Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer.

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships.

The Neu/HER-2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast-derived Neu, the ovarian protein did not display covalent cross-linking to radiolabeled NDF, and was devoid of ligand-induced association with phosphatidylinositol 3'-kinase. Direct binding analysis showed that NDF binds with high affinity (Kd approximately 10(-9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin-like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF-induced phosphorylation and covalent cross-linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene.     

Determination of HER-2/Neu status in hepatocellular carcinoma cases

Specific chromosome abnormalities and genetic changes in hepatocellular carcinoma (HCC) have been demonstrated by conventional cytogenetic studies or molecular cytogenetic approaches like comparative genomic hybridization and loss of heterozygosity analyses. HER-2/Neu amplification and expression has been studied as a molecular target for treatment of HCC, and there are conflicting results. We aimed to determine HER-2/Neu status in archive materials of HCC patients by fluorescence in situ hybridization (FISH). Among the 35 patients, 2 had HER-2/Neu amplification and 3 had increased chromosome 17 copy number. All these patients had grade 2 or 3 tumor with a diameter of 3-12 cm. We conclude that although HER-2/Neu amplification is not the primary mechanism in the development of liver tumors, it might play a role in one of the steps of multistage carcinogenesis.     

Detection by immunohistochemistry of c-erbB2 oncoprotein in breast carcinomas and benign mammary lesions.

The presence of the c-erbB2 oncoprotein was demonstrated by immunohistochemistry in a study involving 173 mammary lesions. The lesions included infiltrating cancers, non-invasive neoplasia, as well as atypical and benign lesions. Our aim was to investigate the correlation between the c-erbB2 oncoprotein overexpression and the morphological features of the different mammary tissues analyzed to obtain a better characterization for the growth potential of certain lesions, with emphasis placed on the non-invasive neoplasia and the atypical lesions. Nearly 30% of infiltrating ductal carcinomas (27/89 cases) and 2 out of 24 infiltrating lobular carcinomas were positive. The comedocarcinomas were mostly stained (83%). In contrast, the intraductal carcinomas of cribriform or papillar patterns were consistently negative. No staining was observed in the atypical epithelial hyperplasia located in the vicinity of positive cancers for anti-oncoprotein c-erbB2 antibody. Furthermore, the only 5 positive cases for c-erbB2 out of 32 cancer-free cases were three fibroadenomas and two fibrocystic diseases with atypical ductal hyperplasia. A close correlation was thus observed between c-erbB2 oncoprotein overexpression and cancerous cell morphology, characterized by a marked nuclear hypertrophy often associated with cellular pleomorphism. However, predictive abnormalities of malignant transformation in non-neoplastic epithelial proliferation was difficult to identify, considering only the c-erbB2 expression. A group of tumors with little nuclear abnormalities were found positive for c-erbB2 immunostaining. These probably corresponded to a particular cellular phenotype. Further studies involving other oncogenes should lead to a better characterization of the different tumor phenotypes and help to clarify breast carcinogenesis.     

TOP2A and HER-2 gene amplification in fine needle aspirates from breast carcinomas

The TOP2A gene is located on chromosome 17 close to the HER-2 gene. It encodes an enzyme involved in the regulation of cell proliferation. Using fluorescence in situ hybridization (FISH), we have examined fine needle aspiration smears from 42 cases of breast carcinoma with probes for TOP2A, HER-2 and chromosome 17. We found that amplification of TOP2A is a frequent finding in breast cancer and is often but not exclusively accompanied by HER-2 gene amplification. It is associated with high histological grade and oestrogen receptor (ER) negativity. TOP2A deletions may also be associated with high histological grade and loss of ER. TOP2A amplification in the absence of HER-2 amplification may be associated with lower histological grade and ER positivity. Testing for TOP2A aberrations may be useful in the search for individually tailored treatment regimes for breast cancer.     

Expression of cyclooxygenase-2 in invasive breast carcinomas and its prognostic impact

Representative tumour sections from 468 patients with invasive breast cancer were immunostained for cyclooxygenase-2 (COX-2) and evaluated. The relationships between COX-2 expression, clinical outcome and various clinicopathological variables, including tumour vascularity and disseminated tumour cells (DTC) in the bone marrow were examined. COX-2 expression in invasive breast carcinoma cells was positively associated with oestrogen receptor and/or progesterone receptor positivity (p<0.001). Triple-negative tumours showed no/low COX-2 expression more frequently than other tumour types (p<0.001). Expression of COX-2 was not associated with breast cancer-specific survival (p=0.49, log-rank) or distant disease-free survival (p=0.67, log-rank) for all patients, including lymph node-negative, untreated patients (p>0.14, log-rank). There was also no significant association between COX-2 expression and histological grade, tumour size, nodal status, DTC in bone marrow, p53, HER2, or tumour vascularity. In conclusion, COX-2 expression in this series was associated with the presence of hormone receptors. Low COX-2 expression was observed in triple-negative breast carcinomas.     

Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using Real Time RT-PCR and immunohistochemistry

Purpose

Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes.

Methods

Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2^− ΔΔCt method.

Results

23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%.

Conclusion

Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique.     

Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification.

Amplification and overexpression of the HER-2 (neu/ erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification. This indicates that inhibition of HER-2/HER-3 heterodimer function may be an especially effective and unique strategy for blocking the HER-2-mediated transformation of breast cancer cells. Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominant negative form of HER-3 in which most of the cytoplasmic domain was removed for introduction into cells. By using a bicistronic retroviral vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations. Breast carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/ HER-3 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. Responses to exogenous HRG were strongly inhibited by dominant negative HER-3. In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture. Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.     

Quantitation of HER-2/neu oncoprotein overexpression in invasive breast cancer by image analysis: a study comparing fresh and paraffin-embedded material.

HER-2/neu oncoprotein overexpression was compared in fresh frozen and paraffin-embedded formalin-fixed invasive breast cancer material from the same patients. The HER-2/neu protein was detected by an immunohistochemical staining method, and the average amount of protein staining per cell was measured using the CAS-200 image analysis system and expressed relative to the amount of HER-2/neu protein of calibration cells of the SKBR3 cell line which are known to have amplification of the HER-2/neu gene and overexpression of the HER-2/neu protein. There was a significant correlation between degree of HER-2/neu protein overexpression and DNA-hyperdiploidy (P less than 0.01, chi 2 test). No significant correlation could be demonstrated between degree of HER-2/neu overexpression and tumor size, lymph node status, number of positive nodes or morphometric features. There was in general a good concordance (r = 0.83) in HER-2/neu expression values between fresh and paraffin-embedded material. Pairwise comparison of the two series (Wilcoxon signed ranks test) revealed no significant differences, indicating that there were no systematic differences between HER-2/neu assessments in fresh and paraffin material. When analysing the HER-2/neu expression values according to thresholds used earlier for overexpression, comparable results for fresh and paraffin material were obtained for most cases. In the fresh and paraffin material a different staining pattern was observed (more membrane staining in the fresh material in contrast to a more diffuse staining pattern in the paraffin material). It was concluded that both fresh-frozen and paraffin-embedded, formalin-fixed material is suitable for assessment of HER-2/neu protein overexpression by image analysis and provides comparable HER-2/neu expression values in most cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trastuzumab (Herceptin) in the adjuvant treatment of HER-2-positive early breast cancer

The prognosis of HER-2-positive breast cancer (characterized by amplification of the HER-2 oncogene and/or overexpression of HER-2 receptor) is unfavourable. Trastuzumab (Herceptin), a monoclonal antibody against HER-2 receptor can improve the outcome of HER-2-positive breast cancer. Up to now this was proven only in advanced disease. Recently five large multicentric phase III adjuvant trials gave level one evidence on the benefit of adjuvant treatment with Herceptin, concerning disease-free survival (DFS) and overall survival (OS). Herceptin has decreased the relative risk of recurrence with about 50% and that of death with nearly 30% in HER-2-positive early breast cancer. Based on these results Herceptin, together with chemotherapy, has been recently approved for the adjuvant treatment of HER-2-positive breast cancer.     

HER-2/neu oncogene expression, DNA ploidy and proliferation index in breast cancer.

HER-2/neu gene expression, DNA ploidy and proliferation index were studied in 250 cases of breast cancer. Expression of HER-2/neu was determined by using an antibody to the HER-2/neu receptor. Ki-67 antibody was used to determine the proliferation index of the breast cancers, and the Feulgen method was used to assess DNA amounts in the tumor cells. Histochemical staining was quantitated by image analysis. Of the cancers studied, 72 were positive for overexpression of HER-2/neu protein; of these, 62 (86%) possessed near-tetraploid DNA content, and 47 (65%) had more than one G0G1 stem line (polyploid) of DNA distribution. Cells from the cases negative for HER/2-neu overexpression contained DNA amounts that ranged from diploid to varying degrees of aneuploid. A significant difference in the amounts of cellular proliferation in HER-2/neu overexpressing cancers was found between those that expressed the HER-2/neu receptor on their membranes and those that exhibited mainly cytoplasmic receptors.     

Semiquantitative assessment of c-erbB-2 (HER-2) status in cytology specimens and tissue sections from breast carcinoma

OBJECTIVE: To determine whether various methods of fixation of surgical pathology specimens from breast carcinomas would influence the outcome of evaluation of the expression levels of c-erbB-2 (HER-2). For this, comparisons were made between (1) alcohol-fixed (95%) and air-dried smears from fresh surgical pathology specimens of breast carcinomas, and (2) formalin-fixed, paraffin-embedded tissue sections of the same specimens. STUDY DESIGN: Alcohol-fixed and air-dried smears or touch preparations were made from 30 fresh mastectomy/lumpectomy surgical pathology specimens from breast carcinomas. Immunohistochemistry was performed using the c-erbB-2 primary antibody against the extracellular domain of the c-erbB-2 gene product. Staining was simultaneously performed on formalin-fixed, paraffin-embedded tissue sections of the same specimens. A semiquantitative approach was used for evaluation of immunostaining by three independent investigators, and a consensus was reached. RESULTS: A total of 30 cases were reviewed. Tissue positivity was determined for c-erbB-2 in: 73% of alcohol-fixed specimens (n = 13 [3+] and n = 9 [2+]), 67% of air-dried smears (n = 9 [+3] and n = 11 [+2]) and 47% (n = 8 [+3]) and n = 6 [+2]) of formalin-fixed, paraffin-embedded tissue specimens. All formalin-fixed tissue specimens that were determined to positively express c-erbB-2 were also found to be positive on the alcohol-fixed smears. CONCLUSION: The incidence of c-erbB-2 expression in fresh cytologic material is significantly higher (P < .05) than in formalin-fixed, paraffin-embedded tissue. Alcohol-fixed smears demonstrate a slightly higher percentage of cell staining and stronger intensity of c-erbB-2 expression than the matched, air-dried smears. This is a sensitive and simple processing method that can be routinely applied in surgical pathology or fine needle aspiration biopsy specimens for the detection of c-erbB-2 (HER-2), with clinical implications.     

Discordant results obtained for different methods of HER-2/neu testing in breast cancer--a question of standardization, automation and timing

BACKGROUND: HER-2/neu positivity is required for the selection of stage IV breast cancer patients for trastuzumab therapy. We compared the results of the recommended immunohistochemistry (IHC) evaluation with the automated ACIS IHC system and with fluorescence in situ hybridization (FISH). These HER-2/neu tissue results were correlated with the serum HER-2/neu (sHER-2/neu) levels at the time of metastatic spread. PATIENTS AND METHODS: A total of 61 IHC slides from 30 patients were stained using the HercepTest. HER-2/neu gene amplification was determined using the Ventana FISH assay. sHER-2/neu levels were measured with the Oncogene Science" ELISA kit. The concordance of HER-2/neu results was determined using the concordance index Kappa (kappa). RESULTS: The best concordance between any IHC and FISH was found for the automated ACIS system (88.5%, kappa=0.68, category "good"). The comparison between the manual interpretations and the automated IHC was categorized as "very good" (95.1%, kappa=0.85). The median sHER-2/neu level of FISH positive patients was significantly higher (67 ng/mL) than that of FISH negative patients (17 ng/mL, p=0.018). The increase in HER-2/neu positivity comparing tissue to stage IV serum was statistically significant (p=0.001). CONCLUSIONS: The concordance between conventional IHC and computerized analysis was very good. The number of patients with stage IV breast cancer with an elevated sHER-2/neu level was much higher than HER-2/neu positivity in tissue. This discrepancy is only partially explained by the influence of tumor load. Patients with an elevated sHER-2/neu level and no tissue overexpression should be considered for retesting of tissue or a new biopsy.     

HER-2/neu gene in primary and local metastatic axillary lymph nodes in human breast tumors.

In order to verify whether the HER-2/neu gene is involved in the initial phases of neoplastic disease or in its progression, we evaluated the amplification and overexpression of this gene in the primary tumor and in synchronous metastatic axillary lymph nodes of 26 women with operable breast cancer. HER-2/neu was amplified in 35% and overexpressed in 33% of the primary sites; similar percentages were found in lymph nodes. The clear correlation between the two disease sites regarding gene, mRNA and protein levels, supports the hypothesis that this gene is involved in the initial and invasive phases of neoplasia. Its actual role with respect to other biological tumor characteristics during the metastatic process should be investigated further.     

