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10.1245/ASO.2004.02.022
Annals of Surgical Oncology 11:1045-1055 (2004)
© 2004 Society of Surgical Oncology
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ORIGINAL ARTICLES

Differential Association of BRCA1 and BRCA2 Genes with Some Breast Cancer–Associated Genes in Early and Late Onset Breast Tumors

Neelanjana Chunder, MSc, Syamsundar Mandal, PhD, Anup Roy, MD, Susanta Roychoudhury, PhD and Chinmay Kumar Panda, PhD

From the Department of Oncogene Regulation (NC, CKP) and Department of Medical Records (SM), Chittaranjan National Cancer Institute, Kolkata, India; Department of Pathology, Bankura Sammilani Medical College (AR), Bankura, West Bengal, India; and Department of Human Genetics and Genomics, Indian Institute of Chemical Biology (SR), Kolkata, India.

Correspondence: Address correspondence and reprint requests to: Chinmay Kumar Panda, MD, Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata 700026, India; Fax: 91-33-2475-7606; e-mail: ckpanda{at}vsnl.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Accumulating evidence indicating more aggressive features of breast carcinoma (BC) in young women than their older counterparts have raised the question of whether these differences are present at the genetic level.

Methods: For this purpose, we performed a comparative analysis of the frequency of deletions of BRCA1, BRCA2, BRCAX, TP53, ATM, and RB1 and amplification of Cyclin D1 and also studied the interrelation and prognostic significance of these genetic alterations in 30 early onset (≤40 years) and 33 late onset (>40 years) cases of BC. These gene alterations were also studied in 11 other types of breast lesions.

Results: A differential pattern of alterations (deletion/amplification) was observed in the two age groups, with the sequence in younger women being BRCA1 (72%), TP53 (71%), ATM (64%), BRCA2 (62%), RB1 (60%), Cyclin D1 (43%), and BRCAX (24%) and that in the older group being TP53 (66%), RB1 (63%), BRCA1 (56%), ATM (53%), BRCA2 (45%), Cyclin D1 (24%), and BRCAX (23%). Similar, differential correlations were also seen with several clinicopathological parameters, prognosis, and combinations of alterations among these genes in the two age groups.

Conclusions: Differential frequencies and interrelationships of genetic alterations and prognoses in these two age groups indicate that the molecular pathways for the development of tumors in both age groups may not be similar, though the ultimate effect is deregulation of cell cycle checkpoints and defects in the DNA repair pathway.

Key Words: BRCA1 • BRCA2 • Early onset breast cancer • Late onset breast cancer • LOH • Survival


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast carcinoma (BC) is the most common malignancy among females worldwide, and more than 1,000,000 new cases are diagnosed every year.1 BC may be of early or late onset, depending on age at onset. However, the cutoff value ranging from 35 to 50 years for early onset BC varies among investigators.2–10 Despite this discrepancy, younger women with BC exhibit more aggressive pathological features, including larger tumor size, tumors of higher grade (grade III), presence of positive lymph nodes, absence of steroid receptors, and a high S-phase fraction, than do older women with BC.2–4 Moreover, poorer prognosis has been reported for BC in young women,5,6 a finding that suggests that early onset BC may be of a biologically different origin and may be regarded as a separate disease.5 Though several reports have suggested BC affecting younger women may be of a different nature, to date most studies concerning genetic aberrations in BC have not considered the age distribution of patients; hence, there are only a few reports on comparative studies of different age groups at molecular levels. A detailed molecular characterization is required that could shed some light on the difference in the biology and behavioral features of BC among the different age groups.

Most BC cases are sporadic, and 15% to 20% of cases are due to inherited mutations in several BC–predisposing genes.11 A minority of hereditary BC cases are attributable to germline mutations in high-penetrance BC-susceptibility genes BRCA1 and BRCA2. Germline mutations of additional multiple high- to moderate-penetrance genes such as TP53, ATM, PTEN, LKB1, CHK2, and MLH1/MSH2 also predispose individuals to BC, but these cases are rare and are usually part of a multicancer and/or developmental abnormality syndrome.12 Increased risk of other types of cancers has been reported to be associated with BRCA1 and BRCA2 mutation carriers and their family members.11 Efforts are being made to identify additional candidate penetrant genes that could explain most non-BRCA1/BRCA2 BC cases. Genetic linkage analysis has identified several chromosomal regions,13 of which one such BC susceptibility gene locus (BRCAX) is at chromosome 13q21.14 Though separate studies15,16 have found no evidence of linkage for BRCAX at 13q21, this chromosomal region is involved in sporadic BC and other types of cancers.14 However, there are no reports on the role of this locus in early and late onset BC.

