Optical Genome Mapping for Detection of BCR::ABL1—Another Tool in Our Toolbox
by
, , , , and
Zhenya Tang
1,2,*
,
Wei Wang
2, 
Gokce A. Toruner
2
,
Shimin Hu
2,
Hong Fang
2,
Jie Xu
2,
M. James You
2,
L. Jeffrey Medeiros
2,
Joseph D. Khoury
1
and
Guilin Tang
2,* 1
Department of Pathology, Microbiology and Immunology, University of Nebraska Medical Center, Omaha, NE 68198, USA
2
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
*
Authors to whom correspondence should be addressed.
Genes 2024, 15(11), 1357; https://doi.org/10.3390/genes15111357
Submission received: 28 September 2024
/
Revised: 18 October 2024
/
Accepted: 18 October 2024
/
Published: 22 October 2024
(This article belongs to the Special Issue Clinical Molecular Genetics in Hematologic Diseases)
Abstract
:Background: BCR::ABL1 fusion is mostly derived from a reciprocal translocation t(9;22)(q34.1;q11.2) and is rarely caused by insertion. Various methods have been used for the detection of t(9;22)/BCR::ABL1, such as G-banded chromosomal analysis, fluorescence in situ hybridization (FISH), quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and optical genome mapping (OGM). Understanding the strengths and limitations of each method is essential for the selection of appropriate method(s) of disease diagnosis and/or during the follow-up. Methods: We compared the results of OGM, chromosomal analysis, FISH, and/or RT-PCR in 12 cases with BCR::ABL1. Results: BCR:ABL1 was detected by FISH and RT-PCR in all 12 cases. One case with ins(22;9)/BCR::ABL1 was cryptic by chromosomal analysis and nearly missed by OGM. Atypical FISH signal patterns were observed in five cases, suggesting additional chromosomal aberrations involving chromosomes 9 and/or 22. RT-PCR identified the transcript isoforms p210 and p190 in seven and five cases, respectively. Chromosomal analysis revealed additional chromosomal aberrations in seven cases. OGM identified extra cytogenomic abnormalities in 10 cases, including chromoanagenesis and IKZF1 deletion, which were only detected by OGM. Conclusions: FISH offers rapid and definitive detection of BCR::ABL1 fusion, while OGM provides a comprehensive cytogenomic analysis. In scenarios where OGM is feasible, chromosomal analysis and RT-PCR may not offer additional diagnostic value.
1. Introduction
The chimeric BCR::ABL1 is a diagnostic hallmark of chronic myeloid leukemia (CML), and a recurrent cytogenetic abnormality used for subclassification of B-lymphoblastic leukemia (B-ALL), mixed-phenotype acute leukemia (MPAL), and acute myeloid leukemia (AML) by the 5th edition of the World Health Organization Classification of Hematolymphoid Tumors (WHO-HAEM5) [1,2] and the International Consensus Classification (ICC) guidelines [3,4]. BCR::ABL1 is mostly derived from a reciprocal t(9;22)(q34.1;q11.2), with the active BCR::ABL1 located in the derivative chromosome 22, der(22) or the Philadelphia (Ph) chromosome [1,5], whereas the ABL1::BCR in the derivative chromosome 9 or der(9) remains inactive. An active BCR::ABL1 can be formed through the insertion of partial ABL1 into BCR or vice versa [6]. On rare occasions, the BCR::ABL1 may be located in a chromosome other than der(22) or der(9) [6]. G-banded chromosomal analysis (karyotyping) [7], fluorescence in situ hybridization (FISH) [8], and quantitative real-time PCR [9] are three major methods that have been applied for detecting BCR::ABL1 fusion in clinical diagnostic laboratories in the past decades.
Karyotyping is usually performed on cultured bone marrow or peripheral blood specimens and detects the BCR::ABL1 that resulted from t(9;22) by chromosomal morphology [7]. This method also can detect aberrations involving other chromosomes, if any. It usually takes 2–3 days for the cultured specimen to be ready for analysis and 1 to 2 additional days to have a final chromosomal analysis report. The turnaround time (TAT) is usually around 5 workdays or possibly longer. FISH tests often employ fluorescence-labeled DNA probes specifically against ABL1, BCR, and/or their flanking region(s) [8]. FISH signal pattern(s) will change if an abnormality involving the target(s) (e.g., ABL1, BCR) and/or their flanking regions covered by the FISH probes. However, any aberrations beyond the probe-covered regions will not be detected by FISH. A FISH test can be performed on specimens with and/or without cell culture, e.g., aspirate smears, touch prep, and so on. The FISH results can be made ready within 4 h and are definitive for BCR::ABL1. A quantitative real-time PCR (or RT-PCR, hereafter) usually applies both ABL1 and BCR-specific primer pairs (even exon-specific for individual isoform type) to amplify cDNA synthesized from purified RNA. Quantification is often performed via comparison with an internal control (e.g., ABL1 level) or external reference material. This method is extremely specific and sensitive and is capable of differentiating the BCR::ABL1 isoforms [9]. However, it is not capable of detecting aberrations other than a BCR::ABL1 transcript. Therefore, RT-PCR plays a significant role in monitoring the level of BCR::ABL1, especially for a measurable residual disease. Due to the involvement of multiple steps/procedures, such as the extraction of RNA, cDNA synthesis, PCR amplification and detection, and requirement of setting up as many controls as for each step/procedure, a quantitative RT-PCR is usually performed on a batch of specimens that, in return, can impact the TAT of this method.
