Optical Genome Mapping: Clinical Validation and Diagnostic Utility for Enhanced Cytogenomic Analysis of Hematological Neoplasms

Abstract

Hematological neoplasms are predominantly defined by chromosomal aberrations that include structural variations (SVs) and copy number variations (CNVs). The current standard-of-care (SOC) genetic testing for the detection of SVs and CNVs relies on a combination of traditional cytogenetic techniques that include karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarrays (CMA). These techniques are labor-intensive, time and cost-prohibitive, and often do not reveal the genetic complexity of the tumor. Optical genome mapping (OGM) is an emerging technology that can detect all classes of SVs in a single assay. We report the results from our clinical validation (in a CLIA setting) of the OGM technique for hematological neoplasms. The study included 92 sample runs (including replicates) using 69 well-characterized unique samples (59 hematological neoplasms and 10 controls). The technical (QC metrics and first-pass rate) and analytical performance [sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV)] were evaluated using the clinical samples. The reproducibility was evaluated by performing inter-run, intra-run, and inter-instrument comparisons using six samples run in triplicates. The limit of detection (LoD) for aneuploidy, translocation, interstitial deletion, and duplication was assessed. To confirm the LoD, samples at 12.5%, 10%, and 5% allele fractions (theoretical LoD range) were run in triplicates. The technical performance resulted in a 100% first-pass rate with all samples meeting the minimum QC metrics. The analytical performance showed a sensitivity of 98.7%, specificity of 100%, accuracy of 99.2%, PPV of 100%, and NPV of 98%, which included the detection of 61 aneuploidies, 34 deletions, 28 translocations, 11 duplications/amplifications, 15 insertions/additional material not identified with karyotyping, 12 marker chromosomes, and one each of ring chromosome, inversion and isochromosome. OGM demonstrated robust technical and analytical inter-run, intra-run, and inter-instrument reproducibility. The LoD was determined to be at 5% allele fraction for all the variant classes evaluated in the study. In addition, OGM demonstrated higher resolution to refine breakpoints, identify the additional genomic material, marker, and ring chromosomes. OGM identified several additional SVs, revealing the genomic architecture in these neoplasms that provides an opportunity for better tumor classification, prognostication, risk stratification, and therapy selection. This study is the first CLIA validation report for OGM for genome-wide structural variation detection in hematological neoplasms. Considering the technical and analytical advantages of OGM compared to the current SOC methods used for chromosomal characterization, we highly recommend OGM as a potential first-tier cytogenetic test for the evaluation of hematological neoplasms.

Optical genome mapping in acute myeloid leukemia: a multicenter evaluation

“…This becomes particularly relevant when addressing the lower limit of detection (LOD). In this study the LOD appeared to be around a 5% allele fraction which is consistent with the LOD reported by Sahajpal and colleagues [36] who empirically demonstrated the LOD for several classes of SVs from 25% down to 5% allele fraction [36]. In cases 24 and 25, non-sentinel karyotype abnormalities were present at 10% and 11% respectively.…”

“…Since OGM is performed without culturing of cells, and given the significantly higher number of molecules analyzed, the true VAF in the extracted bulk DNA used for OGM (pre-culture) in cases 24 and 25 may have actually been below 5%. Discrepancies in variant detection between karyotyping and OGM at the 5% allele fraction boundary have also been reported by other groups [22,36]. Since empirical experiments using OGM detect SVs down to a 5% allele fraction, the karyotype events missed by OGM most likely represents culture bias of allele fractions that initially were below 5% but artificially inflated during the cell culture process.…”

High-resolution structural variant profiling of myelodysplastic syndromes by optical genome mapping uncovers cryptic aberrations of prognostic and therapeutic significance

“…Standard run quality control parameters [total DNA ( ≥150 kbp), N50 ( ≥150 kbp), map rate (≥150 kbp), effective coverage (>300x), average label density (per 100 kbp)] were evaluated per manufacturer's guidelines. Details regarding limit of detection, reproducibility and precision are described in Supplementary Material [41,42].…”

Multisite Study of Optical Genome Mapping of Retrospective and Prospective Constitutional Disorder Cohorts

“…Multiple studies published by one of the senior authors of this study demonstrate that OGM allows for a streamlined workflow and consolidation of multiple SOC methods for the manner 15,44,45 . By reducing the number of cytogenomic/molecular tests to a single platform with an estimated increase in diagnostic yield by 10-15% (over SOC), one may expect overall healthcare savings and faster time to results thereby ending the diagnostic odyssey of these patients and families.…”