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.