Achaete-scute family bHLH transcription factor 2 activation promotes hepatoblastoma progression.

Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.

Successful treatment of young childhood standard-risk hepatoblastoma with cisplatin monotherapy using a central review system.

BACKGROUND: The JPLT3-S (Japanese Study Group for Pediatric Liver Tumors-3) study, conducted cisplatin (CDDP) monotherapy for young children (<3 years old) with standard-risk hepatoblastoma (HB) using a central review system in Japan. In the previous JPLT2 study, cases with resectable tumors without any annotation factors in the PRETEXT (PRETreatment EXTent of disease) classification (standard-risk HB) showed favorable outcomes with treatment consisting of CDDP and pirarubicin, but showed toxicities and late complications. In the JPLT3-S trial, a less intense regimen consisting of CDDP alone was evaluated. METHODS: Patients who were less than 3 years of age and with PRETEXT I, II, or III HB without any annotation factors (e.g., E1, E1a, E2, E2a, H1, N1, P2, P2a, V3, and V3a) were eligible for inclusion in this study. In this trial, the central radiological and pathological features of all patients were reviewed. The primary outcome was the 3-year progression-free survival (PFS). RESULTS: A total of 38 patients (23 female) were included. The median patient age was 12 months (range: 2-34). Two patients discontinued treatment because of progressive disease, and five patients discontinued treatment for other reasons. The 3-year PFS rate was 93.9% (95% confidence interval [CI]: 86.4%-100%). All 38 patients survived (follow-up period 38-98 months), and the OS rate was 100% (CI: 100). Eighteen of the 38 patients (47.4%) experienced ototoxicity as a late complication. CONCLUSION: CDDP monotherapy regimen is feasible in young patients with localized HB, as classified by a central review.

Single-cell next-generation sequencing of circulating tumor cells in patients with neuroblastoma.

Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2+ CD90+ CD45- CD235a- DAPI- cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.

Determination of the umbilicus position in Japanese children.

BACKGROUND: Although there have been many reports concerning the normal position of the umbilicus, the measurements were performed from the surface of the body in all cases. We examined computed tomography (CT) images to determine the accurate position of the umbilicus in children. METHODS: We retrospectively examined the CT data of 120 Japanese children (60 boys, 60 girls). The angle between both iliac crests to the umbilicus (IU angle), the angle between both anterior superior iliac spines and the umbilicus (AU angle), and the ratio of the length from the xiphoid process to the umbilicus and length from the umbilicus to the pubic symphysis were measured. RESULTS: The mean AU angle was 33.7° ± 5.1°, showing the least data variations. A significant difference was noted in the AU angle between boys and girls (32.7° ± 4.6° and 34.6° ± 5.4° respectively; p = 0.04). When we defined the position of the umbilicus as an AU angle of 33° in boys and 35° in girls, 115 children (95.8%) fell within ±10°. CONCLUSIONS: The AU angle is the preferable predictor of the umbilicus position in children.

A new surgical technique for short bowel syndrome.

OBJECTIVE: Short bowel syndrome (SBS) is a severe intestinal disease that causes malabsorption. Long-term parental nutrition therapy induces infection and liver failure. For the surgical management of intestinal rehabilitation, the intestinal loop lengthening method and serial transverse enteroplasty (STEP) method have been reported, although their effects have proven limited. We herein report a new surgical technique, Saeki-Spiral-Shark (3S) method for SBS using biomimetics of shark intestine. METHODS: In the 3S method, a spiral valve is formed inside the intestine by external sutures. Using a 25 cm length intestinal organ model, we performed both the 3S method and STEP procedure. We then compared the length and fluid passage times of the subsequently formed intestine. RESULTS: After the 3S method was performed, the length of the intestinal model changed to 22 cm, and after the STEP procedure, that was elongated to 30 cm. Although the water passage times did not change markedly, the semi-digestive nutritional supplement passage time slowed down in the model with the 3S method. There was slight leakage in the STEP procedure model. CONCLUSIONS: The 3S method is a unique method of treating SBS based on biomimetics. This procedure does not require an incision of the intestine, which thereby enabling clean and less-invasive surgery. We plan to conduct animal experiments in the future.

