Human Molecular Genetics, 2001, Vol. 10, No. 18 1907-1913
© 2001 Oxford University Press
Genetic model of multi-step breast carcinogenesis involving the epithelium and stroma: clues to tumour–microenvironment interactions
1Clinical Cancer Genetics and Human Cancer Genetics Programs, Comprehensive Cancer Centre, and Division of Human Genetics, Department of Internal Medicine, 2Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics and 3Center for Biostatistics, Comprehensive Cancer Centre, The Ohio State University, Columbus, OH 43210, USA, 4Department of Pathology, University of Manitoba Health Sciences Centre, Winnipeg, Manitoba R3E 0W3, Canada and 5CRC Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 2QQ, UK
Received April 18, 2001; Revised and Accepted June 21, 2001.
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ABSTRACT |
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Although numerous studies have reported that high frequencies of loss of heterozygosity (LOH) at various chromosomal arms have been identified in breast cancer, differential LOH in the neoplastic epithelial and surrounding stromal compartments has not been well examined. Using laser capture microdissection, which enables separation of neoplastic epithelium from surrounding stroma, we microdissected each compartment of 41 sporadic invasive adenocarcinomas of the breast. Frequent LOH was identified in both neoplastic epithelial and/or stromal compartments, ranging from 25 to 69% in the neoplastic epithelial cells, and from 17 to 61% in the surrounding stromal cells, respectively. The great majority of markers showed a higher frequency of LOH in the neoplastic epithelial compartment than in the stroma, suggesting that LOH in neoplastic epithelial cells might precede LOH in surrounding stromal cells. Furthermore, we sought to examine pair-wise associations of particular genetic alterations in either epithelial or stromal compartments. Seventeen pairs of markers showed statistically significant associations. We also propose a genetic model of multi-step carcinogenesis for the breast involving the epithelial and stromal compartments and note that genetic alterations occur in the epithelial compartments as the earlier steps followed by LOH in the stromal compartments. Our study strongly suggests that interactions between breast epithelial and stromal compartments might play a critical role in breast carcinogenesis and several genetic alterations in both epithelial and stromal compartments are required for breast tumour growth and progression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Breast cancer is the most common and second most lethal cancer in women in Western countries. Numerous studies have focused on the role of chromosome abnormalities and gene mutations in sporadic breast cancer, but to date no clear model of the critical events or delineation of primary abnormalities has emerged. Various chromosome arms have been observed to be affected by a high frequency of structural or numerical abnormalities (1–5). Although several of these chromosome arms appear to be the sites of putative tumour suppressor genes (TSGs), the number and identity of TSGs relevant for mammary carcinogenesis is unknown. At the molecular level, several somatic mutations in genes residing in these regions have been described (1,6–9). Despite this abundance of data, the relevance, role and timing of most of the described genetic abnormalities in sporadic breast cancer are still unclear. It is also not known whether specific mutations play relevant roles as causative factors or are the consequence of the general genomic instability and progression in breast tumours.
A few studies have previously demonstrated that loss of heterozygosity (LOH) identified at various chromosomal loci at high frequency in invasive cancer is already present in in situ carcinoma, atypical ductal hyperplasia, non-atypical hyperplasia of the breast, and perhaps adjacent normal epithelial cells, although these studies predated laser capture microdissection (LCM), hence, contamination from clearly malignant tissue cannot be excluded (10–15). These observations are also found in colonic adenomas (16), Barrett oesophageal metaplasia (17,18) and lung hyperplasias (19). These reports indicate that the majority of premalignant or precursor lesions share their LOH phenotypes with invasive disease in the same organs, providing novel biologic evidence that they are genetically and perhaps evolutionarily related. Nonetheless, until recently, any cancer such as breast cancer was treated as a single amorphous entity. Most such genetic studies were uniformly performed on the entire tumour without regard to its components, despite the fact that a few groups were quite aware of both epithelial and stromal components of tumours, and the cell biology of the tumour ‘microenvironment’ has been described for the last 20 years. Thus, until now, when a genetic alteration, be it intragenic mutation, regional amplification or deletion manifested by LOH, is attributed to a breast cancer, it is unclear if the alteration is actually occurring in the epithelial compartment, the surrounding stromal compartment or both.
