OncoKids: A Comprehensive Next-Generation Sequencing Panel for Pediatric Malignancies.

The OncoKids panel is an amplification-based next-generation sequencing assay designed to detect diagnostic, prognostic, and therapeutic markers across the spectrum of pediatric malignancies, including leukemias, sarcomas, brain tumors, and embryonal tumors. This panel uses low input amounts of DNA (20 ng) and RNA (20 ng) and is compatible with formalin-fixed, paraffin-embedded and frozen tissue, bone marrow, and peripheral blood. The DNA content of this panel covers the full coding regions of 44 cancer predisposition loci, tumor suppressor genes, and oncogenes; hotspots for mutations in 82 genes; and amplification events in 24 genes. The RNA content includes 1421 targeted gene fusions. We describe the validation of this panel by using a large cohort of 192 unique clinical samples that included a wide range of tumor types and alterations. Robust performance was observed for analytical sensitivity, reproducibility, and limit of detection studies. The results from this study support the use of OncoKids for routine clinical testing of a wide variety of pediatric malignancies.

Copy number alterations determined by single nucleotide polymorphism array testing in the clinical laboratory are indicative of gene fusions in pediatric cancer patients.

Gene fusions resulting from structural rearrangements are an established mechanism of tumorigenesis in pediatric cancer. In this clinical cohort, 1,350 single nucleotide polymorphism (SNP)-based chromosomal microarrays from 1,211 pediatric cancer patients were evaluated for copy number alterations (CNAs) associated with gene fusions. Karyotype or fluorescence in situ hybridization studies were performed in 42% of the patients. Ten percent of the bone marrow or solid tumor specimens had SNP array-associated CNAs suggestive of a gene fusion. Alterations involving ETV6, ABL1-NUP214, EBF1-PDGFRB, KMT2A(MLL), LMO2-RAG, MYH11-CBFB, NSD1-NUP98, PBX1, STIL-TAL1, ZNF384-TCF3, P2RY8-CRLF2, and RUNX1T1-RUNX1 fusions were detected in the bone marrow samples. The most common alteration among the low-grade gliomas was a 7q34 tandem duplication resulting in a KIAA1549-BRAF fusion. Additional fusions identified in the pediatric brain tumors included FAM131B-BRAF and RAF1-QKI. COL1A1-PDGFB, CRTC1-MAML2, EWSR1, HEY1, PAX3- and PAX7-FOXO1, and PLAG1 fusions were determined in a variety of solid tumors and a novel potential gene fusion, FGFR1-USP6, was detected in an aneurysmal bone cyst. The identification of these gene fusions was instrumental in tumor diagnosis. In contrast to hematologic and solid tumors in adults that are predominantly driven by mutations, the majority of hematologic and solid tumors in children are characterized by CNAs and gene fusions. Chromosomal microarray analysis is therefore a robust platform to identify diagnostic and prognostic markers in the clinical setting.

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Disrupting LIN28 in atypical teratoid rhabdoid tumors reveals the importance of the mitogen activated protein kinase pathway as a therapeutic target.

Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.

SWI/SNF chromatin remodeling complexes and cancer.

The identification of mutations and deletions in the SMARCB1 locus in chromosome band 22q11.2 in pediatric rhabdoid tumors provided the first evidence for the involvement of the SWI/SNF chromatin remodeling complex in cancer. Over the last 15 years, alterations in more than 20 members of the complex have been reported in a variety of human tumors. These include germline mutations and copy number alterations in SMARCB1, SMARCA4, SMARCE1, and PBRM1 that predispose carriers to both benign and malignant neoplasms. Somatic mutations, structural abnormalities, or epigenetic modifications that lead to reduced or aberrant expression of complex members have now been reported in more than 20% of malignancies, including both solid tumors and hematologic disorders in both children and adults. In this review, we will highlight the role of SMARCB1 in cancer as a paradigm for other tumors with alterations in SWI/SNF complex members and demonstrate the broad spectrum of mutations observed in complex members in different tumor types.

Diagnostic application of high resolution single nucleotide polymorphism array analysis for children with brain tumors.

