Ductal and Lobular Breast Cancer

1Ductal and lobular breast cancer Combinations of tumor markers; prognosis and differencesDocent Dan Hellberg, Center for clinical research Docent Tibor Tot, Department of pathology and clinical cytology Falun 2Table of contentsPage 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 3HYPOTHESES AND AIMS OF THE STUDY1. 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 REVIEWBreast 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). 4According 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 5 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 signatureThere 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 receptorsThe 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 6 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 7Three 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 8 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). 9Insulin-like growth factors are involved in normal breast development, but might also play a role in malignancies. IGFs have mitogenic effects. Buslund et al. (35) reported that IGFBP 2 was not significantly expressed in normal glandular cells or hyperplasia. An increasingly cytoplasmic expression was observed in atypical hyperplasia, carcinoma in situ and invasive cancer, respectively. No differences were found between ductal and lobular cancers. The results indicated that IGFBP 2 is involved in the growth of mammary cancers, but it is unclear if expression has any prognostic importance.Transglutaminase-2 is a protein involved in cell growth and is detected in all human tissues. In normal breast tissue it has been observed in 13% of cases in normal epithelial and stromal breast tissue. In the same study epithelial expression was found in 63% of 41 ductal cancers and in all 6 of the lobular subtypes. Stromal TG-2 was found in 34% of ductal, compared to 67% of lobular subtypes, which was a significant difference. Interestingly TG-2 expression has also been associated with cell adhesion, metastasis and extracellular modulation. The prognostic role of TG-2 in breast cancer is unknown (36).The C35 protein is a novel biomarker and its functions are poorly investigated. Expression is similar in ductal and lobular carcinoma and increases from 32% to 66% in grade 1 compared to grade 2-3 cancer. A similar increase is seen from stage I to stage IV carcinomas, giving a high expression in metastases. Although the expression in normal breast tissue varies from 13 to 35%, the staining intensity is low. A possible correlation to prognosis is unknown (37).Ski-related novel protein N (SnoN) is a negative controller of Transforming Growth Factor-β. TGF- β is a tumor suppressor and arrests growth of breast tumors. High levels of subcellular (cytoplasmic) localization of SmoN was seen in ductal, but not lobular carcinomas and was associated with shorter disease-free survival in the former, but not the latter cancer subtype. High levels of SmoN were also associated with unfavourable factors in ductal carcinomas, such as absence of hormone receptors, and high levels of COX-2, Ki-67, p53 and erb-B-2 (38).Receptor-type tyrosine kinases, like c-met, are important in cell signal transduction and proliferation. Abnormal expression often leads to malignant transformation. An association between high levels of c-met and β-catenin has been observed. β -catenin/E-cadherin complexes have key roles in cell adhesion. Loss of cell adhesion is essential for dissemination 10 of cancer cells. C-met expression was observed at significantly higher levels in lobular than in ductal cancer, which is in accordance with loss of E-cadherin in the former type. Conflicting results on the prognostic value of c-met have been reported (39).Minichromosome maintenance protein 2 is believed to have an important role for epithelial cell cycle entry by unwinding DNA thus allowing for replication. In one study, Mcm-2 was a strong prognostic factor, independent and superior of histological grade, lymph node stage and Ki-67 but not of Nottingham Prognostic Index (NPI). Mcm-2 was positively correlated to tumor size, mitotic index, histologic grade and NPI. Whether Mcm-2 has a prognostic role, in addition to clinical parameters, is at present unclear (40).Extracellular-regulated kinases (ERK 1/2) regulate genes involved in cell proliferation and differentiation after activation by phosphorylation (pERK 1/2). After activation they are translocated from the cytoplasm to the nucleus. They have been extensively studied in breast cancer cell lines, but there are fewer clinical studies. Expression of pERK was in one study correlatet with a longer disease-free and overall survival independent of clinical prognostic findings. No significant associations were found with 21 cancer markers considered to represent proliferation and invasion-associated proteins, and transcription factors (41). Another study reported that while cytoplasmic ERK1/2 were independently associated with a favourable prognosis, nuclear ERK1/2 were independently associated with poor prognosis (42). Fibroblast growth factor 8 induce proliferation and differentiation of epithelial, and cells of mesodermal and neuroectodemal origin. FGF-8 has been associated with prostate and breast cancer. In breast, no association has been found to lobular versus ductal cancer, presence of estrogen and progesterone receptors and nodal status. There is no evidence that FGF-8 could be of clinical value in breast cancer and the highest levels have in fact been in the lactating breast (43).Nerve growth factor is not restricted to neural cells and has reported to be a tumor suppressor. NGFR/p75 has been identified in the myoepithelial layer in most carcinoma in situ and invasive cancer, but was rarely expressed in malignant breast tumor cells. I a subgroup of basal-like carcinomas NGFR/p75 was associated with good prognosis. NGFR/p75 seems to be of limited value in routine breast cancer investigations (44). 