Feasibility of 18F-DOPA and 18F-FDG PET/CT for guiding decision-making for localized incidental neuroblastoma in infants under 18 months of age.

BACKGROUND: Neuroblastoma varies widely in risk. Risk indicators in infants with incidental neuroblastoma refine treatment confidence for observation or intervention. The potential of functional imaging, particularly PET/CT, remains to be defined. PROCEDURE: A retrospective review of infants under 18 months diagnosed with incidental neuroblastoma from 2008 to May 2022 in our institute was conducted. Before October 2015, incidental patients were treated similarly to symptomatic cases, undergoing biopsy or surgical excision upon diagnosis (early cohort). Post October 2015 (late cohort), treatment decisions were guided by PET/CT findings, with 18F-DOPA PET/CT confirming diagnosis and staging. For tumors with low 18F-FDG uptake, an expectant observation approach was considered. Patient characteristics, diagnostic methods, image findings at diagnosis, treatment courses, and responses were compared between cohorts. RESULTS: Thirty infants less than 18 months were identified with incidental neuroblastoma and completed PET/CT at diagnosis. The early and late cohorts each comprised 15 patients. In the late cohort, nine out of 15 patients (60%) presented with localized FDG non-avid tumors were offered the option of expectant observation. Of these, seven patients opted for observation, thereby avoiding surgery. Treatment outcomes were comparable between early and late cohorts, except for one mortality of a patient who, despite showing 18F-FDG activity, declined treatment. CONCLUSIONS: This study demonstrates the potential utility of 18F-DOPA and 18F-FDG PET/CT scans in aiding clinical decision-making for infants with localized, incidental neuroblastoma. Given the concerns regarding radiation exposure, such imaging may be valuable for cases with suspected metastasis, initial large tumor size, or growth during follow-up.

Higher tumor mutational burden is associated with inferior outcomes among pediatric patients with neuroblastoma.

INTRODUCTION: Neuroblastoma is a pediatric malignancy with heterogeneous clinical outcomes. Our aim was to identify prognostic genetic markers for patients with neuroblastoma, who were treated with the Taiwan Pediatric Oncology Group (TPOG) neuroblastoma N2002 protocol, to improve risk stratification and inform treatment. METHODS: Our analysis was based on 53 primary neuroblastoma specimens, diagnosed pre-chemotherapy, and 11 paired tumor relapse specimens. Deep sequencing of 113 target genes was performed using a custom panel. Multiplex ligation-dependent probe amplification was performed to identify clinical outcomes related to copy-number variations. RESULTS: We identified 128 variations associated with survival, with the number of variations being higher in the relapse than that in the diagnostic specimen (p = .03). The risk of event and mortality was higher among patients with a tumor mutational burden ≥10 than that in patients with a lower burden (p < .0001). Multivariate analysis identified tumor mutational burden, MYCN amplification, and chromosome 3p deletion as significant prognostic factors, independent of age at diagnosis, sex, and tumor stage. The 5-year event-free survival and overall survival rate was lower among patients with high tumor burden than in patients with low tumor burden. Furthermore, there was no survival of patients with an ALK F1147L variation at 5 years after diagnosis. CONCLUSIONS: Genome sequencing to determine the tumor mutational burden and ALK variations can improve the risk classification of neuroblastoma and inform treatment.

The genetic basis and cell of origin of mixed phenotype acute leukaemia.

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

Prognostic implications of distinctive imaging characteristics in primary intracranial germ cell tumors: A retrospective analysis.

