ATM
Expression Patterns of the ATM Gene in Mammary Tissues and Their Associations With
Breast Cancer Survival
Chuanzhong Ye,MD,PhD1
Qiuyin Cai,MD,PhD1
Qi Dai,MD,PhD1
Xiao-ou Shu,MD,PhD1
Aesun Shin,MD,PhD1
Yu-Tang Gao,MD2
Wei Zheng,MD,PhD1
1Department of Medicine,Vanderbilt Epidemiol-ogy Center,Vanderbilt Ingram-Cancer Center, Vanderbilt University School of Medicine,Nash-ville,Tennessee.
2Department of Epidemiology,Shanghai Cancer Institute,Shanghai,China.BACKGROUND.The ataxia-telangiectasia mutated(ATM)gene plays a critical role in cell-cycle arrest,apoptosis,and DNA repair.However,to date,no study has directly investigated the association between ATM gene expression and breast cancer survival.
METHODS.ATM gene expression levels were evaluated in tumor and adjacent nor-mal tissue from patients diagnosed with primary breast cancer or BBD using quantitative real-time reverse transcription polymerase chain reaction(RT-PCR) assays.Cox regression models were used to evaluate the association of ATM gene expression and survival in a cohort of471breast cancer patients.
RESULTS.In breast cancer cases,ATM expression in cancer tissues was decreased by approximately50%compared with adjacent normal tissues from the same patients.In BBD cases,the expression level of the ATM gene was similar in be-nign tumor tissue and adjacent normal tissues.No apparent difference was found in ATM gene expression levels in adjacent normal tissues obtained from cancer patients or BBD https://www.wendangku.net/doc/d57250188.html,pared with patients with the lowest tertile of the ATM mRNA,patients in the upper2tertiles had more favorable disease-free sur-vival(hazard ratio[HR]?0.46,95%confidence interval[CI]:0.30–0.73and HR?0.52,95%CI:0.33–0.81,respectively)and overall survival(HR?0.56,95% CI:0.35–0.92and HR?0.70,95%CI:0.43–1.13,respectively). CONCLUSIONS.The ATM gene expression was down-regulated in breast cancer tissues and a high ATM gene expression level in breast cancer tissue was asso-ciated with a favorable prognosis.Cancer2007;109:1729–35.ó2007American Cancer Society.
KEYWORDS:ATM,mRNA,breast cancer,real-time PCR,Chinese women,survival. T he ATM(ataxia-telangiectasia mutated)gene is located on chro-mosome11q22$23,encoding a protein kinase of350kDa,which is involved in the maintenance of genome integrity.1–6Many in vitro and in vivo studies suggest that the ATM gene may play an important role in breast carcinogenesis.Most previous studies in humans have focused on the evaluation of the association between ATM gene var-iants and breast cancer.1,3,5,7–11Only a few small studies have directly evaluated ATM gene expression in breast tissues.6,12Low ATM expres-sion in breast cancer tissue has been shown to be related to a high rate of DNA mutation in cancer cells and,thus,a progressive cancer phenotype.8,13–15In addition,low ATM expression in breast carci-noma has also been correlated with increased neoangiogenesis.16 However,to date no study has directly investigated the association between ATM gene expression and breast cancer survival.In this study we first evaluated the expression patterns of the ATM gene in
Supported by NIH grants RO1CA64277and RO1
CA90899from the National Cancer Institute.
We thank Drs.Wanqing Wen and Qiong Li for
their contributions in data analysis,Brandy Venuti
for technical assistance in the preparation of the
article,and all of the study participants and
research staff of the Shanghai Breast Cancer
Study for their support.
Address for reprints:Wei Zheng,MD,PhD,Van-
derbilt Epidemiology Center,S-1121A,Medical
Center North,116121st Ave.S,Nashville,TN
37232-2587;Fax:(615)322-1754;E-mail:wei.
zheng@https://www.wendangku.net/doc/d57250188.html,
Received November21,2006;revision received
January4,2007;accepted January11,2007.
a2007American Cancer Society
DOI10.1002/cncr.22592
Published online15March2007in Wiley InterScience(https://www.wendangku.net/doc/d57250188.html,).
1729
breast tissues from patients diagnosed with breast cancer or benign breast disease(BBD).To investigate the clinical utility of ATM gene expression patterns, we examined the associations between levels of ATM mRNA expression and breast cancer survival. MATERIALS AND METHODS
Study Subjects and Tissue Samples
Included in this study was a subset of patients who were recruited as a part of the Shanghai Breast Cancer Study.17,18These patients were diagnosed with breast cancer or BBD between1996and1998and were iden-tified through a network of major hospitals that treat over80%of breast cancer patients in urban Shanghai.
A total of1602women who were diagnosed with a pri-mary breast cancer were identified.Among them, 1459(91.1%)participated in the Shanghai Breast Can-cer Study.During surgery,tumor tissue samples were obtained from the tumor and the adjacent normal tis-sue samples were obtained from the distal edge of the resection.These samples were snap-frozen in liquid nitrogen as soon as possible,typically within10min-utes.Samples were stored atà708C until the relevant assays were performed.
All patients were interviewed at the time of recruitment.A structured questionnaire was used in this study to collect information on demographic fac-tors,menstrual and reproductive history,hormone use,previous disease history,family history of cancer, physical activity,tobacco and alcohol use,and usual dietary habits.All participants were measured for cur-rent weight,circumferences of the waist and hips,and sitting and standing heights.Medical charts were reviewed using a standard protocol to obtain informa-tion on cancer treatment,clinical stages,and cancer characteristics,such as estrogen and progesterone re-ceptor status.Two pathologists reviewed pathology slides to confirm the diagnosis for breast cancer or BBD.BBDs were classified based on the published cri-teria developed by Page et al.19
All breast cancer patients were followed through July2005either in person or via phone contact and through record linkage to the death certificates kept by the Shanghai Vital Statistics Unit.In all,89.2%of patients successfully completed the follow-up inter-view either in person or by telephone.For those who could not be contacted in person or by phone,linkage to the death certificate data was conducted to obtain information on the date and cause of death.Subjects who had no match in the death registry were assumed to be alive on December30,2004,6months before the linkages in order to allow for a possible delay of entry of the death certificates into the https://www.wendangku.net/doc/d57250188.html,boratory Assays
Total RNA was extracted from tissue specimens by ho-mogenization in TRIzol solution(Invitrogen,Carlsbad, Calif),phase separation,precipitation,and washing following the manufacturer’s instructions.The quality and quantity of RNA was measured by spectrophoto-metric analysis.TaqMan Reverse Transcription Reagents(N8080234)or High Capacity cDNA Archive Kits(4322171)were obtained from Applied Biosystems (Foster City,Calif).RNA was reverse-transcribed(RT) in a final volume of15l L containing0.15l g RNA and 13RT polymerase chain reaction(RT-PCR)buffer, 5.5mM MgCl2,500l M each dNTP,2.5l M Random Hexamers,0.4U/l L RNase inhibitor,and3.125U/l L MultiScribe reverse transcriptase(Applied Biosys-tems).The mixture was incubated at258C for10min-utes,378C for120minutes,and958C for5minutes.
The primers and probes for the A TM gene(Hs00175892) and the b-actin gene(Hs99999903)were obtained from Applied Biosystems.Quantitative real-time PCR was performed using a384-well optic tray on an ABI PRISM7900HT Sequence Detection System(Applied Biosystems).A total reaction volume of5l L contain-ing2.2l L cDNA template at different dilutions,13 TaqMan Universal PCR Master Mix(without UNG), and13Gene Expression Assay Mix,including the pri-mers and marked probes from Applied Biosystems Assay-on-Demand services.The thermal cycling con-ditions were as follows:958C for10minutes to activate the AmpliTaq Gold enzyme,followed by40cycles of 958C for15seconds and608C for1minute.Every sam-ple was tested in triplicate.Two control samples were used in each plate to monitor interplate variation, which was found to be smaller than5%in our study. The threshold cycle(Ct)was determined at0.1based on the amplification linear area of the target genes and the b-actin gene(an internal control).The nor-malized quantity of the target gene was calculated as 2àD Ct,where D Ct was obtained directly by subtracting Ct for the target gene from Ct for the b-actin gene. The final result was expressed as2àD Ct*1000.20 The initial samples included in the current analy-sis contained tumor tissues from128primary breast cancer patients and39BBD patients,along with the paired adjacent normal tissues from43cancer pa-tients and39BBD patients.For survival analysis,tu-mor tissue samples for an additional343breast cancer patients were included(batch2)to enhance the statistical power of the study.
Statistical Analysis
The data were skewed to the high value,and thus medians(25th percentile,75th percentile)were used and compared across study groups using the Mann-
1730CANCER May1,2007/Volume109/Number9
Whitney test.Paired data were evaluated using the Wilcoxon signed rank test.The primary outcomes used for this study were overall survival(OS)and dis-ease-free survival(DFS).The endpoint for the analysis of OS was any death and for the analysis of DFS was cancer recurrence/metastasis or death related to breast cancer.Survival time was calculated as the time from cancer diagnosis to the endpoints of the study, censoring at the date of last contact or noncancer death(for DFS only).For subjects who had died of breast cancer without information on the date of re-currence or metastasis the total survival time was sub-stituted for the DFS time.18The5-year survival rate was estimated using the Kaplan-Meier method.The log-rank test was applied to test the differences in sur-vival across comparison groups.Cox proportional hazard models were applied for calculating hazard ratios(HRs)using the lowest tertile as the reference after adjusting for age,menopausal status,TNM stages,and estrogen receptor/progesterone receptor (ER/PR)status.All P-values are2-sided.SAS software was used for statistical analysis(v.9.1;SAS Institute, Cary,NC).
RESULTS
Table1presents the characteristics of study partici-pants,which included471breast cancer patients and 39BBD patients.The mean age was47.5years for breast cancer patients and43.7years for BBD patients.In BBD patients,53.8%were nonproliferative lesions,whereas among proliferative benign breast disease fibroadenoma was the most common,
accounting for33%of the total benign diseases diag-nosed,and only1subject had atypical hyperplasia. There were23.8%breast cancer patients diagnosed at an early stage(0or I),40.3%at stage IIa,26.3%at stage IIb,and9.6%at stage III or IV.Most of the breast cancer patients(66.6%)had a stage II cancer,and vir-tually all patients received surgery(100%)or chemo-therapy(96.8%).Radiotherapy was given to45.2%of patients,whereas76.9%received tamoxifen therapy. The reason for the high chemotherapy percentage in our study is due to the recommendation in China that neoadjuvant chemotherapy is a major adjunctive treatment for breast cancer.21,22
Table2compares the expression levels of the ATM gene in tumor tissues and adjacent nonneoplastic tis-sues in cancer or BBD patients.ATM expression levels were significantly lower in cancer tissue than in paired adjacent normal tissues(P<.001),whereas ATM expression in benign tumor tissues were similar to their corresponding adjacent tissues(P?.433).Fur-ther,there was no significant difference between tu-mor tissues and the adjacent normal tissues in either nonproliferative or proliferative cases among BBD patients(P?.061and P?.463,respectively).When cancer patients and BBD patients were compared, cancer tissues showed a remarkably reduced ATM expression compared with benign tumor tissues (P<.001).The expression levels of this gene in the ad-jacent normal tissues from cancer patients and BBD patients,however,were comparable(Table2).We analyzed the ATM mRNA expression in breast cancer tissue with respect to breast cancer stage.However, no significant association was observed between ATM expression level and TNM stage(data not shown).In addition,ATM expression levels were not related to radiotherapy in either adjacent normal tissues or in tumor tissues of breast cancer patients(data not shown).This is not surprising because tissue samples were collected before any cancer therapies.
The median follow-up time for breast cancer patients was approximately7years.Table3presents HRs and95%CIs after adjusting for potential TABLE1
Characteristics of Study Participants
Participant characteristics No.of subjects Percentage
Breast cancer patients(n?471)
Age,y
<4518038.2
45–4912526.5
!5016635.3
Mean?SD?47.5?7.6
TNM stage
0$I11223.8
IIa19040.3
IIb12426.3
III$IV459.6
ER/PR status*
ER-positive24966.2
PR-positive25067.0 Cancer therapy received
Surgery471100.0
Chemotherapy45697.0
Radiotherapy18945.3
Tamoxifen30376.9 Benign breast disease patients(m?39)
Age,y
<452051.3
45–491435.9
!50512.8
Mean?SD?43.7?5.7 Histopathologic classification
Nonproliferative2153.8
Proliferative/atypical hyperplasia1846.2
SD indicates standard deviation;ER,estrogen receptor;PR,progesterone receptor.
