Pathological examination has been the gold standard for diagnosis in cancer and its role has also included the elucidation of etiology, pathogenesis, clinicopathological correlation, and prognostication. The advent of newer technologies and the realization that breast cancer is heterogeneous has shifted the focus to prognostication, with increased attention being paid to the identification of morphological features and immunohistochemical markers of prognostic relevance. However, despite the massive efforts invested in the identification of immunohistochemical biomarkers in breast cancer the majority have not proven to be of value in multivariate analyses and only estrogen receptor, progesterone receptor, and Her2/neu expression have remained essential components of pathological examination. These 3 markers were initially employed for prognostication but their role in treatment also rendered them of predictive value. Newer molecular methods, especially high-throughput technologies, have shown that even morphologically similar subtypes of breast cancer can show molecular heterogeneity; moreover, infiltrating ductal carcinoma can be separated into at least 4 molecular subtypes designated luminal (ER+, PR+, and Her2/neu-), Her2 overexpressing (ER-, PR-, and Her2/neu+), basal-like (ER-, PR-, Her2/neu-, and CK5/6+, EGFR+), and normal breast-like (ER-, PR-, and Her2/neu-), each with different clinical outcomes. The importance of proliferative gene expression in these subtypes has been demonstrated and surrogate immunohistochemical markers include ER, PR, Her2/neu, and Ki67 for the more expensive molecular tests. Molecular technologies, importantly, have not only provided further insights into the heterogeneity of breast cancer but have also opened new avenues for treatment through the identification of signaling molecules important in the proliferation and survival of the neoplastic cells. The treatment of cancer thus shifts from the conventional approach of 'one size fits all' to one of personalized treatment tailored to the specific characteristics of the tumor. Pathologists continue to play their traditional role in diagnosis but, as purveyors of the excised tissue, pathologists now have the additional role of identifying biomarkers responsive to therapeutic manipulation, thus playing an inextricable role as diagnostic oncologists in the management of breast cancer.

The rapid acceptance of immunohistology as an invaluable adjunct to morphologic diagnosis has been possible because of the development of new and more sensitive antibodies and detection systems that allow its application to formalin-fixed, paraffin-embedded tissue (FFPT). More importantly, antigen-retrieval techniques have resulted in some degree of consistency allowing immunohistology to be used reliably as a diagnostic tool. The advent of prognostic and predictive biomarkers, and the desire for individualized therapy has resulted in mounting pressure to employ the immunohistological assay in a quantitative manner. While it was not a major issue when the technique was employed in a qualitative manner, the numerous variables in the preanalytical and analytical phases of the test procedure that influence the immunoexpression of proteins in FFPT become critical to standardization. Tissue fixation is pivotal to antigen preservation but exposure to fixative prior to accessioning by the laboratory is not controlled. Antigen retrieval, crucial in the analytical phase, continues to be employed in an empirical manner with the actual mechanism of action remaining elusive. There is great variation in reagents, methodology, and duration of tissue processing and immunostaining procedure, and the detection systems employed are not standardized between laboratories. While many of these variables are offset by the application of antigen retrieval, which enables the detection of a wide range of antigens in FFPT, the method itself is not standardized. This myriad of variables makes it inappropriate to provide meaningful comparisons of results obtained in different laboratories and even in the same laboratory, as in current practice, each specimen experiences different preanalytical variables. Furthermore, variables in interpretation exist and cutoff thresholds for positivity differ. Failure to recognize false-positive and false-negative stains leads to further errors of quantitative measurement. Many of the problems relating to the technology and interpretation of immunostaining originate from failure to recognize that this procedure is different from other histological stains and involves many more steps that cannot be monitored until the end result is attained. While several remedial measures can be suggested to address some of these problems, accurate and reproducible quantitative assessment of immunostains presently remains elusive as important variables that impact on antigen preservation in the paraffin-embedded biopsy -cannot be standardized.

The integration of immunostaining as an often-essential component of morphologic assessment makes it necessary that pathologists are familiar with the many technical and interpretive pitfalls that may arise because of the wide variety of factors that can significantly influence the ability to demonstrate relevant antigens in paraffin-embedded tissue sections. Pre-analytical variables that affect immunostaining include fixation, type of fixative, duration, temperature, and pH of fixation, variables in tissue processing, antigen loss resulting from delays in fixation, tissue necrosis, and levels of antigen expression. Analytical factors relate to the complex and sometimes capricious immunolabeling procedure that has as variables the specificity and sensitivity of the antibody clone, reagent dilution, detection system, and chromogen, and importantly, the method of antigen retrieval, which has its own set of variables such as time, temperature, method of heat generation, retrieval solution pH and molarity, and the synergistic action of proteolytic digestion. To obtain the highest diagnostic yield from immunostaining the correct questions must be asked and the pathologist must have familiarity with the characteristics of the antibody, its cross reactivity, if any, and localization of antigen in the cell to understand thresholds and cut-off levels and to recognize false-positive staining. Proper utilization of immunostaining requires that it is employed as a morphology-based technique and not interpreted in isolation.

The present study was designed to investigate the clinical/prognostic relevance of immunohistochemical expression of p53-targeted genes mdm-2, p21WAF1 and bcl-2 alone and in combination with p53 for the indirect assessment of p53 gene status in breast cancer. 141 archival breast carcinomas were immunostained, and the putative mutational status of the p53 gene was defined in 21 of them, as a control for immunohistochemistry, using the polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) analysis. Genetic changes of p53 correlated significantly with p53 protein overexpression (p = 0.01) but did not do so with any of the related molecules. Immunohistochemical p53 status was directly correlated with mdm-2 (p = 0.0001), p21 (p = 0.0004) and inversely with bcl-2 (p = 0.005) expression. bcl-2 proved to be an independent marker of prognosis, p53 only in the group of node-positive carcinomas, whereas bcl-2-/p53+ tumours revealed the worst prognosis. Mdm-2 and p21 expression was of prognostic significance neither alone nor in combination. We conclude that the detection of down-stream regulators of p53 does not increase the efficacy of immunohistochemistry in assessing the functional status of p53 in breast cancer; however, their combined analysis may help to select subgroups of patients at the extremes of risk for recurrence, or those with greater chances for survival.

p53, c-erbB-2, and tumor microvascular density have been shown to be potential prognostic tools in female breast cancer. Our objective was to assess the significance of these biomarkers as prognostic factors in infiltrating male breast cancer.

A retrospective study of expression of p53, c-erbB-2, and tumor microvascular density was done on a group of 26 male breast cancer patients. Biotin-streptavidin immunohistochemical study with specific anti-p53, anti-c-erbB-2, and anti-CD34 antibodies was carried out on paraffin sections of breast carcinoma. The data of expression of the biomarkers were merged with clinicopathological data such as tumor grade, T class, TNM stage, estrogen receptor status, tumor recurrence, and patient survival.

p53 and c-erbB-2 were expressed in 46% and 39% of carcinomas, respectively. No correlation was found between positive immunoreactivity of p53, and tumor grade, size, T class, TNM stage, and survival. Nor was any relation found between tumor size, T class, TNM stage, survival, and c-erbB-2 overexpression. c-erbB-2 overexpression was significantly higher in high grade carcinomas. Estrogen receptor (ER) were positive in 21 out of 26 of tumors (81%). No trends were observed between estrogen receptor status and clinicopathological parameters or survival (data not shown). There was a positive correlation between mean microvascular density (MVD), advanced T class, and survival: higher MVD counts were found in patients with advanced tumors and in those who had tumor relapses or died of metastatic disease.

This study suggests that tumor microvascular density may serve as a potential prognostic tool in male breast carcinoma.

Diagnosis coupled with prognostication is the challenge for and charge of the pathologist. In this time of rapidly developing basic knowledge and increasing sophistication in the evaluation of prognostic information, there has also been an important re-evaluation of the validity, reliability, and relevance of classic histopathology. Also, the precision of and criteria for evaluating tumor size and status of regional lymph nodes is under study. Our emphasis in this review is tissue pathology and further, its practical relevance to patient management. Histopathology remains the basis of diagnosis universally; the addition of other elements will increase precision of prediction, particularly of responsiveness to individual therapies. Histologic grade may be integrated to substratify high and low stage cases into prognostically more useful subsets. Histologic types also interact with size and nodal status to predict patients with excellent prognosis. Further refinement of these parameters may occur by analysis within clinical, pathologic, or therapeutic subsets.

Fodrin, E-cadherin, and beta-catenin immunolocalization was studied in 54 cases of infiltrating ductal carcinoma of the breast and compared with an in vitro model in order to study the dynamic relationship between these components of an adhesion complex. In low-grade tumours, the staining patterns were similar for both fodrin and E-cadherin, with localization of these proteins to the cell membranes. beta-Catenin showed reduced membrane staining compared with non-neoplastic epithelium. High-grade tumours displayed strong membranous as well as cytoplasmic immunolocalization of fodrin, while E-cadherin staining was fragmented or lost from the membranes, with only occasional weak intracellular staining. beta-Catenin showed fragmented membrane staining and cytoplasmic accumulation. In addition, nuclear staining of beta-catenin was occasionally observed. In a v-src-transformed MDCK cell line, following 15min of src activation, beta-catenin began to detach from the cell membrane and localize to the cytoplasm, while fodrin and E-cadherin remained unchanged. After 30-45min of src activation, the cells lost their cuboidal shape and began to lose cell-to-cell contact. Fodrin staining remained mostly membranous while that of E-cadherin and beta-catenin was fragmented and spiky. After 60min of src activation, fodrin localized completely in the cell cytoplasm, while E-cadherin and beta-catenin were partly cytoplasmic with fragmented and spiky membranous staining. Occasionally, beta-catenin was seen in the nucleus. Both in vivo and in vitro findings clearly demonstrated a disruption of the E-cadherin/beta-catenin/fodrin/cytoskeleton linkage concomitant with the loss of cell-to-cell adhesion and change in cell shape, from epithelioid to a fibroblastoid phenotype. Membranous localization of E-cadherin showed a positive correlation with oestrogen and progesterone expression, whereas loss of membranous E-cadherin and cytoplasmic accumulation of fodrin was more often observed in high-grade carcinomas and showed a positive correlation with p53 expression.

The genes for p53, neu (c-erbB-2) and nm23 are all located on chromosome 17. Abnormal expression of their protein products is an important prognostic parameter. The aim of this study was to investigate if numerical aberrations of chromosome 17 are reflected in the expression of these markers. The immunohistochemical expression was analysed on histological specimens from 33 breast carcinomas. In situ hybridization (ISH) was performed on interphase cell nuclei in air-dried fine-needle aspirates from the same cases using a digoxigenin-labelled alpha-satellite probe for chromosome 17. ISH for chromosome 6, 7 and 12 was used additionally to give an estimate of ploidy. Of the carcinomas 76% were aneuploid, and numerical abnormalities of chromosome 17 were found in 34%. Abnormal p53 protein was expressed in 15% (five cases). All of these were aneuploid, but only one of them revealed aneusomy of chromosome 17. Neu overexpression was found in 18% of the tumours (six cases). Five of these were aneuploid, whereas two were aneusome for chromosome 17. Four cancers showed full (normal) expression of nm23 protein, whereas 29 had reduced expression. Reduced expression was found in 23 of 25 aneuploid tumours. Numerical aberrations of chromosome 17 were found equally in carcinomas with reduced and full nm23 protein expression. Abnormal numbers of chromosome 17 seem only to have a minor impact on these markers and are not reflected significantly in their expression.

Current analytic methodologies allow the extraction, even from small tumor masses, of extensive information on the biologic characteristics of malignant lesions, such as tumor aggressivity, metastatic potential, drug resistance, and host interactions. Clinical practice now offers a wide range of therapeutic strategies. Information technological advances offer the opportunity to refer to very large data bases of patient anamnestic data, response to treatment and clinical outcome. There is a need to formulate therapy and prognosis for each individual case. Case based reasoning is a knowledge based methodology where the outcome for complex situations can be predicted by referring to a large data base of cases of known outcomes. The preliminary data obtained from this study suggest that case based reasoning may offer a promising approach to individual targeted prognosis.