Investigating Breakpoint Cluster Region-Abelson 1(BCR-ABL) (BCRABL1) signal patterns across chronic myeloid leukemia morphologic phases and its impact on molecular response

Treatment regimen

All CML-CP cases were risk-stratified using the EUTOS score and were given either first-generation tyrosine kinase inhibitor (TKI) (imatinib) for low-risk or second-generation TKI (Dasatinib) for high-risk cases. For CML myeloblastic crisis, the 7+3 regimen (cytarabine daunorubicin) with imatinib, and for lymphoblastic crisis, the BFM-90 protocol was given with imatinib.

FISH Procedure

Dual color DUAL Fusion FISH for BCR-ABL1 was performed on bone marrow aspirate samples at diagnosis. At least 200 well-visualized cells with well-delineated signals were evaluated. The type of signal pattern in FISH was reported in each case, and the presence of a fusion signal in any number of cells was considered as Philadelphia positive(t(9;22)).

Molecular response assessment using real-time PCR

Post-treatment blood/bone marrow samples after one year were collected to evaluate for molecular response using quantitative real-time PCR. The attainment of major molecular response (MMR) was assessed based on the ratio of fusion copy numbers.

Statistical analysis

The data was collected in a Microsoft Excel master sheet. Both Microsoft Excel and the statistical package for Social Sciences (SPSS) version 20 software were used for statistical analysis. The continuous data, like age, is expressed as a median with a range. The association and significance between discrete data have been determined using the Chi-square test.

Morphological subtyping among the cases

It revealed 70(19%) cases of blast crisis and 299 (81%) cases of chronic phase [Figures 1 and 2]. Among the 299 cases presenting with CML-CP on bone marrow biopsy, increased fibrosis was noted in 29 cases (9.7%). Out of 70 cases of blast crisis, 16 cases had extramedullary blast proliferation (22.8%) (Illustration: 1). 53 of these cases had myeloid blast crisis, while 17 cases presented with lymphoblastic crisis Among the 16 extramedullary blast crises, lymph nodes and cerebrospinal fluid were the common sites to be involved (25% each), followed by bone, ascitic, and pleural fluid [Figure 3]. Out of 299 cases of CML-CP, 69 cases (23.1%) were categorized as low risk and 230 cases (76.9%) as high risk based on the EUTOS scoring system.

FISH findings in these cases

FISH was performed in all 369 cases, which revealed presence of the Philadelphia chromosome (t(9;22)) in all these cases. Also further based on the fluorescent signals, the patternswere reported as typical or atypical [Figure 4]. Out of 369 cases, 239 (64.8%) had typical FISH patterns, and remaining 130 cases (35.2%) had a typical FISH patterns [Table 1]. 10 FISH signal patterns in CML, including one typical [Figure 4a and b] and 9 atypical ones [Figure 4c-h]. The exact number of cases in each of the morphological subgroups with their specific FISH pattern is given in the table.

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Figure 1:

(a) Peripheral blood leucocytosis, granulocytic left shift, basophilia and eosinophilia - CML in chronic phase (20x, Wright stain). (b) and (c) Bone marrow aspirate and biopsy showing myeloid bulge with prominent myeloblasts- CML in myeloblastic crisis (b) - 40X, Wright stain. (c) - 40X, Hematoxylin and Eosin). (d) Bone marrow aspirate showing prominent blasts in the background of myeloid series proliferation- CML in Lymphoblastic crisis (40X, Wright).

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Figure 2:

(a and b) CML-CP; (a) Bone marrow biopsy showing hypercellular marrow with no fat spaces in the marrow fragments (4x, H and E). (b) Granulocytic hyperplasia with all stages of maturation and scattered dwarf megkaryocytes (40x, H and E). (c) Bone marrow biopsy shows fibrosis with streaming of cells. (10x, H and E). (d) Reticulin stain in the same case demonstrated increased reticulin fibres with many intersections- Grade 2 fibrosis (40x, Reticulin). H and E: Hematoxylin and eosinó

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Figure 3:

(a) Lymph node showing granulocytic sarcoma(40X, Hematoxylin and eosin). The blasts are positive for Myeloperoxidase (MPO). (b) and CD34 (c) (40X, Immunohistochemistry). (d) Cerebrospinal Fluid shows numerous myeloblasts in a clear background 40x, May-Grunwald-giemsa stain (MGG). (e) Pleural fluid cytology - highly cellular with sheets of myeloblasts – Extramedullary blast crisis (40X, Papanicolaou stain)

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CML: Chronic myeloid leukemia, CML CP: Chronic myeloid leukemia- chronic phase, CML BC: Chronic myeloid leukemia- Blast crisis

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Figure 4:

(a)-FISH images showing Typical and atypical patterns. (b)-101G2F (Typical), (c)- 1O1G1F, (d)- 1O1G3F, (e) - 1O2G1F, (f)-2O1G3F , (g)- 2O1G1F and (h) - 2O2G1F (White arrow in each figure points to the classical appearance of the respective FISH pattern).

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Correlation of FISH patterns with morphology

Table 2 illustrates the distribution of typical and atypical FISH patterns among each diagnostic subgroup.

MR status by RT-PCR

At the end of 12 months of therapy, out of 369 cases, RTPCR was performed in 324 of these cases. In the remaining 45 cases, RT-PCR was not done as they were lost to follow-up. Out of 324 cases, major molecular response was attained in 132 (40%) cases at the end of 12 months of therapy, and the remaining 192 (60%) cases failed to achieve MMR.

Baseline characteristics

In our study, 369 cases of CML were reported during the period of 18 months. The median age at presentation was 41 years. Most of our patients presented between 40-60 years of age. This is concordant with many previous studies and review articles.^[9–11] The youngest presenting age was 9 years in our study. Male to female ratio in our study was 2:1. This is also in concordance with the available literature, which stated that males are more likely to be affected than females.^[12]

Morphological subtyping

The 2022 WHO Classification adopted a biphasic scheme for the natural history of CML by only recognizing the chronic phase (CP) and Blast phase (BP) with an intermediate group called high-risk chronic phase.^[12,13] Many literature studies stated that approximately 85% of people are in the chronic phase of CML.^[14] Similarly, in our study, morphological subtyping was done in all 369 cases, and it revealed that 81% of cases were in the chronic phase and 19% of cases were in blast crisis.

Bone marrow biopsies among the CP cases were examined for fibrosis, which revealed that 29 cases (9.7%) had increased fibrosis. BM fibrosis occurs in 40% of patients with CML at diagnosis.^[15,16] Bone marrow fibrosis (grade 2 or more) at diagnosis of CML, or its later development during the course of the disease was regarded as a poor prognostic factor. However, according to recent WHO updates, the degree of bone marrow fibrosis in CML does not have a bad prognosis when treated with imatinib.^[17]

Extramedullary involvement (cutaneous/bone/lymph node/CSF/other body fluids involvement) has been reported in only 04% to 16% of cases of CML-BC.^[18] In our study, 16 cases (23%) had extramedullary(EM) blast proliferation out of 70 cases of blast crisis.

FISH patterns

FISH was performed in all 369 cases, and it revealed 65% of cases had typical FISH patterns, and 35% of cases showed atypical FISH patterns. This was roughly similar to the previous reported incidence of 71%-88%.^[19] Atypical FISH patterns represent small deletions within the BCR and ABL1 hybridization sites and some adjacent DNA sequences, and also duplication of the Philadelphia chromosome.^[20,21]

In this study, we found 9 atypical FISH patterns apart from he typical one (1O1G2F). The most common atypical FISH pattern was 1O1G1F. The majority of chronic phase CML cases showed typical FISH patterns (70.3%), whereas 57.4% of the blast crisis cases showed atypical FISH patterns in our study, thus indicating a more aggressive course of presentation. 65% of the CML cases with fibrosis showed atypical FISH patterns while only 29.7% of CML cases without fibrosis showed atypical FISH patterns. Also, among the extramedullary BC cases, 62.5% cases showed atypical FISH patterns. From these findings, our study has proven that there is a significant difference between CML-CP, CML-BC-BC- BC and CML-CP with fibrosis with respect to the type of FISH pattern. Atypical patterns were found to be far more common in CML-BC and CMLCP with fibrosis (p-0.007 and p<0.001, respectively) than in CML-CP without fibrosis.^[22,23] Overall, CML cases with atypical FISH patterns showed faster progression to advanced phases with unfavourable outcomes.^[24,25] There are only very few studies that highlighted the impact of atypical FISH patterns with disease outcome. Our study is one of the few studies which mainly focused on the importance of detecting atypical FISH patterns and their correlation with various phases of CML. This highlights that any case with variant BCR–ABL1 fusion warrants a close follow-up, and a normal or noncomplex karyotype detected in these cases by conventional karyotyping is misleading in terms of risk stratification, response categorization, and clinical follow-up.

Determination of molecular response through RT-PCR

RT-qPCR quantifies the amount of BCR-ABL1 mRNA in the blood, thereby allowing precise measurement of leukemic burden. The results were expressed as the ratio of BCRABL1 to ABL1(housekeeping gene) and expressed on the International scale (IS). In our study, MMR was evaluated using qRT-PCR at the end of 12 months of therapy with TKIs with or without chemotherapy. Out of 369 cases, 45 cases were lost to follow-up. In the remaining 324 cases, 40% cases attained MMR and 60% failed to achieve MMR. Several studies have pointed out that achieving MMR early is associated with better long-term survival and a low risk of disease progression.^[26,27]IRIS Study (International Randomized Study of InterferonVs. STI571) demonstrated that approximately 40% of patients treated with imatinib achieved MMR at the end of 1 year, similar to our study.^[28] A study by Zhimei Cai et al, based on molecular evaluation of 79 cases of CML, showed that 51.9% cases achieved MMR at the end of 1 year of imatinib therapy.^[29]

Correlation of Phases of CML and EUTOS Risk Stratification With MMR Status

In our study, the percentage of CML cases in chronic phase that attained MMR was significantly higher than those in blast phase and CML with increased fibrosis. 48% of CMLCP attained MMR out of 324 cases, whereas only 22.4 % and 12.5% cases of BC and CML with increased fibrosis attained MMR, respectively. These differences were clinically significant (p< 0.001). This also demonstrates that, with disease progression(either fibrosis or blast crisis), the time required to achieve a major molecular response increases.^[30] Few studies have also pointed out that patients diagnosed with chronic phase CML, who have started therapy early, have a better prognosis and a higher probability of achieving durable molecular responses than those in advanced phases.^[31] We also observed that 55% of low EUTOS risk cases attained MMR while only 42% of high-risk cases attained MMR, which was significant (p<0.05), and this finding corresponds with previously published literature.^[2]

Association Between FISH Patterns and RT-PCR Status

While comparing the FISH patterns with MMR status, 70% of cases with a typical FISH pattern attained MMR, but only 7.7% of cases with an atypical FISH pattern attained MMR at the end of 12 months. This significant difference in molecular responses might be attributed to increased complexity of chromosomal abnormalities, such as additional clonal evolution leading to a more aggressive disease course and resistance to standard therapies, with an overall poor prognosis. Even though a detailed analysis comparing atypical FISH patterns with molecular response has not been performed till now, a few of the studies have stated that atypical FISH patterns are linked to slower achievement of molecular response.^[19,32]. A study by Zhanglin et al compared heterogeneous BCR-ABL1 signal patterns in 270 cases of CML with leukemic clonal evolution. This study showed that 52.9% cases with an atypical FISH pattern presented in blast phase were associated with additional clonal evolution and failure to molecular milestones, similar to our study.^[4]

Even though CML-CP cases with typical and atypical FISH patterns did not show a significant difference in EUTOS risk assessment, they showed a significant difference in attainment of MMR. A significantly higher proportion of cases with typical FISH patterns attained MMR compared to cases with atypical patterns (65.9% vs 9.1%). This strongly suggests that FISH patterns are independently associated with treatment response, irrespective of EUTOS score. Further studies on a large scale are needed for validation of this finding.

Recent studies have shown that there is no significant difference in prognosis between CML-CP and CML-CP with fibrosis.^[33] However, our study proves that CML-CP with fibrosis is significantly associated with atypical FISH patterns and also associated with delayed achievement of MMR compared to CML-CP. Further studies with long-term follow-up are required to assess the prognosis and long-term survival among cases with CML-CP with fibrosis.

Our study has also proven that atypical FISH patterns in CML are significantly associated with faster disease progression and are less likely to attain MMR by one year than typical FISH patterns. From the above findings, it can be concluded that CML cases with atypical patterns need a closer follow-up to monitor disease progression and have a worse prognosis with significantly delayed attainment of major molecular response (MMR) compared to CML cases with typical FISH patterns.

Further studies are required to assess the response of CML cases with atypical patterns when treated with the next generation of TKIs and also to assess any change in treatment protocol required in these cases to evoke a better response.

Author contributions

US: Design, drafting of manuscript, acquisition of data; KD: Drafting of manuscript, analysis of data; PW: Study design, critical revision of manuscript; SJG: Technical supervision; statistical analysis; PP: Study design; BP: Technical supervision; PT: Technical supervision; DMP: Technical supervision.