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.

DNA ploidy, S-phase, and steroid receptors in more than 127,000 breast cancer patients.

Several potential prognostic factors are available today for patients with breast cancer, and many more are being identified and studied. To evaluate the clinical utility of these factors, it will be necessary to measure them on a large number of patients, and then follow these patients so that multivariate survival analyses can be performed. The Oncology Research Network was established in 1986 by the University of Texas Health Science Center at San Antonio and Nichols Institute Reference Laboratories in order to evaluate the clinical utility of new prognostic factors for patients with primary breast cancer. The first generation of prognostic factors included steroid receptors, along with DNA ploidy and S-phase fraction determined by flow cytometry. Currently, laboratory results have been obtained from more than 127,000 patients, and follow-up information is available on a subset of more than 25,000 of these patients. S-phase fraction was related to the ploidy status of the tumor. An increased incidence of aneuploidy and higher S-phase fractions were found in estrogen and progesterone receptor negative tumors, tumors from patients with positive axillary lymph nodes, tumors greater than 2 cm in diameter, and patients younger than 35 years of age. Preliminary survival analyses suggest that S-phase fraction and DNA ploidy, in combination with other prognostic factors, are powerful predictors of early disease relapse. The Oncology Research Network provides an important resource for examining the clinical significance of new laboratory assays and for expediting improvements in existing laboratory techniques.

Glutathione transferase GST pi in breast tumors evaluated by three techniques.

The glutathione transferases are involved in intracellular detoxification reactions. One of these, GST pi, is elevated in some breast cancer cells, particularly cells selected for resistance to anticancer agents. We evaluated GST pi expression in 60 human breast tumors by three techniques, immunohistochemistry. Northern hybridization, and Western blot analysis. There was a significant positive correlation between the three methods, with complete concordance seen in 64% of the tumors. There was strong, inverse relationship between GST pi expression and steroid receptor status with all of the techniques utilized. In addition, there was a trend toward higher GST pi expression in poorly differentiated tumors, but no correlation was found between tumor GST pi content and DNA ploidy or %S-phase. GST pi expression was also detected in adjacent benign breast tissue as well as infiltrating lymphocytes; this expression may contribute to GST pi measurements using either Northern hybridization or Western blot analysis. These results suggest that immunohistochemistry is the method of choice for measuring GST pi in breast tumors.

Insulin-like growth factor-I receptors are overexpressed and predict a low risk in human breast cancer.

IGF-I receptor (IGFR) content and its prognostic significance were evaluated in human breast cancer specimens using a sensitive and specific radioimmunoassay (V. Pezzino et al., Metabolism, 40: 861, 1991). The prognostic significance of IGFR expression was investigated by two different approaches: (a) detectable IGFR content was measured in 82% of specimens in a consecutive series of 184 human breast cancers and in 32% of 19 normal breast tissues. The average IGFR content in breast cancer was nearly 10-fold higher than the value observed in normal breast tissue (7.6 +/- 0.8 versus 0.8 +/- 0.1 ng/0.1 mg protein, mean +/- SEM; P < 0.001). IGFR content was positively correlated with estrogen (ER) and insulin receptor content (r = 0.269 and 0.515, respectively, Pearson correlation) but not with progesterone receptors (PR). No significant correlation was observed between IGFR content and a variety of tumor parameters (tumor size, lymph node involvement, grade) and host characteristics (age, body mass index, menopausal status); (b) IGFR content was measured in a noncontinuous series of 265 primary breast cancer specimens subdivided into 136 high-risk and 129 low-risk specimens on the basis of being either negative (ER-/PR-/aneuploid/high S-phase) or positive (ER+/PR+/diploid/low S-phase) for four well-established prognostic factors. IGFR levels were significantly higher in the low-risk group (6.4 +/- 0.4 ng/0.1 mg protein, mean +/- SEM) than in the high-risk group (3.6 +/- 0.5; P < 0.0001, Wilcoxon sum rank test). In summary, our data indicate that there is an elevated IGFR content in most human breast cancers compared with normal breast tissue and that an elevated IGFR content is a favorable prognostic indicator.

The 28-kDa protein whose phosphorylation is induced by protein kinase C activators in MCF-7 cells belongs to the family of low molecular mass heat shock proteins and is the estrogen-regulated 24-kDa protein.

We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status.

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E2 treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2-6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.

Estrogen receptor, progesterone receptor, and HER-2/neu protein in breast cancers from pregnant patients.

BACKGROUND: Because the occurrence of breast cancer during pregnancy is uncommon and because the high levels of estrogens and progestins associated with pregnancy could cause false-negative results from ligand binding assays (LBA), the actual incidence of steroid hormone receptor positivity in tumors from this subset of women is unclear. METHODS: Estrogen receptor (ER) and progesterone receptor (PgR) were determined using LBA methods in 15 tumors from 15 pregnant patients with breast cancer. In addition, immunohistochemistry was done for ER, PgR, pS2, heat shock protein 27 (hsp27), and HER-2/neu on 12 of the 15 tumors. RESULTS: Five of 15 (33%) tumors were positive for ER by LBA, compared with 52% of tumors from age-matched nonpregnant patients. Six of 12 (50%) were ER-positive by immunohistochemistry. For PgR, 7 of 15 (47%) tumors were positive by LBA, compared with 42% of tumors from nonpregnant patients. Ten of 12 (83%) stained positive for PgR. By LBA, 67% of tumors studied were positive for ER or PgR or both, as opposed to 57% of tumors from the nonpregnant comparison group. Two other estrogen receptor-mediated proteins, pS2 and hsp27, were present by staining in 8 of 12 (67%) and 10 of 12 (83%) of tumors, respectively. Seven of 12 tumors (58%) had positive staining for HER-2/neu, whereas only 16% of age-matched nonpregnant patients had positive-staining tumors. CONCLUSION: By LBA, the incidence of ER and PgR in breast tumors from pregnant women was not significantly different from that of tumors from nonpregnant age-matched patients. Some ER-negative tumors were PgR, pS2, or hsp27 positive, indicating that an intact estrogen response system was operative although ER was not detectable by standard LBA.

Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications.

BACKGROUND: Cell synthesis of heat shock (stress-response) proteins is increased by a variety of environmental and pathophysiological stressful conditions. The 70-kd heat shock protein (hsp70) is thought to be involved in protein-protein interactions including those of the protein products of the human c-myc oncogene and the p53 (also known as TP53) tumor suppressor gene. PURPOSE: The purpose of this study was to investigate whether elevated hsp70 expression may be an indicator of biological stress experienced by a breast cancer and may, therefore, predict disease outcome. METHODS: Levels of hsp70 were determined by Western blot analysis in primary breast tumors from patients with negative axillary lymph nodes. We performed exploratory data analyses on a set of 162 primary breast cancers and constructed prognostic indexes of hsp70 expression levels. The optimal cutpoint for hsp70 expression was considered to be the value yielding the greatest separation for disease-free survival for the resulting two groups of patients. That cutpoint was then validated in a set of 345 tumors by univariate and multivariate analyses. Data were analyzed for overall survival, disease-free survival, tumor size, and patient age, as well as estrogen receptor and progesterone receptor status, ploidy (DNA content), and percentage of cells in S phase as determined by flow cytometry. RESULTS: Expression of hsp70 emerged as a useful prognostic factor, both in univariate and in multivariate analyses. Patients whose tumors had high expression of hsp70 had significantly shorter disease-free survival (P = .006). The other statistically significant factors were S-phase fraction (P = .008) and tumor size (P = .01). For patients who received adjuvant therapy, hsp70 was the only independent predictor of disease recurrence (P = .05). For those with tumors 1-3 cm in diameter, hsp70 (P = .008) and S-phase fraction (P = .02) were statistically significant predictors of recurrence. CONCLUSIONS: Measurement of hsp70 expression in primary tumors from patients with node-negative breast cancer may be useful in identifying patients at high risk for disease recurrence and thus may affect decisions regarding treatment after surgery. IMPLICATIONS: Future studies should be performed to determine if detection of hsp70 by immunohistochemistry can be used to predict clinical outcome and to better understand the relationships between hsp70 and the effects of various treatment modalities.

Prognostic indicators in early breast cancer.

Approximately 115,000 new cases of axillary node negative breast cancer were diagnosed in this country last year. Since about 20-30% of these patients will ultimately relapse and die of their disease, adjuvant systemic therapy has been advocated for this group to decrease the relapse rate and prolong survival. However, although most clinical trials have demonstrated a modest impact on disease recurrence, the available data have failed to show consistent improvements in overall survival and does not justify the generalized use of systemic treatment in this patient subgroup. For this reason, a plethora of prognostic factors have been described to identify those patients with a higher risk of recurrence to concentrate therapeutic options in this specific group. Of all the disease prognosticators studied, tumor size, nuclear grade, and proliferative indexes appear to correlate well with tumor recurrence. In addition, biologic characteristics of primary tumors such as the presence of hormone and growth factor receptors, secretion of specific polypeptides and proteases, expression of proto-oncogenes, and abnormalities in tumor suppressor genes have been shown to be potentially useful as prognostic indicators in patients with early breast cancer. Despite these provocative data, larger clinical trials are necessary before incorporating these parameters in the routine evaluation of patients with axillary node negative breast cancer.

How to integrate steroid hormone receptor, flow cytometric, and other prognostic information in regard to primary breast cancer.

A large group of patients with node-positive breast cancer was divided into a training set (n = 851) and a validation set (n = 432) to demonstrate techniques for integrating steroid hormone receptor status, DNA flow cytometric findings, and other prognostic factors to predict patient survival. Multivariate analyses showed that estrogen receptor status, the number of involved axillary lymph nodes, patient age, S-phase fraction, progesterone receptor status, and tumor size were significant predictors of survival in patients with node-positive breast cancer. Techniques for optimizing and validating a cut point for a new prognostic factor and for examining alternative representations of prognostic factors were demonstrated. Prognostic indexes were created that could be used to identify patients with very good or very poor prognoses.

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.

Measurement of steroid hormone receptors in breast cancer patients on tamoxifen.

Estrogen (ER) and progesterone receptor (PgR) positive breast tumors often respond to tamoxifen, but ultimately progress as they become tamoxifen resistant. An accurate assessment of receptor status in specimens from tamoxifen-resistant patients could help to understand potential mechanisms of resistance and to predict response to second line hormonal therapies. However, since tamoxifen itself can affect ER and PgR determinations, assay results can be misleading. We measured ER and PgR by both ligand binding (LBA) and immunohistochemical (IHC) assays in 34 tumors from patients on tamoxifen, 30 of whom were displaying resistance to the drug. These tumors were classified into several receptor phenotypes. Eleven patients, 8 of whom were clearly progressing, expressed both receptors while on tamoxifen. ER was significantly less often negative when measured by IHC, suggesting that ER status by LBA was falsely negative in this group due to receptor occupancy by tamoxifen. Six patients had no detectable ER by LBA or IHC but still expressed PgR. The presence of PgR suggests that ER could still be functional, though undetectable, in these tumors, or that PgR is constitutively expressed by them. Finally, 12 patients were ER and PgR-negative by both assays, suggesting hormonal independence as the mechanism for resistance in this group. In a subset of patients with receptor assays both prior to tamoxifen and at the time of progression while taking the drug, we found that most ER-positive tumors converted to an apparent ER-negative status when assayed by LBA, while PgR status frequently remained unchanged. The continued expression of ER and/or PgR in many patients with tumor progression on tamoxifen indicates that mechanisms for resistance other than receptor loss are common in breast cancer.

Prognostic significance of p53 gene alterations in node-negative breast cancer.

UNLABELLED: Mutations in the p53 gene can play a role in the transformation of normal to malignant cells. Because these mutations are more frequently reported later in the course of transformation, their presence could reflect a greater malignant potential of the tumor and, thus, an increased probability of metastasis and recurrence after local therapy. In a pilot study using single-stranded conformation polymorphism analysis (SSCP), 200 node-negative breast tumors were examined for mutations in the region encompassing exons 5 through 9 of the p53 gene. Exons 5 through 9 were tested because they contain 80-90% of known p53 gene mutations. The tumors ranged in size from 1 to 3 cm. 28 tumors were found to have an abnormal band pattern on both initial and repeat analysis. 4 of these tumors were sequenced; 3 contained a p53 mutation and the 4th had a rare neutral polymorphism. Disease-free survival (DFS) at 5 years for women with tumors having an abnormal SSCP analysis was 57% (+/- 10%), compared to a 79% (+/- 3%) DFS for the group with a normal pattern. By the log rank test, this difference was highly significant, p < or = 0.01. The relative risk of recurrence for the group with an abnormal SSCP pattern was 2.2. In a multivariate analysis including ER, PgR, ploidy, S-phase, age, and tumor size, an abnormal p53 by SSCP analysis and patient age were the only factors that independently predicted DFS at 5 years. CONCLUSION: Women with node-negative breast cancer who have tumors with alterations in the p53 gene, as indicated by SSCP analysis, have a significantly poorer prognosis and a higher rate of relapse at 5 years. The prognostic significance is maintained in a multivariate analysis including many established prognostic factors.

Neural Network Analysis of DNA flow cytometry histograms.

A pattern recognition system based on Neural Network Analysis, a form of artificial intelligence, was used to search DNA flow cytometry histograms for features that correlated with breast cancer patients' risk of relapse. DNA flow cytometry histograms and clinical follow-up information from 796 breast cancer patients were used to train a Neural Network to predict the clinical outcome of patients in a separate independent set of 794 patients. Median follow-up in this patient data base was short, 23 months. Neural Network Analysis resulted in a model that evaluated DNA flow cytometry histograms differently than conventional analysis, which categorizes the histograms by ploidy and S-phase fraction. Neural Network Analysis appeared to identify low risk and high risk subsets of patients as accurately as conventional analysis. Neural Network Analysis placed heavy emphasis on the region to the right of the diploid G2/M peak, where a subpopulation of nuclei with high DNA content is seen even in many histograms scored as diploid by conventional techniques. The number of nuclei in this region was found to be a powerful predictor of patient outcome, and multivariate analysis showed that the number of nuclei in this region and the S-phase fraction both were independently predictive of relapse. This pilot study suggests that conventional analysis (based on a mechanistic interpretation of regions in flow cytometry histograms) might be used in conjunction with and improved by pattern recognition systems or insights derived from them.

The ER-positive/PgR-negative breast cancer phenotype is not associated with mutations within the DNA binding domain.

We have used in vitro DNA binding assays as a measure of estrogen receptor (ER) function in human breast tumors. We found that the majority of ER+ (25 ER+/progesterone receptor [PgR]+, and 25 ER+/PgR-) tumors we examined were capable of binding consensus estrogen response element (ERE) oligonucleotides in this assay system. We found significant proteolytic activity in many of the tumors such that protease inhibitors were found to be essential during the preparation of tumor extracts. We next applied direct sequence analysis of the ER DNA binding domain of several of these tumors, and determined that the ER+/PgR- breast tumors did not contain mutations within the DNA binding domain which might explain their apparent discordant receptor phenotype. We did identify an alternatively spliced ER variant missing exon 3 of the DNA binding domain. This variant was unable to function as a transcriptional inducer of an estrogen-responsive reporter in a yeast assay system. Furthermore, the exon 3 ER deletion variant was expressed at equivalent levels in all of the ER+ breast tumors, so that it does not appear to be involved in the evolution of the ER+/PgR- breast cancer phenotype.

Prognostic factors and therapeutic decisions in axillary node-negative breast cancer.

The incidence of node-negative breast cancer is increasing, and it is becoming a more commonly encountered clinical problem. Prognostic factors such as tumor size, histopathological characteristics, receptor status, proliferative markers, biochemical alterations, and gene abnormalities can be useful in assessing the risk of cancer recurrence after primary local therapy. By accurately assessing the risk of recurrence, informed decisions can be made about whether or not to treat with adjuvant systemic therapy.

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.

Regulation of insulin-like growth factor-binding protein (IGFBP) expression by breast cancer cells: use of IGFBP-1 as an inhibitor of insulin-like growth factor action.

BACKGROUND: The insulin-like growth factors (IGFs) play an important role in normal growth and development. Evidence suggests they may also regulate the growth of several cancer cell types. This regulation is mediated by interactions between the receptors and ligands. There is now ample evidence to suggest that these interactions are also influenced by extracellular IGF-binding proteins (IGFBPs). Six different IGFBPs have been cloned. Some species may act to inhibit the mitogenic effects of the IGFs. Since breast cancer cells are responsive to the IGFs, it is possible that regulated expression of the IGFBPs affects tumor growth. Furthermore, inhibitory binding proteins could be used as neutralizers of IGF action. PURPOSE: We conducted this study to fully characterize the expression and hormonal regulation of IGF-binding protein expression in human MCF-7 breast cancer cells and to test the ability of purified IGFBP-1 to inhibit IGF-I action. METHODS: We used ribonuclease protection assays and Western ligand blotting to examine IGFBP expression in MCF-7 cells. The effect of IGF-I, IGFBP-1, and 17 beta-estradiol on serum-free cell growth was also studied. RESULTS: MCF-7 cells expressed IGFBP-2, IGFBP-4, and IGFBP-5 RNA and protein. These cells are dependent on estrogen for growth. In short-term culture, IGF-I can substitute for estrogen. Concomitant addition of IGF-I and estrogen enhanced stimulation above the level achieved by either factor alone. Estrogen also increased IGFBP production, making it unlikely that the IGFBPs induced by estrogen in MCF-7 cells could function as major inhibitors of IGF action. In contrast, exogenous addition of IGFBP-1 could block IGF-I-induced mitogenesis; this effect was reversible by excess IGF-I. CONCLUSIONS: The studies suggest that cancer cell growth may be regulated by endogenous IGFBP expression. Furthermore, the exogenous addition of the IGFBP-1 blocked IGF-I action and potentially could be used as a pharmacologic inhibitor of IGF action.