A Novel Functional Assay Using Etoposide Plus Nutlin 3, FISH and Mutational Analysis Detects Heterogeneity of ATM/p53 Pathway Alterations in CLL.
Authors: Best Giles , Gardiner Anne , Ibbotson Rachel , Majid Aneela , Walewska Renata , Austen Belinda , Stankovic Tanja , Dyer Martin , Oscier David Graham 
Published: October 2019

Abstract
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.
Category No of Patients Function No Loss No Mutation 6 All Normal p53 Loss no mutation 5 All normal Loss and mutation 8 All type A No Loss with mutation 6 Normal, 3 type A ATM Loss no mutation 6 All normal Loss and mutation 9 All type B No Loss with mutation 3 All type B