Retrospective analysis of own long-term experience in studying the BCR::ABL kinase domain mutational status in patients with chronic myeloid leukemia

Background. Most patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors achieve durable optimal responses. Loss of the achieved molecular response is observed in 15–30 % of patients. Mutations in the BCR::ABL kinase domain are one of the most common mechanisms for the development of resistance to tyrosine kinase inhibitors.

Aim. To conduct a retrospective analysis of the BCR::ABL kinase domain mutational profile in patients with CML observed at the Russian Research Institute of Hematology and Transfusiology from 2012 to 2023. To assess the impact of mutations type and number on the rate of achieving a major molecular response (MMR). To study the risk of MMR loss depending on the therapy line and existing mutational status.

Materials and methods. 1831 patients with CML were examined at different times. The mutational status of the BCR::ABL kinase domain was analyzed by direct Sanger sequencing. A standard cytogenetic study was carried out using GTG banding technology with the analysis of at least 20 metaphase plates.

Results. Mutations in the BCR::ABL kinase domain were identified in 27.6 % of the total studied patients. The most common mutation, 6.3 % in the overall group or 22.7 % among patients with mutations, was the T315I mutation. Additional chromosomal aberrations (ACAs) were detected in Ph-positive cells in 20.5 % of patients, in Ph-negative clones in 3.9 % of cases (p = 0.0001). The frequency of ACAs detection did not statistically significantly differ (p = 0.25) between patients with BCR::ABL mutations (23.5 %) and with a negative mutation status (17.7 %), and the presence of mutations in the kinase domain did not correlate with ACAs in Ph-positive clones (p = 0.73). However, the frequency of T315I mutation detection in Ph-positive cells had significant differences: 40.9 % in combination with ACAs and 21 % without ACAs (p = 0.032). Patients with the T315I mutation had significantly worse MMR than patients with mutations in other BCR::ABL regions (p = 0.04) and patients without mutations (p = 0.02). The probability of MMR achieving did not differ significantly between patients with different numbers of BCR::ABL mutations (p = 0.14). Loss of MMR occurred more often in patients with mutations (p = 0.04) and not depend on the line of therapy (p = 0.03).

Conclusion. For complete monitoring and optimal choice of therapy, CML patients require not only monitoring of BCR::ABL relative expression level, but also standard cytogenetic and analysis of the mutational status.

1. Shuvaev V.A., Martynkevich I.S., Sidorkevich S.V. Myeloproliferative neoplasms. Moscow, 2023. 336 p. (In Russ.).

2. Flis S., Chojnacki T. Chronic myelogenous leukemia, a still unsolved problem: pitfalls and new therapeutic possibilities. Drug Des Devel Ther 2019;13:825–43. DOI: 10.2147/DDDT.S191303

3. Weerkamp F., Dekking E., Ng Y.Y. et al. Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients. Leukemia 2009;23(6):1106–17. DOI: 10.1038/leu.2009.93

4. McWhirter J.R., Wang J.Y. An actin-binding function contributes to transformation by the Bcr-Abl oncoprotein of Philadelphia chromosome-positive human leukemias. EMBO J 1993;12(4): 1533–46. DOI: 10.1002/j.1460-2075.1993.tb05797.x

5. Peiris M.N., Li F., Donoghue D.J. BCR: a promiscuous fusion partner in hematopoietic disorders. Oncotarget 2019;10(28): 2738–54. DOI: 10.18632/oncotarget.26837

6. Quintás-Cardama A., Cortes J. Molecular biology of bcr-abl1positive chronic myeloid leukemia. Blood 2009;113(8):1619–30. DOI: 10.1182/blood-2008-03-144790

7. Vinhas R., Lourenço A., Santos S. et al. A novel BCR-ABL1 mutation in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia. Onco Targets Ther 2018;11:8589–98. DOI: 10.2147/OTT.S177019

8. Hochhaus A., Baccarani M., Silver R.T. et al. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia 2020;34(4):966–84. DOI: 10.1038/s41375-020-0776-2

9. Frazer R., Irvine A.E., McMullin M.F. Chronic myeloid leukaemia in the 21st century. Ulster Med J 2007;76(1):8–17.

10. Lomaia E.G., Konopleva M.Yu., Romanova E.G., Zaritzkiy A.Yu. Chronic myeloid leukemia – before and after imatinib (Third part). Onkogematologiya = Oncohematology 2010;(1):5–20. (In Russ.)

