|Year : 2019 | Volume
| Issue : 2 | Page : 51-60
Fluorescence In situ hybridization signal patterns and intrachromosomal breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 amplification analysis in imatinib-resistant chronic myelogenous leukemia patients using tricolor dual fusion probe
Karthik B K. Bommannan1, Shano Naseem1, Neelam Varma1, Jogeshwar Binota1, Pankaj Malhotra2, Subhash Varma2
1 Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Web Publication||10-Jul-2019|
Dr. Shano Naseem
Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
BACKGROUND: Cytogenetic evaluation is required till a complete cytogenetic remission is achieved in chronic myelogenous leukemia (CML) patients on tyrosine kinase inhibitor (TKI) therapy. The routine dual colour fluorescence in situ hybridization (FISH) probes are less sensitive in identifying der(9) abnormalities. BCR/ABL/ASS1 tri-colour dual fusion (TCDF) probe is highly sensitive and specific in identifying der(9) deletions and random signal overlaps.
METHODS: Peripheral blood interphase FISH analysis was performed on imatinib-resistant CML patients using TCDF probe.
RESULTS: On analyzing 37 adult patients, all had residual Philadelphia (Ph) chromosome. Classic Ph fusion pattern was seen in 33 (89%), derivative chromosome 9 [der(9)] abnormalities in 25 (67.5%) and supernumerary Ph chromosomes in 11 (30%) patients. Coexistence of classical fusion and der(9) abnormalities was seen in 21 patients (57%); and classical fusion, der(9) abnormalities and supernumerary Ph chromosome in 8 patients (22%). None of the patients had BCR-ABL1 gene amplification. There was significant difference in the der(9) abnormal cell percentages between patients with e13a2 and e14a2 transcripts (P = 0.008) and patients with disease transformation (P = 0.007).
CONCLUSION: A high frequency of der(9) abnormalities and absence of BCR-ABL1 gene amplification was seen in imatinib-resistant CML patients analyzed. The use of TCDF probe for cytogenetic follow-up in CML patients was found to be useful in identifying BCR-ABL1 related aberrations. The identified patterns in this study, can serve as a reference material for I-FISH signal interpretation using TCDF probe.
Keywords: Breakpoint cluster region/Abelson murine leukemia viral oncogene homolog/arginosuccinate synthetase 1 tricolor dual fusion probe, chronic myelogenous leukemia, fluorescence in situ hybridization, imatinib resistant
|How to cite this article:|
K. Bommannan KB, Naseem S, Varma N, Binota J, Malhotra P, Varma S. Fluorescence In situ hybridization signal patterns and intrachromosomal breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 amplification analysis in imatinib-resistant chronic myelogenous leukemia patients using tricolor dual fusion probe. J Appl Hematol 2019;10:51-60
|How to cite this URL:|
K. Bommannan KB, Naseem S, Varma N, Binota J, Malhotra P, Varma S. Fluorescence In situ hybridization signal patterns and intrachromosomal breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 amplification analysis in imatinib-resistant chronic myelogenous leukemia patients using tricolor dual fusion probe. J Appl Hematol [serial online] 2019 [cited 2019 Nov 21];10:51-60. Available from: http://www.jahjournal.org/text.asp?2019/10/2/51/262543
| Introduction|| |
Chronic myelogenous leukemia (CML) is the most common myeloproliferative neoplasm and represents 15% of all adult leukemias. Philadelphia (Ph) chromosome, the hallmark of CML, is the derivative chromosome 22 [der(22)] resulting from t(9;22)(q34;q11). In Ph chromosome, the breakpoint cluster region (BCR) gene on chromosome 22 is juxtaposed with Abelson murine leukemia viral oncogene homolog 1 (ABL1) gene on chromosome 9. The resultant BCR-ABL1 fusion oncoprotein has constitutional tyrosine kinase activity.
Conventional cytogenetics is the most common modality to diagnose CML but may show false negativity in around 5% of cases having cryptic translocations. Polymerase chain reaction (PCR) testing for BCR-ABL1 fusion transcript is an effective alternative; however, in rare cases with alternate transcripts, translocations can be missed by routine analysis. However, by fluorescence in situ hybridization (FISH) technique, CML case with alternate transcripts can also be identified.
