The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center: A Unique Resource for Defining the “Molecular Histology” of the Breast

Breast cancer accounted for approximately 200,000 incident cases and 40,000 deaths in the United States in 2009 (1). Mammographic screening and improved treatments have contributed to reductions in breast cancer mortality (2, 3), but progress towards improving outcomes for biologically aggressive cancers remains limited. Many lethal cancers occur before initiation of screening, elude mammographic detection, or fail to completely respond to available therapies (4–6). Accordingly, developing better means of risk assessment, detection, and prevention of aggressive breast cancers is an important goal.

Most mechanistic and biomarker research in humans has taken a “backward looking” approach; markers and mechanisms are identified in cancer and secondarily tested at earlier stages of carcinogenesis. Though productive, this approach is limited by effects of the carcinogenic process itself, which results in many changes (e.g., passenger mutations, genetic instability) that are neither causal nor early. These complexities might be avoided by taking a “forward looking” strategy in which the effects of risk factors on morphology and molecular markers in normal breast are assessed.

Investigations in animal models have revealed substantial insights into the transition between physiology and early carcinogenesis, but vast interspecies differences in biology have made translation of these findings to women challenging. Until recently, the lack of optimally prepared normal human breast tissues annotated with risk factor data have precluded observational studies of these changes in women. Thus, the “molecular histology” of the normal human breast, conceptualized as encompassing the range of morphologic and molecular characteristics throughout the life span, is largely undefined, and therefore, limits our ability to discern and characterize the earliest stages of carcinogenesis. Given that many such changes are probably reversible, identifying these processes may offer opportunities for successful prevention. To enable studies of breast molecular histology in humans, Indiana University in collaboration with Susan G. Komen for the Cure has developed a novel specimen bank of annotated normal breast tissues (komentissuebank.iu.edu). In this commentary, we present the rationale for developing the Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (“Komen Tissue Bank”; Indianapolis, IN), highlight its potential value in breast research, and describe its characteristics.

“Normal breast,” as defined by physical examination, radiological methods, histologic study, or molecular analysis, varies widely in its implications. A normal physical examination roughly corresponds to the absence of a mass, a normal appearing nipple areola complex and unremarkable skin, but offers little information about cellular content, apart from relative adiposity. Radiological methods are useful for identifying masses, asymmetries, and calcifications, and for estimating the percentage of fibroglandular tissue (mammographic density), but neither distinction of epithelium from stroma nor characterization of benign epithelium is easily achievable routinely. Finally, microscopic identification of unremarkable terminal duct lobular units (TDLUs), the source of nearly all breast cancer, may only partially satisfy criteria for normal. Studies have shown that TDLUs near breast cancers may share some molecular alterations with their associated cancers such as loss of heterozygosity and altered expression of p53 or HER2 (7), DNA methylation of CpG islands (8), and altered mRNA expression profiles (9) that resemble cancer. Similarly, “normal” tissues associated with breast cancer may show expression profiles consistent with wound responses, which are specific for molecular subtypes and augur a poor prognosis (10).

Beyond the recognition that definitions of normal breast differ by technique of assessment, it is also clear that singular definitions of normal lack broad biologic relevance, given the remarkable changes that occur over a woman's life span or even a single menstrual cycle. Normative ranges of histology and marker expression (“molecular histology”), rather than rigid universal definitions, are needed to capture the interindividual variation that results from the interplay of aging, genetics, and environmental and lifestyle factors (Fig. 1). In fact, such characteristics may be population specific, reflecting the differences in the frequency of breast cancer risk factors and cancer incidence rates. Whereas data suggest that age and other breast cancer risk factors influence the molecular histology of the breast (11–13), we also speculate that molecular histology modifies the effect of risk factors.

Observational data suggest that the state of the breast at the time of an exposure influences the net effect of that exposure with regard to carcinogenesis. This may partly account for why many breast cancer risk factors show “qualitative age interactions,” reducing cancer risk at some ages whereas increasing risk at others (14). Similarly, “quantitative age interactions,” reflecting a difference in magnitude of an effect (e.g., “window of vulnerability”), may also result from the action of identical risk factors on different normal histologic substrates. This view is supported by animal studies showing that prior pregnancy or treatment with exogenous agents that mimic pregnancy confers resistance to carcinogenic challenges and the observation that radiation exposure before a first pregnancy increases risk among women more than later exposure (15). These observations are also consistent with the view espoused by Boyd and colleagues that high mammographic density at early ages is a key determinant of breast cancer risk (16). Thus, the effect of carcinogenic exposures may vary by the amount and state of the tissue substrate upon which they act.