11. Zhuravlev A.V., Knysh O.I. Key principles of drug therapy in patients with chronic myeloid leukemia. Farmakoekonomika. Sovremennaya farmakoekonomika i farmakoepidemiologia = Farmakoekonomika. Modern Pharmacoeconomics and Pharmacoepidemiology 2023;16(2):332–44. (In Russ.). DOI: 10.17749/2070-4909/farmakoekonomika.2023.166

12. Rosti G., Castagnetti F., Gugliotta G., Baccarini M. Tyrosine kinase inhibitors in chronic myeloid leukaemia: which, when, for whom? Nat Rev Clin Oncol 2017;14(3):141–54. DOI: 10.1038/nrclinonc.2016.139.

13. Zabriskie M.S., Eide C.A., Tantravahi S.K. et al. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia. Cancer Cell 2014;26(3):428–42. DOI: 10.1016/j.ccr.2014.07.006

14. Wylie A., Schoepfer J., Jahnke W. et al. The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1. Nature 2017;543(7647):733–7. DOI: 10.1038/nature21702

15. Alves R., Gonçalves A.C., Rutella S. et al. Resistance to tyrosine kinase inhibitors in chronic myeloid leukemia – from molecular mechanisms to clinical relevance. Cancers 2021;13(19):4820. DOI: 10.3390/cancers13194820

16. Jabbour E., Kantarjian H. Chronic myeloid leukemia: 2020 update on diagnosis, therapy and monitoring. Am J Hematol 2020;95(6):691–709. DOI: 10.1002/ajh.25792

17. Cortes J., Lang F. Third-line therapy for chronic myeloid leukemia: current status and future directions. J Hematol Oncol 2021;14(1):44. DOI: 10.1186/s13045-021-01055-9

18. Poudel G., Tolland M.G., Hughes T.P., Pagani T.S. Mechanisms of resistance and implications for treatment strategies in chronic myeloid leukaemia. Cancers (Basel) 2022;14(14):3300. DOI: 10.3390/cancers14143300

19. Balabanov S., Braig M., Brümmendorf T.H. Current aspects in resistance against tyrosine kinase inhibitors in chronic myelogenous leukemia. Drug Discov Today Technol 2014;11:89–99. DOI: 10.1016/j.ddtec.2014.03.003

20. Patel A.B., O’Hare T., Deininger M.W. Mechanisms of resistance to ABL kinase inhibition in chronic myeloid leukemia and the development of next generation ABL kinase inhibitors. Hematol Oncol Clin North Am 2017;31(4):589–612. DOI: 10.1016/j.hoc.2017.04.007

21. Soverini S., Hochhaus A., Nicolini F.E. et al. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. Blood 2011;118(5):1208–15. DOI: 10.1182/blood-2010-12-326405

22. Soverini S., Colarossi S., Gnani A. et al. Contribution of ABL kinase domain mutations to imatinib resistance in different subsets of Philadelphia-positive patients: by the GIMEMA working party on chronic myeloid leukemia. Clin Cancer Res 2006;12(24):7374–9. DOI: 10.1158/1078-0432

23. Meenakshi Sundaram D.N., Jiang X., Brandwein J.M. et al. Current outlook on drug resistance in chronic myeloid leukemia (CML) and potential therapeutic options. Drug Discov Today 2019;24(7):1355–69. DOI: 10.1016/j.drudis.2019.05.007

24. Braun T.P., Eide C.A., Druker B.J. Response and resistance to BCR-ABL1-targeted therapies. Cancer Cell 2020;37(4):530–42. DOI: 10.1016/j.ccell.2020.03.006

25. Cang S., Liu D. P-loop mutations and novel therapeutic approaches for imatinib failures in chronic myeloid leukemia. J Hematol Oncol 2008;1:15. DOI: 10.1186/1756-8722-1-15

26. Shahrin N.H., Wadham C., Branford S. Defining higher-risk chronic myeloid leukemia: risk scores, genomic landscape, and prognostication. Curr Hematol Malig Rep 2022;17(6):171–80. DOI: 10.1007/s11899-022-00668-2

27. Liehr T. International system for human cytogenetic or cytogenomic nomenclature (ISCN): some thoughts. Cytogenet Genome Res 2021;161(5):223–4. DOI: 10.1159/000516654

28. Van Dongen J.J., Macintyre E., Gabert J. et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13(12):1901–28. DOI: 10.1038/sj.leu.2401592

29. Alikian M., Gerrard G., Subramanian P.G. et al. BCR-ABL1 kinase domain mutations: methodology and clinical evaluation. Am J Hematol 2012;87(3):298–304. DOI: 10.1002/ajh.22272

30. Branford S., Rudzki Z., Walsh S. et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003;102(1):276–83. DOI: 10.1182/blood-2002-09-2896

31. Soverini S., Martinelli G., Rosti G. et al. ABL mutations in late chronic phase chronic myeloid leukemia patients with up-front cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival: a study by the GIMEMA Working Party on Chronic Myeloid Leukemia. J Clin Oncol 2005;23(18):4100–9. DOI: 10.1200/JCO.2005.05.531