As a novel powerful technology for detecting both structural variants (SVs) and copy number variants (CNVs) across the whole genome, optical genome mapping (OGM) is becoming popular in clinical diagnostic laboratories. OGM can detect many small but clinically relevant SVs and CNVs beyond the karyotyping detection and, more importantly, provide information on putative fusion genes [10,11,12,13,14,15,16,17,18,19]. In this study, we compare OGM with three other traditional methods (karyotyping, FISH, and RT-PCR) for the detection of BCR::ABL1 and other cytogenomic alternations in twelve cases with BCR::ABL1. We also discuss how these methods should be selected for clinical diagnostic purposes.
2. Materials and Methods
2.1. Case Selection
This study cohort included 12 cases positive for BCR::ABL1 that were concurrently evaluated with four methods, karyotyping, FISH, RT-PCR, and OGM, in the Clinical Cytogenetics Laboratory at the University of Texas MD Anderson Cancer Center. The clinicopathologic and laboratory data were collected by electronic medical chart review. This study was approved by the Institutional Review Board and was conducted in compliance with the Declaration of Helsinki.
2.2. Chromosomal Analysis
Conventional G-banded chromosomal analysis was routinely performed on unstimulated 24 h and 48 h bone marrow (BM) aspirate and/or peripheral blood (PB) specimens using standard techniques [6,16]. A total of 20 metaphases were analyzed for each case, and the findings were reported according to the International System for Human Cytogenetics Nomenclature (ISCN 2020) [20]. A complex karyotype is defined as ≥3 chromosomal abnormalities with at least one of them being a structural abnormality.
2.3. FISH Analysis
FISH analysis was performed primarily on PB or BM smears using the Vysis LSI BCR/ABL1 ES Dual Color Fusion probe set (referred to as “ES probe”) (Abbott Molecular, Des Plaines, IL, USA). This probe set is designed to distinguish major breakpoints (M-BCR, p210 isoform) and minor breakpoints (m-BCR, p190 isoform) when typical signal patterns are observed (2R1G1F for M-BCR and 1R1G2F for m-BCR; R: red, G: green, F: fusion/orange). For cases with atypical signal pattern(s), such as 1R1G1F, which suggested del(9q), a reflexed FISH with Vysis BCR/ABL1/ASS1 Tri-Color DF FISH probe set (referred to as “tricolor probe”, Abbott Molecular, Des Plaines, IL, USA) was followed [6,16]. Metaphase FISH on G-banded slides was reflexed for cases with complex rearrangements.
2.4. Quantitative BCR::ABL1 RT-PCR Assay
A quantitative RT-PCR assay was used routinely to measure BCR::ABL1 at initial diagnosis and in follow-up studies as described previously [6]. Briefly, 1 µg of total RNA extracted from BM or PB was reversed to cDNA using random primer and superscript II reverse transcriptase (Gibco-BRL) according to the manufacturer’s instructions. The resulting cDNA subsequently underwent amplification with isoform-specific BCR and ABL1 primer sets. The quantitative BCR::ABL1 mRNA levels are presented as the percent ratio of BCR::ABL1 to ABL1 transcript levels. The sensitivity of this assay is between 1 in 10,000 and 1 in 100,000.
2.5. Optical Genome Mapping (OGM) Analysis
OGM was performed on fresh PB or BM aspirates using the procedures described previously [16,17,18,19,21,22,23]. Briefly, ultra-high molecular weight (UHMW) genomic DNA (gDNA) was extracted from approximately 1.5 million nucleated cells following the manufacturer’s protocols (Bionano Prep SP-G2 DNA Isolation Kit, Catalog# 90151; Bionano Genomics, San Diego, CA, USA); 750 ng UHMW gDNA was applied for a sequence-specific direct label and stain (Bionano Prep DLS-G2 Labeling Kit, Catalog# 80046). The purified, fluorescence-labeled gDNA molecules were loaded on Saphr G3.3 Chip and then linearized and imaged through massively parallel nanochannel arrays in a Saphr instrument. The Bionano Access software (v1.7) was employed for data analysis, utilizing the Annotated Rare Variant Analysis platform and the Genome Reference Consortium GRCh38/hg38 as the reference genome. The analysis was conducted in two steps, applying two sets of feature files, HemeTargets and hg38-primary_transcripts, along with their corresponding filters. The HemeTargets feature file is custom-designed and encompasses over 500 genes, loci, and fusion genes pertinent to hematologic malignancies. This file was constructed in accordance with guidelines by WHO-HAEM5 [1,2], the International Consensus Classification (ICC) [3,4], National Comprehensive Cancer Network (NCCN) [24,25,26,27], and the National Health Service (NHS) of the UK. Using this file, we screened for critical SVs and CNVs by using the manufacturer (Bionano)-recommended confidence without imposing a minimum size restriction. Concurrently, a 200 Kbp overlap precision was applied to capture gene rearrangements for genes with wide and variable breakpoints. After the initial “hotspot” screening, we shifted to using the hg38-primary_transcripts feature file. For this step, a minimum size of 500 Kbp was set as the filter for both SVs (comprising insertions, deletions, inversions, or duplications) and CNVs.
3. Results
3.1. Patient Information
This cohort included 12 patients, five men and seven women, aged 36 to 73 years at the time of testing. Seven patients (cases #1–#7) had chronic myeloid leukemia (CML): three at the chronic phase (CP), one at the high-risk chronic phase (CP), and three at the blast phase (BP). Five patients (cases #8–#12) had B-lymphoblastic leukemia (B-ALL) (Table 1). The main-line treatments, patients’ response to treatments (based on NCCN criteria [26,28]), follow-up time, and outcomes are summarized in Table 1. In this cohort, six cases (cases #1, #5, #6, and #8–#10) were initially tested with chromosomal analysis, FISH, and/or RT-PCR as a routine work-up. They were deliberately selected for OGM validation to resent a variety of BCR::ABL1 positive cases (CML vs. ALL, p210 vs. p190 BCR::ABL1 transcripts; cryptic variants, etc.). In the remaining six cases in this cohort, OGM was performed as a clinical diagnostic test per requests by clinicians; after this, a new test was implemented for clinical services.
