Genetic changes in bilateral breast cancer by comparative genomic hybridisation

Abstract The aim of this study is to find any specific genetic defect occurring frequently in bilateral breast cancer by examining the genetic changes of each chromosome using comparative genomic hybridisation (CGH). CGH was conducted for 36 breast cancer tissues taken from patients treated with surgery for bilateral breast cancer. Tumour and control DNAs were hybridised to metaphase chromosome with differential staining with fluorescein and rhodamine-dUTP. An average rate of green (DNA of tumour cell) against red (DNA of a normal peripheral blood lymphocyte) was calculated in these captured metaphase chromosomes and a ratio of more than 1.17 was defined as an acquisition, less than 0.85 as a loss and, finally, more than 2 as amplification. Twenty-six out of 36 cases (72.2%) showed a change in the number of DNA copies by CGH in one or more regions of gene. On average, 5.3 alterations for each chromosome (range, 1–14) were found, and gain was present more than loss at a ratio of 1.3:1. Loci that showed amplification were X, 17q, Xq, 8q, 14q11-21 and 17q22-qter. The locus showing the most gain was the X chromosome, which was observed in 15 (57.7%) out of 26 cases. Loss was most frequently observed in the short arm of chromosome 8. The concordance of genetic transformation of primary cancer and secondary cancer in bilateral breast cancer was an average of 18.7% in synchronous and 10.7% in metachronous cancer, showing higher similarity in synchronous breast cancer.     

Genetic characterisation of invasive breast cancer: a comparison of CGH and PCR based multiplex microsatellite analysis

AIMS: Comparative genomic hybridisation (CGH) is a reliable tool to gain an overview of all unbalanced chromosomal alterations within a tumour. Nevertheless, the high numbers of tumour cells required and the comparatively low resolution are drawbacks of this technique. Polymerase chain reaction (PCR) based multiplex microsatellite analysis represents a semi-automated, highly reproducible method, which requires small amounts of tumour cells. This is a comparative study of CGH and microsatellite analysis. METHODS: Eighty one samples of invasive breast cancer were investigated by two sensitive multiplex PCRs containing three microsatellites each of six markers (D6S261, D11S907, D6S300, D11S927, D8S272, and D11S925), and two additional microsatellite markers located within intron 1 of the epidermal growth factor receptor gene (egfr) and p53 (p53CA). RESULTS: At least one example of loss of heterozygosity was detectable in all breast cancer tissues. However, the overall rate of accordance between the two methods tested was only 61%. An increasing rate of the number of genetic alterations in each case was mirrored by a constantly increasing fractional allelic loss index. CONCLUSIONS: PCR based multiplex microsatellite analysis using this panel of eight microsatellite markers not only enables the characterisation of cells that have malignant potential in a high frequency of patients with breast cancer, but can also give an estimate of the degree of genetic progression.     

The pattern of gene copy number changes in bilateral breast cancer surveyed by cDNA microarray-based comparative genomic hybridization

The nature of genetic alterations in bilateral breast cancer (BBC) associated with the distinctive development of a second primary tumor or a metastatic lesion is not clearly established. In this study, patterns of promoter methylation and gene copy number changes were assessed for their utility in the distinction of two types of BBC (synchronous and metachronous). Seven cases of synchronous and five cases of metachronous breast cancer tissues were used in X chromosome inactivation assay to assess the methylation pattern of human androgen receptor gene. X chromosome inactivation assay alone did not provide enough information to distinguish the genetic origins of synchronous and metachronous BBC. When four pairs of paraffin-embedded BBC tissues were used in cDNA array-based CGH with placenta DNA as a reference, higher DNA copy number changes were observed from metachronous pairs (9.0%) than from synchronous pairs (3.1%). From the two cases of metachronous pairs tested, 44 genes were found to be commonly modulated in gene copy numbers in a cancer detected later.     

