Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Exploring Collagen Parameters in Pure Special Types of Invasive Breast Cancer.

One of the promising tools to evaluate collagen in the extracellular matrix is the second-harmonic generation microscopy (SHG). This approach may shed light on the biological behavior of cancers and their taxonomy, but has not yet been applied to characterize collagen fibers in cases diagnosed as invasive breast carcinoma (BC) of histological special types (IBC-ST). Tissue sections from 99 patients with IBC-ST and 21 of invasive breast carcinoma of no special type (IBC-NST) were submitted to evaluation of collagen parameters by SHG. Tissue microarray was performed to evaluate immunohistochemical-based molecular subtype. In intratumoral areas, fSHG and bSHG (forward-SHG and backward-SHG) collagen parameters achieved their lowest values in mucinous, papillary and medullary carcinomas, whereas the highest values were found in classic invasive lobular and tubular carcinomas. Unsupervised hierarchical cluster analysis and minimal spanning tree using intratumoral collagen parameters allowed the identification of three main groups of breast cancer: group A (classic invasive lobular and tubular carcinomas); group B (IBC-NST, metaplastic, invasive apocrine and micropapillary carcinomas); and group C (medullary, mucinous and papillary carcinomas). Our findings provide further characterization of the tumor microenvironment of IBC-ST. This understanding may add information to build more consistent tumor categorization and to refine prognostication.

Changes in caveolae, caveolin, and polymerase 1 and transcript release factor (PTRF) expression in prostate cancer progression.

BACKGROUND: Caveolae are specialized invaginations in the cell membrane involved in the regulation of cell transport and signal transduction. The aims of this study were to investigate the number of caveolae and expression of caveolae-associated proteins, caveolin-1 and -2, and polymerase 1 and transcript release factor (PTRF) with development of prostate cancer. METHODS: Transmission electron microscopy was used to investigate the number of caveolae in normal human prostate stromal, epithelial cells, and androgen-dependent (LNCaP) and androgen-independent (PC3) cancer cell lines. Surgical tissue was obtained from patients with benign prostatic hyperplasia (BPH), well-differentiated and poorly differentiated prostate cancer. Caveolin-1, caveolin-2, and PTRF expression was identified by immunohistochemistry in tissue samples and quantified by Western blot analysis in cell lines. RESULTS: Caveolae were identified in normal epithelial and stromal prostate cells. The number of caveolae was significantly reduced in LNCaP and PC3 cells (P < 0.0001). PTRF was localized to stromal and epithelial cells in tissue from patients with BPH and in normal stromal and epithelial cells in vitro. PTRF expression was significantly decreased in LNCaP and PC3 cells and also in cancer tissue. In prostate tissue, caveolin-1 and -2 expression appeared to increase in prostate cancer. Caveolin-1 and -2 expression was decreased in LNCaP cells but caveolin-2 expression was significantly increased in PC3 cells compared to normal epithelial cells. CONCLUSIONS: This study demonstrates that changes in the cell membrane involving loss of caveolae and PTRF expression occur with the development of prostate cancer. These changes are accompanied by an up-regulation of caveolin-2.

Centrosome impairments and consequent cytokinesis defects are possible mechanisms of taxane drugs.

Taxol and taxotere are two of the most promising anticancer drugs. To determine the mechanisms responsible for cell death after exposure to low doses of taxane, PC3 cells were treated with taxol and taxotere, and observed with immunofluoroscence microscopy. Pericentriolar material dissociation and blockage of normal centrosome separation were found to result in two different abnormal spindle types; multipolar and monopolar spindles, respectively. The majority of abnormal spindles induced by taxol were monopolar spindles, whereas taxotere mostly induced abnormal multipolar spindles. Consequently, monopolar spindle mitosis resulted in cleavage failure, while multipolar spindle mitosis led to the formation of both cleavage failure and multipolar cell division. Multinucleation characterized interphase cells which had undergone cytokinesis defects. These cells subsequently became giant multinucleated cells after several rounds of cell cycle with sustained cleavage failure, and were gradually eliminated through cell death.

