Translational practice of fluorescence in situ hybridisation to identify neuroblastic tumours with TERT rearrangements.

Recently, telomerase reverse transcriptase (TERT) gene rearrangements have been identified in neuroblastoma (NB), the typical pathological type of neuroblastic tumours (NTs); however, the prevalence of TERT rearrangements in other types of NT remains unknown. This study aimed to develop a practical method for detecting TERT defects and to evaluate the clinical relevance of TERT rearrangements as a biomarker for NT prognosis. A TERT break-apart probe for fluorescence in situ hybridisation (FISH) was designed, optimised, and applied to assess the genomic status of TERT in Chinese children with NTs at the Beijing Children's Hospital from 2016 to 2019. Clinical, histological, and genetic characteristics of TERT-rearranged NTs were further addressed. Genomic TERT rearrangements could be effectively detected by FISH and were mutually exclusive with MYCN amplification. TERT rearrangements were identified in 6.0% (38/633) of NTs overall, but 12.4% (31/250) in high-risk patients. TERT rearrangements identified a subtype of aggressive NTs with the characteristics of Stage 3/4, high-risk category, over 18 months old, and presenting all histological subtypes of NB and ganglioneuroblastoma nodular. Moreover, TERT rearrangements were significantly associated with elevated TERT expression levels and decreased survival chances. Multivariable analysis confirmed that it was an independent prognostic marker for NTs. FISH is an easily applicable method for evaluating TERT defects, which define a subgroup of NTs with unfavourable prognosis. TERT rearrangements would contribute to characterising NT molecular signatures in clinical practice.

Ganglioneuroblastoma intermixed: Clinicopathological implications of diagnosis at presentation and genomic correlations.

BACKGROUND: Ganglioneuroblastoma intermixed (GNBI) is classified as "favorable" histology by International Neuroblastoma Pathology Classification system. However, the International Neuroblastoma Risk Group (INRG) stratifies patients using wider clinicopathological and cytogenetic/molecular parameters. While the diagnosis of GNBI is typically made on resected tumor, it may sometimes be rendered on initial biopsy. We studied GNBI noted at diagnosis to evaluate its correlation with INRG staging and other clinicopathological and molecular features. METHODS: In this retrospective study, clinical, radiological, pathological, cytogenetic, and molecular information from patients with GNBI at diagnosis seen between 1995 and 2021 was analyzed. INRG staging was performed. RESULTS: Of the 15,827 neuroblastoma specimens, GNBI was noted in 237 patients. Of these, 53 had the initial pathological diagnosis of GNBI; median follow-up 3.5 (range: 0.2-14) years. Disease was locoregional in 41 (77%, 16 stage L1 and 25 L2); none relapsed. Twelve (23%) had metastatic disease at presentation; six (50%) relapsed, and two died of disease. MYCN was amplified in two metastatic tumors. Six of 31 (19%) tumors tested had recurrent cytogenetic abnormalities and nonrecurrent somatic gene mutations in 10/23 (43%). The presence of any adverse molecular/cytogenetic findings was associated with metastatic disease (p < .05). For patients with localized GNBI undergoing both biopsy and resection, GNBI was diagnosed in both in 17/19 (90%). CONCLUSIONS: Localized GNBI at diagnosis has excellent long-term clinical outcome even without cytotoxic therapy. For localized GNBI, a biopsy sample is adequate to make the diagnosis. When associated with metastasis at diagnosis, prognosis is poorer, possibly due to associated adverse biological features.

A Rare Case of a Solitary Fibrous Tumor in a Child, Presenting as a Congenital Mass, Displaying NAB2ex4::STAT6ex2 Gene Fusion with an Incidental Ganglioneuroblastoma.