11OncoproteinsThe c-myc oncogene and its proto-oncogene are among the oldest known and most studied factors associated with a poor prognosis in human cancer. C-myc protein is multifunctional and more than 200 target sites are known. A specific main function is thus difficult to establish. C-myc is frequently amplified and overexpressed in breast cancer and in adjacent areas (45). Despite numerous studies, there is no consensus about c-myc as a marker of poor prognosis. There are indications that expression of both c-myc and erbB-2 might be more clinically useful (46).HER-2/ErbB-2/neu, expression has been used for prediciting tumor size, differentiation, lymph node metastases and prognosis, and is overexpressed in 20% to 30% of breast cancers. Studies on erbB-2 and prognosis have been conflicting and different subgroups, in particular the presence of lymph node metastases have been studied. At present, erbB-2 expression is clinically important, as these tumors appear to be less responsive to treatment with tamoxifen or conventional chemotherapy, in contrast to treatment with trastuzumab (Herceptin), an antibody specific for erbB-2 (47-49).The proto-oncogene PCPH is expressed in normal epithelium and decreases markedly in benign tumors, ductal cancer in situ and becoming almost undetectable in invasive cancers. Simultaneously, increased alterations in PCPH expression increase with increasing malignancy grade. The prognostic significance is unknown, but it has been suggested that PCPH expression might be useful for studying malignant progression (50).Mutations of the PIK3CA gene have been reported in several cancer types. When these mutations were studied in different breast tumor types, a much higher frequency was found in lobular cancer (46%), compared to ductal cancer (22%). Mutations of the PIK3CA gene was associated with low levels of Ki-67 and erbB-2 proteins. As this is also a characteristic of lobular tumors, Ki-67 and erbB-2 was adjusted for in multivariate analyses, but the results were unchanged. Mutations of the gene were not correlated to overall survival, but the 12 material did not allow for separate analyses of lobular cancers. PIK might help in differentiating cancer subtypes, but prognosis prediction has given discrepant results in other studies (51).Tumor suppressorsP53 is one of the first tumor suppressors discovered in the 1970s. It is activated in response to DNA damage and p53 protein causes cell cycle arrest by blocking the cell at the G1 and G2 phase prior to DNA replication and mitosis, respectively, and thereby aid in the DNA repair process and prevents mutations. Paradoxically, presence of p53 in breast cancer in general is associated with aggressive tumors and poor prognosis. That is because of the common mutations of p53, leading to inactivation and loss of suppressor function. Mutated p53 is present in about one third of breast malignancies. Mutation seems to precede invasion (52), and in carcinoma in situ p53 is associated with large nuclear size and aneuploidy (53). In invasive cance,r overexpression of p53 has generally also been correlated to large tumor size, grade, and positive lymph nodes. Although p53 has been a negative prognosis predictor in most breast cancer studies, there might be possible different roles in ductal and lobular cancer although this is at present controversial. Mottolese et al. (54) found no differences in survival between subtypes, but cases with lobular cancer were small. Middleton et al. (55) reported high mutated p53 expression (48%) in the aggressive pleomorphic lobular carcinoma, but only in 3% of infiltrating lobular cancer.P16 is a cycline-dependent kinase inhibitor (CDKI) and is expressed in the G1 phase, causing cell cycle inhibition, and thus having a role as tumor suppressor. Several studies have found overexpression correlated to poor prognostic indicators such as stage, grade, tumor size, lymph node involvement and increased mortality. In contrast to p53, p16 has a long half-life. It has been speculated that a high proliferative rate in the tumor gives accumulation of p16. Another suggestion is that cytoplasmic, with or without nuclear, expression indicates inactive p16 (56).Another CDKI is p27 that inhibits cyclin-complexes in the early G1 phase. Activation of p27 results e.g. by anti-proliferative signals such as transforming growth factor-β mobilize stored p27. In a majority of studies low p27 expression were associated with poor prognosis (22). In one study there was a significant positive correlation between p27 expression and positive 13 lymph nodes in patients with advanced breast cancer (57). Results are, however, difficult to compare which generally also is the case for most studies on tumor markers. Results for different subgroups often deviate.Reduction of the fragile histidine triad (FHIT) protein expression has been observed in various cancers. The FHIT gene encompasses the common chromosomal fragile site FRA3B that are commonly deleted in cancer. This is a tumor suppressor gene that is involved in the control of apoptosis and proliferation. FHIT was detected quite recently but has gained much attention in breast cancer research. FHIT protein is strongly expressed in normal epithelium but is reduced in 50-70% of breast cancers. Deletions of the FHIT gene was in one study found to occur in 59% of ductal, as compared to 16% in lobular, carcinoma (58). Reduced expression has been observed in CIS and in adjacent