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.

Mammary carcinoma: a specific biochemical defect in autonomous tumors.

Rat mammary carcinoma (R3230AC) which does not regress after ovariectomy has a markedly reduced amount of cytoplasmic estradiol binding protein. Cytoplasm from the tumor fails to interact with estradiol sufficiently to permit estradiol binding to tumor chromatin. This defect can be corrected in vitro by substituting cytoplasm, containing the binding protein, from rat uterus, thus permitting estradiol binding to tumor chromatin. The results indicate that the hormonal autonomy of this carcinoma is due to a lack of estradiol binding protein and not to the inability of estradiol to interact with the cell genome.

Human breast cancer: biologically active estrogen receptor in the absence of estrogen?

The human breast cancer cell line MCF-7 does not require estrogen for growth, but paradoxically its growth is inhibited by antiestrogens. Our results show that, unlike normal target cells, MCF-7 cells carry most of their estrogen receptors in their nuclei even when these receptors are not charged with estrogens. The receptors for androgen and for progesterone, on the other hand, are localized in the cytoplasm as usual. Therefore, it is possible that the growth of these abnormal cells is stimulated by estrogen receptor in spite of the absence of the hormone and that the binding of antiestrogen molecules antagonize this stimulation.

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.

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.

Progesterone receptor gene restriction fragment length polymorphisms in human breast tumors.

We examined the progesterone receptor (PgR) gene in tissue from both primary human breast tumors and normal placentas, detecting restriction fragment length polymorphisms (RFLPs) with the restriction endonucleases Pst I/Sst I and HindIII. There was a general agreement of the Pst I and Sst I polymorphisms in any individual tumor, suggesting that they define two alleles in the human PgR locus, one being characterized by a deletion of about 300 base pairs with respect to the other. Both primary human breast tumor specimens (n = 36) and human term placentas (n = 48) displayed similar allele frequencies and typical mendelian distribution of these Pst I/Sst I alleles. The previously reported HindIII PgR RFLP was also investigated in 132 breast tumors. The HindIII PgR gene RFLP did not display typical mendelian distribution in the breast tumors; the factors affecting the HindIII allele frequencies are presently unknown. Neither the HindIII RFLP nor the deletion defined by Pst I and Sst I correlated with PgR expression as determined by a ligand-binding assay, suggesting that neither is related to the heterogeneity of PgR expression seen in breast tumors.

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.

Estrogen receptor in very small breast tumor specimens: a modified charcoal-gelatin assay.

We described recently a modified charcoal-gelatin (MCG) assay for measuring progesterone receptor activity in low-protein cytosols. We showed that pre-mixing of gelatin with sample cytosols (final gelatin concentration 0.1%) and removal of unbound steroid by a 1% charcoal suspension with 0.1% gelatin but without dextran preserves the progesterone receptor activity in dilute cytosol. For estrogen receptor (ER), as for progesterone receptor, the efficiency of the standard dextran-coated charcoal (DCC) assay drops rapidly as samples are diluted much below 1 mg protein per ml. We have therefore applied the MCG procedure to the assay of ER in breast tumor cytosols. We find that MCG is far more efficient for ER at low protein concentrations than either the DCC method or three other methods recommended previously for dilute samples, retaining at least 60% efficiency even at 0.01 mg protein per ml. The measured Kd of the receptor for estradiol is the same by MCG as by DCC. A series of human breast tumor biopsies assayed by MCG at 0.1 mg protein per ml gave about the same ER values (fmol/mg) as at 1 mg/ml, while the DCC efficiency for ER at the lower concentration averaged only 32%. In combination with 125I-labeled estradiol, this MCG method should allow accurate ER assays of extremely small breast cancer specimens.

Measurement of progesterone receptor in human breast cancer biopsies.