Targeted therapy in breast cancer: the HER-2/neu gene and protein

The HER-2/neu oncogene, a member of the epidermal growth factor receptor or erb gene family, encodes a transmembrane tyrosine kinase receptor that has been linked to prognosis and response to therapy with the anti-HER-2-humanized monoclonal antibody, trastuzumab (Herceptin, Genentech, South San Francisco, CA) in patients with advanced metastatic breast cancer. HER-2/neu status has also been tested for its ability to predict the response of breast cancer to other therapies including hormonal therapies, topoisomerase inhibitors, and anthracyclines. This review includes an analysis of 80 published studies encompassing more than 25,000 patients designed to consider the relative advantages and disadvantages of the various methods of measuring HER-2/neu in clinical breast cancer specimens. Southern blotting, PCR amplification detection, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is reviewed along with the current studies designed to test whether HER-2/neu status can predict the response to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review will also evaluate the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.     

Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry

Immunohistochemistry and activity histochemistry were used to study the localization of dipeptidyl peptidase (DPP) IV in rats. For immunohistochemistry, polyclonal as well as monoclonal anti-DPP IV antibodies were employed. The pattern of DPP IV immunoreactivity, determined with polyclonal anti-DPP IV antibody, corresponds to the histochemical pattern found for the enzymic activity of DPP IV. Immunoreactivity was present, in addition, in nerve cells, lateral membranes of certain surface epithelia, e.g., Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland. The monoclonal antibodies against DPP IV recognized four different epitopes (A-D) of the DPP IV molecule, and revealed that certain epitopes were not detectable by immunohistochemistry in some organs. Generally, the staining intensities for epitopes A, B, C and D decreased in that order. Usually, the monoclonal antibodies against epitopes A and B showed similar reaction patterns to those as obtained with the polyclonal antibody. Epitope D was recognized in the lumen of the duct system of exocrine glands and the intestine. Furthermore, high reactivity of this epitope was detected in goblet cells of the intestine, where no DPP IV activity was present.     

Clinical implications of HER-2/neu overexpression and proteolytic activity imbalance in breast cancer

The aggressive biological behavior of invasive and metastatic cancer is considered to be the most insidious and life-threatening aspect for breast cancer patients. It is mostly the result of changes in many molecular characteristics of tumor cells, including alterations in gene expression and the balance of proteolytic activity. The objective of this study was to determine the level of MMP-2, its natural inhibitor TIMP-2, their ratio and HER-2/neu as diagnostic and prognostic factors. Markers were analyzed in 240 tissue samples categorized into 96 benign breast disease and 144 breast cancer patients. Enzyme linked immunosorbent assay procedure was used to evaluate the level of MMP-2 and TIMP-2 in the cell lysate, HER-2/neu in the membrane fraction, and steroid hormone receptors (ER and PgR) in the cytosol fraction. Breast cancer patients were followed-up for three years. Receiver operating characteristic curves were used to determine the cutoff points for the investigated factors. Positive values for all investigated factors were significantly increased in breast cancer patients compared to benign ones. Mean levels for all investigated factors were significantly correlated with lymph node and hormone receptor status, while MMP-2 and TIMP-2 were correlated with tumor grade (P < 0.05). In Univariate analysis, positive MMP-2, MMP-2/TIMP-2, HER-2/neu overexpression, higher tumor grade, late clinical stages and positive lymph nodes status were significantly associated with relapse. By multivariate analysis, all aforementioned factors apart from tumor grade were independent variables. Thus, the investigated markers are constructive for biologic aggressiveness of breast cancer and MMP-2/TIMP-2 ratio might be a new significant marker in early diagnosis and estimate prognosis in breast cancer.     

Preliminary study on oncogene product immunohistochemistry (c-erbB-2, c-myc, ras p21, EGFR) in breast pathology.

The expression of oncogene products related to cell growth (c-erbB-2, c-myc, ras p21, EGFR) was investigated in benign (15 cases) and malignant breast lesions (20 cases) by means of immunohistochemistry using the avidin-biotin-peroxidase technique with polyclonal and monoclonal antibodies. The aim of this study was to evaluate the relationship between the staining positivity and various morphological and biological features, such as tumour type, grading, hormone receptor status and cell kinetic parameters. In benign breast lesions, as expected, the kinetic parameters were low, both for Ki-67 and LI. All the specimens showed a diploid condition (the DI being equal to 1) and we found a limited degree of immunoreactivity for all the growth factors and oncogene products. In breast cancer we studied the distribution of immunohistochemical positivity for EGFR, c-erbB-2, c-myc, ras p21 and Ki-67, which was related to age, nodal status, ER and PgR receptor status, LI, DI and histopathological grading. A significant positive correlation was found both between ras p21 expression and nodal status and ER-ICA positivity. We observed a strong correlation between LI and Ki-67 and an inverse relation between Ki-67 and ER expression. These findings suggest the importance of studying the relationship between prognostic factors which may provide preoperative prediction in the biological behaviour of breast cancer, not only on biopsy specimens, but also on fine needle aspirates.