*Excluding95patients with missing data on ER status and98patients with missing data on PR status.
ATM Expression and Breast CA Survival/Ye et al.1731
confounding factors,including TNM stage,ER/PR sta-tus,radiotherapy,menopausal status,and https://www.wendangku.net/doc/d57250188.html,-pared with patients with the lowest tertile of ATM expression in cancer tissue,patients within the upper 2tertiles of ATM expression had a more favorable DFS and,to a lesser extent,OS.Results from stratified analyses by TNM stage are also presented in Table3, showing that the inverse association of breast cancer survival with ATM expression exists in both earlier and later stage cancer patients.The curves for OS and DFS are shown in Figure1.As expected,patients in the upper2tertiles of ATM expression in breast cancer tissue had better OS and DFS compared with patients with the lowest ATM expression level throughout the study period.
We conducted additional analyses treating ATM expression as a continuous variable.Because the ATM expression data were skewed to the right,log-trans-formed ATM expression values were used.An inverse association between ATM expression and breast can-cer risk was observed.The adjusted HRs were0.80(95%CI:0.42–1.51)for OS and0.66(95%CI:0.36–1.24)for DFS.These results were consistent with those based on categorical data analysis(data not shown in table).
DISCUSSION
In this population-based study we found that the ATM mRNA level was significantly lower in breast cancer tissue than the adjacent normal tissue from the same patients or breast tissues from patients with BBD. Furthermore,high ATM expression levels in breast cancer tissue were associated with increased DFS and OS.This finding is new and consistent with the hy-pothesis that the ATM gene may play an important role in breast cancer development and progression.
Only2small studies have examined ATM mRNA expression levels in breast cancer tissues using semi-quantitative competitive RT-PCR methods.6,12In the first study conducted in German women,ATM mRNA expression was evaluated in39breast cancer patients,
TABLE2
Comparison of ATM mRNA Expression Levels Between Tumor Tissues and Tumor Adjacent Normal Tissues in Patients Diagnosed With Breast Cancer or Benign Breast Disease(BBD)
Study groups No.of patients
Median(25th,75th Percentile)
P* Tumor tissue Adjacent nonneoplastic tissue
Breast cancer patients43y 2.9(1.8,5.3) 5.6(3.9,8.3)<.001
BBD patients39 6.0(4.4,7.7) 6.6(5.0,7.9).433
*Derived from paired t-tests.
y TNM stage:0-I:14;IIa:18;IIb:9;III-IV:2.
TABLE3
Association of ATM Gene Expression in Tumor Tissues With Overall and Disease-Free Survival Among Breast Cancer Patients
No.of patients
Overall survival Disease-free survival
Event HR(95%CI)HR*(95%CI)Event HR(95%CI)HR*(95%CI)
All patients
T115842 1.00(reference) 1.00(reference)57 1.00(reference) 1.00(reference) T2156280.63(0.39–1.02)0.56(0.35–0.92)310.51(0.33–0.79)0.46(0.30–0.73) T3157340.74(0.47–1.17)0.70(0.43–1.13)370.59(0.39–0.89)0.52(0.33–0.81) Patients with stage0,I,or IIa cancer
T19716 1.0(reference) 1.0(reference)28 1.0(reference) 1.0(reference) T2102130.77(0.37–1.60)0.77(0.37–1.61)140.44(0.23–0.85)0.43(0.22–0.82) T3103150.81(0.40–1.65)0.87(0.42–1.81)160.49(0.26–0.90)0.50(0.26–0.95) Patients with stage IIb,III,or IV cancer
T16126 1.0(reference) 1.0(reference)29 1.0(reference) 1.0(reference) T254150.56(0.29–1.07)0.58(0.30–1.11)170.63(0.34–1.15)0.64(0.35–1.17) T354190.73(0.41–1.33)0.72(0.39–1.33)210.72(0.41–1.27)0.72(0.40–1.29)
HR indicates hazard ratio;CI,confidence interval;ER,estrogen receptor;PR,progesterone receptor.
*Adjusted for age,menopausal status,TNM stage,radiotherapy,and ER/PR status.