A solitary fibrous tumor (SFT) is a ubiquitous tumor that occurs across all ages. It is rarely reported in pediatric patients, especially as a congenital mass. A 1 year 7-month old male child presented with a slow-growing gluteal mass since birth along with a recent episode of bleeding. Magnetic resonance imaging (MRI) showed a well-defined, T1-hypointense and T2-hyperintense mass in the skin and the subcutaneous plane. Subsequently, he underwent a surgical resection that revealed a 5.7 cm-sized fleshy mass. On histopathologic examination, there was a fairly well-circumscribed, cellular spindle cell tumor, with cells arranged in intersecting fascicles and hemangiopericytomatous pattern with areas of hemorrhage and fibrinoid necrosis. Cells revealed mild nuclear atypia. Mitotic figures were up to 8/10 hpf. Immunohistochemically, the tumor cells were diffusely positive for CD34 and STAT6. Furthermore, the tumor revealed NAB2 exon 4:: STAT6 exon 2 fusion by RT-PCR and Sanger sequencing. Post-excision, during follow-up, the patient developed an adrenal mass that was histopathologically proven as a neuroblastic tumor, not further specified on biopsy and finally as ganglioneuroblastoma, intermixed type on excision. This constitutes one of the rare cases of SFT in a pediatric patient, presenting as a congenital mass, confirmed by STAT6 immunostaining and further, molecular testing. A review of literature of similar cases, including treatment-related implications, is presented.

Single-cell profiling of peripheral neuroblastic tumors identifies an aggressive transitional state that bridges an adrenergic-mesenchymal trajectory.

Peripheral neuroblastic tumors (PNTs) represent a spectrum of neural-crest-derived tumors, including neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Malignant cells in PNTs are theorized to interconvert between adrenergic/noradrenergic and mesenchymal/neural crest cell states. Here, single-cell RNA-sequencing analysis of 10 PNTs demonstrates extensive transcriptomic heterogeneity. Trajectory modeling suggests that malignant neuroblasts move between adrenergic and mesenchymal cell states via an intermediate state that we term "transitional." Transitional cells express programs linked to a sympathoadrenal development and aggressive tumor phenotypes such as rapid proliferation and tumor dissemination. Among primary bulk tumor patient cohorts, high expression of the transitional gene signature is predictive of poor prognosis compared with adrenergic and mesenchymal expression patterns. High transitional gene expression in neuroblastoma cell lines identifies a similar transitional H3K27-acetylation super-enhancer landscape. Collectively, our study supports the concept that PNTs have phenotypic plasticity and uncovers potential biomarkers and therapeutic targets.

Genetic and Histopathological Heterogeneity of Neuroblastoma and Precision Therapeutic Approaches for Extremely Unfavorable Histology Subgroups.

Peripheral neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma) are heterogeneous and their diverse and wide range of clinical behaviors (spontaneous regression, tumor maturation and aggressive progression) are closely associated with genetic/molecular properties of the individual tumors. The International Neuroblastoma Pathology Classification, a biologically relevant and prognostically significant morphology classification distinguishing the favorable histology (FH) and unfavorable histology (UH) groups in this disease, predicts survival probabilities of the patients with the highest hazard ratio. The recent advance of neuroblastoma research with precision medicine approaches demonstrates that tumors in the UH group are also heterogeneous and four distinct subgroups-MYC, TERT, ALT and null-are identified. Among them, the first three subgroups are collectively named extremely unfavorable histology (EUH) tumors because of their highly aggressive clinical behavior. As indicated by their names, these EUH tumors are individually defined by their potential targets detected molecularly and immunohistochemically, such as MYC-family protein overexpression, TERT overexpression and ATRX (or DAXX) loss. In the latter half on this paper, the current status of therapeutic targeting of these EUH tumors is discussed for the future development of effective treatments of the patients.

Primary uterine ectomesenchymoma harboring a DICER1 mutation: case report with molecular analysis.

