Fibroadenoma Arising in Axillary Ectopic Breast Tissue. A Diagnostic Challenge.

Ectopic or accessory breast tissue may occur in primitive embryonic milk lines or locations other than the milk line. The same pathology arising in breast tissue may occur less frequently in ectopic breast tissue. Fibroadenomas rarely occur in ectopic breast tissue, with less than 50 reported cases in the English literature, despite being the most common benign breast neoplasms. Diagnosing fibroadenoma in ectopic breast tissue can be challenging due to the lack of clinical suspicion and the atypical findings in imaging studies. Treatment consists of surgical excision. In this manuscript, we present a case of a 24-year-old patient with a fibroadenoma of the left axilla arising in bilateral axillary ectopic breast tissue, and we comprehensively review the literature.

Integrating Microstructured Electrospun Scaffolds in an Open Microfluidic System for in Vitro Studies of Human Patient-Derived Primary Cells.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Matrix-producing Breast Carcinoma: A Rare Subtype of Metaplastic Breast Carcinoma.

Matrix-producing carcinoma (MPC) is a rare subtype of metaplastic breast carcinoma (MBC) that was first described in 1989 by Wargotz and Norris. It accounts for less than 1% of breast carcinomas and has distinctive clinical, morphological, and immunohistochemical features. Histologically it consists of invasive carcinoma of no special type with transition to cartilaginous or osseous matrix without a spindle cell component. Data on this entity are limited with the literature consisting mostly of case reports and a small number of case series. We report a case of matrix-producing breast carcinoma, with excellent clinical outcome. We also discuss the histogenesis, imaging, histological, and immunohistochemical characteristics, treatment, and focus on the differential diagnosis of this rare tumor.

Kallikrein-related peptidase 6 (KLK6) expression differentiates tumor subtypes and predicts clinical outcome in breast cancer patients.

Novel molecular markers that address the heterogeneity of breast cancer (BC) and provide meaningful prognostic information for BC patients are needed. Kallikrein-related peptidase 6 (KLK6) is aberrantly expressed and functionally implicated in BC and, like other members of the KLK family, may prove a useful molecular tool for clinical management. Our objective was to assess, for the first time, the clinical relevance of KLK6 mRNA expression in BC. Total RNA was isolated from 165 breast tumors, as well as 100 adjacent non-cancerous tumor specimens. After cDNA synthesis, and following quality control, quantitative real-time PCR for KLK6 expression analysis took place. Receiver operating characteristic curves were constructed in order to assess the ability of KLK6 mRNA expression levels to differentiate between molecular BC subtypes. Survival analyses, using DFS as endpoint, were performed at the univariate and multivariate levels. Publicly available BC databases and online survival analysis tools were used to validate our findings. A significant downregulation of KLK6 mRNA expression was observed in BC tissue sections compared to the non-cancerous component (P < 0.001). The expression of KLK6 is positively associated with tumor grade (P = 0.038) and is overexpressed in TNBC and HER2-positive tumors (P < 0.001). Aberrant KLK6 expression predicts the clinical outcome of BC patients in terms of DFS, independently of currently used prognostic markers (HR = 7.11, 95% CI = 1.19-42.45). The differential expression of KLK6 and its association with unfavorable outcome in BC patients was validated via in silico analyses. Although an independent external cohort is necessary to confirm our findings, we proved for the first time that KLK6 can provide independent prognostic information for BC patients.

Psychosocial Adjustment to Illness Scale: Factor structure, reliability, and validity assessment in a sample of Greek breast cancer patients.

The study and measurement of psychosocial adjustment is important for evaluating patients' well-being, and assessing the illness's course, treatment's success, and patients' recovery. In this study, internal consistency reliability and construct validity of the Greek version of the Psychosocial Adjustment to Illness Scale-Self-Report (PAIS-SR) were examined. Demographic and psychosocial data were collected from a sample of 243 women with breast cancer, recruited from September 2011 to December 2012. With some exceptions in specific items, the original conceptually-derived PAIS-SR subscales emerged in a seven-factor solution. Social Environment, Job and Household Duties, and Psychological Distress accounted for more of the total variance than other subscales. PAIS-SR showed good internal consistency reliability, with Cronbach's alpha coefficients >0.62. Correlations of PAIS-SR domains with measures of quality of life and posttraumatic stress symptoms supported the convergent validity of the PAIS-SR and its significance for cancer research. The Greek version of the PAIS-SR has acceptable internal consistency reliability and construct validity, as well as satisfactory convergent validity. Results provide some suggestions for the development of programs to evaluate adjustment status and implement psychosocial interventions among breast cancer survivors.