The effects of the metabolism of the tumour stroma, locally as well as systemically, are largely unknown. Although the stroma has generally been considered a silent bystander during epithelial carcinogenesis, the concept that the microenvironment is central to maintenance of cellular function and tissue integrity provides the rationale for the idea that its disruption can contribute to neoplasia (20). Several studies performed in vivo and in vitro indicated that the growth and invasive potentials of carcinoma cells are influenced through interactions with host stromal cells (21–23). Despite these progressive cell biological studies, the precise genetic mechanisms leading to tumour progression remain unclear. Even less clear is the role of differential genetic alterations in the epithelial neoplastic component and its surrounding stroma.
Recently, Moinfar et al. (5) reported the high frequency of LOH in the mammary stroma with breast cancer. They examined 11 patients with ductal carcinoma in situ, including five cases with invasive ductal carcinoma, and found LOH in the stromal cells. Although these investigators identified frequent genetic alterations in the mammary stroma, each component of the breast carcinoma was manually microdissected; thus cross-contamination cannot be excluded. Further, they examined a limited number of samples and a limited number of microsatellite markers on only five chromosome arms. In this present study, we sought to systematically examine for genetic alterations in the epithelial and stromal components of invasive adenocarcinomas of the breast with the DNA extracted from cells from each compartment obtained by LCM. We found frequent LOH in both epithelial and/or stromal components of breast cancer and identified associations among LOH at various chromosomal regions, suggesting that genetic alterations in the epithelial and surrounding stromal cells are involved in the breast tumorigenesis through concurrent and independent pathways.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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LCM of each specimen was performed to selectively obtain normal epithelial or stromal cells, carcinomatous epithelial cells and stromal cells surrounding the epithelial carcinoma (e.g. Fig. 1A). LOH at each of the 13 chromosomal regions was detected in epithelial and/or stromal compartments among the 41 invasive adenocarcinomas of the breast (e.g. Fig. 1B). Table 1 summarizes the frequencies of LOH observed at 13 loci in neoplastic epithelial and surrounding stromal cells. Among the 13 microsatellite markers examined, the LOH frequency ranged from 25% (9/36) at D3S1581 (3p14–q21) to 69% (22/32) at D17S796 (17p13) in the neoplastic epithelial compartment, and from 17% (6/36) also at D3S1581 to 61% (20/33) at D2S156 (2q34) in the surrounding stromal component (Table 1). D2S156 (2q34) and perhaps D6S437 (6q25) were the only two markers demonstrating a higher frequency of LOH in the surrounding stromal compartment compared with the neoplastic epithelial cells. In contrast, the great majority of markers showed a higher frequency of LOH in the neoplastic epithelial compartment compared with the surrounding stromal cells (Table 1).
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On further inspection of the differential LOH data, it can be noted that for certain markers, LOH predominates in the neoplastic epithelial compartment, for others, LOH predominates in the stromal compartment, and for yet other markers, it occurs in both compartments (Table 1). For instance, LOH at 16q24.3 (D16S413) was identified more frequently in only the neoplastic epithelial cells (11 tumours) than in only the stromal cells (three tumours; P = 0.0325, McNemar’s test). The number of tumours that showed LOH at 17p13 (D17S796) only in stromal cells (two tumours) was significantly fewer than that having LOH at 17p13 in both epithelial and stromal cells or only in the neoplastic epithelial cells (12 or 10 tumours; P = 0.0209, McNemar’s test).
We then looked for pair-wise associations of dependency or independency of particular genetic alterations with one another in either epithelial or stromal compartments (Fig. 2). We found that there were statistically significant associations in 17 pairs (17/325; 5.3%) of markers (Fig. 2). Fifteen pairs (15/17; 88%) showed positive associations and two pairs (2/17; 12%) showed negative associations (Fig. 2). LOH at four markers, D10S1765, D11S912 and D17S579 in the surrounding stromal compartment and D2S156 in the neoplastic epithelial compartment, were associated with three or more sites of LOH (Fig. 2). Of note, LOH at D10S1765 in the stromal compartment was associated with LOH at D17S579 and D22S277 in the stromal compartments and also with LOH at D2S156 in the neoplastic epithelial compartments. LOH at D17S579 in the stromal compartment, in turn, was found to be associated with LOH at D2S156 and D13S155 in the neoplastic epithelial compartment and at D10S1765 in the surrounding stromal compartment. In contrast, LOH at D8S264 (8p23.2) in the neoplastic epithelial cells is negatively correlated with LOH at D1S228 (1p36) in the surrounding stromal cells. Interestingly, LOH at D8S264 in the surrounding stromal compartment is negatively correlated with LOH at D1S228 in the neoplastic epithelial compartment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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In this present study, we have found frequent LOH in both neoplastic epithelial and surrounding stromal cells of invasive adenocarcinomas of the breast. In our series of 41 breast cancer samples, we identified LOH in the neoplastic epithelial compartment, ranging from 25% (9/36) to 69% (22/32), and in the stromal compartment, ranging from 17% (6/36) to 61% (20/33), respectively. Of note, the great majority of markers demonstrated a higher frequency of LOH in the neoplastic epithelial compartment compared with the surrounding stromal cells (Table 1). Further inspection of the differential LOH data indicated that the frequency of LOH only in the stromal compartment occurred among fewer tumours than that in both neoplastic epithelial and surrounding stromal compartments, or that in only the neoplastic epithelial cells (Table 1). In the field of human cancer genetics, it has been shown that markers with the highest frequency of LOH represent those with the earliest genetic alterations, the so-called first ‘hits’, and the one with the lowest frequency of LOH represents the latest ‘hit’ in carcinogenesis (16). Thus, under this assumption, from our data, we can propose a genetic model of multistage, stepwise carcinogenesis in the breast, according to the relative frequencies of LOH in the epithelial and stromal compartments (Fig. 3). Our proposed model encompasses data that shows a higher frequency of LOH in breast epithelial cells occurs earlier than in the stromal compartment (Fig. 3). Although Moinfar et al. (5) worked without the advantage of LCM and used markers representing only five chromosomal regions and a sample size of 11, their observations of relative frequencies of LOH in the neoplastic epithelium (occurring in 1/4 to 3/3 tumours) compared with presumably surrounding stroma (occurring in 1/4 to 4/5 tumours) might be interpreted as concurrent with ours. These data together with our observations suggest that genetic alterations in the epithelial compartment, at least in some chromosomal regions, precede the genetic changes in the surrounding stromal cells. If in fact we may extrapolate that each region of LOH represents at least one putative TSG, then it is possible that the same putative gene involved in epithelial carcinogenesis plays some role in the stroma at a later stage, with the possible exception of D2S156. In our study, the earliest genetic alterations occurred at D8S264, D16S413 and D17S796 in the epithelium as well as D2S156 in the stroma (Fig. 3). It is almost certain that the D17S796 marker represents TP53, and indeed, TP53 alterations have been noted amongst the most frequent and earliest somatic alterations in prior studies involving ‘whole’ breast carcinomas (24,25). The regions of D8S264 and D16S413 have yet to yield convincing TSGs involved in breast carcinogenesis. Our data would strongly support the existence of one or more TSGs residing in these two regions which when mutated participate in the initiation of cancer within the breast epithelium. Of interest, LOH at D2S156 in the stromal cells is also scored as an early event (Fig. 3). This has not been a region noted to have LOH in whole breast cancers. Nonetheless, our data suggest that there will be at least one important gene residing in that interval which plays a prominent role in the initiation of breast carcinogenesis, possibly from a microenvironmental or ‘landscaper’ point of view (26). The genetic model proposed (Fig. 3) assumes for simplicity that frequency of occurrence reflects temporal sequence. This in turn assumes an equivalent effect on tumorigenesis and early progression between all alterations. However, it is recognized that an alternative possibility is that some alterations may be dominant while others may require the cooperation of parallel or multiple complex alterations at other sites to facilitate progression, and that this would influence the prevalence of the alteration in advanced tumours. In the case of these different assumptions, a lower frequency of stromal alterations could reflect the fact that stromal alterations are not dominant and only exert an indirect effect on the adjacent epithelium, or only exert an effect in collaboration with others, to influence the overall process of tumorigenesis.
