Open AccessArticle SNP-Based Chromosomal Microarray Analysis in the Era of Optical Genome Mapping: An Enriched Case-Series Evaluating Copy-Neutral Events by Alexander R. Marr Alexander R. Marr SciProfiles Scilit Preprints.org Google Scholar , Patrick R. Gonzales Patrick R. Gonzales SciProfiles Scilit Preprints.org Google Scholar and Shivani Golem Shivani Golem SciProfiles Scilit Preprints.org Google Scholar * Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA * Author to whom correspondence should be addressed. Cancers 2026, 18(11), 1841; https://doi.org/10.3390/cancers18111841 (registering DOI) Submission received: 5 May 2026 / Revised: 27 May 2026 / Accepted: 28 May 2026 / Published: 4 June 2026 Simple Summary Cytogenetic testing plays a key role in diagnosis and treatment selection for many blood cancers. Several laboratory methods are currently used, each with different strengths and limitations. A new technology called optical genome mapping has the potential to replace older methods by providing a comprehensive and streamlined view of complex genetic changes. However, it may miss smaller but clinically important alterations that can affect treatment decisions. In our study, we reviewed patient cases performed at our institution to determine how frequently these small changes occur and whether they remain clinically important. We found many of these alterations to involve key cancer genes and often occur alongside other mutations that influence disease behavior. Our findings suggest that combining multiple testing methods provides the most complete and reliable genetic assessment for cancer patients until technology improves. Abstract Background/Objectives: Chromosomal microarray analysis (CMA) is an essential tool in modern cytogenetics for detecting copy number alterations and copy-neutral loss of heterozygosity (CN-LOH). As optical genome mapping (OGM) emerges as a potential replacement for traditional cytogenetic methods, the extent to which CMA remains necessary in routine diagnostic workflows remains to be elucidated. Methods: We retrospectively reviewed 53 primary neoplastic cases, selected from a larger cohort of 327 hematologic malignancy specimens, in which CMA identified one or more CN-LOH events. Event size, genomic content, and correlation with next-generation sequencing (NGS) findings were assessed. A separate cohort of newly diagnosed B-cell acute lymphoblastic leukemia (B-ALL) was analyzed to evaluate disease-specific CN-LOH frequency. Results: Nearly half of CN-LOH events detected were 500 bp in size [ 16, 17, 18]. Further, cases with complex karyotypes, defined as ≥3 clonal abnormalities [ 19, 20], can be easily resolved, and workflows are streamlined compared to conventional karyotyping. However, OGM also has limitations, including reduced detection performance in repetitive genomic regions, limited sensitivity for low-frequency variants, and the requirement for high-molecular-weight DNA, which can be technically challenging to achieve [ 21]. Additionally, the initial cost of instrumentation may limit the adoption of OGM at low-resource centers [ 21]. For these reasons, conventional cytogenetic approaches remain relevant and are still routinely used in diagnostic workflows. Despite these limitations, the strengths of OGM position it as an attractive alternative or complementary technology to traditional cytogenetic techniques, with the potential to consolidate multiple workflows into a single assay. Despite its potential advantages, OGM’s sensitivity for CN-LOH detection is a key limitation and could be particularly problematic in disorders where biallelic inactivation is significant [ 15, 22]. For example, homozygosity for driver mutations in genes such as JAK2, CBL, TET2, or TP53 can be essential findings for clinical management, particularly when NGS detects pathogenic or likely pathogenic variants within the affected region [ 23, 24, 25, 26]. While OGM excels at characterization of structural rearrangements, its lack of allelic resolution represents a critical diagnostic and prognostic constraint. Accurate detection of CN-LOH remains vital for comprehensive genomic interpretation and optimal clinical management in the age of precision medicine. As laboratories transition to modern genomic technologies, the question remains whether CMA should still have a place in current workflows. To address this, we conducted a retrospective, enriched case-series analysis of 53 neoplastic CMA cases from the University of Kansas Health System (TUKHS) that included at least one CN-LOH event. Our findings demonstrate that, although OGM would enhance structural-variant detection, clinically significant focal copy-neutral events may go undetected. These results highlight the continued importance of performing CMA as a complementary modality in the ascendant era of OGM. 2. Materials and Methods 2.1. Patient Samples This study was designed as a retrospective, enriched case-series analysis focusing on