Section 5 -

The Progenitor Cell Concept Werner Boecker Muenster, Germany

Click here to download handout in pdf format for the current section (178 KB) Although a great wealth of experimental data suggests the existence of a self-renewing mammary stem cell, its characteristics have never been satisfactorily resolved. Based on in situ and biochemical studies we have postulated that the Ck5/14+ cells are phenotypically and behaviorally progenitor (committed adult stem) cells of the human breast epithelium [1]. This hypothesis has been elegantly confirmed by in vitro studies of Dontu et al [2]. This talk addresses the questions of how (a) this concept might help to unravel the cellular mechanisms that govern benign and malignant pathological proliferation of breast epithelium and of how (b) this data can be applied as a differential diagnostic tool to proliferative breast disease lesions. Normal Breast Epithelium In H&E sections the whole ductal-lobular system consists of basal and luminal epithelium. Recently, phenotypic characterization of breast epithelial cells using high molecular weight cytokeratins 5/14 and lineage-specific markers such as cytokeratins 8/18 and sm-actin has displayed considerable cellular heterogeneity and, furthermore, provided evidence of the existence of common progenitor cells. Thus, double immunofluorescence techniques reveal that Ck5/14+ cells differentiate into lineage-specific glandular (Ck8/18+) or myoepithelial cells (sm-actin+). Based on these findings, we proposed a cell biological concept as depicted in the schematic drawing of Figure 1 [1, 3]. Recently data of elegant cell culture studies of human breast epithelium by Dontu et al [4] provided further support for this model. Interestingly, in situ studies showed that these Ck5/14+ cells are located both within the luminal layer, and, more rarely in the basal layer. This contrasts with other glandular tissues such as the prostate or the salivary glands, where these cells are located exclusively in the basal layer [5, 6, 7, 8]. Figure 1: Progenitor cell model of normal human breast epithelium. Progenitor cells of the breast epithelium give rise to both the glandular and myoepithelial cell lineages. The progenitor cell and its progeny are phenotypically characterized by specific expression of cytokeratins and/or sm-actin as shown in this schematic drawing (from: Preneoplasia of the Breast, Sa und ers, 2006) [9] . In contrast to the ductal system, the differentiation status of lobular epithelium in the resting breast may differ considerably from case to case, and even within the same section. Thus, lobules may contain either a very immature glandular epithelium or a mature epithelium composed of differentiated glandular cells. In the lactating breast nearly all glandular cells differentiate into terminal, milk producing Ck8/18+ secretory end cells. Our contention is, therefore, that the lobular and ductal epithelium is subject to independent regulatory control, the mechanisms of which are as yet unknown. Benign Proliferative Breast Disease Lesions Within the conceptual framework of the term benign proliferative breast disease (BPBD) we encompass and classify the following proliferative lesions without atypia: usual ductal hyperplasia (UDH), sclerosing adenosis, papillomas, fibroadenomas, tubular adenoma, nipple adenoma and benign adenomyoepithelial tumors [10, 11]. With the exception of microglandular adenosis, all these lesions show differentiated uni/bilineage characteristics of their epithelial components comparable to normal breast epithelium. This is best exemplified by comparing the cellular organization of normal breast epithelium with two classic forms of BPBD: sclerosing adenosis and UDH. In sclerosing adenosis the "benign tubule" is the prototypic structure consisting of Ck5/14+ cells and their myoepithelial and glandular progeny cells. Compared to normal glands the glandular-myoepithelial layering is preserved, the architecture, however, of these epithelial structures is disturbed (Figure2). With some modifications these principles hold true for all the other benign lesions listed above [9]. In UDH, Ck5/14+ cells and their glandular progeny cells proliferate and lose contact with the