Ultrasound for the evaluation of the urethra in pediatric patients.

Urethral lesions in pediatric patients can be visualized using ultrasonography. Therefore, sonographers and physicians should be familiar with the technique.

Point-of-Care Ultrasound for Mimicker Lesions of Incarcerated Inguinal Hernia.

Inguinal hernia is the most common surgical disease in pediatric patients, and urgent intervention such as manual reduction is needed for incarcerated inguinal hernia. Torsion of undescended testes, inguinal herniated ovarian torsion, and purulent lymphadenitis are mimickers of this condition. If these mimicker lesions are misdiagnosed as incarcerated inguinal hernia, manual reduction usually fails, and edematous and erythematous changes may occur in these mimicker lesions due to manual reduction. For physicians in the emergency department, prompt decisions and familiarity with the sonographic appearance of different contents within an inguinal hernia are important to accurately diagnose these mimickers. In this case series, we present sonographic images of a typical case of incarcerated inguinal hernia (an 11-month-old male with right incarcerated inguinal hernia) and three cases of mimicker lesions (a 7-month-old female with herniated ovarian torsion, a 7-year-old boy with undescended testicular torsion, and a 2-month-old male with purulent lymphadenitis). The incidence of incarcerated inguinal hernia is reported to be higher in males (80%), on the right side (60%), and in infants and toddlers. This information is important for diagnosing mimicker lesions. In addition, to prevent manual reduction in mimicker diseases, point-of-care ultrasound before manual reduction in suspected cases of incarcerated inguinal hernia is important.

Prediction of testicular atrophy based on sonographic findings immediately after successful manual detorsion.

Purpose: To investigate and determine the sonographic findings obtained from manually distorted testes to predict testicular atrophy following manual detorsion. Materials and methods: Twenty-two patients who had been diagnosed with testicular torsion and undergone manual detorsion were included. These patients were classified according to the presence or absence of testicular atrophy. The duration of symptoms, presence or absence of hyperperfusion within the entire affected testis, and echogenicity (homogeneous or heterogeneous) within the affected testis were compared using the Mann-Whitney U-test or Fisher's exact test, as appropriate. Results: Testicular atrophy was detected in seven patients. There was a significant difference in the frequency of hyperperfusion within the entire affected testis (with atrophy [present/absent] vs. without atrophy [present/absent] = 0/7 vs. 8/7, P = 0.023) between patients with and without testicular atrophy. No significant differences in the duration of symptoms (with atrophy vs. without atrophy = 7 ± 3.3 h vs. 4.7 ± 3.6 h, P = 0.075) or frequency of echogenicity within the testis (with atrophy [heterogeneous/homogeneous] vs. without atrophy [heterogeneous/homogeneous] = 2/5 vs. 2/13, P = 0.565) were observed between the groups. Conclusions: This small cohort study suggests that the presence of hyperperfusion within the entire affected testis immediately after successful manual detorsion is useful in predicting the avoidance of testicular atrophy.

Recovery of bowel function after transperitoneal or retroperitoneal laparoscopic pyeloplasty. A multi-center study.

AIM: To document the recovery of bowel function (BF) in children after transperitoneal (TP) or retroperitoneal (RP) laparoscopic pyeloplasty. METHODS: Data were obtained retrospectively from four centers between 2008 and 2019 for TP (n = 51) and RP (n = 58). Each surgeon chose which technique to perform. RESULTS: Subject demographics were not significantly different. Differences in operative times were not significant (RP: 241 min versus TP: 225 min). Mean duration/requirement for postoperative epidural/intravenous analgesia were not significantly different (TP: 1.4 days versus RP: 1.3 days) and (TP: 66.7% versus RP: 67.2%), respectively. Postoperative nasogastric (NG) intubation was more common in RP (TP: 19.6% versus RP: 44.8%; p < .05). NG aspiration (TP: 0.15 mL/kg/hr versus RP: 0.16 mL/kg/hr), nausea (TP: 31.4% versus RP: 17.2%), and vomiting (TP: 19.6% versus RP: 15.5%) were not significantly different. There were no perioperative complications (including ileus). Abdominal distention was problematic in one case per group (TP: 2.0% versus RP: 1.7%). Times for oral liquid (TP: 0.69 day versus RP: 0.83 day), solid food (TP: 0.88 day versus RP 1.07 days), and the first bowel movement (TP: 2.86 days versus RP: 2.79 days), were not significantly different. CONCLUSIONS: BF recovery would appear to be consistent, independent of technique.

