A novel TP53 tandem duplication in a child with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is one of the most common cancer predisposition syndromes that affects both children and adults. Individuals with LFS are at an increased risk of developing various types of cancer over their lifetime including soft tissue sarcomas, osteosarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Heterozygous germline pathogenic variants in the tumor suppressor gene TP53 are the known causal genetic defect for LFS. Single-nucleotide variants (SNVs) including missense substitutions that occur in the highly conserved DNA binding domain of the protein are the most common alterations, followed by nonsense and splice site variants. Gross copy-number changes in TP53 are rare and account for <1% of all variants. Using next-generation sequencing (NGS) panels, we identified a paternally inherited germline intragenic duplication of TP53 in a child with metastatic osteosarcoma who later developed acute myeloid leukemia (AML). Transcriptome sequencing (RNA-seq) demonstrated the duplication was tandem, encompassing exons 2-6 and 28 nt of the untranslated region (UTR) upstream of the start codon in exon 2. The inclusion of the 28 nt is expected to result in a frameshift with a stop codon 18 codons downstream from the exon 6, leading to a loss-of-function allele. This case highlights the significance of simultaneous identification of both significant copy-number variants as well as SNVs/indels using NGS panels.

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.

Unique Familial MLL(KMT2A)-Rearranged Precursor B-Cell Infant Acute Lymphoblastic Leukemia in Non-twin Siblings.

BACKGROUND: Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the ∼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes. PROCEDURES: Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array. RESULTS: The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5'-MLL-AFF1-3' fusion and a non-productive rearrangement of 3'MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 10(9) families. CONCLUSIONS: Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.

Acquired isochromosome 12p, somatic TP53 and PTEN mutations, and a germline ATM variant in an adolescent male with concurrent acute megakaryoblastic leukemia and mediastinal germ cell tumor.

Previous reports have described an association between hematologic malignancies (HMs) and extragonadal germ cell tumor (GCT). Most patients have been adolescent males with mediastinal nonseminomatous GCT. Although a variety of HMs have been reported, there is a striking predilection toward acute megakaryoblastic leukemia (AMKL). Shared cytogenetic anomalies--particularly isochromosome 12p [i(12p)]--have suggested common clonal origins to the tumors. We report the case of a 17-year-old boy presenting with AMKL and a synchronous mediastinal GCT, with the characteristic i(12p) in both neoplasms. The common clonal origin of the AMKL and GCT was further confirmed with massively parallel sequencing, which identified somatic TP53 and PTEN mutations, as well as a rare germline ATM variant. Although these represent commonly mutated genes in cancer, this combination of mutations is not typically associated with either GCT or AMKL, suggesting that these tumors may represent unique biologic entities when they co-occur.

CLL/SLL diagnosed in an adolescent.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a disease of older adults. Pediatric CLL/SLL is vanishingly rare in the literature. We present a case of CLL/SLL diagnosed in a 17-year-old male. The pathologic findings of this case were those of classic CLL/SLL with an ATM deletion, a characteristic genetic abnormality in CLL/SLL. Management guidelines for CLL/SLL are tailored to older adults making determination of the optimal therapy for this patient a unique challenge.

Implementation of high resolution single nucleotide polymorphism array analysis as a clinical test for patients with hematologic malignancies.

Single nucleotide polymorphism-based oligonucleotide arrays have been used as a research tool to detect genomic copy number changes and allelic imbalance in a variety of hematologic malignancies and solid tumors. The high resolution, genome-wide coverage, minimal DNA requirements, and relatively short turnaround time are advantageous for use in a clinical setting. We validated the Illumina HumanHap550 BeadChip array for clinical use by analyzing 127 pediatric leukemia and lymphoma samples that had previously been characterized by means of standard cytogenetic analysis and fluorescence in situ hybridization. A higher resolution Illumina HumanHap610 BeadChip array was ultimately used for clinical testing. To date, 180 samples from children with a suspected or confirmed hematologic malignancy have been analyzed. Of the 180 clinical samples, 130 (72%) bone marrow or lymphoma specimens had aberrations revealed by the array that were not seen in the karyotypes. These typically included deletions in genes associated with B- or T-cell malignancies, such as CDKN2A/B, PAX5, and IKZF1. There were also 75 regions of copy number neutral loss of heterozygosity (>5 Mb threshold) detected in 49 samples in this cohort, which could be categorized as constitutional or acquired abnormalities. On the basis of our experience in the last 2 years, we suggest that single nucleotide polymorphism arrays are a valuable addition to, but not a replacement for, standard cytogenetic approaches for hematologic malignancies.