Japanese Journal of Clinical Oncology 33:556-562 (2003)
© 2003 Foundation for Promotion of Cancer Research
Loss of Heterozygosity Analyses of Asynchronous Lesions of Ductal Carcinoma in situ and Invasive Ductal Carcinoma of the Human Breast
1 Division of Surgical Oncology, 2 Department of Pathology, and 3 Division of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Ductal carcinoma in situ (DCIS) of the breast is known to possess characteristics of the pre-invasive stage of breast cancer and is the precursor to invasive ductal carcinoma (IDC). However, the natural history of the progression from DCIS to IDC remains unknown at the molecular level.
Methods: We investigated the loss of heterozygosities (LOHs) in tumors of seven patients with a history of breast biopsy. The seven specimens were diagnosed as DCIS on histopathological re-examination. These patients were diagnosed with ipsilateral breast cancer a few years after biopsy. We used thirteen selected microsatellite markers that were mapped to and/or very close to the tumor suppressor genes or regions with frequent LOHs in breast cancer. DNA isolated from microdissected formalin-fixed, paraffin-embedded tissues was subjected to a PCR-LOH analysis for these chromosome loci, and the pattern of LOHs was compared between the two asynchronous lesions for the seven cases.
Results: In all patients except one, the LOHs were concordant at 91% as the informative chromosome loci in cases 1 to 6 were 56, and the concordance in LOH pattern between DCIS and IDC was detected at 50 loci. The LOHs had accumulated in accordance with the tumor progression from DCIS to IDC. The recurrent lesion occurred at or near the site of the primary biopsy and had similar or identical histopathologic features.
Conclusions: These recurrences observed were probably residual disease rather than true recurrences. Our results suggest the following: (i) genetic alternations accumulate during cancer progression from DCIS to IDC, (ii) DCIS is a lesion that has a high risk of developing invasive transformation and (iii) after approximately 5 years without treatment, DCIS may develop into IDC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Genetic alternations, including activation of oncogenes and inactivation of tumor suppressor genes, are involved in the development of human breast cancer (1,2). Several studies have reported the loss of heterozygosities (LOHs) in invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS) (3–6). LOHs is also frequently found on several chromosome arms in IDC and DCIS. To date, specific allelic losses have been reported in chromosome arms 6q, 8p, 11q, 13q, 16q, 17p and 17q.
Many IDCs have histopathologically spread as a result of intraductal components (DCIS) (7). IDC is believed to generally arise from DCIS. Therefore, it is important to understand the genetic alternations that lead to the transformation of DCIS into IDC. We had previously analyzed the LOHs in patients harboring synchronous atypical ductal hyperplasia (ADH), DCIS and IDC and found that higher frequencies of LOHs were observed in parallel with the tumor progression from ADH to DCIS and then to IDC (8). Fujii et al. analyzed LOH in patients harboring synchronous DCIS and IDC and found that LOH in DCIS was also observed in synchronous IDC (9). The data supported the idea of a stepwise progression from DCIS to IDC.
An understanding of the natural history of DCIS is thus the basis of prognostication and therapeutic recommendation. Despite the abundant molecular data on breast cancer in general, the natural history of the progression from DCIS to IDC remains unknown at the molecular level because it is difficult to follow the pathological progression from DCIS to IDC in the same patient. The diagnosis of DCIS cannot be established without first removing a breast lesion for microscopic examination. A follow-up of such patients after the biopsy would be the best way to predict the clinical outcome of the disease. We, therefore, decided to evaluate and compare the molecular and clinicopathologic features of a selected number of patients with primary DCIS, who were treated only by biopsy and later had recurrent IDC lesions (asynchronous lesions) of the breast, using the LOH analysis to characterize the relationship between the two events at the molecular level.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Samples Analyzed
We selected patients with a history of breast biopsy, which was histopathologically diagnosed as benign. These patients were diagnosed with ipsilateral breast cancer, IDC, approximately 2 years after the biopsy. In each case, paraffin-embedded, hematoxylin–eosin stained sections were re-examined by three authors (MA, TM, NO) in accordance with the Consensus conference on the classification of DCIS (10). In this study, we examined samples from seven patients diagnosed with DCIS (non-comedo type).
