Serum Protein Profiling Distinguishes BCR-ABL+ CML from Normal and Neutrophilia Cases.

We sought to identify additional biomarkers for chronic myelogenous leukaemia (CML) that could be an aid to early diagnosis and also yield novel antigens for immunotherapy. To this end, we screened patient serum samples at presentation against hematologically normal controls as well as patients with neutrophilia using Surface Enhanced Laser Desorption/Ionization technology (SELDI; Ciphergen ProteinChip series 4000). A total of 84 retrospective and prospective serum samples were analysed: presentation −28, reactive neutrophilia (>15x10⁹ neutrophils/L) − 24 and hematopoietic normal controls − 33. Patients were initially screened by routine cytogenetics and in some cases with qPCR for the BCR-ABL breakpoint. The sera samples were evaluated on 4 different array surfaces and the Immobilised Metal Affinity (IMAC) array was chosen as it bound serum proteins that distinguished CML from normal controls. As little as 1 μl serum was sufficient for each analysis. Biomarker artefacts due to variations in sample collection procedures were ruled out by analysing sera (n=4) from each group at the time of collection and 3 and 6 hours post collection. There were no significant differences in any of the biomarkers at any of the time points. The spectrum of proteins obtained from each of the 84 serum samples was averaged from duplicate runs per experiment. Using the Ciphergen Express program, a panel of 5 proteins were significantly differentially expressed in CML versus the reactive neutrophilia and normal hematopoietic controls (p<0.001). These proteins were identified by a combination of purification techniques using Q HyperD F columns, desalting using reverse phase C-18 beads and isolating the biomarker by 1D-SDS PAGE. The biomarkers were identified by peptide mass fingerprinting and confirmed by Tandem MS sequencing. These were

1. Albumin fragment − 2.8Kd (p< 3.5 x 10⁻⁵, ROC=0.78),

2. Fibrinogen fragments − 5.3Kd (p< 6.25 x 10⁻¹⁰, ROC=0.07) and 5.9Kd (p< 9.6 x 10⁻⁸, ROC=0.14),

3. Complement 3a precursor fragment − 8.9Kd (p< 0.0015, ROC= 0.70),

4. Platelet basic protein precursor − 10.2Kd (p< 1.5 x 10⁻⁴, ROC=0.73) and

5. Lysozyme − 14.6Kd (p=0, ROC=0.92).

Biomarkers 3, 4 and 5 were also verified by antibody capture experiments using NP-20 arrays. In a blinded test set of sera, CML, normal and neutrophilia samples were correctly classified 27/28 (96%), 32/32 (100%), 20/24 (83%) respectively using a combination of the 5.3Kd, 10.2 Kd and the 14.6 Kd markers (Biomaker Pattern software). The algorithm correctly classified 21 new samples as CML (7/8) and control (10/13). The 1/8 CML was misclassified for technical reasons. Therefore, a small number of serum biomarkers in as little as 1 μl serum can be used to distinguish between patients with CML and neutrophilia or hematopoietic normal controls. Similar analyses may be applicable to other more heterogeneous hematological malignancies.