Cytogenetic differences in breast cancer samples between German and Japanese patients

BACKGROUND: Japanese and German breast cancer cases differ substantially in the frequency of egfr amplification. AIMS: To unravel further the cytogenetic differences between Japanese and German breast cancer cases. METHODS: Forty one Japanese breast cancer cases were evaluated by means of comparative genomic hybridisation (CGH). The results were compared with the CGH results from 161 German breast cancer cases. RESULTS: The mean number of genetic alterations/case was significantly higher in German premenopausal patients with breast cancer than in their Japanese counterparts. Japanese breast cancer cases revealed a higher number of chromosome 17p losses. Losses of 8p were associated with oestrogen receptor (ER) negativity in Japanese patients with breast cancer, whereas in the German patients gains of 3q and 6q were associated with the lack of ER expression. CONCLUSIONS: The interethnic differences of invasive breast cancer are reflected by cytogenetic aberrations, which are also associated with the differential expression of the ER.     

Bilateral carcinomas of the breast with local recurrence: analysis of genetic relationship of the tumors.

Local recurrence of bilateral breast carcinomas is rare, but of biological interest, since it is unclear as to which tumor the local recurrence is related to, the ipsilateral or the contralateral, or whether it is an independent neoplasm. The aim of this study was to investigate the genetic relationship of bilateral breast carcinomas to each other and to their local recurrences. Eight cases of bilateral breast carcinomas, five with and three without local recurrence were analyzed using a microsatellite assay for 13 microsatellite loci. The presence of loss of heterozygosity and microsatellite instability in the various tumors was used for clonal analysis. All eight bilateral breast carcinomas showed divergent alterations in at least two microsatellite loci, which ruled out a genetic relationship. Four of five local recurrences were genetically related to the ipsilateral tumor and unrelated to the contralateral tumor. Only one local recurrence that occurred 11.8 years after the surgery of an infiltrative lobular carcinoma simultaneously with distant metastases was genetically related to the contralateral breast carcinoma. Although the number of cases in our study is limited, there is evidence that local recurrence of bilateral breast carcinoma frequently arises from the ipsilateral tumor.     

Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast.

AIMS: The mutual relation of lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) of the breast, as accepted precursor lesions of invasive breast cancer, is controversial. Because they display genetic heterogeneity, it is not clear how genetically advanced these entities are and what causes the transition to an invasive carcinoma. METHODS: Six cases of LCIS, four of them with associated lobular invasive carcinoma, four cases of intermediately differentiated DCIS with an associated invasive lobular carcinoma, and nine cases of intermediately and poorly differentiated DCIS with associated ductal invasive carcinoma were investigated by means of comparative genomic hybridisation (CGH) after microdissection and immunohistochemical staining of E-cadherin. RESULTS: LCIS was characterised by a low average rate of copy number changes, no evidence of amplifications, and a high rate of gains and losses of chromosomal material at 1q and 16q, respectively. A high degree of genetic homology with well differentiated DCIS was obvious, as reported previously. The cases of intermediately differentiated DCIS with associated lobular invasive components and lobular differentiation revealed striking homologies, and a significant difference of E-cadherin expression. The comparison of preinvasive and invasive breast lesions, irrespective of differentiation within the same patient, revealed no specific alteration that might be associated with invasion. Genetic alterations seen in invasive carcinoma were not necessarily seen in the adjacent precursor lesions. CONCLUSIONS: These results provide strong evidence that invasive breast cancer is a disease with multiple cytogenetic subclones already present in preinvasive lesions. Moreover, specific CGH alterations associated with invasion were not observed. Furthermore, the close genetic association between well differentiated and a subgroup of intermediately differentiated DCIS and LCIS led to the hypothesis that LCIS and a subgroup of DCIS are different phenotypic forms of a common genotype.     