PIP: An improved sucrose gradient procedure for measuring progesterone receptor (PR) status was compared with other methods in an attempt to improve the assay of PR, a possibly important determinant in predicting endocrine therapy responses. Tritiated 3H-labeled R 5020 PR assay was modified to determine the actual nature of the competitive binding detected using sucrose density gradient centrifugation. An underestimation of 8S receptor was the result when using the swinging bucket technique, perhaps due to the prolonged run time (16 hours) involved. Hence, a vertical tube rotor was used which eliminated the problem. The 4S component was shown not to be PR, but probably an aberration due to failure to adsorb to hydroxylapatite. Dextran-coated charcoal and hydroxylapatite Scatchard plots confirmed this aberrant finding in that values derived from these methods correlated well with the 8S component of the gradient profile and not with the sum (8S + 4S) in a series of 27 human breast cancer specimens. In the same series the use of dextran-coated charcoal or hydroxylapatite single-saturating-dose assays correlated well with the 8S component of sucrose gradients. However, it is cautioned that until more experiments with these types of correlations are compiled, the 8S component of sucrose density gradient profiles as obtained from vertical tube rotor centrifugation yields the most reliable data on PR concentrations in human breast tumors.^ieng

Evaluation of estrogen receptor assays in human breast cancer tissue.

Standard dextran-coated charcoal (DCC) and sucrose gradient centrifugation assays for estrogen receptor were compared in a series of human breast cancer tissues. From a quantitative standpoint the results were remarkably similar. A simplified version of the DCC assay compared to the sucrose gradient assay yielded acceptable results. We conclude that, in spite of the lack of specificity controls inherent in the sophisticated standard assays, the simplified DCC assay might be useful if the biopsy specimen is too small to provide the number of aliquots for a standard DCC assay or sufficient protein for a sucrose gradient analysis. It also might be useful in research laboratories attempting to develop assays for multiple receptors or other constituents in a single tumor biopsy specimen.

Failure of estradiol immunofluorescence in MCF-7 breast cancer cells to detect estrogen receptors.

An indirect immunofluorescence assay was used to detect estradiol in MCF-7 breast cancer cells to determine if the estradiol-specific fluorescence observed represented estrogen receptor-bound estradiol. Appropriate controls were used to demonstrate the immunological specificity of our assay procedures. Initial studies of estradiol binding in MCF-7 cells were performed at 20 degrees for 1 hr with different concentrations of estradiol. Cytoplasmic and nuclear staining were observed following treatment with 10 nM estradiol, but not with lower concentrations which were nevertheless still sufficient to saturate estrogen receptor. The staining intensity increased with higher estradiol concentration, which is consistent with estradiol binding to lower-affinity binding sites. In order to further determine if estradiol binding by estrogen receptor was being detected, we pretreated MCF-7 cells with 5 nM diethylstilbestrol at 37 degrees for 1 hr to translocate all estrogen receptor to the nucleus and then administered estradiol at varying concentrations for 4 hr at 4 degrees. The estradiol was still primarily detected in the cytoplasm, although virtually all of the estrogen receptor was found to be present in the nucleus by standard [3H]estradiol binding assays. Additional immunochemical studies using sucrose gradient analysis to detect antibody-estradiol-receptor complexes clearly established that these complexes could not be detected. The present results suggest that, although immunocytochemical assays can specifically detect estradiol in MCF-7 cells, the estradiol is bound to lower-affinity binding sites rather than to estrogen receptor. Saturation analyses of intact viable MCF-7 cells performed at 37 degrees for 30 min using [3H]estradiol at concentrations ranging from 0.1 to 93 nM revealed an additional lower-affinity estradiol-binding site besides the receptor, perhaps analogous to the Type II sites reported in the rat uterus and human breast cancers.

Association of the 323/A3 surface glycoprotein with tumor characteristics and behavior in human breast cancer.