PURPOSE: Primary central nervous system (CNS) germ cell tumors (GCTs) are rare brain tumors that encompass two subtypes: germinomas and non-germinomatous germ cell tumors (NGGCTs), NGGCTs have less favorable outcome and require multi-modality treatment. Biopsy is recommended for disease diagnosis, the specimen may not adequately reflect the entire tumor. This study aimed to assess distinct imaging characteristics to differentiate between GCT subgroups and to identify possible initial image and subgroup features that influence survival. METHOD: This retrospective study, conducted from January 2006 to March 2023, analyzed patient data and MRI findings of primary CNS GCTs. It evaluated tumor characteristics including cysts, seeding, multifocality, and hemorrhage. Tumor volumes and apparent diffusion coefficient (ADC) values of both tumoral and normal-appearing contralateral white matter were measured. These factors were correlated with overall and 5-year survival rates. RESULTS: This study included 51 participants with CGTs, comprising 19 germinoma and 32 NGGCTs cases. GCTs with hemorrhage had worse overall (P = 0.03) and 5-year (P = 0.01) survival rates. No survival difference between germinoma and non-hemorrhagic NGGCT. NGGCTs were more likely to bleed (P < 0.001) than germ cell tumor, especially those with choriocarcinoma or yolk sac tumor components (P = 0.001). The ADC ratios of germinomas were significantly lower than those of NGGCTs (P = 0.03 for whole tumor; P < 0.01 or solid part), The ADC ratios of choriocarcinoma were also lower than mixed tumor (P = 0.01; P = 0.02). CONCLUSION: Hemorrhage indicates worse prognosis. Intratumoral hemorrhage and ADC ratios differentiate germinoma from NGGCTs. Larger cohorts and advanced MR techniques are needed for future study.

Clinical impact of minimal residual disease and genetic subtypes on the prognosis of childhood acute lymphoblastic leukemia.

BACKGROUND: This study analyzed data from two consecutive protocols for children newly diagnosed with acute lymphoblastic leukemia (ALL) to determine the clinical impact of minimal/measurable residual disease (MRD) and recently identified tumor genetic subtypes. METHODS: Genetic subtypes were determined by sequential approaches including DNA indexing, reverse transcriptase-polymerase chain reaction, multiplex ligation-dependent probe amplification, and RNA-sequencing. MRD was assessed by flow cytometry. The Taiwan Pediatric Oncology Group TPOG-ALL-2013 study enrolled patients who received MRD-directed therapy. RESULTS: The 5-year event-free survival (EFS) and overall survival rates in the 2013 cohort were 77.8% and 86.9% compared to those of the 2002 cohort, which were 62.4% and 76.5%. Among patients treated with MRD-guided therapy, those with ETV6-RUNX1 fusion and high hyperdiploidy had the highest 5-year EFS (91.4% and 89.6%, respectively). The addition of dasatinib improved outcomes in patients with BCR-ABL1 ALL. Recently identified subtypes like DUX4-rearranged, ZNF384-rearranged, MEF2D-rearranged, and PAX5alt subtypes were frequently positive for MRD after remission induction, and these patients consequently received intensified chemotherapy. Treatment intensification according to the MRD improved the outcomes of patients presenting DUX4 rearrangements. In high-risk or very-high-risk subtypes, the TPOG-ALL-2013 regimen did not confer significant improvements compared to TPOG-ALL-2002, and the outcomes of BCR-ABL1-like, MEF2D-rearranged, and KMT2A-rearranged ALL subtypes (in addition to those of T-cell ALL) were not sufficiently good. Novel agents or approaches are needed to improve the outcomes for these patients. CONCLUSIONS: The TPOG-ALL-2013 study yielded outcomes superior to those of patients treated in the preceding TPOG-ALL-2002 study. This study provides important data to inform the design of future clinical trials in Taiwan. PLAIN LANGUAGE SUMMARY: MRD-directed therapy improved the outcomes for pediatric ALL, especially standard-risk patients. Genomic analyses and MRD might be used together for risk-directed therapy of childhood ALL. Our work provides important data to inform the design of future clinical trials in Taiwan.