3.2. Chromosomal Analysis Results
The karyotypic information is summarized in Table 2. Five cases (#1–#5) showed t(9;22) as the sole abnormality, and the other seven cases showed additional abnormalities. Eight patients showed a balanced t(9;22) (#1–#5, #7, #8, and #11), two cases (#10, #12) showed additional Ph chromosome(s), and two cases (#6 and #10) exhibited more complex aberrations involving der(9), der(22), and other chromosomes. One case (#9) had an ins(22;9), which was cryptic for chromosomal analysis. It is worth mentioning that a three-way translocation, t(1;9;22), was reported previously by another institution in case #6 (Figure 1A). However, the der(22) did not resemble a typical Ph chromosome in this case. Instead, our FISH studies on metaphase cells (see below) demonstrated the complexities of chromosomal rearrangements.
3.3. FISH Analysis Results
All 12 cases showed high percentages of cells with BCR::ABL1 (82% to 96.5%) by FISH (Table 2). Cases #2–#3 and #5–#6 showed a signal pattern of 2R1G1F, suggesting a BCR::ABL1 p210 isoform, while cases #7–#8 and #11 presented a signal pattern of 1R1G2F, indicating a p190 isoform [6]. The signal patterns in cases #1 and #4 (1R1G1F) and case #9 (2R2G1F) were considered atypical, and they usually indicate additional aberrations involving 9q34 and/or 22q11.2 or a rare mechanism for BCR::ABL1, such as insertion (see OGM analysis results below) [6]. The signal patterns in case #10 (2R1G2F and 2R1G3F) and case #12 (1R1G3F and 1R1G2F) were indicative of the presence of two clones with approximately one to two extra copies of the Ph chromosome, which was consistent with chromosomal analysis.
Reflex FISH on previously G-banded metaphases using the BCR/ABL1/ASS tri-color probe set on case #6 demonstrated that the BCR::ABL1 fusion signal was located on the der(22), but there was no FISH signal on chromosome 1 (Figure 1B, upper). Furthermore, whole chromosome painting for chromosome 22 (wcp22) showed that only the normal chromosome 22 and the der(22) were stained (Figure 1B, lower). Therefore, a three-way translocation t(1;9;22) was excluded, and very likely, the BCR::ABL1 is derived from an insertion of a partial 9q34 containing 3′-ABL1 in the BCR on the der(22) (see OGM analysis below).
3.4. Quantitative RT-PCR Analysis Results
Compared with the ABL1 level in the same specimen, the BCR::ABL1 fusion expression was high in this cohort; e.g., 31.73% in case #1 and >100% in all other cases (Table 2). The transcript type was the p210 isoform in seven cases (cases #1–#6, #10) and the p190 isoform in five other cases. Interestingly, a mixture of e13a2 and e14a2 (both encode for p210 isoform) was detected in three cases (#1–#2, #4).
3.5. Optical Genome Mapping (OGM) Analysis Results
A t(9;22)/BCR::ABL1 rearrangement was called directly by OGM in 11 cases (#1–#8, #10–#12). However, in case #9, OGM did not call a BCR::ABL1. Instead, it detected a ~109 Kbp deletion on 9q34 that included partial ABL1 (breakpoint was between exon 1 and exon 2) and its 3′-flanking region (Figure 2A). Additionally, complex rearrangements including insertion, deletion, and duplication were detected on 22q, which involved BCR and its flanking regions (Figure 2B). Manual inspection revealed a fragment exhibiting BCR::ABL1 recombinant patterns (Figure 2C), suggesting that the BCR::ABL1 fusion was formed through an ins(22;9) in this case.
In addition to the t(9;22) or ins(22;9) mentioned above, the OGM assay also detected various additional chromosomal abnormalities involving 9q34 and 22q11.2. These included gains of 9q34 and/or 22q11.2 in cases #10 and #12, losses in cases #1 and #4, and complex rearrangements in case #6 (Table 2). As shown in the circos plot, t(1;9) and t(9;22) were detected in case #6 (Figure 1C). Further analysis identified two distinct types of t(9;22): t(9;22)(q34.12;q11.23)/BCR::ABL1 and t(9;22)(q34.3;q11.23)/EHMT1::BCR. The breakpoints at 9q34 involving ABL1 and EHMT1, as indicated by the green arrows (Figure 1D), were spaced approximately 6.8 Mbp apart. When correlated with the FISH results, these data strongly suggest that a DNA fragment of approximately 6.8 Mbp in size, ranging from the 3′ABL1 to 5′EHMT1, was inserted into BCR located at 22q11.23. Similarly, the t(1;9) also included two types, t(1;9)/UBAP2L::EHMT1 and t(1;9)/ABL1::UBAP2L, with two breakpoints on 9q34, involving ABL1 and EHMT1. The detailed rearrangements that occurred on der(1), der(9), and der(22) are summarized in Figure 1E.