Therapeutic potential of mature adipocyte-derived dedifferentiated fat cells for inflammatory bowel disease.

PURPOSE: Our previous studies demonstrated that mature adipocyte-derived dedifferentiated fat (DFAT) cells possess similar multipotency as mesenchymal stem cells. Here, we examined the immunoregulatory potential of DFAT cells in vitro and the therapeutic effect of DFAT cell transplantation in a mouse inflammatory bowel disease (IBD) model. METHODS: The effect of DFAT cell co-culture on T cell proliferation and expression of immunosuppression-related genes in DFAT cells were evaluated. To create IBD, CD4+CD45RBhigh T cells were intraperitoneally injected into SCID mice. One week later, DFAT cells (1 × 105, DFAT group) or saline (Control group) were intraperitoneally injected. Subsequently bodyweight was measured every week and IBD clinical and histological scores were evaluated at 5 weeks after T cell administration. RESULTS: The T cell proliferation was inhibited by co-cultured DFAT cells in a cell density-dependent manner. Gene expression of TRAIL, IDO1, and NOS2 in DFAT cells was upregulated by TNFα stimulation. DFAT group improved IBD-associated weight loss, IBD clinical and histological scores compared to Control group. CONCLUSION: DFAT cells possess immunoregulatory potential and the cell transplantation promoted recovery from colon damage and improved clinical symptoms in the IBD model. DFAT cells could play an important role in the treatment of IBD.

Intestinal Failure-Associated Liver Disease and Eicosapentaenoic Acid/Arachidonic Acid Ratio.

Intestinal failure-associated liver disease (IFALD) is a fatal complication of short bowel syndrome managed with parenteral nutrition. A clinical cohort study reported the usefulness of parenteral administration of fish-derived omega-3 fatty acids in improving IFALD; however, no biomarker has been developed as yet. The authors report the case of a preterm infant with IFALD complicated by extensive short bowel syndrome. Intravenous administration of omega-3 fatty acids were introduced using Omegaven®at the age of 4 mo for IFALD. The IFALD improved with an increase in Eicosapentaenoic acid (EPA)/ Arachidonic acid (AA) ratio (from 0.08 to 1.99) 7 d after the intravenous treatment. It is important to administer omega-3 fatty acids intravenously at an early stage for IFALD associated with extensive short bowel syndrome. A low EPA/AA ratio may be a serum marker of disease activity in IFALD.

Obliterative cholangiopathy in acquired cystic biliary atresia type III after cyst perforation: a case report.

BACKGROUND: In biliary atresia, the disease process of obliterative cholangiopathy may begin in the perinatal period; however, no chronological evidence exists on how the cholangiopathy progresses to biliary obliteration. This is the first acquired case with the final diagnosis of type III cystic biliary atresia with an extrahepatic biliary cyst which showed the progression of obliterative cholangiopathy in chronological order after birth. CASE PRESENTATION: An 81-day-old girl presented with acute abdominal distress due to bilious peritonitis caused by biliary cyst perforation, for which she underwent emergency biliary drainage. Postoperative images showed a dilated common bile duct and hepatic ducts bilaterally, with flow of the contrast medium to the duodenum through the dilated common bile duct. Biochemistry of the bile collected during and after the operation revealed elevated levels of pancreatic enzymes in the bile from the gallbladder. The patient was diagnosed as having a congenital choledochal cyst and underwent laparotomy at 120 days of age which revealed that she had pancreaticobiliary maljunction. The biliary cyst was resected at the narrow portion just above the junction with the main pancreatic duct. During dissection up to the hepatic hilum, we found that the hilar hepatic ducts were bilaterally replaced by fibrous tissue and were obstructed, leading to a diagnosis of type III a1, µ biliary atresia. The fibrous tissue was excised, and hepatic portoenterostomy was performed according to the Kasai procedure. The patient's postoperative course was uneventful and the jaundice resolved within 1 month. She has had normal liver function tests with no episode of cholangitis for 3 years after discharge. CONCLUSIONS: We demonstrated the process of acquired type III biliary atresia in a patient with cystic biliary atresia and biliary cyst perforation. To the best of our knowledge, this is the first case of acquired cystic biliary atresia showing chronological progression of the course of obliterative cholangiopathy, providing a better understanding of the development of type III biliary atresia as an acquired disease.

