An Integrated Approach Including CRISPR/Cas9-Mediated Nanopore Sequencing, Mate Pair Sequencing, and Cytogenomic Methods to Characterize Complex Structural Rearrangements in Acute Myeloid Leukemia.

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach's value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Concomitance of a novel RMDN2-ALK fusion and an EML4-ALK fusion in a lung adenocarcinoma.

The anaplastic lymphoma kinase (ALK) fusions/rearrangements in non-small cell lung cancer (NSCLC) act as oncogenic driver mutations. ALK tyrosine kinase inhibitors have anti-tumor activities in ALK-positive NSCLC. Although the EML4-ALK fusion is common in NSCLC, concomitance of an additional ALK fusion together with an EML4-ALK fusion is not common. Here, we present a lung adenocarcinoma with two ALK fusions, a novel RMDN2-ALK fusion accompanied by an EML4-ALK fusion, detected by a targeted next generation sequencing assay. The genomic translocation breakpoints of the RMDN2-ALK fusion were mapped to intron 2 for RMDN2 and exon 15 for ALK, and EML4-ALK breakpoints were mapped to intron 13 for EML4 and intron 19 for ALK. ALK break-apart FISH detected multiple ALK rearrangements, a gene fusion panel (NanoString) test confirmed the EML4-ALK fusion, and RNA-sequencing revealed two ALK fusions. The RMDN2 gene locates at the short arm of chromosome 2 between ALK and EML4 genes. The intact ALK kinase domain fused to RMDN2. Genome-wide copy number variants were found in multiple chromosome arms and the short arm of chromosome 2, suggestive of complex rearrangements. Further detailed analyses of breakpoints and copy number variants may shed light on mechanisms of their formation and pathogenesis in lung malignancies.

Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies.

Copy number variants (CNVs) and gene mutations are important for diagnosis and treatment of myeloid malignancies. In a routine clinical setting, somatic gene mutations are detected by targeted next-generation sequencing (NGS) assay, but CNVs are commonly detected by conventional chromosome analysis and fluorescence in situ hybridization (FISH). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both somatic mutations and CNVs. Herein, we sequenced 406 consecutive patients with myeloid malignancies by targeted NGS and performed a head-to-head comparison with the results from a myelodysplastic syndrome (MDS) FISH and conventional chromosome analysis to detect CNVs. Among 91 patients with abnormal MDS FISH results, the targeted NGS revealed all 120 CNVs detected by MDS FISH (including -5/5q-, -7/7q-, +8, and 20q-) and 193 extra CNVs detected by conventional chromosome analysis. The targeted NGS achieved 100% concordance with the MDS FISH. The lower limit of detection of MDS CNVs by the targeted NGS was generally 5% variant allele fraction for DNA, based on the lowest percentages of abnormal cells detected by MDS FISH in this study. This proof-of-principle study demonstrated that the targeted NGS assay can simultaneously detect both MDS CNVs and somatic mutations, which can provide a more comprehensive genetic profiling for patients with myeloid malignancies using a single assay in a clinical setting.

Lymph node metastases of melanoma: challenges for BRAF mutation detection.

Detection of B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations is required to predict response to BRAF or mitogen-activated protein kinase kinase 1 and 2 inhibitors in metastatic melanoma. Lymph node (LN) specimens carrying melanoma cells intermingled with abundant lymphocytes often contain low tumor cellularity. This study is aimed to examine challenges in the clinical detection of BRAF mutations in LN specimens with metastatic melanoma and to illustrate characteristic features of p.V600E and non-p.V600E mutations. In this retrospective study for quality assessment of the pyrosequencing assay, we compared characteristics of 53 LN and 135 non-LN formalin-fixed, paraffin-embedded specimens with metastatic melanoma submitted for BRAF mutation detection over a 40-month period. LN specimens showed a significantly higher incidence of p.V600E mutations than non-LN specimens (49% versus 22%, P < .01) but a significantly lower tumor cellularity, particularly in the case of subcapsular or infiltrative metastases. Mutant allele-specific imbalance of the p.V600E mutation was predominantly present in specimens with distant organ metastases (79% versus 27% in LN metastases versus 13% in primary cutaneous tumors or adjacent soft tissue, P < .001). p.V600K was detected in 23% of men older than 60 years old, compared with 6% in women older than 60 years old and 2% in both men and women younger than 60 years old (P < .001). LN specimens with low tumor cellularity due to numerous adjacent lymphocytes may pose a challenge to clinical detection of BRAF mutations of melanoma. The higher incidence of p.V600E mutations in LNs may prompt further studies to elucidate if the p.V600E mutation in primary tumors is associated with a higher risk of LN metastasis.