Rearrangements involving the ABL1 gene at chromosome 9q and the BCR gene at 22q result in formation of the Philadelphia (Ph) chromosome, t(9; 22)(q34; q11), and are present in all cases of chronic myeloid leukemia (CML) and in some cases of acute lymphoblastic leukemia (Ph+ ALL). The BCR-ABL1 fusion gene, characteristic for these patients, encodes an oncoprotein that has an activated tyrosine kinase domain in the ABL region. This infamous protein promotes cell proliferation leading to loss of stromal adhesion and inhibits apoptosis by activating downstream pathways, including Janus kinase-signal transducer and activator of transcription (Jak-Stat) and Myc [1, 2].

Introduction and widespread clinical use of the tyrosine kinase inhibitor (TKI), imatinib in CML patients, has resulted in much better overall survival compared to interferon [3]. Furthermore, imatinib has reduced the need for allogeneic stem cell transplantation in these patients [4]. Despite the extraordinary success of imatinib for the treatment of CML, up to 40% of patients experience intolerance or resistance. The most common reason for treatment failure is the acquisition of mutations within the kinase domain (KD) of BCR-ABL1 that impair drug binding. Although more developed second-generation TKIs were designed to target most imatinib-resistant mutations, some mutations show resistance even to these new generation drugs (T315I) [5, 6]. Almost 25% of imatinib-resistant patients were reported to have more than one BCR-ABL1 KD mutation, and the presence of multiple mutations defines a poor-risk subgroup of chronic phase (CP) CML patients [7]. These patients have a higher likelihood of acquiring new resistant mutations and poor response to treatment with second-generation TKIs, irrespective of the resistance profile of the mutations [7].

Currently, TKIs applied to treat CML strongly improve the overall survival (OS) of patients [2, 8]. The majority of patients achieve a life expectancy close to that of the general population [9]. However, there are still some treatment-related problems present: 1) The disease still progresses to an acceleration phase (AP) or blast crisis (BC) following treatment with TKIs; 2) a number of patients are resistant to TKIs; and 3) patients cannot endure the side effects of TKIs [10].

Patients frequently develop primary or secondary resistance to TKIs, despite the high response rates (primary or intrinsic resistance is a lack of initial response, whereas secondary or acquired resistance (relapse) is a loss of an established response) [11]. According to the European LeukemiaNet (ELN) criteria, resistance is defined as failure of first-line TKI therapy: less than a complete hematologic response and/or no cytogenetic response (CyR; defined as Ph+ bone marrow metaphases >95%) at 3 months, BCR-ABL1 transcript levels above 10% and/or less than a partial CyR (PcyR; defined as ≤35% Ph+ metaphases) at 6 months, BCR-ABL1 transcript levels above 1% and/or less than a complete CyR (CCyR; defined as no Ph+ metaphases) at 12 months, or loss of a complete hematologic response or complete CyR or confirmed loss of MMR, mutations, or clonal chromosome abnormalities in Ph− cells at any subsequent time during therapy [1, 12]. According to the National Comprehensive Cancer Network (NCCN) guidelines, on the other hand, resistance is defined as the detection of BCR-ABL1/ABL1 transcript levels above 10% on the International Scale or failure to achieve PCyR at 3 months, or failure to achieve CCyR at 12 months or 18 months [13]. Several mechanisms have been associated with resistance, including BCR-ABL1-dependent and BCR-ABL1-independent mechanisms [14]. BCR-ABL1 KD mutations and BCR-ABL1 genomic amplification are well-known mechanisms of resistance to TKI therapy [15].

Mutation analysis is usually performed after patients experience TKI treatment resistance; the results of mutation analysis may guide the selection of subsequent TKIs [1, 12, 13, 16]. Imatinib resistance, regardless of aetiology, necessitates a change in treatment strategy. However, the choice of second-generation TKI should take into account ABL1 KD mutation status to ensure optimum efficiency of TKI. Imatinib resistance often results from ABL1 mutations in the P-loop (G250E or E255K/V) and in the ATP-binding site (F317L) or from the panresistant mutation T315I [17–20]. Only few mutations are known to cause clinical resistance to nilotinib (Y253H, E255K/V, and F359V/C/I) [21, 22] or dasatinib (V299L, T315A, F317L/I/V/C) [22–25] or both (T315I). Patients already having mutations have higher likelihood of developing new mutations [15, 22] and multiple mutations can be associated with poor prognosis [24, 26, 27]. BCR-ABL1 KD mutation analysis can be done either by direct Sanger sequencing or Pyrosequencing which has been largely used to-date [1, 16]. Although newer technologies with greater sensitivity are available, most of them are limited by their specificity and limited spectrum of mutations, next-generation sequencing (NGS), nevertheless, is an exception.

The aim of this study is thus to analyse the association of identified BCR-ABL1 KD mutations with resistance to first-line TKI imatinib.

Data from a cohort of 163 CML patients, subjected to mutation screening, were rigorously examined, revealing that 119 individuals (73%) were classified as being in the CP, 26 individuals (15.9%) in the AP, and 18 individuals (11.1%) in the BC. Remarkably, we identified 5 mutations in CP, 5 in AP, and 12 in BC. Our statistical analysis unveiled a pronounced association between mutations in the ABL kinase domain (ABL KD) and the respective disease phases, with a statistically significant p-value of <0.05.

Notably, the T315I mutation emerged as the predominant genetic aberration and exhibited a significant correlation with overall survival (OS). Patients afflicted with the T315I mutation (n = 7), T315I/Y253H (n = 1), Y253H (n = 2), and E255K (n = 1) mutations faced adverse clinical outcomes, culminating in mortality. By the end of the study, the majority of patients with T315I, Y253H and E255K mutations had progressed to the BC phase and they failed to respond to combination chemotherapy and second-line TKIs.

Our investigation conclusively established that these mutations were significantly associated with resistance to both first- and second-line TKIs. Within the cohort of patients in whom mutations were identified, four individuals demonstrated the presence of multiple mutations. Three patients exhibited co-occurrence of F359V/F359C mutations, while one patient manifested both T315I/Y253H mutations. However, due to the retrospective design of the study, it was not possible to confirm whether these mutations were complex or polyclonal. Furthermore, the limited representation of such patients in our study impeded the assessment of the potential impact of compound mutations on overall survival.

43-month survival was noted in 92% (142) of resistant patients without the T315I mutations. 30 (21%) out of 142 patients were treated with imatinib dose escalation, and the rest 112 (79%) patients received second-line TKIs. At the culmination of a 24 month interval, 48 patients (43%) subjected to second-line TKI therapy manifested MMR, while 64 individuals (57%) exhibited concurrent hematological response and achieved CCyR.

Subsequently, the cohort was stratified into low-, medium-, and high-risk groups based on their clinical characteristics at the time of initial CML diagnosis, revealing no significant associations between risk stratification, patients’ blood groups, Rh-factor, seasonality of morbidity, and ABL KD mutations. Additionally, the analysis failed to reveal any significant correlations between initial HB, WBC, PLT, eosinophil count, basophil count, spleen size at the time of diagnosis, and the presence of mutations. Nevertheless, a substantial association was observed between baseline blast cell count and the occurrence of ABL KD mutations, with a statistically significant p-value of <0.05.

The emergence of resistance to tyrosine kinase inhibitors (TKIs) can be dichotomized into BCR-ABL-dependent and BCR-ABL-independent processes. BCR-ABL-dependent mechanisms encompass BCR-ABL kinase overexpression and mutations within the BCR-ABL domain, while BCR-ABL-independent processes involve the activation of alternative signaling pathways, such as JAK/STAT, as well as the upregulation of efflux transporters or downregulation of influx transporters, resulting in reduced intracellular TKI levels [29]. Among these mechanisms, mutations in the BCR/ABL KD are of paramount importance. Intriguingly, our study affirmed that mutations in the KD, which confer resistance to TKI therapy, exhibit a markedly higher incidence in patients in the BC phase compared to those in the CP. Furthermore, patients with mutations in the CP category demonstrated an elevated propensity for progressing to AP or BC. Consequently, these mutations bear clinical significance by providing insights into patients who fail to achieve optimal responses to TKIs [30, 31]. Notably, the current therapeutic landscape offers a plethora of alternative options for TKI treatment contingent upon the specific mutations associated with TKI resistance [32, 33]. Acquired mutations, in particular, interfere with TKI binding or result in diminished TKI sensitivity [34]. Notably, distinct mutations display varying degrees of sensitivity to TKIs, underscoring the importance of mutation detection in guiding TKI selection and influencing patient prognosis. These principles align with recommendations from both the European LeukemiaNet (ELN) and the National Comprehensive Cancer Network (NCCN), advocating the consideration of mutational profiles in the determination of subsequent therapy [13, 35].

It is noteworthy that, owing to the contribution of TKIs and an enhanced understanding of the influence of the BCR-ABL mutational profile on drug efficacy, CML is presently recognized as a chronic ailment. However, it remains pertinent to acknowledge that a subset of patients continues to experience disease progression. Consequently, there is an imperative need for further investigations elucidating the intricate interplay between genes and drugs, with the aim of achieving optimal coverage in the treatment of CML.