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Background: We previously showed in HER2+ models of breast cancer (BC) that potent inhibition of the HER receptor layer can lead to re-expression of estrogen receptor (ER) or activation of the ER pathway. Consequently, the anti-apoptotic ER gene product Bcl2 is upregulated, resulting in enhanced tumor cell survival and treatment resistance. In this study, we investigated whether Bcl2 and ER expression levels are simultaneously increased by neoadjuvant treatment with the dual HER1/2 tyrosine kinase inhibitor lapatinib in HER2+ BC patients. Methods: In a neoadjuvant phase II clinical trial 49 HER2+ BC patients were treated with lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before surgery. Tumor specimens were prospectively collected at different time-points during lapatinib treatment (baseline, and weeks 2 and 4). Bcl2, ER, progesterone receptor (PR), total (t) and phosphorylated (p)-HER2, and Ki67 were assessed by immunohistochemistry. Spearman correlation was used to evaluate the association among the biomarkers at baseline, and the correlation of their changes over time. Fisher's Exact test and non-parametric Wilcoxon rank sum test were used respectively to determine if the frequency and the magnitude of Bcl2 expression changes were associated with baseline ER status. Results: 35/49 HER2+ tumor specimens (71%) were available for baseline evaluation of Bcl2 and ER. Of those, 12 (34%) were ER-positive (Allred score 3) and 23 (66%) ER-negative. Baseline Bcl2 expression correlated positively with ER (r = .75; p < .0001) and PR (r = .53; p = .0015), and inversely with t-HER2 (r = -.43; p = .0097). ER baseline expression correlated positively with PR (r = .57; p = .0004), and inversely with t and p-HER2 (r = -.55; p = .0005, and r = -.37; p = .0282, respectively) and Ki67 (r = -.39; p = .0271). Bcl2 changes at week 2 (w2) positively correlated with changes in both ER and PR levels (r = .70; p = .0002 and r = .57; p = .0076, respectively). Additionally, the increase in Bcl2 expression, observed in 9 of the 23 (39%) tumors with tissue available at w2, was significantly more frequent (p = .0147) and of greater magnitude (p = .0001) in ER-pos vs. ER-neg tumors -- 8/9 ER-pos tumors at w2 (including 3 converted from ER-neg by lapatinib) had increased Bcl2, while only 1 of the 14 (7%) ER-neg tumors (at baseline and w2) had increased Bcl2. The expression of ER itself at w2 also increased in 3 out of the 6 (50%) tumors which were originally ER-pos and had tissue available at w2, and in all of them Bcl2 increased in parallel. Of note, the single baseline ER-pos tumor that showed a reduction in ER at w2 had a parallel decrease in Bcl2. Similar observations or trends were found at week 4. Conclusion: Our study suggests that Bcl2 is upregulated as a result of enhanced/restored ER activity upon anti-HER2 therapy with lapatinib in HER2+ tumors. This further supports the use of endocrine along with anti-HER-2 therapy to block this escape pathway which could otherwise cause treatment resistance. In addition, the ER re-expression with lapatinib treatment observed in this study emphasizes the need to re-biopsy HER2+/ER--patients receiving anti-HER2 therapy and to add endocrine therapy if the tumor becomes ER-positive. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We have used the screening techniques of CMC and SSCP of selected PCR fragments, and also gel retardation assays, to discover a number of ER variants in clinical breast cancer tissues. We have found base pair insertions, transitions, and deletions, and deletions of exons 3, 5 and 7. Using a yeast transactivation assay, we have discovered receptors with outlaw function consisting of both dominant positive receptors that were transcriptionally active in the absence of oestrogen and dominant negative receptors that were transcriptionally inactive themselves but prevented normal ER function. Future efforts should focus in particular on such dominant positive and dominant negative variants. With regard to positive variants, we should like to know whether they stimulate tumour growth and, if so, whether they can be turned off. With regard to dominant negative variants, we should like to determine whether they can inhibit tumour growth and, if so, whether they can be turned on.

We have used the screening techniques of chemical mismatch cleavage, single stranded conformational polymorphism, and gel retardation to discover a number of estrogen receptor RNA variants in clinical breast cancer tissues. We have found basepair insertions, transitions, and deletions as well as alternative splicing, yielding deletions of exon 3, 5, or 7. Using a yeast transactivation assay we have discovered receptors with outlaw function, including both a dominant-positive receptor, which is transcriptionally active in the absence of estrogen, and a dominant-negative receptor, which is itself transcriptionally inactive, but prevents the action of normal estrogen receptor. These variants could have clinical significance, helping to explain breast tumor behavior and patient outcome.

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.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.

The presence of androgen receptor (AR) in prostate cancer has been linked to the androgen-dependent nature of the tumor and has also been shown to have prognostic significance; it also appears to be a positive prognostic indicator in breast cancer. However, due to the relatively low AR concentrations in most tumors and the inherently low specific activity of tritium, the assay of AR based on available 3H-ligands is not sensitive enough to measure accurately the amount of receptor in small specimens. A 125I-ligand like those available for the estrogen and progesterone receptors would be helpful, but development of such a ligand for AR has not been very successful. Although several androgen analogues containing iodine, bromine, or selenium have been synthesized specifically as potential probes for AR, none have shown any significant affinity or specificity for the receptor. We therefore undertook the synthesis of new potential AR ligands which could be radioiodinated, and determined their affinities for AR (from rat uterus and MCF-7 human breast cancer cells) by using a competition assay. We have examined both 5 alpha-dihydrotestosterone (5 alpha-DHT) and 19-nortestosterone analogues and have identified two such compounds which showed high AR affinity: (17 alpha,20E)-17 beta-hydroxy-21-iodo-5 alpha-pregn-20-en-3-one (17 alpha-[E)-iodovinyl)-5 alpha-DHT, 9) and 17 beta-hydroxy-7 alpha-methyl-(17 alpha,20E)-21-iodo-19-norpregna-4,20-dien-3- one (7 alpha-methyl-17 alpha-[E)-iodovinyl)-19-nortestosterone, 11). In fact, the affinity of the latter for human AR was found to be superior to that of 5 alpha-DHT itself. These iodovinyl analogues could be easily prepared in the radioiodinated form, and should prove to be extremely useful in assaying low levels of AR in small specimens.

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.

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.

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.