Complex post-transcriptional regulation of EGF-receptor expression by EGF and TGF-alpha in human prostate cancer cells.

The epidermal growth factor receptor (EGFR) plays an important role in the development and progression of prostate cancer and its overexpression is associated with decreased survival. With progression, prostate cancer cells switch from epidermal growth factor (EGF) to transforming growth factor alpha (TGF-alpha) synthesis, which contributes to autocrine growth and unrestrained proliferation. To define the molecular mechanisms involved in the regulation of EGFR expression by EGF and TGF-alpha we studied three human prostate cancer cell lines, androgen-responsive (LNCaP) and -unresponsive (DU145 and PC3). Here we show that TGF-alpha stabilized EGFR mRNA two- to threefold in all three cell lines, whilst EGF stabilized EGFR mRNA approximately twofold in LNCaP and DU145 cells, but not in PC3 cells. Both ligands increased EGFR transcription in LNCaP and DU145 cells, with less effect in PC3 cells. In all three cell lines EGF reduced total EGFR protein levels more than TGF-alpha, but this was associated with a greater increase in de novo protein synthesis with EGF compared to TGF-alpha. Only EGF, however, shortened EGFR protein stability (half-life decreased from 5 h to 120 min), resulting in rapid disappearance of newly synthesized EGFR protein. Both ligands increased total LNCaP and DU145 cell numbers. These studies demonstrate that the EGF- and TGF-alpha-induced upregulation of EGFR mRNA and protein in human prostate cancer cell lines is complex and occurs at multiple, transcriptional and post-transcriptional levels. Taken together, these data provide novel insight into the molecular mechanisms by which TGF-alpha would preferentially maintain an autocrine loop in human prostate cancer cells. Furthermore, this work suggests that in human prostate cancer cells ligand-specific differential intracellular trafficking of the EGFR plays a major role in regulating its expression.

[meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl) ethylenediamine]dichloroplatinum(II), a compound with a specific activity on hormone-sensitive breast cancers--evidence for a diethylstilbestrol-like mode of action.

[meso-1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]- dichloroplatinum(II) (meso-1-PtCl2), an estrogenic and cytotoxic platinum complex, shows activity against ER+ but not against ER- breast cancers in vivo (ER: estrogen receptor; ER+ and ER- indicate the presence or absence of the ER). To clarify whether its estrogenic or its cytotoxic potency or both properties are the cause of this specific inhibitory effect, we tested meso-1-PtCl2 comparatively in vivo on an ER+ and an ER- murine breast cancer (MXT-M-3.2 MC and MXT-M-3.2(ovex) MC, respectively), and in vitro on two cell lines derived from the former in vivo models (MXT+ and MXT-, respectively). The estrogens diethylstilbestrol (DES) and the ligand of meso-1-PtCl2 (meso-1), responsible for the hormonal effect of meso-1-PtCl2, and the cytotoxic drug cisplatin (cDDP) were used as comparative substances. Meso-1-PtCl2. DES and cDDP showed a strong and comparable activity on the ER+ MXT-M-3.2 MC in vivo, meso-1 being somewhat less inhibitory. In experiments on the murine, ER- MXT-M-3.2(ovex) MC only cDDP caused a marked inhibitory effect. The other compounds were inactive or only marginally active. In accordance with the in vivo results cDDP was also very active on the MXT+ and MXT- breast cancer cell line. In contrast to this meso-1-PtCl2, meso-1, and DES proved to be only weakly active or inactive on both cell lines. From these results it can be concluded that there is only little if any contribution of the cytotoxic PtCl2 moiety of meso-1-PtCl2 to the anti-breast cancer activity in vivo. On the ER+ MXT-M-3.2 MC transplanted into ovariectomized mice meso-1-PtCl2 yielded a biphasic dose activity curve, i.e. an increase of the tumor growth at low doses followed by a decrease at high doses, identical with those of the estrogens DES and meso-1. These results indicate that meso-1-PtCl2 inhibits ER+ breast cancers by its estrogenicity in the same manner as meso-1 and DES. The complex mechanism of anti-breast cancer active estrogens involves presumably the endocrine and/or the immune system. Its investigation is the subject of further studies.

Telomere length, telomerase activity and telomerase RNA expression during mouse mammary tumor progression.

To investigate the roles of telomere length (mean length of the terminal restriction fragments; TRFs), telomerase activity (TA) and telomerase RNA (mTR) expression in relation to mouse mammary tumor progression, we examined a pregnancy-dependent mouse mammary tumor line (TPDMT-4) and its four autonomous sublines (T4-OI320: non-metastatic; and T4-OI165, -OI96, and -OI145: artificial metastatic) of DDD/1 mouse origin, and an autonomous growing mammary tumor (JYG-MC) showing spontaneous lung metastasis developed in BALB/c mice infected with a Chinese feral mice (Sub-Jyg)-derived mouse mammary tumor virus (JYG-MTV). Compared with normal (pregnant) mammary tissue, the TA was elevated in the TPDMT-4 tumor and in the non-metastatic subline tumor (T4-OI320) (x10 fold, respectively), and was further increased (x13-15 fold) in parallel with the acquisition of metastatic potential (T4-OI165, -OI96, and -OI145). The mTR level was upregulated (x2.7-2.8 fold) in all autonomous growing tumors compared to the normal counter-part, but not in TPDMT-4. The TRF was shorter in accord with tumor progression (normal mammary tissue, 48 kb; TPDMT-4, 45 kb; T4-OI320, 37 kb; T4-OI165, -OI96 and -OI145, mean 37.7 kb; and JYG-MC, 21 kb). These results suggest that the activation of TA occurs as an early event at the stage of hormone-dependent tumorigenesis, followed by the up-regulation of mTR expression in accordance with the acquisition of autonomous growth, and then further activation of TA occurs when the tumor acquires metastatic potential. The TRF shortening was in parallel with the tumor progression.

Decrease in the level and mRNA expression of LH-RH and EGF receptors after treatment with LH-RH antagonist cetrorelix in DU-145 prostate tumor xenografts in nude mice.

Using radioligand binding, RT-PCR, and Southern blot analyses, we evaluated whether agonist [D-Trp6]LH-RH and antagonist Cetrorelix could affect the levels of receptors for LH-RH and EGF and expression of mRNA for these receptors in DU-145 human androgen-independent prostate cancers xenografted into nude mice. Radioligand binding studies showed the presence of specific high affinity receptors for LH-RH and EGF in DU-145 prostate tumors. Cetrorelix, but not [D-Trp6]LH-RH significantly inhibited tumor growth. The concentration of LH-RH receptors was reduced by 22% (p<0. 05) and 67% (p<0.01) after 4 weeks of treatment with [D-Trp6]LH-RH and Cetrorelix respectively. The concentration of EGF receptors fell by 48% (p<0.05) in the [D-Trp6]LH-RH group, whereas Cetrorelix led to a 66% reduction (p<0.01). The expression of LH-RH and EGF receptor mRNA was investigated by RT-PCR analysis followed by Southern blotting. Densitometric analysis of the developed bands showed that the antagonist Cetrorelix decreased the expression of LH-RH receptor mRNA by 55% (p<0.01) compared to control group while the 20% reduction after treatment with the LH-RH agonist was non-significant. Treatment with [D-Trp6]LH-RH and Cetrorelix also reduced the expression of EGF receptor mRNA by 35% and 68% respectively (both, p<0.01) compared to control group. In conclusion, these data demonstrate that growth inhibition of DU-145 prostate tumors induced by prolonged administration of LH-RH antagonist Cetrorelix is accompanied by a marked decrease in the concentration of LH-RH and EGF receptors as well as in their mRNA levels.

Melatonin: receptor-mediated events that may affect breast and other steroid hormone-dependent cancers.

