T-cell large granular lymphocyte (T-LGL) leukemia is a rare chronic lymphoproliferative disorder of cytotoxic T cells of unknown etiology. Chronic antigenic stimulation of T-lymphocytes in autoimmune diseases or viral infection has been postulated to play a role in the pathogenesis of this condition. Surface-enhanced laser desorption/ionization (SELDI) mass spectrometry of plasma and serum specimens was applied as a useful screening method to determine pathophysiologic protein/peptide markers, which may be useful in the diagnosis of occult malignancies. In contrast to traditional diagnostic principles, the SELDI method enables screening of protein patterns without knowing the identity of targets. Patterns of up- and down-modulation of peptides and proteins (resulting from the presence of abnormal products of diseased cells, pathogens or immunogenetic factors) may signal various clinical features and diagnostic clues. We used SELDI mass spectrometry to study proteomic patterns in the plasma of patients with T-LGL leukemia. After initial screening and optimization we selected a hydrophobic H4 ProteinChip surface for further systematic screening. Deviation of mass values and peak intensities were <0.5% and <20%, respectively.

Reproducibility was monitored by random duplication of samples with an average of 70 peaks per spectrum (mass/charge ratio from 1500–50,000 Da). Our study included plasma samples from 28 LGL patients, 9 patients with idiopathic neutropenia (without the presence of LGL clone) and 36 healthy blood donors. The diagnosis of T-LGL leukemia included the presence of monoclonal TCR rearrangement and flow cytometric detection of abnormal CD8+CD3+CD57+ cell population. Spectra were analyzed using Biomarker Patterns Software, which allows for internal cross validation for the prediction of the accuracy in a blinded test set. To separate LGL patients from patients with chronic neutropenia, intensity cut-off for 2050 Da peak was found in 22/28 LGL patients, resulting in 100% specificity and 67% sensitivity. Based on the intensities of 3 mass peaks (2274, 5870, 2660 Da), the sensitivity and specificity of LGL distinction from healthy controls was 96% and 61%, respectively. By direct digestion with carboxypeptidase Y on the ProteinChip surface, the C-terminal sequence of the 2050 Da mass peak has been identified. From the sequencing analysis and the search in the ExPASy protein database, we concluded that the 2050 Da mass peak corresponded to the fragment of complement C3 precursor. Additional 2 kDa mass peaks may represent various other C3 fragments. Interestingly, the intensity of the mass peak corresponding to the C3 fragments decreased with the application of immunosuppressive therapy when plasma samples from 3 LGL patients were serially analyzed. The pathophysiologic significance of the identified peak remains unclear, but increased expression of the C3 receptor on LGL cells has been reported. Our data in LGL leukemia indicate that SELDI mass spectrometry can be used to identify immune biomarkers for malignant lymphoproliferations.

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