Clinical validation of the Ion Torrent Oncomine Myeloid Assay GX v2 on the Genexus Integrated Sequencer as a stand-alone assay for single-nucleotide variants, insertions/deletions, and fusion genes: Challenges, performance, and perspectives.

OBJECTIVES: Myeloid neoplasms require comprehensive characterization of genetic abnormalities, including single-nucleotide variants, small insertions and deletions, and fusions and translocations for management. The Oncomine Myeloid Assay GX v2 (Thermo Fisher Scientific) analyzes 17 full genes, 28 hotspot genes, 30 fusion driver genes, and 5 expression genes. METHODS: The validation set included 192 DNA samples, 28 RNA samples, and 9 cell lines and contrived controls. The DNA and RNA were extracted from both peripheral blood and bone marrow. Library preparation, templating, and sequencing was performed on the fully automated Genexus Integrated Sequencer (Thermo Fisher Scientific). The sequencing data were analyzed by manual curation, default Oncomine filters and the Oncomine Reporter (Thermo Fisher Scientific). RESULTS: Of the 600 reference pathogenic DNA variants targeted by the assay, concordance was seen in 98.3% of unfiltered variant call format files. Precision and reproducibility were 100%, and the lower limit of detection was 2% variant allele frequency for DNA. Inability to detect variants in long homopolymer regions intrinsic to the Ion Torrent chemistry led to 7 missed variants; 100% concordance was seen with reference RNA samples. CONCLUSIONS: This extensive clinical validation of the Oncomine Myeloid Assay GX v2 on the Genexus Integrated Sequencer with its built-in bioinformatics pipeline and Ion Torrent Oncomine Reporter shows robust performance in terms of variant calling accuracy, precision, and reproducibility, with the advantage of a rapid turnaround time of 2 days. The greatest limitation is the inability to detect variants in long homopolymer regions.

Prothrombin deficiency with recurrent subretinal hemorrhage.

Ocular hemorrhage has been recorded in congenital factor deficiencies like hemophilia A, but it has never been documented in prothrombin deficiency. Here, we describe an unusual case of sudden vision loss in the right eye caused by subretinal hemorrhage following a coughing episode in a 67-year-old woman. Notably, the patient underwent left eye enucleation 12 years previously under similar circumstances due to subretinal hemorrhage. During the interview, it was discovered that the patient had a history of prothrombin deficiency, which was subsequently confirmed through laboratory testing. Aside from recurrent ocular bleeding and 1 instance of bleeding following dental extraction in childhood, there is no other history of bleeding. Subsequent molecular studies revealed a homozygous missense mutation at G1499A (Arg500Gln), a variant previously identified as R457Q. Although the likelihood of prothrombin deficiency initiating subretinal hemorrhage is low, it is likely to worsen retinal hemorrhage and contribute to difficulty in controlling bleeding. A comprehensive coagulation workup is essential in patients with ocular hemorrhage. Determining factor II activity should be included in individuals exhibiting variably prolonged prothrombin time and activated partial thromboplastin time with correction in mixing studies. Additional investigations, such as genetic sequencing and family studies, are advised for those with isolated low prothrombin levels.

MPO Expression of Background Neutrophils in MPO Negative Acute Promyelocytic Leukemia, An Easy Clue to Corroborate a Challenging Diagnosis: A Case Report and Review of Literature.

Acute promyelocytic leukemia (APL) is characterized by the pathogenic driver fusion transcript PML-RARA resulting from the t(15;17) translocation. Early recognition of APL with prompt ATRA induction has a decisive impact on the early death rate. The preliminary diagnosis of APL relies heavily on cytomorphology and flow cytometry. In APL with variant morphology, such as the microgranular variant, immunophenotype, especially the bright MPO positivity is the basis of diagnosis. Till date, only five cases of APL with reduced/absent MPO have been described in literature. The identification of MPO deficiency based on genetic testing would involve at the least a MPO gene scanning with NGS, followed by microarray to identify somatic uniparental disomy in heterozygotes. This testing is not only redundant given the scant clinical implications of heterozygous MPO deficiency but also time consuming. An easy way to identify background MPO deficiency confounding the immunophenotype of a myeloid neoplasm is the MPO expression in background neutrophils gated on the initial flow cytometry. A dim MPO in the background neutrophils, in the morphological setting of APL, can identify underlying MPO deficiency, clarifying the immunophenotypic ambiguity and thus establishing an unequivocal diagnosis as seen in the current case.