Sutureless enterostomy for extremely low birth weight infants.

OBJECTIVE: In recent years, improved survival rates of extremely low birth weight infants (ELBWIs) have led to an increasing number of enterostomy performed for those with meconium obstruction of prematurity (MOP)1,2, spontaneous intestinal perforation (SIP)3,4. To prevent serious stoma-related complications such as stoma side perforation, prolapse, fall and surgical site infection, we introduce our new "sutureless enterostomy" technique. METHODS: We present the procedures in detail. We reviewed the medical records of twelve patients who underwent "sutureless enterostomy" in our neonatal intensive care unit from 2007 to 2013. Patient attributes, surgery-related items, stoma-related complications and outcomes were investigated. RESULTS: Mean birth weight was 671±158g (mean±S.D.). Six cases of MOP, three cases of SIP and three cases of NEC were diagnosed. Mean operative time was 75±35min (mean±S.D.) None of them presented any of early stoma-related complications (necrosis, fall, and surgical site infection). However the parastomal hernia occurred in one patient as late complication. Three deaths occurred postoperatively as a result of exacerbations of their conditions. CONCLUSIONS: Based on our preliminary observations, our new "sutureless enterostomy" was done safely and reduced the risk of stoma-related complications. It may be an ideal procedure for the ELBWI with MOP or SIP.

Depletion of TFAP2E attenuates adriamycin-mediated apoptosis in human neuroblastoma cells.

Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system and accounts for approximately 15% of all pediatric cancer-related deaths. To newly identify gene(s) implicated in the progression of neuroblastoma, we investigated aberrantly methylated genomic regions in mouse skin tumors. Previously, we reported that TFAP2E, a member of activator protein-2 transcription factor family, is highly methylated within its intron and its expression is strongly suppressed in mouse skin tumors compared with the normal skin. In the present study, we analyzed public data of neuroblastoma patients and found that lower expression levels of TFAP2E are significantly associated with a shorter survival. The data indicate that TFAP2E acts as a tumor suppressor of neuroblastoma. Consistent with this notion, TFAP2E-depleted neuroblastoma NB1 and NB9 cells displayed a substantial resistance to DNA damage arising from adriamycin (ADR), cisplatin (CDDP) and ionizing radiation (IR). Silencing of TFAP2E caused a reduced ADR-induced proteolytic cleavage of caspase-3 and PARP. Of note, compared with the untransfected control cells, ADR-mediated stimulation of CDK inhibitor p21WAF1 was markedly upregulated in TFAP2E­knocked down cells. Therefore, our present findings strongly suggest that TFAP2E has a pivotal role in the regulation of DNA damage response in NB cells through the induction of p21WAF1.

Timing and outcome of stoma closure in very low birth weight infants with surgical intestinal disorders.

PURPOSE: Very low birth weight infants (VLBWIs) are at risk of surgical intestinal disorders including necrotizing enterocolitis (NEC), focal intestinal perforation (FIP), and meconium-related ileus (MRI). We conducted this study to verify whether the timing of stoma closure and that of enteral nutrition establishment after stoma closure in VLBWIs differ among the most common disorders. METHODS: A retrospective multicenter study was conducted at 11 institutes. We reviewed the timing of stoma closure and enteral nutrition establishment in VLBWIs who underwent stoma creation for intestinal disorders. RESULTS: We reviewed the medical records of 73 infants: 21 with NEC, 24 with FIP, and 25 with MRI. The postnatal age at stoma closure was 107 (28-359) days for NEC, 97 (25-302) days for FIP, and 101 (15-264) days for MRI (p = 0.793), and the postnatal age at establishment of enteral nutrition was 129 (42-381) days for NEC, 117 (41-325) days for FIP, and 128 (25-308) days for MRI (p = 0.855). The body weights at stoma closure were 1768 (620-3869) g for NEC, 1669 (1100-3040) g for FIP, and 1632 (940-3776) g (p = 0.614) for MRI. There were no significant differences among the three groups. CONCLUSIONS: The present study revealed that the time and body weights at stoma closure and the postoperative restoration of bowel function in VLBWIs did not differ among the three diseases.

Risk factors for surgical intestinal disorders in VLBW infants: Case-control study.