areas in most cases. FHIT expression is successively lost with cancer stage progression. An association was observed between low FHIT expression and increased grade, size, p53 and high proliferation. There is some evidence that FHIT is more useful as a predictor in patients with good prognosis (59-62).Another region of chromosomal breaks, common fragile sites, is associated with the PARK2 gene. Break of PARK2 was found in only 6% of breast cancers while loss of its protein expression was found in 13% of cancers. At present there is little evidence that PARK2 have a clinical or prognostic impact in breast cancer (63).NeoangiogenesisThe growth of tumors beyond 1-2 mm is dependent on formation of new blood vessels, neoangiogenesis. Angiogenesis is a prerequisite for continuous tumor growth and the proliferation of stromal fibroblasts. Vascular endothelial growth factor (VEGF) is the most studied and maybe the major angiogenetic factor. A high VEGF expression has been found to correlate with ER-negativity and erbB-2 positivity, indicating an aggressive, proliferative tumor (64). Studies on VEGF and prognosis has been inconclusive. When VEGF subtypes were analysed separately, different results were accomplished for VEGF-A, VEGF-B and VEGF receptor-1 on correlations to other tumor markers and to prognosis. Thus, VEGF-A expression was not related to prognosis, while VEGF-B seemed to be a prognostic indicator only in node-positive patients. VEGFR-1 was negatively correlated to survival in p53-positive tumors (65). One must always be cautious when a number of subgroups are formed and 14 occasional significant correlations appear. Also, VEGF expression might indicate a tumor with good blood supply, but may also represent a tumor signalling hypoxia.Inhibitor of DNA binding (Id-1) is known to correlate with angiogenesis and possibly also in proliferation and invasion. During embryogenesis it plays a role in differentiation and cell cycle progression. In breast cancer, strong expression of Id-1 was correlated to increased microvessel density in ER-negative and in node-positive tumors. Ductal tumors had significantly higher overexpression than other subtypes. There was no correlation to prognosis (66).Vascular endothelial cadherin (cadherin 5) is an adhesion molecule, only expressed in endothelial cells. In breast cancer it has been correlated to microvessel density and factors related to poor prognosis determined by the Nottingham Prognostic Index. At present it is unclear if cadherin 5 is associated to clinical prognosis (67). Another protein that is involved in angiogenesis is Ets-1. The prognostic importance of Ets-1 is unknown (68).CytoskeletonThe tissue architecture of epithelial cells includes the cytoskeleton and junctional complexes between cells. The cytoskeleton of normal breast epithelium is largely formed by cytokeratin 8, 18 and 19. Expression of CK 18 is down-regulated in metastatic cancer. Woelfle et al (69) reported almost universal finding of CK 18 in normal breast tissue, but reduced expression in 25% of ductal and 17% of lobular carcinomas. Low CK 18 expression was significantly associated with advanced tumor stage, high grade and increased mortality. Expression of the basal type CK 5/6 has been used to distinguish between ductal hyperplasia and cancer in situ (70). In addition, CK 8 stains ductal and lobular carcinoma differently and has been proposed to be a sensitive tool to establish cancer type (71). In invasive cancer cytokeratin immunohistochemistry have been used to diagnose sentinel lymph node metastases. Cote et al (72) reported that metastases was detected in 7% of mammary cancers with haematoxylin and eosin staining, compared to 20% with cytokeratin staining. Cserni et al (73) could detect lymph metastases with cytokeratin staining in 34% of cases, which were negative for haematoxylin/eosin. 15Metalloproteinanses (MMP) are responsible for degradation of the extracellular matrix and are thought to play an important role in invasion and matastases. MMP-7 degrades type-IV collagen, fibronectin and laminin (74). Tissue inhibitors of MMP (TIMP) prevent invasion and metastasis, but paradoxically also promotes cell growth. TIMPs have been evaluated for prognostic significance. Nakopoulou et al. (75), however, found a correlation between TIMP- 2 expression and small tumor size, as well as higher survival rate. In a subsequent study TIMP1 overexpression was observed in 60% of breast cancer. TIMP1 expression was not associated with tumor size, histopathological type, stage or lymph node involvement. There was a significantly increased survival rate, with 75% survival with TIMP1-positive cancers, compared to 60% among TIMP1-negative cases. This difference increased in patients with MMP-2 negative tumors with no nodal metastases (76).Urokinase-type plasminogen activator (uPA) is an enzyme that also plays a role in degradation of extracellular matrix.Dublin et al. (77) showed that uPA in firbroblasts, but not in tumor cells were significantly higher expressed in invasive cancer than in carcinoma in situ. UPA expression was correlated to large tumor size, but no significant correlation to survival was found. Hemsen et al. (78) investigated the role of uPA-receptor in metastases. UPA receptor was expressed in 90% of tumor cells and 75% of fibroblasts. No correlation to lymph node metastases was found, but expression of UPA receptor in tumor cells, but not in fibroblasts, correlated significantly to bone marrow metastases.RhoC is a recently identified ras homology gene that is involved in cytoskeletal reorganisation and adhesion. The RhoC protein is increasingly expressed from normal epithelium, carcinoma in situ, invasive cancer and metastases. Moderate or strong RhoC expression was found in 39% of invasive cancer, compared to 3% in normal epithelium or hyperplasia. In a material of the aggressive inflammatory breast cancer type, 10-year survival was 23% when RhoC expression was high, compared to 53% when the cancers showed less expression (79).Myoepithelial cellsBenign breast epithelium and carcinoma in situ have a peripheral layer of myoepithelial cells and basement membrane. Stromal invasion occurs when malignant epithelial cells extend beyond the myoepithelial cell layer and the basement membrane. Myoepithelial cells are generally absent from invasive tumors. Immunohistochemical staining of myoepithelial 16 markers has been used to distinguish in situ lesions from invasive cancer. Commonly used markers are smooth muscle actin and myosin heavy chain, calponin, cytokeratins and p63 (80).Rakha et al. (81) compared invasive tumors with myoepithelial or basal phenotype. Myoepithelial carcinomas were characterized by expression of smooth muscle actin and/or p63, while the basal type expressed CK5/6 and/or CK14. The basal type was correlated to loss of estrogen receptors and FHIT and expression of p53 and EGFR, while the myoepithelial type showed increased E-cadherin expression. Basal, but not myoepithelial, carcinomas were associated with poor survival in multivariate analyses.14-3-3δ is a candidate tumor suppressor gene transactivated by p53 in response to DNA damage. 14-3-3δ is epressed by myoepithelial cells in normal breast. In invasive carcinomas expression has been found in 19% of cancers. In normal epithelium 14-3-3δ is predominately expressed in cytoplasma and occasionally in the nuclei. Exclusive cytoplasmic localization was correlated to poor prognosis, while a simultaneous nuclear expression was not associated with survival. Inactivation of estrogen receptors might be a reason for this unexpected finding (82).SPARC (osteonectin) is expressed in myothelial cells and modulates cellular interaction with the extracellular matrix. In a study by Jones et al. (83), 5% of breast carcinomas expressed SPARC. Expression was moderately but significantly associated with poor prognosis.Cell adhesionE-cadherin is one of the most studied cell-adhesive molecules. In breast cancer E-cadherin expression has been widely used clinically for differential diagnosis between ductal and lobular carcinoma as it is expressed in almost all ductal, but rarely in lobular cancer (84). Absence of E-cadherin is associated with abnormal cytoplasmic and nuclear localization of p120 catenin. Aberrant staining was rarely seen in ductal carcinomas. P120 may be a mediator of the oncogenic effect seen with E-cadherin inactivation, such as enhanced motility and invasion (85). Lobular carcinoma of pleomorphic type is associated with a poor prognosis. In a series of 27 tumors Palacios et al. (86) reported loss of heterozygosity for E-cadherin in all cases. The results, however, are similar to classic infiltrative lobular carcinomas. 17Bukholm et al. (87) studied expression of E-cadherin in a histopathologically mixed series of breast carcinomas. Reduced expression of E-cadherin was observed in 64% of 90 tumors. T1 (mammographically detected tumors) had significantly less alterations of expression than T2 (clinically palpable tumors). In addition, a correlation between lymph node metastases and reduced expression of E-cadherin was found.Loss of expression of E-cadherin in the large majority of lobular cancer makes is less useful for prognostic information. Rakha et al. (88) investigated a large material (n=1516) cases of invasive non-lobular breast carcinomas. A score was used where both percentage and intensity of staining was evaluated and a score was constructed. Only membraneous E-cadherin was considered. Reduced expression was diagnosed in 759 cases. There were significant correlations to disease-free and overall survival, larger tumor size, higher histological grade, ER-negativity and distant metastases. A scoring method, also considering localization might be an interesting approach for determining prognosis by E-cadherin expression.Soler et al. (89) did, however, not find a correlation between E-cadherin expression and survival in a material largely including ductal tumors. P-cadherin (placental) was expressed in 52% of carcinomas and was a strong predictor of survival. At five years follow-up survival was 90% of patients with P-cadherin negative tumors, compared to 59% of patients with P- cadherin positive tumors. Expression was correlated to negative estrogen/progesterone status. Survival results were independent of tumor size and lymph node metastases.ß-catenin is closely related to E-cadherin and is essential for its adhesive function. In one study, abnormal expression of ß-catenin was slightly higher in lobular than in ductal tumors (77% vs. 64%). Expression was lower in in situ lesions (90). ß-catenin expression was not correlated to survival in the study of Soler et al. (89).Laminin and collagen IV are important components of the basement membrane as well as adhesion glycoproteins. They are involved in the adhesion of neoplastic cells to the basement membrane and are thought to play a role in invasion and correlation to poor prognosis. Laminin expression were reported associated with poor differentiation, positive lymph nodes, non-diploid tumors, and a higher frequency of ER-negativity. Laminin expression was seen 18 more often in recurrences (85%) than in patients with no sign of recurrence (57%). Type IV collagen expression correlated to ER-negativity and non-diploidi, as well as relapse (74% vs. 49% in patients with no relapse) (91).CD44s (standard) is with at least 10 isoforms