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Keywords:

  • BRCA1;
  • methylation;
  • epigenetics;
  • prognosis;
  • non–small cell lung cancer

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

BACKGROUND:

Even after early detection and curative resection of early stage non–small cell lung cancer (NSCLC), a significant fraction of patients develop recurrent disease. Molecular biomarkers that can predict the risk of recurrence thus need to be identified to improve clinical outcomes.

METHODS:

Using the methylation-specific polymerase chain reaction assay, promoter methylation of the breast cancer susceptibility gene 1 (BRCA1) was assessed in cancer tissues from 70 patients with curatively resected stage I NSCLC. The clinical relevance of BRCA1 methylation status was evaluated in terms of outcome of the disease.

RESULTS:

Methylation of the BRCA1 promoter was detected in 13 of 70 patients (18.6%). Multiple logistic regression analysis revealed that BRCA1 methylation was an independent risk factor for recurrence (P = .0197) and that patients with BRCA1 methylation demonstrated significantly poorer recurrence-free survival compared to those without (P = .0139). Cox's proportional hazard regression analysis revealed that BRCA1 methylation was an independent risk factor for recurrence-free survival (P = .0155).

CONCLUSIONS:

Methylated BRCA1 can be a potential biomarker that predicts the prognosis after curative resection of stage I NSCLC. Considering that BRCA1 plays a role in chemotherapy-induced apoptosis, it is plausible that identification of methylated BRCA1 could provide information that is clinically relevant to tailored adjuvant therapy. Cancer 2013. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Lung cancer has become the leading cause of cancer deaths in many countries. The treatment strategy chosen for patients with lung cancer is generally guided by tumor–node–metastasis (TNM) classification. Surgery with curative intent is the standard treatment of choice for patients with stage I non–small cell lung cancer (NSCLC). However, even after complete resection of stage I NSCLC, there is a wide spectrum of outcomes.1,2 The possibility that molecular biomarkers might better predict biological characteristics and outcomes than TNM classification should be investigated.3

A growing body of evidence indicates that aberrant methylation of cytosine-guanine dinucleotide (CpG) islands in the promoter regions of tumor suppressor genes silences these genes by blocking transcription.4–7 Promoter methylation in various tumor suppressor genes has been demonstrated to be involved in the development and/or progression of lung cancer3,8–10 and has thus been used as a molecular biomarker to accurately predict the outcome of disease.11

Germline mutations in the breast cancer susceptibility gene 1 (BRCA1) elevate the risk of breast and ovarian cancer development;12 hypermethylation of the BRCA1 promoter has been demonstrated in sporadic breast and ovarian cancers.13,14 It is now known that BRCA1 is a tumor suppressor gene that encodes a multifunctional protein that is involved in both breast and ovarian cancers.15 In addition, germline mutations in BRCA1 are a statistically significant risk factor in the prognosis of pancreatic and cervical cancers.16 Several studies have investigated the clinical relevance of messenger RNA expression17 and methylation of the BRCA1 promoter18,19 in lung cancer, with emphasis primarily on responses to chemotherapy.20 The relationship between epigenetic modifications of BRCA1 and the prognosis of curatively resected stage I NSCLC has not been elucidated.

In this study, we examined the methylation status of the BRCA1 promoter in 70 patients with curatively resected stage I NSCLC. Using methylation-specific polymerase chain reaction (PCR), we investigated the possible association of BRCA1 methylation with the outcome of this disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Study Population

A total of 70 patients who underwent complete resection for stage I NSCLC at National Hospital Organization Kure Medical Center and Chugoku Cancer Center in Japan between June 2005 and March 2009 were enrolled in this study. The end of the follow-up period was defined as December 2011. No patients received any preoperative chemotherapy or radiation therapy. Patients who underwent surgery for Noguchi Type A or B tumors were excluded from this study. The demographic data and clinicopathological features of enrolled patients were collected using an institutional database. Written, informed consent was obtained from all enrolled patients, and the protocol for this study was approved by the institutional review board.

Sample Preparation

Surgically resected lung tissue samples were immediately snap-frozen and subsequently stored in liquid nitrogen. Genomic DNA was extracted from both primary tumors and adjacent normal lung tissue, through use of proteinase K digestion and phenol-chloroform extraction followed by ethanol precipitation, as described.21

Bisulfite Modification and Methylation-Specific PCR

For sodium bisulfate modification, DNA was digested using BamHI (New England Biolabs, Ipswitch, Mass), and 1 μg of the digested DNA was denatured in 0.3 N NaOH at 37°C for 15 minutes. The samples underwent 15 cycles of 30-second denaturation at 95°C and 15-minute incubation at 50°C in 3.1 N sodium bisulfite (pH 5.0) and 0.5 mM hydroquinone. The product was desalted with the Wizard DNA cleanup system (Promega), and desulfonated in 0.6 N NaOH. The sample was ethanol-precipitated and dissolved in 20 mL of TE (Tris plus ethylenediamine tetraacetic acid) buffer. Methylation-specific PCR was carried out using 1 μL of the sodium bisulfite–modified DNA; primers for amplification were specific for methylated or unmethylated sequences, as described.22–24 The primer sequence and PCR conditions for BRCA1 were as follows: unmethylated forward primer (5′-TGGTAGTTTTTTGG TTTTTGTGGTAATG), unmethylated reverse primer (5′-TCAACAAACTCACACCACACAATCA), methylated forward primer (5′-CGGTAGTTTTTTGGT TTTCGTGGTAACG), methylated reverse primer (5′-TCAACGAACTCACGCCGCGCAATCG), for 37 cycles and annealing at 66°C. All procedures were repeated at least 3 times for each sample.

Immunohistochemical Analysis

Paraffin blocks of resected tumors were cut into 5 -μm slices, then processed using standard deparaffinization and rehydration techniques. A monoclonal antibody against BRCA1 (1:500; Calbiochem, Billerica, Mass) was used as the primary antibody for detecting protein expression. Immunodetection was performed by incubation with a specific biotinylated secondary antibody followed by use of the Vectastain ABC kit (Vector Laboratories, Burlingame, Calif). 3,3′-Diaminobenzidine (BD Biosciences, Franklin Lakes, NJ) was used as the developing reagent followed by a hematoxylin counterstain.

Statistical Analysis

Associations between clinicopathological characteristics and BRCA1 methylation were determined using the Fisher's test. Logistic regression analysis was performed to estimate the odds ratios of independent factors for recurrence. The effect of promoter methylation on time to death or recurrence was estimated using the Kaplan-Meier method, and the log-rank test was used to analyze differences between groups. Overall survival was calculated from the date of surgery to the date of death or the last follow-up. Recurrence-free survival was calculated from the date of surgery to the date of recurrence or the last follow-up. Cox's proportional hazard regression analysis was used to analyze the hazard ratios of independent factors for survival. Differences were considered statistically significant with a P value < .05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

DNA Methylation Profiles of Tumor Tissues in the Evaluation Set

To identify a prognostic marker associated with recurrence, DNA methylation in 14 genes (p16, Cyclin-dependent kinase inhibitor 2A; APC, Adenomatous polyposis coli; CDH1, Cadherin 1; RARB, Retinoic acid receptor beta; BRCA1, Breast cancer susceptibility; TIMP3, Tissue inhibitor of metalloproteinase-3; RASSF1A, Ras association domain family member 1; p27, Cyclin-dependent kinase inhibitor 1B; FHIT, Fragile histidine triad; MGMT, O6-methylguanine-DNA methyltransferase; TERC, Telomerase RNA component; MLH1, Human mutL homolog 1; GSTP1, Glutathione S-transferase P 1; and MASPIN, Serpin peptidase inhibitor) was analyzed in selected patients, who were matched with respect to sex, age, histology, stage, and differentiation as the evaluation set. In this evaluation set, patients with recurrence showed a significantly higher frequency of BRCA1 methylation compared with patients without recurrence, and the methylation status in any of the remaining genes did not show statistically significant association with recurrence in this evaluation set (data not shown).

BRCA1 Methylation in Resected Stage I NSCLC

Figure 1A shows representative results of methylation-specific PCR for BRCA1. The overall frequency of methylation of the BRCA1 promoter was 18.6% (13 of 70 patients, Table 1). The proportion of patients with methylated BRCA1 was quite similar to that observed in previous studies.25 Five of 13 patients with BRCA1 methylation in tumor tissues had BRCA1 methylation in their matched noncancerous tissues as well (Table 1). Three of these 5 patients developed recurrent tumors (multiple lung metastases were diagnosed at 2 years after surgery in 1 patient; bone metastasis was diagnosed at 7 months after surgery in 1 patient; and brain metastasis was diagnosed at 7 months after surgery in 1 patient).

thumbnail image

Figure 1. (A) Analysis of BRCA1 gene methylation is shown in stage I non–small cell lung cancer (NSCLC). Methylation-specific polymerase chain reaction of BRCA1 was performed using primers specific for unmethylated (U) and methylated (M) forms of BRCA1 in tumor tissue. BRCA1 methylation was detected in tumor tissues of patients 2 and 4 (arrow). (B) Immunohistochemical analysis of BRCA1 proteins is shown in resected NSCLC specimens. Positive immunoreactivity is visible as a brown precipitant in cell nuclei. Representative expression of BRCA1 protein in the patient without BRCA1 methylation is shown (panel a), and a concordant lack of immunoreactivity is found in the patient with BRCA1 methylation (panel b). Normal adjacent lung tissue with expression of BRCA1 protein is shown as the positive control (panel c). Original magnification, ×400.

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Table 1. Comparison of BRCA1 Methylation Status Between Cancerous and Matched Noncancerous Adjacent Tissue
StatusCancerous TissueMatched Noncancerous Tissue
(n = 70)(n = 70)
  • a

    These 5 patients showed BRCA1 methylation in both cancerous and matched noncancerous tissues.

BRCA1Methylated13 (18.6%)5a
 Unmethylated57 (81.4%)65

Images showing representative immunohistochemical staining of BRCA1 protein are shown in Figure 1B. Staining within nuclei of tumor cells was considered positive. Immunohistochemical analysis confirmed that all tumors with BRCA1 promoter methylation showed an absence of or marked reduction in BRCA1 expression; tumor cells without methylation contained considerably more BRCA1 protein in their nuclei. As described in previous studies, BRCA1 expression levels were associated with promoter hypermethylation status.19,26

Correlation Between Disease Outcome and BRCA1 Methylation Status

Demographics and clinical characteristics according to BRCA1 methylation status are shown in Table 2. There was no statistically significant relationship between BRCA1 methylation status and any of the clinicopathological features that we analyzed.

Table 2. Analysis of Baseline Demographics and Clinical Characteristics of Study Population by BRCA1 Methylation Status
Characteristic BRCA1P
MethylatedUnmethylated
(n = 13)(n = 57)
  1. Abbreviations: AD, adenocarcinoma; SQ, squamous cell carcinoma.

Age, y 65.2 ± 2.668.7 ± 1.2.2193
SexFemale512 
 Male845.2801
HistologyAD1039 
 SQ213 
 Others15.8204
DifferentiationWell624 
 Moderately, poorly733.7901
Lymphatic duct infiltration+617 
740.3297
Vessel infiltration+720 
637.2258
Pathological stageIA836 
IB521.9132
SmokingCurrent, former946 
 Never411.5141
Chronic lung disease+824 
533.2323
Any prior tumor+013 
1344.1074

Table 3 shows the odds ratio for the risk of recurrence in clinicopathological variables by univariate logistic regression analysis. The degree of differentiation, vessel infiltration, and BRCA1 methylation status were statistically significant as predictors for recurrence (P = .0347, P = .0300, and P = .0369, respectively). Multiple logistic regression analysis of clinicopathological variables that were significant by univariate analysis revealed that BRCA1 methylation status was an independent risk factor for recurrence (P = .0197, Table 4).

Table 3. Univariate Logistic Regression Analysis of Clinicopathological Valuables for Recurrence
Factors RecurrenceOR95% CIP
+; Event−; Unevent
n = 12n = 58
  1. Abbreviations: AD, adenocarcinoma; CI, confidence interval; OR, odds ratio; SQ, squamous cell carcinoma.

Age > 70 yYes/no7/523/352.13040.6079−7.9791.2346
SexFemale/male2/1015/430.57330.0820−2.5028.4844
HistologyAD/SQ, others6/643/160.34880.0948−1.2697.1083
DifferentiationWell/moderately, poorly2/1028/300.21430.0311−0.9030.0347
Lymphatic duct infiltration+/−5/718/401.58730.4207−5.6656.4820
Vessel infiltration+/−8/419/394.10531.1448−17.0146.0300
Pathologic stageIA/IB6/638/201.89990.5310−6.8335.3177
SmokingCurrent, former/never11/144/143.49990.5970−66.7958.1861
Chronic lung disease+/−8/424/342.83330.7974−11.6248.1084
Any prior tumor+/−3/910/481.60000.3142−6.5550.5417
BRCA1 methylation+/−5/78/504.46431.0996−17.8341.0369
Table 4. Multiple Logistic Regression Analysis of Clinicopathological Variables Found to Be Significant by Univariate Analysis
Variables OR95% CIP
  1. Abbreviations: CI, confidence interval; OR, odds ratio.

BRCA1 methylation status(+/−)6.34501.3503−33.4334.0197
Differentiation(Well/moderately, poorly)0.17290.0226−0.8019.0232
Vessel infiltration(+/−)1.54300.3648−6.7321.5500

Prognostic Impact of BRCA1 Methylation

The median follow-up time of patients in this study population was 1350 ± 458 days. Patients without event survived for at least 900 days after surgery by the end of the follow-up period. Three-year survival rates were 89.5% for overall survival and 83.9% for recurrence-free survival. Recurrence-free survival rates in patients with BRCA1 methylation were significantly poorer than in those without BRCA1 methylation (P = .0139, Fig. 2). Overall survival did not reach statistical significance between groups assigned by methylation status (P = .2588, data not shown). Cox's proportional hazard regression analysis revealed that BRCA1 methylation was an independent risk factor for recurrence-free survival (P = .0155, Table 5). These data indicate that BRCA1 methylation could be used as a biomarker for predicting the disease outcome after curative resection of stage I NSCLC. Of the 13 patients with BRCA1 methylation, 5 developed recurrent tumors. Two of these 5 patients received chemotherapy (paclitaxel and carboplatin in 1 patient; gefitinib in 1 patient) after diagnosis of the recurrent tumors. Two patients were followed without any intensive therapy, and 1 patient received radiation therapy for brain metastasis. Two patients (paclitaxel and carboplatin, 1; best supportive care, 1) survived more than 3 years after the diagnosis of recurrence. In contrast, 7 patients without BRCA1 methylation developed recurrent tumors. One of these 7 patients survived more than 3 years after surgical resection of pulmonary metastases that had been diagnosed 1 year after the resection of his primary lung cancer. Four of these 7 patients survived less than 4 months after diagnosis of their recurrent tumors and died of cancer; 2 patients survived less than 6 months, with recurrent tumors by the end of the follow-up period.

thumbnail image

Figure 2. Patient survival is classified by BRCA1 methylation status. Recurrence-free survival differed significantly between patients with BRCA1 methylation and those without (P = .0139). P value was calculated using the log-rank test.

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Table 5. Cox's Proportional Hazard Regression Analysis of Prognostic Variables Found to Be Significant by Univariate Analysis
VariablesUnivariate AnalysisPMultivariate AnalysisP
HR95% CIHR95% CI
  1. Abbreviations: CI, confidence interval; HR, hazard ratio.

BRCA1 methylation status(+/−)3.69341.0923−11.5869.03664.72561.3780−15.0518.0155
Differentiation(Well/moderately, poorly)0.23200.0356−0.8807.03020.18640.0283−0.7219.0129

Correlation Analysis of Combination Genes With BRCA1

Because the methylation status of p16, APC, CDH1, RASSF1A, and FHIT were reported to predict the risk of recurrence in curatively resected stage I NSCLC,3,8 the association between methylation of these genes and recurrence was assessed in all 70 study patients. Promoter methylation was detected in 20.0% for p16, 34.3% for APC, 52.9% for CDH1, 30.0% for RASSF1A, and 27.1% for FHIT. Among these genes, p16 methylation, which was demonstrated to have association with recurrence by Brock et al,3 was not significantly but marginally associated with recurrence in our patients (odds ratio, 3.89; 95% confidence interval (CI), 0.972-15.170; P = .0546). Methylation of the other 4 genes did not show statistically significant association with recurrence. After combination analyses, methylation of either p16 or BRCA1 was associated with an increased risk of recurrence (odds ratio, 13.13; 95% CI, 3.052-91.744; P = .0003), and significantly poorer recurrence-free survival (P = .0004). Methylation of either FHIT or BRCA1 was associated with an increased risk of recurrence (odds ratio, 6.16; 95% CI, 1.628-30.228; P = .0067), and significantly poorer recurrence-free survival (P = .0070).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Hypermethylation of CpG islands in the promoter region of various genes has been reported in lung cancer. Gene silencing through epigenetic alterations has been widely implicated in a variety of pathologic processes, including cancer induction and progression.3,8–10 Although many investigators have studied various clinicopathological features that might predict the outcome of curatively resected lung cancer, the clinical relevance of aberrant promoter methylation to the outcome of disease has been investigated by only a few groups. These investigations have identified aberrant methylation of specific genes or cohypermethylation of several genes as risk factors for recurrence. Recent reports have identified p16, H-cadherin 13 (CDH13), RASSF1A, APC, and FHIT as risk factors for recurrence in patients with stage I NSCLC treated by curative surgery.3,8 Results of multivariate analysis in this study indicate that BRCA1 methylation had significant effects on the outcome of the disease, even in curatively resected stage I NSCLC. Moreover, correlation analysis demonstrated that the combination of BRCA1 with p16 or FHIT seemed to be the better predictor of the outcome of disease; however, because the cohort of this study was small, future prospective studies using larger sample sizes are needed to investigate how methylation of BRCA1 contributes to the outcome of curatively resected stage I NSCLC.

BRCA1 was the first breast cancer susceptibility gene to be identified (in 1990).27 The majority of this gene was cloned in 1994 by Miki and colleagues.28 Previous studies indicate that higher BRCA1 methylation levels in breast cancer correlate with more advanced tumor stages at diagnosis and are associated with a 45% increase in mortality compared with patients who have unmethylated BRCA1 promoters.29 In our patients, the frequency of BRCA1 methylation in resected stage II or III NSCLC was 14.6% (data not shown), compared with 18.6% in stage I patients. These data indicate there is no apparent relationship between BRCA1 methylation level and tumor stage in lung cancer. We demonstrated that methylation of the BRCA1 promoter gene correlates with lower recurrence-free survival in patients with curatively resected pathological stage I NSCLC; however, BRCA1 promoter methylation status was not associated with any clinicopathological features, including pathological stage. Interestingly, 5 patients showed BRCA1 methylation in tumor tissue and adjacent normal lung tissue as well. Three of these 5 patients developed recurrent tumor; however, the characteristics of recurrence status seem to be nonspecific. Although it is not clear whether these 5 patients possessed germline methylation of BRCA1, positive unmethylation status was demonstrated in noncancerous tissue, and the degree of methylation in noncancerous tissue was not strong compared with cancerous tissue. Furthermore, epigenetic alteration initiated by exposure to carcinogens in tobacco smoke is one of the main causes of lung cancer, and tobacco smoking plays a significant role in the prognosis of patients with lung cancer.30 Despite these facts, no association between BRCA1 promoter methylation and smoking status was observed in this study.

Several molecular markers have been identified for use as both prognostic tools and targets for novel therapeutic approaches. Such molecular markers also help to identify patients who would benefit from specific anticancer therapies. A growing body of evidence indicates that BRCA1 plays a central role in DNA repair and in cell cycle control.31,32 A lack of functional BRCA1 leads to increased sensitivity of tumor cells to molecular damage, suggesting that BRCA1 could be used as a predictive molecular marker to identify patients who would benefit from specific anticancer therapies. According to the results of several investigations, BRCA1 confers sensitivity to apoptosis induced by antimicrotubule drugs (eg, paclitaxel and vincristine), but induces resistance to DNA-damaging agents (eg, cisplatin and etoposide) and radiotherapy.33–36 Because adjuvant chemotherapy is currently a matter of great debate, particularly regarding its use in curatively resected NSCLC, BRCA1 methylation could be a promising molecular marker for predicting not only the disease outcome but also the effectiveness of chemotherapy. In this study, the small sample size limited the assessment of differences in the effects of chemotherapy between patients with methylated BRCA1 and those without. Further investigations using a larger sample size are needed to determine whether assessment of BRCA1 methylation status provides clinical information relevant to tailored adjuvant therapy.

In summary, we observed methylation of the BRCA1 promoter in 13 of 70 (18.6%) cases of curatively resected stage I NSCLC and determined that such methylation was an independent risk factor for tumor recurrence. BRCA1 methylation status was not associated with any specific clinicopathological features, including pathological stage. These results indicate that BRCA1 methylation plays an important role in the progression of NSCLC and that BRCA1 methylation is a promising biomarker that predicts the outcome of disease after curative resection of stage I NSCLC. BRCA1 has been recognized as a promising genetic determinant of responses to different types of chemotherapy; therefore, further studies using larger samples are warranted to investigate the usefulness of determining a patient's BRCA1 methylation status. Such studies should be designed with a special emphasis on establishing customized adjuvant treatment strategies for patients with curatively resected stage I NSCLC.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

This work was supported, in part, by Grants-in-Aid for the Third-term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan; Grants-in-Aid for Cancer Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan; Research Funding from Kyowa Hakko; and Research Funding from Daiwa Securities Health Foundation.

CONFLICT OF INTEREST DISCLOSURE

The authors made no disclosure.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES