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Normal-appearing epithelium of cancer patients can harbor occult genetic abnormalities. Data comprehensively comparing gene expression between histologically normal breast epithelium of breast cancer patients and cancer-free controls are limited. The present study compares global gene expression between these groups. We performed microarrays using RNA from microdissected histologically normal terminal ductal-lobular units (TDLU) from 2 groups: (i) cancer normal (CN) (TDLUs adjacent to untreated ER+ breast cancers (n = 14)) and (ii) reduction mammoplasty (RM) (TDLUs of age-matched women without breast disease (n = 15)). Cyber-T identified differentially expressed genes. Quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC), and comparison to independent microarray data including 6 carcinomas in situ (CIS), validated the results. Gene ontology (GO), UniProt and published literature evaluated gene function. About 127 probesets, corresponding to 105 genes, were differentially expressed between CN and RM (p < 0.0009, corresponding to FDR <0.10). 104/127 (82%) probesets were also differentially expressed between CIS and RM, nearly always (102/104 (98%)) in the same direction as in CN vs. RM. Two-thirds of the 105 genes were implicated previously in carcinogenesis. Overrepresented functional groups included transcription, G-protein coupled and chemokine receptor activity, the MAPK cascade and immediate early genes. Most genes in these categories were under-expressed in CN vs. RM. We conclude that global gene expression abnormalities exist in normal epithelium of breast cancer patients and are also present in early cancers. Thus, cancer-related pathways may be perturbed in normal epithelium. These abnormalities could be markers of disease risk, occult disease, or the tissue's response to an existing tumor. © 2007 Wiley-Liss, Inc.
Breast cancer arises as genetic aberrations accumulate in precursor epithelial cells. Considerable information is available about the molecular alterations characterizing breast cancers, but knowledge of alterations in earlier lesions is limited. Recently, abnormalities have been appreciated in histologically normal breast epithelium. These abnormalities include allelic imbalance or loss of heterozygosity,1–8 aberrant methylation of p16INK49 and of RASSF1A,10 cytogenetic changes,11, 12 telomere shortening,13 loss of IGF2 imprinting,14 aberrant response to estrogen,15 loss of RARβ expression,16 aberrant phosporylation of p38,17 upregulation of EZH2.18 Some of these abnormalities have been detected in normal-appearing tissue adjacent to the tumor, and others have been found at a distance from it. Some abnormalities are concordant, and others are discordant, with abnormalities in the tumors themselves.
Despite the evidence supporting the existence of occult abnormalities in normal-appearing breast epithelium of breast cancer patients, the roles these abnormalities play in carcinogenesis is poorly understood. One approach to better understand their significance is to compare histologically normal breast epithelium of breast cancer patients to normal breast epithelium of women without breast cancer. The few studies comparing these groups have examined allelic imbalance, aneuploidy and methylation or expression of specific proteins, and have found abnormalities more frequently in patients with cancer than in controls.7, 8, 10, 12, 17–19 We hypothesized that by taking a comprehensive gene expression approach, we might detect consistent abnormalities in the normal-appearing epithelium of breast cancer patients, compared to controls. These abnormalities might suggest mechanisms predisposing to cancer, or activated early in carcinogenesis. If this hypothesis were true, then elucidating these abnormalities could enhance understanding of important functional alterations present early in carcinogenesis, suggest targets for cancer prevention and improve cancer risk assessment. To begin testing this hypothesis, we undertook the present study to identify gene expression differences in the histologically normal breast epithelium of breast cancer patients, compared to reduction mammoplasty controls.
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The current understanding of events that initiate or predispose to breast carcinogenesis is limited. Therefore, the present study evaluated global gene expression in tumor-adjacent, histologically normal breast TDLUs microdissected from patients with untreated ER+ breast cancers, compared to TDLUs from control patients with no increased breast cancer risk. We identified differences in 127 probesets, corresponding to 105 genes. Most differences were maintained in a set of CIS. The 105 genes included a large group of transcriptional regulators, IE genes, and members of signaling pathways. The majority of these genes were expressed at lower levels in epithelium from women with cancer. One-third of the genes have been implicated previously in breast cancer, another third have been implicated in other cancers, and a final third have not been associated with cancer before. We cannot determine if these changes represent an effect of the tumor or an occult premalignant condition. But taken together, the data suggest that perturbations of key cellular functions are identifiable prior to the development of any histological abnormality, and that these perturbations may play important roles in the early stages of breast carcinogenesis.
Several potential objections could be raised to our study. The number of patients investigated is small, due to practical limits on the number of samples that can be investigated meticulously. However, a counterbalancing strength of the study is its use of primary uncultured epithelium, which eliminates introduction of artifacts inherent in cultured cells. We used amplified RNA, because only nanogram quantities are available from microdissected epithelium; however, we (and others) have shown that this approach yields reliable and reproducible data in which the biological variation between samples is greater than the technical variation between replicates.20 The data may not be generalizeable to ER-breast cancers, but that would not be unexpected, given breast cancers' considerable intrinsic heterogeneity.
Despite these potential objections, the data raise several points for consideration. First, how do our results compare to existing expression data from human breast tissue? Most existing breast tissue expression signatures were derived to predict tumor subtype29, 30 or disease outcome,31–35 or to distinguish luminal from myoepithelial cells in RM tissue,28, 36 as opposed to distinguishing between patients with and without breast cancer, and so are not directly comparable to our data. It is therefore not surprising that few of the genes that we find to vary between CN and RM epithelium have been useful in predicting tumor subtype, disease outcome, or epithelial cell type (analyses not shown).
Other studies are more comparable to ours
One found no gene expression differences between RM and tumor-adjacent normal epithelium by unsupervised hierarchical clustering.37 We also could not discern differences between CN and RM by unsupervised hierarchical clustering or principal component analysis of all genes (results not shown). This may be due to the presence of genes that vary from patient to patient and obscure the consistent differences between CN and RM that we see when comparing directly these 2 sample types. In contrast, there is overlap between our results and those reported to distinguish TDLUs from an early hyperplastic breast cancer precursor.38 There is also overlap between our results and those reported in a study comparing luminal epithelium from RM and cancers.28 These reports, combined with the fact that the CN vs. RM differences are largely preserved in the independent CIS samples we examined, suggest that the CN vs. RM differences are authentic alterations reflecting a breast cancer related process.
Second, although the CN vs. RM differences appear authentic, we cannot distinguish whether they represent cause or effect, i.e., an occult premalignant condition, or secondary changes due to the tumor or its surrounding stroma. We favor the former explanation, because of the similarity of the CN vs. RM differences to cancer microarray data. However, we cannot determine how far the affected area might extend geographically, since all CN TDLUs were tumor-adjacent. Tissue that is adjacent to a breast tumor may harbor more, or different, genomic abnormalities than tissue that is more distant.4
If the CN vs. RM differences represent a primary abnormality, then the identification of genes whose expression varies in normal epithelium from patients with breast cancer, compared to controls, suggests mechanisms that may predispose to breast cancer development or are active early in carcinogenesis. If the CN vs. RM differences represent a secondary abnormality, then they can illuminate direct or paracrine effects occurring in vivo. Regardless, the largest functional category among the 105 genes is transcription factors and regulators, especially members of the composite transcription factor AP-1. Transcription regulators are implicated frequently in breast carcinogenesis (for review see Ref.39). Many transcription-related genes (23/29 (79%)) were underexpressed in CN (and CIS) samples, which may reflect a generalized decrease in transcriptional activity, rather than involvement of a specific family. Also notable among the 105 genes was a large group (n = 16) of IE genes, which are rapidly induced upon cell stimulation and whose transcription is not dependent on protein synthesis. The IE genes were also underexpressed in CN (and CIS) epithelium. In addition, many of the 105 genes participate in signaling pathways. The largest number participates directly in the MAPK pathway, and others may affect MAPK signaling more peripherally. Considerable evidence supports the involvement of MAPK in breast cancer (for reviews see Refs.39 and40). Increased MAPK activity is usually reported, but the tumors examined have been mainly ER-negative and ERBB2-overexpressing.41, 42 In contrast, we found decreased expression of MAPK components, which could be related to using tissue from ER-positive tumors, or may reflect an initial step in the pathway's perturbation.
A final consideration is how can these data be utilized. If validated in future studies, the genes or pathways implicated here could identify new targets for chemoprevention, or help prioritize those already being studied.43 If differential expression of these genes can be detected in women without evident breast cancer, and associated with future disease, then they may be pertinent to risk assessment, since breast cancer risk is not thought to be uniform across all women.44, 45 DNA structural variants46–48 or single nucleotide polymorphisms might alter RNA expression49 and be associated with risk of disease.
To our knowledge, this is the first study to find gene expression differences between histologically normal epithelium of breast cancer patients and breast-cancer free controls. Our findings suggest that cancer-related pathways are already perturbed in normal epithelium of breast cancer patients. These perturbations could be markers of disease risk, of occult disease, or of the tissue's response to an existing tumor. Future studies should expand upon these results by examining expression of these genes in additional samples from breast cancer patients and controls, manipulating these genes' expression in model systems and developing clinically useful disease and risk classifiers.