Detection of MYCN amplification using blood plasma: noninvasive therapy evaluation and prediction of prognosis in neuroblastoma.

PURPOSE: Amplification of neuroblastoma derived (avian)v-myc myelocytomatosis viral related oncogene (MYCN) is an important risk-stratified indicator in neuroblastoma. To evaluate the feasibility of noninvasive measurement of MYCN amplification, we analyzed MYCN amplification in stored blood plasma samples. METHODS: We used quantitative real-time PCR to determine MYCN copy numbers in plasma-derived DNA of 10 healthy volunteers and 50 neuroblastoma cases. The copy number was calculated as the ratio of copies of MYCN to those of a reference gene. Plasma samples obtained after surgery or neoadjuvant therapy were also analyzed in five cases and four cases, respectively. RESULTS: In 34 neuroblastoma cases, MYCN was nonamplified in both tumor tissue and blood plasma. In 16 neuroblastoma cases, MYCN was amplified in both tumor tissue and blood plasma; 13 of the 16 cases showed poor outcomes. MYCN amplification was undetectable in blood plasma shortly after surgery or neoadjuvant therapy. The correlation coefficient between MYCN copy numbers in tumor tissue and in blood plasma was approximately 0.9. CONCLUSION: We can detect MYCN amplification of tumor tissue noninvasively and quantitatively by measuring the MYCN copy number in blood plasma. Determination of MYCN copy number in plasma may be useful when evaluating surgery and neoadjuvant chemotherapy.

A cisplatin plus pirarubicin-based JPLT2 chemotherapy for hepatoblastoma: experience and future of the Japanese Study Group for Pediatric Liver Tumor (JPLT).

INTRODUCTION: The Japanese Study Group for Pediatric Liver Tumor (JPLT) has conducted cooperative treatment studies on hepatoblastoma (HBL) since 1991. The JPLT2 protocol was launched in 1999 to evaluate the efficacy of cisplatin/pirarubicin (CITA) under risk stratification. European and North American groups showed the improvement of HBL patients by pre- and postoperative chemotherapeutic regimens. Therefore, we evaluated the results of JPLT study and considered the future aspect of JPLT. METHODS: A total of 389 children with malignant hepatic tumors were enrolled in JPLT-2 until 2010. Data from 331 HBL cases were analyzed. RESULTS AND DICUSSION: Of the 331 patients enrolled, their 5-year overall survival and event-free survival rates were 83.3 and 68.0%, respectively. While outcomes of standard-risk cases (tumors involving 3 or fewer sectors of the liver) were excellent, those of high-risk cases (tumors involving 4 sectors of the liver or with distant metastases) remained poor. For 26 high-risk or relapse/refractory HBL cases, high-dose chemotherapy (HDC) with stem cell transplantation (SCT) was carried out. Among them, 6 of 12 relapse or refractory cases died. Compared with other regimens, the CITA regimen achieved similar or superior rates of survival among children with standard-risk HBL, while HDC with SCT was not effective in patients with high-risk HBL. Presently, a global Children's Hepatic Tumor International Consortium (CHIC) project is ongoing, with a focus on international cooperation and risk stratification in the field of rare liver cancers in children. More promising strategies, including liver transplantation and new targeting drugs under global risk stratification, are being proposed.

Surgical intervention for patent ductus venosus.

Patent ductus venosus (PDV) is a rare condition, which usually presents secondary to hepatic atrophy and hepatic failure. We have treated eight cases of PDV, all with hypergalactosemia and hyperbilirubinemia. Ultrasonography and three-dimensional computed tomography demonstrated communication between the portal vein and the inferior vena cava. Of the eight PDV cases, three from the older age group (ages 9, 11, and 14 years) had high-density lesions in their brain nucleus, and one case (age 19 years) had undergone prior Kasai portoenterostomy for biliary atresia. Six PDV patients underwent ligation of PDV and the remaining two cases underwent partial banding of PDV with intraoperative monitoring to maintain portal vein pressure (PVP) under 30 cm H(2)O. Improvement of the intrahepatic portal vein flow was achieved by ligation or banding of PDV. Postoperatively, serum galactose and bilirubin fell to normal ranges, but portal thrombus occurred postoperatively in the first case. We subsequently administered postoperative anticoagulation in the remaining cases and experienced no major complications. These results suggest that PDV ligation and banding are effective surgical approaches for patients with PDV. Close postoperative monitoring to avoid portal thrombus is imperative in these cases.

Evaluation of genes identified by microarray analysis in favorable neuroblastoma.