High frequency of heat shock protein 27 overexpression is a highly effective, high-coverage marker for minimal residual disease detection in children with B-cell acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Minimal residual disease (MRD) detection is the most powerful prognostic tool for monitoring treatment efficacy and predicting clinical outcomes. We aimed to identify key leukemia-associated markers, the proportions of differential expression in patients, and the most effective marker combination for MRD detection by flow cytometry. METHODS: Bone marrow samples were collected from 132 pediatric patients with newly diagnosed (n = 115) or relapsed (n = 17) B-cell precursor (BCP)-ALL. We used CD19, CD10, CD34, CD45 as backbone markers to identify immature B cells and analyzed the differential expression of 18 leukemia-associated markers using seven-color multiparameter flow cytometry. RESULTS: Leukemic cells in all 132 patients expressed leukemia-associated markers. The most commonly overexpressed marker was heat shock protein 27 (Hsp27) (108 patients, 81%), followed by CD73 (102 patients, 77%) and CD123 (80 patients, 60%). CD38 was underexpressed in 64 patients (48%). Hsp27 overexpression persisted in 50 out of 57 follow-up MRD bone marrow samples (87%) and was associated with older age at diagnosis. Hsp27 overexpression was not associated with MRD levels or genetic abnormalities including hyperdiploidy, t(12;21)/ETV6-RUNX1, t(1;19)/TCF3-PBX1, t(9;22)/BCR-ABL1, or 11q23/KMT2A rearrangements. Four remaining leukemia-associated markers (Hsp27, CD73, CD58, CD24) after in silico deletion from the original panel could collectively detect leukemia-associated cell profiles in 100% of cases in this cohort and 98% of cases in a validation cohort. CONCLUSION: Hsp27 combined with CD73, CD58, CD24, and backbone markers allows monitoring MRD in virtually all patients with BCP-ALL.

Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity.

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

Surgical treatment confers prognostic significance in pediatric malignant mediastinal germ cell tumors.

BACKGROUND: Primary malignant mediastinal germ cell tumors (GCTs) are rare pediatric tumors that have a poorer prognosis compared to GCTs occurring elsewhere in the body. The current study aimed to assess the prognostic factors and treatment outcomes of children with primary malignant mediastinal GCT in Taiwan. METHODS: The authors retrospectively reviewed children 0-18 years old who were newly diagnosed with primary malignant mediastinal GCT between January 1, 2005 and December 31, 2019 and were registered in the Taiwan Pediatric Oncology Group patient registry. The impact of presenting characteristics, including sex, age, tumor stage, histology subtype, surgical treatment, and chemotherapy regimens of the patients were analyzed. RESULTS: This study enrolled 52 children with malignant mediastinal GCT who had a median age of 16.0 (range, 6.0-17.9) years at diagnosis. The most common histological subtypes were mixed GCTs (n = 20) and yolk sac tumors (n = 15). Advanced disease stage and choriocarcinoma histology subtype were associated inferior outcomes. Children who received surgical treatment exhibited better outcomes compared to those who did not (5-year overall survival, 78% vs. 7%, p < .001). After comparing patients who received first-line cisplatin- and carboplatin-based chemotherapy, no difference in treatment outcomes was observed. Multivariate analysis showed that surgical management was the only independent predictor for superior OS. CONCLUSIONS: Surgical treatment is recommended for mediastinal GCT. Cisplatin-based chemotherapy was not superior to carboplatin-based chemotherapy as first-line treatment and may be avoided due to toxicity concerns.

Acute pancreatitis in children with acute lymphoblastic leukemia correlates with L-asparaginase dose intensity.

BACKGROUND: L-Asparaginase (L-Asp) is an important therapeutic for childhood acute lymphoblastic leukemia (ALL). Asparaginase-associated pancreatitis (AAP) is a severe complication of L-Asp related to the dosage. We investigated the incidence of, and risk factors for, AAP in pediatric patients with ALL. METHODS: From January 2002 to December 2018, pediatric patients with ALL treated at National Taiwan University Hospital were enrolled in this study. The diagnosis of AAP was based on the criteria of the Ponte di Legno Toxicity Working Group. RESULTS: Of the 353 patients enrolled in this study, 14 (4.0%) developed AAP. The incidence of AAP in ALL patients was significantly higher after treatment with the 2013 protocol compared with the 2002 protocol of the Taiwan Pediatric Oncology Group (9.5% vs. 1.3%). Multivariate analysis showed that a high peak L-Asp dose intensity (>45,000 U/m2/month) and older age at diagnosis (>6.8 years) were independently predictive of AAP development. CONCLUSIONS: The incidence of acute pancreatitis in childhood ALL was correlated more strongly with the peak dose intensity than with the cumulative dose of L-Asp. These results could be used to reduce the treatment-related complications of ALL. IMPACT: L-Asparaginase is an important therapeutic for childhood acute lymphoblastic leukemia, and the accumulated dosage of L-asparaginase is considered as a major risk factor of asparaginase-associated pancreatitis. This article demonstrated that the incidence of pancreatitis correlates with the dose-intensity of L-asparaginase, but not the accumulated dosage. Identification of patient group with high risk of pancreatitis could lead to early diagnosis and reduce the complication. This finding could aid in developing further new protocol or therapeutic strategy design to reduce treatment-related complications and improve clinical outcomes of childhood acute lymphoblastic leukemia.

Integration of immunohistochemistry, RNA sequencing, and multiplex ligation-dependent probe amplification for molecular classification of pediatric medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is commonly classified into four molecular groups, that is, WNT, SHH, group 3, and group 4, for prognostic and therapeutic purposes. METHODS: Here we applied immunohistochemistry (IHC) and RNA sequencing (RNA-seq) for the molecular classification of MB, and utilized multiplex ligation-dependent probe amplification (MLPA) to determine chromosomal alterations and specific gene amplifications. RESULTS: We retrospectively enrolled 37 pediatric MB patients. Twenty-three had genomic material available for gene/RNA analysis. For IHC, ß-catenin, GAB1, and YAP were the biomarkers to segregate MB into three subgroups, WNT (1/23), SHH (5/23), and non-WNT/non-SHH (17/23). However, four cases (17.3%) were found to be misclassified after analysis by RNA-seq. The result of MLPA revealed two group 3 tumors carrying MYC amplification, and three SHH tumors harboring MYCN amplification. While IHC provided rapid subgroup stratification, it might result in incorrect subgrouping. Thus, validation of the IHC result with genomic data analysis by RNA-seq or other tools would be preferred. In addition, MLPA can detect important genetic alterations and is helpful for the identifications of high-risk patients. CONCLUSIONS: Our study revealed that integration of these diagnostic tools can provide a precise and timely classification of MB, optimizing an individualized, risk-directed postoperative adjuvant therapy for these patients. This workflow can be applied in a countrywide fashion to guide future clinical trials for patients with MB.

Characteristics and outcomes of second cancers in patients with childhood cancer: A report from the Taiwan Pediatric Oncology Group.

BACKGROUND: Patients with childhood cancer are at increased risk for the development of second cancers. METHODS: A national multicenter survey of second cancers conducted by the Taiwan Pediatric Oncology Group retrieved retrospective data from the database at the Children Cancer Foundation in Taiwan beginning in 1995. The characteristics of second cancers and associations of patient demographic and clinical characteristics with time to death due to a second cancer were analyzed. RESULTS: We examined the records of 8782 patients with a primary cancer diagnosed between January 1, 1995 and December 31, 2013, and a total of 99 patients with a second cancer were identified. The most common type of second cancer was acute myeloid leukemia (n = 35), followed by acute lymphoblastic leukemia (n = 15), central nervous system (CNS) tumors (n = 15), and sarcomas (n = 10). Secondary hematological malignancies occurred earlier than other secondary cancers. The frequencies of second CNS tumors and second bone cancers and sarcomas were notably increased when prior radiation doses increased from zero, low dose to high dose. The overall 5-year survival of patients with a second cancer was poor (33.7%). Multivariate survival analysis revealed that the year of primary diagnosis ≤2002, secondary hematological malignancies, and age at second cancer diagnosis ≤9.3 years or >26.8 years increased the risk of death following second cancer. CONCLUSION: Children who develop a second cancer have an unfavorable outcome. Early detection and improved treatment for second cancers are needed.

Childhood acute lymphoblastic leukemia mercaptopurine intolerance is associated with NUDT15 variants.

