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Clinical Characteristics, Disease Evolution, and Survival in Patients with Chronic Myeloid Leukemia, BCR-ABL1 (+), and T315I Mutation

Introduction: The T315I mutation in patients with chronic myeloid leukemia (CML) has been associated with therapeutic resistance and an unfavourable prognosis. Aim: To study the frequency of T315I mutation in patients with CML, BCR-ABL (+), their clinical characteristics, disease evolution, and median survival. Patients and methods: We studied 75 patients with CML and BCR-ABL1 (+). T315I mutation was detected by digital droplet PCR and BCR-ABL1 was analyzed by RT-PCR. A comparative analysis was performed by sex, age, disease phase, risk group, treatment, molecular response (MR), and median survival in T315I (+) and T315I (−) patients. Results: T315I mutation was detected in 11 patients (14.7%). No significant difference was found in the phase, risk group, and firstline therapy. A significantly higher proportion of T315I (+) did not achieve MR >3.5 log: 8 (72.7%) vs. 22 (34.4%) (p=0.023). The lowest mean BCR-ABL1 levels were significantly higher in the CML T315I (+) group compared to the CML T315I (−) group: 12.1±6.0 vs. 3.77±1.28 (p=0.009). The median survival of T315I (+) patients was significantly shorter: 73 months vs. 175 months (p<0.0001, CI 95%). Conclusions: Our data confirm the world experience on the frequency of T315I mutation, including the unfavourable evolution, resistance to TKI treatment and short survival. ddPCR is a highly sensitive method for early detection of genetic mutations which gives the chance for effective treatment.


INTRODUCTION
The discovery of the Philadelphia chromosome in patients with chronic myeloid leukemia (CML) is a fundamental achievement in medical science. As a result of the successful targeted inhibition of its fusion protein, the mutant tyrosine kinase BCR-ABL1, today patients with CML have an expected survival comparable to that of people without CML in the same age group. However, as a result of additional mutations in the kinase domain of the BCR-ABL1 gene, resistance to the effect of tyrosine kinase inhibitors (TKI) occurs. 1,2 The T315I mutation in the BCR-ABL1 gene affects the common ABL kinase binding site and results in high-grade cross-resistance to TKIs. Currently, five TKIs are used in clinical practice, with different drugs covering a different spectrum of genetic mutations. The T315I mutation is one of the most difficult to treat. 3 It is associated with resistance to treatment, failure or early loss of molecular response (MR), progression to the acceleration phase and blast crisis, and shortened survival. 4 Currently, RTq-PCR, Sanger sequencing, and next-generation sequencing are the golden standard for monitoring of MR and mutation analysis in patients with CML. 5 In recent years, emulsion digital PCR (ddPCR) has become increasingly important as a highly informative, fast and cost-effective methodology. 6

AIM
To investigate the frequency of T315I mutation detected by ddPCR in patients with CMR BCR-ABL1 (+), to analyze their clinical features, disease evolution, and median survival, and to compare those in CML patients without the T315I mutation.

PATIENTS AND METHODS
We studied 75 patients with CML, BCR-ABL1 (+), who did not achieve an optimal therapeutic response within the defined timeframes, or with a loss of the achieved molecular response. Patients were diagnosed, treated and monitored between March 2000 and December 2018 in the Clinic of Clinical Hematology and Clinic of Medical Oncology, St George University Hospital, Medical University of Plovdiv. The detection and quantitative measurement of the T315I mutation was performed by ddPCR using the QX200 Droplet Digital PCR system in the Department of Medical Genetics, Medical University of Plovdiv under the PERI-MED project. Digital droplet PCR is based on the division of a standard PCR reaction into tens of thousands of nano-droplets that contain one (or more) or do not contain the target sequence. Amplification is performed in each droplet and the number of the sequences is calculated from the proportion of positive and negative droplets using the Poisson distribution. 7 Molecular genetic tests for detection and quantitative measurement of fusion transcripts BCR-ABL1 were performed by RT-PCR in the Specialized Hospital for Active Treatment of Hematological Diseases in Sofia and presented on an international scale, according to the criteria of European Leukemia Net. Table 1 presents the characteristics of the studied contingent of patients at the time of diagnosis. The male to female ratio was 1.2/1.0, the mean age was 54.64±14.4 years. In patients with T315I mutation (CML T315I +), a comparative analysis was performed by sex, age, disease phase, risk group, treatment, type, and duration of molecular response and median survival calculated from the time of diagnosis and compared to patients with CML without T315I mutation (CML T315I −). Statistical data processing was performed with descriptive analysis, correlation analysis and the Independent Sample t-test for comparison of mean values, at a level of significance p<0.05. Survival analysis was performed by the Kaplan-Meier method with a log-rank test. 8