Epidemiological studies have suggested a possible link between extremely low frequency electromagnetic fields (EMFs) and increased rates of certain cancers. One cancer that has been postulated to be associated with EMF exposure is breast cancer, for which increased rates have been reported among electricians. These cancer associations are weak, and the link to EMF exposures remains tenuous. Understanding the mechanisms by which EMFs could have biological effects will help in elucidating the risk, if any, from EMFs. One hypothesis that has received considerable attention involves reduction of melatonin levels by EMFs. This hypothesis suggests that loss of melatonin affects a variety of hormonal processes such as estrogen homeostasis and thereby may increase breast cancer rates. Since this theory was first presented, putative melatonin receptors have been cloned, providing new tools with which to examine melatonin's mechanism of action and the melatonin hypothesis. These receptors are found in nuclear and membrane fractions of cells. The nuclear receptors (retinoid Z receptors) are found both in the brain and in non-neural tissues, whereas the membrane-bound receptors are found primarily in neural tissue and have a higher affinity for melatonin. These receptors may control a variety of hormonal and immunological functions, including the release of gonadotropins from the hypothalamus and pituitary and 5-lipoxygenase activity in B lymphocytes. This Working Hypothesis briefly reviews our current knowledge of melatonin receptors and then provides theories on how the inactivation of melatonin receptors may cause cancer and suggests areas of research for addressing this question.

Synthesis and biological evaluation of 4-(hydroxyalkyl)estradiols and related compounds.

A series of synthetic estrogens containing hydroxyalkyl side chains at the C-4 position of the A ring were designed as metabolically stable analogs of 4-hydroxyestradiol, a catechol estrogen. These synthetic steroids would facilitate investigations on the potential biological role of catechol estrogens and also enable further examination of the structural and electronic constraints on the A ring in the interaction of estrogens with the estrogen receptor. Catechol estrogens are implicated as possible causative agents in estrogen-induced tumorigenesis. 4-Hydroxyestradiol has weaker affinity for the estrogen receptor and exhibits lower estrogenic activity in vivo; on the other hand, the catechol estrogens are prone to further oxidative metabolism and can form reactive intermediates. This report describes the synthesis and initial biochemical evaluation of 4-(hydroxyalkyl)estrogens and 4-(aminoalkyl)estradiols. The 4-(hydroxyalkyl)estrogens were prepared by oxidative hydroboration of 4-alkenylestradiols. The alkenylestradiols were obtained via a Stille cross-coupling between a MOM-protected 4-bromoestradiol and an alkenylstannane. The (4-aminoalkyl)estrogens were prepared from the hydroxyalkyl derivatives with phthalimide under Mitsunobu conditions. The substituted estradiols were evaluated for estrogen receptor binding activity in MCF-7 human mammary carcinoma cells, and 4-(hydroxymethyl)estradiol 1 exhibited the highest affinity with an apparent EC50 value of 364 nM. The relative activities for mRNA induction of the pS2 gene in MCF-7 cell cultures by the 4-(hydroxyalkyl)estrogens closely parallel the relative binding affinities. 4-(Hydroxymethyl)estradiol 1 did not stimulate the growth of MCF-7 cells at concentrations up to 1 microM. Thus, 4-(hydroxymethyl)estradiol 1 exhibited similar estrogen receptor affinity as the catechol estrogen, 4-hydroxyestradiol, and may prove useful in the examination of the biological effects of 4-hydroxyestrogens.

Ploidy level determination and quantitative chromatin pattern description in pregnancy-associated breast cancers.

The present study deals with the characterization of hormone-sensitivity in pregnancy-associated breast cancers (PBCs). This characterization was carried out in 22 PBCs as opposed to 88 non-pregnancy-associated breast cancers (NPBCs). For this study, we used the digital cell image analysis of Feulgen-stained nuclei to assess the type of hormone-sensitivity. In a previous study it was demonstrated that the chromatin pattern in breast cancers is related to the amounts of estrogen receptors they contain. Our results demonstrated that the quantitative description of the chromatin pattern by means of 15 parameters (relating to morphometric, densitometric, and textural features) made it possible to identify typical cell nuclei populations in the PBC and NPBC groups. The use of specific statistical analyses (principal-components and discriminant) made it possible to quantify the proportion of each cell nucleus type in the PBCs. Furthermore, of the 22 PBCs under study, 13 contained a large majority of cell nuclei whose chromatin pattern was characteristic of hormone-sensitive cells, while 5 cases contained a large majority of typically hormone-insensitive ones. The remaining 4 cases contained a relatively similar proportion of typically hormone-sensitive and insensitive cell nuclei. The quantitative chromatin pattern description thus made it possible to characterize the hormone-sensitivity level in PBCs, whereas DNA ploidy level determination did not enable any such characterization to be carried out. The chromatin pattern assay described here, which enables hormone-sensitive pregnancy-associated breast cancers to be identified from hormone-insensitive ones independently from biochemical assays, should help the physician regarding therapy adaptation.

