Complex/cryptic EWSR1::FLI1/ERG Gene Fusions and 1q Jumping Translocation in Pediatric Ewing Sarcomas.

Ewing sarcomas (ES) are rare small round cell sarcomas often affecting children and characterized by gene fusions involving one member of the FET family of genes (usually EWSR1) and a member of the ETS family of transcription factors (usually FLI1 or ERG). The detection of EWSR1 rearrangements has important diagnostic value. Here, we conducted a retrospective review of 218 consecutive pediatric ES at diagnosis and found eight patients having data from chromosome analysis, FISH/microarray, and gene-fusion assay. Three of these eight ES had novel complex/cryptic EWSR1 rearrangements/fusions by chromosome analysis. One case had a t(9;11;22)(q22;q24;q12) three-way translocation involving EWSR1::FLI1 fusion and 1q jumping translocation. Two cases had cryptic EWSR1 rearrangements/fusions, including one case with a cryptic t(4;11;22)(q35;q24;q12) three-way translocation involving EWSR1::FLI1 fusion, and the other had a cryptic EWSR1::ERG rearrangement/fusion on an abnormal chromosome 22. All patients in this study had various aneuploidies with a gain of chromosome 8 (75%), the most common, followed by a gain of chromosomes 20 (50%) and 4 (37.5%), respectively. Recognition of complex and/or cryptic EWSR1 gene rearrangements/fusions and other chromosome abnormalities (such as jumping translocation and aneuploidies) using a combination of various genetic methods is important for accurate diagnosis, prognosis, and treatment outcomes of pediatric ES.

HER2 amplification by next-generation sequencing to identify HER2-positive invasive breast cancer with negative HER2 immunohistochemistry.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) positive breast carcinomas due to HER2 amplification are associated with aggressive behavior and a poor prognosis. Anti-HER2-targeted therapies are widely used to treat HER2-positive breast carcinomas with excellent outcomes. Accurate identification of HER2 amplification status in breast carcinomas is of important diagnostic and treatment value. Currently, HER2 amplification status is routinely determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) testing. This study will review our past HER2 data to determine and characterize discordant results between HER2 IHC and FISH. It will also determine a potential impact of HER2 amplification status by next-generation sequencing (NGS) on these patients. METHODS: We reviewed a total of 4884 breast carcinomas with coexisting HER2 IHC and HER2 FISH performed at our institution from 2010 to 2022. 57 cases also had a Next-Generation-Sequencing-based (NGS) gene panel performed. Given the advances in biostatic analysis pipelines, NGS methods were utilized to provide results on HER2 amplification status along with somatic mutations. RESULTS: While the majority (ranging from 98.5% with IHC score of 0 and 93.1% with IHC score of 1 +) of 4884 breast carcinomas had concordant results from HER2 IHC and HER2 FISH testing, a small percentage of patients (ranging from 1.5% in those with IHC score of 0, to 6.9% with IHC score of 1 +) had discordant results, with negative HER2 IHC and positive HER2 FISH results. These patients could be reported as HER2-negative breast carcinomas if only HER2 IHC testing has been performed according to a current cost-effective HER2 test strategy. 57 patients had HER2 amplification status determined by NGS, and all patients had concordant results between HER2 NGS and FISH tests. A HER2-amplified breast carcinoma by NGS had a negative IHC and a positive HER2 FISH result. This case was classified as a HER2-positive breast carcinoma, had anti-HER2-targeted therapy, and achieved a complete clinical response. CONCLUSIONS: A small percentage of HER2-positive breast carcinomas are unidentified because of a negative HER2 IHC based on our current cost-effective HER2 test strategy. It is not feasible and affordable in routine clinical practice to perform HER2 FISH for the cases with negative HER2 IHC (IHC score 0 and 1 +). Therefore, NGS assays capable of simultaneously detecting both somatic mutations and HER2 amplification could provide a more comprehensive genetic profiling for breast carcinomas in a clinical setting. Identification of HER2 amplification by NGS in HER2-positive breast carcinomas with negative HER2 IHC results is important since these cases are concealed by our current cost-effective HER2 test strategy with IHC first (for all cases) and FISH reflex (only for cases with IHC score of 2 +), and would offer the opportunity for potentially beneficial anti-HER2-targeted therapies for these patients.

Utility of targeted next-generation sequencing assay to detect 1p/19q co-deletion in formalin-fixed paraffin-embedded glioma specimens.

Molecular classification of brain neoplasms is important for diagnosis, prognosis, and treatment outcome of histologically similar tumors. Oligodendroglioma is a glioma subtype characterized by 1p/19q co-deletion and IDH1/IDH2 mutations, which predict a good prognosis, responsiveness to therapy, and an improved overall survival compared to other adult gliomas. In a routine clinical setting, 1p/19q co-deletion is detected by interphase-FISH and SNP microarray, and somatic mutations are detected by targeted next-generation sequencing (NGS). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both 1p/19q co-deletion and somatic mutations. Among 247 consecutive patients with formalin-fixed paraffin-embedded brain tumors with various subtypes, NGS revealed 1p/19q co-deletion in 26 oligodendrogliomas and an IDH-wildtype astrocytoma, and partial loss across chromosomes 1p and 19q/whole-arm loss of 1p or 19q/copy neutral loss of heterozygosity in 11 nonoligodendrogliomas. For this 247 brain-tumor cohort, the overall sensitivity, specificity, and accuracy of detecting 1p/19q co-deletion by NGS in oligodendrogliomas were 96.2%, 99.6%, and 99.2%, respectively. The oligodendroglioma cohort had more mutations in IDH1/IDH2, CIC, FUBP1, and TERT, and fewer mutations in ATRX and TP53 than the nonoligodendroglioma cohort. This proof-of-concept study demonstrated that targeted NGS can simultaneously detect both 1p/19q co-deletion and somatic mutations, which can provide a more comprehensive genetic profiling for patients with gliomas using a single assay in a clinical setting.

Diagnostic Utility of Gene Fusion Panel to Detect Gene Fusions in Fresh and Formalin-Fixed, Paraffin-Embedded Cancer Specimens.

Somatic gene fusions are common in leukemias/lymphomas and solid tumors. The detection of gene fusions is crucial for diagnosis. NanoString fusion technology is a multiplexed hybridization method that interrogates hundreds of gene fusions in a single reaction. This study's objective was to determine the performance characteristics and diagnostic utility of NanoString fusion assays in a clinical diagnostics laboratory. Validation using 100 positive specimens and 15 negative specimens by a combined reference standard of fluorescence in situ hybridization (FISH)/RT-PCR/next-generation sequencing (NGS) assays achieved 100% sensitivity in leukemias/lymphomas and 95.0% sensitivity and 100% specificity in solid tumors. Subsequently, 214 consecutive clinical cases, including 73 leukemia/lymphoma specimens and 141 formalin-fixed, paraffin-embedded solid tumor specimens, were analyzed by gene fusion panels across 638 unique gene fusion transcripts. A variety of comparator tests, including FISH panels, conventional karyotyping, a DNA-based targeted NGS assay, and custom RT-PCR testing, were performed in parallel. The gene fusion assay detected 31 gene fusions, including 16 in leukemia/lymphoma specimens and 15 in solid tumor specimens. The overall sensitivity, specificity, and accuracy of gene fusions detected by the gene fusion panel in all 329 specimens (validation and consecutive clinical specimens) tested in this study were 94.8%, 100%, and 97.9%, respectively, compared with FISH/RT-PCR/NGS assays. The gene fusion panel is a reliable approach that maximizes molecular detection of fusions among both fresh and formalin-fixed, paraffin-embedded cancer specimens.

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.