Successful mediastinal teratoma resection in a child by assisted VATS: a case report.

BACKGROUND: Video-assisted thoracic surgery (VATS) is widely used for thoracic lesions in pediatric patients. VATS is also applied to pediatric mediastinal tumors if there is no adhesion or invasion between the tumor and adjacent neuronal and cardiovascular structures. Here, we present a pediatric case of mediastinal teratoma in which the tumor adhered to the superior vena cava, and resection was safely completed using assisted VATS, an integrated surgical approach comprising mini-thoracotomy with video assistance. CASE PRESENTATION: A 9 year-old girl presented with right shoulder pain. Chest radiography and computed tomography revealed a 5.4 × 5.1 × 5.8 cm mass in the right upper anterior mediastinum. She was presumed with a mature teratoma, and resection was performed by assisted VATS with muscle sparing axillar skin crease incision (MSASCI) for a mini-thoracotomy. The procedure was safely completed, with the patient discharged on postoperative day 5. At 1 year postoperatively, there was no recurrence with excellent motor and cosmetic results. CONCLUSIONS: The combination of MSASCI and VATS would be useful not only for mediastinal teratomas but also for other mediastinal tumors and almost all other thoracic lesions in pediatric patients.

Reduction en masse of Inguinal Hernia in a 2-Month-Old Male Infant.

Reduction en masse is the reduction of the hernial sac into the preperitoneal space, with a loop of bowel remaining trapped at the neck of the hernial sac. This complication is rare, usually associated with inguinal hernias, and is characterized by the absence of a noticeable bulge in the groin. The patient was a 2-month-old male infant and presented with a nonreducible bulge in his left groin, and incarceration of the left inguinal hernia was diagnosed. Manual reduction was performed, and the hernia bulge became less noticeable. He was admitted, and laparoscopic percutaneous extraperitoneal closure was scheduled for the next day. The laparoscopy revealed remarkably dilated intestines, serous ascites, and an ischemic intestine in the left groin. A laparotomy was performed and revealed reduction en masse of the left inguinal hernia with a strangulated ileum at its neck. We made an incision at the neck, followed by the resection of 20-cm long strangulated ileum. The patient's condition was unstable on the day of operation, but the postoperative period was uneventful, and the left inguinal hernia was repaired, 11 months after the operation. Reduction en masse in pediatrics is significantly rare but when it occurs, the diagnosis can be delayed and occasionally the patient will be life-threatening. To avoid reduction en masse, it is crucial to perform the reduction gently and confirm the absence of a hernia sac in the preperitoneal space containing a loop of bowel by ultrasound scanning. Moreover, contrary to common practice, overnight observation and close monitoring will avoid missing a late presentation, leading to timely interventions.

A formula for determining the standard liver volume in children: a special reference for neonates and infants.

Accurately evaluating the ratio of GV to the SLV (GV/SLV) is important in successful pediatric liver transplantation. However, the formula used to calculate the SLV of children, including neonates and infants, has not yet been established. The aim of the current study was to estimate the SLV of children, including neonates, and to establish an accurate formula. The LV of 100 children (including 7 neonates and 15 infants) were measured using thin slice (3-5 mm) helical CT images. Their BSA was calculated from height and weight. A new formula to estimate the SLV was established as follows: SLV (mL) =689.9 × BSA (m(2)) -24.7. The SLV of children was significantly lower than that in previous reports (p < 0.001). A formula for calculating the SLV of children including neonates was established. This new formula will be useful in pediatric liver transplantation.

Intracranial hemorrhage associated with vitamin K-deficiency bleeding in patients with biliary atresia: focus on long-term outcomes.