Biochemical, genetic, and cytological studies have implicated multiple roles for BRCA1 gene product in the regulation of gene transcription, DNA damage repair, proliferation, and apoptosis. Accumulating data reveal an important role of BRCA1 protein in cell cycle checkpoints following DNA damage.17,18 Many proteins, notably p53, pRB, and BRCA2, have been found to interact with BRCA1.19 In response to DNA damage, the BRCA1 is phosphorylated by the ATM and localizes to the nucleus, where it becomes part of a multiprotein DNA repair complex (BRCA1-BRCA2-BARD1-RAD51) to mediate DNA repair.20 Moreover, cells that lack BRCA1 and BRCA2 accumulate chromosomal abnormalities, including chromosomal breaks, severe aneuploidy, and centrosome amplification.19 The BRCA1 functions as a transcriptional coactivator of p53 by physically associating with it.21 ATM also stabilizes p53 by its phosphorylation through CHK2 that leads to the activation of cell cycle regulator p21. The p21 protein inhibits cyclin E-cdk2, leading to inhibition of pRB phosphorylation and blockage of G1-S phase checkpoint. The pRB phosphorylation is also regulated by cyclin D1-cdk4/6 complex.18 The pRB also regulates the expression of BRCA1 gene through its ability to modulate E2F transcriptional activity.22 Similarly, Cyclin D1 gene expression is reported to be downregulated by induction of BRCA1.23 Contrary to BRCA1, the role of BRCA2 protein in different cellular pathways is less certain. Functional analyses of BRCA2 gene products have established its dual participation in DNA damage repair, along with additional DNA repair proteins such as BRCA1 and RAD51, and transcription regulation that remains largely unexplored.24

Though germline mutations in BRCA1 and BRCA2 are common in women with early onset BC, with BRCA1 and BRCA2 mutations being found in 4% to 6% and 2% to 3%, respectively, of women diagnosed with BC at or before age 35 years,25,26 the frequency of somatic mutations is rare in these two genes. Evidence is emerging to suggest that BRCA1 may be involved in the pathogenesis of sporadic BC by gene underexpression rather than mutation.27 In sporadic BC, frequent loss of heterozygosity (LOH) of BRCA1 in chromosome 17q21.31 and BRCA2 on chromosome 13q13.1 has been found at frequencies ranging from 22% to 70%6,28–32 and 20% to 63%,28,31–35 respectively. Frequent LOH of TP53 (chromosome 17p13.1), ATM (chromosome 11q22.3), and RB1 (chromosome 13q14.2) have been observed in 24% to 67%,6,7,29,31,32,36,37 33% to 48%,28,38,39 and 7% to 61%,33,40 respectively, in sporadic primary BC. The Cyclin D1 gene located at chromosome11q13.3 is frequently amplified (10%–30%) in BC.8 However, Cyclin D1 amplification is reported to be absent in breast tumors of BRCA1 mutation carriers, suggesting distinct separate mechanism of breast tumorigenesis in BRCA1 mutation carriers.41 It may be observed that frequencies of alterations of these genes cover a wide range as reported by different investigators. This may be due to heterogeneity of breast tissues and to differences in the ethnicity, etiology, sample preparation, and methodologies used. Two independent studies suggest that differences in LOH frequencies involving TP53, BRCA1, and BRCA2 exist between early and late onset BC.9,10

To understand the molecular differences in early and late onset breast tumorigenesis, attempts have been made in the present study to analyze the alterations (deletion/amplification) of BRCA1, BRCA2, BRCAX, TP53, ATM, RB1, and Cyclin D1, with use of intragenic markers in 30 early (≤40 years of age) and 33 late onset (>40 years of age) BCs. We have also assessed the alterations of these genes in 11 other types of breast lesions in younger and older women. Potential relationships between alterations at these loci and the prognostic significance and histopathological features of the carcinomas are examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Collection and Clinical Data
Seventy-four primary tumors and their corresponding normal tissues or peripheral blood leukocytes (PBLs) were obtained from 70 unrelated patients with breast tumors who were undergoing surgery at the hospital section of Chittaranjan National Cancer Institute, Kolkata, India. Tissues were frozen immediately and stored at –80°C until further use. Informed consent from both patients and hospital authorities were obtained for sample collection.