The seven cases comprised Japanese patients with sporadic breast cancer. Three of them were from Tohoku University Hospital in Sendai, two from other hospitals in Sendai and two were from Kurashiki. Informed consent regarding the usage of their samples for genetic experiments was obtained from the patients who were treated in Tohoku University Hospital. All the tumor specimens (primary DCIS and recurrent IDC) were fixed in formalin and embedded in paraffin. These paraffin-embedded tissues were sectioned at 3 µm, stained with hematoxylin and eosin and then re-examined histopathologically. In recurrent IDCs, intraductal components of IDC were also examined and evaluated and then compared with primary DCISs. The clinical characteristics and pathologic data of the seven cases are summarized in Table 1.
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Microdissection and DNA Extraction
In each case, the formalin-fixed and paraffin-embedded tissues were sectioned at 10 µm. In situ cancer (primary DCIS) cells, invasive cancer cells (recurrent IDC) and normal cells were collected by microdissection using the Laser Captured Microdissection system, LM100 (Olympus, Japan) (11). DNA was extracted by a standard procedure, according to methods described previously (12).
LOH Analysis
Thirteen selected microsatellite markers, which were mapped to and/or very close to the tumor suppressor genes or regions with frequent LOHs in breast cancer, were used in this study. Primers for PCR amplification of these markers were designed based on the nucleotide sequences obtained from the GenBank database. Nucleotide sequences of the primers and annealing temperatures of PCR amplifications have been described previously (8). One out of each pair of primers was labeled with Cy 5 (Pharmacia Biotech, Uppsala, Sweden), and PCR amplifications and electrophoreses were performed according to methods described previously (13) using an ALFred DNA Sequencer with Fragment Manager software (Pharmacia Biotech, Piscataway, NJ). An allelic loss was defined as a more than a 50% reduction in the area of a peak calculated in the tumor as compared to that of a corresponding normal tissue. PCR reactions were performed twice per marker to confirm LOHs in both primary DCIS and recurrent IDC.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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All the seven primary lesions were histopathologically diagnosed as a benign proliferative disease, but the re-examined diagnosis for all revealed DCIS (non-comedo type), van Nuys classification Group 1 (14). A total of seven cases with primary DCIS and recurrent IDC were analyzed using 13 selected microsatellite markers that are the predicted loci for localization of tumor suppressor genes (TSGs) for breast cancer. Microscopic examination of the biopsy specimens for primary DCIS revealed that the surgical margins of these specimens were clearly positive in cases 1 to 4. However, the surgical margins in cases 5 to 7 were undetermined because only one slice of the specimen was used and other aspects of the surgical edge were unclear. The time interval between the data of the first biopsy (primary DCIS) and that of the curative operation (recurrent IDC) was 28~89 months; the mean time was 55 months. None of the women, who had a positive family history of breast cancer, received any form of radiation therapy, hormonal therapy or chemotherapy after the biopsy of the primary lesions.
LOHs were observed at one or more of the tested loci in all seven cases. The incidence of LOH in each locus is shown in Table 2. Typical examples of LOH analyses are shown in Fig. 1. In all cases except one (case 7), the LOHs were concordant at 91% (50/56) as the informative chromosome loci in cases 1 to 6 were totally 56, and the concordance in LOH pattern between DCIS and IDC was detected at 50 loci. Even if the LOH pattern differed, the LOHs had accumulated in accordance with the tumor progression from DCIS to IDC. The recurrent lesion occurred at or near the site of the primary biopsy and had similar or identical histopathologic features.
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Case 1 showed one LOH at 13q in primary DCIS and an additional LOH at p53 in recurrent IDC. Recurrent IDC appeared at the edge of the primary operation scar; the time interval from progression of primary DCIS to recurrent IDC was 28 months. Comedo-necrosis was not observed, and the nuclear grade was low (NG 1) in both primary DCIS and recurrent IDC. Similarly, the architectural pattern of DCIS was cribriform + solid in both primary DCIS (Fig. 2A) and recurrent IDC (Fig. 2B).
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Case 2 showed no allelic loss in primary DCIS, however, a new allelic loss was observed at 16q in recurrent IDC. Recurrent lesions appeared at the edge of the biopsy scar, and the time interval was 33 months. Comedo-necrosis was not observed, the nuclear grades were intermediate (NG 2), and the architectural patterns were papillary + cribriform in both lesions (Fig. 2D, E).
