Actualizări legate de fază în managementul diagnostic şi monitorizarea pacienților cu leucemie mieloidă cronică

Introduction

The updated scientific references reiterate that chronic myeloid leukemia (CML) embraces 15% of all leukemias totality and 20-25% of leukemias population in adults, being the most commonly recorded myeloid neoplasm^(1-7). The incidence of CML is higher in people aged 35-65 years old (average age: 50-53 years old), indicating a prevalent involvement of the workable and socially active inhabitants^(3,8,9). The narrative review of the recent references pointed out that roughly 7.4% of CML subjects were in the 20-34 age group and 33% in the under-40 age group, bearing out, thus, the significant disease burden amongst relatively young population^(10,11). Since the implementation into clinical practice of imatinib mesylate, the mortality rate from CML has declined from 20% to 1-2%^(11-13). However, the epidemiological patterns and management approaches in CML are equivocally demonstrated in literature, especially after COVID-19 pandemic, and continue to be of both scientific and practical issues. The increased lactate dehydrogenase (LDH) level is admitted as a biochemical marker of neoplastic proliferation and anaerobic metabolism, and it has been asserted as an unfavorable prognostic factor in leukemias and malignant lymphomas^(14-16). LDH is an engaging monitoring indicator in myelodysplastic syndromes and multiple myeloma, which may assist in earlier diagnosis of disease progression and, thus, be helpful in risk grouping and patient selection for groundbreaking high-tech therapies⁽¹⁴⁾. Scanty studies were performed in order to evaluate the plasmatic concentrations of LDH, alkaline phosphatase (ALP) and the detectability of the BCR::ABL1 transcripts in CML patients and healthy subjects. It was advocated that the sequential assessment of LDH and BCR::ABL1 transcript during the follow-up of CML patients could be subsequently considered a marker for decision-making regarding the continuation of medication. Meantime, 30-50% of chromosome impairments in CML represent fused genes that can only be revealed by molecular genetics methods, such as FISH or qualitative RT-PCR^(17-19). These laboratory options are more selective and important not only for the diagnosis and estimation of the short- and long-term therapy outcomes, but also for the differential diagnosis with other chronic myeloid neoplasms. A remarkable aspect of molecular diagnosis of CML has been recently spotlighted, as RT-PCR detects BCR::ABL1 chimerism in 25-30% of allegedly healthy population⁽²⁰⁾. It is supposed that BCR::ABL1 may not serve as a sole genetic impairment, which triggers the development of CML. It is worth emphasizing that in 10% of cases relapses develop within the first five years of treatment and follow-up, including 10% of subjects in whom a complete cytogenetic response was achieved⁽²¹⁾. Taking into account the disclosed challenging diagnosis and management issues, the diagnostic and tracking significance of LDH and ALP levels, as well as of T315I mutation of the BCR::ABL1 transcripts continues to serve as an actual and deep concern for the ongoing research, especially in relapses and resistant cases, with the intentions to upgrade therapeutic tactics.

Objective

This research aimed to assess and update the biochemical and molecular-genetic profile of chronic phase (CPh) of CML at diagnosis and during the follow-up.

Materials and method

Our analytical and cohort study included 193 prospective and retrospective patients with CPh-CML treated and monitored within the period of 2016-2025 in the Hematology Division of the Oncological Institute from Chișinău, Republic of Moldova. The control group comprised CML cases with early CPh. The inclusion criteria were: age over 18 years old, morphological ± cytogenetics ± molecular genetics confirmation of CML diagnosis, patient’s consent and adherence for participation in the research, and the opportunity of dynamic follow-up. The enrolled study participants underwent complex examinations: clinical, laboratory and imaging investigations in order to appraise the CML phase and the response to medication.

During the karyotype examination of each patient, at least 20 metaphases of the bone marrow cells obtained in direct and non-stimulated cultures were analyzed. Molecular-cytogenetic investigations were fulfilled by FISH test on the bone marrow specimens^(28,29). The abnormal mixed signal, detected in nuclei, indicated the presence of t(9;22)(q34;q11)-bearing cells. Quantitative RT-PCR was applied to evaluate the expression of BCR::ABL1 p210 and p190 transcripts^(28-32,47). Peripheral blood samples were collected for biochemical tests (ALP, LDH) and molecular-genetic determination of T315I mutation of the BCR::ABL1 gene, with the intention of appreciating the diagnostic and prognostic significance of these biochemical parameters and molecular aberration.

Bibliographic references to the scientific topic and discussion were searched in the international medical databases, including Google Search, PubMed, NCBI, Medscape, by keywords: “chronic myeloid leukemia”, “diagnosis”, “alkaline phosphatase”, “lactate dehydrogenase”, “BCR::ABL1 transcripts”, “mutations”, “relapse”, “survival”.

The collected data were processed by medical statistician, using Microsoft Excel, Anova, Epi Info 7.2 and IBM SPSS Statistics version 26.0. The research protocol and the study design, the patient informed consent and the confidentiality commitment forms along with the information sheet were validated by the Ethics Committee and Scientific Council of the “Nicolae Testemițanu” State University of Medicine and Pharmacy, Chișinău. The research was realized at the state comprehensive cancer infrastructure – Institute of Oncology, within the frame of Postdoctoral Programs under the auspice of the National Agency for Research and Development from the Republic of Moldova. The study project was performed with the support of Fundeni Clinical Institute, Bucharest, and the “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.

Results

The studied cohort included 193 CPh-CML patients, with the age range between 21 and 82 years old. The average age was 47.5 years old (m=1.02; F=0.251). The age ranged between 25 and 60 years old in the majority of CML patients (79.4%) included in the research, which assumed the current references data on the prevalent exposure of the workable population^(10,32-34). Over the last decades, the basic quantitative hematological indicators showed to be phase-related in CML. Nevertheless, the transformative changes of biochemical parameters are not comprehensively studied, and may serve as an additional diagnosis and prognosis indicators, especially in cases of CML progression or relapse. Our study revealed a phase-related trend of the increase of uric acid (UA), ALP and LDH values (Tables 1 and 2). In the overwhelming majority of CML cases (151; 78.2%), the elevated levels of LDH were found in the venous blood samples, while the ALP levels increased in 48 cases (30%).

The statistically significant increase was attributed to ALP (p<0.05) and LDH (p<0.01) values, which rose noteworthy up to 1516 U/L and 2509 U/L in the late CPh, correspondingly. An expanding trend was identified in the rate of patients with elevated levels of UA (p>0.05) and ALP (p<0.05) as CML progressed into the late CPh (27.1% and 30%, respectively). Meanwhile, a significant increase of the rate of patients with elevated LDH levels (p<0.001) was spotlighted during the transformation of CML from the early CPh (60%) into the late ChP (78.2%). It can be summarized that the LDH and ALP levels demonstrate the activity of CML, and should be weighed for phase assessment at diagnosis confirmation and follow-up during the treatment.

The expression range of the BCR::ABL1 p210 transcript was 5-100% IS (Table 3) in the whole cohort. In most cases (approximately 72%), the expression of BCR::ABL1 transcripts exceeded 65%. Thus, these expression values indicated an extended leukemic myeloproliferation. The percentage rate of BCR::ABL1 fusion gene expression below 25% was recorded only in 11.3% of patients, being commonly unveiled in cases with the early CPh (94.1%).

The increasing dynamics (p>0.05) of the percentage of the BCR::ABL1 transcripts-bearing cells was observed during the clinical-hematological transformation of CML from the early CPh (M=47.06%) to the late chronic phase (M=60.78%), which asserted a versatile diagnostic value of this molecular-genetic indicator for tracking disease phases. Therefore, the increase of the expression values of the BCR::ABL1 fusion gene matches the CML progression and, thus, remain more sensitive diagnostic indicators in relation to the percentage of metaphases with the t(9;22) translocation. Positive correlations were established between LDH values and BCR::ABL1 transcripts expression (r=0.72; p<0.05). Of scientific and practical interest is the study of the rates of BCR::ABL1 gene transcripts in patients with CML depending on the phase of the disease (Table 4). The b3a2 transcript proved to be the most frequent at diagnosis in early CPh (five cases; 50%), as well as in the late CPh (65 cases; 53.3%) (X2=27.08; p<0.001). The b3a3 transcript was identified only in one (12.5%) case with early CPh (X2=34.38; p<0.001). The e1a2 transcript was revealed in only 12 (9.2%) patients with late CPh, which can assert it as the factor accelerating the disease progression.

The global survival (GS) indices were significantly higher in the group of CPh patients (Figure 1) with LDH levels ≤414 U/L, being: over one year – 100%; over five years – 93.7%; and over 10 years – 84.4% (M=221.090 months; 95% Confidence Interval; 187.788-254.392). The GS indices were found to be the lowest in the patients groups with LDH 1001-1250 U/L (over one year – 100%; over five years – 55.6%; and over 10 years – 0%; M=110.977 months; 95% Confidence Interval; 79.915-142,039) and ≥1250 U/L levels (over one year – 96.6%; over five years – 65.5%; and over 10 years – 38.3%; M=70.500 months; 95% Confidence Interval; 44.464-96.536).

Our study searched for the novel impact factors which might influence the disease-free survival (DFS) in CPh-CML. The DFS indices turned out to be higher in the group of patients (Figure 2) with ALP levels ≤270 U/L, being: over one year – 91.3%; over five years – 81.6%; and over 10 years – 75% (M=167.507 months; 95% Confidence Interval; 148.185-186.828). The DFS indices proved to be lower in the patient group with ALP levels >270 U/L (over one year – 84%; over five years – 62.9%; and over 10 years – 62.9%; M=114.618 months; 95% Confidence Interval; 84.403-144.834). The DFS indices proved to be superior in the group of patients (Figure 3) with LDH levels ≤414 U/L (over 36 months – 95.2%; over 60 months – 90.2%; over 120 months – 84.9%; M=187.934 months; m=10.729; 95% Confidence Interval; 166.905-208.963). The DFS indices proved to be lower in the patient group with LDH levels >1000 U/L (over 36 months – 71.4%; over 60 months – 47.62%; over 120 months – not achieved; M=58.648 months; m=10.310; 95% Confidence Interval; 27.738-89.558; Mdn=42.500 months).

Thirty-six TKIs-resistant and relapsed patients included 21 males (58.3%) and 15 females (41.7%). The diagnosis of CML was established in the early CPh in two cases (5.6%), in the late CPh in 32 cases (88.8%), and in the accelerated and acute phases in two cases (5.6%). Quantitative RT-PCR revealed broad ranges of the BCR::ABL1 p210 transcript expression (23.4-100%), with the average value of 63.4±2.89%. Nevertheless, the lowest BCR::ABL1 p210 transcript value proved to be higher than the lowest BCR::ABL1 p210 transcript value in the whole cohort of CML patients. The leukocyte count ranged between 21.9 and 358x10⁹/L (average: 154.6x10⁹/L). The leukocyte count was ≤100x10⁹/L in 14 cases (38.9%), between 100x10⁹/L and 200x10⁹/L in seven cases (19.4%), between 200x10⁹/L and 300x10⁹/L in 12 cases (33.3%), and exceeded 300x10⁹/L in three cases (8.4%). No correlation was demonstrated between BCR::ABL1 p210 transcript expression and white blood cell elevation degree. The thrombocyte count varied between 46x10⁹/L and 1313x10⁹/L (average: 364.9x10⁹/L), and was increased in 14 cases (38.89%). LDH values at diagnosis ranged from 169 to 1960 U/L (M=630.6 U/L). LDH raised in 29 subjects (80.6%). Upper LDH values occurred in 16 cases (47.1%), with the highest leukocytes number (≥150x10⁹/L). ALP values in blood ranged between 68 and 398 U/L (M=221.6 U/L). ALP content was uplifted in 25 subjects (86.2%). The superior ALP values were noticed in 18 cases (72%), with white blood cell count over 100x10⁹/L and shifted between 176 and 398 U/L (M=249 U/L; p<0.05). The discovered findings may signify that the LDH and ALP concentration rises with the expansion of circulating leukocytes pool, thus certifying the progressive CML course. T315I mutation occurred in 14 cases (38.9%). In these subjects, the LDH concentration accounted 275-1960 U/L, being higher (M=692.4 U/L) as compared to CML cases lacking such mutation (M=565.7 U/L; range: 169-1609.4 U/L).

The increased ALP concentration emerged in 13 subjects (92.8%) with T315I mutation. Hence, the prognostic correlation is revealed by the raised LDH and ALP concentration, and the evidence of T315I mutation. Relapses emerged in 18 cases (62.1%) with initially elevated LDH levels and in nine (64.3%) of 14 cases with T315I mutation. The overall survivor of subjects without resistance mutations varied between 73 and 298.4 months (median value: 159.7±3.42). The overall survivor of subjects with resistance mutations shifted from 54.7 to 289.3 months (median value: 151.5±4.21). The lack of a significant survivor difference amongst these two studied subcohorts with and without resistance mutations was due to response to the newer line TKIs treatment of refractory patients.

Discussion

In 2020, acute and chronic leukemias were estimated to be the 15^th and the 11^th leading cause of cancer-related morbidity and mortality worldwide, accounting for 474,519 new cases and for 311,594 deaths⁽³⁵⁾. The CML burden tendencies released by the Global Burden Disease (GBD) study provided essential data for promoting health services and public health. The GBD study showed that the burden of CML varied significantly across countries due to different laboratory opportunities for early detection and monitoring, the availability of new antineoplastic agents, and to medical resources⁽³⁷⁾. Therefore, the conventional PCR testing is considered a complex diagnostic option and involves intensive skilled personnel efforts, as well as a lot of reagents and high-priced laboratory tools. Quality supervision is crucial to achieve and preserve the selectivity and preciseness of the test, which demands technical expertise background and methodological training. At the beginning of the program, BCR::ABL1 testing was performed on the expanded basis by transporting blood to the outside diagnostic centers, which was a costly, time-consuming and unapproachable process.

According to the recent communications, the estimated PCR tests split in monitoring capacity was $29,143,083 over the five-year period across all regions covered by the access initiatives⁽³⁸⁾. Due to the combination of country- and patient-specific trends, the estimated CML patients population increased in these regions from 26,915 to 30,071 patients by the end of 2021 owing to the improved access to PCR testing. Taking into account the important repercussion of point-of-care systems in patients diagnosing and monitoring in resource-poor areas, the Max Foundation established a cooperation with Cepheid to enhance the availability of the PCR equipment and testing in over 60 states with low- and middle-income economies. The BCR::ABL1 assay was developed in collaboration with universities in the USA, and it is currently used in many medical and research centers in the USA and Europe for the surveillance of patients with CML in clinical trials and in the community, being FDA-approved in the USA. The system is widely applicable in low- and middle-income countries for infectious pathologies (tuberculosis, AIDS etc.), and it is increasingly used in a number of oncological applications, including BCR::ABL1^(38,39). The Cepheid platform has many valuable attributes, especially for the states with low- and middle-income economies: simplicity and cost-efficiency of usage (requiring less technical training, with flexible use between batches or individual assessments), optimal transporting solutions (cartridges rather than multiple test solution reservoirs), enhanced replicability and precision in results reporting.

With the widespread practical implementation of various commercially available TKIs and the increasing prevalence of CML, more patients develop drug refractoriness⁽¹¹⁾. Substantial boosts in molecular-genetic and biochemical surveillance can facilitate the opportune identification of CML resistance and relapses, so that TKIs resistance eradication means could be started within the rational timelines. TKIs progressively inhibit the ATP binding locus of the BCR::ABL1 protein, making it efficient for BCR::ABL signal transduction, which results in losing the downstream pathways in BCR::ABL transduction activity⁽¹⁾. Imatinib mesylate was produced as a common medication for CML due to its higher activity and lower adverse events rates. Nevertheless, it is predicted that roughly 30% of patients will finally develop refractoriness to treatment.