Ectomesenchymoma is an exceedingly rare biphasic malignant tumor characterized by the presence of mesenchymal and neuroectodermal elements. The majority of patients are infants or children. We describe the first case of this entity diagnosed as a primary uterine tumor. A 72-year-old female presented with post-menopausal bleeding. Dilatation and curettage showed irregular mesenchymal proliferation of uncertain nature. In the hysterectomy specimen, a myxoid spindle cell tumor with areas of skeletal muscle and neural differentiation was found in the uterus, with direct invasion of the small intestine, and biphasic differentiation into rhabdomyosarcoma and ganglioneuroblastoma was unequivocally seen in a lymph node metastasis. The morphological findings were validated by immunohistochemistry. Massive parallel sequencing identified TP53, PTEN, and DICER1 mutations in the tumor. This report describes the presence of ectomesenchymoma in an unusual primary organ and in an uncharacteristic age and presents novel data regarding the genetic characteristics of this tumor.

Association of image-defined risk factors with clinical features, histopathology, and outcomes in neuroblastoma.

BACKGROUND: Clinical, molecular, and histopathologic features guide treatment for neuroblastoma, but obtaining tumor tissue may cause complications and is subject to sampling error due to tumor heterogeneity. We hypothesized that image-defined risk factors (IDRFs) would reflect molecular features, histopathology, and clinical outcomes in neuroblastoma. METHODS: We performed a retrospective cohort study of 76 patients with neuroblastoma or ganglioneuroblastoma. Diagnostic CT scans were reviewed for 20 IDRFs, which were consolidated into five IDRF groups (involvement of multiple body compartments, vascular encasement, tumor infiltration of adjacent organs/structures, airway compression, or intraspinal extension). IDRF groups were analyzed for association with clinical, molecular, and histopathologic features of neuroblastoma. RESULTS: Patients with more IDRF groups had a higher risk of surgical complications (OR = 3.1, p = 0.001). Tumor vascular encasement was associated with increased risk of surgical complications (OR = 5.40, p = 0.009) and increased risk of undifferentiated/poorly differentiated histologic grade (OR = 11.11, p = 0.013). Tumor infiltration of adjacent organs and structures was associated with decreased survival (HR = 8.90, p = 0.007), MYCN amplification (OR = 9.91, p = 0.001), high MKI (OR = 6.20, p = 0.003), and increased risk of International Neuroblastoma Staging System stage 4 disease (OR = 8.96, p < 0.001). CONCLUSIONS: The presence of IDRFs at diagnosis was associated with high-risk clinical, molecular, and histopathologic features of neuroblastoma. The IDRF group tumor infiltration into adjacent organs and structures was associated with decreased survival. Collectively, these findings may assist surgical planning and medical management for neuroblastoma patients.

Central nervous system ganglioneuroblastoma harboring MYO5A-NTRK3 fusion.

Central nervous system (CNS) ganglioneuroblastoma is a rare neuroectodermal neoplasm and little is known about its clinical and biological features. Herein, we report a pediatric case of CNS ganglioneuroblastoma harboring MYO5A-NTRK3 fusion. The patient, a 4-year-old boy, underwent a partial resection of a supratentorial tumor that was histopathologically diagnosed as a CNS ganglioneuroblastoma. Treatment with radiotherapy was started per the St Jude Medulloblastoma 03 (SJMB03) protocol; however, the tumor progressed rapidly and radiotherapy was temporally discontinued. Meanwhile, the patient underwent a second surgery, in which a gross total resection was successfully performed, following which he completed the remaining protocol-based therapy. Although an early focal recurrence was detected for which he received additional radiotherapy and oral temozolomide, the patient remained in complete remission for 14 months after the completion of the treatment. A central pathological review and molecular analysis were performed that revealed a MYO5A-NTRK3 fusion. Interestingly, the MYO5A-NTRK3 fusion has been recurrently detected in melanocytic tumors but not in other types of tumors. Therefore, it can be speculated that our case might partly share tumorigenesis mechanisms with MYO5A-NTRK3-positive melanocytic tumors. In addition, our case may enable an improved understanding of the pathogenesis and clinical features of CNS ganglioneuroblastomas.