PURPOSE: The biological heterogeneity of neuroblastoma results in a varied outcome ranging from spontaneous regression to fatal tumor progression. Microarray expression profiling and genetic polymorphism arrays may help identify key genes that differ in aggressive neuroblastomas from those observed in tumors associated with a favorable outcome. METHODS: Total RNA was extracted from 16 neuroblastomas obtained from patients who subsequently died of the disease and from 16 favorable neuroblastomas and analyzed using a human whole genome oligomicroarray (55K CodeLink). Genes overexpressed in favorable tumors were subsequently analyzed in 121 neuroblastoma tumors obtained before chemotherapy using real-time RT-PCR. And among these cases, expression levels of these genes were also analyzed in 20 tumors obtained after chemotherapy. RESULTS: Oligomicroarray analysis revealed the overexpression of 283 genes in favorable tumors that were associated with either regressing or maturing tumors. Three candidate genes, including DHRS3, NROB1, and CYP26A1, were selected that were significantly overexpressed in favorable tumors by quantitative real-time RT-PCR (P < 0.01). No cases with overexpression of all three genes showed poor outcomes. In 20 post-chemotherapeutic tumors, the expression levels of these genes increased in the cases where patients survived but decreased in the fatal cases. CONCLUSIONS: Using microarray expression profiling, we identified genes that exhibit altered gene expression in neuroblastoma tumors associated with a favorable outcome. These candidates warrant further study for use in risk assessment and/or as therapeutic targets in neuroblastoma.

Paratesticular Rhabdomyosarcoma Presenting With a Giant Abdominoscrotal Hydrocele in a Toddler.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in infants and children. Paratesticular RMS accounts for approximately 7% of all RMSs. It commonly presents as a painless scrotal mass, which is usually distinct from the testis. We report an unusual case of paratesticular RMS presenting with a giant abdominoscrotal hydrocele in a toddler. To the best of our knowledge, this is the first case of paratesticular RMS presenting with an abdominoscrotal hydrocele.

A Case of Bifid Phallus and Bladder Neck Incompetence: Is This a Variant of Epispadias or Hypospadias?

Diphallia or duplication of the penis is an extremely rare congenital anomaly. Based on the presence of one or two corpora cavernosa in each of the penises, diphallia is classified into two major groups: bifid phallus and true diphallia. Because true diphallia associated with various anomalies is dominant in published studies, little is known about bifid phallus or isolated cases. Here we present a 9-month-old boy with an isolated bifid phallus. After successful reconstruction of the penis and urethra, urinary incontinence became apparent. To the best of our knowledge, this is the first case of complete bifid phallus associated with bladder neck incompetence.

Resectability and tumor response after preoperative chemotherapy in hepatoblastoma treated by the Japanese Study Group for Pediatric Liver Tumor (JPLT)-2 protocol.

BACKGROUND/PURPOSE: We aimed to clarify whether surgical resectability and tumor response after preoperative chemotherapy (preCTx) represented prognostic factors for patients with hepatoblastoma (HBL) in the JPLT-2 study (1999-2012). METHODS: Patients (N=342) with HBL who underwent preCTx were eligible. PRETEXT, CHIC risk stratification (standard [SR], intermediate [IR] and high risk [HR]) at diagnosis, POST-TEXT, and tumor resectability were evaluated by imaging. Tumor response was classified into responders (CR or PR) and nonresponders (NC or PD) according to RECIST criteria. RESULTS: There were 7 PRETEXT I, 106 II, 143 III, and 86 IV, including 71 metastatic HBLs. In POST-TEXT, 12 PRETEXT II, 42 III, and 58 IV were down-staged. The 5-year EFS/OS rates of 198 SR, 73 IR, and 71 HR-HBLs were 82/94%, 49/64%, and 28/34%, respectively. In 198 SR, 154 of 160 responders and 24 of 38 nonresponders survived event-free (P<0.01). In 73 IR, 12 of 24 whose tumors remained unresectable experienced recurrence, 9 of whom were nonresponders (P<0.01). In 71 HR, chemoresponders and tumor resectability after preCTx correlated with favorable outcomes (P<0.05). CONCLUSIONS: Evaluation of response and tumor resectability after preCTx is useful for predicting prognosis in HBLs. To improve outcomes, we should reconsider surgical procedures according to resectability and chemoresponsiveness. LEVEL OF EVIDENCE: Level II.

Balloon-occluded retrograde transvenous obliteration successfully performed for a large gastric varix in combination with temporary occlusion of the splenic artery in a child.