1732CANCER May1,2007/Volume109/Number9
14benign breast lesion patients,and 4unmatched control individuals.6ATM transcript levels were the lowest in breast cancer tissue,followed by benign tu-mor tissue and normal breast tissue specimens.The reduced ATM expression in cancer tissues,however,was not observed in the second study,a hospital-based study of 89Caucasian women with breast cancer.12Both studies used competitive RT-PCR.Although the sensitivity of competitive RT-PCR was high,it may have increased the likelihood of contami-nation and detection of false-positives.23,24We used real-time quantitative RT-PCR method,a more accu-rate and reliable method in assessing gene expression levels.24Our findings were consistent with the first study described above as well as 2other studies,in which ATM protein expression levels were found to be lower in breast cancer tissue than in matched control samples.2,25These findings indicate that ATM mRNA expression is substantially down-regulated in breast cancer tissues,but not in adjacent normal or benign breast tumor tissue.The cellular mechanisms that lead to the deregulation of ATM expression in breast cancer tissue remain to be determined.Losses on chromosome 11q,where the ATM gene is located,are frequently observed in breast cancer cells,which may explain the reduced mRNA level of the ATM gene in some cancer patients.4,6Alternatively,a reduced level of ATM protein expression may be due to methylation of the bidirectional ATM promoter region.26
More favorable survival was found in our study among breast cancer patients whose cancer tissues expressed a high level of ATM mRNA.However,we did not find the interaction between ATM expression and the adjuvant radiotherapy in breast cancer survival.Our findings are novel and biologically plausible.Pre-vious in vitro studies have found that cells defective in the ATM gene appear to be more resistant to irradia-tion-induced apoptosis.27–29In addition,ATM protein activates and stabilizes p53tumor suppressor protein,which results in the induction of G 1,S,and G 2cell cycle arrest,and DNA repair and activation of cell death pathways.3,4,30,31Because radiation and chemo-therapy results in extensive DNA damage,a signal to ATM activation,cancer cells with no ATM or reduced ATM expression may respond poorly to radiation and/or chemotherapy.This,in turn,influences the progno-sis of breast cancer.4,28,31,32Our finding is also sup-ported by a recent study in which reduced ATM expression in breast cancer tissue was found to be correlated with increased microvascular parameters,a prognostic factor for breast cancer.16However,our findings are not consistent with data from some in vitro studies.33,34One study found high ATM gene expression was observed in methotrexate-resistant mouse fibroblast cell lines.33In another study,silen-cing of ATM protein enhanced radiation-mediated cell killing of human cervical cancer cells.34These findings suggest that patients who have a higher ATM expression level may be less sensitive to radiotherapy or chemotherapy,and more likely to have a poor sur-vival rate compared with those who possessed a lower ATM expression.The possible explanation for this inconsistency is that ATM may play an important role in neoangiogenesis and activation of cell death path-ways instead of affecting the responses to adjuvant therapy,16,35,36or ATM may have a different role in dif-ferent cancer types.Therefore,further studies are war-ranted to replicate these findings.
The large sample size of our study provides sta-ble estimates of gene expression patterns and
their
FIGURE 1.Kaplan-Meier survival curve for (A)overall and (B)disease-free survival among breast cancer patients by tertiles of ATM mRNA in tumor tissue.ATM Expression and Breast CA Survival/Ye et al.1733
correlations with cancer survival.In addition,we included both tumor tissue and adjacent normal tis-sue from patients diagnosed with breast cancer or BBD,which enabled a systematic evaluation of expression patterns of the ATM gene based on the type of tissues and diseases.However,it should be noted that although the median length of the follow-up period(84months)is considered adequate,reeva-luation of the data after extension of the follow-up pe-riod might provide additional information.Although we did not adjust for each of the specific prognostic factors,such as invasiveness of cancer,metastatic sta-tus,and node involvement,TNM stages were adjusted in our study and used in stratified analyses,showing that these prognostic factors are unlikely to affect the results from this study.
In conclusion,we found that ATM expression was down-regulated in breast cancer tissues and that a high level of ATM expression in breast cancer tissue was linked to improved survival in breast cancer patients.Our findings,if confirmed,may have signifi-cant clinical implications in identifying high-risk breast cancer patients for cancer treatment and fol-low-up.
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