BACKGROUND: Mercaptopurine-induced neutropenia can interrupt chemotherapy and expose patients to infection during childhood acute lymphoblastic leukemia (ALL) treatment. Previously, six candidate gene variants associated with mercaptopurine intolerance were reported. Herein, we investigated the association between the mean tolerable dose of mercaptopurine and these genetic variants in Taiwanese patients. METHODS: In total, 294 children with ALL were treated at the National Taiwan University Hospital from April 1997 to December 2017. Germline variants were analyzed for NUDT15, SUCLA2, TPMT, ITPA, PACSIN2, and MRP4. Mean daily tolerable doses of mercaptopurine in the continuation phase of treatment were correlated with these genetic variants. RESULTS: Mercaptopurine intolerance was significantly associated with polymorphisms in NUDT15 (P value < 0.0001). Patients with SUCLA2 variants received lower mercaptopurine doses (P value = 0.0119). The mean mercaptopurine doses did not differ among patients with TPMT, ITPA, MRP4, and PACSIN2 polymorphisms (P value = 0.9461, 0.5818, and 0.7951, respectively). After multivariable linear regression analysis, only NUDT15 variants retained their clinically significant correlation with mercaptopurine intolerance (P value < 0.0001). CONCLUSION: In this cohort, the major genetic determinant of mercaptopurine intolerance was NUDT15 in Taiwanese patients. IMPACT: NUDT15 causes mercaptopurine intolerance in children with ALL. The NUDT15 variant is a stronger predictor of mercaptopurine intolerance than TPMT in a Taiwanese cohort. This finding is similar with studies performed on Asian populations rather than Caucasians. Pre-emptive genotyping of the patients' NUDT15 before administering mercaptopurine may be more helpful than genotyping TPMT in Asians.

Clinical outcomes of childhood Langerhans cell histiocytosis in Taiwan: A single-center, 20-year experience.

BACKGROUND/PURPOSE: The Taiwan Pediatric Oncology Group (TPOG) initiated two consecutive protocols for treating pediatric patients with Langerhans cell histiocytosis (LCH) since 1994. However, the results have not been analyzed and reported. This study aimed to investigate the survival outcomes of childhood LCH at the National Taiwan University Hospital over the past 20 years. METHODS: Treatment of pediatric patients with LCH according to TPOG protocols at the National Taiwan University Hospital began in 1994. During 1994-2003, patients were treated using the TPOG LCH-94 protocol. After 2003, patients were treated using the TPOG LCH-2003 protocol. Clinical data of these patients were obtained retrospectively by reviewing electronic medical records. Patients were followed up until July 31, 2018. RESULTS: Fifty-three newly diagnosed pediatric patients with LCH were treated at National Taiwan University Hospital during 1994-2015. Twenty-nine (54.7%) were treated with the TPOG LCH-94 protocol, and 24 (45.3%) were treated with the TPOG LCH-2003 protocol. The 5-year event-free survival and overall survival rates were 96.2 ± 2.6% standard error (SE) and 98.1 ± 1.9% (SE), respectively. Overall survival and 5-year event-free survival between patients treated with the TPOG LCH-94 and TPOG LCH-2003 protocols showed no significant difference. Multisystem, liver, or spleen diseases were associated with significantly bad survival outcomes. Among at-risk-organ involvement in LCH, liver involvement was an independent factor for poor prognosis. CONCLUSION: Clinical outcomes of children with LCH in Taiwan was good. The results of this study may help in the better classification of risk grouping for protocol designs in the future.

Outcomes and prognostic factors associated with 180-day mortality in Taiwanese pediatric patients with Hemophagocytic Lymphohistiocytosis.

BACKGROUND/PURPOSE: Hemophagocytic lymphohistiocytosis (HLH), a rarely occurring syndrome with various triggers, is associated with early mortality. Owing to a lack of sufficient corresponding data in Taiwan, this study aimed to identify the outcome and potential factors associated with 180-day mortality in pediatric HLH. METHODS: This retrospective study analyzed clinical and laboratory data on pediatric patients diagnosed with HLH at our institute (1995-2019). Logistic regression analysis was conducted to determine the associations between various factors and 180-day mortality. RESULTS: Overall, 48 patients had HLH; their median age at diagnosis was 5 years (interquartile range: 2-11 years). Clinical presentations and laboratory parameters required for diagnosis included fever (98%), splenomegaly (79%), hyperferritinemia (98%), hemophagocytosis (94%), thrombocytopenia (90%), anemia (63%), hypertriglyceridemia (68%), and neutropenia (57%). The 5-year overall survival (OS) rate was 49%. Of 22 patients who had died at the last follow-up, 15 (68%) died within 180 days after diagnosis. In the multivariate analysis, hemoglobin (odds ratio [OR]: 0.564, p = 0.024) and triglyceride (OR: 1.004, p = 0.049) were significantly associated with 180-day mortality. Higher triglyceride levels at diagnosis were related to significantly lower 180-day OS rates (52.9% vs. 86.1%, p = 0.018). CONCLUSION: The overall outcome in our cohort was similar to that reported in some of the largest international cohorts. Hypertriglyceridemia and anemia may be indicative of poor prognoses in pediatric HLH patients independently and may be used to guide treatment strategy formulations for better outcomes.

