PHD SUMMARY

Identificarea de noi markeri moleculari pentru predicţia răspunsului sau a recidivei în leucemia cronică mieloidă, la pacienţi trataţi cu inhibitori de tirozin kinază

 Identification of new molecular markers for response or relapse prediction in chronic myeloid leukemia, in patients treated with tyrosine-kinase inhibitors

First published: 24 noiembrie 2015

Editorial Group: MEDICHUB MEDIA

DOI: 10.26416/OnHe.33.4.2015.4346

Abstract

The article represents the doctoral dissertation of Dr. Ana Manuela Crisan. It has a general part, in which there is made an update of knowledge in the topic of chronic myeloid leukemia(CML) - from diagnosis criteria, risk group classification, describing lab techniques used for diagnosis and response monitoring, Tyrosine kinase inhibitors(TKI) resistance mechanisms , international trial results which established tyrosine kinase inhibitors as standard treatment in low risk CML, to the the role and importance of allogeneic bone marrow transplant in CML. In the special part (methodology and findings), the author developed the topic of new molecular markers used for response or relapse prediction in chronic myeloid leukemia, in patients treated with tyrosine-kinase inhibitors , such as : clinical evolution and TKI response in correlation to standard prognosis factors; evolution and survival of CML patients which presented cytogenetic abnormalities in Philadelphia positive and negative cells; evolution and survival of CML patients who benefit from Imatinib blood level testing; evolution and survival of CML patients who benefit from mutation analysis and evolution and survival of CML patients who received allogenic bone marrow transplant - all data from the patient group being correlated  to international literature.
 

Keywords
chronic myeloid leukemia, molecular markers, tyrosin kinase inhibitors

Rezumat

Lucrarea reprezintă teza de doctorat a Dr. Ana Manuela Crişan. Cuprinde o parte generală, în care sunt aduse actualizări ale cunoştinţelor în domeniul leucemiei mieloide cronice(LMC) - de la criterii de diagnostic, clasificare pe grupe de risc, tehnici de laborator folosite pentru diagnostic şi monitorizarea răspunsului la tratament, mecanisme de rezistenţă la tratamentul cu inhibitori de tirozin kinază (TKI), rezultate ale studiilor care au validat utilizarea TKI ca tratament standard în LMC cu risc redus până la rolul şi importanţa transplantului alogenic de măduvă hematogenă în LMC. În partea specială, autoarea a căutat să dezvolte principalele metode de diagnostic molecular cu rol predictiv pentru răspuns sau a rezistenţei la tratamentul cu TKI disponibile, cum ar fi: evoluţie clinică şi răspunsul la TKI în corelaţie cu factorii de prognostici clasici, evoluţia şi supravieţuirea pacienţilor cu LMC cu anomalii citogenetice în celule cu cromozom Philadelphia prezent sau absent, evoluţia şi supravieţuirea pacienţilor care beneficiază de dozarea nivelurilor serice de Imatinib, evolutia şi supravieţuirea pacienţilor care beneficiază de analiza prezenţei mutaţiilor şi evoluţia şi supravieţuirea pacienţilor care au primit transplant alogenic de măduvă - toate datele obţinute în cadrul lucrării, pe lotul de pacienţi, fiind corelate cu datele din literatura internaţională.
 

Introduction

Chronic myeloid leukemia (CML) is a clone neoplasia of pluripotent stem cell according to 2008 WHO classification of chronic myeloid neoplasms (MPN). In aproximatively 90% of cases, CML is due to reciprocal translocation between 9 and 22 chromosomes known as Philadelphia chromosome. The molecular equivalent of genetic material exchange between 9 and 22 chromosomes is BCR-ABL1 which leads to expansion of erithroid, myeloid and megakaryocytic populations and reduction of progenitor sensibility to haematopoiesis. The CML evolution without treatment could evolve in 3 phases: chronic phase (CP) with years duration, accelerated phase (AP) and blast (BP) crisis similar to acute leukemia but with unfavourable prognosis due to treatment resistance. BP could be announced or not by accelerated phase (AP).

Once Imatinib was developed, the first tyrosine kinase inhibitor which specifically targets BCR-ABL1, the prognosis of CML patients has changed and natural evolution improved immensely. The time showed that, the treatment although efficient in achieving levels under molecular testing detection, does not cure the disease, the clone will persist in some quiescent stem cells how could re-enter cellular cycle and re-establish CML. The studies showed that a small number of patients will develop intolerance and resistance and a new generation of tyrosine kinase inhibitors was developed in the attempt of controlling  the disease. Those new molecules were tested in different studies for assessing the best management of CML patients. Although the last decade represented a true progress in CML, the cure is still a distant target, which lead to scientific research in identification of mechanisms of resistance to tyrosine kinase inhibitors and means to overcoming them. The introduction of qRT-PCR technique was an important step in diagnosis and monitoring of CML patients. In Romania, qRT-PCR technique became available in the year 2006 in the Molecular Department of Fundeni Clinical Institute, Bucharest, conducted by Prof. Coriu Daniel who worked together with other European labs to standardize and establish a conversion factor which allows unification of results. Since 2007, the same lab used Sanger technique for mutation analysis. The data was used in the general study and in the mutation analysis patients. This represents the biggest CML patients database which were assessed through cytogenetic, molecular, blood level testing and after bone marrow transplant. This analysis integrates cytogenetic and molecular response, mutation analysis, blood level testing, additional cytogenetic abnormalities and bone marrow transplant data from Romanian CML population in the international CML literature.

