Keratin 6a marks mammary bipotential progenitor cells that can give rise to a unique tumor model resembling human normal-like breast cancer.

Progenitor cells are considered an important cell of origin of human malignancies. However, there has not been any single gene that can define mammary bipotential progenitor cells, and as such it has not been possible to use genetic methods to introduce oncogenic alterations into these cells in vivo to study tumorigenesis from them. Keratin 6a is expressed in a subset of mammary luminal epithelial cells and body cells of terminal end buds. By generating transgenic mice using the Keratin 6a (K6a) gene promoter to express tumor virus A (tva), which encodes the receptor for avian leukosis virus subgroup A (ALV/A), we provide direct evidence that K6a(+) cells are bipotential progenitor cells, and the first demonstration of a non-basal location for some biopotential progenitor cells. These K6a(+) cells were readily induced to form mammary tumors by intraductal injection of RCAS (an ALV/A-derived vector) carrying the gene encoding the polyoma middle T antigen. Tumors in this K6a-tva line were papillary and resembled the normal breast-like subtype of human breast cancer. This is the first model of this subtype of human tumors and thus may be useful for preclinical testing of targeted therapy for patients with normal-like breast cancer. These observations also provide direct in vivo evidence for the hypothesis that the cell of origin affects mammary tumor phenotypes.

DNA repair signature is associated with anthracycline response in triple negative breast cancer patients.

We hypothesized that a subset of sporadic triple negative (TN) breast cancer patients whose tumors have defective DNA repair similar to BRCA1-associated tumors are more likely to exhibit up-regulation of DNA repair-related genes, anthracycline-sensitivity, and taxane-resistance. We derived a defective DNA repair gene expression signature of 334 genes by applying a previously published BRCA1-associated expression pattern to three datasets of sporadic TN breast cancers. We confirmed a subset of 69 of the most differentially expressed genes by quantitative RT-PCR, using a low density custom array (LDA). Next, we tested the association of this DNA repair microarray signature expression with pathologic response in neoadjuvant anthracycline trials of FEC (n = 50) and AC (n = 16), or taxane-based TET chemotherapy (n = 39). Finally, we collected paraffin-fixed, formalin-embedded biopsies from TN patients who had received neoadjuvant AC (n = 28), and tested the utility of the LDA to discriminate response. Correlation between RNA expression measured by the microarrays and 69-gene LDA was ascertained. This defective DNA repair microarray gene expression pattern was significantly associated with anthracycline response and taxane resistance, with the area under the ordinary receiver operating characteristic curve (AUC) of 0.61 (95% CI = 0.45-0.77), and 0.65 (95% CI = 0.46-0.85), respectively. From the FFPE samples, the 69-gene LDA could discriminate AC responders, with AUC of 0.79 (95% CI = 0.59-0.98). In conclusion, a promising defective DNA repair gene expression signature appears to differentiate TN breast cancers that are sensitive to anthracyclines and resistant to taxane-based chemotherapy, and should be tested in clinical trials with other DNA-damaging agents and PARP-1 inhibitors.

Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo.

BACKGROUND: Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth. METHODS: Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun NH(2)-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided. RESULTS: Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P=.004) and GST (twofold; P=.004) activity and statistically significantly reduced glutathione levels (greater than twofold; P<.001) and HMS activity (10-fold; P<.001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P=.04) and phosphorylated c-Jun and JNK levels (P<.001) were statistically significantly increased in the tamoxifen-resistant tumors. CONCLUSION: Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth.

Prognostic value of p53 for local failure in mastectomy-treated breast cancer patients.

