Clinical investigation: breast
ATM mutations in female breast cancer patients predict for an increase in radiation-induced late effects

Presented at the ASTRO Annual Meeting, San Francisco, CA, Nov. 4–8, 2001.
https://doi.org/10.1016/S0360-3016(01)02684-0Get rights and content

Abstract

Purpose: Mutation of the ATM gene may be associated with enhanced radiosensitivity and increased radiation-induced morbidity. Denaturing high performance liquid chromatography (DHPLC) is a powerful new technique proven to be sensitive and accurate in the detection of missense mutations, as well as small deletions and insertions. We screened female breast cancer patients for evidence of ATM gene alterations using DHPLC. This study attempted to determine whether breast cancer patients who develop severe radiotherapy (RT)-induced effects are more likely to possess ATM mutations than patients who display normal radiation responses.

Methods and Materials: Forty-six patients with early-stage breast carcinoma underwent limited surgery and adjuvant RT. DNA was isolated from blood lymphocytes, and each coding exon of the ATM gene was amplified using polymerase chain reaction. Genetic variants were identified using DHPLC by comparing test patterns with a known wild-type pattern. All variants were subjected to DNA sequencing and compared with wild-type sequences for evidence of a mutation. A retrospective review was performed, and the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer acute and late morbidity scoring schemes for skin and subcutaneous normal tissues were applied to quantify the radiation-induced effects.

Results: Nine ATM mutations were identified in 6 patients (8 novel and 1 rare). The median follow-up was 3.2 years (range 1.3–10.3). A significant correlation between ATM mutation status and the development of Grade 3–4 subcutaneous late effects was found. All 3 of the patients (100%) who manifested Grade 3–4 subcutaneous late sequelae possessed ATM mutations, whereas only 3 (7%) of the 43 patients who did not develop this form of severe toxicity harbored an ATM mutation (p = 0.001). One ATM mutation carrier developed Grade 4 soft tissue necrosis after RT and required hyperbaric oxygen. All 3 patients manifesting Grade 3–4 late subcutaneous responses in fact harbored 2 ATM mutations. In contrast, none of the 3 ATM carriers who had a single mutation developed a severe subcutaneous reaction. ATM mutation status did not predict for a significant increase in early effects. Of the 23 patients with Grade 2–3 moist desquamation, 4 (17%) had an ATM mutation compared with 2 (9%) of 23 patients without desquamation (p = 0.7).

Conclusion: Possession of an ATM mutation, particularly when 2 are present, may be predictive of an increase in subcutaneous late tissue effects after RT for breast cancer and may subsequently prove to be a relative contraindication to standard management. These patients may be better served with reduced doses of radiation. Equivalent local control remains to be tested, but this germline alteration may radiosensitize normal tissues, as well as the tumor itself. DHPLC is effective in the identification of these patients. A larger study is required to confirm these findings.

Introduction

Ataxia-telangiectasia (AT) is a rare autosomal recessive disorder that results from mutations in both copies of the ATM gene. This disorder was first described almost 50 years ago and is characterized by a pleiotropic phenotype and multiple organ system involvement (1). Cerebellar degeneration, ocular telangiectasias, immunodeficiency, and enhanced sensitivity to ionizing radiation are typical manifestations (2). Devastating radiation responses in these patients have also been known for decades (3). A single mutated copy of the ATM gene (ATM heterozygosity) occurs in approximately 1% of the general population (4). These individuals do not express the classic symptoms of AT but may be at an increased risk of carcinogenesis and radiosensitivity because of differences in ATM protein function or transcription levels 5, 6, 7. It has also been postulated that this ATM heterozygous state is more common in breast cancer patients than in the general population 8, 9.