General part

This chapter contains general informations about CML diagnosis criteria, risk group classification, describes lab techniques used for diagnosis and response monitoring (conventional cytogenetic exam, FISH exam, qualitative and quantitative assessment through RT-PCR method, Imatinib blood level testing in CML), Tyrosine kinase inhibitors resistance mechanisms (oral bioavailability, plasmatic protein binding, intracellular availability of tyrosine kinase inhibitors, hOCT-1, clone evolution, SRC supraexpression, quiescent stem cells, increasing BCR-ABL level, Abl kinase domain mutations), international trial results which established tyrosine kinase inhibitors as standard treatment in low risk CML (Imatinib, Dasatinib, Nilotinib, Bosutinib, Ponatinib and Omacetaxin) and the role and importance of allogeneic bone marrow transplant in CML.

Special part

This chapter contains the thesis objectives (clinical evolution and TKI response in correlation to standard prognosis factors; evolution and survival of CML patients which presented cytogenetic abnormalities in Philadelphia positive and negative cells; evolution and survival of CML patients who benefit from Imatinib blood level testing; evolution and survival of CML patients who benefit from mutation analysis; evolution and survival of CML patients who received allogeneic bone marrow transplant; correlation of our risk group data to international literature), inclusion and exclusion criteria of CML patients in our analysis, material and methods used for CML diagnosis and treatment response monitoring and parameters and statistical methods used in our study.

The Ist Study: The impact of prognostic scores at diagnosis, cytogenetic, molecular and to tyrosine kinase inhibitors response on overall survival in our study group

In our group, the patient’s distribution was in favour of males with median diagnosis age of 50 years, EUTOS score was the most important score and was followed by Sokal and Hasford score. At CML diagnosis, the patients were in CP 87,8%, in AP 7,4% and in BP 4,8%. The median survival time for CP was 85 months, for AP was 77 months and for BP was 4,66 months. Survival rate at 8 years for CP was 84,9% and for AP was 71,4%. Imatinib in dose of 400 mg daily was the most used first line TKI for low and intermediary risk CML patients. CCyR at 12 months was achieved by 64,2 % and during monitoring, the patient`s CCyR improved and overall survival was better. Achievement of MMR at 18 months improved overall survival.

The IInd Study: The resistance mechanisms to tyrosine kinase inhibitors treatment: TKI inability to Abl kinase domain coherence (the role of mutations in Abl kinase domain), the TKI inability to enter into CML cells (the role of blood level testing) and clone evolution (the role of additional cytogenetic abnormalities).

1. TKI inability to Abl kinase domain coherence: The role of mutations in Abl kinase domain

In our group, all patients were categorised as treatment failure according to ELN recommendations, standard indication for mutation status analysis which showed that 22% of patients expressed one or more mutations in Abl kinase domain, the obtained percentage being in compliance to general media according to international studies. Four of ten identified mutations: M244V, Q252H, F359V, T315I are the most frequent identified mutations according to recent 2013 ELN study. The identified mutations are situated in four regions of Abl kinase domain: P loop (M244V, E255K, Q252H), bounding domain (T315 and F317), catalytic domain (F359V) and activation loop (L387M). After mutation status analysis, all available options were used: TKI escalation dose, TKI switch and allogeneic bone marrow transplant.

2. TKI inability to enter into CML cells: The role of blood level testing

In our group, Imatinib blood level testing was not influenced by patient demographic characteristics as gender, body surface area and age. The patient`s distribution according to Sokal score was: low (15%), intermediary (50%) and high (35%). All patients were categorised as treatment failure according to ELN recommendations after medium Imatinib treatment of 43,5 months. Although in our study, BLT was not performed in day 8 and/or 29 of TKI treatment, the test results allowed us to adapt treatment in real time, improved MMR rates and overall survival.

3. Clone evolution: The role of additional cytogenetic abnormalities

In our group, cytogenetic abnormalities at diagnosis were observed at 5,8% cases and the frequency of major and minor cytogenetic abnormalities in Philadelphia positive cells were similar to international literature. The presence of cytogenetic abnormalities is associated with lack of optimal response to TKI (especially Imatinib), progression to advance phase and shorter overall survival. CCyR rate was lower, time to CCyR was longer and overall survival was inferior but no important differences compared to general CML population were noticed. The presence of major cytogenetic abnormalities in Philadelphia positive cells during TKI treatment was confirmed as a sign of acceleration of CML evolution. No cytogenetic abnormalities in Philadelphia negative cells with poor prognosis were identified as chromosome 7 abnormalities and in the absence of dysplasia, they did not influence TKI response. Survival in CCyR was observed in 89,3% of cases and exitus was observed in 10,7% of cases due to progression in BP after a medium time of 4 months.

