Severe reaction to radiotherapy for breast cancer as the presenting feature of ataxia telangiectasia

Abstract

Background: Severe early and late radiation reaction to radiotherapy is extremely rare in breast cancer patients. Such a reaction prompted an investigation into a 44-year-old mother (patient A-T213).

Methods: A neurological examination was performed and blood lymphocytes and skin fibroblasts were assessed for radiosensitivity chromosomally and by colony-forming assay. The ATM gene was sequenced and ATM mutations modelled by site-directed mutagenesis. The ATM kinase activity was also assessed.

Results: Patient A-T213 was normally ambulant with no ataxia and minimal other neurological features. T lymphocytes and skin fibroblasts were unusually radiosensitive, although less sensitive than in classical ataxia telangiectasia (A-T). A lymphoblastoid cell line and skin fibroblasts expressed ATM protein with some retained kinase activity. One missense ATM mutation c.8672G>A (p.Gly2891Asp) and a c.1A>G substitution were identified. In the modelling system, the p.Gly2891Asp mutant protein was expressed and shown to have residual ATM kinase activity.

Conclusion: Patient A-T213 has a milder form of A-T with biallelic ATM mutations, which may have contributed to breast cancer development, and certainly caused the severe radiation reaction. Ataxia telangiectasia should be investigated as a potential cause of untoward severe early and late radiation reactions in breast cancer patients.

Figure 1

Appearance of right breast following radiotherapy.

Figure 2

Colony-forming assay following exposure of cells to ¹³⁷Cs γ-rays. The proportion (%) of surviving colonies was plotted against dose of γ-rays for normal TERT-immortalised cells, TERT-immortalised skin fibroblasts from patient A-T213 and TERT-immortalised fibroblasts from a classical A-T patient A-T1BR. The assay was repeated at least three times for each cell strain. Error bars show the s.e.m. survival at each dose.

Figure 3

Electropherograms of mutations in genomic DNA in A-T213. Top panels show the wt sequence from a normal individual and the lower panels the mutations at these positions in patient A-T213; (A) the c.1A>G mutation, and (B) the c.8672G>A mutation. The boxed sequence triplet in (A) indicates the translation initiation codon.

Figure 4

ATM from A-T213 LCL and fibroblast lysates shows some retained ATM kinase activity. (A) A-T213 LCL lysate (lanes 3 and 4) shows the presence of some ATM kinase activity towards the ATM targets Smc1, KAP1, Nbs1 and CREB after exposure to IR, but this is reduced compared with lysate from a normal LCL (lanes 1 and 2). LCL lysate from a classical A-T patient A-T236, which does not express ATM, is shown in lanes 5 and 6. (B and C) Time course of phosphorylation of different ATM downstream targets by A-T213 fibroblast ATM protein, showing the presence of kinase activity at 1 h compared with the classical A-T patient (A-T248) but reduced activity compared with normal ATM (normal 2).

Figure 5

Full-length ATM is expressed in an LCL from patient A-T213, and immunoprecipitated ATM from this LCL has kinase activity in vitro. (A) Western blot (top and second panel; OE=overexposed) showing expression of ATM in a normal LCL (lanes 1 and 2), absence of ATM in an LCL from a classical A-T patient (A-T183, lanes 3 and 4) and reduced expression of ATM in LCL from A-T213 (lanes 5 and 6). Whereas the ATM in patient A-T213 appears to be full length, the ATM in an LCL from patient A-T86 with the c.2T>C mutation (and also null mutation c.7665delinsGTGA;p.His2555Gln ins^*) is slightly truncated (lanes 7 and 8). It is likely that truncated ATM from c.2T>C in patient AT187 is obscured by the p.Arg3047X (c.9139C>T) mutant protein (lanes 9 and 10) and the truncated ATM from c.1A>G in patient A-T213 is obscured by the p.Gly2891Asp (c.8672G>A) mutant protein (lanes 5 and 6). Immunoprecipitated ATM was used to phosphorylate the p53_1–72 substrate in vitro (third and fourth panel) following activation of ATM by IR. The level of normal radiation-induced ATM kinase activity is shown in lane 2. There is absence of induced activity in cells from classical A-T patient A-T183 (lane 4) with no ATM protein. Lane 6 shows induced activity of ATM from the LCL of A-T213 but not from ATM of patient A-T86 with a c.2T>C mutation and a second truncating mutation (lane 8) or from patient A-T187 with a c.2T>C mutation and c.9139C>T (p.Arg3047X). (B) Histogram of relative levels of ATM in vitro kinase activity from the immunoprecipitates in Figure 5A.

Figure 6

The p.Gly2891Asp protein has residual ATM kinase activity and a higher level of expression than the protein expressed from the c.1A>G mutation. (A) ATM protein without tags. ATM protein expression after zinc induction in the LCL stably transfected with pMEP4-ATM constructs was very low. Overexposure of the western blot showed that the c.1A>G derived protein (1A>G, lane 4) is truncated and accumulates to a lower level relative to the wt protein (pTAM2, lane 2) or the p.Gly2891Asp protein (8672G>A, lane 5). No ATM protein was expressed from the LCL transfected with vector alone (pMEP4, lane 3). Aprataxin was used as a loading control. (B) ATM protein with N-terminal tags. The induced levels of both wt protein (pTAM2, lane 2) and p.Gly2891Asp protein (8672G>A, lane 4) were increased when the ATM constructs were N-terminally tagged (compare with Supplementary Figure 2A). The p.Gly2891Asp (8672G>A) protein was expressed at a similar level to the wt protein. Aprataxin was used as a loading control. (C) Comparison of ATM kinase activity in lysates from LCLs stably transfected with pMEP4-ATM wt (pTAM2), pMEP4-ATM 8672G>A (8672G>A) or pMEP4 vector alone (pMEP4) all with N-terminal tags. Expression of the p.Gly2891Asp (8672G>A) protein (lane 5) was comparable to that of wt ATM (pTAM2, lanes 1 and 7). The p.Gly2891Asp (8672G>A) protein autophosphorylated Ser1981 following IR (lane 6) to levels comparable to that of ATM wt protein (lanes 2 and 8). Levels of IR-induced phosphorylation of ATM targets Smc1, KAP-1 and Nbs1 by the p.Gly2891Asp (8672G>A) protein were substantially lower than those for the ATM wt protein (lanes 2 and 8) but higher than for vector alone.