The TDLU is the basic milk-producing structure within the breast and represents the anatomic source of nearly all breast cancer precursors and cancers (15). TDLUs are not present at birth; the prepubertal breasts of both girls and boys consist mainly of small ducts embedded in fibrous tissue. At puberty, ducts lengthen and branch, and then develop a cap of small acinar units that is enveloped by specialized stroma, constituting the TDLU (17). Data from a large Danish cohort found that high birth weight, greater height or lower body mass index (BMI) at age 14 years, and peak growth at an early age increase risk for adult breast cancer, suggesting that the pace or degree of breast development is related to breast cancer risk (18). Furthermore, it is notable that breast development begins a year earlier among African-American than among Caucasian girls (19) and that rates of early onset breast cancer are higher among African-American women (20).

Ducts and acini are lined by an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells that are bound by a basement membrane. The TDLU is a highly dynamic structure, which undergoes cyclic changes during the menstrual cycle (21) and progressive changes with aging (22). During pregnancy, TDLUs first expand and later differentiate into milk-secreting structures, which persist until weaning and postpartum involution.

Pregnancy and lactation permanently alter the molecular histology of the breast. In rodent models, parity alters the molecular histology of the breast, rendering it relatively resistant to carcinogenic challenges. Limited data also suggest that parity and other factors related to breast cancer risk permanently alter the molecular histology of the normal breast among women and reduce susceptibility to malignant transformation (15, 17). Specifically, the transcriptome of normal breast of nulliparous and parous women differs for genes involved in a diverse range of processes (15). Although pregnancy-related changes and postpartum involution seem to permanently alter breast biology and confer resistance to cancer, the breast maintains sufficient plasticity to support the development of milk production at subsequent pregnancies.

Postpartum involution and remodeling is an active process that may exert a promotional effect on breast cancer development (23). Data suggest that breast cancers that are detected in mothers after birth are particularly aggressive and may include etiologically and biologically distinctive tumors such as “basal cancer”. In the years immediately following birth, breast cancer risk is increased. The long-term effects of parity vary by age at first live birth (17). A first live birth at younger ages is associated with long-term reduced breast cancer risk, whereas at older ages a first birth is related to increased risk. In addition, new data suggest that parity may not reduce risk for basal breast cancers (24).

In contrast to postpartum involution, which is comparatively rapid and at least partly reversible, age-related involution seems to evolve more slowly and contributes to the variable appearance and functional state of TDLUs among older women. However, as originally conceptualized by Pike and colleagues (25), differences in risk factor exposures may slow or hasten “breast tissue aging” as compared with a woman's chronological age, and thereby influence cancer risk through a number of incompletely understood mechanisms (26). In the Mayo benign breast disease cohort, the identification of nonatrophic TDLUs was a marker of increased breast cancer risk (27). Lee and colleagues (28) have suggested that hyperplastic TDLUs represent an early change in the development of cancer. Additional data from the Mayo study has shown that involution of TDLUs, a microscopic feature, is associated with lower mammographic density, a radiological characterization (29). Thus, high mammographic density, a strong breast cancer risk factor, may be associated with molecular histology, suggesting that macroscopic radiological features reflect microscopic and submicroscopic changes in tissues.

Approaches for studying breast tissues of young women are limited because neither imaging nor biopsy is commonly carried out until screening is initiated, typically at age 40 years in the United States or 50 years elsewhere. Most strategies for studying “molecular histology” are suboptimal because well-preserved–annotated tissues obtained from normal women have been lacking. Accordingly, various alternative sources of “low-risk” breast tissue have been studied, each of which has strengths and limitations (Table 1).

Prospective collection of fresh normal tissue, especially TDLUs, is difficult because normal epithelium, normal stroma, and most examples of benign breast disease are indistinguishable grossly. Although fresh tissue has advantages for molecular analysis, researchers often use formalin-fixed tissues, which presents limitations. Notably, analysis of normal-appearing structures in formalin-fixed, paraffin-embedded tissue blocks identified by reviewing hematoxylin and eosin–stained sections cut from such blocks may be flawed because such structures are part of specimens that were removed for a clinical indication (e.g., a radiographic finding or palpated abnormality) and thus may reflect molecular changes characteristic of the associated disease state. This concern also applies to mammoplasty tissues. Pathologic review of reduction mammoplasty specimens has shown that 88% contain benign breast disease, including 17% with proliferative changes, whereas in the Komen Tissue Bank, 35% of tissues show benign changes and only 3.3% show proliferative changes, suggesting possible differences between these groups of women (30).

Currently, most suspicious lesions are assessed using radiologically guided biopsies, which are typically small, available only as fixed tissue, and largely consumed in preparing diagnostic pathology sections. Limited data suggest that histologically normal tissue identified in surgical excisions already shows molecular alterations, either reflecting early carcinogenic alterations surrounding the tumor (“field effect”) or modification secondary to the effects of the nearby cancer (“reverse causality”). These tissues may have value for comparison of cases, but are less useful for defining baseline normal parameters. Reduction mammoplasty specimens are commonly studied as a source of nondiseased tissue, but this approach belies the reality that these women often have extremely large fatty breasts, suggesting that neither the patients nor their organs are normal. A recent review of 516 mammoplasty specimens found that 18% of women had proliferative lesions, 5% had ductal carcinoma in situ (DCIS), and 3% had lobular neoplasia, further calling into question the normalcy of these samples (31). Finally, even biopsies that do not show clinically significant pathology were prompted by clinical findings and may be at least subtly abnormal (e.g., radiological calcification or asymmetry).

Analysis of postmortem breast tissues has been explored, including a novel historical effort in which consecutive forensic procedures were subjected to detailed sampling (32). This work yielded seminal information about the morphology of the breast, including appearances characteristic of different phases of the menstrual cycle (21) and aging, the contrasting prevalence of benign breast lesions among women of different ethnic backgrounds, and the composition of tissues associated with radiological density. However, access to postmortem tissues is often limited or delayed by the need to locate and consent next of kin; as a result, tissue degradation limits the value of such specimens, especially for molecular studies. Medical postmortem studies, even using rapid autopsy techniques, are generally restricted to hospitalized women with chronic illnesses. Nonetheless, the ability to conduct extensive sampling of the entire body may allow new insights about breast biology and pathophysiology from a systemic perspective.

Ductal lavage and nipple aspiration represent potential means of obtaining normal epithelial cells and fluid; however, yields are often low, stroma is not represented and architectural context is lost. Only structures that communicate with the duct system can be sampled and the inevitable occurrence of fibrocystic changes with aging, therefore, often renders aspects of the breast inaccessible. Random fine-needle aspiration represents another cytologic sampling technique, which may suffer from limitations similar to lavage, especially in nonexpert hands. Finally, analysis of cells and fluid-comprising breast milk represents a promising approach for understanding postpartum breast biology through analysis of fresh cells and fluid. Using immunomagnetic methods to isolate breast epithelial cells, Wong and colleagues have shown DNA methylation of tumor suppressor genes in breast epithelium of healthy young women (33). However, analysis of milk is limited to studying the postpartum breast, and methods for collection and fractionation of milk have not been completely developed and tested.

Amassing experience shows that multiple factors such as medications, hypoxia, and devascularization occurring both intra- and postoperatively may affect molecular analyses (34). Most surgeries are preceded by biopsies that induce wounds and fat necrosis, which may alter not only the sampled tissues but also the systemic milieu. Ideally, normal tissues should be collected through nonoperative procedures and frozen immediately.

The full value of research on normal breast tissue remains incompletely defined. A few applications are noted below:

• Determining the molecular histologic footprint of nongenetic and genetic risk factors as a means of assessing the impact of these exposures;

• Identifying mechanisms that mediate the effects of risk factors and protective factors, with the goal of developing prevention strategies that inhibit the former and mimic the latter;

• Assessing levels of candidate biomarkers for risk assessment or early detection in normal tissue to assess their specificity for cancer and its precursors and determine optimal cutoff points for sensitivity and specificity;

• Providing evidence for causal effects of circulating biomarkers by showing changes in molecular histology that would suggest increased risk (e.g., increased proliferation, decreased apoptosis);

• Assessing the expression of molecular targets that represent candidates for therapeutic or preventive interventions;

• Understanding the breast stroma and its interaction with TDLUs;

• Furthering the understanding of “stem cells” including number, distribution, and genomic integrity.

In 1997, the National Cancer Institute convened a meeting of basic and clinical researchers from academia, industry and government, and representatives of the patient advocate community for the purpose of identifying barriers to progress in the treatment and prevention of breast cancer. Thirteen deficiencies were identified, the first of which was as follows:

“Our limited understanding of the biology and developmental genetics of the normal mammary gland is a barrier to progress. …it is now clear that a more complete understanding of the normal mammary gland at each stage of development—from infancy through adulthood—will be a critical underpinning of continued advances in detecting, preventing, and treating breast cancer.”

The Komen Tissue Bank is a unique biorepository established expressly to acquire healthy breast tissue from volunteer donors. Approximately 5 tissue collection events are held each year, most of them at the IU Simon Cancer Center in Indianapolis, IN. Tissue samples are obtained from approximately 100 women at each event. Donors have been recruited primarily by word of mouth and newsletters, as well as by occasional coverage in news media. Donors interested in future donation events can contact A.M. Storniolo at the Tissue Bank.

The collections are carried out under the approval of the Indiana University Institutional Review Board, even when they occur at other locations. The donors provide a broad consent, allowing the tissue to be used for unspecified future breast cancer research. Virtually all donors have consented to future contact as necessary. Abiding by HIPAA mandate that the donors' personal identifying information not be revealed, the aforementioned contact will be done by tissue bank personnel.

The tissue and blood samples, as well as the clinical annotation, are available to researchers worldwide, via a proposal application process which is described in detail on the Komen Tissue Bank website. Proposals are reviewed by an independent panel that assesses the quality of the science and also ensures nonduplication of effort and appropriate utilization of such a limited resource.

As of November 2010, the Bank has acquired breast tissue from 1,174 unique donors. Self-described African-Americans comprise 5.2% of donors. The Bank's specimens are annotated with details of the donors' reproductive, medical, and family histories and current medication usage. These tissue specimens are a significantly limited resource. To mitigate this limitation, the Komen Tissue Bank has produced 33 epithelial and 36 stromal cell lines from the tissue specimens; 8 of the epithelial and 10 of the stromal lines were derived from the breast tissue of African-American women. The cell lines have been characterized with karyotyping, interphase FISH mapping, immunohistochemistry, and flow cytometry. A subset of the cell lines has been assayed for single-nucleotide polymorphisms (SNP), DNA copy number variation (i.e., greater than or less than diploid), and gene expression.

Volunteer tissue donors are asked to complete a questionnaire detailing demographics and most established breast cancer risk factors. Following a blood draw, donors undergo a tissue acquisition procedure in which up to 4 cores of breast tissue are obtained. Cores of tissue of the upper outer quadrant of either breast are acquired with 10-gauge needles and immediately processed as snap-frozen tissues and as formalin-fixed paraffin-embedded tissues. The standardization of the procedures ensures rapid, consistent, and optimal handling of the tissues.

Though the Komen Tissue Bank provides clear advantages over some of the other sources of “normal” breast tissue, it too has some shortcomings. The ideal normal breast tissue bank would include thoroughly annotated specimens obtained from a representative population-based sample of women who have provided fresh breast tissue sufficient to fully characterize their breasts bilaterally. Furthermore, repeated sampling and extended follow-up of a large cohort of such women could provide a comprehensive picture of breast carcinogenesis from initiation to progression. However, developing such a resource is challenging. The Komen Tissue Bank represents a step forward by obtaining multiple breast core biopsies from women who provide a risk factor questionnaire and blood. However, core biopsies are also limited in terms of their size and representativeness of the breast. Some samples consist entirely of fat, and therefore, are not useful for evaluating epithelium or nonfatty stroma. Finally, biopsies conducted at one time point do not reveal the evolution of dynamic processes (e.g., proliferation, involution, neoplasia, etc.), and without follow-up one cannot guarantee that a subject does not have prevalent occult cancer or a precancerous lesion.

Selected characteristics of the first 521 enrolled volunteers without a personal history of cancer are shown in Table 2. Subjects are predominantly White non-Hispanic women who are somewhat better educated than the U.S. population overall. The women range in age from 18 to 83 years and the majority are premenopausal. Most women are parous, including about 50% of the premenopausal subjects. About 25% of participants report having a first-degree relative with breast or ovarian cancer. The mean BMI of subjects is 28 kg/m² indicating a large proportion of women are overweight or obese, similar to the general U.S. population. About 16% of the women have had a history of prior breast biopsies.

The resource has been established to provide broad access to the research community, predicated on approval based upon scientific merit, feasibility, and external funding as required. Upon completion, results of analyses will be made available on the Komen Tissue Bank's virtual tissue bank website.

Some of the initial projects will relate patient characteristics, nongenetic breast cancer risk factors, and relationships of susceptibility loci to characterization of the morphology of TDLUs, cellular composition, assessment of markers of proliferation, apoptosis and hormone receptors, and mRNA profiling.

The Komen Tissue Bank has been established to address the research need for “normal” breast tissue specimens. These specimens, paired with detailed clinical annotation, will serve to characterize the spectrum of normal. The molecular histology of the breast—from early adulthood to menopause and across different races and ethnicities—can now be defined. This knowledge will allow further investigations into the molecular changes leading to carcinogenesis. The Komen Tissue Bank represents a critical tool in the ongoing efforts to find new strategies for the treatment and prevention of breast cancer.

No potential conflicts of interest were disclosed.

The authors thank the work of hundreds of volunteers who have made this dream a reality. Finally, words alone cannot capture the significance of the gift that 1,174 women thus far have made to science, and to a future without breast cancer.

This study was supported by Susan G. Komen for the Cure; the Breast Cancer Research Foundation; Oracle Giving; the Catherine Peachey Fund, Inc.; and National Cancer Institute, Intramural Research Program.