CML patients are effectively treated by tyrosine kinase inhibitors (TKIs). Quantitative TKI response assessment is feasible by monitoring residual BCR-ABL1 transcript levels and percentage of Ph chromosomes, through molecular and cytogenetic techniques, respectively. During therapeutic response assessment, residual Ph chromosomes detected by conventional cytogenetics is frequently used. However, for conventional cytogenetics, it is mandatory to analyze at least 20 metaphases prepared from bone marrow (BM) aspirates (BMAs) cultured at 24 and 48 h. The technique is, therefore, laborious and time-consuming, and in cases with <20 metaphases, the results have to be validated by FISH or BCR-ABL1 transcript levels by real-time PCR (RQ-PCR).
Although cytogenetic follow-up is done interchangeably with either BM cytogenetics or peripheral blood (PB) interphase FISH (I-FISH), there is no consensus regarding this approach. Till date, there is no concrete evidence to establish the long-term survival of patients being followed up with I-FISH alone. Head-to-head comparisons between conventional BM cytogenetics and I-FISH during CML follow-up are scanty but encouraging for the later.
National Comprehensive Cancer Network recommends conventional BM cytogenetics as the best modality for follow-up cytogenetic evaluation, and PB I-FISH is to be considered only if BMA is not feasible. This is due to the difficulty in differentiating random signal overlaps and split signals from true signal patterns by dual-color single fusion (DCSF) or dual color dual fusion (DCDF) I-FISH. Although the diagnosis of classical BCR-ABL1 fusion pattern is not cumbersome, DCSF and DCDF probes demand a higher cutoff of positive cells to confidently identify derivative chromosome 9 (der) abnormalities.,, In this regard, BCR/ABL/ arginosuccinate synthetase 1 (ASS1) tricolor dual fusion (TCDF) probe is highly specific in identifying der(9) abnormalities with a false positivity rate of <1%.,,,, Of late, there are few studies which have used TCDF probe to describe the signal patterns and confirm der(9) abnormalities detected by DCDF probes.,, To our knowledge, the isolated use of TCDF probe in TKI-resistant CML patients has not been exclusively studied.
Despite the impressive track record of imatinib mesylate ( first-generation TKI) in CML treatment, 16%–33% of patients develop resistance to the drug.,, The resistance could be due to BCR-ABL1-dependent or -independent mechanisms., ABL tyrosine kinase domain (TKD) mutations and intrachromosomal amplification of BCR-ABL1 (iAMP-BCR-ABL), i.e., presence of multiple copies of BCR-ABL1 in the same Ph chromosome, are the BCR-ABL1-dependent mechanisms of imatinib resistance. The presence of multiple Ph chromosomes has been associated with disease progression, but iAMP-BCR-ABL has been associated with TKI resistance and rapid development of TKD mutations. Since its first description in 2000, iAMP-BCR-ABL is the least common and least explored mechanism of TKI resistance.,,,
In this study, we aimed to analyze the PB I-FISH signal patterns and look for iAMP-BCR-ABL amplification in baseline high-grade (accelerated phase [AP]/blast crisis [BC]) and imatinib-resistant CML patients, using a highly specific BCR/ABL/ASS1 TCDF FISH probe (Abbott Laboratories, Abbott Park, Illinois, USA).
| Methodology|| |
For the morphological diagnosis of CML and the disease phase, evaluation of May–Grunwald–Giemsa-stained PB and BM smears was done. The diagnosis was then confirmed by qualitative reverse transcriptase PCR (RT-PCR) to detect BCR-ABL1 fusion (e13a2, e14a2, and e1a2 transcripts) using specific primers. All newly diagnosed CML patients were on daily doses of imatinib (400 mg for chronic phase [CP] and 600–800 mg for AP and BC). The follow-up was done with clinical examination, hemogram, and residual BCR-ABL1 transcript load assessment in an international scale (BCR-ABL1IS) by RQ-PCR. For the current study spanning from January 2015 to June 2016, all treatment–compliant patients were selected as per the European LeukemiaNet 2013 recommendations (morphologic and molecular genetics criteria alone) for TKI resistance testing.