We have earlier described a monoclonal antibody (323/A3) against a Mr 43,000 surface glycoprotein of MCF-7 human breast cancer cells which shows considerable specificity for primary and metastatic breast tumors (Cancer Res., 46: 1306-1317, 1986). Here we report the occurrence of the 323/A3 antigen in a large cohort of primary breast tumors (m = 384) and its interrelationship with several clinically important variables. Frozen, stored tumor tissues were examined by a Western blot procedure, and the level of 323/A3 protein in individual tumors was calculated in arbitrary units based on the integrated Mr 43,000 signal in tumors compared with an MCF-7 internal standard. Thirty-six % (139 of 384) of tumors were found to be positive for 323/A3. Higher frequencies of 323/A3 protein were found in tumors larger than 2 cm (P = 0.03), tumors with infiltrated lymph nodes (P = 0.01), and tumors without estrogen receptor (P = 0.006). No significant relationship was found with patient age, menopausal status, or progesterone receptor status. Of the newer clinical determinants proliferative rate (% S phase), DNA ploidy, and the lysosomal protease cathepsin D, but not the HER-2/neu oncogene protein, were significantly correlated with 323/A3. The presence of 323/A3 protein was also related to increased recurrence (P = 0.003) and mortality (P = 0.036) after primary treatment. As an exposed surface antigen, this glycoprotein might be a useful target in radioimaging and immunotherapy of some human breast tumors, especially those having large size, infiltrated lymph nodes, deficient estrogen receptor, high proliferative rate, abnormal DNA content, and high levels of cathepsin D, all of which are ominous indicators of tumor behavior.

Multiple progesterone receptor assays in human breast cancer.

A review of assay results from more than 5500 patients revealed 283 patients in whom multiple breast cancer specimens were analyzed for progesterone receptor (PGR). All assays were performed in a single laboratory between 1975 and 1982 using the sucrose gradient technique. We considered only the 8S fraction of PGR. Simultaneous assays in 109 patients yielded 14% discordance [one assay with greater than 10 fmol/mg cytosol protein (PGR+) and one assay with less than 5 fmol/mg protein (PGR-)]. Among 161 sequential assays, there was an overall discordance of 19%: 8% (nine of 106) when the initial assay was PGR-, but 44% (24 of 55) when the initial assay was PGR+. Among PGR+ patients initially assayed at the time of diagnosis, there was a tendency to greater receptor loss in patients with positive axillary lymph nodes (44 versus 11%). The length of time between biopsies did not increase the discordance, but endocrine therapy within this interval did increase it (56% of initially PGR+ patients who received interim endocrine therapy were PGR- at second biopsy). to evaluate the significance of interval loss of PGR, we compared survival from first biopsy in initially PGR+ patients who subsequently lost their receptor versus those whose receptor persisted. The latter group experienced a significantly longer survival (p less than 0.02). In summary, we observed an ominous loss of PGR in sequential biopsies, particularly with intervening endocrine therapy, and those patients whose tumor cells lost PGR experienced poorer survival than did patients retaining PGR. Therefore, patients with PGR+ primary tumors require repeat biopsy for PGR upon disease recurrence for optimal treatment planning.

Estrogen receptor expression in human breast cancer associated with an estrogen receptor gene restriction fragment length polymorphism.

Estrogen receptor (ER) content is a well-known predictor of clinical outcome in human breast cancer. The recent cloning of a human ER complementary DNA has made possible the characterization of the ER gene on a molecular level. We have examined in human breast cancers a single, two-allele restriction fragment length polymorphism using the restriction enzyme PvuII. Initial studies in human breast cancer cell lines suggested a possible association between the absence of one allele and the absence of ER expression; subsequent analysis of allele distribution and frequency in 188 primary human breast tumor biopsies did indeed show a significant but not complete correlation between the absence of one allele and the failure to express ER. Preliminary data suggest that this restriction fragment length polymorphism is located within gene sequences coding for the putative DNA or hormone-binding domains of the ER.

Variant human breast tumor estrogen receptor with constitutive transcriptional activity.