myoepithelial cells / basement membrane. Figure 2: Schematic drawing of the epithelial components of sclerosing adenosis and usual ductal hyperplasia lesions and their architectural patterns compared to normal breast epithelium. In "benign tubules" of BPBD lesions and in epithelial hyperplasia the same cellular organization as in normal breast epithelium is fo und . However, the architectural order of the glandular structures and/or of the glandular (epithelial) proliferations is deranged (from: Preneoplasia of the Breast, Sa und ers, 2006 [9] ). Our conclusion is that the cell biology of benign breast lesions can only be explained by assuming that Ck5/14+ cells and their progeny are the found ing cells of these lesions. Malignant Proliferative Breast Disease Lesions 'Precursor lesions' as defined by the WHO are biologically and clinically heterogeneous neoplastic lesions with the potential for progression to invasive breast cancer [12]. Precursor lesions include flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and its subtypes, atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS) and its variants. The potential for progression of these lesions to invasive breast carcinoma and thus the clinical implications of a given diagnosis vary considerably from entity to entity [13, 14]. Here we discuss some basic principles of neoplastic proliferative processes with the aim of defining and clarifying the above-mentioned entities in the context of the progenitor cell concept. The overwhelming number of breast malignancies display a purely glandular phenotype expressing Ck8/18 and lacking Ck5/14 expression in their tumor cells (Figure3). This phenotype is found in 70 - 90% of invasive breast malignancies and is observed consistently in FEA, ADH, ALH, low, intermediate and most high-grade DCIS [15, 16, 17, 18]. Only a minority of invasive malignancies, including a small group of extremely rare carcinomas, express the basal cytokeratins 5/14 typically fo und in benign lesions. These cells show lineage differentiation to glandular cells coexpressing Ck8/18. This immunophenotype is observed in less than 5% of high-grade DCIS and less than 10-30% of invasive carcinoma, most of them grade 3. The existence of such basal-type tumors has been confirmed by immunohistochemical analyses, Western blotting experiments and gene expression studies [15, 16, 17, 18, 19]. In addition to expressing Ck5/14, these neoplastic cells are also characterized by specific cytogenetic alterations and protein expression patterns, including a positive correlation with EGFR, p53 and cyclin A expression and an inverse correlation with c-erbB2 [20, 21]. Interestingly, a large subgroup of these tumors seem to be associated with germline BRCA1 mutations [22, 23, 24, 25, 26]. Ck5/14+ carcinomas with bilineage, myoepithelial, squamous or heterologeous differentiation with matrix formation are extremely rare. Breast Carcinogenesis Based on the accumulated data, we propose a model of breast carcinogenesis, which takes the analogies between normal breast epithelium and tumors into consideration [1]. The different breast tumors observed in the human female breast tend to recapitulate stages of normal epithelial differentiation so that, to a certain extent, they can be classified according to a corresponding normal stage. We believe that the differentiation characteristics of various breast carcinomas and their precursors are currently the best guess of what might be the possible target cells of malignant transformation (Figure 3). Our model implies that different types of breast carcinomas derive from different target cells. Figure 3: Hypothetical model of breast carcinogenesis, based on our modified progenitor cell concept. Note that the differentiation characteristics of specific tumors can be correlated with the different subtypes of the myoepithelial and glandular cell lineages (for details see text) (from: Preneoplasia of the breast, Sa und ers, 2006 [9] ). The most interesting tumors within this conceptual framework are carcinomas that display progenitor cell features with or without any of the differentiated characteristics observed in normal breast epithelium. Such Ck5/14+ tumors are usually characterized by