Mononuclear cell suspensions were prepared from heparin anticoagulated PB samples and were fixed in Carnoy's fixative. As per the manufacturer's protocol, the cells were dropped on a glass slide, aged, and hybridized with BCR/ABL/ASS1 TCDF FISH probe (Abbott Laboratories, Illinois, USA) in ThermoBriteStatSpin hybridizer (Abbott Laboratories, Illinois, USA). The fluorescent signals were analyzed in GenASIS cytogenetics workstation comprising motorized BX60 fluorescent microscope (Olympus, Hamburg, Germany). As per the manufacturer's design for this TCDF probe, the green (G) probe identifies entire BCR sequence in chromosome 22. In chromosome 9, the orange (O) probe spans the entire ASS1 and ABL1 sequences, including the sequences present between these two genes. The aqua probe (A) covers ASS1 and the sequences between ASS1 and ABL1. The probe design is such that the isolated green-orange (GO) fusion signal is considered as genuine BCR-ABL1 fusion, i.e., Ph-positive. In a Ph-positive cell, the presence of a green-orange-aqua (GOA) fusion signal is identified as reciprocal ABL1-BCR fusion in der(9). Classic fusion pattern is represented by 1G 1OA 1GO 1GOA pattern. The presence of GOA signal in a Ph-negative cell indicates random overlap of the probes and has to be ignored. The probe performance was standardized on 700 interphase nuclei prepared from PB mononuclear cells of RT-PCR-proven BCR-ABL1-negative controls and the TCDF signals were interpreted as per the recommendations.,, In brief, dual band pass filters (spectrum orange and spectrum green) were used in tandem to screen for GO fusion (Ph-positive cell). If GO fusion signals were present, spectrum aqua filter was used to detect aqua signal overlap at the GO fusion sites. Isolated GO signals separated by more than one signal diameter were counted as separate BCR-ABL1 fusion signals, i.e., supernumerary Ph chromosomes. Clustering of multiple GO fusion signals (each within one signal diameter from the other) was considered for iAMP-BCR-ABL1. A false positivity rate of 0.4% was determined during our standardization, which was comparable to the established false positivity of 0.6% for this specific probe., In the analysis cohort, a minimum of 200 nonoverlapping interphase cells were analyzed per case and any signal pattern which was present in ≥1% of the cells was considered positive. The signal patterns identified in our cohort and their interpretation are depicted in [Table 1]. The results were compiled in Microsoft Excel 2007 and analyzed using SPSS (version 19) software (Chicago, Illinois, USA).
|Table 1: Interpretation of triple color dual fusion interphase fluorescence in situ hybridization signals|
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| Results|| |
The cohort consisted of 37 CML patients. The median age was 40 years (range, 24–75), with male: female ratio of 1.5:1. On molecular genetic analysis, p210 BCR-ABL1 e13a2 and e14a2 transcripts were seen in 19 and 18 patients, respectively. Apart from one baseline BC patient who was included before imatinib therapy, the remaining 36 patients had 42 months (range, 3–171) of median imatinib therapy. The indications for imatinib resistance analysis in these patients were as follows – baseline BC in 1 patient; progression to AP in 2 patients; progression to BC in 4 patients; loss of complete hematologic response in 3 patients; and loss of major molecular response in 28 patients.
On analyzing 7642 cells from these 37 patients, classical fusion signals were seen in 33 patients (89%) [Figure 1]. Atypical fusion signals in the form of der(9) abnormalities and supernumerary Ph chromosomes were seen in 25 (67.5%) and 11 (30%) patients, respectively. Four patients (11%) did not have any classical fusion cells but had only BCR-ABL1 fusion cells with der(9) abnormalities [Figure 2]. Coexistence of classical fusion and der(9) abnormalities was seen in 21 patients (57%), whereas 8 patients (22%) had the mutual existence of classical fusion, der(9) abnormalities, and supernumerary Ph chromosomes. None of our cases had Ph amplification. During analysis, a median of 23% (range, 5–45) cells with random signal overlap were encountered and ignored. The constellation of signal patterns encountered in each patient is described in [Table 2].
|Figure 1: Interphase fluorescence in situ hybridization signal patterns using tricolor dual fusion probe. A 46.year.old male who had loss of major molecular response at 83 months of tyrosine kinase inhibitor. Yellow arrow: Classical Philadelphia fusion pattern; Red arrow: Random signal overlap|
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|Figure 2: Interphase fluorescence in situ hybridization signal patterns using tricolor dual fusion probe. A 43-year-old male diagnosed with chronic myelogenous leukemia-chronic phase and progressed to blast crisis at the 72nd month of imatinib therapy. Red arrow: Normal cell; Yellow arrow: Philadelphia-positive cell with loss of residual Abelson murine leukemia viral oncogene homolog 1 on der(9); White arrow: Random signal overlap|
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|Table 2: Percentages cells with different fusion signal patterns in each case of our cohort|
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Derivative chromosome 9 abnormalities
Among the der(9) abnormalities seen in our cohort, complete loss of reciprocal ABL1-BCR fusion was the most common, seen in 14 patients (38%), followed by loss of translocated BCR in 12 patients (32%) and loss of residual ABL1 in 8 patients (22%). The above observation includes 4 patients (11%) who had coexistence of cells with residual ABL1 loss and loss of ABL1-BCR fusion and 2 patients (5%) with all three der(9) anomalies.
A median of 16.5% (range, 0–81) and 11% (range, 0–48) cells with der(9) abnormalities were seen among patients with b2a2 and b3a2 BCR-ABL1 transcript types, respectively, and this difference was statistically significant (P = 0.008). There was also a significant difference (P = 0.007) in the disease transformation status with respect to the percentage of der(9) atypical cells. In this regard, patients with progressive disease (AP and BC) had a median of 59% (range, 0–81) der(9) atypical cells in comparison to a median of 13% (range, 0–69) such cells in patients without disease transformation.
Supernumerary Philadelphia chromosomes
Duplication and triplication of Ph chromosome were seen in 11 (30%) and 3 (8%) patients, respectively, with two of these patients having both these abnormalities. Interestingly, these supernumerary Ph chromosomes were observed as the addition of extra Ph chromosomes to the patient-specific atypical fusion patterns [case numbers: 1, 2, 14, 18, 19, 24, 29, and 34 in [Table 2]. Patients with Ph duplication/triplication had a median BCR-ABL1IS level of 67.5% (range, 5.0–100), in comparison to median BCR-ABL1IS levels of 12.0% (range, 1.0–92.0) in patients without these anomalies and this difference was statistically significant (P = 0.003).
Loss of breakpoint cluster region and Abelson murine leukemia viral oncogene homolog 1 in uninvolved chromosomes
Loss of ABL1 in nontranslocated chromosome 9 was seen in 5 cases (13.5%). Loss of BCR in nontranslocated chromosome 22 and presence of both these anomalies were seen in one patient each.
| Discussion|| |
The young age of Indian CML patients is well documented and our cohort's median age (40 years) is in line with the same.,, Our entire imatinib-resistant cohort had residual Ph chromosomes indicating disease persistence. The classical BCR-ABL1 fusion pattern seen in 89% of our cohort is concordant with the reported frequency of 71%–88% from Western and Indian studies.,,,,
The high frequency of der(9) abnormalities (67.5%) described in our study is from patients who are already on imatinib therapy and their baseline der(9) status is not known. There are previous studies which have documented der(9) abnormalities at the time of baseline diagnosis, rather than their emergence at later time points.,, Based on the reported literature, we also hypothesize that our cohort must have had a very high frequency of baseline der(9) abnormalities. This would be in contrast to both Western and Indian studies reporting a lower frequency (10%–20%) of baseline der(9) abnormalities in treatment-naive CML patients, using dual color probes.,,,,, We hypothesize that high frequency of der(9) abnormalities observed in our cohort might be imatinib induced or inherent to the biology of disease progression. Imatinib-induced TKD mutations are well-established cause for treatment failure in CML; however, there are no data documenting co-existing imatinib-induced cytogenetic changes in patients with kinase domain mutations. Our study opens scope for further research in this concept. Limited by the lack of baseline banding cytogenetics/FISH profile, our study was not powered to prove clonal cytogenetic evolution, though multiple clones (presence of both classic fusion and classic fusion with der(9) abnormalities together) of tumor cells coexisted in 57% of our patients. Interestingly, in patients with Ph duplications/triplications, it is observed that these supernumerary Ph chromosomes have evolved from their respective classical or atypical BCR-ABL1 fusion clones, further favoring our hypothesis of heterogeneous clonal evolution in these patients.
