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Introduction Mutations affecting p53 or its upstream activator Chk2 are associated with resistance to DNA-damaging chemotherapy in breast cancer. ATM (Ataxia Telangiectasia Mutated protein) is the key activator of p53 and Chk2 in response to genotoxic stress. Here, we sought to evaluate ATM's potential role in resistance to chemotherapy. Methods We sequenced ATM and assessed gene expression levels in pre-treatment biopsies from 71 locally advanced breast cancers treated in the neoadjuvant setting with doxorubicin monotherapy or mitomycin combined with 5-fluorouracil. Findings were confirmed in a separate patient cohort treated with epirubicin monotherapy. Each tumor was previously analyzed for CHEK2 and TP53 mutation status. Results While ATM mutations were not associated with chemo-resistance, low ATM expression levels predicted chemo-resistance among patients with tumors wild-type for TP53 and CHEK2 ( P = 0.028). Analyzing the ATM-chk2-p53 cascade, low ATM levels (defined as the lower 5 to 50% percentiles) or mutations inactivating TP53 or CHEK2 robustly predicted anthracycline resistance ( P -values varying between 0.001 and 0.027 depending on the percentile used to define "low" ATM levels). These results were confirmed in an independent cohort of 109 patients treated with epirubicin monotherapy. In contrast, ATM-levels were not suppressed in resistant tumors harboring TP53 or CHEK2 mutations ( P > 0.5). Conclusions Our data indicate loss of function of the ATM-Chk2-p53 cascade to be strongly associated with resistance to anthracycline/mitomycin-containing chemotherapy in breast cancer.

Purpose: The cyclin-dependent kinase inhibitor p21 acts as a main executor of p53-induced growth arrest. Recently, a second transcript, p21B, was found to code for a protein expressing proapoptotic activity. We investigated p21 and p21B for mutations and epigenetic silencing in locally advanced breast cancers treated with doxorubicin or 5-fluorouracil/mitomycin and correlated our findings with treatment response and TP53 status.Experimental Design: We used reverse transcription-PCR to analyze p21/p21B mutation status in 73 breast cancer samples. The p21 promoter region was sequenced and analyzed for hypermethylations by methylation-specific PCR. In addition, a selection of patients were analyzed for mutations in the p21B promoter.Results: The p21 gene was neither mutated nor silenced by promoter hypermethylation in any of the tumors examined. One patient harbored a novel p21 splice variant in addition to the wild-type transcript. We observed two base substitutions in the p21 transcript, C93A and G251A, each affecting six patients (8.2%). The G251A variant had not been reported previously. In 12 patients (16.4%), we observed a novel base substitution, T35C, in p21B. All three base substitutions were observed in lymphocyte DNA and therefore considered polymorphisms. The polymorphisms did not correlate with p21 staining index, treatment response to doxorubicin or 5-fluorouracil/mitomycin, or TP53 status.Conclusions: Our findings do not suggest that genetic or epigenetic disturbances in p21 or p21B cause resistance to doxorubicin or mitomycin/5-fluorouracil in breast cancer. Future studies should assess potential associations between these novel polymorphisms and breast cancer risk.