BACKGROUND AND AIM: The prophylactic oral administration of vitamin K to newborns has markedly reduced the incidence of vitamin K deficiency (VKD); however, intracranial hemorrhage (ICH) is still one of the complications found in biliary atresia (BA) patients and is associated with VKD bleeding. Therefore, we aimed to investigate the incidence and long-term outcome of ICH in patients with BA who previously received prophylactic vitamin K during the neonatal period. METHODS: Eighty-eight consecutive infants with BA were treated and followed up at Kyushu University Hospital from 1979 to 2009. The clinical records and imaging study results were retrospectively reviewed in the infants with BA who presented with ICH. RESULTS: ICH occurred in 7.95% of patients with BA. The onset of ICH occurred at 47 to 76 days after birth, before the patients underwent surgery for BA (9-37 days after the onset of ICH). Coagulopathy was found upon admission in all of the cases with available data and improved after intravenous administration of vitamin K. A craniotomy was required in 2 cases before the surgery for BA. During the 22 to 278 months of follow-up, some neurologic sequelae persisted in 5 of 7 cases. Follow-up head computed tomography scans showed a low-density area in the left hemisphere in 5 cases. CONCLUSIONS: Although vitamin K prophylaxis had been given during the neonatal period, ICH-associated VKD bleeding was still found in 7.95% of patients with BA. Persistent neurologic sequelae were found in 5 of 7 cases, with low-density area in the left hemisphere.

A morphological study of the removed livers from patients receiving living donor liver transplantation for adult biliary atresia.

BACKGROUND: In liver transplantation (LT) for adult biliary atresia (BA), we often encounter a cirrhotic deformation of the native liver. We aimed to investigate a morphological study of the removed livers and the patient's clinical status. METHODS: We examined 8 BA patients who had undergone LT in adulthood at our hospital. The presence of hypertrophic or atrophic areas of the removed liver was recorded macroscopically. We graded the microscopic findings in the porta hepatis area, a hypertrophic area, and an atrophic area, respectively. Moreover, we investigated the relationship between these morphological findings and the pre-transplant clinical status (MELD score). RESULTS: Macroscopically, a hypertrophic area existed in central liver in all cases (8/8 cases), while an atrophic area was existed in peripheral liver (7/8 cases). Microscopically, an atrophic area was the most severely impaired, while the porta hepatis and hypertrophic area were relatively intact. The pathological score in a compensatory hypertrophic area was strongly correlated with the MELD score. CONCLUSIONS: This study suggests that the partial shrinking is not uncommon in BA cirrhotic liver. It may be due to the imbalance of bile drainage by the different segment. The patient's pre-transplant status depends on the compensatory hypertrophic liver.

Feasibility of Real-Time Central Surgical Review for Patients with Advanced-Stage Hepatoblastoma in the JPLT3 Trial.

In the JPLT3 study, a real-time central surgical reviewing (CSR) system was employed aimed at facilitating early referral of candidates for liver transplantation (LTx) to centers with pediatric LTx services. The expected consequence was surgery, including LTx, conducted at the appropriate time in all cases. This study aimed to review the effect of CSR on institutional surgical decisions in cases enrolled in the JPLT3 study. Real-time CSR was performed in cases in which complex surgeries were expected, using images obtained after two courses of preoperative chemotherapy. Using the cloud-based remote image viewing system, an expert panel consisting of pediatric and transplant surgeons reviewed the images and commented on the expected surgical strategy or the necessity of transferring the patient to a transplant unit. The results were summarized and reported to the treating institutions. A total of 41 reviews were conducted for 35 patients, and 16 cases were evaluated as possible candidates for LTx, with the treating institutions being advised to consult a transplant center. Most of the reviewed cases promptly underwent definitive liver surgeries, including LTx per protocol.

Ischemic preconditioning and remote ischemic preconditioning have protective effect against cold ischemia-reperfusion injury of rat small intestine.

PURPOSE: To investigate the protective effect of ischemic preconditioning (IPC) and remote ischemic preconditioning (RIPC) against cold ischemia-reperfusion injury (IRI) associated with small bowel transplantation (SBT). METHODS: Male Lewis rats weighing 200-300 g were used for this study. The rats were assigned to three groups: control, ischemic preconditioning (IPC), or remote ischemic preconditioning (RIPC). Heterotopic SBT was thereafter performed. The recipient rats were killed 3, 6, 12 and 24 h after transplantation. Specimens from the intestine were histologically scored according to a grading system (Park et al.). Serum lactate dehydrogenase (LDH), aspirate aminotransferase (AST), alanine aminotransferase (ALT) were examined and heme oxygenase-1 (HO-1) were analyzed by ELISA where HO-1 served as an indicator of protection against IRI. RESULTS: The values of tissue injury were significantly lower in the IPC and RIPC groups than in control group at 3 h after SBT. The serum LDH, AST and ALT levels also significantly decreased in the IPC and RIPC groups at 3 h after SBT, but these protective effects against cold IRI diminished by 12 and 24 h after SBT. The serum HO-1 level significantly increased in the IPC and RIPC groups 3 h after SBT. CONCLUSION: Both IPC and RIPC were found to ameliorate ischemia-reperfusion injury after rat SBT in the early phase. HO-1 may therefore play a protective role against cold IRI.

Association of lymphocyte crossmatch and the outcome of intestinal transplantation in swine.

BACKGROUND: The effect of preformed antidonor antibodies have been demonstrated in various types of solid organ transplantation. However, the significance of anti-donor antibodies in intestinal transplantation remains unclear. The aim of this study is to evaluate the impact that the extent of T cell crossmatch has on the outcome of swine intestinal transplantation. MATERIALS AND METHODS: All studies were performed on outbred domestic male pigs weighing from 15 to 20 kg. Intestinal transplantation was performed orthotopically with an exchange of grafts between white and black pigs. FK506 was administered intravenously (0.1 mg/kg per day, POD 0-7) for immunosuppression. A lymphocyte crossmatch test was performed using the direct CDC crossmatch. The results were considered positive when more than 10% of the donor lymphocytes were killed by the recipient's serum. In addition, 0-10, 11-20, 21-30, 31-80 and 81-100% of the killed lymphocytes were classified as grade 1, 2, 4, 6 and 8, respectively. RESULT: A total of 34 intestinal transplantations were performed. All but one case had positive donor specific T cell crossmatches. The number of grade 2, 4, 6 and 8 cases was 11, 14, 6 and 2, respectively. Although there was a tendency towards a decreased survival according to the grade, the survival rate was not statistically different among each different grade. Moreover, the rates of acute cellular rejection and vascular complications were not significantly different among the four grades. CONCLUSION: These results suggest that the extent of positive T cell crossmatch is not associated with the outcome of swine intestinal transplantation.

Design of taste-masked swellable drug particles using dry-coating technology with mechanical curing.

A novel dry coating technique for fine particles that does not require any liquids has been developed. Swellable ordered-mixed drug particles (Swell-OM-spheres, SOS), using a modified starch as the core particle and a drug coating layer have been previously developed. In the present work, SOS particles were further processed to generate 100-µm taste-masking particles using an all dry coating processes. SOS particles were coated with a gastric-soluble powder using a mechanical powder processor. The coated particles (CPs) were subsequently heated while rotating in the same powder processor, completing film formation by a process termed dynamic curing. As a control, conventional film formation (static curing) was performed using a drying oven. The CPs obtained by these two curing processes had distinct appearances, but exhibited equivalent dissolution suppression effects in a medium at pH 6.8 (the pH of the oral cavity). The suppression effect was further improved by adding a plasticizer to the coating powder, even though a lower heating temperature was required. Orally disintegrating tablets incorporating these CPs exhibited excellent taste-masking performance, i.e., suppressing taste in saliva while accelerating dissolution in gastric juice. The dissolution behavior indicated that the CPs can provide an ON/OFF switching function in drug release.