Case 3 showed identical LOHs at 8p, 16q and 17p and no new LOHs in the recurrent lesions. Recurrent lesions appeared at the biopsy scar, and the time interval was 63 months. Comedo-necrosis was not observed, and the nuclear grades and architectural patterns were similar in both lesions (Fig. 2G, H). Case 4 showed identical allelic losses at 3p, 6q, 8p, 9p, 11q and 16q and a new loss at 8p in the recurrent lesion. The recurrent lesion appeared near the primary lesion, and the time interval was 89 months. Comedo-necrosis was not observed, and the nuclear grades and architectural patterns were similar in both lesions (Fig. 2J, K).
Case 5 showed identical allelic losses at 9p, 11q and 16q and new losses at 3p and 17q in the recurrent lesion. The recurrent lesion appeared at the primary scar, and the time interval was 60 months. Comedo-necrosis was not observed, and the nuclear grades and architectural patterns were similar in both lesions (Fig. 3A, B).
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Case 6 showed LOH at 16q and a new LOH at 8p in recurrent IDC. The recurrent lesion appeared at the edge of the primary lesion, and the time interval was 55 months. Comedo-necrosis was not observed, and the nuclear grades and architectural patterns were similar in both lesions (Fig. 3D, E).
Case 7 showed LOHs at 3p, 8p, 11q, 13q and 16q in primary lesions, however, LOHs were observed at 8p, 11q and 16q in the recurrent lesion. The recurrent and primary lesions appeared in different quadrants. Comedo-necrosis was not observed in both lesions. The nuclear grade was NG II in primary DCIS but NG I in IDC. The architectural pattern was micropapillary in DCIS (Fig. 3G) and solid in IDC (Fig. 3H). This case is considered to have different clonality in IDC and DCIS because the appearance of lesions; the nuclear grades, architectural patterns and allelic losses for DCIS and IDC were all different.
In all the cases, the architectural pattern of DCIS associated with IDC was non-comedo, and the nuclear grade was low or intermediate. Cases 1 to 6 not only showed the same clonality, but also exhibited similar nuclear grades, architectural patterns and appearance of lesions between the primary DCIS and recurrent IDC. Furthermore, all the allelic losses observed in the primary DCIS were also observed in the recurrent IDC. In all these cases, the time interval between diagnosis of primary DCIS and the recurrent IDC was 28~89 months, and the mean time interval was 55 months.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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The natural history of breast carcinogenesis, progression from DCIS to IDC, has important prognostic and therapeutic implications. This is the first report on longitudinal molecular analysis from primary DCIS (non-comedo type) to recurrent IDC. We investigated LOHs using 13 microsatellite markers in seven cases of breast cancer. These chromosomal loci were selected on the basis of various previous studies (3–6). In all cases except one, LOHs were observed in parallel with the tumor progression from DCIS to IDC, suggesting a common genetic pathway for the development of both lesions and the continuous proliferation of the residual disease. These cases occurred at or near the site of the primary biopsy and had similar or identical histopathologic features. The observed recurrences were probably residual disease rather than true recurrences because surgical margins were clearly positive in four of the six cases.
According to the data obtained from this study, it is important to discuss the genetic criteria for differentiating between locally recurrent tumors and metachronous multicentric tumors. All the tumors except that of case 7 were considered to originate near the primary lesion, suggesting continuous genetic accumulation from the residual disease. They carried a maximum of two out of nine chromosomal loci (22%, case 5) with different LOH patterns at the asynchronous lesions. However, the lesion in case 7, which originated in another quadrant with different histological characteristics, showed a different LOH pattern at five (45%) of 11 chromosomal loci, suggesting that the threshold of discordance should lie between 22% and 45%.
In conclusion, hypothetically, recurrent IDC develops from residual primary DCIS that has accumulated one or more additional genetic alternations. Lininger et al. reported a comparison of LOH in primary DCIS and recurrent DCIS, in three cases with ipsilateral recurrent DCIS. LOH observed in primary DCIS was also observed in recurrent DCIS, and at least one additional LOH was observed in recurrent DCIS (15). Page et al. reported that the natural history of small, non-comedo DCIS lasts for at least two decades, with invasive carcinoma developing at the site of biopsy (16). However, our results showed that the natural history of small, non-comedo DCIS developed into IDC after approximately 5 years of treatment by biopsy only.