Of these 70 patients, 61 presented with carcinoma in at least one breast. Two patients (numbers 2972 and 5287) presented with carcinoma in both breasts, and one patient (number 3924) presented with carcinoma in the right breast and multiple calcified fibroadenoma nodules in the left breast. Two patients (numbers 2610 and 5393) presented with fibroadenosis, and the former had two lumps (T1 and T2) in the same breast at the time of diagnosis. There were three patients with malignant phyllodes, two with benign phyllodes, one with fibroadenoma, and one with atypical ductal hyperplasia (Figs. 1, 2, and 3GoGo).



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  		FIG. 1. Schematic representation of the pattern of alterations of the different genes observed in group A (≤40 years). LOH = loss of heterozygosity; ROH = retention of heterozygosity; NI = noninformative; MA1 = microsatellite size alteration of one allele; L = left tumor; R = right tumor; IDC = invasive ductal carcinoma; ILC = invasive lobular carcinoma; WD = well-differentiated; MD = moderately differentiated; PD = poorly differentiated; ND = not determined; NA = not applicable. Family history ++ = history of breast carcinoma only; Family history + = history of carcinomas other than breast carcinoma.

 


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  		FIG. 2. Schematic representation of the pattern of alterations of the different genes observed in group B (>40 years). HMA1 = microsatellite size alteration of one allele while constitutively noninformative; LMA = loss of one allele and microsatellite alteration of the other allele; other symbol definitions as in Figure 1 footnote.

 


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  		FIG. 3. Schematic representation of the pattern of alterations of the different genes observed in different breast diseases apart from carcinoma. T1, T2 = two lesions from same breast; ADH = atypical ductal hyperplasia; ND = not determined; NA = not applicable.

 
The 61 carcinoma patients were categorized as group A, 28 patients aged ≤40 years at diagnosis (median age, 34 years; range, 20–40 years), and group B, 33 patients aged >40 years at diagnosis (median age, 52 years; range, 42–72 years) (Figs. 1 and 2 Go). Of the 28 patients in group A, two (numbers 2972, 5287) had bilateral carcinomas. All 28 patients in this group were premenopausal females. The distribution of 30 carcinomas in these 28 patients included 28 instances of invasive ductal carcinoma and 2 of invasive lobular carcinoma (patients 3365 and 4734). The distribution of carcinoma stages in this group were stage I, 1 case; stage II, 4 cases; stage III, 22 cases; and stage IV, 3 cases. Among these 30 carcinomas, 20 were found to have lymph node involvement. The 30 carcinomas in this group were classified as grade I (3), grade II (16), grade III (9), or none available (2). One patient (number 5364) had a family history of BC, and two (numbers 2737 and 4262) reported a history of other cancers (uterine, lung, oral) among first-degree relatives.

Among the 33 patients in group B, one (number 3924) had bilateral lesions (discussed earlier in this section). Four of the 33 were premenopausal females and the others were postmenopausal females, with the exception of two males (numbers 5164, 5596). The stage distribution in this group included stage II (10), stage III (22), and stage 1V (1). Among the 33 carcinomas, 28 were found to have positive lymph nodes. The 33 carcinomas were classified as grade I (4), grade II (20), grade III (7), and none available (2). Two patients (numbers 1099 and 3924) reported a positive family history of BC at presentation, and five (numbers 2434, 4832, 4671, 5337, and 6189) reported a history of other types of cancer (colorectal, gastric, oral) among the family members. All these carcinomas were graded and staged according to the UICC TNM classification.42 In the present study, the prevalence of stage III carcinomas in both age groups is due to the fact that in developing countries such as India, most cases are reported in the advanced stages (T3 and T4) because of poor health consciousness among the population.43