involved in cell-cell adhesion and cell- extracellular matrix adhesive interactions. Different isoforms have been linked to tumor progression in a variety of cancer types, but like in breast cancer, results have been contradictory. In lobular breast cancer only CD44v5 showed some correlation to poor prognostic factors when CD44s, v3, v5, v7 and v3-10 were investigated. Thus, cytoplasmic staining of CD44v5 correlated with lymph node negativity, wheras membranous staining correlated with lymph node positive patients (92). At present CD44 isoforms seem to be of limited value for the use as prognostic markers.Mucins are present at the surface of most epithelial cells. More than 20 mucins have been characterized on preneoplastic and neoplastic cells. In breast cancer overexpression and underglycolysation has been observed in MUC1 and has been correlated to poor prognosis. Sonora et al. (93) investigated MUC5B and reported expression in a majority of invasive cancer, but also in non-malignant breast disease. None of the five normal breast tissues showed mucin expression. Interestingly, expression was found in 42% of normal-appearing breast epithelia adjacent to the tumor. Baldus applied numerous different monoclonal antibodies to MUC1 and MUC2. Staining detecting overexpression of sialylated short chain isoform, but no others, was associated with a better prognosis in both ductal and lobular carcinomas (94).Gap junctions are fundamental structures for cell-cell communication and signal transduction. Connexins have key roles in forming these junctions. Phosphorylation inactivates connexin and has been observed in increasing expression from normal breast epithelium to invasive cancer. It believed to reflect proliferation but is unclear if intensity of expression has prognostic significance (95).Psoriasin is a member of the S100 gene family and is known for its involvement in psoriasis. Psoriasin is a possible immune regulator and expressed at low levels in normal breast tissue. Expression in cancer is associated with decreased adhesion, loss of attachment to extracellular matrix, increased growth and invasiveness. In breast cancer, lobular carcinomas have slightly 19 increased levels compared with ductal tumors. Increased RNA transcripts were in one study reported strongly correlated to higher grade and poor prognosis (96).ApoptosisImmortality is the key attribute of cancerous cells. Increased apoptosis, programmed cell death, would theoretically lead to better prognosis. A number of tumor markers have been associated with apoptosis. Bcl-2 and Bax have important regulatory roles and have been widely studied. The ratio between bcl-2 (suppressing apoptosis), and Bax (promoting cell death) expression has been used as an apoptotic index. In breast cancer presence of bcl-2 is paradoxically often associated with a favourable outcome. Ioachim et al. (97) studied correlations of expression of a number of tumor markers. A strong relation between overexpression of bcl-2, and Bax, estrogen and progesterone receptor expression was found, while there was an inverse correlation to p53 expression. This favourable tumor marker expression might be one explanation for the correlation between bcl-2 expression and prognosis.In another study, an inverse association between Bcl-2 and Cyclin A expression was observed. Both reduced bcl-2 and Bax expression was associated with lymph node metastases, and low bcl-2 expression correlated to a better survival rate, but lost its significance after control for Cyclin A expression (98). The authors conclude that other mechanisms than apoptosis is important for metastatic spread. A large number of markers that had some association with apoptosis were studied in beast-, colon-, lung- and prostate cancer. In the whole material, p27, p16 and bcl-2 was associated with better survival, and expression of bcl-2 seemed to be the strongest predictor of survival (99).Other results have, however, been reported. Mottolese at al. (54) found no association between bcl-2 and survival in ductal carcinomas, but a worse prognosis in women with the lobular subtype. Fas is a cell membrane receptor that is positively correlated to apoptosis after binding to its ligand, FasL. FasL is present in normal epithelial cells of the breast, but show a marked increase in expression in invasive carcinoma. Fas protein expression has, on the other hand, 20 been reported decreased in cancer tissue, compared to normal breast. The clinical significance of Fas and FasL expression is unclear (100).Caspases belong to a family of cysteine proteases and mediates cellular functions leading to apoptotic cell death. Mitochodrical disruption appears to be a critical point in this process. Nakopoulou et al. (101) studied the relation between caspase-3 and the antiapoptotic proteins bcl-2 and mutant p53. Caspase-3 was overexpressed in 75% of cancerous tissue, and showed a correlation between low p53 expression and high bcl-2-positive immunostaining. In addition, tissue inhibitor of matrix metalloproteinases (TIMP-2) expression in both cancer cells and stromal cells was positively correlated to caspase-3 expression. The correlations to bcl-2, p53 and TIMP-2 would give expectations of good prognosis with presence of caspase- 3. A significance correlation to a poor prognosis was, however, found demonstrating the complexity of tumor marker expression, and the need for further studies.Immunological markersImmunological mechanisms have been studied extensively in cancer including large number of markers. The importance of CD T-cells has possibly raised most attention. Ben-hur et al. (102) investigated immune response in stroma and included CD4+ T-cells, CD8+ T-cells, CD20+ B-cells and macrophages. A sharp increase of CD8+ T-cells was shown during progression from normal epithelium to invasive cancer, while expression of other markers showed lower or no increase.CD4+ CD25+ regulatory T-cells are involved in inhibiting immune response through their ability to suppress T-cell proliferation, thus enabling tumors to escape the immune defence. Tumor-infiltrating FOXP3 is regarded as a more specific regulatory T-cell marker than CD4+ CD25+. Leong et al. (103) investigated presence of T-cells expressing CD4+, CD8+, CD25+, CD28+, CD56+ and their combinations. FOXP3 was present in in 43% of CD4+ CD25+ T- cells. No correlation to prognosis was found, but the entire study included only 47 patients. Bates et al. (104) reported increasing FOXP3 expression form normal epithelium, ductal cancer in situ to invasive cancer. FOXP3 expression was correlated to relapse of cancer in situ, shorter disease-free survival in invasive cancer and identification of high-risk patients when high number of regulatory T-cells was found within ER-positive patients. 21Other markersOnly one or two markers below are presented and will not presented in a separate section. Some markers represent multiple functions in carcinogenesis and could be difficult to choose a ‘main’ mechanism, others are recently discovered and functions have not been clearly elucidated.Peroxisome proliferator activated receptor gamma (PPARλ) and its co-activator PGC-1 belongs to a family of nuclear receptors. Staining has been weaker in cancer tissue than in normal breast epithelium. PPARλ is involved in apoptosis induction and inhibiting cell growth. Staining intensity is lower in lobular than ductal tumors (105). PPARλ and PGC-1 may be useful as prognosis markers, but this is at present unclear.Increased expression of hypoxia-inducible factor (HIF-1α) is considered to be the strongest signal for angiogenesis, in particular to activate VEGF. High levels of HIF-1 α has been associated with high microvessel density, increased EGFR, a well as platelet-derived growth factor, expression. In this study, there was only a non-significant correlation to increased VEGF expression (106). High HIF-1α intensity has been correlated to poor prognosis and it has been speculated if HIF-1 α could be a target for pharmaceutical anti-cancer therapy. Kronblad et al. (107) observed a moderate correlation between HIF-1α expression and impaired survival in premenopausal women with stage II breast cancer. Various subgroups were analysed and a strong correlation between HIF-1 α positive tumors and survival was found in node-positive patients with Nottingham histological grade ½ tumors. One must however be aware of that this latter subgroup was small.Nm23 H1 has been considered to be a metastasis suppressor gene, mainly because of in vitro studies. Nakopoulou et al. (108) studied the Nm23 H1 peptide in breast cancer, but found no significant correlation to most factors suggesting a poor prognosis, i.e. ER, grade, tumor size and lymph node metastases. Nm23-H1 expression was more common in ductal tumors, and was associated with PR-positivity and with c-ErbB-2 negative tumors. Prognosis, however, was not investigated.Gluthathione is one of the most important intracellular antioxidants and reacts against reactive oxygene species (ROS). ROS are involved in processes causing cellular damage and genomic 22 alterations contributing to carcinogenesis. Gluthatione is synthesized by glutamate-L-cysteine ligase (GLCL), that consists of a catalytic and a regulatory subunit, and glutathione synthesase. In breast cancer, expression of the two subunits has been observed in 50% and 44%, respectively, and is more often seen in cancer in situ and in lobular cancer, than in ductal cancer. Expression has been inversely correlated to lymph node metastases and to a favourable prognosis. In patients receiving chemotherapy, however, no prognostic differences were observed (109).COX-2 expression has been shown correlated with a poor prognosis in different cancer types. Among its many functions COX-2 has been suggested to be involved in angiogenesis, invasion and metastases. In breast cancer, an association with MMP-2 expression has been shown. Approximately on third of breast cancers are COX-2 positive, and a correlation to large tumor size negative ER and PR status and unfavourable prognosis has been observed (110).Xanthine oxidoreductase (XOR) is an oxidant involved in the hypoxanthine metabolism. XOR is strongly expressed in normal breast epithelium, in particular during lactogenesis. In breast cancer absent expression was reported in 7% of tumors, while the remaining are distributed among moderate or strong expression. Absence of XOR has been associated with poor differentiation, positive axillary lymph nodes, high COX-2 expression and decreased survival. An unfavorable prognosis was reported irrespective of lymph node metastases and tumor size. Clinical use of XOR might be limited as the XOR negative population were small (111).The oncofetal antigen Tn is a widely studied tumor marker. In breast cancer light to moderate staining is positive in most cells. There are few, if any, evidences that Tn could be useful as a prognostic factor in breast cancer. Preliminary results show, however, that normal tissue adjacent to cancer in situ or invasive cancer show Tn expression in 40-45% (112). Protein kinase C is associated with the loss of differentiation. In a study on breast cancer, high heterogenous staining was correlated to the presence of positive lymph nodes, but frequent homogenous staining was observed in normal epithelium (113).Telomeres are repetitive DNA sequences at the ends of chromosomes that are shortened at each cell division. Progressive shortening of DNA may cause chromosome instability and cell 23 death. Telomerase is able to synthesize repetitive sequences onto chromosomal ends maintaining telomere length thereby contributing to immortilization of cancer cells. Expression of the catalytic subunit of human telomerase reverse transcriptase, hTERT, is used for immunohistochemical detection. Telomerase activity has been shown in both in situ and invasive cancer lesions (114). Expression of hTERT has been associated with poor prognosis in a number of studies, but there are contradictory results (115).The most striking difference between ductal and lobular cancers by tumor markers is the absence of E-cadherin in lobular cancer. Mathieu et al. speculated that the poor responsiveness to neoadjuvant chemotherapy in infiltrating lobular carcinomas compared to ductal was different biological profiles. Factors that were associated a poor response, i.e. ER, Ki67 and p53, were more frequent in infiltration lobular carcinomas than in ductal. Overall survival was, however, similar in both types of cancer. The authors question the use of neoadjuvant chemotherapy in the former tumors (116). Finally, he higher frequency of metastases in infiltrating lobular carcinoma have been proposed to be caused by the lack of e- cadherin expression resulting in defect cell adhesion. The paradoxal finding in many studies that prognosis is similar to that of ductal carcinomas might be due to low proliferation rate and high expression of ER (117).Serum Estradiol and ProgesteroneBreast cancer is strongly hormone-related as evident by presence of ER and PR and the favourable effect of inhibitors such as tamoxifen. Less studied are molecular effects, on breast cancer cells by female sex hormones and androgens. It is well known that ER and PR levels increase with increasing serum estradiol and progesterone (118). Occasionally, studies have included both serum steroid hormone levels and breast cancer tissue tumor markers. Ki-67 expression was in one study inversely correlated with high serum estradiol levels (119), while no correlation between serum estradiol and progesterone, and proliferation, measured as Ki- 67 labeling index, was found in another study (118). In the former study (119), high estradiol levels in postmenopausal patients correlated inversely with c-erb-B2 expression. Supplementation with progesterone in breast cancer cell cultures was associated to down- regulation of p53 expression, while estradiol had the reverse effect (120). It has also been reported that progestins stimulate early cancer growth with concomitant increased c-myc expression in cell cultures, and a progestin regulatory region was found in the c-myc gene 24(121). The mitotic effect of progesterone might also be caused by increasing levels of insulin- like growth factor-I after progesterone was added, in particular in PR positive cancers. The results were reported in a cell culture study (122). There is evidence that the mitotic effect on breast cancer cells by estrogens may at least partly be mediated by transactivation of EGFR. (123).It is evident that in vivo data on correlations between sex steroid hormones and tumor markers in breast cancer are sparse. Further studies within the area are warranted to learn about the tumor biology.MATERIAL AND METHODS1. Tumor marker study1a. Material. During 1996 to 2007 data on 2304 women with invasive breast cancer have been computerized at the Department of Pathology and Clinical Cytology at Falun Hospital. Approximately 70% of the tumors were of ductal subtype, 15% of lobular subtype and the remaining 15% were of other subtypes.The entire study material will be characterized using the following data set: Personal identification code Preoperative cytology Tumor size Tumor growth pattern (unifocal, multifocal or diffuse) In situ component (type, grade size) Location of tumor Histological subtype (invasive component) Differentiation, ploidy S-phase fraction Lymph node metastases Distant metastases 25The missing data will be completed through patient records. The prognoistic significance of these parameters will be tested in relation to overall survival and disease free survival.Tissue micro array (TMA) will be made on 350 cases of each ductal and lobular tumors (cases matched according to tumor stage and size) from a representative area of the original blocks. Each TMA-block will include 24 2 mm biopsies, to cut down costs for antibodies and diagnostics. In cases with local recurrence or metastasis the recurrent or metastatic invasive tumor will also be sampled to TMA. Diagnostics will be made by a experienced pathologist familiar with immunohistochemistry and blinded for all clinical and histopathological details1b. Time schedule. End-point of the study is ten-year survival. Collection of data will thus close at the end of 2017. Final analyses of data and preparation of articles will be made before the end of 2020. Five-year survival will be used for intermediate analyses and articles for women with their diagnosis before 2003, and will be established through the patient records and the register of the Regional Oncology Center at Uppsala University.Analyses of distribution of tumor markers according to histopathological variables, subtype, growth pattern etc. will start immediately.1c. Tumor markers. Initially, expression of three tumor markers was used in clinical routine. At present, seven tumor markers are included.Initial tumor markers: 1. Estrogen receptor 2. Progesterone receptor 3. c-erb-2/her-2.Routinely used tumor markers at present: 1. Estrogen receptor 2. Progesterone receptor 263. c-erb-2/her (oncoprotein) 4. E-cadherin (cell-cell adhesion) 5. Cytokeratin 5/6 (cytoskeleton) 6. Cytokeratin 14 (cytoskeleton) 7. Epidermal Growth Factor Receptor (proliferation)Tumor markers to be included in the study:1. Smooth muscle actin (stroma) 2. Tenascin C (stroma) 3. Collagen 4 (stroma) 4-6. CD 3, CD 4, CD 8, CD 20, CD1a, CD68 and CD138 (3-4 of these immunological markers will be selected) 7. CD 44v5 (cell-cell adhesion) 8. Ki-67/MIB1 (proliferation) 9. Cyclin E (cell cycle regulation) 10. P53 (tumor suppressor) 11. Ski-related novel protein (SnoN) (proliferation) 12. Fragile histidine triad (FHIT) (tumor suppressor) 13. Bcl-2 (apoptosis suppressor) 14. COX-2 (multiple functions) 15. Telomerase (hTERT) (immortilization)Tumor markers have been selected by their significance as prognosis predictors in accordance to the literature review. It was also taken into account that the choice of tumor markers represents different mechanisms in carcinogenesis.Purpose:1. Expression of each tumor marker, or in combination, will be investigated for correlation to:1. Precense of positive lymph nodes 2. Local recurrences 3. Disease-free survival 274. Five-year survival and eventually ten-year survival 5. Expression in apparently normal epithelium of the same breast lobe, and in an adjacent lobe that is not affected by cancer. 6. Histological growth pattern 7. Histological distribution and extent of the tumor2. Serological study With the women´s approval, 5 mL serum will be sampled and frozen in –70 C in consecutive patients surgically treated for carcinoma in situ or invasive breast cancer during two years. This material will include approximately 400 patients.Analyses will be made for serum levels of estradiol, progesterone and androstenedione, the latter as a marker of androgenicity. Purpose: Correlations with the same clinico-pathologically variables and tumor markers as above will be searched for.RESULTSStudy 1.The entire study material of 2304 cases will be characterized regarding the above listed clinical and basic histopathology dataset. This will allow delineating the cases of lobular carcinomas and forming a corresponding group of ductal carcinomas of matching clinical characteristics and basic morphologic parameters. Five year overall survival will be used as outcome to test the significance of the listed parameters, which will be necessary for comparison when the significance of biomarkers will be tested. 28Study 2.Initially, correlation between expression of the individual tumor markers and prognosis, other clinical characteristics, and growth pattern will be investigated. The aim is then to find combinations of individually significant tumor markers that could identify aggressive tumors and strengthen prognosis prediction. Comparisons of different histological subtypes of the tumor and their expression of tumor markers will provide evidence if these are different cancers that share in common that they affect the breast, or that different subtypes show many similarities and are only variants of the same cancer. Lack of E-cadherin expression in lobular cancer indicates that subtypes are at least partly different cancers.The search for expression of tumor markers in normal epithelium of the same breast lobe and in a normal lobe is made to investigate the theory of the ‘sick lobe’. Findings of expression of one or more tumor markers simultaneously in the tumor and the lobe, but no expression in a healthy breast lobe will support this theory. Additional genetic analysis of unifocal, multifocal and diffuse lobular versus ductal tumors is also planned.Study 3.In sharp contrast to expression of estrogen- and progesterone receptors, analyses of serum sex steroid hormone levels and their association with tumor marker expression in breast cancer tissue is an area of investigation that has been done sparsely. The aim of this study is mainly to increase the knowledge about breast cancer mechanisms. The biological roles of androgens in breast cancer are virtually unknown. Serum androstenedione might be a better marker of androgenicity in women, but testosterone could be added to the analyses. One could speculate that finding a specific role of androgens in breast cancer might open new ideas of pharmaceutical treatment as a supplement to tamoxifen. 29StatisticsA chi2 test (likelihood ratio) will be used for crude comparisons of dichotomous, categorical variables. T-test will be used for continuous variables, such as age. Multifactorial analyses by logistic regression (loglikelihood test) will be used for adjustment for possible confounding factors, and for estimation of odds ratios (OR) and 95% confidence intervals (95% CI).Each of the tumor markers will be analysed separately for correlation to survival. Analysis will include crude significance testing, odds ratios with confidence intervals, and multivariate analysis including possible confounders to determine ‘independent’ correlations, and to compare odds ratios.Expression of tumor markers will be compared to find interactions. Combinations of tumor markers will be evaluated to analyze survival. Cox regression (proportional hazard) will be used for survival, and include confounders such as stage. Kaplan Meier survival curves will be used for graphics.PowerBased on a 35% mortality with expression of a tumor marker in one group of women and 25% mortality in the remaining women, 348 subjects will be required in each group to achieve significance with 80% power.PhD projectThe project was initially designed for two PhD students, who has accepted, and will be formally recruited before the project is about to start. 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Ductal and Lobular Breast Cancer

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