Exome sequencing identifies predisposing and fusion gene in ganglioneuroma, ganglioneuroblastoma and neuroblastoma.

This study intended to gain new insight into the genetic basis underlying ganglioneuroma (GN), ganglioneuroblastoma (GNB), and neuroblastoma (NB). Three fresh-frozen surgically resected tumor tissues (GN1, GNB1, and NB1) and matched blood samples (GN2, GNB2, and NB2) were respectively obtained from three pediatric patients with GN, GNB, and NB. After exome sequencing, we predicted the somatic single nucleotide variants (SNV) and insertion and deletion (InDel), and screened the predisposing genes. Finally, we detected and filtered the fusion gene using Fusionmap. Exome sequencing identified 815, 985, and 884 somatic SNV, and 56, 43, and 34 InDel for GN, NB, and GNB respectively. Total 29, 19 and 37 predisposing genes were identified from GN, GNB and NB samples, such as PIK3CA (GN), MUC4 (GN), PML (NB), TFR2 (GNB), and MAX (GNB). Additionally, four common fusion genes, such as HOXD11-AGAP3 and SAMD1-CDC42EP5, were identified from three tumor samples. Moreover, SAMD1-CDC42EP5 was also a common fusion position in three blood samples. These previously unrecognized predisposing genes, such as PIK3CA, MUC4, PML, TFR2 and MAX, and fusion genes, like HOXD11-AGAP3, and SAMD1-CDC42EP5 may have the potential to impact the progression and development of neuroblastic tumors.

Frequency of ALK and GD2 Expression in Neuroblastoma.

Background: The aim of this study was to elucidate the significance of immunohistochemical staining patterns of ALK and GD2 in peripheral neuroblastic tumors with different stages and favorable/unfavorable features. Materials and methods: 32 neuroblastomas, 7 ganglioneuroblastomas, and 1 ganglioneuroma cases were immunohistochemically stained with ALK and GD2, and the expressions were graded and correlated with differentiation, size, and favorable/unfavorable histology. Results: There was no statistically significant correlation between ALK immunopositivity and tumor differentiation or stage. Although there was no statistically significant correlation between GD2 immunopositivity and stage, the intensity and prevalence of GD2 immunostaining were statistically significantly higher in the well differentiated group and in tumors which were smaller than 10 cm. Conclusion: GD2 immunostaining levels correlated with tumor differentiation and size. ALK immunostaining was not related to tumor differentiation or stage.

High Oct4 expression: implications in the pathogenesis of neuroblastic tumours.

BACKGROUND: Neuroblastic tumours (NBTs) are paediatric solid tumours derived from embryonic neural crest cells which harbour their own cancer stem cells (CSC). There is evidence indicating that CSC may be responsible for tumour progression, chemotherapy resistance and recurrence in NBTs. Oct4 is a transcription factor which plays a key role in mammal embryonic development and stem cell fate regulation. The aim of the study is to elucidate the clinical significance of Oct4 in NBTs. METHODS: We studied Oct4 expression in 563 primary NBTs using digital image quantification. Chi-square test was applied to analyse the correlation between histopathology and the Oct4+ cell percentage. Survival analysis was carried out with Kaplan-Meier curves and log-rank test. Additionally, a multivariate Cox regression analysis with the stepwise backwards (Wald) method was undertaken to calculate the impact of Oct4 expression level on survival. RESULTS: We found that tumours with a high proportion of cells expressing Oct4 correlated statistically with undifferentiated and poorly differentiated neuroblastoma / nodular ganglioneuroblastoma, and that Oct4 expression was not present in ganglioneuroma (p < 0.05). Statistical analysis also indicated a relationship between high Oct4 expression levels, high-risk patients according to the International Neuroblastoma Risk Group pre-treatment classification parameters, larger blood vessels and low survival rates. CONCLUSIONS: These results suggest that the Oct4 gene may regulate NBT pathogenic differentiation pathways, and should thus be considered as a target for knockdown when developing novel therapies for high-risk NBT patients.