We encountered a 10-year-old boy with a gastric varix that occurred after long-term treatment by Kasai's hepatic portoenterostomy for biliary atresia for which balloon-occluded retrograde transvenous obliteration (B-RTO) was performed. With the standard B-RTO procedure, sufficient distribution in the entire gastric varix could not be obtained. However, the gastric varix was successfully treated by the addition of occlusion of the splenic artery by inflation of a balloon catheter during B-RTO.

Circulating free DNA as non-invasive diagnostic biomarker for childhood solid tumors.

PURPOSE: Our aims are to determine circulating free DNA (cfDNA) in childhood solid tumor patients who underwent surgical intervention and to analyze any relationships with clinical parameters. METHODS: Fourty-four consenting children admitted with solid tumors between 2010 and 2014 were recruited. CfDNAs isolated from 0.5mL plasma obtained before and 1-30days after surgery were analyzed by next-generation sequencing (NGS: IonTorrent Cancer Hotspot panel) and by gene amplification analysis using a digital PCR (dPCR) platform. RESULTS: Total amounts of cfDNA were 54-825ng and were significantly associated with stage of disease. In cfDNA, 15 mutations or deletions (2 ALK, 2 TP53, 1 WT1, 3 CTNNB1, 1 APC, 1 KIT, 1 RET, 1 CDNK2AT, and 3 SMARCB1) were identified. In 10 neuroblastoma suspected cases, 2 showed high copy numbers of MYCN using dPCR. The positive rate in our cohort was 36%, and all of these aberrations were detected in the original tumors. None of the aberrations were detectable in cfDNA after surgery except for three cases whose tumors remained after surgery. CONCLUSIONS: These data demonstrate the feasibility and potential utility of mutation/deletion/amplification screening in cfDNA using NGS and dPCR for the detection of tumor biomarkers in children with solid tumors. These markers also have the potential utility to evaluate complete resection after surgery.

Mortality and morbidity in primarily resected hepatoblastomas in Japan: Experience of the JPLT (Japanese Study Group for Pediatric Liver Tumor) trials.

BACKGROUND: In the Japanese Study Group for Pediatric Liver Tumor (JPLT) protocols (JPLT-1 and 2) for evaluating the cure rate of risk-stratified hepatoblastoma, primary resection was permitted in PRETEXT I and II cases, followed by postoperative chemotherapy. METHODS: In approximately 500 enrolled cases, resection was performed as the initial treatment in 60 cases, including all 18 PRETEXT I, 30 PRETEXT II, and 12 ruptured cases. The clinical features, surgical procedures, complications, and survival rates were compared in these three groups. RESULTS: All 18 PRETEXT I cases underwent complete resection by lobectomy or segmentectomy (n=14) or nonanatomical partial hepatectomy (NPH) (n=4). The 30 PRETEXT II cases underwent primary resection by right or left lobectomy (n=16), NPH (n=10), or other procedures (n=4). Of these 30 cases, operational death occurred in 1 newborn, and recurrence occurred in 7 cases (14.6%), including 6 NPH cases and 4 older cases (aged >3years). Of the 12 ruptured cases, 7 (58.3%) showed recurrence. Event-free survival rates at 5years in the 3 groups were 88%, 70%, and 32%, respectively. CONCLUSIONS: Primary resection for PRETEXT I or II HB cases should be performed by anatomical resection according to strict surgical guidelines. More intensified chemotherapy is required for primary resected cases whose tumors have ruptured.

Clinical features of ATRX or DAXX mutated neuroblastoma.

PURPOSE: Previously, we reported that alternative lengthening of telomere (ALT) may be a biomarker for chemo-sensitivity and late recurrence in neuroblastoma (NBL). In this study, alterations of ATRX or DAXX, which both encode chromatin remodeling proteins in telomeric region, and their relationship to ALT were examined in NBLs. METHODS: Our previous report on 121 NBLs revealed 11 NBLs with elongated telomeres by ALT. In these NBLs, ATRX or DAXX gene alterations were identified using next-generation sequencing and compared to clinical and other biological factors. RESULTS: In 11 ALT cases, DAXX mutations were detected in one case, and ATRX alterations were detected in 10 cases. Except for one case, no DAXX or ATRX alterations were detected in 110 tumors with normal or shortened telomeres. MYCN amplification was not detected in ATRX altered tumors. In ALT cases, three infants showed ATRX deletions, and all seven cases detected after 18months of age showed poor prognosis. CONCLUSIONS: In NBLs, ALT was caused by ATRX or DAXX alterations. ATRX altered cases without MYCN amplification detected at greater than 18months showed poor prognosis, suggesting that ATRX or DAXX alterations are a particular NBL subtype. Since these tumors showed chemo-resistance and late recurrence, complete resection in a surgical approach should be performed to improve patient prognosis.

