Shan Zheng

Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex

Hepatoblastoma (HB) is the most common primary liver tumor in children. Mutations in the β‐catenin gene that lead to constitutive activation of the Wnt pathway have been detected in a large proportion of HB tumors. To identify novel mutations in HB, we performed whole‐exome sequencing of six paired HB tumors and their corresponding lymphocytes. This identified 24 somatic nonsynonymous mutations in 21 genes, many of which were novel, including three novel mutations targeting the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) genes in the Wnt pathway, and genes previously shown to be involved in the ubiquitin ligase complex (SPOP, KLHL22, TRPC4AP, and RNF169). Functionally, both the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) were observed to be gain‐of‐functional mutations, and the CAPRIN2 (R968H/S969C) was also shown to activate the Wnt pathway in HB cells. These findings suggested the activation of the Wnt pathway in HB, which was confirmed by immunohistochemical staining of the β‐catenin in 42 HB tumors. We further used short hairpin RNA (shRNA)‐mediated interference to assess the effect of 21 mutated genes on HB cell survival. The results suggested that one novel oncogene (CAPRIN2) and three tumor suppressors (SPOP, OR5I1, and CDC20B) influence HB cell growth. Moreover, we found that SPOP S119N is a loss‐of‐function mutation in HB cells. We finally demonstrated that one of the mechanisms by which SPOP inhibits HB cell proliferation is through regulating CDKN2B expression. Conclusion: These results extend the landscape of genetic alterations in HB and highlight the dysregulation of Wnt and ubiquitin pathways in HB tumorigenesis. (Hepatology 2014;;60:1686–1696)

Genome-Wide Analysis of Long Noncoding RNA (lncRNA) Expression in Hepatoblastoma Tissues

Long noncoding RNAs (lncRNAs) have crucial roles in cancer biology. We performed a genome-wide analysis of lncRNA expression in hepatoblastoma tissues to identify novel targets for further study of hepatoblastoma. Hepatoblastoma and normal liver tissue samples were obtained from hepatoblastoma patients. The genome-wide analysis of lncRNA expression in these tissues was performed using a 4×180 K lncRNA microarray and Sureprint G3 Human lncRNA Chips. Quantitative RT-PCR (qRT-PCR) was performed to confirm these results. The differential expressions of lncRNAs and mRNAs were identified through fold-change filtering. Gene Ontology (GO) and pathway analyses were performed using the standard enrichment computation method. Associations between lncRNAs and adjacent protein-coding genes were determined through complex transcriptional loci analysis. We found that 2736 lncRNAs were differentially expressed in hepatoblastoma tissues. Among these, 1757 lncRNAs were upregulated more than two-fold relative to normal tissues and 979 lncRNAs were downregulated. Moreover, in hepatoblastoma there were 420 matched lncRNA-mRNA pairs for 120 differentially expressed lncRNAs, and 167 differentially expressed mRNAs. The co-expression network analysis predicted 252 network nodes and 420 connections between 120 lncRNAs and 132 coding genes. Within this co-expression network, 369 pairs were positive, and 51 pairs were negative. Lastly, qRT-PCR data verified six upregulated and downregulated lncRNAs in hepatoblastoma, plus endothelial cell-specific molecule 1 (ESM1) mRNA. Our results demonstrated that expression of these aberrant lncRNAs could respond to hepatoblastoma development. Further study of these lncRNAs could provide useful insight into hepatoblastoma biology.

Comment on: Steroid-resistant Kaposiform Hemangioendothelioma: A retrospective study of 37 patients treated with vincristine and long-term follow-up