Statistical Framework in Support of a Revised Children's Oncology Group Neuroblastoma Risk Classification System.

PURPOSE: The International Neuroblastoma Risk Group (INRG) Staging System (INRGSS) was developed through international consensus to provide a presurgical staging system that uses clinical and imaging data at diagnosis. A revised Children's Oncology Group (COG) neuroblastoma (NB) risk classification system is needed to incorporate the INRGSS and within the context of modern therapy. Herein, we provide statistical support for the clinical validity of a revised COG risk classification system. PATIENTS AND METHODS: Nine factors were tested for potential statistical and clinical significance in 4,569 patients diagnosed with NB who were enrolled in the COG biology/banking study ANBL00B1 (2006-2016). Recursive partitioning was performed to create a survival-tree regression (STR) analysis of event-free survival (EFS), generating a split by selecting the strongest prognostic factor among those that were statistically significant. The least absolute shrinkage and selection operator (LASSO) was applied to obtain the most parsimonious model for EFS. COG patients were risk classified using STR, LASSO, and per the 2009 INRG classification (generated using an STR analysis of INRG data). Results were descriptively compared among the three classification approaches. RESULTS: The 3-year EFS and overall survival (± SE) were 72.9% ± 0.9% and 84.5% ± 0.7%, respectively (N = 4,569). In each approach, the most statistically and clinically significant factors were diagnostic category (eg, NB, ganglioneuroblastoma), INRGSS, MYCN status, International Neuroblastoma Pathology Classification, ploidy, and 1p/11q status. The results of the STR analysis were more concordant with those of the INRG classification system than with LASSO, although both methods showed moderate agreement with the INRG system. CONCLUSION: These analyses provide a framework to develop a new COG risk classification incorporating the INRGSS. There is statistical evidence to support the clinical validity of each of the three classifications: STR, LASSO, and INRG.

Irinotecan-temozolomide with temsirolimus or dinutuximab in children with refractory or relapsed neuroblastoma (COG ANBL1221): an open-label, randomised, phase 2 trial.