The OGM assay also detected multiple chromosomal aberrations beyond the 9q34 and 22q11.2 regions, which were not detected by karyotyping, FISH, or RT-PCR. These included the recurrent abnormalities, del(9p)/CDKN2A/CDKN2B loss (cases #10 and #12), del(7p)/IKZF1 loss (cases #7 and #12), and chromoanagenesis (defined as ≥5 SVs and/or CNVs involving the same chromosome in this cohort) (e.g., cases #9 and #10) (Table 2). Notably, IKZF1 deletion, a high-risk factor for B-ALL, is often cryptic for chromosomal analysis due to its small size. Multiple non-recurrent SVs and CNVs were detected in single cases (such as in cases #5–#12) (Table 2). For case #7, the chromosomal analysis suggested a karyotype 45,XY,t(9;22)(q34;q11.2),psu dic(13;12)(q34;p11.1)[19]/46,XY[1] (Figure 3A), and OGM detected del(12p), t(5;12), and t(5;13), as well as an interstitial deletion del(5)(q35.1q35.3) (Figure 3B). These findings very likely indicated an unbalanced three-way translocation t(5;12;13), but the alternations in 5q were cryptic for chromosomal analysis. Additionally, the t(5;12)(q33.3;p11.1) resulted in a putative EBF1::SYT10 gene fusion. For case #8, a karyotype of 47,XX,-7,+8,t(9;22)(q34;q11.2),+mar[20] was detected by chromosomal analysis (Figure 4A). The OGM assay confirmed the presence of t(9;22), -7, and +8, and additionally showed four copies of the 9p24.3 to 9q13 region (Figure 4C), clarifying that the “marker” chromosome was actually idic(9)(q13). This finding was subsequently confirmed by FISH using probes for CDKN2A and CEP9 (Figure 4B).
4. Discussion
OGM is a novel technology with a demonstrated power to detect SVs and CNVs throughout the whole genome with high resolution. More and more clinical laboratories are interested in adopting this new technology for patient care. In this study, we performed a head-to-head comparison of OGM versus chromosomal analysis, FISH, and quantitative RT-PCR analyses for the detection of BCR::ABL1 as well as other cytogenomic aberrations in 12 BCR::ABL1+ cases. The latter three methods have been widely applied in clinical laboratory diagnosis for decades. The detection power of each method is listed in Table 3. For detecting BCR:ABL1, all four methods can be effective in most cases, although the ins(22;9)/BCR::ABL1 in case #9 was not detected by karyotyping and OGM at initial analysis due to the small-sized fragment insertion, which is cryptic for chromosomal analysis [6] and complex rearrangements.
The prevalence of BCR:ABL1 fusion isoforms is different between CML (p210 predominant) and B-ALL cases (p190 predominant). Therefore, identification of the isoform type may provide a clue for the differential diagnosis between the CML blast phase and de novo B-ALL at initial diagnosis [1,2,3,4]. It has been reported that a p190 isoform in CML cases may indicate an inferior response to TKI therapy [29,30,31]. Although both the e13a2 and e14a2 subtypes code a p210 isoform, CML cases with the e13a2 subtype showed a poorer response to imatinib treatment than that with e14a2 subtypes [32,33,34]. Therefore, quantitation and determination of the BCR:ABL1 isoform has become the standard-of-care for all BCR:ABL1-positive cases. Among the four methods applied in this cohort, karyotyping was no help in determining the BCR::ABL1 isoform. In contrast, typical FISH signal patterns may provide clues for p210 vs. p190 but cannot distinguish e13a2 and e14a2 subtypes of p210. As a novel technology, the OGM assay provides both SVs and CNVs with estimated breakpoints at nucleotide (nt) coordinates. The coordinates in six cases in this cohort (Table 3) have been applied to the UCSC GenomeBrowser to map the breakpoints in BCR and ABL1 (Figure 5). The putative BCR::ABL1 isoforms calculated from these breakpoints involving BCR and ABL1 from OGM data analyses were all p210, with other co-existing “unknown” isoforms (e.g., e1a4 in case #8 and e3a2 in case #10) (Table 3). Certainly, the putative isoforms from the OGM data were obtained from DNA-level analysis, and they could present differently at the RNA or cDNA levels. In general, putative BCR::ABL1 isoforms from OGM data should be considered questionable at this stage (Table 3, Figure 5). The RT-PCR using isoform-specific primer pairs is still the gold standard for determining the BCR::ABL1 isoforms.
Other facts regarding the detection power of each method are also important for an overall evaluation of each case (Table 4). For example, the detection of additional chromosomal aberrations, including additional BCR::ABL1, is associated with clonal evolution and the complexity of genome, and thus affects the patient management and outcome [30,31]. The OGM assay has shown the power of this aspect. In addition, the OGM assay detected a few putative novel gene fusions. The rearrangements of UBAP2L [35,36] and EHMT1 [35,36,37] have been reported in liver, lung, breast, ovary, and prostate cancer but not in hematological malignancies, and ABL1 or BCR has not been reported as their partner genes [35,36,37]. Therefore, the function of the novel fusions of EHMT1::BCR, UBAP2L::EHMT1, and ABL1::UBAP2L detected in case #6 remains unknown. Similarly, EBF1 rearrangement has been reported in B-ALL [38,39,40,41], but was not partnered with SYT10, as detected in case #7. The findings of novel fusion genes pave the way for future research into the roles these genetic alterations play in hematological malignancies.
Selecting the appropriate tests from a list of available technologies/platforms currently plays an important role in clinical practice. Several facts need to be considered before making a selection, such as the turnaround time (TAT), sensitivity, and specificity of the method; the purpose of the test (qualitative and/or quantitative determination, further characterization of isoforms, complexity of aberrations, and clonal diversity); and cost-effectiveness (Table 4) [42]. In cases with a high percentage of lymphoblasts, distinguishing the CML blast phase and de novo B-ALL is clinically important but sometimes can be challenging. Screening for Ph+ segmented nuclei on FISH slides can be very helpful for this purpose [2,4,30] and has become a standard procedure in our Clinical Cytogenetics Laboratory.
In summary, we evaluated four testing methodologies, karyotyping, FISH, RT-PCR, and OGM, for their efficacy in detecting BCR::ABL1 and other cytogenomic aberrations across 12 cases. While OGM effectively identified the BCR::ABL1 fusion, it had a limitation in potentially missing the fusion if it occurred through the insertion of a very small fragment. Despite this, OGM demonstrated significant advantages by detecting additional aberrations in approximately two-thirds of the cases. These additional findings included novel gene fusions and high-risk cytogenetic aberrations such as chromoanagenesis and IKZF1 loss, and the latter has a clinical implication in disease progression, prognosis prediction, and modification of therapy regimens in both myeloid and lymphoid neoplasm [43,44,45].