Single-cell DNA and RNA sequencing of circulating tumor cells.

Single-cell sequencing of circulating tumor cells can precisely represent tumor heterogeneity and provide useful information for cancer treatment and research. After spiking TGW neuroblastoma cells into blood derived from healthy volunteer, the cells were isolated by fluorescence-activated cell sorting. DNA and mRNA were amplified by four different whole-genome amplifications (WGA) and three whole-transcriptome amplifications (WTA) methods, followed by single-cell DNA and RNA sequencing. Multiple displacement amplification (MDA)-based WGA methods showed higher amplification efficiency than other methods with a comparable depth of coverage as the bulk sample. The uniformity of coverage greatly differed among samples (12.5-89.2%), with some samples evaluated by the MDA-based WGA method using phi29 DNA polymerase and random primers showing a high (> 80%) uniformity of coverage. The MDA-based WTA method less effectively amplified mRNA and showed non-specific gene expression patterns. The PCR-based WTA using template switching with locked nucleic acid technology accurately amplified mRNA from a single cell. Taken together, our results present a more reliable and adaptable approach for CTC profiling at the single-cell level. Such molecular information on CTCs derived from clinical patients will promote cancer treatment and research.

The risk factors of persistent thrombocytopenia and splenomegaly after liver transplantation.

PURPOSE: The occurrence of thrombocytopenia in the perioperative period after liver transplantation (LT) in patients with portal hypertension is not uncommon. In most cases, thrombocytopenia in the early period after LT recovers with the restoration of the graft hepatic function. In contrast, the recovery of pre-transplant thrombocytopenia differs among patients, and some patients experience persistent thrombocytopenia and splenomegaly even several years after LT. METHODS: We retrospectively reviewed the clinical records of 38 liver transplant patients who had at least a 1-year follow-up in our institute. The serial platelet counts and the spleen volumes estimated by the CT scans were obtained before LT and at 1 month, 1 year, and 3 years after LT. In cases with persistent thrombocytopenia (less than 100,000/µl beyond 1 year after LT) and splenomegaly after LT, the associated clinical factors were reviewed. RESULTS: The platelet counts increased and the spleen volumes decreased continuously in most cases after LT. However, six patients (15.8%) were categorized as persistent thrombocytopenia. The spleen volumes of these six patients decreased more slowly after LT, and, in some cases, showed even re-increased volumes. The factors such as the age at LT, GRWR, the existence of hepatopulmonary syndrome, and the existence of biliary complication after LT were associated with persistent thrombocytopenia after LT. CONCLUSIONS: The evaluation of the pre-transplant spleen volumes and the platelet counts before LT thus makes it possible to predict the persistent thrombocytopenia after LT.

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.

Features and Techniques of Laparoscopic Percutaneous Extraperitoneal Closure for Ovarian Hernia.

PURPOSE: The purpose of this report was to evaluate the safety and efficacy of the simple laparoscopic percutaneous extraperitoneal closure (LPEC) method for treating ovarian hernia patients, including newborns and low-birth-weight infants. METHODS: We retrospectively reviewed the cases of ovarian hernia that were treated in our institution from May 2012 to September 2017. RESULTS: Thirty-four infants were included in this study (right side, n = 8; left side, n = 22; bilateral, n = 4). The mean corrected age was 2.3 ± 2.8 months and the mean body weight was 4.4 ± 1.6 kg. The contralateral positive rate was 53%, and 34% of the cases showed sliding of the fallopian tube. Umbilical hernias were observed in 24 cases (71%). The mean operative time was 53 ± 20 minutes. The LPEC procedure was successful in 32 cases; two cases were converted to open surgery. CONCLUSION: Small infants with ovarian hernia have some specific features such as a shortened round ligament and the presence of peritoneum cavities that require skillful techniques when being surgically repaired. The simple LPEC procedure can be performed safely with a low risk of recurrence, even in infants with sliding of the fallopian tube.