BACKGROUND: Very low-birthweight (VLBW) infants (VLBWI) are at increased risk for surgical intestinal disorders including necrotizing enterocolitis (NEC), focal intestinal perforation (FIP) and meconium-related ileus (MRI). The aim of this study was to identify disease-specific risk factors for surgical intestinal disorders in VLBWI. METHODS: A retrospective multicenter case-control study was conducted at 11 institutes. We reviewed VLBWI who underwent laparotomy for intestinal disorders including perforation and intractable bowel obstruction. The surgical disorders were classified into four categories (NEC, FIP, MRI, others) based on the macroscopic findings at operation. In order to identify risk factors, two matched controls for each subject were chosen based on gestational age and birthweight. OR and 95%CI were calculated using a conditional logistic regression model and a multivariate model. RESULTS: A total of 150 cases (NEC, n = 44; FIP, n = 47; MRI, n = 42; others, n = 17) and 293 controls were identified. The cases and controls were similar in terms of gestational age and birthweight (cases/controls, 26.7 ± 2.5/26.5 ± 2.6 weeks; 790 ± 256/795 ± 257 g). On multivariate modeling, disease-specific risk factors were as follows: female (OR, 0.23; 95%CI: 0.06-0.89), respiratory distress syndrome (OR, 35.7; 95%CI: 2.48-514) and patent ductus arteriosus (OR, 10.9; 95%CI: 1.51-79.3) for NEC; outborn delivery (OR, 5.47; 95%CI: 1.48-20.2) for FIP; and twin pregnancy (OR, 4.25; 95%CI: 1.06-17.1), PROM (OR, 6.85; 95%CI: 1.33-35.4) and maternal steroid (OR, 0.23; 95%CI: 0.07-0.79) for MRI. CONCLUSIONS: Different risk factors were identified for NEC, FIP and MRI, suggesting that each disease has a different etiology, and that different strategies are required to prevent these diseases.

Establishment of a new anal sphincter injury model in rats based on cardiotoxin.

BACKGROUND/PURPOSE: Mainstream models for anal sphincter injury use large animals. We developed a simple and stable anal sphincter injury model in a small animal (i.e., rats) to obtain manometry measurements by using a miniaturized probe and applying cardiotoxin. METHODS: The histological structure of the anal canal was evaluated by using manometry in normal rats (n=40). We damaged the internal and external anal sphincters by locally administering snake poison (cardiotoxin; 20 uM, 100µL 8 points). We evaluated the anal canal function through manometry measurements (n=5) and examined the histology using hematoxylin-eosin staining (at each time point, n=3; total n=15). RESULTS: The manometry parameters and structure of the anal canal of normal rats were similar to those of humans, because rats have resting pressure, rectoanal reflex in the manometry, and an external and internal anal sphincter. After inducing injury, the following findings were observed: rhythmic wave loss and a remarkable reduction in the anal sphincter resting pressure; and local bleeding and advanced infiltration of the inflammatory cells (day 1) and the loss of muscle fibers (day 3). CONCLUSION: This new rat model will contribute to increasing the knowledge on the anal canal.

Outcome in VLBW infants with surgical intestinal disorder at 18 months of corrected age.

BACKGROUND: Surgical intestinal disorders, such as necrotizing enterocolitis (NEC), focal intestinal perforation (FIP), and meconium-related ileus (MRI), are serious morbidities in very low-birthweight infants (VLBWI). The aim of this study was to compare the composite outcomes of death or neurodevelopmental impairment (NDI) in VLBWI with surgical intestinal disorders and assess independent risk factors for death and NDI at 18 months of corrected age. METHODS: A retrospective matched-cohort study was conducted at 11 institutes. We included VLBWI who had undergone laparotomy for NEC, FIP, and MRI. Two control subjects were chosen for every surgical patient and matched for gestational age and birthweight to form the comparison group. Death and neurodevelopmental outcome at 18 months of corrected age were evaluated. RESULTS: The number of infants in the NEC, FIP, MRI, and control groups was 44, 47, 42, and 261, respectively. In-hospital mortality was higher in infants with NEC and MRI relative to those in the control group (P < 0.001). The incidence rate for NDI at 18 months of corrected age was higher in infants with MRI relative to those in the control group (P = 0.021). On logistic regression analysis, low gestational age, male sex, small for gestational age, intraventricular hemorrhage, and MRI were associated with increased risk of death or NDI at 18 months of corrected age. CONCLUSIONS: NEC and MRI were associated with in-hospital mortality, and MRI was associated with NDI or death at 18 months of corrected age.

