins associated with gap junction. Connexin43 knockout mice die after birth owing to hypoplastic lungs. The purpose of this study was to analyze the hypoplastic lung of Cx43 knockout mice to clarify the role of the Cx43 during lung development. METHODS: Adult hetero Cx43 mice were mated. Newborn mice were divided into the following groups: wild, hetero, and knockout. Total RNA was extracted from the right lung, and the left lung was fixed for immunohistochemical staining. The mRNA expression of surfactant protein C, aquaporin-5, and alpha-smooth muscle actin were analyzed by reverse transcriptase polymerase chain reaction. H&E and immunohistochemical staining for those markers were performed. RESULTS: The mRNA expression of aquaporin-5, surfactant protein C, and alpha-smooth muscle actin was significantly lower in knockout mice than that in the wild and hetero mice. H&E staining in the knockout mice showed narrow airspaces and thicker interalveolar septae. Immunohistochemical staining in all markers showed the formation of alveoli to be delayed in the knockout mice. CONCLUSION: Based on these findings, Cx43 is closely related to alveolar and vascular formation during lung development.

Clinical features of Hirschsprung's disease associated with Down syndrome: a 30-year retrospective nationwide survey in Japan.

PURPOSE: To identify the clinical features in diagnosis and treatment of Hirschsprung's disease (HD) associated with Down syndrome (DS), the authors retrospectively analyzed data for patients with DS from the past 3 nationwide surveys in Japan. This survey was already approved by the Japanese Society of Pediatric Surgeons. METHODS: Patient data were collected in 3 phases-phase I (1978-1982), n = 47; phase II (1988-1992), n = 79; and phase III (1998-2002), n = 90. In total, data on 216 patients (5.6%) of 3852 were collected and analyzed. RESULTS: The incidence of DS in patients with HD was 2.9%, 7.1%, and 8.2% in phases I, II, and III, respectively, with a corresponding male/female ratio of 5:1, 2.4:1, and 5:1. The ratio of the extent of aganglionosis was nearly consistent across all phases. In phases I, II, and III, the incidence of total colonic aganglionosis was 2.1%, 0%, and 2.2%; and that of cardiovascular anomalies, 36.1%, 45.6%, and 55.6%; and that of preoperative enterocolitis, 31.0%, 26.6%, and 24.4%. The 2 most common surgical procedures were the Soave procedure, including transanal endorectal pull-through, and Duhamel procedure including Z-shaped anastomosis. The mortality rate decreased over time, from 26.1% in phase I to 11.4% in phase II and 7.8% in phase III. Almost all mortality cases were associated with cardiovascular anomalies: 54.5%, 62.5%, and 85.7% in phases I, II, and III, respectively. CONCLUSIONS: The incidence of HD with DS has increased over time. The number of male patients and cardiac anomalies has also increased in the last 10 years. Total colonic aganglionosis was rare. A marked decrease in the overall mortality rate was observed.

Hirschsprung's disease patients diagnosed at over 15 years of age: an analysis of a Japanese nationwide survey.

PURPOSE: Hirschsprung's disease (HD) is usually diagnosed in patients who are under 1 year of age, however, there are still several reports of adult HD cases. We herein analyzed the data of HD patients collected over 30 years according to a nationwide survey in Japan. METHODS: The data of HD patients over 15 years of age were thus selected in three phases, namely from 1978 to 1982, from 1988 to 1992, and from 1998 to 2002. A total of 27 patients (0.7%) out of 3,852 were thus analyzed. RESULTS: The male/female was 15/11. The age at diagnosis was as follows: 10 patients were teenagers (37.0%), 14 patients were in their 20s (51.9%), 1 patient was in his 40s (3.7%), and 2 patients were in their 50s (7.4%). The extent of aganglionosis was as follows; the lower rectum: 12 patients (44.4%), the sigmoid colon: 14 patients (51.9%), and the descending colon 1 patient. As a definitive operation, the Duhamel's procedure including Z-shaped anastomosis was performed on 14 patients (51.9%), Swenson's procedure on 5 patients (18.5%), Soave's procedure on 2 patients (7.4%), and Myectomies on 2 patients (7.4%). No mortalities were reported among these cases. CONCLUSIONS: Twenty-seven HD patients diagnosed over 15 years of age were analyzed. The number of patient diagnosed over 15 years of age has decreased over time. However, further attention is still required in adult patients who are present with persistent intestinal obstruction of unknown etiology.

Duodenal stenosis resulting from a preduodenal portal vein and an operation for scoliosis.