5. Conclusions
Our study demonstrated that OGM provides a comprehensive cytogenomic analysis and FISH offers rapid and definitive detection of BCR::ABL1 fusion. In scenarios where OGM is feasible, chromosomal analysis and RT-PCR may only provide limited diagnostic values.
Author Contributions
Conceptualization, Z.T. and G.T.; Data curation, Z.T., W.W., G.A.T., S.H. and G.T.; Formal analysis, Z.T. and G.T.; Investigation, Z.T. and G.T.; Methodology, Z.T. and G.T.; Validation, Z.T. and G.T.; Writing—original draft, Z.T. and G.T.; Writing—review and editing, W.W., G.A.T., S.H., H.F., J.X., M.J.Y., L.J.M. and J.D.K. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
This study is performed under a research protocol approved by the Institutional Research Bureau (IRB).
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.
Acknowledgments
All authors acknowledge Alexandra Reynolds, Christopher Danos, and all our team members in the Clinical Cytogenetic Laboratory for their dedicated work in optical genome mapping (OGM) assay development. We also thank Jeff Robinson, James Yu, Erika Headrick, and Alka Chaubey from Bionano Genomics, Inc. for their strong support.
Conflicts of Interest
The authors declare no conflicts of interest.
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Figure 1.
Cytogenetic analyses by three methods in case #6. (A) Chromosomal analysis indicated aberrations involving chromosomes 1, 9, and 22 (indicated with blue arrow) and their homologs, respectively. (B) FISH analysis using BCR/ABL1/ASS tri-color dual-fusion probes indicated neither BCR nor ABL1 signal translocated to chromosome 1 (upper). Whole chromosome painting (wcp) 22 indicated that only the normal and abnormal chromosomes 22 were stained, indicating no chromosome 22 material translocated to chromosomes 1 or 9 (lower). (C) Circus plot of OGM showing t(1;9), t(9;22) and del(16q). (D) Breakpoints of BCR on chromosome 22 (red arrows) and breakpoints of ABL1 and EHMT1 on chromosome 9 (green arrows), indicating that the BCR::ABL1 is derived from an insertion of DNA segment from 3′ABL1 to 5′EHMT1 (about 6.8 Mbp) into BCR. (E) Details of der(1), der(9), and der(22).
Figure 1.
Cytogenetic analyses by three methods in case #6. (A) Chromosomal analysis indicated aberrations involving chromosomes 1, 9, and 22 (indicated with blue arrow) and their homologs, respectively. (B) FISH analysis using BCR/ABL1/ASS tri-color dual-fusion probes indicated neither BCR nor ABL1 signal translocated to chromosome 1 (upper). Whole chromosome painting (wcp) 22 indicated that only the normal and abnormal chromosomes 22 were stained, indicating no chromosome 22 material translocated to chromosomes 1 or 9 (lower). (C) Circus plot of OGM showing t(1;9), t(9;22) and del(16q). (D) Breakpoints of BCR on chromosome 22 (red arrows) and breakpoints of ABL1 and EHMT1 on chromosome 9 (green arrows), indicating that the BCR::ABL1 is derived from an insertion of DNA segment from 3′ABL1 to 5′EHMT1 (about 6.8 Mbp) into BCR. (E) Details of der(1), der(9), and der(22).
Figure 2.
Unusual OGM findings in case #9. (A) A deletion involving 3′ABL1 (breakpoint was between exon1 and exon 2) and its flanking region. (B) A deletion of about 2.7 Mbp involving BCR and its flanking region. However, a gain of 22q11.2 was also observed (the purple bar on the top). (C) Manual alignment showing that molecules contained BCR::ABL1, derived from an insertion of approximately 100 Kbp fragment containing 3′ABL1 and its flanking region.
Figure 2.
Unusual OGM findings in case #9. (A) A deletion involving 3′ABL1 (breakpoint was between exon1 and exon 2) and its flanking region. (B) A deletion of about 2.7 Mbp involving BCR and its flanking region. However, a gain of 22q11.2 was also observed (the purple bar on the top). (C) Manual alignment showing that molecules contained BCR::ABL1, derived from an insertion of approximately 100 Kbp fragment containing 3′ABL1 and its flanking region.
Figure 3.
A comparison of chromosomal analysis and OGM assay in case #7. (A) Chromosomal analysis indicated a balanced t(9;22)(q34;q11.2) and a psu dic(13;12)(q34;p11.1), as indicated by arrows. (B) Circos plot of OGM assay indicated a t(9;22), a t(5;12), and a t(5;13) at the same 5q33.3 band level, an apparent del(12p) and a del(5q) of small size, likely suggesting unbalanced three-way translocation t(5;12;13) in addition to the t(9;22)/BCR::ABL1 aberration.
Figure 3.
A comparison of chromosomal analysis and OGM assay in case #7. (A) Chromosomal analysis indicated a balanced t(9;22)(q34;q11.2) and a psu dic(13;12)(q34;p11.1), as indicated by arrows. (B) Circos plot of OGM assay indicated a t(9;22), a t(5;12), and a t(5;13) at the same 5q33.3 band level, an apparent del(12p) and a del(5q) of small size, likely suggesting unbalanced three-way translocation t(5;12;13) in addition to the t(9;22)/BCR::ABL1 aberration.
Figure 4.
Cytogenetic analyses by three methods in case #8. (A) Chromosomal analysis showed a karyotype of 47,XX,-7+8,t(9;22)(q34;q11.2),+mar, indicated by arrows (m: marker chromosome). (B) FISH analysis using CDKN2A/CEP9 probe set showed the marker chromosome containing two copies of CDKN2A/CEP9 signals (red arrow). (C) OGM confirmed all the abnormalities of t(9;22), -7, and +8, as well as four copies of 9p24.3 to q21.11, an idic(9)(q21.11) aberration for the marker chromosome by chromosomal analysis.