Evaluation of the efficacy of incision and drainage versus hainosankyuto treatment for perianal abscess in infants: a multicenter study.

PURPOSE: We retrospectively compared the short-term outcomes between incision and drainage (ID) and hainosankyuto (TJ-122, Tsumura & Co, Tokyo, Japan) treatment for perianal abscess (PA) in infants. METHODS: We retrospectively examined 48 consecutive patients (median age 129 days; range 19-330 days) who presented with PA over a 3 year period. Group 1 comprised 26 patients who were treated with ID at presentation, and Group 2 comprised 22 patients who were treated with oral TJ-122 at presentation; oral treatment was continued until the disappearance of purulent discharge and resolution of induration at the abscess site. RESULTS: PAs were identified in all 48 patients at presentation. The median duration of follow-up was 26 months (range 13-40 months). At presentation, there were no differences in the gender, age, birth weight, duration of symptoms, skin erosion or prevalence of diarrhea between the two groups. Purulent discharge resolved within a median period of 26 days (range 7-42 days) in Group 2, but persisted for 40 days (range 4-196 days) in Group 1. The induration resolved within a median period of 39 days (range 7-91 days) in Group 2, but persisted for 70 days (range 4-308 days) in Group 1 (p = 0.04). CONCLUSIONS: TJ-122 treatment was more beneficial than ID in treating PA in infants.

Effectiveness of Interval Appendectomy After Conservative Treatment of Pediatric Ruptured Appendicitis with Abscess.

The management of patients with acute perforated appendicitis with abscess is controversial. The aim of the present study was to assess the outcomes of treatment in patients with this condition. We retrospectively analyzed 31 patients (16 men and 15 women with a mean age of 8.4 years) with appendicitis presenting with abscess. Patients were divided into two groups (emergency operation group and interval operation group), and clinical characteristics and outcomes of treatment were investigated. On presentation, no differences in gender, age, body weight, duration of symptoms, temperature, white blood cell count, C-reactive protein level, or maximum size of the abscess in the axial view were detected between the two groups. Fifteen patients (48.4 %) underwent emergency surgery. The remaining 16 patients (51.6 %) were initially treated conservatively with antibiotics. All 16 patients underwent planned operations after receiving conservative treatment, and two (12.5 %) of these patients underwent appendectomy before the planned operation day because of recurrent appendicitis without abscess. There were no differences in the length of hospital stay. In the emergency operation group, six (40 %) patients presented with wound infection and four (26.7 %) developed a postoperative intra-abdominal abscess. No infective complications were reported in the interval operation group. Interval appendectomy after conservative treatment of pediatric ruptured appendicitis with abscess significantly reduced postoperative infection rates.

Long-term results of seton placement for fistula-in-ano in infants.

BACKGROUND: The present study aimed to assess the long-term results of seton placement for fistula-in-ano (FIA) in infants. METHODS: Data of patients aged <1 year who presented to our department with perianal abscess (PA) between January 2006 and February 2010 were retrospectively reviewed. Our standard initial treatment for PA was incision and drainage. Patients with systemic diseases and inflammatory bowel diseases were excluded. RESULTS: Ninety-five patients were treated for PA and/or FIA during the 5-year period, and follow-up data were available for 90 patients. The mean follow-up duration in these patients was 49.8 ± 11.4 months, and mean age at presentation was 3.1 ± 2.7 months. Of the 90 patients, 36 (40%) developed FIA (39 lesions) and underwent seton placement. The condition healed in a mean period of 6.3 ± 4.0 weeks after the placement of a cutting seton. Healing of the fistula was achieved in 35 (97.2%) of 36 patients after the initial seton procedure, and one patient who showed recurrence underwent a second seton placement, resulting in successful healing of the FIA after 5 weeks. CONCLUSIONS: The long-term success of seton placement indicates that this procedure should be a treatment option for FIA in infants.

[Clinical features of solid malignant tumors in childhood].