PURPOSE: The loss of p53 function is a recognized adverse prognostic factor in invasive breast cancer. Several studies have shown a relationship between the nuclear accumulation of p53 protein (a surrogate marker of p53 inactivation) and poor disease-free and overall survival. In general, however, these studies did not report the prognostic value of p53 for local failure, which we have therefore assessed retrospectively here. MATERIALS AND METHODS: Accumulation of p53 protein was evaluated by immunohistochemistry in 1,530 mastectomy-treated breast cancer patients (259 radiation therapy [RT]- and 1,271 mastectomy only [No RT]-treated patients). Statistical comparisons were made between p53 protein accumulation, estrogen/progesterone receptors, nodal status, tumor size, and local failure rate (LFR). Local failure was defined as tumor recurrence involving the chest wall and/or the ipsilateral supraclavicular/axillary lymph nodes. The median follow-up period was 62 months. RESULTS: In the No RT group, the LFR was 9.1% and 16. 5% in p53-negative and p53-positive patients, respectively (P<.001). Multivariate analysis revealed that p53 protein accumulation was significantly associated with an increased risk of local relapse (relative risk [RR], 1.7; 95% confidence interval [CI], 1.2 to 2.4). Nodal status and tumor size were also significant factors. In the RT group, the LFR was 9.3% and 21.5% in p53-negative and p53-positive patients, respectively (P = .009). Multivariate analysis revealed that p53 protein accumulation was significantly associated with an increased risk of local relapse (RR, 2.5; 95% CI, 1.1 to 5.7), as was nodal status. CONCLUSION: Nuclear accumulation of p53 protein is independently associated with a significantly increased local failure rate in breast cancer patients treated with mastectomy, with or without radiation.

Time-dependence of hazard ratios for prognostic factors in primary breast cancer.

Some prognostic factors, such as steroid receptors, appear strongly related to outcome in early studies with short follow-up, but as follow-up matures the relationships appear to weaken. We investigated this phenomenon for several factors (tumor size, axillary lymph nodes, S-phase fraction, estrogen receptor (ER) status, and adjuvant therapy) in a large sample of breast cancer cases (N=2,873) with up to 17 years of follow-up for disease-free survival (DFS). Subjects in the study were identified from patients who had hormone receptor assays performed in our laboratory. Analysis of DFS included fitting a multivariate Cox proportional hazards model, testing for nonproportionality, and examining diagnostic plots. The assumption of proportional hazards was violated for several factors including ER, tumor size, and S-phase fraction. For ER, the hazard ratio was initially less than 1.0, indicating a good effect on prognosis, but increased at later times to values greater than 1.0, indicating a bad effect on prognosis. In contrast, the hazard ratios for tumor size and S-phase were initially high and decreased asymptotically toward 1.0 over time. Analysis of p53 expression in a subset of cases yielded qualitatively similar results. We conclude that several standard prognostic factors (ER, tumor size, S-phase fraction) and possibly other investigational factors have important but nonproportional effects on hazard. It is likely that violation of proportional hazards is common and not limited to breast cancer. Failure to recognize violations of proportional hazards can lead to both over- and under-estimation of the effects of important prognostic factors.

Mitosin (a new proliferation marker) correlates with clinical outcome in node-negative breast cancer.

Tumor proliferation rate is an important prognostic factor in breast cancer, and S-phase fraction (SPF), as measured by flow cytometry, is the most clinically validated of several methods for measuring it. However, flow cytometry is not well suited to evaluating the formalin-fixed, paraffin-embedded tumors that are routinely available or to the increasing number of small breast cancers. These and other limitations have motivated research into alternative methods for measuring proliferation, including immunohistochemistry (IHC) against cell cycle-related antigens, which are better suited for the evaluation of small archival tissue samples. Mitosin is a recently described 350 kD nuclear phosphoprotein that is expressed in the late G1, S, G2, and M phases of the cell cycle but not in G0. Using a new monoclonal antibody (14C10), this pilot study evaluated mitosin expression by IHC in a series of 386 node-negative, formalin-fixed, archival breast cancers and correlated the results with several prognostic factors and clinical outcome (median follow-up, 78 months; range 3-214 months). The median and range of mitosin positive cells were 7% and 1-47%, respectively. There was a strong positive correlation between mitosin and SPF (r = 0.57; P = 0.0001), and there were significant negative correlations with estrogen receptor, progesterone receptor, and patient age. Mitosin was not related to overall survival in this pilot study. However, in a univariate cutpoint analysis of disease-free survival (DFS), patients with high levels of mitosin (>9% positive cells) had significantly worse DFS than did patients with lower levels (68% versus 84% at 5 years, respectively). In a multivariate analysis of DFS, large tumor size (>2 cm) and high mitosin were the only independently significant predictors of recurrence (relative risks = 2.47 and 1.72, respectively) in a model containing the additional factors estrogen receptor, progesterone receptor, patient age, and SPF. These preliminary results suggest that mitosin as assessed by IHC may be superior to SPF as a prognostic factor in node-negative breast cancer, but additional studies are necessary to validate these promising findings.

The small heat shock protein HSP27 is not an independent prognostic marker in axillary lymph node-negative breast cancer patients.

Heat shock protein 27 (hsp27) belongs to the family of heat shock proteins and is thought to be involved in thermotolerance, cell proliferation, drug resistance, and chaperone processes. The aim of this study was to investigate whether hsp27 levels are correlated with clinical outcome in axillary lymph node-negative breast cancer patients. We describe a Western blot study measuring hsp27 levels in 425 patients and an immunohistochemistry (IHC) study analyzing 788 patients. Results obtained by both methods were concordant. Univariate survival analysis was performed considering hsp27 either as an optimally dichotomized variable or as a continuous variable. Additional data include age at biopsy, tumor size, estrogen receptor (ER) and progesterone receptor status, tumor ploidy and percentage of cells in S phase, and adjuvant therapy. hsp27 levels correlated positively with ER status (P = 0.0001 in Western blot and IHC study), progesterone receptor status (P = 0.0001 in Western blot and IHC study), and aneuploidy (Western blot study, P = 0.0012; IHC study, P = 0.0004) but not with tumor size (Western blot study, P = 0.69; IHC, P = 0.53) or S phase (Western blot study, P = 0.19; IHC study, P = 0.38). Overall, there was no relationship between hsp27 expression and disease-free survival (Western blot study, P = 0.70/0.54; IHC, P = 0.47/0.30) or overall survival (Western blot study, P = 0.16/0.15; IHC, P = 0.46/0.78). Exploratory subset analyses defined by ER status and use of adjuvant treatment indicated that in ER+/untreated patients, high hsp27 levels correlated modestly with shorter disease-free survival (Western blot, P = 0.04/0.04; IHC, P = 0.11/0. 03). hsp27 is not a useful prognostic marker for the clinic in axillary lymph node-negative patients. However, the finding of modest prognostic value of hsp27 in the subgroup of ER+/untreated patients raises new questions about the biological function of hsp27 in breast cancer.

The c-erbB-2 proto-oncogene as a prognostic and predictive marker in breast cancer: a paradigm for the development of other macromolecular markers--a review.

Seven years after the initial studies of the prognostic value of the proto-oncogene c-erbB-2 in breast cancer, its role is still being defined. The interpretation of studies on the use of this gene and its protein product in prognostic and predictive tests for breast cancer is complicated by multiple methodologies and the inherent difficulties in the studies. The work has moved beyond the stage at which small studies with short follow-up (useful for hypothesis generation) are of value, to the stage in which large studies with sufficient statistical power to find significant correlations are central. These larger studies do not lend support for the use of c-erbB-2 in the evaluation of axillary-node-negative patients, the group of breast cancer patients for whom refinement of prognostic estimates is now most important. There are, however, hints that c-erbB-2 may have value in predicting response to certain treatments, though the studies so far are too few, often too small and too conflicting to reliably confirm this.

Rising levels of estrogen receptor in breast cancer over 2 decades.

