Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Abstract 1629

Poster Board I-655

Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia.