Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Even though the risk-group adjusted therapies have dramatically improved the outcome, approximately 25% of ALL patients experience relapse of the disease. Recent gene microarray studies showed distinct gene expression profiles for immunophenotypically and genetically defined ALL subgroups and their association with treatment outcome. However, not only transcriptional control, but also a variety of post-transcriptional/post-translational modifications, play an important role in regulating protein expression and function, and ultimately may prove informative for predicting tumor behavior. In this pilot study, we hypothesize that in childhood precursor B ALL surface enhanced laser desorption/ionization mass spectrometry can identify proteins discriminating between age and WBC defined risk-groups. Ultimately, specific proteins may emerge that will provide predictive information beyond currently known prognostic factors. Our pilot series consisted of 78 pediatric precursor B ALL patients. The protein samples were obtained from archival Wright-Giemsa stained bone marrow aspirate smears. Initial experiments, using CM10 and IMAC30 chips demonstrated that the latter provided superior resolution and presented more numerous protein peaks. The protein mass spectra were originally generated using laser intensities of 150 and 160 arbitrary units. Data obtained at laser intensity of 160 was used for further analysis. Twenty-three protein peaks common to all samples were identified within examined range (up to 30 kDa; CiphergenExpressData Manager software, Ciphergen, Fremont, CA). These proteins span the spectrum between 1.0 to 15.2 kDa. The comparison of protein profiles between standard- and high-risk groups (SR and HR, respectively), WBC subgroups (less and more than 50 K/ul) and in the prognostic age groups was performed. Two protein peaks, 3.0 and 1.7 kDa, discriminated between SR and HR (p=0.001 and p=0.04, respectively). Both proteins showed significantly higher intensity in SR group. The protein peak of 3.0 kDa was further confirmed to discriminate between patients with low and high presentation WBC (p=0.01). Our results demonstrate that reliable quality protein profiles can be obtained using archival Wright-Giemsa stained bone marrow aspirate smears. Using this technology, specific proteins were found to discriminate between prognostic risk-groups. We are currently analyzing precursor B-cells sorted from non-neoplastic bone marrows to compare protein profiles to our pilot precursor B ALL series in a cluster analysis. The use of archival material facilitates analysis of large patient series and identification of protein panels with prognostic power, which can be validated in prospective studies.

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