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The apparent asynchronous LOH at each marker between epithelium and stroma might suggest that while the neoplastic epithelium is clonal, as is the stroma, these observations may support one viewpoint that epithelium and stroma derive from different cellular origins. However, there are advocates of a common cellular origin of both epithelium and stroma (27). If this latter is true, then in the context of our observations, the LOH in epithelium and stroma occurred after the divergence of epithelial and stromal cell from the presumed common cell.
Despite a relatively small sample size, we were able to examine pair-wise associations between regions and compartments where LOH occurred (Fig. 2). For example, LOH at D17S579 in the neoplastic epithelial cells was associated with LOH at D17S796 and D10S1765 in the neoplastic epithelial compartments. These chromosomal loci contain several putative TSGs. The polymorphic markers, D17S579 (17q21), D17S796 (17p13) and D10S1765 (10q23.3), are in proximity to the BRCA1, TP53 and PTEN genes, respectively. Previous reports have identified that there are significant associations between LOH of BRCA1 and TP53 (28,29) or PTEN gene (28) in sporadic heterogeneous breast cancer samples. Crook et al. (30) found a high proportion of ‘whole’ breast and ovarian tumours from BRCA1 mutation carriers had TP53 mutations. Our proposed genetic model does suggest that LOH at D17S796 in the neoplastic epithelial cells is the earliest hit, LOH at D17S579 in the neoplastic epithelial cells is the second one, then LOH at D10S1765 in the neoplastic epithelial cells is the third hit in these consequences (Table 1 and Fig. 3). The association between LOH of BRCA1 and PTEN is, therefore, one of the genetic alterations that might be expected to occur as a consequence of the loss of BRCA1 and TP53. LOH at D2S156 in the neoplastic epithelial compartment was associated with LOH at three markers, D10S1765, D11S912 and D17S579, in the surrounding stromal compartments. The reciprocal interaction between epithelial and stromal cells plays a key role in the morphogenesis, proliferation and differentiation of epithelial cells (31–33). Most of the intercellular substances, extracellular matrix (ECM) molecules that are required for tumour growth and progression are produced by the stromal cells (34). It is well demonstrated that altered gene expression occurs between normal and neoplastic breast stroma (35) and that stromal cells play a critical role in the production and possible dissolution of the ECM (36,37). Thus, genetic alterations in the stromal cells may change the interaction between epithelial cells and ECM molecules and influence the tumour invasion and dissemination (22,23). Our results suggest that LOH at 2q34 (D2S156) might precede LOH of three chromosomal loci in the stromal compartments (Figs 2 and 3). Therefore, we hypothesized that loss of a putative TSG on 2q34 might play an important role in genetic alterations of stromal compartments, which in turn might influence tumour invasion and dissemination through ECM remodelling. If this hypothesis is correct, then we can further hypothesize that genetic alterations in the stroma might predict for poorer prognosis due to increased tumour invasiveness.
In summary, we have found frequent LOH in both neoplastic epithelial and surrounding stromal compartments in invasive adenocarcinomas of the breast and statistically significant associations among the LOH at various chromosomal regions. We also propose a multi-step genetic model of breast carcinogenesis involving epithelium and stroma, which can help build further hypotheses and guide future studies of reciprocal interactions between breast neoplastic epithelial and stromal cells in tumour initiation, progression, invasion and metastases. Such studies might eventually lead to novel therapeutic strategies, which selectively target epithelium or stroma.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Breast cancer samples
Forty-one archival (formalin-fixed and paraffin-embedded) tissues that were distinct cases of clinically sporadic primary invasive adenocarcinomas of the breast were used. Twenty-six samples were obtained from the National Cancer Institute of Canada Manitoba Breast Tumour Bank and 15 were from the Department of Pathology of The Ohio State University.
Microdissection of tissue and DNA extraction
Microdissection of carcinomatous epithelial cells, surrounding stromal cells, and normal epithelial or stromal cells from fixed, paraffin-embedded sections of breast was performed using an Arcturus PixCell II Laser Capture microdissecting microscope (Arcturus Engineering Inc., Mountain View, CA). This system utilizes a transparent thermoplastic film applied to the surface of the tissue section on standard histopathology slides. The breast cancer epithelial, surrounding stromal, and normal epithelial or stromal cells to be microdissected were identified and targeted through a microscope, and a narrow (
15 uM) carbon dioxide laser-beam pulse specificity activated the film above these cells. The resulting strong focal adhesion allowed selective procurement of only the targeted cells (38) (Fig. 1A). The cells removed in Figure 1A are the neoplastic epithelial component. The surrounding stromal fibroblasts are immediately adjacent to the removed cells (Fig. 1A, arrow). It is acknowledged that while LCM minimizes cross-contamination of cell types, it does not guarantee against it. However, the very ‘clean’ LOH (virtually all or none) which we have obtained does suggest that any cross-contamination is not significant.