BACKGROUND: Outcomes for children with relapsed and refractory neuroblastoma are dismal. The combination of irinotecan and temozolomide has activity in these patients, and its acceptable toxicity profile makes it an excellent backbone for study of new agents. We aimed to test the addition of temsirolimus or dinutuximab to irinotecan-temozolomide in patients with relapsed or refractory neuroblastoma. METHODS: For this open-label, randomised, phase 2 selection design trial of the Children's Oncology Group (COG; ANBL1221), patients had to have histological verification of neuroblastoma or ganglioneuroblastoma at diagnosis or have tumour cells in bone marrow with increased urinary catecholamine concentrations at diagnosis. Patients of any age were eligible at first designation of relapse or progression, or first designation of refractory disease, provided organ function requirements were met. Patients previously treated for refractory or relapsed disease were ineligible. Computer-based randomisation with sequence generation defined by permuted block randomisation (block size two) was used to randomly assign patients (1:1) to irinotecan and temozolomide plus either temsirolimus or dinutuximab, stratified by disease category, previous exposure to anti-GD2 antibody therapy, and tumour MYCN amplification status. Patients in both groups received oral temozolomide (100 mg/m2 per dose) and intravenous irinotecan (50 mg/m2 per dose) on days 1-5 of 21-day cycles. Patients in the temsirolimus group also received intravenous temsirolimus (35 mg/m2 per dose) on days 1 and 8, whereas those in the dinutuximab group received intravenous dinutuximab (17·5 mg/m2 per day or 25 mg/m2 per day) on days 2-5 plus granulocyte macrophage colony-stimulating factor (250 µg/m2 per dose) subcutaneously on days 6-12. Patients were given up to a maximum of 17 cycles of treatment. The primary endpoint was the proportion of patients achieving an objective (complete or partial) response by central review after six cycles of treatment, analysed by intention to treat. Patients, families, and those administering treatment were aware of group assignment. This study is registered with ClinicalTrials.gov, number NCT01767194, and follow-up of the initial cohort is ongoing. FINDINGS: Between Feb 22, 2013, and March 23, 2015, 36 patients from 27 COG member institutions were enrolled on this groupwide study. One patient was ineligible (alanine aminotransferase concentration was above the required range). Of the remaining 35 patients, 18 were randomly assigned to irinotecan-temozolomide-temsirolimus and 17 to irinotecan-temozolomide-dinutuximab. Median follow-up was 1·26 years (IQR 0·68-1·61) among all eligible participants. Of the 18 patients assigned to irinotecan-temozolomide-temsirolimus, one patient (6%; 95% CI 0·0-16·1) achieved a partial response. Of the 17 patients assigned to irinotecan-temozolomide-dinutuximab, nine (53%; 95% CI 29·2-76·7) had objective responses, including four partial responses and five complete responses. The most common grade 3 or worse adverse events in the temsirolimus group were neutropenia (eight [44%] of 18 patients), anaemia (six [33%]), thrombocytopenia (five [28%]), increased alanine aminotransferase (five [28%]), and hypokalaemia (four [22%]). One of the 17 patients assigned to the dinutuximab group refused treatment after randomisation; the most common grade 3 or worse adverse events in the remaining 16 patients evaluable for safety were pain (seven [44%] of 16), hypokalaemia (six [38%]), neutropenia (four [25%]), thrombocytopenia (four [25%]), anaemia (four [25%]), fever and infection (four [25%]), and hypoxia (four [25%]); one patient had grade 4 hypoxia related to therapy that met protocol-defined criteria for unacceptable toxicity. No deaths attributed to protocol therapy occurred. INTERPRETATION: Irinotecan-temozolomide-dinutuximab met protocol-defined criteria for selection as the combination meriting further study whereas irinotecan-temozolomide-temsirolimus did not. Irinotecan-temozolomide-dinutuximab shows notable anti-tumour activity in patients with relapsed or refractory neuroblastoma. Further evaluation of biomarkers in a larger cohort of patients might identify those most likely to respond to this chemoimmunotherapeutic regimen. FUNDING: National Cancer Institute.

Maturation toward neuronal tissue in a Ewing sarcoma of bone after chemotherapy.

BACKGROUND: Ewing sarcoma is the second most common bone tumor, occurring mainly in children and young adults. It shows a typical primitive, small round cell morphology and a characteristic fusion oncogene involving EWSR1 and members of the ETS family in most of the cases. Neuronal maturation after chemotherapy is a rare phenomenon and we herein describe such an exceptional case. CASE PRESENTATION: An 8-year old boy was diagnosed with a Ewing sarcoma in the left femur. On biopsy the morphology was typical and there was an EWSR1-FLI1 gene fusion. He underwent neo-adjuvant chemotherapy and resection of the tumor. On microscopic evaluation, part of the tumor showed ganglioneuroblastoma-like differentiation with expression of neuronal markers. The continued presence of EWSR1 rearrangement in both the blue round cell component and the ganglioneuroblastoma-like component was shown by FISH analysis. CONCLUSIONS: In conclusion, this case describes the possibility of a Ewing sarcoma to differentiate into a ganglioneuroblastoma-like lesion after neo-adjuvant chemotherapy treatment; the prognostic value of this phenomenon remains questionable.

Oncologic Phenotype of Peripheral Neuroblastic Tumors Associated With PHOX2B Non-Polyalanine Repeat Expansion Mutations.

BACKGROUND: Germline non-polyalanine repeat expansion mutations in PHOX2B (PHOX2B NPARM) predispose to peripheral neuroblastic tumors (PNT), frequently in association with other neurocristopathies: Hirschsprung disease (HSCR) or congenital central hypoventilation syndrome (CCHS). Although PHOX2B polyalanine repeat expansions predispose to a low incidence of benign PNTs, the oncologic phenotype associated with PHOX2B NPARM is still not known in detail. METHODS: We analyzed prognostic factors, treatment toxicity, and outcome of patients with PNT and PHOX2B NPARM. RESULTS: Thirteen patients were identified, six of whom also had CCHS and/or HSCR, one also had late-onset hypoventilation with hypothalamic dysfunction (LO-CHS/HD), and six had no other neurocristopathy. Four tumours were "poorly differentiated," and nine were differentiated, including five ganglioneuromas, three ganglioneuroblastomas, and one differentiating neuroblastoma, hence illustrating that PHOX2B NPARM are predominantly associated with differentiating tumors. Nevertheless, three patients had stage 4 and one patient had stage 3 disease. Segmental chromosomal alterations, correlating with poor prognosis, were found in all the six tumors analyzed by array-comparative genomic hybridization. One patient died of tumor progression, one is on palliative care, one died of hypoventilation, and 10 patients are still alive, with median follow-up of 5 years. CONCLUSIONS: Based on histological phenotype, our series suggests that heterozygous PHOX2B NPARM do not fully preclude ganglion cell differentiation in tumors. However, this tumor predisposition syndrome may also be associated with poorly differentiated tumors with unfavorable genomic profiles and clinically aggressive behaviors. The intrafamilial variability and the unpredictable tumor prognosis should be considered in genetic counseling.

Rapid-Onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD): exome sequencing of trios, monozygotic twins and tumours.

BACKGROUND: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is thought to be a genetic disease caused by de novo mutations, though causative mutations have yet to be identified. We searched for de novo coding mutations among a carefully-diagnosed and clinically homogeneous cohort of 35 ROHHAD patients. METHODS: We sequenced the exomes of seven ROHHAD trios, plus tumours from four of these patients and the unaffected monozygotic (MZ) twin of one (discovery cohort), to identify constitutional and somatic de novo sequence variants. We further analyzed this exome data to search for candidate genes under autosomal dominant and recessive models, and to identify structural variations. Candidate genes were tested by exome or Sanger sequencing in a replication cohort of 28 ROHHAD singletons. RESULTS: The analysis of the trio-based exomes found 13 de novo variants. However, no two patients had de novo variants in the same gene, and additional patient exomes and mutation analysis in the replication cohort did not provide strong genetic evidence to implicate any of these sequence variants in ROHHAD. Somatic comparisons revealed no coding differences between any blood and tumour samples, or between the two discordant MZ twins. Neither autosomal dominant nor recessive analysis yielded candidate genes for ROHHAD, and we did not identify any potentially causative structural variations. CONCLUSIONS: Clinical exome sequencing is highly unlikely to be a useful diagnostic test in patients with true ROHHAD. As ROHHAD has a high risk for fatality if not properly managed, it remains imperative to expand the search for non-exomic genetic risk factors, as well as to investigate other possible mechanisms of disease. In so doing, we will be able to confirm objectively the ROHHAD diagnosis and to contribute to our understanding of obesity, respiratory control, hypothalamic function, and autonomic regulation.

The neuroblastoma and ganglion components of nodular ganglioneuroblastoma are genetically similar: evidence against separate clonal origins.

