Neuroblastoma patient-derived cultures are enriched for a mesenchymal gene signature and reflect individual drug response.

Ex vivo evaluation of personalized models can facilitate individualized treatment selection for patients, and advance the discovery of novel therapeutic options. However, for embryonal malignancies, representative primary cultures have been difficult to establish. We developed patient-derived cell cultures (PDCs) from chemo-naïve and post-treatment neuroblastoma tumors in a consistent and efficient manner, and characterized their in vitro growth dynamics, histomorphology, gene expression, and functional chemo-response. From 34 neuroblastoma tumors, 22 engrafted in vitro to generate 31 individual PDC lines, with higher engraftment seen with metastatic tumors. PDCs displayed characteristic immunohistochemical staining patterns of PHOX2B, TH, and GD2 synthase. Concordance of MYCN amplification, 1p and 11q deletion between PDCs and patient tumors was 83.3%, 72.7%, and 80.0% respectively. PDCs displayed a predominantly mesenchymal-type gene expression signature and showed upregulation of pro-angiogenic factors that were similarly enriched in culture medium and paired patient serum samples. When tested with standard-of-care cytotoxics at human Cmax -equivalent concentrations, MYCN-amplified and non-MYCN-amplified PDCs showed a differential response to cyclophosphamide and topotecan, which mirrored the corresponding patients' responses, and correlated with gene signatures of chemosensitivity. In this translational proof-of-concept study, early-phase neuroblastoma PDCs enriched for the mesenchymal cell subpopulation recapitulated the individual molecular and phenotypic profile of patient tumors, and highlighted their potential as a platform for individualized ex vivo drug-response testing.

De novo trisomy 12p in twin girls with different levels of mosaicism.

We report on a pair of twins with trisomy 12p diagnosed postnatally. The girls were referred for dysmorphism and global developmental delay and have been followed from 10 months of age. They have different levels of mosaicism for both buccal cells and lymphocytes. Although their phenotypic features were similar, there were different degrees of severity which correlate with the different levels of mosaicism.

Clinicopathological and treatment response characteristics of updated rhabdomyosarcoma histomolecular subtypes: An Asian population-based study.

AIM: New histomolecular subtypes of rhabdomyosarcoma have recently been defined but their corresponding clinical characteristics are not well described. Also, these clinical phenotypes vary greatly by age and ethnicity but have not been profiled in Asian populations. Thus, we sought to determine the landscape of rhabdomyosarcoma subtypes in a national Asian cohort and compare clinical characteristics among age groups and molecular subtypes. METHODS: We performed a retrospective population-based study of all rhabdomyosarcoma patients in Singapore public hospitals from 2004 to 2014 (n = 67), and assigned histomolecular subtypes according to the updated 2020 WHO classification of soft tissue tumors following central pathology review and molecular profiling. RESULTS: Age-specific prevalence followed a tri-modal peak. There were significantly more embryonal and alveolar (p = 0.032) and genitourinary (non-bladder/prostate) tumors (p = 0.033) among children. Older age was associated with complete resection among spindle cell/sclerosing tumors (p = 0.027), with the omission of chemotherapy among embryonal tumors (p = 0.001), and with poorer survival among embryonal and alveolar tumors (p = 0.026, p = 0.022, respectively). Overall survival differed with stage, group, and surgical resection, adjusted for age group (p = 0.004, p = 0.001, p = 0.004, respectively). Spindle-cell/sclerosing tumors showed an indolent phenotype with a significantly lower incidence of nodal metastasis (p = 0.002), but two of 15 patients with MYOD1 mutations had a contrastingly aggressive disease. CONCLUSION: Disease and treatment response profiles of rhabdomyosarcoma subtypes vary significantly between adults and children, especially surgical resectability. In our Asian population, poorer outcomes were observed in adults with embryonal and alveolar tumors, while activating mutations influence the behavior of otherwise favorable spindle cell/sclerosing tumors.

Validation of first trimester screening for trisomy 21 in Singapore with reference to performance of nasal bone.

INTRODUCTION: The aim of this study is to describe the performance of first trimester screening (FTS) for trisomy 21 using maternal age, serum biochemistry and fetal nuchal translucency (NT) in a single center and to evaluate the effect of nasal bone on screening performance. MATERIAL AND METHODS: In 12,585 singleton pregnancies, the NT and nasal bone were examined. The majority of these mothers also had their serum biochemical markers analyzed. Risk was computed using different combinations of maternal age, biochemistry, NT and nasal bone. Down syndrome cases were confirmed by karyotyping. RESULTS: There were 12,519 normal pregnancies, 31 with trisomy 21 and 35 with other chromosomal abnormalities. Without considering the nasal bone, the combined FTS detected 87.1% of trisomy 21 fetuses (false positive rate 5.1%), using 1:300 as the risk threshold, and this was further improved to 96.8% with the policy that classifies all fetuses with an absent nasal bone as high risk. Subgroup analysis showed that the detection rate would be 90.9%, with a false positive rate of 3.7%, if nasal bone was incorporated in the risk algorithm, compared to 81.8% and a false positive rate of 5.4% if it was not used. DISCUSSION: FTS is very effective in early detection of trisomy 21 in Singapore. The nasal bone is a useful marker that can substantially improve the screening performance.