The IIIrd Study: the role and place of allogeneic bone marrow transplant in CML patients

In our group, the time for bone marrow transplant was 12 months after CML diagnosis due to need for HLA matched unrelated bone marrow donors. At transplant time, most patients were in CP. The type of bone marrow conditioning regimen was decided according to EBMT score, patient characteristics and HLA matched donor availability. After bone marrow transplant, most patients developed infectious complications. Early transplant related mortality was high due to infectious complications with unpredicted evolution and different from acute and chronic GVHD. Survival after transplant in CP and AP patients was inferior to international data due to high early transplant related mortality. All survivors achieved MMR at 6 months after allogeneic bone marrow transplant.

Conclusions

This chapter contains our study results from general CML population, mutation analysis group, blood level testing group, cytogenetic abnormalities in Philadelphia positive and negative cells and allogeneic bone marrow group.

1. In our study, the patient`s distribution was in favour of males with median diagnosis age of 50 years, EUTOS score was the most important.

2. At CML diagnosis, the patients were in CP (87,8%), in AP (7,4%) and in BP (4,8%).

3. The median survival time for CP was 85 months, for AP was 77 months and for BP was 4,66 months. Survival rate at 8 years for CP was 84,9% and for AP was 71,4%.

4. Imatinib in dose of 400 mg daily was the most used first line TKI for low and intermediary risk CML patients.

5. CCyR at 12 months was achieved by 64,2% and during monitoring, the patient’s CCyR improved and overall survival was better.

6. Achievement of MMR at 18 months improved overall survival.

7. In our group, all patients were categorised as treatment failure according to ELN recommendations, standard indication for mutation status analysis

8. 22% of patients expressed one or more mutations in Abl kinase domain, the obtained percentage being in compliance to general media according to international studies and four of ten identified mutations: M244V, Q252H, F359V, T315I are the most frequent identified mutations according to recent 2013 ELN study.

9. After mutation status analysis, all available options were used: TKI escalation dose, TKI switch and allogeneic bone marrow transplant.

10. In our group, cytogenetic abnormalities at diagnosis were observed at 5,8% cases.

11.The presence of cytogenetic abnormalities is associated with lack of optimal response to TKI (especially Imatinib), progression to advance phase and shorter overall survival. CCyR rate was lower, time to CCyR was longer and overall survival was inferior but no important differences compared to general CML population were noticed.

12. The presence of major cytogenetic abnormalities in Philadelphia positive cells at diagnosis did not change first line TKI treatment and the presence of major cytogenetic abnormalities in Philadelphia positive cells during TKI treatment was confirmed as sign of acceleration of CML evolution.

13. No cytogenetic abnormalities in Philadelphia negative cells with poor prognosis were identified as chromosome 7 abnormalities and in the absence of dysplasia, they did not influence TKI response.

14. In our group, the time for bone marrow transplant was 12 months after CML diagnosis due to need for HLA matched unrelated bone marrow donors. At transplant time, most patients were in CP.

15. The type of bone marrow conditioning regimen was decided according to EBMT score, patient characteristics and HLA matched donor availability.

16. Early transplant related mortality was high due to infectious complications with unpredicted evolution and different from acute and chronic GVHD.

17. Survival after transplant in CP and AP patients was inferior to international data due to high early transplant related mortality. All survivors achiev­ed MMR at 6 months after allogeneic bone marrow transplant.

Bibliografie

1. Baccarani M, Deininger MW, Rosti G, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia.Blood.2013;122 (6): 872-84. 
2. Marzocchi G, Castagnetti F, Luatti S, et al. Gruppo Italiano Malattie EMatologiche dell’Adulto (GIMEMA) Working Party on Chronic Myeloid Leukemia. 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. 
3. Branford S, Fletcher L, Cross NC, Muller MC, Hochhaus A, Kim DW, et al. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials.Blood. 2008;112(8):3330-8. 
4. Mahon F-X, Bouchet S, Etienne G, et al. Management of chronic myelogenous leukemia using therapeutic drug monitoring of imatinib: the French experience of a centralized laboratory. J Clin Oncol. 2008;26(Suppl.):Abstract 7087. 
5. Branford S, Goh H-G, Izzo B, et al. A review of mutation analysis in the TOPS trial of standard versus high dose IM in CML suggests that refinements to the ELN recommendations for mutation screening may be appropriate. Blood.2010;116(21): Abstract 889. 
6. Saglio G., et al. Nilotinib is associated with fewer treatment failures and suboptimal responses vs. imatinib in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP). Results from ENESTnd.Haematol.2011; 96(s2):58-9.Abstract 145. 
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