Clinical proteomics of myeloid leukemia

Abstract

Myeloid leukemias are a heterogeneous group of diseases originating from bone marrow myeloid progenitor cells. Patients with myeloid leukemias can achieve long-term survival through targeted therapy, cure after intensive chemotherapy or short-term survival because of highly chemoresistant disease. Therefore, despite the development of advanced molecular diagnostics, there is an unmet need for efficient therapy that reflects the advanced diagnostics. Although the molecular design of therapeutic agents is aimed at interacting with specific proteins identified through molecular diagnostics, the majority of therapeutic agents act on multiple protein targets. Ongoing studies on the leukemic cell proteome will probably identify a large number of new biomarkers, and the prediction of response to therapy through these markers is an interesting avenue for future personalized medicine. Mass spectrometric protein detection is a fundamental technique in clinical proteomics, and selected tools are presented, including stable isotope labeling with amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantification (iTRAQ) and multiple reaction monitoring (MRM), as well as single cell determination. We suggest that protein analysis will play not only a supplementary, but also a prominent role in future molecular diagnostics, and we outline how accurate knowledge of the molecular therapeutic targets can be used to monitor therapy response.

Figure 1

Myeloid leukemia and normal hematopoiesis. Acute myeloid leukemia (AML; red box) is a hematological disease characterized by a block in differentiation and promotion of proliferation or survival. Recurrent genetic abnormalities include t(8;21)(q22;q22), inv(16)(p13.1q22), t(16;16)(p13.1q22), t(15;17)(q22;q21), and t(9;11)(p22;q23). Chronic myeloid leukemia (CML; green box) is characterized as a stem cell disease with hyperplastic myeloid cells, including both immature and mature myeloid cells. The disease is defined by occurrence of the BCR-ABL fusion gene in the Philadelphia chromosome. Juvenile myelomonocytic leukemia (JCMML) and chronic myelomonocytic leukemia (CMML) (blue box) are hematological diseases with features of a myeloproliferative neoplasm and a myelodysplastic syndrome. Characteristics are peripheral blood monocytosis > 1 × 10⁹/l, no Philadelphia chromosome or BCR-ABL fusion gene, no rearrangement of platelet-derived growth factor receptor alpha polypeptide, or platelet-derived growth factor receptor beta polypeptide, and > 20% blasts in the blood and bone marrow. The figure was kindly provided by Dr Line Wergeland, University of Bergen.

Figure 2

Workflow for identification, verification and application of biomarkers in clinical diagnostics. Stable isotope labeling with amino acids in cell culture (SILAC), difference gel electrophoresis (DIGE) and isobaric tags for relative and absolute quantification (iTRAQ) are all powerful tools for finding differences in protein production in separate samples. A marker is added to the samples during either the experiment or the preparation for analysis; the samples are then analyzed together and in the resulting data can be told apart on account of the different markers. The aim is to find proteins that significantly differ in expression between the samples. For further validation, reverse-phase protein array (RPPA), and especially multiple reaction monitoring (MRM), are highly sensitive methods that can detect subtle differences in production of proteins identified as potential biomarkers. RPPA is an antibody-based assay that detects and quantifies protein production. MRM allows detection and absolute quantification of protein based on internal standard peptides. A suitable peptide, fulfilling the criteria to enable optimal analysis, is chosen from within the target protein and then produced with a heavy isotope amino acid incorporated. This synthetic peptide is added in known amounts to the sample. In the triple quadruple instrument (QQQ), the peptides of interest are selected (Q1), fragmented (Q2) and the resulting target peptide ions selected (Q3) for detection. As the amount of standard peptide added to the sample is known, peak comparison allows calculation of the amount of the target protein present in the sample. To apply identified and validated biomarkers in clinical diagnostics, the analytical method must be highly reproducible. Flow cytometry is a well-established method of analyzing hematological samples. With the application of mass spectrometry detection after flow cytometry selection (ICPTOF-MS), problems with multiplexing are overcome, and this method enables detection of up to 20 biomarker proteins. The nanofluidic proteomic assay (NIA) method allows quantitative detection of protein production in very limited material. The proteins are separated according to isoelectric point inside capillary glass tubes before immobilization and antibody detection.