Microdissection and DNA Isolation
Usually the normal cells present as contaminants create difficulties in accurate interpretation of results. Thus, these contaminant normal cells in the specimen were removed by a microdissection procedure. More than 50 serial sections (10–20 µm) were taken on glass slides with use of a cryostat (Leica CM 1800, Germany). The representative 5-µm sections from different regions of the specimens were stained with hematoxylin and eosin for diagnosis as well as for marking of tumor-rich regions. These marked regions were meticulously dissected by microdissection procedure. The samples containing >60% tumor cells after microdissection were further analyzed.4

High-molecular-weight DNA from the microdissected tumors and their corresponding normal tissue or PBLs were extracted by proteinase K digestion, followed by phenol-chloroform extraction.44

Molecular Analyses
Deletion analysis of the genes with microsatellite markers
The deletion of the genes was analyzed with use of intragenic microsatellite markers by polymerase chain reaction (PCR) analysis. Three intragenic markers each for BRCA1 (D17S855, D17S1322, D17S1323) and BRCA2 (D13S1699, D13S1701, D13S1695) and one each for TP53 (TP53. PCR15), RB1 (D13S153), ATM (D11S2179), and BRCAX (D13S1308) were used.14,39,45,46,47 The location of the genes/markers was obtained from http://genome.ucsc.edu (Human, assembly July 2003) and primer sequences of these markers were obtained from the Genome Database.

PCR analysis
The isolated DNA were amplified by PCR in a 20-µL reaction mixture that contained 1x PCR buffer (67 mM Tris-HCl [pH, 8.7], 16.6 mM [NH4]2 SO4, 0.01% Tween-20), 1 to 1.5 mM MgCl2, 4 pmol of each primer, 0.2 mM of each dNTP (Gibco-BRL, USA), 0.5 to 1 unit of Taq polymerase (Gibco-BRL), and 25 to 50 ng genomic DNA. One of the paired primers in the reaction mixture was end-labeled with [{gamma}32P] ATP (specific activity, 3000 ci/mmol; Perkin Elmer Life Sciences, USA) with use of T4-polynucleotide kinase (Gibco-BRL).4 The cycle conditions were 95°C for 3 minutes and then 30 cycles of 95°C for 1 minute, 47°C to 62°C (annealing temperature varied with each primer) for 1 minute, and 72°C for 2 minutes, with a final extension step of 7 minutes at 72°C in a thermal cycler (Bio-Rad, USA). The labeled PCR products were electrophoresed on 7% denaturing polyacrylamide gel containing 8 M urea and then autoradiographed.4

Interpretation of LOH and MA
In our analysis, LOH was determined by densitometric scanning (Shimadzu CS-9000) of the autoradiographs. For informative (heterozygous) cases, allelic loss was scored if there was a complete loss of one allele or if the relative band intensity of one allele was reduced at least 50% in the tumor, compared with the same allele in the corresponding normal control DNA.4,48 The value was calculated as the ratio of the band intensities of the larger to the smaller alleles in the tumor DNA, divided by the same ratio in the corresponding normal DNA sample. A LOH index of >1.5 (loss of the smaller allele) or <0.67 (loss of the larger allele) corresponded to at least 50% reduction in the relative band intensities.4,49

Microsatellite size alteration (MA) was scored as present if one or both alleles at a given locus showed variation (either expansion or contraction) in comparison with the same allele in normal control DNA. In calculations of LOH, samples showing homozygosity (NI) and MA only were not considered. Those samples showing both LOH and MA at the same locus were scored as LMA and considered for calculations of both LOH and MA. In case of BRCA1 and BRCA2 genes, three intragenic markers span the entire gene locus. If all three of these markers show LOH or LOH + NI, the entire gene locus is said to be either deleted in one chromosome or partially deleted in both chromosomes. However, overall LOH was considered for BRCA1 and BRCA2 if any one of the intragenic markers exhibited LOH.