In screening searching for dynamic prognostic determinants in malignant lymphomas and acute leukemias, LDH proved to be a suitable observational indicator. The association between LDH levels and 30-day mortality was analyzed in 238 patients with acute and chronic leukemias and intracranial hemorrhage during the induction phase⁽²²⁾. The overall 30-day mortality rate was 61.5%, with 56 of the 91 patients succumbing. Among those with LDH levels ≥570 U/L, the mortality rate was 74.4%, which was higher than the 50% mortality rate of the LDH

Nevertheless, the diagnostic and prognostic relevance of LDH in CML remains not widely studied and seldom narrated in academic research tools and resources. Furthermore, the molecular-genetic test for BCR::ABL1 transcripts is expensive, and may be difficult to access in countries with scanty financial resources and socio-economic disparities⁽⁴⁰⁾. These constraints indicate the need of examination of the other cost-effective and applicable diagnostic and prognostic biological markers. A pilot study analyzed 22 CML patients and 56 healthy subjects⁽²⁰⁾. LDH concentration in plasma was higher in patients with CML. All CML patients underwent the treatment, but, even so, four patients exhibited the b3a2 transcript in the venous blood samples. Two out of the four patients with b3a2 showed higher LDH (486 U/L and 589 U/L). Thus, even though the study was realized in the less numerous cohorts, it was possible to adjust therapy for two patients with b3a2 transcript in the blood specimens, whose LDH concentration was high, in order to favor the disease outcomes. It was suggested that LDH>330 U/L was an important adverse risk factor for survival, and should be included in future studies of risk performed on larger patient groups. Regarding the laboratory findings, the BCR::ABL1 oncoproteins were revealed in the venous blood samples in CML patients who were enrolled for antineoplastic medication and exhibited superior LDH levels⁽²⁰⁾.

Another widely cited research on CML enrolled 46 (56.8%) patients from the medically treated group and the remaining 35 (43.2%) treatment-naive patients⁽⁴⁰⁾. As in our study, considerable positive correlations were observed between LDH and BCR::ABL1 transcripts expression (r=0.79; p<0.001). The correlation coefficient value of UA (r=0.295; p<0.008) with BCR::ABL1 transcripts showed a weak correlation between these two biological parameters. There was a statistically relevant (p<0.05) difference in the median level of BCR::ABL1 expression and LDH among patients in the treatment group (median=21%, 350 U/L) and the treatment-naive cohort (median=57%; 1246 U/L), respectively. The area under the curve for LDH, basophil count and leukocyte count were 0.881, 0.889 and 0.748, respectively, which demonstrated better performance for surveillance of CML patients than UA (0.695) and thrombocytes count (0.70). These findings can serve as an alternative fitting option for confirming the capability of LDH as an additional widely-applicable cost-efficient diagnostic, prognostic biomarker, and a new therapeutic target in CML⁽⁴⁰⁾.

Resistance and intolerance to ATP-competitive TKIs have been described in about 25% of cases⁽⁴¹⁾. A common mechanism of resistance is reported to involve point mutations in the BCR::ABL1 kinase domain, which impairs the activity of available TKIs. Second-generation TKIs overcome most of the mutations that confer resistance to imatinib, although new mutations have emerged that render leukemia resistant to second-generation TKIs^(11,42). A major T315I mutation, known as the “gatekeeper”, loans resistance to most available TKIs, except for ponatinib. Before defining a patient as having TKIs resistance and switching therapy, the compliance and drug-drug interactions are required to be analyzed. Adherence and compliance rates to imatinib were estimated to range from 75% to 90%, and lower adherence rates correlated with inferior results^(11,42).

Among the variety of resistance mechanisms that may underpin a nonoptimal response to TKIs therapy in CML, secondary point mutations in the BCR::ABL1 kinase domain and T315I represent those actionable ones. T315I mutations occur in around 5% of patients treated with second-generation TKIs and confer resistance by restoring or increasing ABL1 kinase activity. BCR::ABL1 mutations testing asserts clinical decision-making whenever response is unsatisfactory. In case of response failure, a change of therapy is mandatory and the main aim of BCR::ABL1 kinase domain and T315I mutations testing is to help exclude the TKIs unlikely to be effective⁽⁴³⁾. T315I-bearing CML patients have an unfavorable prognosis. Long-term outcome depends on phase of disease when T315I mutation occurs, with five-year global survival estimated at 70% in CPh and 10% in acute phase^(43,44). Patients with CPh-CML treated with ponatinib and/or asciminib had a five-year global survival of 77% compared with 50% in those who received other treatments (conventional chemotherapy, second-generation TKIs, homoharringtonine and investigational drugs) (p=0.14)⁽⁴⁴⁾. Consequently, the data obtained in our study proved to be consistent with the above-mentioned reports. Two patients (5.7%) with elevated LDH at diagnosis and T315I mutations progressed to the acute phase, achieved only a short partial hematological response after the treatment with ponatinib and asciminib, and passed away because of the uncurable complications.

According to the recently published data from high-throughput sequencing studies, CPh-CML is a genetically heterogeneous leukemia, although it shows less heterogeneity compared with acute leukemia at diagnosis^(45,46). Non-silent mutations affecting cancer-related genes have been identified in approximately 35% of patients with CPh-CML (range: 29-50%), either by targeted sequencing or whole-exon sequencing. In the aforementioned studies, ASXL1 was the most frequently mutated gene in approximately 10% of CPh-CML patients. Other frequently mutated genes included mutations and deletions in IKZF1 (4%), RUNX1 (2%), TET2 (2%) and DNMT3A (2%). Typical AML-related mutations have rarely been identified in chronic phase CML, including IDH1/2, FLT3, EZH2 and NRAS mutations. In cases with atypical transcripts e1a2, e8a2 or e19a2, the reliable monitoring by routine RT-qPCR is not achievable, therefore an assessment of the individual molecular response with specific RT-qPCR primers is recommended^(41,47).

Provable refractoriness mutations may extend, or at least maintain, if the treatment is not upgraded. These mutations constitute presumably a marker of the degree of genetic instability. Thus, irrespective of their actual role in resistance, positivity for any mutation can indicate higher-risk patients demanding more cautious and frequent surveillance⁽³⁷⁾. If a mutant clone is not promptly eliminated, and if inefficiently inhibited BCR::ABL1 transcripts are preserved at increased levels, further acquisition of mutations, emerging into compound mutations, can develop⁽³⁷⁾. In cases of uncertain clinical relevance of a mutation (regardless of either due to the lack of sensitivity data or to a low mutational burden), closer follow-up of BCR::ABL1 transcripts expression may be useful in deciding whether therapy should be switched. Mutations data from trials and the “real-world” clinical experience with asciminib remain still reserved, which require the forthcoming investigations for the characterization of the full spectrum of asciminib refractory mutations.

Conclusions

The expression of the BCR::ABL1 transcripts exhibits elevation during the CML recurrence or transformation in the advanced phases, and remains a more sensitive biological marker as compared to the percentage of metaphases with Ph-chromosome. The increased ALP and LDH values demonstrate the activity of CML, and may be used for asserting the phase at diagnosis confirmation and monitoring under treatment. Positive correlations were ascertained between BCR::ABL1 transcripts expression and LDH values. Maintenance and endorsement of molecular-genetic and biochemical surveillance may thus facilitate the opportune identification of CML resistance and relapses, so that TKIs resistance overcoming management could be started within the rational timelines. The general survival proved to be higher in CPh-CML patients with lower LDH values at diagnosis and, thus, certified LDH value as a promising prognostic factor. Disease-free survival was also higher in CPh-CML patients with lower ALP and LDH levels at diagnosis.

Conflict of interests. None of the authors has any financial or personal conflicts of interests to disclose.

Informed consent statement. The informed consent was obtained from the patients enrolled in this research.

Ethical considerations. The authors declare that all the methods and procedures of this research are realized according to the ethical standards of the revised Helsinki Declaration 2024 and match the national laws.

Authors contributions. Vasile Musteață – conceptualization, writing – original draft, writing – review and editing, project administration, methodology, investigations, follow-up; Valentina Stratan – methodology, investigations, data supervision and validation, draft revie; Daniel Coriu – data validation, draft review.

Corresponding author: Vasile Musteață E-mail: vasile.musteata@usmf.md

Conflict of interests: none declared.

Financial support: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.