The ATM gene product has been the subject of intensive investigation and appears to act as a central component in cell cycle regulation and genomic stability. It is located primarily in the nucleus of dividing cells (10) and displays protein kinase activity in response to double-strand DNA damage 11, 12, 13. The downstream effects of ATM are critical to the genetic integrity of the cell. This phosphorylation activity has been well characterized at the G1-S checkpoint and phosphorylation of p53 by ATM is a necessary step in cell cycle arrest in response to genomic damage. There is evidence that the ATM protein is also involved at the other cell cycle regulation points 14, 15, 16. Cells lacking the ability for proper genomic surveillance and DNA double-strand damage repair are at risk of mutagenesis and enhanced radiation responses 17, 18.

The hypothesis that cells with an ATM mutation exhibit enhanced radiosensitivity has been demonstrated on a cellular level 6, 7. Studies have examined cell lines from normal patients, ATM heterozygotes, and AT patients, and the D0 values for ATM heterozygotes falls between that of patients with a normal genotype and those with AT 6, 19. It is predicted that this inherent radiosensitivity would result in enhanced normal tissue damage after a course of radiotherapy (RT). In vivo studies in female breast cancer patients have failed to correlate ATM mutation status with enhanced normal tissue damage 20, 21, 22, 23, 24. However, these studies have relied on the protein truncation test, a test that only detects genetic alterations that result in protein truncation. Mutations that result in early termination of the ATM gene product are characteristic of both allelic products in the disorder AT, but it has been hypothesized that missense mutations, which result in amino acid substitutions rather than protein truncation, are more prevalent in ATM heterozygous breast cancer patients 25, 26, 27. The degree to which these missense mutations may have an impact on the function of the ATM protein can only be deduced by assays that can quantify differences in protein function. We refer to them as mutations by virtue of a change in the genetic material, but they may be newly identified polymorphisms and have little or no biologic significance.

Denaturing high performance liquid chromatography (DHPLC) is a robust technique that can be used to screen any gene in a large population for single nucleotide substitutions 28, 29, 30, 31, 32, 33, 34, 35, 36. This mutation detection method has proved to be highly sensitive and specific and will allow for identification of the missense mutations that appear to be most prevalent in ATM carriers (9).

This study involved the screening of a group of female breast cancer patients after breast conservation management and attempted to correlate the ATM germline mutation status with acute and/or late radiation normal tissue effects using DHPLC technology.

Section snippets

Patients

Peripheral venous blood samples were collected from 46 female patients with AJCC (37) Stage 0–II breast carcinoma who underwent limited surgery and adjuvant RT between 1992 and 1997. Nine patients also received adjuvant sequential chemotherapy. The patient demographics and tumor characteristics are presented in Table 1. RT was delivered with 6 MV photons using either opposed tangential portals alone or 3 fields that included an additional supraclavicular portal if the axillary lymph nodes were

Results

Six patients were found to have 8 novel ATM missense mutations and 1 rare ATM genetic variant. The ATM database that contains all previously identified genetic alterations was reviewed (www.vmresearch.org/atm.htm) before our classification of the genetic alterations. Eight had not been previously described, and 1 was estimated be found in <1% of the population, characterizing it as a rare genetic variant rather than a polymorphism. The specific location of these genetic alterations and their

Discussion

It has been hypothesized that ATM heterozygotes may exhibit enhanced radiosensitivity and may be at an increased risk of radiation-induced normal tissue toxicity. However, no statistically significant correlation between ATM mutation status and normal tissue effects has been reported 20, 21, 22, 23, 24. To our knowledge, aside from case reports, this represents the first study that suggests such an association exists in the female breast cancer population.

Several studies have been performed in

Conclusion

Possession of ATM mutations in the female breast cancer population predicts for an increase in subcutaneous tissue effects after RT. DHPLC is effective for the identification of patients with ATM mutations. These results suggest that the presence of such mutations may be a relative contraindication to breast conservation management using conventional doses. Alternatively, germline ATM mutation carriers may prove ideal candidates for radiation dose reduction trials. A larger study is required to

Acknowledgements

We gratefully acknowledge support from the Mount Sinai Ruttenberg Cancer Center that enabled the purchase of the WAVE Nucleic Acid Fragment Analysis System.

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    Supported by the Breast Cancer Research Fund of the Department of Defense, Grant DAMD17-97-1-7208.

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