The pathogenesis of pediatric malignant tumors is associated with congenital abnormalities. Oncogenes and antioncogenes are identified in some of these cases. Neuroblastoma arises from the adrenal medulla and sympathetic ganglia. Most neuroblastomas produce catecholamine. Urinary vanillylmandelic acid(VMA)and homovanillic acid(HVA), metabolites of catecholamine, are sensitive tumor markers. Risk stratification according to tumor stage and a combination of prognostic factors helps determine the appropriate therapeutic strategy in clinical settings. Nephroblastoma(Wilms tumor)is the most common pediatric renal tumor and is often accompanied by congenital anomalies. Surgical resection of the tumor and the involved kidney is the initial treatment recommendation in the US and Japan. Consecutive chemotherapy and radiotherapy are administered after surgical staging and a definite histopathological diagnosis. Prognosis is relatively good for most nephroblastoma cases with a favorable histology. In addition to nephroblastoma, clear cell sarcoma of the kidney, characterized by a tendency to metastasize to the bone, is a renal tumor with poor prognosis. Rhabdoid tumor of the kidney is another tumor type; however, its pathogenesis is still unknown and it is associated with extremely poor prognosis because of the lack of effective therapeutic measures. Hepatoblastoma is the most common malignant liver tumor. The serum alpha-fetoprotein level is the most effective tumor marker. Complete surgical resection of the involved liver lobe is the definitive approach for cure. Preoperative chemotherapy increases the possibility of complete surgical resection. High-risk patients have a poor prognosis.

Non-promoter DNA hypermethylation of Zygote Arrest 1 (ZAR1) in neuroblastomas.

BACKGROUND: The comprehensive methylation analysis of tumor-specific differently methylated regions in malignant melanomas and brain tumors has led to the identification of non-promoter hypermethylation of zygote arrest 1 (ZAR1). To search the non-promoter ZAR1 hypermethylation in neuroblastomas, we analyzed the levels of the methylation and transcript expression of ZAR1. METHODS: The MassARRAY® EpiTYPER (Sequenom Inc., San Diego, CA, USA) system was optimized to determine the quantitative methylation levels of ZAR1 for 12 neuroblastoma cell lines, 23 neuroblastoma samples and four adrenal samples. ZAR1 expression levels were evaluated through a quantitative, real-time reverse transcription-polymerase chain reaction. The quantitative methylation levels of ZAR1 were subjected to correlation studies with the established markers of progressive disease and outcome. RESULTS: Strikingly, the hypermethylation of ZAR1 regions and ZAR1 expression levels was observed in the neuroblastoma cell lines and neuroblastoma samples, compared to the adrenal samples. Somatic changes in ZAR1 methylation and ZAR1 expression were found in all three neuroblastoma patients. In the ZAR1 regions, poor-outcome tumors that were MYCN-amplified and/or Stage 3 or 4 and/or the age at diagnosis was≥18months, and/or showed an unfavorable histology were frequently hypermethylated. CONCLUSION: Our results indicate that the hypermethylation of ZAR1 regions is extremely frequent in neuroblastomas and correlates with established markers of progressive disease and outcome.

Identification of aberrant methylation regions in neuroblastoma by screening of tissue-specific differentially methylated regions.

BACKGROUND: The identification of tissue-specific differentially methylated regions (tDMRs) is key to our understanding of mammalian development. Research has indicated that tDMRs are aberrantly methylated in cancer and may affect the oncogenic process. PROCEDURE: We used the MassARRAY EpiTYPER system to determine the quantitative methylation levels of seven neuroblastomas (NBs) and two control adrenal medullas at 12 conserved tDMRs. A second sample set of 19 NBs was also analyzed. Statistical analysis was carried out to determine the relationship of the quantitative methylation levels to other prognostic factors in these sample sets. RESULTS: Screening of 12 tDMRs revealed 2 genomic regions (SLC16A5 and ZNF206) with frequent aberrant methylation patterns in NB. The methylation levels of SLC16A5 and ZNF206 were low compared to the control adrenal medullas. The SLC16A5 methylation level (cut-off point, 13.25%) was associated with age at diagnosis, disease stage, and Shimada classification but not with MYCN amplification. The ZNF206 methylation level (cut-off point, 68.80%) was associated with all of the prognostic factors analyzed. Although the methylation levels at these regions did not reach statistical significance in their association with prognosis in mono-variant analysis, patients with both hypomethylation of SLC16A5 and hypermethylation of ZNF206 had a significantly prolonged event-free survival, when these two variables were analyzed together. CONCLUSIONS: We demonstrated that two tDMRs frequently displayed altered methylation patterns in the NB genome, suggesting their distinct involvement in NB development/differentiation. The combined analysis of these two regions could serve as a diagnostic biomarker for poor clinical outcome.