Ductal and Lobular Breast Cancer
27
Ductal and lobular breast cancer
Combinations of tumor markers; prognosis and differences
Docent Dan Hellberg, Center for clinical research
Docent Tibor Tot, Department of pathology and clinical cytology
Falun
Table of contents
Page
Hypothesis and aims of the study 3
Literature review 3
Immunohistochemically detected diagnostic factors 4
Gene signature 5
Hormone receptors 5
Growth factors 6
Oncoproteins 11
Tumor suppressors 12
Neoangiogenesis 13
Cytoskeleton 14
Myoepthelial cells 15
Cell adhesion 16
Apoptosis 19
Immunological markers 20
Other markers 21
Serum estradiol and progesterone 23
Material and Methods 24
Results 27
Statistics 28
Power 28
PhD project 29
Ethical approval 29
References 30
HYPOTHESES AND AIMS OF THE STUDY
1. The use of molecular markers, tumor markers, in addition to E-cadherin, as well as tracing genetic alterations in breast cancer tissue will add information on the biological differences between cancer subtypes, in particular between invasive lobular and ductal cancer and in relation to their distribution within the breast tissue.
2. Breast cancer is a disease of an entire breast lobe. Tumor marker expression in apparently normal epithelium will provide evidence for this theory.
3. Studying epithelial – stromal interaction in different tumor subtypes may provide additional information on differences in tumor growth pattern
4. Combinations of expression of tumor markers with prognostic significance individually will refine prognosis prediction.
5. Serum levels of estradiol, progesterone and androstenedione influence the expression of tumor markers in breast cancer.
LITERATURE REVIEW
Breast cancer is by far the most common cancer in women, comprising more than 20% of all female cancers. With more than 150,000 research articles listed in medline, it is also one of the most studied cancer types. It has been estimated that there are more than 1,000000 new cases per year worldwide. Breast cancer is more common in the developed world with age standardised incidence rates of approximately 100 cases per 100,000 women, compared to about 20/100,000 in many developing countries. Breast cancer rates are steadily increasing in all parts of the world (1). In Sweden, more than 7,000 new cases per year are diagnosed (2). Mortality in the developing countries is less than 20%. Increasing incidence has been explained by an increase of exposure to risk factors such as decreased childbearing and breastfeeding, increased exogenous hormone exposure, and detrimental dietary and lifestyle changes, including obesity and less physical activity. The introduction of mammography screening has also resulted in detection of small, early stage cancers and carcinoma in situ, giving an increase in breast cancer diagnoses. Ductal carcinoma in situ (DCIS) has increased seven-fold, while lobular (LCIS) has increased two-fold since 1980. Only four to six per cent of ductal and lobular CIS progress to invasive cancer but are registered as breast cancer, giving inadequately high incidence rates when historical comparisons are made (3).
According to the theory of the ´sick lobe’, breast cancer is a lobar disease, where genetic malformations in early embryonic life and/or increased pool of cancer stem cells, increase the risk of malignant transformation (4). Breast development from embryonic to mature breasts can be studied through expression of cytokeratins. The ‘sick lobe´ theory postulate an early malconstruction of the lobe. A clinical implementation would mean that breast surgery should not only include the tumor and its margins, but the complete lobe. Breast cancer is typed in a variety of subgroups, where ductal (approximately 70%) and lobular (approximately 15%) cancer dominates. A considerable proportion of breast cancer is multifocal. This is particularly true for invasive lobular cancer (ILC). It has been reported that 60% of ILC was multifocal, diffuse or combined (5) and therefore more often undetectable on mammography than invasive ductal cancer (IDC).
Clinically, some of the factors indicating a poor prognosis in breast cancer are histological type, grade, multifocal location or diffuse tumors, tumor size and metastases in lymph nodes and elsewhere.
Immunohistochemically detected diagnostic factors
Immunohistochemistry for detecting proteins involved in diagnosis and prognosis has been increasingly used during the last decades, and an ever-increasing number of those are commercially available. Estrogen and progesterone receptors have become standard in breast tumor specimen investigations. Immunohistochemistry is useful in determination of stromal invasion, distinction between IDC and LDC, and evaluation of lymph node metastases including sentinel lymph nodes (6).