an aborted lineage differentiation and are ER-. We believe that these tumors can best be explained when the target cells of transformation are Ck5/14+/ER- stem cells. On the other hand we hypothesize that malignancies with a purely glandular phenotype might be derived from glandular cells of the resting breast. This does, of course, not exclude that initiating mutational events might have taken place at the progenitor cell level. Differential Diagnosis The differential diagnosis of sclerosing lesions includes tubular and other invasive carcinomas. The most characteristic clue helpful in distinguishing between these lesions is the type of epithelial structure. "Benign tubules" preserve a glandular-myoepithelial layer and a basement membrane and contain the whole spectrum of Ck5/14+ cells and their progeny cells. Epithelial malignancies show a glandular phenotype and lack the myoepithelial layer (Figure 4). In difficult cases, immunostaining for myoepithelial markers such as sm-actin, calponin or sm-actin may help to highlight the myoepithelial layer. Furthermore, most carcinomas display a complete lack of Ck5/14 immunostaining in contrast to the Ck5/14+ sclerosing lesions. It should, however, be borne in mind that myofibroblasts may be a component of the desmoplastic stroma of a carcinoma and would likely interfere with interpretation of sm-actin immunohistochemistry, leading to a mistaken diagnosis of malignancy. Figure 4: Comparison between"benign tubules" and epithelial malignancies of the breast. Diagnosis is not a problem in most cases of usual ductal hyperplasia cases due to the presence of a heterogeneous cell proliferation with a fenestrating growth pattern. They can be easily distinguished from low-grade lesions, with its monotonous cell growth and cribriform pattern, and from high-grade DCIS, with its overtly malignant nuclear features. However, there may be a considerable overlap both in cellular features and growth patterns between usual ductal hyperplasia and some cases of intermediate-grade DCIS. The results published in the literature so far confirm the practical importance of Ck5/14 immunohistochemistry in this area of breast pathology [15]. Usual ductal hyperplasia is defined by its Ck5/14 mosaicism, whereas ductal precursor lesions – with the exception of some high grade lesions- are characterized by its Ck8/18 positivity, lacking Ck5/14. Atypical Proliferation (ADH/DCIS) ex BPBD The vast majority of ductal carcinomas arise de novo from glandular cells of the TDLUs that do not show the mixed cellular features of any type of BPBD [27]. Nevertheless every pathologist is aware of benign lesions that clearly show a transformation to either malignant ductal or lobular-type in situ malignancy, which may even progress to invasive carcinoma. Although such malignant transformations have been noted in every type of BPBD lesions, such occurrences are – with the exception of papillary lesions - rare, instead representing a chance finding. We introduce the term atypical proliferation of ductal or lobular type ex BPBD to define such a process. One important feature of atypical ductal proliferations in this context is that their cells resemble the phenotype of the normal glandular cell – most of them express Ck8/18 in the absence of Ck5/14. Thus the evolution of atypical proliferation ex BPBD can be unveiled using Ck5 and/or Ck14 immunostaining. Conclusions In normal breast epithelium Ck5/14+ progenitor cells give rise to both the glandular Ck8/18+) and myoepithelial cell lineages (sm-actin+, calponin+, etc). This cell model provides a basis for a better und erstanding of proliferative breast disease lesions. With the exception of microglandular adenosis, benign lesions show differentiated uni/bilineage characteristics comparable to normal breast epithelium. The "benign tubule" and usual hyperplasia are the most frequently encountered prototypic structures in this context. Most breast malignancies display a purely glandular phenotype expressing Ck8/18 and lacking Ck5/14 expression in their tumor cells. Malignancies that do express Ck5/14 are either high-grade lesions or lesions with a very specific morphology. The fundamental principles of this concept and its implications in differential diagnosis context of benign and malignant lesions are discussed. References Boecker W, Moll R, Poremba C, et al. Common Adult Stem Cells in the Human Breast Give Rise to Glandular and Myoepithelial Cell Lineages: A New Cell Biological Concept. Lab Invest 2002;82:737-46. Dontu G, Al Hajj M, Abdallah WM, Clarke MF, Wicha MS. Stem cells in normal breast development and breast cancer. Cell Prolif 2003;36 Suppl 1:59-72. Boecker W, Moll R, Dervan P, et al. Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J Pathol 2002;198:458-67. Dontu G, El Ashry D, Wicha MS. Breast cancer, stem/progenitor cells and the estrogen receptor. Trends Endocrinol Metab 2004;15:193-7. Foster CS, Dodson A, Karavana V, Smith PH, Ke Y. Prostatic stem cells. J Pathol 2002;197:551-65. Bonkhoff H. Role of the basal cells in premalignant changes of the human prostate: a stem cell concept for the development of prostate cancer. Eur Urol 1996;30:201-5. Wernert N, Seitz G, Achtstatter T. Immunohistochemical investigation of different cytokeratins and vimentin in the prostate from the fetal period up to adulthood and in prostate carcinoma. Pathol Res Pract 1987;182:617-26. Verhagen AP, Ramaekers FC, Aalders TW, et al. Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. Cancer Res 1992;52:6182-7. Boecker W. Preneoplasia of the Breast. A New Conceptual Approach to Proliferative Breast Disease. Muenchen: Saunders Elsevier; 2006. Dupont WD, Page DL. Risk factors for breast cancer in women with proliferative breast disease. The New England Journal of Medicine 1985;312:146-51. Page DL, Dupont WD, Rogers LW, Rados AM. Atypical Hyperplastic Lesions of the Female Breast. A Long-Term Follow-Up Study. Cancer 1985;55:2698-708. Tavassoli FA, Hoefler H, Rosai J, et al. Intraductal proliferative lesions. In: Tavassoli FA, Devilee P, editors. Tumours of the Breast and Female Genital Organs. Lyon: IARC-Press; 2003. p.63-73. Page DL, Dupont WD. Anatomic markers of human premalignancy and risk of breast cancer. Cancer 1990;66:1326-35. Page DL, Dupont WD. Premalignant conditions and markers of elevated risk in the breast and their management. Surg Clin North Am 1990;70:831-51. Otterbach F, Bankfalvi A, Bergner S, et al. Cytokeratin 5/6 immunohistochemistry assists the differential diagnosis of atypical proliferations of the breast. Histopathol 2000;37:232-40. Abd El-Rehim DM, Pinder SE, Paish CE, et al. Expression of luminal and basal cytokeratins in human breast carcinoma. J Pathol 2004;203:661-71. Wetzels RHW, Holland R, van Haelst UJGM, et al. Detection of Basement Membrane Components and Basel Cell Keratin 14 in Noninvasive and Invasive Carcinomas of the Breast. Am J Pathol 1989;134:571-9. Wetzels RHW, Kuijpers HJH, Lane EB, et al. Basal Cell-specific and Hyperproliferation-related Keratins in Human Breast Cancer. Am J Pathol 1991;138:751-63. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000;406:747-52. Korsching E, Packeisen J, Agelopoulos K, et al. Cytogenetic alterations and cytokeratin expression patterns in breast cancer: integrating a new model of breast differentiation into cytogenetic pathways of breast carcinogenesis. Lab Invest 2002;82:1525-33. Wang X, Southard RC, Kilgore MW. The increased expression of peroxisome proliferator-activated receptor-gamma1 in human breast cancer is mediated by selective promoter usage. Cancer Res 2004;64:5592-6. Foulkes WD, Stefansson IM, Chappuis PO, et al. Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst 2003;95:1482-5. Jacquemier J, Padovani L, Rabayrol L, et al. Typical medullary breast carcinomas have a basal/myoepithelial phenotype. J Pathol 2005;207:260-8. Lakhani SR, Reis-Filho JS, Fulford L, et al. Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res 2005;11:5175-80. Lakhani SR, van de Vijver MJ, Jacquemier J, et al. The pathology of familial breast cancer: predictive value of immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in patients with mutations in BRCA1 and BRCA2. J Clin Oncol 2002;20:2310-8. Jones C, Nonni AV, Fulford L, et al. CGH analysis of ductal carcinoma of the breast with basaloid/myoepithelial cell differentiation. Br J Cancer 2001;85:422-7. Azzopardi JG. Problems in Breast Pathology. 1st ed. London: W.B. Saunders; 1979.