ALBERT

Description: Background: Genomic resources within the phylum Arthropoda are largely limited to the true insects but are beginning to include unexplored subphyla, such as the Crustacea and Chelicerata. Investigations of these understudied taxa uncover high frequencies of orphan genes, which lack detectable sequence homology to genes in pre-existing databases. The ticks (Acari: Chelicerata) are one such understudied taxon for which genomic resources are urgently needed. Ticks are obligate blood-feeders that vector major diseases of humans, domesticated animals, and wildlife. In analyzing a transcriptome of the lone star tick Amblyomma americanum, one of the most abundant disease vectors in the United States, we find a high representation of unannotated sequences. We apply a general framework for quantifying the origin and true representation of unannotated sequences in a dataset and for evaluating the biological significance of orphan genes. Results: Expressed sequence tags (ESTs) were derived from different life stages and populations of A. americanum and combined with ESTs available from GenBank to produce 14,310 ESTs, over twice the number previously available. The vast majority (71%) has no sequence homology to proteins archived in UniProtKB. We show that poor sequence or assembly quality is not a major contributor to this high representation by orphan genes. Moreover, most unannotated sequences are functional: a microarray experiment demonstrates that 59% of functional ESTs are unannotated. Lastly, we attempt to further annotate our EST dataset using genomic datasets from other members of the Acari, including Ixodes scapularis, four other tick species and the mite Tetranychus urticae. We find low homology with these species, consistent with significant divergence within this subclass. Conclusions: We conclude that the abundance of orphan genes in A. americanum likely results from 1) taxonomic isolation stemming from divergence within the tick lineage and limited genomic resources for ticks and 2) lineage-specific genes needing functional genomic studies to evaluate their association with the unique biology of ticks. The EST sequences described here will contribute substantially to the development of tick genomics. Moreover, the framework provided for the evaluation of orphan genes can guide analyses of future transcriptome sequencing projects.

Description: Abstract 4885 Chondroitin sulfate proteoglycan-4 (CSPG4) is a membrane-bound proteoglycan that is expressed on the surface of differentiated malignant cells, progenitor cells, and cancer initiating cells in various types of solid tumors. CSPG4 is highly conserved through evolution; its structure, amino acid sequence and functional properties show a high degree of homology with its rat counterpart, named neuron-glial antigen 2 (NG2). CSPG4 has been shown to be involved in the activation of several signaling pathways that play an important role in tumor progression. Because of its high levels of expression on malignant cells and its restricted distribution in normal tissues, CSPG4 is potential candidate tumor marker and target for immune- and targeted therapy. CSPG4 has been shown previously to be expressed on AML cells using an antibody raised against the rat counterpart and levels of expression were correlated with the clinical course of the disease. In this report we extend these findings by identifying a monoclonal antibody (mAb) directed at a human CSPG4 epitope and then used this antibody to examine co-expression with other common markers in AML as well as important molecular/cytogenetic abnormalities. Initially we screened a panel of 15 CSPG4-specific mAb which recognize 7 distinct epitopes for reactivity with leukemic blasts. mAb 225.28 was shown to have the highest reactivity and was selected for further studies. CSPG4 expression using mb225.28 was detected in 18/18 peripheral blood samples containing AML blasts at levels ranging from 0.2% to 98%. 7 samples showed less than 10% CSPG4 positive cells, 2 showed 10 – 20% positivity, 4 showed 20 – 30% positivity, and 5 showed greater than 30% positivity. CSPG-4 expression was not confined to any single blast sub-population defined by co-staining with CD34, CD117 and CD33. The expression of CSPG4 was shown on leukemic cells with mixed lineage leukemia (MLL) gene rearrangements, FTL3 mutation, NPM1 mutation, and complex cytogenetic abnormalities. These results indicate that the expression of CSPG4 is not restricted to any sub-type of AML or confined to one developmental compartment and using the mAb 225.28 CSPG4 may constitute a potential marker for AML. Disclosures: No relevant conflicts of interest to declare.

Description: Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34+ stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients, we now demonstrate that some, but not all, of these parameters correlate with subsequent clinical response to IM therapy. CD34+ cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P 〈 .001) and direct sequencing of cloned transcripts from CD34+ cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders, P 〈 .003). In contrast, CD34+ cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly, one BCR-ABL mutation (V304D), predicted to destabilize the interaction between p210BCR-ABL and IM, was detectable in 14 of 20 patients. T315I mutant CD34+ cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus, 2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management.