BACKGROUND: The incidence of estrogen receptor (ER)-positive breast cancer apparently is increasing. It remains unclear whether this increase is due to an improvement in receptor assay sensitivity, a change in patient characteristics, or a change in tumor biology. METHODS: The distribution of ER, tumor size, and patient age for 11,195 tumor specimens gathered from patients nationwide from 1973 to 1992 were analyzed. All assays were performed in a single laboratory. A single-label, dextran-coated charcoal (DCC) method was used from 1973 to 1984, and a dual-label, DCC method, which allows the determination of both ER and progesterone receptor levels in the same assay, was used from 1985 to 1992. RESULTS: The median level of ER has increased steadily from 14 fmol per milligram of protein in 1973 to 58 fmol per milligram of protein in 1992 (P < 0.0001). The percentage of ER-positive tumors also rose from 73-78% during the same period (P = 0.008). When the assay method was modified from single to dual label, no abrupt or stepwise increase occurred. Tumor size decreased over the same period (P < 0.0001). From 1973 to 1977, 48% of tumors were larger than 2 cm, and 15% were larger than 5 cm, compared to 60% and 9%, respectively, from 1988 to 1992. The percentage of women older than 50 years of age remained relatively constant over time. After adjusting for tumor size, age, number of positive lymph nodes, and change in assay method, a sustained rise in ER level remained. In a multivariate analysis that included age, age group, year of biopsy, tumor size, and number of positive nodes, the year of biopsy still was independently predictive of ER level (P < 0.0001). CONCLUSION: The measured level of ER in primary breast cancers has increased during the last 2 decades. It is unlikely that technical improvements or changes in tumor size, age, or nodal status fully explain this increase. The rising level of ER may reflect a change in breast cancer biology and in hormonal events that influence breast cancer genesis and growth.

Tumor biologic factors and breast cancer prognosis among white, Hispanic, and black women in the United States.

BACKGROUND: In the United States, prognosis and survival after the diagnosis of breast cancer is poorer among black patients and, to a lesser extent, among Hispanic patients, compared with white patients. Patients who are black or Hispanic have been reported to present with higher stage or more advanced disease. Even after adjusting for stage, however, survival rates are lower for blacks but not for Hispanics. PURPOSE: Our purpose was to compare survival, age, tumor size, nodal status, estrogen-receptor (ER) and progesterone-receptor (PgR) status, histologic type, S-phase fraction, DNA ploidy status, HER-2/neu protein expression, and p53 protein status, along with systemic treatment, in a large group of white, black, and Hispanic U.S. women. METHODS: From 1970 to 1991, breast tumor specimens were submitted to The University of Texas Health Science Center from 31 contributing hospitals throughout the United States for ER and PgR assay. A total of 4885 white, 1016 black, and 777 Hispanic women were eligible for this study. Median follow-up was 57 months. RESULTS: Overall, white women were significantly more likely to be older and to have smaller tumors, have less lymph node involvement, have tumors with positive ER and PgR status, and have a lower S-phase fraction compared with Hispanic or black women. There were no clinically important differences in DNA ploidy, histologic type, HER-2/neu, and p53 expression among the three groups. Considering all stages, white women had the best overall survival (date of diagnosis to date of death) at 5 years--75% +/- 1% (means +/- SE), with a median survival of 166 months, but Hispanic women had an intermediate survival--70% +/- 2% (median survival, 156 months), and black women had the worst survival--65% +/- 2% (median survival, 117 months) (P < .0001). For node-negative patients, there was no significant difference in disease-free survival (date of diagnosis to date of first recurrence) or overall survival, although blacks tended to have a worse prognosis. For node-positive or locally advanced disease and for metastatic disease, blacks had significantly (P < .0001) worse disease-free and overall survival than did white or Hispanic women. Differences in the use of systemic therapy did not explain these outcomes. CONCLUSION: A number of biologic factors associated with poor prognosis are found with a significantly increased frequency in breast tumors from Hispanic and, particularly, from black women. Tumors with a more aggressive biology could lead to a higher stage at diagnosis and a poorer survival for the group as a whole.

Cathepsin D by western blotting and immunohistochemistry: failure to confirm correlations with prognosis in node-negative breast cancer.

PURPOSE: We attempted to replicate and improve on our previous study (N Engl J Med 322:297-302, 1990) that showed that 34-kd cathepsin D levels as determined by Western blotting strongly correlated with disease-free survival (DFS) and overall survival (OS) of axillary node-negative (N-) breast cancer patients. We also examined the prognostic significance of cathepsin D measured by immunohistochemistry (IHC) in these patients. PATIENTS AND METHODS: Western blotting was performed on cytosols from frozen tumor specimens of 927 N- breast cancer patients in the San Antonio Breast Tumor Bank. The monoclonal antibody M1G8 was used to detect cathepsin D (in previous study, a polyclonal antiserum had been used). The same monoclonal antibody was also used for frozen-section IHC staining of tumor specimens from 562 N- patients from the same tumor bank. Levels of cathepsin D expression were then correlated with DFS and OS. RESULTS: Although the levels of cathepsin D expression as measured by Western blotting and IHC correlated with each other and with levels of cathepsin D measured in previous work using Western blotting with the polyclonal antiserum, in this present study, using the monoclonal antibody M1G8, we were unable to demonstrate that cathepsin D expression (measured by either Western blotting or by IHC) correlates with DFS or OS. CONCLUSION: In this study, cathepsin D expression as determined by either Western blotting or IHC using the monoclonal antibody M1G8 failed to improve the prognostic evaluation of N- breast cancer patients. The role of cathepsin D expression as a prognostic factor is still not precisely defined, raising questions about its use in the routine evaluation of N- breast cancer patients.

Constitutive overexpression of the 27,000 dalton heat shock protein in late passage human breast cancer cells.

We present evidence that the mechanisms controlling induction of heat shock transcription factors (HSFs) and mRNA expression of the 27,000 molecular weight heat shock protein, hsp27, are diverse in human breast cancer cells. Heat shock accumulation of hsp27 RNA is associated with the activation of HSF in MDA-MB-231 cells. We have later passage MCF-7 breast cancer cell lines with elevated, constitutive expression of hsp27 mRNA, perhaps due to hsp27 gene amplification. Estradiol and heat shock treatment no longer affect the level of hsp27 mRNA in these cells. Heat induction of HSF is inhibited in cells overexpressing hsp27, although metal ions and amino acid analogs are still capable of activating HSF. These cells will provide a useful system for characterizing alternative pathways in HSF inhibition and activation.

Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review.

Heat shock and other environmental and pathophysiologic stresses stimulate synthesis of heat shock proteins (Hsps). These proteins enable the cell to survive and recover from stressful conditions by as yet uncompletely understood mechanisms. Hsp27 is an important small Hsp (molecular weight, 27,000) found in human cells--both cancer cells and normal cells. This protein, besides its putative role in thermotolerance, is of special clinical interest because of recent data suggesting it may also play a role in drug resistance. In adults, Hsp27 is found particularly in several cell types such as breast, uterus, cervix, placenta, skin, and platelets. Although low-molecular-weight (small) Hsps have been found to be involved in embryogenesis of Xenopus and Drosophila, they have not been detected in human fetal organs. Regulation of expression of the Hsp gene (also known as HSPB1) has been considered a paradigm of gene regulation and is actively being studied in both prokaryotes and eukaryotes. In prokaryotes, the major Hsp genes are transcriptionally regulated by positively and negatively acting transcription factors. In eukaryotes, the genes encoding Hsps contain a regulatory DNA motif (inverted repeats of the pentameric sequence nGAAn) known as the heat shock element. Hsp27 may function as a molecular chaperone and in signal transduction pathways of different cell regulators, and Hsp27 and other Hsps may be active in development of resistance to stressful conditions and agents including cytotoxic drugs. Study findings indicate that some but not all estrogen-positive breast cancers express Hsp27, and overexpression of Hsp27 has been associated with both good and poor prognosis. In endometrial carcinomas, the presence of Hsp27 is correlated with the degree of tumor differentiation as well as with the presence of estrogen and progesterone receptors. Studies suggest, however, that detection of Hsp27 should not be considered to be a method for identifying hormone-responsive tumors or detecting estrogen receptors. Hsp27 seems to be a biochemical marker of estrogenic endometrial response. In patients with cervical cancer, Hsp27 is predominantly expressed in well-differentiated and moderately differentiated squamous cell carcinomas. In addition, expression of Hsp27 seems to be a negative prognostic factor for gastric cancer. Different isoforms of Hsp27 have been found in lymphoid tissue of patients with acute lymphoblastic leukemia, and the protein has also been associated with viral infections. These aspects are summarized and discussed in the present review.

Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer.

BACKGROUND: The p53 (also known as TP53) tumor suppressor gene encodes for a nuclear phosphoprotein thought to regulate proliferation of normal cells. Most p53 mutations result in a nonfunctional protein that accumulates in tumor cell nuclei. These common mutations appear to be involved in the development and/or progression of several neoplastic diseases including human breast cancer. PURPOSE: Our purpose was to investigate the relationships between levels of mutant p53 protein expression, tumor cell proliferation rate, and clinical outcome in patients with node-negative breast cancer. METHODS: Expression of mutant p53 protein was evaluated by frozen-section immunohistochemistry (IHC) and light microscopy in 700 breast cancers from axillary lymph node-negative patients with long-term follow-up (median, 54 months). The immunostaining signal was expressed as the sum of scores representing the proportion and staining intensity of negative and positive tumor cell nuclei (ranges, 0 and 2-8, respectively). Statistical comparisons were made between levels of p53 protein expression and disease-free survival, overall survival, and tumor proliferation rate expressed as the percentage of cells in the S phase (%S phase) as determined by flow cytometry. RESULTS: Of the 700 tumors, 362 (52%) showed positive nuclear immunostaining (IHC score > 0). Proliferation rates were significantly higher (P = .0001) in positive tumors (median %S phase, 7.1%) than in negative tumors (4.1%). In a univariate cutpoint analysis, negative tumors (n = 388) versus low-positive tumors (IHC score = 2-6; n = 263) versus high-positive tumors (IHC score > 6; n = 99) showed progressively reduced disease-free survival (80% versus 72% versus 58% at 5 years, respectively; P < or = .05 for all pairwise comparisons). Analogous results for overall survival were 88% versus 84% versus 74%; only the result for negative versus high positive tumors was significant (P = .003). In a multivariate analysis, expression of p53 protein and high %S phase were independently associated with reduced disease-free survival (P = .008 and .01, respectively). CONCLUSIONS: Expression of mutant p53 protein was associated with high tumor proliferation rate, early disease recurrence, and early death in node-negative breast cancer. Despite the strong direct correlation between accumulation of p53 protein and tumor proliferation rate, both factors were independently associated with poor prognosis, suggesting that p53 may have other biological functions in addition to cell-cycle regulation. IMPLICATIONS: This test, when combined with other prognostic factors, may enhance our ability to identify node-negative breast cancer patients at high risk for early disease recurrence and/or death, for whom the use of adjuvant chemotherapy is unequivocally justified.

Estrogen receptor mutations in breast cancer.

It is fairly well accepted that the presence of estrogen receptor (ER) identifies those breast cancer patients with a lower risk of relapse and better overall survival [Clark and McGuire, 1988], and the measurement of ER has become a standard assay in the clinical management of breast cancer. Receptor status also provides a guideline for those tumors which may be responsive to hormonal intervention [McGuire 1978; Osborne et al., 1980; Rose et al., 1985]. But only about half of ER-positive patients will respond to the various hormonal therapies available, and of those who do initially respond, most will eventually develop hormonally unresponsive disease following a period of treatment even though ER is often still present. Loss of ER from initially ER-positive tumors biopsied again at a later date has been estimated at only 19% [Gross et al., 1984]. Obviously the simple measurement of ER presence by ligand-binding assays does not provide us with an adequate estimate of the functional state of the receptor. In 1985 Sluyser and Mester hypothesized that the loss of hormone dependence of certain breast tumors may be due to the presence of mutated or truncated steroid receptors that activate transcription even in the absence of hormone [Sluyser and Mester, 1985]. Based on the recent identification of several ER sequence variants in human breast cancer cell lines and tumor specimens, we would now like to propose that some of these identified mutations play a role in receptor dysfunction in vivo, and will review those ER mutations which may prove to be important in breast cancer progression.

