Proteomics-based discovery of biomarkers for paediatric acute lymphoblastic leukaemia: challenges and opportunities

Abstract

There are important breakthroughs in the treatment of paediatric acute lymphoblastic leukaemia (ALL) since 1950, by which the prognosis of the child majority suffered from ALL has been improved. However, there are urgent needs to have disease-specific biomarkers to monitor the therapeutic efficacy and predict the patient prognosis. The present study overviewed proteomics-based research on paediatric ALL to discuss important advances to combat cancer cells and search novel and real protein biomarkers of resistance or sensitivity to drugs which target the signalling networks. We highlighted the importance and significance of a proper phospho-quantitative design and strategy for paediatric ALL between relapse and remission, when human body fluids from cerebrospinal, peripheral blood, or bone-marrow were applied. The present article also assessed the schedule for the analysis of body fluids from patients at different states, importance of proteomics-based tools to discover ALL-specific and sensitive biomarkers, to stimulate paediatric ALL research via proteomics to 'build' the reference map of the signalling networks from leukemic cells at relapse, and to monitor significant clinical therapies for ALL-relapse.

Keywords: acute lymphoblastic leukaemia; biomarkers; paediatrics; proteomics.

Fig. 1

Possible flow-through – proteomic strategies – which may be useful for ALL-relapse research. The proteins must be purified from body-human fluids, subsequently high abundant proteins must be depleted as they can artefact the biomarkers-identification of ALL-relapses. The resulting purified proteins can be loaded into SIMAC chromatography to purify phosphorylated proteins. Finally, the peptides can be labelled via iTRAQ to quantify the level expression of phosphorylated proteins for a given clinical sample. The resulting potentially identified biomarkers by mass spectrometry, can be validated via ELISA and SRM/MRM. In addition, just by using iTRAQ plus mass spectrometry tools, the reference map of the proteome (ALL paediatric relapses) can be achieved studying the differential expressed proteins. Thus, new therapy targets can be discovered to improve current therapies for paediatric ALL relapses.

Fig. 2

: Schedule for the analysis of sample-body fluids according to the different states of the paediatric ALL patients suffering relapses. The body-fluid samples, such as cerebrospinal fluid, peripheral blood and bone marrow from ALL paediatric patients at different states -at the diagnose, during chemotherapy and at the end of chemotherapy- must be analysed by using replicates (coming from different vials) to ensure reproducible data. First, the samples must be tested by using replicates from a biobank to get reproducibility and ensure the best conditions during all the work-flow, as the sample preparation is a critical step for achieving efficient data. Secondly, the lymphocytes from bone marrow and peripheral blood can be isolated via Ficoll, while the cells from the cerebrospinal fluid can be isolated by centrifugation. All cells from the three detailed kinds of samples must be lisated by using kits for human cells (i.e. RIPA) and the resulting proteins can be digested via trypsin to get the complex-mixture of peptides from the clinical sample. Finally, the resulting peptides can be loaded into SIMAC to isolate the phosphorylated peptides. Subsequently the purified phosphopeptides can be desalted, cleaned and concentrated (i.e. via POROS R3 reverse-phase chromatography) and injected in the mass spectrometer. The resulting data by mass spectrometry, permits improving the knowledge of signalling networks related to paediatric ALL relapses, which permits obtaining the phosphoproteome reference map of paediatric ALL-relapses, thus important clues of ALL-relapses can be unravelled. (Note: using 150 µg of proteins per sample (individual or via pools) is enough to carry out the complete proteomic strategy and identify biomarkers of paediatric ALL-relapses).