Since progesterone receptor (PgR) is normally induced by estrogen, breast cancer lacking estrogen receptor (ER) would also be expected to lack PgR. However, a small percentage of breast cancers are ER- yet PgR+. These tumors might possess an ER which is defective in estrogen binding but is still functional in stimulating estrogen-responsive genes such as PgR. We have now detected such a variant, lacking exon 5 of the hormone-binding domain, using complementary DNA amplified by the polymerase chain reaction. This variant was the predominate ER RNA expressed in three ER-/PgR+ tumors. Furthermore, the variant ER constitutively activates transcription of a normally estrogen-dependent gene construct in yeast cells. The variant ER could explain the expression of PgR in certain tumors and have therapeutic implications.

Estrogen receptor gene methylation in human breast tumors.

DNA methylation is known to be involved in eukaryotic gene control; it may thus exert effects during development and tumorigenesis. We have examined the methylation status of the estrogen receptor (ER) gene in different human tissues. The ER gene was found to be methylated in placental tissues, but normal breast tissues exhibited a different methylation pattern. In addition, specific sites in the hormone-binding domain of the ER gene were observed to be differently methylated in different human breast tumor specimens. We did not detect, however, any association between the ER status of a tumor and ER gene methylation at these sites. Interestingly, a difference in the methylation status between normal and adjacent breast tumor tissues was observed. Thus, DNA methylation may be considered an additional molecular measure of the genetic heterogeneity in breast cancer.

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.

Multiple estrogen receptor assays in human breast cancer.

A review of assay results from more than 6000 patients revealed 232 patients in whom multiple breast cancer specimens were analyzed for estrogen receptor (ER). All assays were performed in a single laboratory. Specimens were considered estrogen receptor positive (ER+) if the ER level was greater than 10 fmol/mg protein and estrogen receptor negative (ER-) if the ER level was less than 3. ER values between 3 and 10 fmol/mg protein were considered borderline. Simultaneous assays were performed in 58 patients with 3% major discordance (i.e., one assay ER- and one assay ER+). Major discordance for sequential biopsies was 19% (16 of 82) when the initial assay was ER+ and 13% (eight of 63) when the initial assay was ER-. (Apparent change from ER- to ER+ status was observed in five of nine patients with primary tumors less than 2 cm in diameter, suggesting that an inadequate amount of tissue may have been submitted for initial ER analysis.) There was no significant relationship between the time interval between sequential biopsies and the rate of discordance. Marked decreases in ER levels and 78% discordance were seen if rebiopsy was performed within 2 months of tamoxifen treatment. When these tamoxifen cases were excluded from the analysis, neither intervening endocrine therapy nor chemotherapy significantly altered discordance rates.

Estrogen and progesterone receptor proteins in breast cancer.

PIP: This article reviews the contribution of steroid hormone receptor studies to the resolution of a basic clinical problem: how to determine which cancers are hormone dependent without an actual treatment trial. Previously published studies on hormone receptor assays and analyses are reviewed, the current status of knowledge in the area is summarized in terms of its relevance to breast cancer cells, and future scenarios are proffered. Assay methods and available clinical results for cytoplasmic estrogen receptor identification in human breast cancer comprise the first section. Information on assaying nuclear estrogen receptor, and its relative clinical importance, is presented in Part 2. Assays to determine the presence of progesterone receptor in human breast cancer; the estogen regulation of such progesterone receptors; and clinical findings comprise Part 3. Studies of the mechanisms of estrogen action in the MCF-7 human breast cancer cell line are the subject of Part 4. Recent experiments in animals on the efficacy of antiestrogen treatments, such treatments in human in vitro cell cultures, and the few clinical trials available are presented in Part 5. It is emphasized that the simple presence or absence of estrogen receptors in tumors does not absolutely indicate whether growth of a particular tumor is sensitive to estrogens. Experimental appraoches, designed to further delineate this problem, are outlined, based on observations such as the finding that the probability of tumor regression correlates better with quantitative rather than with qualitative assessment of estrogen receptors; that the specific end product of hormone action is unknown and without this information an ideal biochemical marker to a tumor's sensitivity of hormones is unavailable; and that the complex sequence of biochemical events in the actions of estrogen needs more complete elucidation.^ieng