Different genetic pathways in the evolution of invasive breast cancer are associated with distinct morphological subtypes

Invasive breast cancer shows a wide range of morphological differentiation, associated with differences in prognosis, but as yet, the underlying genetic mechanisms cannot be accounted for. In order to establish a model of the possible progression from the different subtypes of ductal carcinoma in situ (DCIS) to invasive breast cancer, 77 selected cases of invasive breast cancer representing distinct morphological subtypes were investigated by means of comparative genomic hybridization (CGH). There was a high degree of genetic homology between tubular and tubulo-lobular carcinoma and well-differentiated DCIS, and between ductal invasive carcinoma G3 and poorly differentiated DCIS. Highly differentiated invasive breast cancers were characterized by a loss of 16q and a low average number of aberrations per case. In high-grade tumours, losses of this chromosomal region were seen with a much lower frequency in cases with evidence of an aneuploid tumour status. These data demonstrate the close genetic similarity of well-, intermediately, and poorly differentiated DCIS and distinct morphological types of invasive breast carcinoma, providing further evidence that DCIS is a direct precursor lesion of invasive breast cancer and that various evolutionary genetic pathways exist.     

Comparative evaluation of microsatellite marker, AP-PCR and CGH studies in primary renal cell carcinoma

Renal cancer consists of a heterogeneous tumor group which is characterized by complex cytogenetic and molecular genetic abnormalities. In this study, three different techniques were applied to screen renal cancers for genetic alterations. We studied 99 primary, sporadic renal cancers (96 renal cell carcinomas and three other renal cancers) by a comparative evaluation of different microsatellite markers, comparative genomic hybridization (CGH) and AP-PCR. The AP-PCR produces a genomic fingerprint after an AluEI DNA restriction digest of tumor DNA samples. Microsatellite alterations were investigated using nine microsatellite markers spanning well-known regions of FhiT and VHL (3p14.2, 3p26) but also of oncogenes and tumor suppressor genes like Myc-L1 and TP53Alu (1p32, 17p13.1). To receive a genomic fingerprint, AP-PCR was carried out for all patient samples. Performing AP-PCR, only one case out of 99 displayed genomic imbalance. Seven of 99 investigated primary renal cancers showed alterations in up to four microsatellite loci (TP53Alu, Myc-L1, D3S1300, D3S1560, D3S1317, D3S4260). Three markers (Bat25, Bat26, REN) did not reveal any aberrations within the tested tumor samples. Six cases with microsatellite alterations and four without were examined by CGH. Five samples yielded aberrations, four of them were positive for microsatellite alterations. Only one tumor sample displayed microsatellite alterations, shift patterns in AP-PCR and alterations analyzed by CGH. Our data suggest that genomic aberrations found by microsatellite analysis are also detectable by CGH with the restriction of a minimum of alterated DNA of >10 Mb. Based on this study of RCC and in contrast to other reports for solid tumors, we conclude that AP-PCR is far less informative in investigation of renal cancers.     

Comparative genomic hybridization analysis of benign and invasive male breast neoplasms

Comparative genomic hybridization (CGH) analysis was performed for the identification of chromosomal imbalances in two benign gynecomastias and one malignant breast carcinoma derived from patients with male breast disease and compared with cytogenetic analysis in two of the three cases. CGH analysis demonstrated overrepresentation of 8q in all three cases. One case of gynecomastia presented gain of 1p34.3 through pter, 11p14 through q12, and 17p11.2 through qter, and loss of 1q41 through qter and 4q33 through qter. The other gynecomastia presented del(1)(q41) as detected by both cytogenetic and CGH analysis. CGH analysis of the invasive ductal carcinoma confirmed a gain of 17p11.2 through qter previously detected by cytogenetic analysis. These regions showed some similarity in their pattern of imbalance to the chromosomal alterations described in female and male breast cancer.     

Genetic losses in breast cancer: toward an integrated molecular cytogenetic map

Breast cancer is the most common malignant disease in Caucasian women, but is less frequent in Chinese women. The molecular basis for such ethnical difference in disease pathogenesis remains unknown. To address this issue, we performed allelotyping analysis of formalin-fixed, paraffin-embedded samples from 21 Chinese patients with breast cancer using 59 fluorescently tagged oligonucleotide primers amplifying microsatellite loci. Loss of heterozygosity (LOH) was found in all tumor samples. Frequent allelic losses were identified at markers D3S1578 (56%); D7S507 (55%); D1S2766 (50%); D17S789 and D17S946 (43% each); D19S814 (35%); D2S162, D13S158 and D13S296 (33% each); D1S551 and D1S2800 (29% each); D3S1597 and D6S260 (22% each); and D1S1588 (21%). To compare our data to previous reports, we determined the band-specific frequency of chromosomal imbalances in breast cancer karyotypes reported in the Mitelman database, and from the CGH results of cases accessible through the Progenetix website. Furthermore, published LOH analyses of breast cancer cases were compared to our own LOH results, demonstrating the most common chromosomal regions affected by allelic losses. The combined results provide a comprehensive view of genetic losses in breast cancers, indicating the comparability of these different techniques and suggesting the presence of a distinct subset of breast cancers with high-frequency LOH at chromosomes 1 and 2p in Chinese patients.     

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1-4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33 approximately p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21 approximately q32 (2 patients), 20q21 approximately q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance (MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing--at the level of the light microscope--an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.     

Genetic alterations in gastric cancers from British patients

Twenty-six gastric carcinoma and matching normal tissue DNAs, which had previously been analyzed for alterations of the APC (adenomatous polyposis coli) and MCC (mutated in colorectal cancer) genes were further investigated for the following genetic alterations: mutation and loss of heterozygosity (LOH) of the p53 gene, replication error (RER) and LOH at 12 microsatellite repeat loci, and mutation of the hMSH2 gene. In addition, 9 of the 26 gastric carcinomas were analyzed for genetic alterations using comparative genomic hybridization (CGH). Somatic mutations of the p53 gene were found to be frequent being detected in 31% of gastric carcinomas while LOH at the p53 locus was observed in 37.5% of informative cases. Loss of wild type p53 allele was detected in the majority (7 of 8) tumors found to be harboring a mutation. In the hMSH2 gene, an intronic 4 base pair insertion at 31 base pairs upstream of the beginning of exon 13 was detected in both tumor and normal tissue from one gastric carcinoma case. RER was detected in 11.5% of gastric carcinomas, at one or more microsatellite repeat loci. Of the 12 microsatellite repeat loci analyzed LOH was most frequently observed at D22S351 (30% informative cases) suggesting that a tumor suppressor gene on 22q may be important in gastric carcinogenesis. In support of this, CGH analysis carried out on 9 of the gastric carcinomas identified loss of chromosome 22 in 5 of these tumors.     

Allelic imbalance analysis of chromosome 16q shows that grade I and grade III invasive ductal breast cancers follow different genetic pathways.

It remains controversial as to whether low- and high-grade invasive ductal breast cancers are related by progression through grade, or generally follow distinct genetic pathways. It has previously been shown by comparative genomic hybridization (CGH) that the latter is more likely to be correct, based on the high frequency of chromosome 16q deletion in grade I cancers, but not in grade III. This study used microsatellite markers on 16q to confirm the differences between grade I and grade III tumours and to exclude the possibility that grade III tumours generally arise from grade I tumours through a process involving regain of 16q. The concordance between the CGH and microsatellite data is good but imperfect, probably reflecting the different sensitivities and specificities of the two techniques and the varying mechanisms of allele loss in each tumour type.     

Simultaneous bilateral malignant breast neoplasms in Nigerian women.

This study reviews the clinicopathological features and survival of 18 Nigerian women with simultaneous bilateral breast cancer. Twelve (67%) of these patients had bilateral Burkitt''s lymphoma of the breast. The average age of these patients was 22 years, and all of the women were pregnant or lactating at the time of initial clinical presentation. The remaining six patients (33%) had bilateral malignant epithelial neoplasms, with lobular carcinoma being present in four cases. The average age of patients with bilateral malignant epithelial tumors was 37 years. None of the 18 patients with simultaneous bilateral breast cancer survived for up to 2 years after diagnosis, indicating that bilateral synchronous breast cancer in Nigerian women is a rapidly progressive and aggressive disease.     