Figure 4.
Cytogenetic analyses by three methods in case #8. (A) Chromosomal analysis showed a karyotype of 47,XX,-7+8,t(9;22)(q34;q11.2),+mar, indicated by arrows (m: marker chromosome). (B) FISH analysis using CDKN2A/CEP9 probe set showed the marker chromosome containing two copies of CDKN2A/CEP9 signals (red arrow). (C) OGM confirmed all the abnormalities of t(9;22), -7, and +8, as well as four copies of 9p24.3 to q21.11, an idic(9)(q21.11) aberration for the marker chromosome by chromosomal analysis.
Figure 5.
The reported breakpoints of ABL1 and BCR obtained using OGM assay were shown in UCSC Genome Browser in five cases in this cohort (case #1: purple; case #5: green; case #6: orange; case #8: blue; and case #10: red), and their putative isoforms were postulated in each case (Table 3).
Figure 5.
The reported breakpoints of ABL1 and BCR obtained using OGM assay were shown in UCSC Genome Browser in five cases in this cohort (case #1: purple; case #5: green; case #6: orange; case #8: blue; and case #10: red), and their putative isoforms were postulated in each case (Table 3).
Table 1.
The general information of 12 BCR::ABL1-positive cases in this study.
| Case # | Sex | Age (Year) | Diagnosis | Blast | Treatments | Follow-Up (Month) /Outcome | Outcome |
|---|---|---|---|---|---|---|---|
| 1 | M | 53 | CML-CP | 2% | dasatinib, ponatinib, asciminib | 6 | progression |
| 2 | F | 36 | CML, CP | 1% | dasatinib | 9 | PCyR |
| 3 | M | 38 | CML, CP | 2% | ponatinib | 12 | Died |
| 4 | M | 66 | CML, CP * | 15% | ponatinib | 4 | DMR |
| 5 | F | 62 | CML-BP | 90% | imatinib, nilotinib, bosutinib, ponatinib, bosutinib | 10 | CCyR |
| 6 | F | 65 | CML-BP | 93% | dasatinib | 10 | CCyR |
| 7 | M | 68 | CML-BP | 33% | mini-Hyper-CVD, blinatumomab, dasatinib | 1 | CR |
| 8 | F | 64 | B-ALL, Ph+ | 90% | blinatumomab, ponatinib. | 6 | PR |
| 9 | F | 45 | B-ALL, Ph+ | 85% | blinatumomab, ponatinib. | 10 | CR |
| 10 | M | 73 | B-ALL, Ph+ | 95% | blinatumomab, ponatinib. | 9 | CR |
| 11 | F | 60 | B-ALL, Ph+ | 67% | mini-Hyper-CVD, blinatumomab, dasatinib | 5 | CR |
| 12 | F | 41 | B-ALL, Ph+ | 95% | blinatumomab, ponatinib. | 1.5 | CR |
* High-risk CP. M: male; F: female. CML: chronic myeloid leukemia; CP: chronic phase; BP: blast phase. CCyR: complete cytogenetic response; PCyR: partial cytogenetic response; DMR: deep molecular response. ALL: acute lymphoblastic leukemia. CR: complete remission; PR: partial remission. Ph+: Philadelphia chromosome+.
Table 2.
A comparison of test results obtained by four different methods in all 12 cases in this study.
Table 2.
A comparison of test results obtained by four different methods in all 12 cases in this study.
| Case | Karyotype | FISH Results | RT-PCR (Isoform, Level) | SVs by OGM | CNVs by OGM |
|---|---|---|---|---|---|
| 1 | 46,XY,t(9;22)(q34;q11.2)[20] | (ABL1,BCR)x2(ABL1 con BCRx1)[186/200] | e13a2 + e14a2/p210, 31.73% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | 9q34.11q34.12(129120861_130847453)x1 |
| 2 | 46,XX,t(9;22)(q34;q11.2)[20] | (ABL1x3,BCRx2)(ABL1 con BCRx1)[177/200] | e13a2 + e14a2/p210, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | No |
| 3 | 46,XY,t(9;22)(q34;q11.2)[20] | (ABL1x3,BCRx2)(ABL1 con BCRx1)[181/200] | e13a2/p210, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | No |
| 4 | 46,XY,t(9;22)(q34;q11.2)[20] | (ABL1,BCR)x2(ABL1 con BCRx1)[184/200] | e13a2 + e14a2/p210, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | 9q34.11q34.12((128703248_130135561)x1 22q11.23q12.1(23592450_26715684)x1 |
| 5 | 46,XX,t(9;22)(q34;q11.2)[20] | (ABL1x3,BCRx2)(ABL1 con BCRx1)[182/200] | e13a2/p210, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | 2p12p11.2(80212101_84371148)x1 |
| 6 | 46,XX,der(1)t(1;9)(q21;q34),der(9)t(1;9)(q21;q34),der(22)ins(22;9)(q11.2;q34q34)[20] | (ABL1x3,BCRx2)(ABL1 con BCRx1)[186/200] | e13a2/p210, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 t(1;9)(q21.3;q34.12)/UBAP2L::ABL1 t(1;9)(q21.3;q34.3)/UBAP2L::EHMT1 | 16q11.1q11.2(38277017_46457433)x1 |
