Genes and Pathways Dysregulated by Nilotinib Treatment in Bone Marrow CD34+/Lin- Cells of Patients with Chronic-Phase Chronic Myeloid Leukaemia - European Medical Journal

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Genes and Pathways Dysregulated by Nilotinib Treatment in Bone Marrow CD34+/Lin- Cells of Patients with Chronic-Phase Chronic Myeloid Leukaemia

1 Mins
Hematology
Authors:
*Alessandra Trojani,1 Ester Pungolino,1 Alessandra Dal Molin,2 Milena Lodola,1 Gabriella De Canal,3 Giuseppe Rossi,4 Mariella D’Adda,4 Alessandra Perego,5 Ester Orlandi,6 Malato Simona,7 Mauro Turrini,8 Lorenza Borin,9 Alessandra Iurlo,10 Maria Luisa Latargia,11 Maria Cristina Carraro,12 Francesco Spina,13 Salvatore Artale,14 Michela Anghilieri,15 Francesco Lanza,16 Enrica Morra,17 Roberto Cairoli1

1. Division of Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy
2. Department of Information Engineering, University of Padova, Italy
3. Pathology Department, Cytogenetics, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy
4. Department of Hematology, ASST Spedali Civili, Brescia, Italy
5. Internal Medicine-Haematology, Desio Hospital, Desio, Italy
6. Hematology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
7. Hematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milano, Italy
8. Division of Hematology, Department of Internal Medicine, Valduce Hospital, Como, Italy
9. Hematology Division, San Gerardo Hospital, Monza, Italy
10. Oncohematology Division, IRCCS Ca’ Granda – Maggiore Policlinico Hospital Foundation, Milano, Italy
11. ASST Valle Olona Ospedale di Circolo, Busto Arsizio, Italy
12. Hematology & Transfusion Medicine, Sacco Hospital, Milano, Italy
13. Division of Hematology – Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
14. ASST Valle Olona Sant’Antonio Abate, Gallarate, Italy
15. ASST Lecco, Lecco, Italy
16. Division of Hematology, Hospital of Cremona, Cremona, Italy
17. Executive Committee, Rete Ematologica Lombarda, Milano, Italy
*Correspondence to [email protected]

Disclosure:

The authors have declared no conflicts of interest.

Citation:
EMJ Hematol. ;6[1]:52-54. Abstract Review No. AR6.
Keywords:
Bone marrow CD34+/Lin- cells, chronic myeloid leukaemia (CML), genes and pathways, nilotinib

Each article is made available under the terms of the Creative Commons Attribution-Non Commercial 4.0 License.

BACKGROUND

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. Specifically, it presents a reciprocal association between chromosomes 9 and 22, yielding the BCR-ABL fusion protein with overacting tyrosine kinase activity.1 CML is treated with tyrosine kinase inhibitors (TKI), which have dramatically improved the long-term survival of CML patients to approximately 80%.2 Among TKI, nilotinib is very effective for the treatment of imatinib-resistant patients in the clinic.3 However, despite the remarkable success of TKI in controlling CML in the chronic phase, some patients develop drug resistance. The incapability of TKI to eradicate the disease completely is best explained by intrinsic and acquired drug resistance in leukaemic stem cells.4 The main research focus in the CML field is therefore to identify pathways and genes that contribute to the survival of leukaemic stem cells.

AIMS

We performed gene expression profiling (GEP) of selected bone marrow (BM) CD34+/Lin- cells of patients with chronic phase (CP)-CML at diagnosis versus 12 months of nilotinib treatment to gain new insights into the molecular mechanisms of nilotinib treatment in CML.

METHODS

We selected and counted BM CD34+/Lin- cells of 30 CP-CML patients at diagnosis and during 3, 6, and 12 months of first-line nilotinib treatment. BM CD34+/Lin- cells were isolated by immunomagnetic separation technology and tested by standard fluorescence in situ hybridisation (FISH) for all 30 patients at diagnosis and after 12 months of nilotinib treatment. GEP analyses of selected BM CD34+/ Lin- cells of the CP-CML patients at diagnosis and after 12 months of nilotinib treatment were performed by AffymetrixTM (Thermo Fisher Scientific Ltd., Waltham, Massachusetts, USA) microarray analysis. Data were preprocessed using the ComBat method to adjust for batch effects and quantile normalisation.5 The significance analysis of microarrays (SAM) test was used for expression analysis at diagnosis versus 12 months of treatment.6 Selection was performed using the R statistical computing software. False discovery rate-adjusted p values <5% were considered significant.7

RESULTS AND CONCLUSION

We demonstrated that the number of BM CD34+/Lin- cells dramatically decreased after 3, 6, and 12 months of nilotinib treatment (Figure 1). FISH analysis detected CD34+/Lin- Ph+ cells in 30 CP-CML patients at diagnosis, while no Ph+ nuclei were detected in CD34+/Lin- cells after 12 months of nilotinib. GEP detected 264 statistically significant differentially expressed genes at diagnosis versus 12 months of nilotinib treatment. Functional enrichment analysis showed that groups of genes belonging to 14 pathways were significantly dysregulated in CP-CML patients after 12 months of nilotinib. Lipid, glucose, and sphingolipid metabolism, insulin resistance, complement and coagulation, platelet activation, the cytoskeleton, cell adhesion, cellular transport, B cell differentiation, the RAS signalling pathway, proliferation, growth factors, and apoptosis were all shown to be significantly altered after 12 months of nilotinib in comparison with diagnosis in CP-CML patients.8,9

Figure 1: The number of bone marrow CD34+/Lin- cells at diagnosis and after 3, 6, and 12 months of nilotinib treatment in 30 chronic myeloid leukaemia patients.

References
Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer. 2007;7(6)441-53. Jabbour E et al. Long-term outcome of patients with chronic myeloid leukemia treated with second-generation  tyrosine kinase inhibitors after imatinib failure is predicted by the in vitro sensitivity of BCR-ABL kinase domain mutations. Blood. 2009;114(10):2037-43. Kantarjian H et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N Engl J Med. 2006;354(24):2542-51. Jiang X et al. Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies. Leukemia. 2007;21(5):926-35. Johnson WE et al. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics. 2007;8(1):118-27. Tusher VG et al. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2011;98(9):5116-21. Storey JD. A direct approach to false discovery rates. J R Stat Soc. 2002;64(3)479-98. Trojani A et al. Wide-transcriptome analysis and cellularity of bone marrow CD34+/lin- cells of patients with chronic-phase chronic myeloid leukemia at diagnosis vs. 12 months of first-line nilotinib treatment. Cancer Biomark. 2017;21(1):41-53. Pungolino E et al. Nilotinib induced bone marrow CD34+/lin-Ph+ cells early clearance in newly diagnosed CP-chronic myeloid leukemia. Am J Hematol. 2018. [Epub ahead of print].

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