A preduodenal portal vein (PDPV) is known to be a rare cause of duodenal stenosis. We treated a 22-year-old male patient with malnutrition as a result of PDPV and a previously performed operation for scoliosis, who showed an improvement in quality of life after being treated with a combination of nutritional support and surgery. The patient with PDPV had been admitted to our department with duodenal stenosis, ranging from the first to third portions. He had suffered from vomiting since 1 year of age, and he developed malnutrition during the last 6-mo period after orthopedic surgery for scoliosis. The stenosis was related to both the PDPV and the previously performed operation for scoliosis. After receiving nutritional support for 6 mo, a gastrojejunostomy with Braun's anastomosis for the first portion and a duodenojejunostomy for the second and third portions were performed. The postoperative course was almost uneventful. Three months later, he was discharged and able to attend university. In patients with widespread duodenal stenosis, there may be a complicated cause, such as PDPV and duodenal stretching induced by previous spinal surgery.

Improvement in the outcome of patients with antenatally diagnosed congenital diaphragmatic hernia using gentle ventilation and circulatory stabilization.

BACKGROUND/OBJECTIVES: No definitive treatment strategy has been established for patients with an antenatal diagnosed congenital diaphragmatic hernia (AD-CDH). From 1997 to 2003 in this department fetal stabilization (FS) was administered using both morphine and diazepam via the placenta just before delivery of the fetus by cesarean section. In contrast, from 2004 to the present, a combination of gentle ventilation (GV) and a delayed operation was selected, which was performed when the patient's circulatory stabilization (CS) was achieved. PATIENTS AND METHODS: This study included 22 patients in the FS group and 16 patients in the GV + CS group, respectively. The outcomes in both groups were compared and the outcome in AD-CDH patients with a patch repaired operation, liver-up or lower lung-to-thorax transverse area ratio (L/T, <0.10) was further investigated in both groups. RESULTS: The overall survival rate (SR) was 93.8% in the GV + CS group and 59.1% in the FS group, respectively (P = 0.04). For the patients with the lower L/T, the SR was 85.7% in GV + CS group and 53.8% in the FS group (P = 0.33). Regarding the patients using a patch and liver-up, the SR in GV + CS group was better than that in the FS group (patch: FS 44.4%, GV +/- CS 87.5%, P = 0.18; liver-up: FS 57.8 and 87.5%, P = 0.30). CONCLUSION: Our strategy of using GV +/- CS might thus be considered to be more effective than that using FS in the treatment of AD-CDH patients.

Need for thiamine in peripheral parenteral nutrition after abdominal surgery in children.

BACKGROUND: Thiamine blood concentrations of pediatric patients receiving peripheral parenteral nutrition change during the postoperative period. In addition, the need to administer thiamine after surgery has not yet been fully studied in children receiving peripheral parenteral nutrition. OBJECTIVE: The objective of this prospective study is to clarify whether pediatric patients require the administration of thiamine while receiving peripheral parenteral nutrition after abdominal surgery. PATIENTS: Fifteen children were divided into 2 groups; 1 group received peripheral parenteral nutrition without thiamine after surgery (n = 7), whereas the other group received peripheral parenteral nutrition with thiamine after surgery (n = 8). In both groups, thiamine blood concentrations were measured on the preoperative day, and changes in thiamine concentration over time were measured during the starvation period from the first to the fifth postoperative day. RESULTS: Preoperative thiamine blood concentrations were within the normal range in both groups. In the group receiving peripheral parenteral nutrition without thiamine, the thiamine concentration gradually decreased with time after the operation, whereas the concentration remained within the normal range in the group receiving peripheral parenteral nutrition with thiamine. Among the 7 patients receiving peripheral parenteral nutrition without thiamine, the thiamine concentration in 3 patients was below the normal range on the fifth postoperative day. CONCLUSION: During the starvation period after abdominal surgery, thiamine blood concentrations decreased in pediatric patients receiving peripheral parenteral nutrition without thiamine. Therefore, clinicians treating pediatric patients should add thiamine to the peripheral parenteral nutrition solution during the short starvation period after abdominal surgery.

A new method of transanal catheter fixation for neonates with long segment-type Hirschsprung's disease.

We managed long-segment aganglionosis in two neonates by performing colonic irrigation through an indwelling transanal catheter for 65 and 30 days, respectively, until a laparoscopy-assisted primary pull-through operation could be performed. The catheter was fixed by tying it to the tubes, criss-crossed at the anus, and securing the opposite ends of the tubes away from the diaper. This form of management with our devised method of transanal catheter fixation and simple frequent tube washouts improved the preoperative quality of life of both the babies and their parents remarkably.