Expression of HER-2/neu oncoprotein in human breast cancer: a comparison of immunohistochemical and western blot techniques.

Three hundred and one primary breast cancers from patients with tumor infiltrated lymph nodes were analyzed for the presence of HER-2/neu oncoprotein by two procedures: Western blot (WB) and immunohistochemistry (IHC). Overexpression of this protein was found by WB in 16.6% of the tumors, and by IHC in 16.3%. Concordance between the two methods was found in 95% of tumors (286/301). In 7 cases we found HER-2/neu by IHC but not by WB, while the opposite was found in the remaining 8 patients. This discrepancy was found mainly in samples with HER-2/neu values just above the cut points and were therefore close to the sensitivity limits of the procedures used here. This study helps to define the parameters that should be considered to evaluate the immunostaining for HER-2/neu as positive (i.e., membrane staining, IHC score of 2 or more). The results obtained by both techniques were correlated with several currently used prognostic factors. Higher HER-2/neu protein expression was found in tumors lacking estrogen or progesterone receptors, in tumors with high S-phase fraction and in patients with more than 3 positive lymph nodes. In contrast, no relationship was found between overexpression of this protein and tumor size, ploidy, or age of the patient. Patients with elevated HER-2/neu expression showed a significantly worse overall survival by both methods, IHC (p = 0.05) and WB (p = 0.001). In conclusion, there is very high agreement between IHC and WB when measuring expression of HER-2/neu and both techniques showed prognostic significance.

Abnormal estrogen receptor in clinical breast cancer.

We have discovered a number of estrogen receptor variants in clinical breast cancer tissues. We have base-pair insertions, transitions, and deletions of exons 3, 5 and 7. Using a transactivation assay we have discovered receptors with outlaw function consisting of both dominant-positive receptors which are transcriptionally active in the absence of estrogen, and dominant-negative receptors which are transcriptionally inactive themselves but prevent normal estrogen receptor function.

Prognosis and treatment decisions in patients with breast cancer without axillary node involvement.

BACKGROUND: Every month, treatment decisions must be made for more than 6000 patients with breast cancer without axillary node involvement in the United States. Approximately 70% of these patients will survive more than 10 years after surgery and/or radiation treatment without additional systemic adjuvant therapy. If we had good methods to identify patients who are destined to have a recurrence of their disease, only those patients should receive adjuvant therapy. METHODS: The authors reviewed the literature supporting the use of currently available prognostic factors for patients with node-negative breast cancer, and formulated a framework on which prognostic factor information can be based to help make these treatment decisions. RESULTS: The steps involved in making treatment decisions are: use prognostic factors to determine the recurrence probability; calculate the expected treatment benefit; and weigh the expected benefits against the potential risks. CONCLUSIONS: Prognostic factors can be used to help make treatment decisions for patients with breast cancer without axillary node involvement. However, the final treatment decision must take into account all aspects of the patient and her disease, and the physician must help the patient evaluate her prognostic factors, arrive at an understanding of her particular risk of recurrence, and weigh the potential benefits and risks of adjuvant therapy.

Treatment decisions in axillary node-negative breast cancer patients.

Treatment decisions must be made on 9000 axillary node-negative breast cancer patients each month in the United States. Which of these patients will benefit from adjuvant therapy is a major question. Valid methods are needed to distinguish those patients who are "cured" from those who will suffer a cancer recurrence. A complex network of prognostic variables enters into the treatment decision, together with a risk-versus-benefit assessment. We are using a neural-network-based form of artificial intelligence that, once "trained" with data representing an event and its outcome, can identify subsets of patients with low recurrence risks. Larger data sets are being evaluated with the hope of introducing the neural-network technique to routine clinical practice.