To the Editor: We read with great interest the report by Wang et al. (Pediatr Blood Cancer 2015; 62:577–580)[1] providing a retrospective review of 37 cases of Kaposiform Hemangioendothelioma (KHE) treated with vincristine, and the accompanying highlight by Francine Blei, pages 551–552).[2] Evidence-based management of vascular anomalies has been impeded by a dearth of prospective clinical trials. Reasons include a lack of a phenotype/genotype classification system, limited numbers of specialists with clinical trial acumen, a historical slant toward surgical and interventional treatments, a paucity of funding options, and rarity of the individual diagnoses. These deficits have pushed patients and families to rally together to demand improved clinical and basic science research and expertise. Presently, the field of vascular anomalies is flourishing with a recently expanded classification system[3] as well as new prospective clinical trials. We believe that collaborative clinical trials among multidisciplinary disease specialists are mandatory for vascular anomalies to emulate the advances made in pediatric oncology by the Children’s oncology group. KHE is a vascular tumor treated by Pediatric Hematologists/ Oncologists. Vincristine treatment is the current consensus-derived treatment for this tumor.[4] After compassionate use of sirolimus for one patient, Cincinnati Children’s Hospital Medical Center (CCHMC) prospectively studied sirolimus as treatment in a total of 60 patients with complicated vascular anomalies.[5] This phase 2 study (NCT# 00975819) was completed in 2014, presented at ISSVA and ASPHO, and recently submitted for publication. The clinical trial proved sirolimus to be safe and efficacious for a majority of vascular anomalies, with 13 of 13 participants with KHE with KMP having partial responses. Preliminary results with sirolimus appear favorable but many questions remain unanswered, including effectiveness compared to standard vincristine-based therapy, time to response, durability of response, and long-term effects. The present retrospective study[1] and the expert opinion consensus paper[4] recommend vincristine and steroids as the first line treatment for KHEwith KMP. Dr. Blei’s thoughtful highlight article[2] provides historical perspective on KHE therapy, but concludes with the suggestion of sirolimus as a first line agent. We suggest caution before considering sirolimus as first line therapy based on small numbers of patients. We strongly recommend that these questions be answered in a randomized controlled trial. This study actually recently opened at CCHMC (NCT # 02110069—“A Randomized Phase 2 Study of Vincristine vs. Sirolimus to treat High Risk Kaposiform Hemangioendothelioma [KHE]”). This FDA-funded and Pfizer-supported trial will open at seven additional sites throughout the United States in coming months, establishing an efficient referral network. This study will establish the most efficacious treatment for KHE with KMP, incorporatingmulti-faceted outcomemonitoring, long-term followup, and the use of adaptive trial design with early stopping rules. Collaborative studies are needed for many of the disease entities within the field of vascular anomalies. Pediatric hematologist/ oncologists are taking leadership roles in the treatment of these diseases. ThroughASPHO, theVascular Anomalies Special Interest Group has drawn tremendous interest and is one example of a collaborative group forming within our specialty to utilize our clinical research expertise to improve outcomes for patients with vascular anomalies.

Steroid-resistant kaposiform hemangioendothelioma: A retrospective study of 37 patients treated with vincristine and long-term follow-up: Vincristine for Steroid-Resistant KHE

Kaposiform hemangioendothelioma (KHE) with Kasabach–Merritt phenomenon (KMP) still remains a particular and life‐threatening disease. The purpose of this study was to evaluate the efficacy of vincristine (VCR) and the possibility of replacement with steroids in the treatment of steroid‐resistant KHE with KMP.

Neonatal suprarenal mass: differential diagnosis and treatment

PurposeTo develop better diagnosis of and treatment strategy for suprarenal masses in the neonates.MethodsA total of 28 neonates with suprarenal mass were treated in the Department of Pediatric Surgery, Children’s Hospital of Fudan University, between May 2003 and August 2010. The medical records of these patients were reviewed. The clinical, radiological, surgical and pathological data were collected.ResultsSix cases were diagnosed as having adrenal tumors, including 1 adrenal teratoma and 5 adrenal neuroblastomas (NB), and 22 cases were diagnosed as having adrenal hemorrhages. Ultrasound showed that the hematoma started to regress after 7xa0days to 6xa0months. Among the NBs, 2 cases were of stage I, 2 cases were of stage IV, and 1 case was of stage III. While 1 stage III patient and 1 stage IV patient underwent adjuvant chemotherapy after the surgery, other cases were cured by surgery alone and currently have no evidence of disease. The suprarenal mass presented cystoids or solid masses detected by ultrasound and CT both in adrenal hemorrhages and in tumors.ConclusionsThe accurate diagnosis of neonatal suprarenal mass depends on prenatal ultrasonography, clinical manifestations, urine VMA test, CT and ultrasound, but dynamic observation of suprarenal mass by CT and ultrasound is an important means of differential diagnosis. While conservative therapy is suitable for adrenal hemorrhage, adrenal tumors need surgical excision. In addition, adrenal mass that is difficult to diagnose can be followed up for 1xa0month without any adverse effects on the therapy and prognosis of the tumor.