Identification of Rad51 alteration in patients with bilateral breast cancer

The human Rad51 gene, HsRAD51, is a homolog of RecA of Escherichia coli and functions in recombination and DNA repair. BRCA1 and BRCA2 proteins form a complex with Rad51, and these genes are thought to participate in a common DNA damage response path-way associated with the activation of homologous recombination and double-strand break repair. Additionally, we have shown that the pattern of northern blot analysis of the Rad51 gene is closely similar to those of the BRCA1 and BRCA2 genes. It is therefore possible that alterations of the Rad51 gene may be involved in the development of hereditary breast cancer. To investigate this possibility, we screened Japanese patients with hereditary breast cancer for Rad51 mutations and found a single alteration in exon 6. This was determined to be present in the germline in two patients with bilateral breast cancer, one with synchronous bilateral breast cancer and the other with synchronous bilateral multiple breast cancer. In both patients, blood DNAs showed a G-to-A transition in the second nucleotide of codon 150, which results in the substitution of glutamine for arginine. As this alteration was not present in any patients with breast or colon cancer examined, we assume that this missense alteration is likely to be a disease-causing mutation. Received: November 9, 1999 / Accepted: December 16, 1999     

Comparative genomic hybridization analysis of bilateral hyperplasia of usual type of the breast

Hyperplasia of usual type (HUT) is part of the spectrum of benign proliferative disease in the breast and confers an increased risk of developing invasive cancer. Its role as a putative precursor of invasive ductal carcinomas in the breast is, however, controversial. HUT is occasionally seen bilaterally but it is not clear whether there is a genetic predisposition to the process. This study has analysed 14 cases of bilateral HUT in the breast (28 independent lesions) by comparative genomic hybridization (CGH) analysis to define DNA copy number changes and to investigate any commonality in these genetic alterations. The mean number of alterations seen was 1.6 (46/28) in all lesions, with common losses at chromosomes 1p, 16p, 17q, and 22q. Bilaterally, only five alterations were seen in common across both lesions in both breasts, but no single locus was altered preferentially. These data indicate that a proportion of HUT in the breast are indeed clonal, neoplastic proliferations which exhibit genetic alterations in common with invasive breast cancer, albeit at very low levels. The limited data from these experiments do not reveal a specific predisposition locus.     

Distinct genetic and epigenetic changes in medullary breast cancer

Genetic instability resulting in chromosome aneuploidy or mismatch repair deficiency characterizes cancer. Medullary carcinoma (MC) of the breast is a specific form of breast cancer with unique clinical, epidemiologic, and prognostic features, suggesting distinctive tumorigenic pathways. To investigate the nature of the genetic changes associated with MC we analyzed a series of 22 tumors. Chromosomal imbalances were assessed by comparative genomic hybridization (CGH) and mismatch repair (MMR) deficiency tested for through assessment of microsatellite instability (MSI) and expression of MLH1 and MSH2 genes. MMR deficiency was detected in only a small proportion of cases. The chromosomal copy number changes showed some similarities to BRCA1-associated tumors. A high level of BRCA1 promoter hypermethylation was detected, suggesting a possible role of this gene in MC development.     