Stromal invasion occurs when the malignant cells extend beyond the myoepitelial layer and the underlying basement membrane. As myoepithelial cells are almost invariably absent from invasive tumor cell nests, myoepthelial markers are used to differentiate between in situ lesions and invasive cancers. Commonly used are smooth muscle actin and myosin heavy chain, calponin and p63. In contrast to DCIS, membraneous staining of E-cadherin is nearly always lost in LCIS. E-cadherin is thus a valuable tool to differentiate between these two types of breast cancer. High molecular cytokeratins, eg. CK 5/6, 10 and 14, show strong antibody staining in benign “usual” hyperplasia, but are almost absent in DCIS, while CK 5/6 is absent in LCIS, aiding in this sometimes difficult differential diagnosis. Lymph node metastases are generally diagnosed with routine haematoxylin and eosin-stained sections. Micrometastases and isolated cancer cells / cellgroups will, however, be easier detected after cytokeratin staining (6).
Gene signature
There has been an increased interest in gene-expression profiles of the entire genome, and correlation to prognosis in cancer. Recently, gene expression in the minority of breast tumors characterized by CD44 but not CD24 expression was compared with normal breast epithelium. The former are known to have a higher tumorigenic capacity than other subtypes of cancer cells. A genetic signature of 186 genes was generated. A group of patients had been delineated having a genetic profile associated with poorer prognosis as compared with other genotypes of breast cancer (7). The method has, however, been criticised. Where several genetic studies are compared, the gene sets are largely non-overlapping. Tumor types, which might be rare, and that are known to be aggressive are selectively studied. The biologic significance and clinical implications have been questioned. When combinations of expression of 186 genes, such as in the study above, are investigated, it will not answer which are the specific biological mechanisms leading to an aggressive cancer (8). Immunohistochemical antibody staining studies detecting over- or underexpression of proteins, encoded by specific genes more directly studies biological mechanisms involved in cancer diagnostics and/or prognosis. The immunohistochemical studies in other cancers indicate that combinations of proteins might be a successful approach (9), but systematic evaluations of biologically plausible combinations in breast cancer have been done only rarely. Markers representing different mechanisms in cancer will be discussed below.
Hormone receptors
The normal breast is a hormone-sensitive organ. Young age at first delivery and multiparity are inversely related to breast cancer risk stressing the importance of final maturation of the breast. There is some evidence that malignant transformation is likely to occur in Ck 8/18, ER negative cells. The evaluation of estrogen (ER) and progesterone receptors (PR) of the breast tumor to predict response to anti-hormonal treatment with tamoxifen has been standard since many years. Estrogen is correlated to proliferation and differentiation (10). ER and PR are significantly increased in benign hyperplastic enlarged lobular units compared to the normal breast (11). The withdrawal of hormone replacement therapy has been found to give and decrease in Ki-67, cyclin D1 (growth factors) and PR expression in women with ER-positive, in contrast to negative tumors. Simultaneously, there will be an increase in p27KIP-1 expression (tumor suppressor) (12).
The presence of ER and PR is independent positive prognosis predictor, irrespective of tamoxifen treatment. In a study of 6000 breast cancers 75% were ER positive while 55% were PR positive. All lobular carcinomas stained for ER, as compared with 74% for ductal. A similar difference was observed for PR (13). A response to tamoxifen therapy is reported in 70-80% of breast cancer containing both ER and PR, while 50-60% of ER positive/PR negative tumors, compared with only 5-10% of ER negative tumors (14). Combinations of ER/PR expressions and other tumor markers have been investigated. ErbB2, p53, and Bcl2 were analysed in different combinations. Presence of ER and absence of ErbB2 seemed to be a slightly better predictor of prognosis than ER alone (15).
The role of ER-β has been increasingly investigated during the past years. The original research group claimed that low ER-β expression was a better predictor of tamoxifen resistance than ER, which is now referred to as ER-α, although ER-β it was found in lesser tumors (50%) (16). A similar frequency of ER-β positive tumors was found in a study comprising 50 tumors (n=27) while 83% were ER-α positive. One tumor was ER-β positive, but ER-α negative. The authors speculated that presence of ER-β might identify a new subgroup where tamoxifen therapy might be inappropriate (17).
The role of androgen receptors in breast cancer is less studied. Androgens have been associated with increased risk, in particular high testosterone levels. In breast tumors there is a close co-expression of androgen receptors, ER and PR. Expression seems to be higher in lobular than ductal tumors, 87% versus 56%, respectively (18). DAX-1 was investigated for a correlation to androgen receptors. DAX-1 functions as a suppressor of steroid synthesis, but its clinical significance is unclear. In breast cancer, DAX-1 was mainly correlated to presence of androgen receptors, but the usefulness, if any, remains to be investigated (19).
Growth factors
Three of the most studied factors associated with proliferation are epithelial growth factor receptor (EGFR), erb-B-2 (also called HER-2 or Neu), Ki-67/MIB-1 and the cyclins. EGFR/erbB-1 belongs to the erbB family of receptor tyrosine kinases alongside with erb-B-2/Her2/Neu, erbB-3 and erbB-4 (20). Ki-67 is absent in quiescent cells, but is expressed in most proliferative cells, and has therefore been used as an indicator of treatment effect (21). Recent trials of drug therapy targeting tumors where erb-B-2 is present have been giving some indications of its usefulness. Several studies have addressed possible prognostic importance for proliferation markers, but results have been contradictory (22).
EGFR, a membrane protein interacts with specific cell-surface receptors and transduce intracellular signals to stimulate DNA synthesis and cell division, and is also involved in malignant transformation and cancer progression in several human tumor types. Approximately 50% of breast tumors are positive for EGFR. Studies on the prognostic importance of EGFR expression have been conflicting. There seem to be an inverse relationship between presence of estrogen receptors and EGFR. These hormone-resistant tumors might represent a subtype where EGFR plays a role, but this theory must be further studied (23).
Ki-67/MIB1 is a nonhistone protein that identifies proliferating cells. Ki-67/MIB1 is generally measured as percentage of stained cells (Ki-67 index). Immunohistochemical measurement is cheap and easy. The antibody was identified in 1983 making it one of the oldest tumor markers. Expression of Ki-67/MIB1 correlates with histopathologic parameters such as mitotic index, the DNA proliferative S-phase fraction and thymidine kinase (TK), that makes expression useful for evaluation of intensity of proliferation (24). A majority of studies have found that increased expression of Ki-67 significantly correlated with overall and disease-free survival (22).
Eukaryotic cells are driven through the cell cycle by successive activation and inactivation of cyclin-dependent kinases. This regulation is dependent of different proteins, among them the cyclins. Expression of cyclins rises and falls at specific stages through the cell cycle. Cyclin E is the limiting factor for G1 phase progression to S-phase. Elevation of cyclin E levels is seen in approximately 40% of breast tumors, with higher levels in ER-negative tumors. Studies on the prognostic importance of cyclin E have given diverging results (22, 25, 26). In a recent meta-analysis overexpression of cyclin E was correlated to poor prognosis (27). The discrepant results between studies have been suggested to be caused by different functions of cyclin E in tumors with different growth pattern. Thus, according the this theory high cyclin E expression in infiltrative tumors would predict a poor prognosis, while cyclin E expression would not be a prognostic marker in tumors with a pushing growth (28).
Cyclin A achieves its peak later in the cycle, and is synthesized during DNA replication and the G2/M transition. It is involved in cellular activities that promote both replication and transcription (29). Overexpression of Cyclin A was in one study associated with reduced survival, even when mutated p53 was included in the analysis (30). A study on advanced breast cancer found Cyclin A expressed in 15% of the tumors, and correlated to a number of other proliferation markers. Cyclin A had the strongest predictive value of poor prognosis of these markers but could not predict response to chemotherapy (31). Several other studies concerning Cyclin A in general have found it useful in predicting prognosis. Expression in only 15% of tumors, however, might hamper its clinical usefulness.
Cyclin D1 is a key regulator protein of the G1 phase progression and thus shortening the G1/S phase transition. Studies on cyclin D1 expression and prognosis in breast cancer have given contradictory, if not confusing, results. In some studies, cyclin D1 expression has shown no correlation to prognosis (32). In another study, cyclin D1 protein expression had features of good prognosis, while cyclin D1 gene overexpression could indicate poor prognosis (33). One study retrospectively investigated a prospective study where the patients were randomly assigned to tamoxifen treatment or not, irrespective of presence of estrogen receptors. In the whole study population, cyclin D1 expression was slightly correlated to better prognosis in untreated patients. In a subgroup of tamoxifen-treated patients with high estrogen receptor expression, however, high expression of cyclin D1 correlated to an unfavourable prognosis (34). Cyclin D1 seems to play a minor role as a marker for breast cancer prognosis.
Among other growth factors that have been investigated in breast cancer are insulin-like growth factor binding protein 2 (IGFBP2), transglutaminase-2 (TG-2), c35, Ski-related novel protein (SnoN), c-met tyrosine kinase receptor, minichromosome maintenance protein 2 (msm-2), keratocyte growth factor (KGF), extracellular-regulated kinases (ERK1/2), fibroblast growth factor 8 (FGF 8) and nerve growth factor receptor (NGFR).