TP53 alterations in relapsed childhood acute lymphoblastic leukemia.

TP53 alterations are frequent relapse-acquired mutations in childhood acute lymphoblastic leukemia (ALL). The present study evaluated the clinical significance of relapsed childhood ALL in Taiwan. Diagnostic and/or relapsed bone marrow or peripheral blood was obtained from 111 children with relapsed ALL who were initially treated by using Taiwan Pediatric Oncology Group (TPOG) ALL protocols from January 1997 to May 2018. Mutations were detected by PCR and sequencing, as well as by multiplex ligation-dependent probe amplification to detect copy number alterations. Copy number and/or sequence alterations of TP53 were detected in 29% (28 of 98) and in 46% (6 of 13) of patients with relapsed B-cell and T-cell ALL, respectively. This incidence was much higher than that in several similar studies conducted in Caucasian populations. Seventy percent of all TP53 alterations were gained at relapse in 67 matched samples by back-tracking matched diagnostic samples. TP53 alterations were associated with lower 5-year event-free survival (EFS) and overall survival (OS) rates (P = .013 and P = .0002, respectively). Multivariate analysis confirmed the prognostic significance of TP53 alterations. Forty-five patients received hematopoietic stem-cell transplantations post-relapse. Patients with TP53 alterations (14/45) had inferior 5-year EFS and OS than patients without TP53 alterations after transplantation (P = .002 and P = .001, respectively). The significance of these TP53 alterations for patients who received transplantations was confirmed by multivariate analysis. In conclusion, TP53 alterations were enriched and useful as prognostic markers in relapsed childhood ALL.

MYCN RNA levels determined by quantitative in situ hybridization is better than MYCN gene dosages in predicting the prognosis of neuroblastoma patients.

The aim of this study was to investigate the prognostic role of MYCN RNA expression by quantitative RNA in situ hybridization and its association with MYCN amplification in neuroblastoma. MYCN RNA expression in 69 neuroblastoma tumors was evaluated by an ultrasensitive quantitative RNA in situ hybridization technique, RNAscope. The correlations between MYCN RNA expression, MYCN amplification, and other clinicopathologic variables of neuroblastoma were analyzed. High expression levels of MYCN RNA were detected 30 of 69 (43%) of neuroblastomas, mainly in those with undifferentiated or poorly differentiated histology. High expression of MYCN RNA was significantly associated with MYCN amplification (P < 0.001) and other adversely prognostic factors, including older age at diagnosis (>18 months, P = 0.017), advanced clinical stage (International Neuroblastoma Staging System stage 3, 4, P = 0.002), unfavorable International Neuroblastoma Pathology Classification tumor histology (P < 0.001), and high-risk Children's Oncology Group risk group (P = 0.001). In Kaplan-Meier analysis, MYCN RNA levels determined by quantitative in situ hybridization were better than MYCN gene dosages determined by chromogenic in situ hybridization in discriminating good and poor prognostic groups of neuroblastoma patients. In multivariate analysis, we further confirmed that high expression of MYCN RNA was an independent adverse prognostic factor for event-free and overall survival. Furthermore, high expression of MYCN RNA predicted unfavorable survival outcomes for neuroblastoma patients with MYCN non-amplification or high-risk Children's Oncology Group risk group. In conclusion, our study is the first report to show the application of MYCN RNA in situ hybridization in neuroblastoma and established that high expression of MYCN RNA could be a better biomarker than MYCN amplification for predicting poor prognosis of neuroblastoma patients.

The Impact of Hepatitis on Clinical Outcomes for Pediatric Patients with Aplastic Anemia.