Frequent genetic alterations in flat urothelial hyperplasias and concomitant papillary bladder cancer as detected by CGH,LOH, and FISH analyses

Flat urothelial hyperplasia, defined as markedly thickened urothelium without cytological atypia, is regarded in the new WHO classification as a urothelial lesion without malignant potential. Frequent deletions of chromosome 9 detected by fluorescence in situ hybridization (FISH) have been previously reported in flat urothelial hyperplasias found in patients with papillary bladder cancer. Using comparative genomic hybridization (CGH) and microsatellite analysis, these hyperplasias and concomitant papillary tumours of the same patients were screened for other genetic alterations to validate and extend the previous findings. Eleven flat hyperplasias detected by 5-ALA-induced fluorescence endoscopy and ten papillary urothelial carcinomas (pTaG1-G2) from ten patients were investigated. After microdissection, the DNA of the lesions was pre-amplified using whole genome amplification (I-PEP-PCR). Loss of heterozygosity (LOH) analyses were performed with five microsatellite markers at chromosomes 9p, 9q, and 17p. CGH was performed using standard protocols. In 6 of 11 hyperplasias and 7 of 10 papillary tumours, deletions at chromosome 9 were simultaneously shown by FISH, LOH, and CGH analyses. There was a good correlation between FISH, LOH, and CGH analyses, with identical results in 6 of 10 patients. In addition to deletions at chromosome 9, further genetic alterations were detected by CGH in 9 of 10 investigated hyperplasias, including changes frequently found in invasive papillary bladder cancer (loss of chromosomes 2q, 4, 8p, and 11p; gain of chromosome 17; and amplification at 11q12q13). There was considerable genetic heterogeneity between hyperplasias and papillary tumours, but a clonal relationship was suggested by LOH and/or CGH analyses in 5 of 10 cases. These data support the hypothesis that flat urothelial hyperplasias can display many genetic alterations commonly found in bladder cancer and could therefore be an early neoplastic lesion in the multistep development of invasive urothelial carcinoma.     

Genomic Instability Is an Early Event during the Progression Pathway of Ulcerative-Colitis-Related Neoplasia

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon associated with a high risk of colorectal cancer. This increased cancer risk is thought to result from the cellular damage induced by the inflammatory field. The aim of this study was to determine the pattern and time course of genomic instability occurring in UC-related neoplasia. Sites of cancer, dysplasia, and nondysplasia from 14 UC colectomy cases containing cancer were analyzed for chromosomal alterations by comparative genomic hybridization (CGH) and for microsatellite instability using a series of 10 microsatellite markers. Clonal chromosomal alterations were present in 85% of cancer sites, 86% of dysplasia sites, and 36% of nondysplasia sites. Losses of chromosome 18 or 18q and chromosome 5 or 5q were common in cancer and dysplasia and were occasionally detected in nondysplasia. High-level microsatellite instability was detected in the cancer and dysplasia of two cases. Samples that demonstrated high-level microsatellite instability were unlikely to have chromosomal alterations demonstrable by CGH. These studies suggest that the predominant type of genomic instability in UC-related neoplasia is associated with chromosomal alterations and that this type of genomic instability frequently occurs before the development of histologically defined dysplasia.     

Genetic imbalances in primary gastric diffuse large B-cell lymphomas: comparison of comparative genomic hybridization, microsatellite, and cytogenetic analysis.

Extranodal malignant non-Hodgkin's lymphomas account for about 40% of lymphoid neoplasms, but few data are available concerning the genetic background of primary gastric diffuse large B-cell lymphoma (DLBCL). A study was performed of 27 primary gastric DLBCLs and 5 gastric DLBCLs with a concomitant low grade component of mucosa-associated lymphoid tissue-type lymphoma using comparative genomic hybridization (CGH), microsatellite studies, classic cytogenetics, and fluorescence in situ hybridization (FISH) to search for specific genetic aberrations. The most frequent aberrations were losses of material on chromosome 6q and gains of parts of chromosome 3. In three cases, a total of six high level DNA amplifications were detected, with five of them involving chromosomal regions not having been reported before in gastric DLBCL. A high overall concordance of 91.4% between microsatellite analysis and CGH was observed using DNA extracted from the same tissue block. The concordance achieved using DNA from different tissue blocks of the same patient was 85%. Microsatellite studies, CGH, FISH, and classic cytogenetics represent complementary techniques that facilitate a comprehensive view of genetic alterations in malignancies such as primary gastric DLBCL.