32. Jabbour E., Kantarjian H., Jones D. et al. Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate. Leukemia 2006;20(10):1767–73. DOI: 10.1038/sj.leu.2404318

33. Nicolini F.E., Corm S., Lê Q.H. et al. Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(phi)-LMC GROUP). Leukemia 2006;20(6):1061–6. DOI: 10.1038/sj.leu.2404236

34. Yohanan B., George B. Current management of chronic myeloid leukemia myeloid blast phase. Clin Med Insights Oncol 2022;16:11795549221139357. DOI: 10.1177/11795549221139357

35. Jabbour E., Morris V., Kantarjian H. et al. Characteristics and outcomes of patients with V299L BCR-ABL kinase domain mutation after therapy with tyrosine kinase inhibitors. Blood 2012;120(16):3382–3. DOI: 10.1182/blood-2012-04-424192

36. Eide C.A., Zabriskie M.S., Savage Stevens S.L. et al. Combining the allosteric inhibitor asciminib with ponatinib suppresses emergence of and restores efficacy against highly resistant BCR-ABL1 mutants. Cancer Cell 2019;36(4):431–43. DOI: 10.1016/j.ccell.2019.08.004

37. Khorashad J.S., Kelley T.W., Szankasi P. et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood 2013;121(3):489–98. DOI: 10.1182/blood-2012-05-431379

38. Shah N.P., Skaggs B.J., Branford S. et al. Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency. J Clin Invest 2007;117(9):2562–9. DOI: 10.1172/JCI30890

39. Yi J.H., Lee G.W., Lee J.H. et al. Multicenter retrospective analysis of patients with chronic lymphocytic leukemia in Korea. Blood Res 2021;56(4):243–51. DOI: 10.5045/br.2021.2021102

40. WHO classification of tumours of haematopoietic and lymphoid tissues. Eds.: S.H. Swerdlow, E. Campo, N.L. Harris et al. 4th edn. Vol. 2. Lyon: IARC Press, 2017.

41. Gorusu M., Benn P., Li Z., Fang M. On the genesis and prognosis of variant translocations in chronic myeloid leukemia. Cancer Genet Cytogenet 2007;173(2):97–106. DOI: 10.1016/j.cancergencyto.2006.10.006

42. Stagno F., Vigneri P., Del Fabro V. et al. Influence of complex variant chromosomal translocations in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Acta Oncol 2010;49(4):506–8. DOI: 10.3109/02841861003660031

43. Marzocchi G., Castagnetti F., Luatti S. et al. Variant Philadelphia translocations: molecular-cytogenetic characterization and prognostic influence on frontline imatinib therapy, a GIMEMA Working Party on CML analysis. Blood 2011;117(25):6793–800. DOI: 10.1182/blood-2011-01-328294

44. Aydin C., Cetin Z., Salim O. et al. Previously unreported chromosomal aberrations of t(3;3)(q29;q23), t(4;11)(q21;q23), and t(11;18)(q10;q10) in a patient with accelerated phase Ph+ CML. Case Rep Genet 2014;2014:582016. DOI: 10.1155/2014/582016

45. Fominykh M.S., Shukhov O.A., Shuvaev V.A. et al. Clinical significance of combined detection of additional chromosomal aberrations in Ph-positive cells and mutations of the BCR-ABL gene in patients with chronic myeloluciasis during therapy with tyrosine kinase inhibitors. Vestnik gematologii = Bulletin of Hematology 2017;8(2):81–2. (In Russ.).

46. Issa G.C., Kantarjian H.M., Gonzalez G.N. et al. Clonal chromosomal abnormalities appearing in Philadelphia chromosome-negative metaphases during CML treatment. Blood 2017;130(19):2084–91. DOI: 10.1182/blood-2017-07-792143

47. Kovitz C., Kantarjian H., Garcia-Manero G. et al. Myelodysplastic syndromes and acute leukemia developing after imatinib mesylate therapy for chronic myeloid leukemia. Blood 2006;108(8):2811–3. DOI: 10.1182/blood-2006-04-017400

48. Jabbour E., Kantarjian H.M., Abruzzo L.V. et al. Chromosomal abnormalities in Philadelphia chromosome negative metaphases appearing during imatinib mesylate therapy in patients with newly diagnosed chronic myeloid leukemia in chronic phase. Blood 2007;110(8):2991–5. DOI: 10.1182/blood-2007-01-070045

49. Hu S., Chen D., Xu X. et al. Targeted next-generation sequencing identifies additional mutations other than BCR-ABL in chronic myeloid leukemia patients: a Chinese monocentric retrospective study. Cancers (Basel) 2022;14(23):5752. DOI: 10.3390/cancers14235752