Abstract The ATM-p53 DNA damage response pathway plays a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as underlying cause of chemoresistance. Also, specific treatments sensitizing TP53-or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Previously we showed that a reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) procedure which quantifies expression levels of the p53-targets, CDKN1A, BBC3 and Bax, in CLL cells following irradiation is able to determine p53 functionality. With the aim of identifying and distinguishing abnormalities of TP53 versus ATM and enabling the identification of additional defects in the DDR, we developed a new RT-MLPA based functional assay. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA-damage using irradiation. A new RT-MLPA probe set (R016-X2, MRC-Holland), which included several p53 and ATM target genes was designed. Genes were selected based on the results of an earlier microarray study [Stankovic et al. Blood 2004;103:291-300]. A cohort of 30 CLL patients was utilized to set-up the RT-MLPA functional assay (training cohort) and an independent second cohort including 67 CLL patients included in the HOVON68 clinical trial was used as a validation cohort. From all these patients genetic analysis of ATM and TP53 was available using FISH and sequencing. First, the assay was performed on the training cohort and was able to identify and distinguish three subgroups of CLL tumors (i.e. TP53-defective, ATM-defective and WT). Next, based on the fold induction factors (i.e. gene expression upon irradiation versus non-irradiation) of 10 selected genes, a support vector machine (SVM) classifier was constructed to enable the classification of CLL samples into 3 different types of response, i.e. ATM/p53 functional, p53-dysfunctional, or ATM-dysfunctional. The RT-MLPA assay and SVM classifier were validated in a separate validation cohort. CLL samples with clear genotypic characteristics (i.e. biallelic defects) were assigned with a high degree of confidence to one of the three categories with sensitivities of 93%, 100% and 78% for TP53/ATM WT, biallelic TP53-defective and biallelic ATM-defective samples, respectively. Interestingly the assay was also able to detect additional samples with a defective DNA-damage response, without molecular aberrations in TP53 and/or ATM (22%; 6 out of 27 WT patients). Additional analysis of these samples showed significantly reduced apoptosis to fludarabine, doxorubicin and irradiation in comparison to the ATM/p53 functional samples, indicating that defects in the DNA-damage response other than TP53/ATM aberrations are indeed present and probably responsible for the observed defective DNA-damage induced apoptotic responses. In conclusion, the newly designed ATM-p53 RT-MLPA assay is able to distinguish three subgroups of CLL tumors (i.e. TP53-defective, ATM-defective and WT) and was also able to detect additional samples with a functional defective DNA-damage response, without molecular defects of TP53 and/or ATM. This indicates that the ATM-p53 RT-MLPA might not only be of additional clinical value over FISH to screen for mutations of TP53 and ATM instead of sequencing, but might also be useful for screening of other defects in the DNA-damage response pathway in addition to ATM and/or TP53 aberrations. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL. Disclosures Hoogendoorn: Novartis: Consultancy; Gilead: Consultancy.