Lines of treatment
Therapeutic choice for first-, second-and ≥ third-line therapy did not differ significantly in the patient groups CML T315I (−) and CML T315I (+), presented in Table 3. Imatinib was most often chosen for a first-line therapy in both groups while nilotinib was the most common therapeutic choice for second line. In patients with CML T315I (+), the mean number of treatment lines was 3.2 compared to 1.1 in the CML T315I (−), (NS). The duration of first-line treatment was significantly shorter in patients with CML T315I (+) mutation compared to the CML T315I (−) group: 13 months (95% CI 3.424-19.710) versus 33 months (95% CI 16.184-76.657) (p=0.001) (Fig. 1).

Depth and duration of the molecular response
Significantly higher proportion of patients with CML T315I (+) did not achieve an optimal molecular res- Table 3. Lines of therapy in patients CML T315 I (+) and CML T315 I (-)

DISCUSSION
In the literature, the frequency of detection of T315I mutation varies in a wide range of 2%-20% depending on the characteristics of the studied contingent and the method of analysis used. In the study of Shi DY et al. on 1093 patients, the T315I mutation was the most common mutation found in imatinib-, nilotinib-, and dasatinib-resistant patients (12.3%, 27.3%, and 34.1%, respectively). 9 Large epidemiological studies on the frequency of T315I mutation confirm that it is the most common mutation in patients with CML. Different authors focus on different aspects of the manifestation of this mutation, but unequivocally prove the T315I mutation is associated with a poor prognosis. 10,11 Nicolini et al. found short overall survival in patients with T315I mutation depending on the phase of the disease: 22.4 months, 28.4 months, 4.0 months for chronic phase, acceleration and blast crisis and progression-free survival of 11.5, 22.2, and 1.8, respectively (calculated from the time of mutation detection). 12 In the study by Shy et al., the detection of a T315I mutation was analyzed in relation to the type of drug administered and was more commonly demonstrated in second-generation TKIs. In addition, according to the same authors, it is more common in men, younger age, lack of concomitant diseases and advanced stage of disease. 9 The analysis of the results of our study did not find a statistical difference between patients with T315I (+) and T315I (−) according to the baseline characteristics of the disease at diagnosisphase and risk group, demographics, sex and age, and the comorbidity profile of patients. Many authors, as well as our analysis, observed adverse clinical evolution, resistance to treatment and short overall survival, reasonably in the course of disease progression. [13][14][15] On the other hand, a large number of researchers hypothesize that if the mutation was detected earlier with the highly sensitive Sanger sequencing, the next generation sequencing or ddPCR, which are able to detect very low expression levels 16,17 , this would be a prerequisite for early change of treatment strategy to the only effective TKI for these patients -ponatinib and better therapeutic results will be expected 18 .
In our cohort of patients harbouring T315I mutation, the switch to ponatinib occurred only recently and the results are not ready to be reported. The analysis of the T315I mutation samples in our study was performed by ddPCR. The method is much cheaper and faster in the search for a particular mutation, but its most significant advantage is its much higher sensitivity (0.01%), which is significantly higher than that of Sanger sequencing (20%) and the next generation sequencing (1-3%). 5,6

CONCLUSIONS
Our results confirm the data from the accumulated world experience on the frequency of T315I mutation, as well as the unfavourable evolution, resistance to TKI treatment, and short survival in patients with CML T315I (+). ddP-CR is a highly sensitive and informative method for early detection of genetic mutations, even at low levels of expression. Timely detection of the T315I mutation, immediately after failure of first-line TKI therapy in CML patients, provides a chance to choose effective treatment, improve their prognosis, and long-term survival.

Author contributions
V.G-M. developed the concept, designed the study, took part in collecting data and samples, and wrote the manuscript. H.I., A.L., I.Zh., and V.S. took part in designing the study and performed the genetic tests for T315I mutation. Zh.G-P. and all co-authors critically revised the manuscript.