Our microsatellite analysis procedure for allelotyping could detect LOH and MA in the presence of 50% and 10% to 30% tumor DNA, respectively.50 Along with normal cell contamination, there is a probability of intratumor heterogeneity in the primary tumor, which could make interpretation of the results difficult. In LOH calculations, we considered the ratio of the upper and lower allele, as mentioned previously.

Cyclin D1 amplification analysis
A quantitative measurement of Cyclin D1 amplification was performed by differential polymerase chain reaction (DPCR) with dopamine D2 receptor gene (DRD2) as a reference sequence.51,52 The primers of both Cyclin D1 and DRD2 genes were coamplified by PCR in the same reaction vessel in a 20-µL reaction mixture containing 1x PCR buffer (67 mM Tris-HCl [pH, 8.7], 16.6 mM (NH4)2 SO4, 0.01% Tween-20), 1 mM MgCl2, 4 pmol of each primer, 0.2 mM of each dNTP (Gibco-BRL), 0.5–1 unit of Taq polymerase (Gibco-BRL), [{alpha}32P] dCTP (specific activity, 3000 ci/mmol; Perkin Elmer Life Sciences), and 50–100 ng template DNA. Amplification cycle conditions were as follows: 95°C for 3 minute and then 30 cycles of 95°C for 30 seconds, 64°C for 1 minute, and 72°C for 1 minute, with a final extension step of 7 minutes at 72°C in a thermal cycler (GeneAmp PCR system 2700, Applied Biosystems, USA). Analysis of radiolabeled PCR products was done with 7% denaturing polyacrylamide gel electrophoresis containing 8 M urea and autoradiography.

Cyclin D1 amplification level (A) in tumor DNA (T) was calculated as follows: A = YT/YN

where YT is the ratio of the peak area (obtained by densitometric analysis of scanned autoradiographs) for the Cyclin D1 PCR product to the peak area for the DRD2 PCR product of tumor DNA and YN is the similar ratio obtained with the corresponding normal DNA. A cutoff value of ≥2.0 average gene copy number, obtained from the ratio of (Cyclin D1/DRD2) index of the tumor and corresponding normal sample, was considered to be positive for Cyclin D1 amplification.51,52

Statistical Analysis of Clinical Data
The association between different variables and the differences in the LOH frequencies between the two age groups were tested with {chi}2 analysis. Survival curves were calculated according to the Kaplan–Meier method. Postoperative overall survival was measured from the date of surgery to the date of last follow-up or death. The log-rank test was used to assess the differences in patient survival between cases with loss and retention of heterozygosity of the genes. Probability value (P value) ≤0.05 was considered statistically significant. A P value between 0.05 and 0.10 was considered to be of borderline significance.53 All the calculations were performed with the statistical program SPSS (SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Alterations (Deletion/Amplification) of the BC-Associated Genes in Early Onset (Group A) and Late Onset (Group B) Carcinomas
The overall incidence and the order of genetic alterations as combined data in the respective gene loci in group A versus group B presented a differential pattern (Figs. 1 and 2Go). The findings are summarized below.

Group A
The overall frequency of genetic alterations in all the investigated gene loci in the carcinomas of this group was 97% (29 of 30) for at least one of the genes/loci tested (Figs. 1 and 4Go). BRCA1 exhibited the highest incidence (72%) of allelic loss, followed by TP53 (71%), ATM (64%), BRCA2 (62%), RB1 (60%), and BRCAX (24%). Cyclin D1 amplification was observed in 43% of the carcinomas.52 Interestingly, of all the carcinomas showing LOH at BRCA1 and BRCA2 loci, 18 (86% of 21) carcinomas exhibited LOH or LOH + NI for all three intragenic markers of BRCA1, and 15 (83% of 18) carcinomas exhibited LOH or LOH + NI for all three intragenic markers of the BRCA2 gene. MA was detected in one tumor (patient 4262) in this age group for BRCA1 only (Figs. 1 and 4Go). It was observed that patient 5364, who reported a family history of BC, exhibited LOH of all the genes tested.



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  		FIG. 4. Representative photograph showing LOH, MA1, and HMA1 in different genes in breast carcinoma samples. T = DNA of tumor cells after microdissection; N = DNA from corresponding normal tissue or PBL; -> indicates loss of the corresponding allele; * indicates size alteration of one allele. The carcinoma numbers and marker loci are indicated above and below each set of lanes, respectively.