Cytostatic and cytotoxic action of Z-1, 1-dichloro-2,3-diphenylcyclopropane in three human breast cancer cell lines.

Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, AII) is known as a potential anti-breast cancer agent and has previously been studied as an antiestrogen (AE). We hypothesized that its activity is independent of estrogen receptor (ER) status. AII and its known and potential metabolites were synthesized and characterized by NMR and MS. ER positive/estrogen and AE sensitive MCF-7, ER positive/estrogen and AE resistant MCF-7/LY2, and ER negative/estrogen and AE resistant MDA-MB231 cells were used in metabolism, cytostasis, cytotoxicity, and serum binding/wash out assays. Bacterial mutation assays were performed with Salmonella typhimurim strain TM677. AII underwent slow solvolysis in culture medium to Z-2-chloro-1,3-diphenyl-2-propen-1-ol and its oxidized form Z-alpha-chlorochalcone (ZCC). ZCC was the major metabolite of AII in all three cell lines. Cytostasis and clonogenic assays showed AII to be cytostatic to each of the lines, and was more potent against MCF-7 and MCF-7/LY2 than MDA-MB231 cells. ZCC was cytotoxic, with IC50 values of 89, 0.5, and 170 nM in MCF-7, MCF-7/LY2, and MDA-MB231 cells, respectively. Cytotoxicity from ZCC was delayed compared to loss in cell viability, suggesting a non-necrotic mechanism. Serum protected against loss of cell viability caused by AII, but had no effect on the action of ZCC. The effects of ZCC could be partially reversed by washing the drug out of cells. The effects of AII persisted after wash out. AII was also shown to be nonmutagenic in forward Salmonella mutation assays both with and without metabolic activation. In conclusion, AII, a chemical with weak antiestrogenicity, anti-breast cancer activity, and low toxicity in whole animals, shows growth inhibitory properties against both ER positive and negative human breast cancer cells in culture. Its direct action appears to be cytostatic and longlived. AII is converted by the cells to a less-retained and -protein bound metabolite, ZCC, that is more cytotoxic. Neither AII nor ZCC appear to have mutagenic activity. Both AII and ZCC thus appear to have potential for use against estrogen-dependent and -independent human breast cancers.

Hormonal control of growth factor receptor expression.

In this report, we have discussed a series of results obtained in our laboratory that, together with data by other authors, demonstrate that the expression of the erbB-2 tyrosine kinase receptor oncogene in breast cancer cells is regulated by multiple factors and hormones, which modulate their growth and differentiation. In particular, we have shown that estrogens specifically inhibit erbB-2 expression by transcriptional repression, which is exerted through a sequence within the erbB-2 gene promoter. Estrogens control mammary cell growth directly, by inducing early gene expression, and indirectly, by increasing autocrine growth factor production or decreasing growth inhibitors. The data presented here suggest that mammary cells respond to estrogen also by modifying the receptor array on their surface, thus setting their own sensitivity to the different autocrine and paracrine factors. As a first consequence, the modulation of erbB-2 expression level by antiestrogen may represent a point to consider when selecting breast cancer patients for hormonal therapy, in those (few) cases where estrogen receptor positivity accompanies erbB-2 amplification. On the other hand, antiestrogen-induced upregulation of erbB-2 may improve tumor targeting of drugs designed to interact or interfere with erbB-2, such as humanized antibodies, immunotoxins, or engineered ligands. These possibilities should be tested in appropriate model systems in the future.