Preliminary evaluation of anorectal manometry in diagnosing Hirschsprung’s disease in neonates

PurposeThe aim of this paper was to assess the clinical value of anorectal manometry (ARMM) in the diagnosing of Hirschsprung’s disease (HD) in neonates.MethodsFrom January 2003 to June 2005, 75 patients in whom HD was clinically suspected were analyzed. ARMM was performed using a desk, high rate gastrointestinal dynamic detection system and the results were compared with barium enema and rectal suction biopsy.ResultsBased on rectal suction biopsies in 52 of 75 patients, the positive, false positive, negative, and false negative rates of ARMM in the diagnosis of HD in neonates were found to be 92.3, 1.9, 1.9, and 3.8%, respectively. Forty-three of 75 patients were diagnosed with HD by both ARMM and barium enema and the diagnoses were validated by pathologic results. The diagnosis of HD was excluded in 18 patients in whom HD was clinically suspected, but in whom the results of ARMM and barium enema were normal. Twelve patients who had ARMM results consistent with HD and a negative barium enema, had serial ARMM performed; a rectoanal inhibitory reflex (RAIR) was elicited in four patients, thereby excluding HD and the remaining eight patients were diagnosed with HD by review of barium enema and pathologic results. One of two patients with a positive barium enema for HD, but an ARMM showing the presence of RAIR was excluded by pathologic results and the other patient was lost to follow-up. The diagnostic accuracies of ARMM and barium enema for HD in neonates were 93.3 and 86.7%, respectively. There was no difference in rectal resting pressure and anal rhythmic wave frequency between neonates with HD and healthy neonates, but neonates with HD had higher anal sphincter pressures than healthy neonates (Pxa0=xa00.0074).ConclusionsARMM is a simple, safe, and non-invasive method with high specificity for the diagnosis of HD in neonates.

Successful treatment of kaposiform lymphangiomatosis with sirolimus.

Generalized lymphatic anomaly (GLA) is a rare and often fatal congenital lymphatic disorder that also commonly affects bone. Kaposiform lymphangiomatosis (KLA) is a novel subtype of GLA with poor prognosis and no proper treatment guidelines. A 9‐year‐old male with recurrent pleural effusion was clinically diagnosed as KLA. Following sirolimus therapy at a dose of 0.8u2009mg/m2 twice daily, pleural effusion was significantly decreased and the general status of the patient markedly improved. The clinical course indicates that sirolimus may present an effective therapeutic option in KLA. Moreover, KLA should be considered in differential diagnosis for cases of GLA with coagulopathy. Pediatr Blood Cancer 2015;62:1291–1293.

Ultrasonographic evaluation in the differential diagnosis of biliary atresia and infantile hepatitis syndrome

ObjectiveTo evaluate the ultrasonographic (US) features distinguishing biliary atresia (BA) from infantile hepatitis syndrome (IHS).MethodsThe US results of infants with obstructive jaundice investigated between 2004 and 2009 were analyzed retrospectively and compared with the clinical and surgical findings, looking for features that distinguished between BA and IHS. US was used to obtain information about the gallbladder shape and the structures of hepatic portal system.ResultsA total of 182 infants were confirmed with intraoperative cholangiogram as having BA (151 cases), HIS (29 cases), or bile duct hypogenesis (2 cases). The gallbladder was visualized by US in 64.24% of BA cases (97/151) and 82.76% of HIS cases (24/29), a difference that was significant (Pxa0<xa00.05). At US, the mean length of gallbladder was 1.28xa0cm in BA case and 2.03xa0cm in HIS cases. The mean volume of the gallbladder was 0.27xa0mL in BA babies and 0.61xa0mL in IHS babies, a significant difference (Pxa0<xa00.05). The diagnostic sensitivity, specificity, and validity of combined visualization of the gallbladder and evaluation of the hepatic portal system were 99.34, 83.87, and 96.7%, respectively.ConclusionsThe ultrasonographic features useful in the differential diagnosis of obstructive jaundice cases are measurement of the gallbladder size, observation of its form, investigations of the triangular cord, and the presence of biliary lake in the area of the hepatic portal system. The validity of a BA diagnosis with these combined ultrasound parameters can, however, be improved effectively.

Interleukin-33 overexpression is associated with gamma-glutamyl transferase in biliary atresia

Interleukin-33 (IL-33) plays a crucial role in inflammation. However, it is not clear whether IL-33 levels are of clinical significance for patients with biliary atresia (BA). The purpose of this study was to determine correlations between serum IL-33 levels and the clinicopathologic features of BA. Serum samples were collected from 18 BA infants, 12 nonicteric choledochal cyst (CC) infants with normal liver function, and 10 healthy controls (HCs). Serum IL-33 levels were measured with an enzyme-linked immunosorbent assay (ELISA). Routine liver function tests were performed on the serum samples. qRT-PCR and Western blot analysis were used to detect IL-33 expression in BA liver biopsy tissues. Hepatic lobule localization of IL-33 expression in the hepatic lobule was conducted by immunohistochemistry (IHC). IL-33 levels in serum collected from BA infants were significantly elevated in comparison with CC and HC patients. Furthermore, the elevated serum levels of IL-33 in BA infants were correlated with gamma-glutamyl transferase (GGT) levels. The expression of IL-33 mRNA and protein levels were up-regulated in BA liver biopsy tissues in comparison with CC patients. IHC analysis revealed increased positive immunostaining for IL-33 in BA liver tissues as compared to that in CC tissues. These results suggest that IL-33 may play an important role in the pathogenesis of BA. In addition, the correlation of serum IL-33 levels with GGT levels may provide a novel marker for the diagnosis of BA.

Single-stage correction of imperforate anus with a rectourethral or a rectovestibula fistula by semi-posterior sagittal anorectoplasty

The aim of this study was to evaluate the clinical advantages of a single-stage correction of imperforate anus with a rectourethral or a rectovestibula fistula in neonates by a semi-posterior sagittal anorectoplasty (PSARP). The medical records of 38 neonates (5 females and 33 males) who had imperforate anus with a rectourethral or a rectovestibula fistula were reviewed and analyzed in Children’s Hospital of Fudan University between January 2004 and July 2007. All patients had an anorectal malformation but without obvious sacral dysplasia. The first group had a single-stage PSARP without a colostomy. All neonates were full-term without severe cardiac, renal anomalies or severe abdominal distension. The second group had a staged Peña procedure; the neonates were not suitable for a single-stage PSARP because of severe abdominal distention or cardiac, renal anomalies. A transverse colostomy was performed, followed 3–6 months later by the Peña procedure, and colostomy closure 2xa0months thereafter. The congenital anomalies, fetal age, birth weight, time and age of the definitive operation, complications, length of hospital stay, cost, and postoperative bowel movements were analyzed between these two groups. Among the 38 patients, 22 had a single-stage definitive operation and 16 had a staged pull-through procedure. There were significant differences in congenital anomalies between the two groups (Pxa0=xa00.0314), but no statistically significant differences between the fetal ages, weight at birth, and time and age at the time of the definitive operation (Pxa0>xa00.05). One patient in the first group had intestinal obstruction and intestinal perforation. For the second group, early complications were related to the colostomy. At after 3xa0years postoperatively, 9 patients in the first group and 12 patients in the second group were followed-up and all had positive voluntary bowel movements. There were no statistically significant differences in soiling and constipation grades between the two groups. The total length of hospital stay was 12.06xa0±xa00.85 and 33.85xa0±xa00.94xa0days and the cost was 10,681.1xa0±xa01,759.5 and 27,355.9xa0±xa01,952.0xa0RMB for the first and second groups, respectively. There was a statistically significant difference in the total length of hospital stay and cost between the two groups (Pxa0<xa00.0001); however, there were no statistically significant differences in the length of hospital stay and cost during the definitive operation between the two groups (Pxa0>xa00.05). This retrospective study shows that it is feasible for correction of imperforate anus with a rectourethral or a rectovestibula fistula in neonates using a modified PSARP without a colostomy.