SELDI Bone Marrow Profiling of B-Lineage Childhood Lymphoblastic Leukemia: Identity of Several Differential Markers.

Background: Childhood Acute Lymphoblastic Leukemia (cALL) is the most common form of pediatric cancer, accounting for 30% of newly diagnosed cases every year. cALL is a heterogeneous disease and the underlying pathways of leukemogenesis have yet to be unraveled. It is known that 30% of cALL is caused by genetic lesions due to chromosomal translocations or abnormalities. SELDI (Surface Enhanced Laser Desorption/Ionization-Time of Flight-Mass Spectrometry) is a promising proteomic platform for the discovery of useful markers to study diagnosis and prognosis of cancers. The protein signatures generated have clinical significance in many cancers. We analyzed 49 B-lineage cALL samples and compared to 4 non-leukemia controls (Immune Thrombocytopenia Purpura, ITP) using this technology.

Materials and Methods: Total cell lysates from 49 cALL bone marrow aspirates were obtained from patients with chromosomal aberrations [hyperdiploidy (n=12), TEL-AML1 (n=13), BCR-ABL (n=5), E2A-PBX1 (n=3) and others (n=1)] and patients without any known/detected chromosomal aberrations (n=15). The MS (mass spectrometry) data were collected, filtered through stringent parameters and was analyzed using Ciphergen® Biomarker Wizard and further confirmed via gel electrophoresis. The significantly differential proteins were then excised, trypsin-digested and further analyzed by TOF-TOF MS to obtain the identities of these markers.

Results: A total of 10 significantly differential protein markers were observed ranging from 6 kDa to 80 kDa. Identities of 6 of the 10 markers have been obtained. Peptide mass fingerprinting of these markers showed significant presence of histone H4 but absence of myeloperoxidase (MPO), lactoferrin (LF), S100A8 calcium binding protein (MRP-8), defensin alpha 1 and neutrophil granule peptide (HP1) in cALL patients.

Discussion: Unsurprisingly, MPO, LF and HP1 are of myeloid origin and therefore not found in cALL. However, histone modification and regulation has been shown to be involved in the development of leukemia since they regulate transcription by chromatin modulation. Histone hyperacetylation and deacetylation are associated with many cancers and hyperacetylated histone H4 was reported in the early stages of squamous cell carcinoma. Histone hyperacetylation might then suggest a disregulation of apoptosis that might be one of the channels of leukemogenesis. It is also postulated that chromatin regulation and control might assist in understanding chromosomal translocations so commonly observed in cALL, since regulation of gene expression through histone acetylation and deacetylation appears to be disrupted by a number of such fusion proteins. The TEL-AML1 fusion protein has been shown to recruit histone deacetylases thereby inhibiting transcription and altering self-renewal capacity and differentiation capacity of hematopoetic stem cells. Similar activity has also been shown in myeloid leukemia and inhibitors of histone deacetylases (anti-tumoral effect) are now being used as drugs in clinical trials. The preliminary results from SELDI are very encouraging and might offer us some insights into leukemogenesis.