Next-generation cytogenetics: Comprehensive assessment of 52 hematological malignancy genomes by optical genome mapping.

Somatic structural variants (SVs) are important drivers of cancer development and progression. In a diagnostic set-up, especially for hematological malignancies, the comprehensive analysis of all SVs in a given sample still requires a combination of cytogenetic techniques, including karyotyping, FISH, and CNV microarrays. We hypothesize that the combination of these classical approaches could be replaced by optical genome mapping (OGM). Samples from 52 individuals with a clinical diagnosis of a hematological malignancy, divided into simple (<5 aberrations, n = 36) and complex (≥5 aberrations, n = 16) cases, were processed for OGM, reaching on average: 283-fold genome coverage. OGM called a total of 918 high-confidence SVs per sample, of which, on average, 13 were rare and >100 kb. In addition, on average, 73 CNVs were called per sample, of which six were >5 Mb. For the 36 simple cases, all clinically reported aberrations were detected, including deletions, insertions, inversions, aneuploidies, and translocations. For the 16 complex cases, results were largely concordant between standard-of-care and OGM, but OGM often revealed higher complexity than previously recognized. Detailed technical comparison with standard-of-care tests showed high analytical validity of OGM, resulting in a sensitivity of 100% and a positive predictive value of >80%. Importantly, OGM resulted in a more complete assessment than any previous single test and most likely reported the most accurate underlying genomic architecture (e.g., for complex translocations, chromoanagenesis, and marker chromosomes). In conclusion, the excellent concordance of OGM with diagnostic standard assays demonstrates its potential to replace classical cytogenetic tests as well as to rapidly map novel leukemia drivers.

High detection rate of clinically relevant genomic abnormalities in plasma cells enriched from patients with multiple myeloma.

Multiple myeloma is a heterogeneous disease, which is characterized by the occurrence of specific genomic abnormalities that are both of diagnostic and prognostic relevance. Since the detection of these abnormalities through molecular-genetic techniques is hampered by the overall low percentage of plasma cells present in primary bone marrow aspirates, we assessed the efficacy of these techniques in enriched plasma cell fractions from 61 multiple myeloma patients. Using interphase FISH, genomic abnormalities could be detected in 96% of the enriched samples as compared to 61% in the cultured whole bone marrow samples. We also found that microarray-based genomic profiling of enriched plasma samples facilitates the detection of additional, possibly clinically relevant, genomic abnormalities. We conclude that the genomic delineation of enriched plasma cells from multiple myeloma patients results in a significantly increased detection rate of clinically relevant genomic abnormalities. In order to facilitate molecular-genetic data interpretation, we recommend morphological assessment of plasma cell purity after enrichment.

Microarray-based genomic profiling as a diagnostic tool in acute lymphoblastic leukemia.

In acute lymphoblastic leukemia (ALL) specific genomic abnormalities provide important clinical information. In most routine clinical diagnostic laboratories conventional karyotyping, in conjunction with targeted screens using e.g., fluorescence in situ hybridization (FISH), is currently considered as the gold standard to detect such aberrations. Conventional karyotyping, however, is limited in its resolution and yield, thus hampering the genetic diagnosis of ALL. We explored whether microarray-based genomic profiling would be feasible as an alternative strategy in a routine clinical diagnostic setting. To this end, we compared conventional karyotypes with microarray-deduced copy number aberration (CNA) karyotypes in 60 ALL cases. Microarray-based genomic profiling resulted in a CNA detection rate of 90%, whereas for conventional karyotyping this was 61%. In addition, many small (< 5 Mb) genetic lesions were encountered, frequently harboring clinically relevant ALL-related genes such as CDKN2A/B, ETV6, PAX5, and IKZF1. From our data we conclude that microarray-based genomic profiling serves as a robust tool in the genetic diagnosis of ALL, outreaching conventional karyotyping in CNA detection both in terms of sensitivity and specificity. We also propose a practical workflow for a comprehensive and objective interpretation of CNAs obtained through microarray-based genomic profiling, thereby facilitating its application in a routine clinical diagnostic setting.

Identification of chromosomal abnormalities relevant to prognosis in chronic lymphocytic leukemia using multiplex ligation-dependent probe amplification.

B-cell chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Characteristic genomic abnormalities provide clinically important prognostic information. Because karyotyping and fluorescence in situ hybridization (FISH) are laborious techniques, we investigated the diagnostic efficacy of the more recently developed multiplex ligation-dependent probe amplification (MLPA) technique. MLPA and interphase FISH data of 88 CLL patients were compared for loci encompassing the 13q14 region, chromosome 12, and the ATM (11q22) and TP53 (17p13) genes. We found a perfect correlation, provided that the abnormal clone was present in at least 10-20% of the cells. Because multiple loci and multiple probes per locus were included in the MLPA assay, additional abnormalities not covered by the FISH probes were detected. Furthermore, in 13 cases deletions partly covering the 13q14.3 locus were observed, including three deletions that remained undetected by FISH. All the deletions included the noncoding RNA locus DLEU1 (previously BCMS), which is considered to be the most likely CLL-associated candidate tumor suppressor gene within the 13q14 region. We conclude that MLPA serves as a comprehensive and reliable technique for the simultaneous identification of different clinically relevant and region-specific genomic aberrations in CLL.

Detection of cryptic subtelomeric imbalances in fetuses with ultrasound abnormalities.

It is known from postnatal diagnosis that imbalances of the subtelomeric regions contribute significantly to idiopathic mental retardation. Consequently, subtelomere screening has been incorporated into the recommendations for the evaluation of individuals with unexplained mental retardation and a normal karyotype. Previous studies suggested that for fetuses with ultrasound abnormalities and a normal karyotype, additional screening for submicroscopic imbalances can be relevant for diagnosis and prognosis. In the present paper, we report the detection of such (subtelomeric) imbalances in three fetuses. Prenatally, the three fetuses presented with ultrasound abnormalities highly suspected of a chromosomal aberration. In two of the fetuses, routine karyotyping showed no aberrations but with MLPA or FISH a small subtelomeric imbalance, that could explain the anomalies, was detected. In the third fetus, a chromosomal abnormality was detected with routine cytogenetic analysis (del(X)(p22.1)), but this abnormality could not explain the ultrasound observations and only with subtelomere screening by MLPA a causative chromosomal aberration was detected. As the three fetuses were already prenatally suspected of a chromosomal aberration, this underlines the potential relevance of subtelomere screening in such fetuses, leading to better clinical diagnosis, prognosis and care. Furthermore, when using MLPA, the analysis can be extended to other regions of known clinical importance.