Gene expression of TRK neurotrophin receptors in advanced neuroblastomas in Singapore--a pilot study.

The clinical hallmark of neuroblastoma is heterogeneity. Biologically, ploidy and N-Myc amplification are currently the only 2 features used to define risk group and to determine therapy. Tyrosine kinase neurotrophin receptors (Trks, including TrkA, TrkB, and TrkC) are important in the clinical and biological behavior of neuroblastomas. The authors aim to study Trks gene expression in their local population of advanced neuroblastoma patients. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay on the expression of TrkA, TrkB, TrkB-truncated, and TrkC was performed on a total of 19 advanced neuroblastoma archival tumors, diagnosed in KK Women's and Children's Hospital between 2003 and 2007. Of the 19 tumors investigated, Trks expression was present in 14 (73.6%) cases. Of these cases, 8 (42.1%), 10 (52.6%), 7 (36.8%), and 6 (31.6%) expressed TrkA, TrkB, TrkB-truncated, and TrkC receptor mRNAs, respectively. Subsequently, the authors compared Trks expression with N-Myc amplification status of the 19 patients. N-Myc was amplified in 5 (26.3%) of the cases. Within the non-N-Myc-amplified group, Trks expression was present in 9 (64%) of the 14 cases. The significant expression of Trk isoforms among advanced neuroblastoma cases as evident from this study support their role as possible risk assessment tools alongside N-Myc amplification status.

Characterization of anaplastic lymphoma kinase-positive medulloblastomas.

Medulloblastomas are the most common pediatric malignant primary brain tumor. To our knowledge, there are no known critical and druggable tyrosine kinases in medulloblastomas, precluding the use of established tyrosine kinase inhibitors that have shown efficacy in other tumor types. We studied the expression of anaplastic lymphoma kinase (ALK), a well-characterized tyrosine kinase and drug target, in a cohort of medulloblastomas by immunohistochemistry, and identified three ALK-positive cases. Mutational analyses did not reveal a definite underlying genetic mechanism for the ALK expression, although one of the cases showed increased ALK copy number. Our findings have clinical implications and warrant further pharmacological and functional studies, as well as evaluation in larger patient cohorts, to fully characterize the value of ALK as a prognostic and predictive therapeutic marker in medulloblastomas.

Chromosome 15q11-q13 copy number gain detected by array-CGH in two cases with a maternal methylation pattern.

BACKGROUND: The 15q11-q13 region contains many low copy repeats and is well known for its genomic instability. Several syndromes are associated with genomic imbalance or copy-number-neutral uniparental disomy. We report on two patients: Patient 1 is a boy with developmental delay and autism; and Patient 2 is a girl with developmental delay, hypotonia and dysmorphism. We performed analyses to delineate their dosage in the 15q region, determine whether the patients' dosage correlates with phenotypic severity, and whether genes in the amplified regions are significantly associated with identified functional networks. RESULTS: For the proximal region of 15q, molecular cytogenetic analysis with Agilent oligonucleotide array showed a copy number of 3 for Patient 1 and a copy number of 4 for Patient 2. Fluorescent in situ hybridization analysis of Patient 2 showed two different populations of cells with different marker chromosomes. Methylation analysis of the amplified region showed that the extra copies of small nuclear ribonucleoprotein polypeptide N gene were of maternal origin. Phenotypic severity did not correlate with the size and dosage of 15q, or whether the amplification is interstitial or in the form of a supernumerary marker. Pathway analysis showed that in Patient 2, the main functional networks that are affected by the genes from the duplicated/triplicated regions are developmental disorder, neurological disease and hereditary disease. CONCLUSIONS: The 15q11-q13 gains that were found in both patients could explain their phenotypic presentations. This report expands the cohort of patients for which 15q11-q13 duplications are molecularly characterized.

De novo 3q22.1 q24 deletion associated with multiple congenital anomalies, growth retardation and intellectual disability.

We describe a boy with a de novo deletion of 15.67 Mb spanning 3q22.1q24. He has bilateral micropthalmia, ptosis, cleft palate, global developmental delay and brain, skeletal and cardiac abnormalities. In addition, he has bilateral inguinal hernia and his right kidney is absent. We compare his phenotype with seven other patients with overlapping and molecularly defined interstitial 3q deletions. This patient has some phenotypic features that are not shared by the other patients. More cases with smaller deletions defined by high resolution aCGH will enable better genotype-phenotype correlations and prioritizing of candidate genes for the identification of pathways and disease mechanisms.