These results suggest that the following: (i) genetic alternations accumulate during cancer progression from DCIS to IDC, and (ii) DCIS is a lesion that has a high risk of developing invasive transformation, and (iii) non-comedo DCIS may develop into IDC after approximately 5 years without radiation therapy, hormonal therapy or chemotherapy.
BRCA1 and BRCA2 are breast cancer susceptibility genes on chromosome bands 17q21 and 13q12-q13, respectively (18,19). LOH on the BRCA1 locus is observed in IDC (case 5). On the other hand, LOH at the BRCA2 locus is observed in DCIS and IDC (case 1). In our previous study (8), inactivation of BRCA1 played an important role in the early stage of breast carcinogenesis, and the inactivation of BRCA2 was a late event.
The loss of D16S422 at the locus for CDH13 was high in DCIS and IDC (cases 3, 4, 6). This gene, located on chromosome bands 16q24, encodes the adhesion molecule H-cadherin (CDH13) and is recognized as a TSG responsible for breast cancer. The expression of CDH13 is significantly reduced in breast cancer (20), and its frequent inactivation in lung cancer has also been reported (21). Further, the introduction of wild-type DNA to breast cancer cells inhibited cell growth (20). Our previous study (8) and results suggested that H-cadherin might play an important role even in the early stages of breast carcinogenesis.
The CDH1 gene, located on chromosome 16q22, encodes the adhesion molecule E-cadherin, which suppresses invasion in vitro. A decreased expression in breast carcinomas correlates with the presence of invasion and a shorter disease-free survival (22). Our previous study (8) suggested that E-cadherin might be a rate event of breast carcinogenesis. In our results, LOH on the E-cadherin locus is observed in IDC (case 1) and in DCIS and IDC (case 5).
The loss of chromosome 8p is also frequently observed not only in IDC, but also in DCIS. Putative TSG in this region probably plays an important role in the early stages of breast carcinogenesis (cases 3, 4). The linkage analysis indicated that a positive LOD score at D8S137 was observed in some familial breast cancer pedigrees that were neither linked to 13q nor to 17q (23). There may be a third breast cancer susceptibility gene (BRCA3) on this chromosome arm, and our results may provide an important clue for identifying the putative BRCA3 gene.
ATM, the gene responsible for ataxia telangiectasia, is located on chromosome 11q22-q23 and the LOH of this gene (cases 4, 5) might be an early event. The frequent LOH of p53 has been reported in a variety of tumors (24). In this study, however, the LOH of p53 was not very frequent (case 1). Many investigators have analyzed the mutations of p53 in breast cancer tumors and have found that this gene was not frequently mutated in this disease (25). Our present study is in agreement with these results.
Though the data on genetic progression of breast cancer is increasing, the mechanisms of carcinogenesis (natural history) from DCIS to IDC at the molecular level are still unknown. The genome-wide search for LOH showed 56 regions with consistent LOH (17). In an LOH analysis of 75 different breast cancers at multiple chromosome loci, every cancer showed a different pattern of deletions. In our previous study on LOH analysis in 70 DCIS lesions at the same chromosome loci, frequent LOH was observed at 8p, 16q and 17q, but the incidences of LOH at loci on 3p, 6q, 9p, 11p, 11q and 17p were marginal. It is not confirmed whether the mutations of genes on these loci play a major role in the genesis of breast cancer. The results of this study also show a different pattern of deletions in each case (Table 2). However, we could not completely exclude these regions as the loci for the responsible genes since the number of samples used in this study were limited.
To summarize, the progression from DCIS to IDC may involve a pathway consisting of stepwise genetic alterations. DCIS is likely to have a high risk of developing invasive transformation. Further studies are necessary to identify the genetic alterations of breast tumor progression from DCIS to IDC in order to develop appropriate clinical management of breast cancer patients.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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We thank Dr Toshiaki Monabe and Dr Hiroshi Sonoo of the Kawasaki Medical School for allowing us to examine and study the cases. This work was supported in part by the Japanese Breast Cancer Society and Grant-in-aid for Cancer Research from the Ministry of Health, Labor and Welfare, Japan.
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FOOTNOTES |
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+ For reprints and all correspondence: Noriaki Ohuchi, Division of Surgical Oncology, Tohoku University School of Medicine, 1–1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. E-mail: noriakio{at}tains.cc.tohoku.ac.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODS RESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Received June 10, 2003; accepted October 9, 2002
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