Single nucleotide polymorphism (SNP) array analysis is currently used as a first tier test for pediatric brain tumors at The Children's Hospital of Philadelphia. The results from 100 consecutive patients are summarized in the present report. Eighty-seven percent of the tumors had at least one pathogenic copy number alteration. Nineteen of 56 low grade gliomas (LGGs) demonstrated a duplication in 7q34, which resulted in a KIAA1549-BRAF fusion. Chromosome band 7q34 deletions, which resulted in a FAM131B-BRAF fusion, were identified in one pilocytic astrocytoma (PA) and one dysembryoplastic neuroepithelial tumor (DNT). One ganglioglioma (GG) demonstrated a 6q23.3q26 deletion that was predicted to result in a MYB-QKI fusion. Gains of chromosomes 5, 6, 7, 11, and 20 were seen in a subset of LGGs. Monosomy 6, deletion of 9q and 10q, and an i(17)(q10) were each detected in the medulloblastomas (MBs). Deletions and regions of loss of heterozygosity that encompassed TP53, RB1, CDKN2A/B, CHEK2, NF1, and NF2 were identified in a variety of tumors, which led to a recommendation for germline testing. A BRAF p.Thr599dup or p.V600E mutation was identified by Sanger sequencing in one and five gliomas, respectively, and a somatic TP53 mutation was identified in a fibrillary astrocytoma. No TP53 hot-spot mutations were detected in the MBs. SNP array analysis of pediatric brain tumors can be combined with pathologic examination and molecular analyses to further refine diagnoses, offer more accurate prognostic assessments, and identify patients who should be referred for cancer risk assessment.

High-Resolution genomic arrays identify CNVs that phenocopy the chromosome 22q11.2 deletion syndrome.

The 22q11 Deletion Syndrome includes the overlapping phenotypes of DiGeorge/Velocardiofacial Syndromes, characterized by conotruncal heart defects, cleft palate, thymus, and parathyroid gland dysplasia. The majority (90%) of patients harbor detectable chr22q11.2 deletions, but a genetic etiology for the remainder of patients without a deletion can remain undefined despite major birth defects. We analyzed DNA from eight patients with normal 22q11 FISH studies by high-density single nucleotide polymorphism (SNP) arrays and identified potentially pathogenic copy number variants (CNVs) in four of eight patients. Two patients showed large CNVs in regions of known genomic disorders: one a deletion of distal chr22q11.2 and the other a duplication of chr5q35. A 3-Mb deletion of chr19p13.3 that includes a gene associated with conotruncal heart defects was found in a third patient. Two potentially pathogenic CNVs were found in a fourth patient: a large heterozygous deletion of chr6p24 and a smaller duplication of chr9p24. Our findings support a recent consensus statement advocating chromosomal microarray analysis as a first-line diagnostic approach for patients with multiple congenital anomalies. In patients with phenotypes suggestive of the 22q11.2 syndrome spectrum and normal FISH, microarray analysis can uncover the molecular basis of other genomic disorders whose features overlap those of 22q11.2 deletions.

Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway.

The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid beta protein (Abeta), a small peptide derived from beta- and gamma-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that beta- and gamma-secretase activities of BACE1 and presenilin, respectively, are concentrated in intracellular lipid raft microdomains. However, the manner in which APP normally traffics to lipid rafts is unknown. In this study, using transient transfection and immuno-precipitation assays, we show that the cytoplasmic domain of low-density lipoprotein receptor-related protein (LRP) interacts with APP and increases Abeta secretion and APP beta-CTF (C-terminal fragment) generation by promoting BACE1-APP interaction. We also employed discontinuous sucrose density gradient ultracentrifugation to show that the LRP cytoplasmic domain-mediated effect was accompanied by greatly increased localization of APP and BACE1 to lipid raft membranes, where beta- and gamma-secretase activities are highly enriched. Moreover, we provide evidence that endogenous LRP is required for the normal delivery of APP to lipid rafts and Abeta generation primarily in the endocytic but not secretory pathway. These results may provide novel insights to block Abeta generation by targeting LRP-mediated delivery of APP to raft microdomains.

Presenilins mediate phosphatidylinositol 3-kinase/AKT and ERK activation via select signaling receptors. Selectivity of PS2 in platelet-derived growth factor signaling.

The Alzheimer's disease-linked genes, PS1 and PS2, are required for intramembrane proteolysis of multiple type I proteins, including Notch and amyloid precursor protein. In addition, it has been documented that PS1 positively regulates, whereas PS1 familial Alzheimer disease mutations suppress, phosphatidylinositol 3-kinase (PI3K)/Akt activation, a pathway known to inactivate glycogen synthase kinase-3 and reduce tau phosphorylation. In this study, we show that the loss of presenilins not only inhibits PI3K/Akt signaling and increases tau phosphorylation but also suppresses the MEK/ERK pathway. The deficits in Akt and ERK activation in cells deficient in both PS1 and PS2 (PS-/-) are evident after serum withdrawal and stimulation with fetal bovine serum or ligands of select receptor tyrosine kinases, platelet-derived growth factor receptor beta (PDGFR beta) and PDGFR alpha, but not insulin-like growth factor-1R and epidermal growth factor receptor. The defects in PDGF signaling in PS-/- cells are due to reduced expression of PDGF receptors. Whereas fetal bovine serum-induced Akt activation is reconstituted by both PS1 and PS2 in PS-/- cells, PDGF signaling is selectively restored by PS2 but not PS1 and is dependent on the N-terminal fragment of PS2 but not gamma-secretase activity or the hydrophilic loop of PS2. The rescue of PDGF receptor expression and activation by PS2 is facilitated by FHL2, a PS2-interacting transcriptional co-activator. Finally, we present evidence that PS1 mutations interfere with this PS2-mediated activity by reducing PS2 fragments. These findings highlight important roles of both presenilins in Akt and ERK signaling via select signaling receptors.

FE65 constitutes the functional link between the low-density lipoprotein receptor-related protein and the amyloid precursor protein.

Increasing evidence has implicated the low density lipoprotein receptor-related protein (LRP) and the adaptor protein FE65 in Alzheimer's disease pathogenesis. We have shown previously that LRP mediates beta-amyloid precursor protein (APP) processing and affects amyloid beta-protein and APP secretion and APP-c-terminal fragment generation. Furthermore, LRP mediates APP processing through its intracellular domain. Here, we set out to examine whether this interaction is of direct or indirect nature. Specifically, we asked whether adaptor proteins such as FE65 influence the LRP-mediated effect on APP processing by forming a protein complex. In coimmunoprecipitation experiments, we confirmed the postulated APP-FE65 and the LRP-FE65 interaction. However, we also showed an LRP-FE65-APP trimeric complex using pull-down techniques. Because FE65 alters APP processing, we investigated whether this effect is LRP dependent. Indeed, FE65 was only able to increase APP secretion in the presence of LRP. In the absence of LRP, APP secretion was unchanged compared with the LRP knock-out phenotype. Using RNA short interference techniques against FE65, we demonstrated that a reduction in FE65 protein mimics the LRP knock-out phenotype on APP processing. These results clearly demonstrate that FE65 acts as a functional linker between APP and LRP.

The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing.

The low-density lipoprotein receptor-related protein (LRP) has recently been implicated in numerous intracellular signaling functions, as well as in Alzheimer's disease pathogenesis. Studies have shown that the beta-amyloid precursor protein (APP) interacts with LRP and that this association may impact the production of amyloid beta-protein (Abeta). In this report, we provide evidence that LRP regulates trafficking of intracellular proteins independently of its lipoprotein receptor functions. We show that in the absence of LRP, Abeta production, APP secretion, APP internalization, turnover of full-length APP and stability of APP C-terminal fragments are affected. Importantly, these changes are not APP isoform dependent. Using deletion constructs, the critical region in LRP that modulates APP processing was mapped to a seven peptide domain around the second NPXY domain (residues 4504-4510). Therefore, we propose a model by which LRP functionally modulates APP processing, including those steps critical for Abeta production, through interactions of the cytosolic domains.