DNA from microdissected tissue was extracted in 50 µl of solution containing 0.04% proteinase K, 1% Tween-20, 10 mM Tris–HCl (pH 8.0), and 1 mM EDTA at 37°C overnight followed by heat inactivation at 95°C for 10 min.
LOH analysis
For purposes of this study, genomic DNA, extracted from paraffin embedded tissues, served as template for PCR amplification of 13 microsatellite markers selected from a comprehensive genetic map of the human genome (39). Fluorescent-labelled polymorphic markers, including D1S228 (1p36), D2S156 (2q34), D3S1581 (3p14.2–p21.2), D3S1286 (3p24.3–p25.1), D6S437 (6q25.3), D8S264 (8p23.2), D10S1765 (10q23.3), D11S912 (11q23), D13S155 (13q14), D16S413 (16q24.3), D17S796 (17p13), D17S579 (17q21) and D22S277 (22q12.2–q13.1), were used for this analysis. All subsequent PCRs were carried out using 0.5 µM each of forward and reverse primer in 1x PCR buffer (Qiagen, Valencia, CA), 1.5 mM MgCl2 (Qiagen), 1x Q-buffer (Qiagen), 1.25 U of HotStar Taq polymerase (Qiagen) and 200 µM of each dNTP (Gibco, Gaithersburg, MD) in a final volume of 25 µl. After a denaturation at 95°C for 14 min, reactions were subjected to 40 cycles of 94°C for 1 min, 55–60°C for 1 min, and 72°C for 1 min followed by 10 min at 72°C. PCR reactions and genotyping were repeated at least a second time to confirm the data. Amplified PCR products were separated by electrophoresis through 6% denaturing polyacrylamide gels, and the signal was detected with an Applied Biosystems model 377xl semi-automated DNA sequencer (Applied Biosystems, Perkin-Elmer Corp., Norwalk, CT). The results were analysed by automated fluorescence detection using the GeneScan collection and analysis software (GeneScan, Applied Biosystems). Scoring of LOH was initially performed by inspection of the GeneScan analysis output. A conservative ratio of peak heights of alleles between germline DNA and somatic DNA 
1.9:1 were used to define LOH in this study (40).
Statistical analysis
Comparisons for statistical significance were performed by using either the standard Fisher’s exact test (2-tailed) or the McNemar’s test for matched pairs at the P = 0.05 level of significance. McNemar’s test was used when interest focused on differences in proportions of patients with LOH in either stromal or epithelial cells, but not both. This test determines whether, in these cases of discordance, there are a disproportionate number of patients with LOH in one of the two sites. McNemar’s test is used in recognition of the fact that the stromal and epithelial cells are taken from the same breast tissue and, hence, are matched. However, the Fisher’s exact test was also employed because it is unclear from a biological point of view whether each data point (i.e. LOH at any one marker) is dependent on the next (i.e. LOH at other markers).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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We thank Fred Wright and Sandya Liyanarchchi for providing statistical assistance. Part of this work was performed in the Laser Capture Microdissection core facility in the Tissue Procurement Shared Resources Service of the Comprehensive Cancer Centre. This work was supported in part by the Jimmy V Golf Classic Award for Basic and Clinical Cancer Research from the V Foundation (to C.E.) and a grant from the National Cancer Institute, Bethesda, MD (P30CA16058 to The Ohio State University Comprehensive Cancer Centre).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Human Cancer Genetics Program, The Ohio State University Comprehensive Cancer Centre, 420 West 12th Avenue, Room 690C TMRF, Columbus, OH 43210, USA. Tel: +1 614 292 2347; Fax: +1 614 688 3582; Email: eng-1{at}medctr.osu.edu
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