The combined expression patterns of Ikaros isoforms characterize different hematological tumor subtypes

Abstract

A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers. In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.

Figure 1. Level of expression of every interexon (I/E) in the assessed pathologies.

(A) expression of the interexon 2-3, (B) expression of the interexon 3-4, (C) expression of interexon 4-5, (D) expression of the inter-exon 56 and (E) expression of interexon 6-7. CML (chronic myeloid leukemia), MM (multiple myeloma), CLL (chronic lymphoid leukemia), BALL ( B acute Lymphoblastic leukemia), AML (acute myeloid leukemia). The control line is the arithmetic media (s.d. <0,01 in all PCR) of three healthy peripherial Blood controls.

Figure 2. Hierarchical cluster analysis of exon differentially expressed in B-ALL, CLL, CML, AML and MM cases.

Mean fluorescence intensity (MFI) values obtained for each PCR were imported and normalized according to mean MFI value observed for that PCR in control cases. Rows represent the B-ALL, CLL, CML, MM cases, while columns correspond to the normalized log₂ ratios of the MFI values obtained for each inter-exon PCR analyzed. The relative level of expression of each PCR is represented in a color code: red represents expression greater than the mean, green represents expression lower than the mean, and the color intensity represents the magnitude of the deviation from the mean; the scale extends from ratios of -3 to +3 (log₂ units). P4-5 and p6-7 columns represent the presence (Y) or absence (N) of non canonical splincing. (A) Cluster without the heterogeneous group of AML, (B) Cluster of all pathologies.

Figure 3. Level of expression of every interexon in Lymphoid vs Myeloid pathologies.

(A) expression of the interexon 2-3, (B) expression of the interexon 3-4, (C) expression of interexon 45, (D) expression of the inter-exon 56 and (E) expression of interexon 6-7.

Figure 4. Ikaros perfil in a case of blastic crisis.

(A) Levels of expression of the patient in B-ALL vs CML, (B) Comparison between the levels of expression of the CML group vs. expression of the patient with progressive pathology in CML stage, (C) Comparison between the levels of expression of the B-ALL group vs. expression of the patient with progressive pathology in B-ALL stage.