Neoplastic transformation by TERT in FGF-2-expanded human mesenchymal stem cells.

The low percentage of human mesenchymal stem cells (hMSCs) in bone marrow necessitates their in vitro expansion prior to clinical use in regenerative medicine. We evaluated the effect of long-term culture of hMSCs on telomere length and transformation capacity by TERT transfection. hMSCs were isolated from the bone marrow aspirates of 24 donors and cultured with fibroblast growth factor-2 (FGF-2). Six cell lines with >500 population doubling levels were considered immortalized. TERT was transfected into two of the six lines for a comparison of telomere length, telomerase activity, differential capacity, colony formation capacity in soft agar and tumorigenicity in immunodeficient (NOD-SCID) mice. hMSC lines exhibited elongated telomeres without the activation of telomerase and retained multi-lineage differentiation potential upon chondrogenic or adipogenic differentiation, while non-immortalized hMSCs showed a marked reduction in telomere length in the differentiation process. Immortalized hMSCs showed anchorage-independence and formed tumors in NOD-SCID mice. Histologically, these tumors consisted of differentiated cells such as fat tissue and cartilage. Two TERT-transfected hMSC lines showed high rates of tumor formation in NOD-SCID mice. These tumors were histologically similar to teratocarcinoma without differentiated cells. These cells may provide a model for the origin of cancer stem cells from adult stem cells, and indicate the possibility that telomerase activation has a major role in the malignant transformation of human stem cells. These data suggest that adult hMSCs have a potential for neoplastic transformation and have implications for the use of hMSCs in tissue engineering and regenerative medicine.

Telomere biology in neuroblastoma: telomere binding proteins and alternative strengthening of telomeres.

PURPOSE: Neuroblastoma (NBL) shows remarkable biologic heterogeneity, resulting in favorable or unfavorable prognoses. Previously, we reported that most unfavorable NBLs express high telomerase activity to maintain telomere length. Recently, telomere binding proteins (TBPs) and alternative lengthening of telomeres (ALTs) have been identified as key factors of telomere maintenance. METHODS: To evaluate the correlation between telomerase activity, telomere length, and the expression levels of TBPs in NBL, we analyzed and quantified these factors in 121 untreated NBLs. RESULTS: Shortened and elongated telomeres were detected in 21 (17.3%) and 11 cases (9.0%), respectively, and there was a significant correlation between telomere length and the length of the 3'-overhang. The tumors with shortened or elongated telomeres showed significant lower expression of TBPs, except for RAP1. Although telomerase activity did not correlate with telomere length, 16 of 22 cases with high telomerase activity and 5 of 9 cases (ALT tumors) that showed long telomeres without high telomerase activity resulted in death. High-dose chemotherapy did not have much effect on these deceased ALT cases, but their survival periods were more than 2 years and relatively long compared with the deceased cases with nonelongated telomeres, suggesting that chemoresistance in ALT tumors may be related to slow growth rates. CONCLUSIONS: High telomerase activity is a poor prognostic factor in NBL. In the cases without high telomerase activity, those with elongated telomere also showed poor outcomes because of chemoresistance. Therefore, ALT and TBPs may be biomarkers for chemosensitivity in NBL. Thus, a better understanding of telomere biology may help define the characteristics of individual NBLs.

Single nucleotide polymorphism array analysis to predict clinical outcome in neuroblastoma patients.

PURPOSE: Neuroblastoma (NB) is a heterogeneous tumor and demonstrates favorable or unfavorable outcomes. In Japan, a nationwide NB mass screening (MS) had been performed on 6-month-old infants for approximately 20 years, which might have detected almost all NB including regressing/maturing tumors. To clarify the heterogeneity of this tumor, we examined genetic alterations in the representative cases using genomewide microarrays. METHODS: Genomic DNA was extracted from 198 NB tissue samples and paired blood samples including 76 MS-detected cases and analyzed by single nucleotide polymorphism arrays. RESULTS: The single nucleotide polymorphism array classified the genetic aberrations into 4 types: whole gain/loss type, partial gain/loss type, MYCN-amplified type, and silent type. Most MS-detecting cases belonged to the whole gain/loss type, whereas unfavorable cases who died of disease showed partial gain/loss, MYCN-amplified, or silent types. CONCLUSIONS: Genomewide genetic analysis is useful to predict the outcome of patients. Although the cases whose tumors showed whole gain/loss may respond well to contemporary therapy, sparing intensive surgery, current therapeutic strategy may be insufficient for the subgroups with partial gain/loss, MYCN-amplified, or silent type. Validation of these results would provide new tools to predict clinical outcome of children with NB.