OBJECTIVES: To assess the prognostic role of hepatitis in pediatric patients with aplastic anemia and the incidence of hepatitis B among patients with hepatitis-associated aplastic anemia in an area with a previously high prevalence of hepatitis B after nationwide hepatitis B vaccination for 30 years. STUDY DESIGN: Pediatric patients (n = 78) with aplastic anemia were enrolled in this study, including 9 with hepatitis-associated aplastic anemia. We collected the clinical characteristics, etiologies of the aplastic anemia, hepatitis B virus serology and serum hepatitis B viral load, response to the treatments, and survival outcome from the participants. We applied univariate and multivariate Cox regression analysis to evaluate the correlations between clinical features and survival outcome. Survival analysis was done using Cox regression model and Kaplan-Meier curves. RESULTS: Patients with hepatitis-associated aplastic anemia were related to significantly worse survival prognosis when compared with patients with non-hepatitis-associated aplastic anemia, and hepatitis-associated aplastic anemia was the only independent prognostic factor to predict a poor survival outcome in our patients with aplastic anemia by multivariable analysis. In none of the total 78 patients was aplastic anemia related to hepatitis B virus infection. CONCLUSIONS: Patients with hepatitis-associated aplastic anemia had a significantly worse prognosis when compared with patients whose aplastic anemia was not hepatitis-associated. This study demonstrates the potential benefit of hepatitis B vaccination in decreasing the incidence of hepatitis-associated aplastic anemia in children.

Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry.

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Children's Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations.

Mosaic paternal haploidy in a patient with pancreatoblastoma and Beckwith-Wiedemann spectrum.

Pancreatoblastoma is a rare type of pancreatic cancer in children. Here, we describe a case in which Beckwith-Wiedemann syndrome (BWS) was first suspected because of placental mesenchymal dysplasia. Although the baby did not show the stigmata characteristic of BWS or abnormal peripheral blood methylation, she developed a massive pancreatoblastoma 2 months later. She survived after partial excision of the tumor and chemotherapy. The methylation pattern of the pancreatoblastoma tissue was typical of BWS. Single nucleotide polymorphism (SNP) array analyzes revealed that the pancreatoblastoma tissue had genome-wide loss of maternal alleles. Peripheral blood and nontumor pancreatic tissue showed normal biparental genomic contribution. Interphase fluorescence in situ hybridization analysis with centromeric probes for chromosomes 2 and 11 revealed haploid pancreatoblastoma cells, whereas the placental mesenchymal dysplasia tissue and nontumor pancreas tissue showed diploidy. SNP genotype analysis suggested the presence of mosaicism with the pancreatoblastoma tissue having a different paternal haplotype than that of the peripheral blood and nontumor pancreatic tissue. We report for the first time mosaic paternal haploidy associated with pancreatoblastoma. Babies with placental mesenchymal dysplasia, even those without a definitive diagnosis of BWS, need to be closely followed for the occurrence of embryonic tumors.

The incidence and risk factors of hepatic veno-occlusive disease after hematopoietic stem cell transplantation in Taiwan.

Hepatic veno-occlusive disease (VOD) is a potentially fatal complication of hematopoietic stem cell transplantation (HSCT). We conducted this study to investigate the incidence and risk factors of hepatic VOD for patients receiving HSCT in Taiwan. We retrospectively analyzed the data from a nationwide registry for patients receiving HSCT, which was collected by the Taiwan Society of Blood and Marrow Transplantation. The data collection period was from 2009 to 2014. A total 2345 patients were reviewed and 39 patients among them were diagnosed as having hepatic VOD. The cumulative incidence of hepatic VOD in the whole cohort of 2345 patients was 1.66%. In multivariate analysis, disease diagnosis of myelodysplastic syndrome, chronic HCV infection, condition regimens of bulsulfan intravenously administered, and antithymocyte immunoglobulin were independent factors to predict higher risk of hepatic VOD. The overall mortality rate for patients with hepatic VOD was 79%. Patients with hepatic VOD had significant worse survival outcomes when compared with those without hepatic VOD (P = 0.00063). In conclusion, although the incidence is low, hepatic VOD remains a serious complication after HSCT in Taiwan. The findings of this study could be the basis for developing prophylactic or early treatment strategies for hepatic VOD.