 
Group B
The overall frequency of genetic alterations in all the investigated gene loci in this group was 88% (29 of 33) for at least one of the genes/loci tested (Figs. 2 and 4Go). The gene that showed highest frequency (66%) of LOH was TP53, closely followed by RB1 (63%), BRCA1 (56%), ATM (53%), BRCA2 (45%), and BRCAX (23%). Cyclin D1 amplification was observed in 24% of the carcinomas.52 Of all the carcinomas showing LOH at BRCA1 and BRCA2 loci, 17 (94% of 18) exhibited LOH or LOH + NI for all three intragenic markers of BRCA1, and 11 (85% of 13) carcinomas exhibited LOH or LOH + NI for all three markers of the BRCA2 gene. MA was detected in two tumors (patients 1144 and 4370) for BRCA2 only (Figs. 2 and 4Go). Patient 1099, who reported a family history of BC, exhibited LOH of all the genes tested. Interestingly, the right carcinoma of bilateral tumor patient 3924 (who had a familial background of BC) showed LOH at TP53, BRCA1, and BRCA2 and also Cyclin D1 amplification (Fig. 2), but the fibroadenoma from the left breast of this patient showed LOH at only the TP53 gene (Fig. 3).

Statistical evaluation of the differences in the LOH/amplification frequencies in the two age groups revealed no significant differences. However, the high incidence of deletion of more than one marker of BRCA1 and BRCA2 in the majority of the carcinomas in both age groups indicates that these genes are either fully deleted in one chromosome or partially deleted in both chromosomes. This might have been due to nondisjunction, with or without reduplication and structural rearrangements of chromosomes containing these genes.

Analysis of Alterations (Deletion/Amplification) of the BC-Associated Genes in Other Types of Breast Lesions
Genetic alterations were also detected in 5 (45% of 11) other breast lesions with at least one of the genes tested, providing evidence of the importance of these genes in different breast lesions (Fig. 3). None of these tumors showed deletion of the ATM gene. Cyclin D1 amplification was seen in a malignant phyllodes tumor (in patient 5585). Deletions were mostly seen at TP53 and BRCA1 genes, with three tumors in each case showing LOH at these loci. Two malignant phyllodes (in patients 1770 and 5892, who reported a family history of BC) did not show any genetic alterations. Similarly, the case with ADH (patient 3101, who had a family history of other cancers) also did not show any change. Of the two patients with benign phyllodes, number 3970 did not show any genetic changes, whereas deletion of RB1 and BRCAX was found in number 6038.

Association with Clinicopathological Variables and Survival Analysis
Among the various clinicopathological parameters tested, group A carcinomas with deletion of TP53 and BRCA1 were significantly associated with lymph node metastasis (P = .013 and .034, respectively), but a trend toward significance was seen in this group between deletion of TP53 and higher grade (P = .074) and between Cyclin D1 amplification and lymph node metastasis (P = .068). In group B, deletion of BRCA1 was significantly correlated with higher grade (P = .048).

The clinical outcome in the two age groups was investigated for a period up to 5 years. Log-rank test uncovered a statistically significant difference in overall patient survival between the cases with LOH and cases with retention of heterozygosity (ROH) for BRCA1 in group A (P = .057) (Fig. 5) and for BRCA2 (P = .039) in group B carcinomas (Fig. 6). Loss at BRCA2 tended to be associated with short overall survival in group A carcinomas, as the differences were of borderline significance (P = .088). No significant correlation was observed between the other genes and shorter survival in the tumors of both the groups.



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  		FIG. 5. Kaplan–Meier plot for the differences in overall survival among cases with retention (ROH) and loss (LOH) of heterozygosity for BRCA1 in early onset breast carcinoma.

 


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  		FIG. 6. Kaplan–Meier plot for the differences in overall survival among cases with retention (ROH) and loss (LOH) of heterozygosity for BRCA2 in late onset breast carcinoma.

 
Interrelation of the Different Genes
Statistical evaluation of association between the combinations of different genes revealed a significant correlation between deletion of BRCA1 and deletion of TP53 (P = .003) as well as between deletion of ATM and deletion of BRCAX (P = .033) in group A (Figs. 7 and 8GoA).