| 7 | 45,XY,t(9;22)(q34;q11.2),psu dic(13;12)(q34;p11.1)[19]/46,XY[1] | (ABL1,BCR)x3(ABL1 con BCRx2)[150/200] | e1a2/p190, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 t(5;12)(q33.3;p11.1)/EBF1::SYT10 t(5;13)(q33.3;q34) | 5q35.1q35.3(172452581_181472714)x1 7p12.2p12.2(50348483_50399656)x1 12p13.2p11.1(11630322_33424504)x1 |
| 8 | 47,XX,-7,+8,t(9;22)(q34;q11.2),+mar[20] | (ABL1,BCR)x3(ABL1 con BCRx2)[189/200] (CDKN2A,CEP9)x4[194/200] | E1a3/p190, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | (7)x1 (8)x3 9p24.3q13(14566_64960054)x4 |
| 9 | 46,XX,r(7)[19]/46,XX,del(7)(q11.2q32)[1] | (ABL1,BCR)x3(ABL1 con BCRx1)[164/200] (D7Z1x2,D7S522x1)[92/200] | e1a2/p190, >100% | ins(22;9)(q11.23;q34.12q934.12)/BCR::ABL1 chromoanagenesis (7) | Numerous segmental loss on chr7 |
| 10 | 48,XY,del(6)(q13q23),der(9)del(9)(p13)t(9;22)(q34;q11.2),+21,der(22)t(9;22),+der(22)t(9;22)[9]/48~49,idem,+der(22)t(9;22),+mar[cp9]/46,XY[2] | (ABL1x5,BCRx4)(ABL1 con BCRx3)[118/200]/ (ABL1x4,BCRx3)(ABL1 con BCRx2)[75/200] | e14a2/p210, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 chromoanagenesis (7,8) | 6p25.3q14.1(76216_77720354)x3 6q14.1q21(77721612_105275332)x1 8q12.3q24.3(64448847_140421018)x3 9p24.3p21.1(14566_31847914)x1 9p21.1p13.1(31858561_38843343)x0 9q34.12q34.3(130755223_138334464)x4 15q14q15.3(33563820_43499262)x1,(21)x3 22q11.21q11.23(18746350_23133605)x4 |
| 11 | 46,XX,t(9;22)(q34;q11.2)[10]/46,idem,del(20)(q11.2q13.1)[8]/46,idem,t(X;6)(q22;p23)[2] | (ABL1,BCR)x3(ABL1 con BCRx2)[184/200] | e1a2/p190, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 t(X;6)(q22.1;p24.1)/TRMT2B t(3;15)(p25.2;q11.2)/RAF1 fus(6;6)(p24.3;p22.3) | 17q22q25(57433782_83246392)x3 20q11.23q13.33(31182877_61861320)x1 |
| 12 | 46,XX,der(8;9)(q10;q10),t(9;22)(q34;q11.2),+der(22)t(9;22)[20] | (ABL1,BCR)x4(ABL1 con BCRx3)[177/200]/ (ABL1,BCR)x3(ABL1 con BCRx2)[16/200] | e1a2/p190, >100% | t(9;22)(q34.12;q11.23)/BCR::ABL1 | 7p12.2p12.2(50273770_50399656)x1 8p23.3p11.2(61805_42571510)x1 9p24.3p12(14566_39591818)x1 9q34.12q34.3(130777258_138334464)x3 22p11.1q12.1(14545087_25515764)x3 |
SVs: structural variants; CNVs: copy number variants.
Table 3.
Detailed information of OGM findings regarding ABL1 and BCR or chromosomes 9 and 22 in five cases in this study.
Table 3.
Detailed information of OGM findings regarding ABL1 and BCR or chromosomes 9 and 22 in five cases in this study.
| Case # | Aberrations | Chr. Involved * | Breakpoints of #1 Chr. | Breakpoints of #2 Chr. | Orientation | Confidence ** | VAF | Putative Gene Fusion | Putative BCR::ABL1 Isoform | Self Molecule Counts | ISCN | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| #1 | #2 | Breakpoint (bp) | Position in Gene | Breakpoint (bp) | Position in Gene | |||||||||
| 1 | transl._interchr. | 9 | 22 | 129,130,483 | PTPA: intron 4 | 23,295,730 | BCR1: intron 15 | +/+ | 0.87 | 0.38 | PTPA::BCR | N/A | 84 | ogm[GRCh38] t(9;22)(q34.11;q11.23) |
| transl._interchr. | 9 | 22 | 130,832,617 | ABL1: intron 1 | 23,295,730 | BCR1: intron 15 | +/+ | 0.98 | 0.53 | ABL1::BCR | e14a2/p210 | 84 | ogm[GRCh38] t(9;22)(q34.12;q11.23) | |
| deletion | 9 | 9 | 129,120,861 | PTPA: intron 1 | 130,847,453 | ABL1: intron 1 | N/A | 0.99 | 0.44 | - | N/A | 80 | ogm[GRCh38] 9q34.11q34.12(129120861_130847453)x1 | |
| 5 | transl._interchr. | 9 | 22 | 130,836,231 | ABL1: intron 1 | 23,295,730 | BCR1: intron 15 | +/+ | 1 | 0.5 | ABL1::BCR | e14a2/p210 | 83 | ogm[GRCh38] t(9;22)(q34.12;q11.23) |
| transl._interchr. | 9 | 22 | 130,847,453 | ABL1: intron 1 | 23,295,730 | BCR1: intron 15 | +/+ | 0.84 | 0.44 | ABL1::BCR | e14a2/p210 | 77 | ogm[GRCh38] t(9;22)(q34.12;q11.23) | |
| 6 | transl._interchr. | 9 | 1 | 137,686,151 | EHMT1: intron 1 | 154,225,651 | UBAP2L: intron 2 | +/+ | 0.95 | 0.28 | UBAP2L::EHMT1 | N/A | 72 | ogm[GRCh38] t(1;9)(q21.3;q34.3) |
| transl._interchr. | 9 | 1 | 137,732,949 | EHMT11: intron 4 | 154,225,651 | UBAP2L: intron 2 | +/+ | 0.97 | 0.28 | UBAP2L::EHMT1 | N/A | 95 | ogm[GRCh38] t(1;9)(q21.3;q34.3) | |
| transl._interchr. | 9 | 1 | 130,836,231 | ABL1: intron 1 | 154,231,851 | UBAP2L: intron 4 | +/+ | 0.98 | 0.51 | UBAP2L::ABL1 | N/A | 128 | ogm[GRCh38] t(1;9)(q21.3;q34.12) | |
| transl._interchr. | 9 | 22 | 130,847,453 | ABL1: intron 1 | 23,261,125 | BCR1: intron 3 | +/+ | 0.98 | 0.63 | ABL1::BCR | E2a2 | 110 | ogm[GRCh38] t(9;22)(q34.12;q11.23) | |
| transl._interchr. | 9 | 22 | 137,667,566 | EHMT1: intron 4 | 23,295,730 | BCR1: intron 15 | +/+ | 0.89 | 0.48 | EHMT1::BCR | N/A | 114 | ogm[GRCh38] t(9;22)(q34.3;q11.23) | |
| 8 | transl._interchr. | 9 | 22 | 130,847,453 | ABL1: intron 1 | 23,225,934 | BCR1: intron 1 | +/+ | 0.99 | 0.59 | ABL1::BCR | e2a2/p210 | 122 | ogm[GRCh38] t(9;22)(q34.12;q11.23) |
| transl._interchr. | 9 | 22 | 130,855,697 | ABL1: intron 3 | 23,219,177 | BCR1: intron 1 | +/+ | 0.96 | 0.61 | ABL1::BCR | e1a4/? | 88 | ogm[GRCh38] t(9;22)(q34.12;q11.23) | |
| 10 | transl._interchr. | 9 | 22 | 130,732,573 | ABL1: intron 1 | 23,295,730 | BCR1: intron 15 | +/+ | 0.93 | 0.48 | ABL1::BCR | e14a2/p210 | 64 | ogm[GRCh38] t(9;22)(q34.12;q11.23) |
| transl._interchr. | 9 | 22 | 130,743,975 | ABL1: intron 1 | 23,261,125 | BCR1: intron 3 | +/+ | 0.98 | 0.34 | ABL1::BCR | e3a2/? | 187 | ogm[GRCh38] t(9;22)(q34.12;q11.23) | |
| transl._interchr. | 9 | 22 | 130,747,294 | ABL1: intron 1 | 23,305,888 | BCR1: intron 15 | +/+ | 0.6 | 0.34 | ABL1::BCR | e14a2/p210 | 61 | ogm[GRCh38] t(9;22)(q34.12;q11.23) | |
* Chromosomes involved in the aberration; ** the highlighted confidence scores were below our cutoff values as described in the materials and methods; Chr: chromosome or chromosomal; transl._interchr.: translocation_interchromosomal; intrachr_fusion: intrachromosomal fusion; N/A: not applicable.
Table 4.
A head-to-head comparison of all four methods applied in this study.
| Chr Analysis | FISH | RT-PCR | OGM | |
|---|---|---|---|---|
| Detection power | ||||
| BCR::ABL1 fusion | Yes | Yes | Yes | Yes |
| Isoforms | No | p210 vs. p190 | Yes | Questionable |
| ACAs on der(9) | Yes | Yes | No | Yes |
| ACAs on der(22) | Yes | Yes | No | Yes |
| Other ACAs * | Yes | No | No | Yes |
| Translocation vs. insertion | Likely | Metaphase FISH | No | Likely |
| Sensitivity | 5% | 0.5–2% | 0.001–0.0001% | 10% |
| Single cell level | Yes | Yes | No | No |
| Turn-around time | 3–5 d | 4 h | 1–7 d | 5–7 d |
| Cost-effectiveness | Yes | Yes | Yes | No |
| Clinical application | ||||
| Initial diagnosis | Yes | Yes, quick result | Yes, isoform | Yes |
| Follow-up studies | Yes | Yes | Yes | Not for MRD |
| Refractory/Relapse | Yes | Yes | Yes | Yes |
* chromosome(s) other than der(9) or der(22). Chr: chromosomal; ACAs: additional chromosomal aberration(s); d: day(s). MRD: minimal residual disease.
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Tang, Z.; Wang, W.; Toruner, G.A.; Hu, S.; Fang, H.; Xu, J.; You, M.J.; Medeiros, L.J.; Khoury, J.D.; Tang, G. Optical Genome Mapping for Detection of BCR::ABL1—Another Tool in Our Toolbox. Genes 2024, 15, 1357. https://doi.org/10.3390/genes15111357
AMA Style
Tang Z, Wang W, Toruner GA, Hu S, Fang H, Xu J, You MJ, Medeiros LJ, Khoury JD, Tang G. Optical Genome Mapping for Detection of BCR::ABL1—Another Tool in Our Toolbox. Genes. 2024; 15(11):1357. https://doi.org/10.3390/genes15111357
Chicago/Turabian StyleTang, Zhenya, Wei Wang, Gokce A. Toruner, Shimin Hu, Hong Fang, Jie Xu, M. James You, L. Jeffrey Medeiros, Joseph D. Khoury, and Guilin Tang. 2024. "Optical Genome Mapping for Detection of BCR::ABL1—Another Tool in Our Toolbox" Genes 15, no. 11: 1357. https://doi.org/10.3390/genes15111357
APA StyleTang, Z., Wang, W., Toruner, G. A., Hu, S., Fang, H., Xu, J., You, M. J., Medeiros, L. J., Khoury, J. D., & Tang, G. (2024). Optical Genome Mapping for Detection of BCR::ABL1—Another Tool in Our Toolbox. Genes, 15(11), 1357. https://doi.org/10.3390/genes15111357
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