Combined effects of 13-cis-retinoic acid, tamoxifen and interferon on the growth of human breast cancer cells.

We studied the effect of 13-cis-retinoic acid (13-cRA) alone and in combination with interferons (IFNs) and tamoxifen (TAM) in two established human breast cancer cell lines: the estrogen-sensitive CG-5 and the estrogen-insensitive MDA-MB-453 cells. 13-cRA (10(-9)-10(-5) M) significantly reduced the growth of both cell lines in a dose-dependent fashion, after 3 and 6 days of treatment. When the retinoid (10(-9)-10(-5) M) was combined with natural beta-IFN (100-1000 IU/ml) for 6 days, we observed a growth inhibition more pronounced than that produced by each of the two single agents in both CG-5 and MDA-MB-453 cells. Only in the former model was the inhibitory effect synergistic at all the drug concentrations used. Association of 13-cRA (10(-9)-10(-5) M) and recombinant alpha2a-IFN (100-1000 IU/ml) or TAM (10(-7)-10(-6) M) did not determine an additive or synergistic effect on the growth of CG-5 cells.

Title aggregation patterns of argyrophilic nucleolar organizer regions induced by 5-fluorouracil in the nuclei of MCF-7 human breast cancer cells.

The effects of tamoxifen and 5-fluorouracil (5-FU) on the patterns of argyrophilic nucleolar organizer regions (AgNORs) in MCF7 human breast cancer cells were studied. Tamoxifen and 5-FU both inhibited the growth of MCF-7 cells by 18% by day 3 of culture, but each had different effects on the AgNORs. Whereas no significant changes were induced by tamoxifen, effects on the AgNORs of MCF-7 cells by 5-FU were dramatic: 5-FU treatment changed the pattern of AgNORs, reducing the number of satellites by aggregation, typically to a single aggregation around nucleoli in a sphenoidal fashion. We named these morphological changes: fluorouracil induced AgNOR aggregations (FAA). Following treatment with 500 ng/ml 5-FU, FAA developed rapidly. AgNORs forming two or three aggregates in 24% (6 h), 24% (12 h), 40% (24 h) and 34% (48 h) of cells, compared to a control rate of 14%. Single large aggregate was rarely found in untreated cultures but after 6, 12, 24 and 48 h treatment with 500 ng/ml 5-FU, AgNORs had formed a single aggregate in 6, 8, 16 and 22% of cells, respectively. FAA were observed at a concentration of 100 ng/ml 5-FU; 48 h treatment resulted in cells in which two or three aggregates were increased by 24% and single aggregate by 16%. These large single aggregates were larger than nucleoli stained by Papanicolau staining.

Tamoxifen aziridine labeling of the estrogen receptor-potential utility in detecting biologically aggressive breast tumors.

Expression of estrogen receptor (ER) is a helpful predictor of response to endocrine therapy and disease free survival in breast cancer patients. The presence of variant estrogen receptors has been demonstrated at the RNA/DNA level and might represent an escape of tumors from hormonal control mechanisms. However, the demonstration that the corresponding peptides do exist is a real challenge. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of covalently bound [3H]tamoxifen aziridine ([3H]TAZ) to ER demonstrates a specific, multiband peptide pattern recognized by anti-ER monoclonal antibodies (anti-ER Mo Abs). The native 66 kDa ER form identified through its hormone binding domain by the H-222 Mo Ab was the most prominent one followed by 50, 35, and 28 kDa forms on fluorography. Such patterns from early human breast tumors were compared to the ones of more advanced disease, namely large primary breast cancers, metastatic lymph nodes, and soft tissue relapses: in these cases, molecular forms of 43 and 35 kDa were identified with a remarkable consistency. The 43 kDa peptide was more frequently identified by the H-226 Mo Ab (which maps a region near the DNA binding domain)-albeit with low labeling intensity as compared to H-222 Mo Ab. In addition, the 43 kDa peptide was inversely correlated to ER levels. This altered ER or related peptide could potentially be a marker of biologically aggressive breast tumors.