CN-LOH-positive cases identified within a larger clinical CMA cohort. CMA was performed on 327 patients with hematological malignancies at TUKHS in 2025. Within this cohort, a subset of 53 patients with CN-LOH events was then selected for full retrospective analysis. The cancer diagnosis was performed on blood or bone marrow by morphological and flow cytometry evaluations, as a standard clinical practice. Next-generation sequencing (NGS) using the QIAseq Targeted DNA Human Myeloid Neoplasms 141 gene Panel (Qiagen, Germantown, MD, USA) was also performed. Clinical significance of NGS variants was reported as per published guidelines [ 27]. The results of these evaluations were retrospectively reviewed from the patient’s medical records. Karyotyping, FISH, and Neoplastic CMA were performed at TUKHS Cytogenetics Laboratory. No patients were excluded. All testing was performed as part of routine clinical care. The retrospective review was approved by an institutional review board (Study ID: STUDY00160638, approved on 20 June 2024). 2.2. Karyotyping and FISH Analysis Conventional karyotyping and FISH analyses were performed on cultured peripheral blood or bone marrow specimens using standard cytogenetic protocols. For conventional karyotyping, a minimum of 20 metaphases were evaluated for each case. FISH testing included acute myeloid leukemia (AML) FISH probes RUNX1T1:: RUNX1, KMT2A, PML:: RARA, MYC, and 7q probes from Abbott (Des Plaines, IL, USA), and 5q, CBFB:: MYH11, MECOM, and PML:: RARA from Cytocell (Tarrytown, NY, USA) and TP53 from MetaSystems (Medford, MA, USA). B-cell acute lymphoblastic leukemia (B-ALL) FISH probes included BCR:: ABL1, KMT2A, CDKN2A, IGH, and MYC from Abbott (Des Plaines, IL, USA), ETV6:: RUNX1, ABL2, PDGFRB, JAK2, EPOR, CRLF2, and P2RY8 from Cytocell (Tarrytown, NY, USA), and IKZF1 from Empire Genomics (Depew, NY, USA). 200 interphase cells were scored for FISH in all cases with new diagnoses. For follow-up cases, 500 interphase cells were scored. Metaphase FISH was not utilized in this analysis. Chromosome and FISH analyses were performed utilizing Cytovision Software version 7.7 (Leica, Teaneck, NJ, USA). Results were interpreted and reported using the International System for Human Cytogenomic Nomenclature (ISCN 2024). 2.3. DNA Extraction Genomic DNA was extracted from peripheral blood or bone marrow using the QIA amp DNA Blood Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. DNA was quantified by Qubit fluorometry. At least 200 ng of total DNA was utilized for CMA. 2.4. Chromosomal Microarray Microarray-based chromosome analysis was performed using the iScan System with the Global Diversity Array-8 (GDACyto) v1.0 Array BeadChip (Illumina, San Diego, CA, USA). Criteria for designating reportable aberrations include gains or losses larger than 50 kb involving clinically significant cancer genes; gains >2 Mb; and losses >1 Mb outside known clinically significant oncology regions that span at least one annotated RefSeq gene. Smaller aberrations are reported only if the regions are likely to be clinically significant. Copy-neutral loss of heterozygosity (CN-LOH) is reported when the region exceeds 3 Mb. CMA and visualization were performed using NxClinical 6.2 (Bionano, San Diego, CA, USA). All NGS and CMA findings are annotated using the human genome reference build GRCh37/hg19. 2.5. Statistics Descriptive statistics, including frequencies, median values, and distribution counts, were generated in Microsoft Excel or R. Figures were generated using R (v4.5.1). 3. Results 3.1. Assessing the Frequency and Impact of CN-LOH Events in Routine Neoplastic Testing TUKHS Cytogenetics Laboratory performed neoplastic CMA on 327 patients aged 18 to 92 years in 2025. 184 patients were male, 142 were female, and one identified as other. Within this cohort, 53 patients were selected for a retrospective, enriched case series analysis to evaluate the prospective clinical value that OGM could have provided in lieu of CMA. The average patient age of the selected cohort was 64 years, with 28 male and 25 female cases. The full demographic, cytogenetic, and molecular results of our patient cohort are summarized in Supplementary Table S1. These cases served as the foundation of our comparative analysis of CMA and potential OGM coverage. A genome-wide map of CN-LOH events from our selected cohort is shown in Figure 1. A total of 85 CN-LOH calls were detected (median ~2 per case), ranging from focal ( 110 Mb) in size. 42 CN-LOH calls (49% of total) were 25 Mb in length. However, focal copy-number changes A p.M155I (VAF: 50%) 5 70 M B-cell ALL arr (X,Y)x1,(1-22)x2 N/A 46,XY,t(9;22)(q34;q11.2)[3]/46,XY[17] Peripheral Blood: nuc ish (ABL1,BCR)x3(ABL1 con BCRx2)[115/200],(IKZF1,7q11.21)x2[200] Bone Marrow: nuc ish (ABL1,BCR)x3(ABL1 con BCRx2)[115/200],(IKZF1,7q11.21)[200],(CDKN2A,CEP9)x2[200] Normal 6 63 M B-cell ALL Cancelled because low-hypodiploid karyotype was detected. N/A 34~36,XY,−2,−3,−7,−9,−12,−13,−15,−16,−17,−20,