It has been observed that CML patients with e13a2 transcript type have a higher propensity for disease transformation (AP/BC). Our cohort had an almost equal number of patients with e13a2 (n = 18) and e14a2 (n = 19) transcript types, and among the six patients who showed disease transformation, there was equal representation (n = 3, each) of both these transcript types. Remarkably, our patients with e13a2 transcript had a significantly higher (P = 0.008) number of der(9) atypical cells (median, 16.5%) than those seen in patients with e14a2 transcript (median, 11%). In addition, all our patients with disease transformation had significantly (P = 0.007) higher number of der(9) abnormal cells (median, 59%) than those seen in patients without disease transformation (median, 13%). Thus, our data demonstrate the association of higher der(9) abnormal cell numbers with adverse factors such as disease transformation and e13a2 transcript type. Although the poor prognosis implicated by der(9) abnormalities have been largely abrogated by TKI therapy,,,, our data suggest that it might be still reasonable to look for der(9) abnormalities as an adverse prognostic factor in imatinib-treated patients.
Since its first description in the millennium year, iAMP-BCR-ABL1 has not been extensively documented.,,, Reported frequency of this rare mechanism of TKI resistance range from 6 to 27%., Though our cohort size is comparable with these earlier studies, none of our patients had iAMP-BCR-ABL1. This finding needs to be further studied on a larger number of patients.
In comparison to conventional banding cytogenetics and metaphase FISH techniques, I-FISH has the advantage of analyzing more cells with a relatively rapid turnaround time (~18 h). The widely used DCSF and DCDF FISH probes cannot differentiate random signal overlaps from a Ph-positive cell with der(9) deletion and also cannot reliably identify supernumerary Ph chromosomes in the presence of coexisting der(9) deletion. The TCDF probe design can accurately recognize der(9) anomalies and can identify supernumerary Ph chromosomes even with coexisting der(9) abnormalities. In addition, TCDF probe can even be used in the cytogenetic follow up of CML patients in whom the baseline der(9) abnormality status is unknown., Due to these advantages, we recommend the use of TCDF I-FISH during follow-up cytogenetic assessment of CML patients. Due to the robustness in differentiating true and false signals at a false positivity of < 1%,, TCDF probe can aid in accurate enumeration of Ph chromosomes and der(9) abnormalities at diagnosis. This would further enhance our understanding of CML biogenesis across all phases of the disease.
In CML diagnosis, TCDF probe has been primarily used as a validation tool to confirm the atypical der(9) signals detected by dual color probes., Except for their occasional use in resolving the discrepancies between molecular-genetic and cytogenetic remission status determined by dual-color FISH, the TCDF probe has not been extensively used in routine cytogenetic follow-up of CML patients. In addition, to the best of our knowledge, none of the studies have compared the I-FISH patterns with molecular genetic parameters such as the baseline BCR-ABL1 transcript type and the BCR-ABL1 transcript load during follow-up.
| Conclusion|| |
In this study, we demonstrated a high frequency of der(9) abnormalities and absence of BCR-ABL1 amplification in an imatinib-resistant CML cohort. Significant association of der(9) atypical cell percentage with e13a2 transcript type and disease transformation status has been identified, which needs to be tested in a larger cohort.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]