Nodular ganglioneuroblastoma is characterized by a macroscopic nodule of neuroblastoma within a ganglioneuromatous component. These two components have been considered to originate from separate clones, with the neuroblastoma clone accounting for the clinical behavior of nodular ganglioneuroblastoma. In order to investigate the clonal origin of the cellular components (neuroblasts, ganglion cells, and Schwann cells) of nodular ganglioneuroblastoma, paraffin-embedded tumor samples from eight cases were analyzed by single nucleotide polymorphism array and in situ hybridization. DNA was extracted separately from neuroblastomatous and ganglioneuromatous areas. By in situ hybridization, MYCN gain (4-10 gene copies/nucleus) was detected in 7/8 neuroblastoma samples. In ganglioneuromatous regions, gains were also detected in ganglion cells but not in Schwann cells. Single-nucleotide polymorphism array studies identified chromosome losses (11q and 14q) and gains (12, 13q, 17q and 18q) in the neuroblastoma component, whereas the ganglioneuromatous component showed fewer or no genetic alterations. There were no unique copy number changes distinguishing nodular ganglioneuroblastoma from other subtypes of neuroblastoma. By in situ hybridization, ganglion cells but not Schwann cells showed the same alterations detected in neuroblasts. Thus, neuroblasts and ganglion cells in nodular ganglioneuroblastoma are genetically related and may arise from the same clone. In contrast, the Schwann cells have a different origin and may be derived from a non-neoplastic neural crest precursor. Our results suggest that the clinical behavior of nodular ganglioneuroblastoma cannot be explained by the presence of separate clones with distinct genetic signatures.

[Molecular genetic abnormalities of N-myc and C-myc in pediatric neuroblastic tumors and clinical pathologic significance].

OBJECTIVE: To investigate the molecular genetic abnormalities of N-myc and C-myc, and their clinical pathological implications in pediatric neuroblastic tumors (NTs). METHODS: Abnormalities of N-myc were detected by interphase fluorescence in situ hybridization (FISH) technique in 246 cases of NTs, including neuroblastoma (NB,188 cases), ganglioneuroblastoma (GNB, 52 cases), ganglioneuroma (GN, 6 cases), and their association with the histological typing of the tumors and prognosis was analyzed. Abnormalities of C-myc were detected by FISH in 133 cases of NTs. RESULTS: Of the 246 cases of NTs, N-myc amplification was only found in 27 cases (11.0%, 27/246) of NB, but not in any cases of GNB or GN (P < 0.05). 89.0% (219/246) N-myc non-amplification were found in NTs, and it included N-myc gain in 175 cases (71.1%, 175/246) and normal N-myc in 44 cases (17.9%, 44/246). Univariate analysis indicated significantly (P = 0.012) poorer outcome in patients with N-myc amplification than N-myc non-amplification. However no significant difference was observed between N-myc gain cases and normal N-myc cases (P = 0.057). C-myc gain was found in 74 of 133 cases (55.6%) of NTs; no C-myc amplification or translocation was detected. Forty percent (6/15) of cases with N-myc amplification and 57.6% (68/118) of cases with N-myc non-amplification were accompanied by C-myc gain. The difference between N-myc amplification and non-amplification with C-myc gain was not significant (P > 0.05). Univariate analysis indicated that the outcome difference was not statistically significant between C-myc gain cases and normal C-myc cases (P = 0.357). CONCLUSIONS: The incidence of N-myc amplification only found in NB is low in pediatric NTs in China. Patients with N-myc amplification predict poorer outcome. No amplification or translocation of C-myc is detected in NTs, whereas C-myc gain is relatively common in NTs. There is no obvious association between N-myc amplification and C-myc gain.

High incidence of MYCN amplification in a Moroccan series of neuroblastic tumors: comparison to current biological data.

MYCN protooncogene status was assessed for the first time in Morocco in peripheral neuroblastic tumors, including neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Correlations with age at diagnosis, stage, mitosis-karyorrhexis index, differentiation, and Shimada histology were evaluated. Thirty-six formalin-fixed, paraffin-embedded peripheral neuroblastic tumor tissue specimens collected between 2007 and 2010 from the Pathology Department were assessed for MYCN amplification using fluorescence in situ hybridization. MYCN amplification was found in 27.8% of cases. An association of MYCN amplification with unfavorable Shimada grading, higher mitosis-karyorrhexis index, and undifferentiated morphologic phenotype was found. We found no correlation with older age, advanced stage, or the presence of metastasis. Our results suggested that the presence of MYCN amplification is a strong biological indicator of a poor outcome and aggressive disease in neuroblastoma and nodular ganglioneuroblastoma.