A Novel Functional Assay Using Etoposide Plus Nutlin 3, FISH and Mutational Analysis Detects Heterogeneity of ATM/p53 Pathway Alterations in CLL.

Abnormalities of the ATM and p53 genes can be detected by interphase FISH, mutational analysis or functional assays. The latter measure up-regulation of p53 or activation of downstream targets following induction of dsDNA breaks. To assess functional integrity of the TP53/ATM pathway we have exposed CLL cells with defined abnormalities of both ATM and TP53 to etoposide alone and to etoposide + an MDM2 inhibitor, nutlin 3 that up-regulates p53/p21 expression in cells with wt p53. Leukemic cells from 98 patients previously screened by FISH for TP53 and ATM loss, del 13q14 and +12 were incubated with etoposide alone. 68 cases were functionally normal; of these, 8 had ATM loss and 6 had TP53 loss. 9 patients had high basal p53 expression and did not up-regulate p53/p21 (type A dysfunction); 7 of these had TP53 loss, none had ATM loss. 20 patients with low basal p53 expression failed to up-regulate p53/p21 (type B dysfunction); of these, 14 had ATM loss and 5 had TP53 loss. 1 case showed a normal p53 response but did not show any p21 up-regulation (type C dysfunction) consistent with a p21 polymorphism. 46 cases were selected for analysis using etoposide/nutlin based on their ATM and TP53 loss and mutational status; see table for summary. 20 of these 46 were cases showing type B dysfunction. Exposure of the 20 type B dysfunctional cases to etoposide/nutlin resulted in normal up-regulation of p53/p21 in cases with ATM loss but not in those with TP53 loss. The expression levels of p53/p21 following exposure to etoposide and nutlin led us to redefine the criteria for identifying the type A and B groups (see table). The assay detected type A or B dysfunction in all 17 cases with 11q/17p loss with associated ATM/TP53 mutation respectively. However, no dysfunction was detected in all 11 patients with either ATM or TP53 loss and no mutation of the remaining allele. In contrast 3/6 patients with TP53 and 3/3 with ATM mutations without associated chromosomal loss were functionally abnormal, suggesting a dominant negative effect of the mutant clone. TP53 loss clone size by FISH was >48% in cases with TP53 mutations and <25% in cases where no mutation was detected. No correlation between mutation and the clone size of ATM loss was observed. These data illustrate a simple functional assay that can reliably detect cases with loss and mutation of ATM and TP53. Data generated using the assay has revealed a previously unsuspected heterogeneity of alterations of the ATM/p53 pathway in CLL; at least 7 different subgroups of disease can be identified using a combination of FISH, mutational analysis and this functional assay. Whether these different subgroups are clinically significant remains to be determined from further studies.