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  		FIG. 7. Association of deletions and amplification of different genes in group A (≤ 40 years) and group B (>40 years).

 


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  		FIG. 8. Schematic representation of the interrelations among different genes (A) in early onset carcinoma and (B) late onset carcinoma. Solid arrows represent significant interrelation between the genes, as observed in Figure 7. Dashed arrows show probable interrelation between the genes not observed in this study.

 
In group B, deletion of BRCA1 was significantly associated with deletion of ATM(P = .02) and Cyclin D1 amplification (P = .031) and also deletion of BRCA2 significantly correlated with deletion of ATM (P = .047) and Cyclin D1 amplification (P = .017), and a trend toward significant association was observed for TP53 gene deletion and loss of BRCAX (P = .064) (Figs. 7 and 8GoB). No statistically significant link between the allelic alterations was detected between other genes in the two age groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we analyzed the alterations (deletion/amplification) of BRCA1, BRCA2, and other BC-associated genes(BRCAX, TP53, ATM, RB1 and Cyclin D1) in a subset of early onset (≤40 years of age) and late onset (>40 years of age) BCs to see if there is any difference in the pattern of alterations of these genes in the two age groups. Moreover, these genetic alterations were also compared in breast lesions other than carcinoma. Genetic alterations are present in both carcinomas and other breast lesions, irrespective of age at onset, but the frequencies of alterations in BC are higher than in other breast tumor types. However, the incidence of genetic alterations in breast lesions other than carcinoma indicates the involvement of these genes in development of these tumors. It is not yet clear whether there is any similarity in the molecular pathways in tumorigenesis between BC and other breast tumor types.

In comparison with the frequencies of genetic alterations in the carcinomas in the two age groups, it is evident from our data that the molecular pathogenesis in early and late onset BCs is not similar. The frequencies of deletion of TP53, BRCA1, BRCA2, and ATM and of amplification of Cyclin D1 are higher in younger women, whereas deletion of RB1 and BRCAX are comparable in both age groups. Similar to our findings, one study showed that both BRCA1 and TP53 are deleted in higher frequencies in early onset BC than in late onset BC.9 Another study further supported the finding that along with both BRCA1 and TP53, BRCA2 is also frequently deleted in younger women.10 In the two age groups, we included patients with a family history of cancer as well as sporadic cases. We did not observe much difference in the deletion pattern among the carcinoma patients with a family history of cancer and sporadic cases, although the number of patients with a positive family history was not sufficient to draw definite conclusions in this study.

Deletion of BRCA1 has been shown to be of more or less equal frequency in both sporadic and familial cases.30 However, a higher incidence of loss of several chromosomal arms, including 3p, 6q, 11p, 11q, 13q, and 17p, and instabilities of chromosome 9p23–24 have been reported to occur in carriers of BRCA2 mutations.36,54,55 Though somatic mutations of BRCA1 and BRCA2 are rare in sporadic BC, somatic mutations along with high frequency of allelic loss of BRCA2 have been observed in sporadic male BC.35 Low incidence of MA is consistent with our previous findings concerning chromosome14 and 1152 that MAs are a rare phenomenon in BC, though we have observed higher frequencies of MAs in primary head and neck squamous cell carcinoma, using the same set of markers of chromosome 11.56

Previous studies have shown that BCs arising in younger women display more aggressive biopathological features than those occurring in older women. In this study, we observed an overall differential association between alterations of the genes and nodal involvement, higher grade, and unfavorable outcome in the two groups. Deletions of BRCA1 and TP53 seem to occur preferably in BC with lymph node metastasis in early onset patients, as it is associated with positive nodal involvement, a finding consistent with that in another study on BC.9 Though we have seen a close trend toward association of Cyclin D1 amplification with positive nodal involvement in early onset tumors, no such age-specific association has been reported earlier. However, positive association of Cyclin D1 amplification with nodal involvement with BC unselected for age has been reported.57 Similarly, deletion of only BRCA1 is significantly associated with higher grade of disease in older women, which is supported by another report.10 Deletion of BRCA1 and BRCA2 was associated with unfavorable clinical outcome in early onset patients and with BRCA2 in late onset patients in our study. There have been no comparative reports regarding the association of poor survival with BRCA1 and BRCA2 deletions in early and late onset BC. However, the significant association of deletion of BRCA1 with poor overall survival in early onset carcinomas has been reported.9 LOH at BRCA2 has been associated with reduced patient survival in sporadic cases unselected for age.34 The association of BRCA2 deletion with poor outcome in both age groups and the association of deletion of BRCA1 with survival in only early onset patients are indicative of age-specific prognostic significance in poorer outcome. More patients need to be screened to provide concrete evidence of these age-specific associations.

No association was found between deletions of TP53, ATM, RB1, and BRCAX and amplification of Cyclin D1 and poor survival in both age groups in our study. This result is supported by lack of association of TP53 LOH with shorter survival in early onset BC7 but is contradicted in another study.9 Similarly, survival is significantly reduced among patients with sporadic tumors that exhibit LOH of ATM.39

The interrelations among the different genes in the two age groups are not similar. In the early onset breast carcinomas, two interrelated molecular pathways, BRCA1-TP53 and ATM-BRCAX, are seen, and these interrelations are not seen in older women. Similarly, in late onset carcinomas, the significantly interrelated pathways ATM-BRCA1-Cyclin D1, ATM-BRCA2-Cyclin D1, and TP53-BRCAX are not seen in younger women. These molecular pathways are shown to be involved in either cell-cycle regulation or DNA repair,18,19,23 but the functional relevance of these pathways in progression of the tumor is not clear. Further study needs to be done to find out if there is any significance of these pathways in tumorigenesis.

Also, the significant association of the deletions of BRCA2 with ATM and Cyclin D1 amplification indicates that there may be some functional interrelations among these genes. On the other hand, the association of deletions of BRCAX with ATM and TP53 indicates that the candidate tumor suppressor genes (TSGs) located in the BRCAX locus may have some role in breast tumorigenesis in both age groups. Chromosomal region 13q21 is implicated in a variety of malignant tumors, including BC,14 and harbors putative TSGs that may play a role in breast tumorigenesis.58,59 However, no candidate TSG(s) have yet been identified from this region. The significant association of the alterations of these genes indicates the deletion/amplification of one of these genes may impose a selective pressure on the other genes to be deleted/amplified for the development of the tumor. However, we have not observed any associations between BRCA1-BRCA2, ATM-BRCA1, and Cyclin D1-BRCA1 in early onset carcinomas and BRCA1-BRCA2 and BRCA1-TP53 in late onset carcinomas, though these genes have established functional linkage in cell cycle regulatory and DNA repair pathways. This may be due to the fact that there are other types of genetic alterations (mutation or methylation) other than deletions occurring in the TSGs analyzed. Thus, the different types of genetic alterations need to be analyzed to determine the interrelations of these genes in breast tumorigenesis.

It can be concluded from our study that the molecular pathways for tumorigenesis in both age groups of patients with breast carcinomas may not be similar. The prognostic significance of BRCA1 and BRCA2 in these two age groups also supported the finding. Though the network of association between the genes in the two age groups is not similar, the resultant effect of the alterations in both age groups may lead to deregulation of the cell cycle checkpoints and the impairment of the DNA repair pathways and gross chromosomal abnormalities. Further analysis of other types of genetic alterations (mutation or methylation) of these genes and their comparative analysis may shed light on their differential roles in the molecular pathogenesis of early and late onset breast carcinoma.


   ACKNOWLEDGMENTS
 
The authors are grateful to the Director, Chittaranjan National Cancer Institute, Kolkata, India, for active support and thank Dr. D. Basu, Dr. J. Biswas, and Dr. J. Majumdar for their help in this work. Neelanjana Chunder is supported by a doctoral studentship from the University Grants Commission, Government of India.


   FOOTNOTES
 
The authors studied molecular alterations of BRCA1 and BRCA2 along with some other breast cancer–associated genes in early and late onset breast carcinoma. Their results suggest that these genes are indeed differentially altered in these two age groups.

Received for publication February 23, 2004. Accepted for publication August 12, 2004.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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