BCL2L12: a promising molecular prognostic biomarker in breast cancer.

OBJECTIVES: BCL2-like 12 (BCL2L12) is a new member of the BCL2 gene family that was discovered and cloned by members of our group and found to be expressed in the mammary gland. Many genes of the BCL2 family were found to be implicated in breast carcinogenesis and to serve as possible prognostic markers. The aim of the present study was the quantification of BCL2L12 mRNA expression in order to assess its value as a prognostic tissue biomarker in breast cancer (BC). DESIGN AND METHODS: BCL2L12 mRNA levels were determined in a statistically significant sample size of cancerous (N=108) and adjacent non-cancerous (N=71) breast tissues using a highly sensitive quantitative real-time polymerase chain reaction (qRT-PCR) method. Relative quantification analysis was conducted using the comparative C(T) (2(-ΔΔC)(T)) method, whereas the association between BCL2L12 expression and clinopathological data, disease-free survival (DFS) and overall survival (OS) were estimated by statistical analysis. RESULTS: BCL2L12 mRNA expression was decreased in malignant samples compared to the histologically normal counterparts (p=0.012). Significant relationships between BCL2L12 expression and TNM stages (p=0.009), metastatic potential (p=0.012), tumor size (p=0.04) and age (p=0.024) were observed. Moreover, Kaplan-Meier and Cox univariate analyses indicated that BCL2L12 expression is associated with longer DFS, whereas multivariate analysis pointed out the independent favorable prognostic value of BCL2L12. CONCLUSIONS: According to our results, BCL2L12 mRNA expression is a favorable prognostic marker of DFS for BC patients, suggesting its possible application as a novel prognostic indicator of this malignancy.

The expression of the CEACAM19 gene, a novel member of the CEA family, is associated with breast cancer progression.

Breast cancer (BC) continues to affect the lives of millions of women worldwide. Several members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subfamily are involved in tumor progression. Notably, the CEACAM subfamily harbors the already established cancer biomarker CEA, as well as other potential molecular markers. CEACAM19, a recently identified gene belonging to CEACAM subfamily, was discovered and cloned by members of our research group. The present study analyzes, quantitatively, the expression of CEACAM19 and evaluates its clinical relevance in BC. Total RNA was extracted from 143 cancerous and 89 normal adjacent breast tissue specimens. Following reverse transcription, quantitative analysis of CEACAM19 mRNA expression levels was performed via real-time PCR and the comparative Ct (2-∆∆Ct) method. CEACAM19 expression and detailed clinicopathological data were used for extensive biostatistical analyses. CEACAM19 was found to be overexpressed in breast cancer tissue specimens compared to normal tissue counterparts (p=0.013). CEACAM19 mRNA expression status was also associated with clinicopathological features indicative of aggressive behavior and poor prognosis in BC, such as high tumor grade (p=0.031) and high Ki67 proliferative index (p=0.038). A significant negative association was documented between CEACAM19 expression and tumor ER status (p=0.018) as well as patients' menopausal state (p=0.016). Our results suggest that CEACAM19 mRNA expression represents a promising, novel and clinically useful tissue biomarker for breast cancer management.

Identification of novel breast cancer-associated transcripts by UniGene database mining and gene expression analysis in normal and malignant cells.

Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation.

Human kallikrein-related peptidase 12 (KLK12) splice variants expression in breast cancer and their clinical impact.

Kallikrein-related peptidases (KLKs) are a group of 15 serine proteases, hormonally regulated, and localized on chromosome 19q13.4. Alternative splicing is a process that plays significant role in the development, physiology, and different diseases, like cancer. Kallikrein family numbers more than 82 alternative transcripts. Understanding the role that those gene transcripts play in various cancer types, could lead to the discovery of diagnostic markers or drug targets. The present study was designed to analyze the expression profile of the splice variants of kallikrein-related peptidase 12 (KLK12) in breast cancer patients and to evaluate their clinical significance. KLK12 splice variants (KLK12sv3 and KLK12sv1/KLK12sv2) were examined in 69 tissue samples of breast cancer using quantitative real-time PCR as well as semi-quantitative PCR. Relative quantitative expression of KLK12 was statistically associated to clinicopathological parameters. From the splice variants examined, statistical associations with clinicopathological parameters were obtained only from KLK12sv3 variant. KLK12sv3 is more frequently expressed in tumors of lower grade (p = 0.040), early patient TNM stage (p = 0.024), and smaller tumor size (p = 0.023). Positive KLK12sv3 expression is associated with longer patient disease-free survival (DFS) (p = 0.042) and higher progesterone receptor concentration (p = 0.008). KLK12sv1/KLK12sv2 expression is statistically associated with KLK12sv3 expression (p = 0.001). KLK12sv3 can be regarded as a marker of good prognosis in breast cancer.

Neoadjuvant sequential epirubicin and docetaxel followed by surgery-radiotherapy and post-operative docetaxel or gemcitabine/vinorelbine combination based on primary response: a multimodality approach for locally advanced breast cancer.

BACKGROUND: Locally advanced breast cancer (LABC) remains a major clinical issue despite progress achieved in recent years. Herein, we present the mature results of a multimodality treatment program tailoring epirubicin (EPI), docetaxel (DOC) and gemcitabine-vinorelbine (GEV) peri-operatively in LABC. PATIENTS AND METHODS: Stage III, Eastern Cooperative Oncology Group-Performance status ≤2 patients were eligible. A biopsy documentation had to be performed before the start of chemotherapy (CT). Treatment consisted of four EPI (100 mg/m(2), d1q2w) followed by three DOC (100 mg/m(2), d1q3w); surgery 3-4 weeks from CT completion, followed by radiation therapy (RT) and CT according to response; partial or complete (PR/CR):DOC, no change or progressive disease (NC/PD):GEV. Primary endpoints were: (a) response and conversion to operability/conservative surgery and (b) overall survival (OS) and time to recurrence (TTR). RESULTS: Fifty-six women, aged 32-75 (median 52 years), 24 IIIA and 32 IIIB were enrolled; 53 patients completed the entire program. Toxicity was acceptable and no treatment-related death was observed. EFFICACY: clinical response rate (RR) 71.4% (40 patients); clinical complete response rate 33.9% (19 patients). Pathological response rate (RR) 67.8% (38 patients); pathological complete response rate 21.4% (12 patients). 33 (58.9%) and 19 (33.9%) patients, respectively, had radical and conservative operations without increased morbidity. After a median follow-up of 62 months, median OS has not yet been reached, while median TTR was 42 months. OS was longer in patients with clinical (p = 0.004) and pathological response (p = 0.002), RT (p < 0.0001) and post-operative DOC (p = 0.038). TTR was favorably affected by pR (p < 0.0001), RT (p < 0.0004) and post-operative DOC (p = 0.005). Pre-operative CT seemed to be equally active throughout all subgroups according to histology, ER/PR and HER2 status. CONCLUSION: The treatment program of the present study allowed for the completion of an effective therapy at the cost of acceptable toxicity. The results of this study suggest a central role of CT for LABC and the value of eventually dose-dense, EPI- and DOC-based CT in a large proportion of LABC patients, regardless of biological tumor profile. Furthermore, tumor response (cR, pR) is an important surrogate for patients survival and further therapy management.

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy.

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828-836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu (+) tumor cell lines. HER-2/neu(828-836), [HER-2(9(828))], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9(828)) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8(+) T-lymphocytes specifically recognizing HER-2(9(828)) in 8 out of 20 HLA-A*0201(+) HER-2/neu (+) breast cancer patients. Moreover, HER-2(9(828))-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9(828)) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9(828)) as a promising candidate for peptide-based cancer vaccines.

Identification of a novel immunogenic HLA-A*0201-binding epitope of HER-2/neu with potent antitumor properties.

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for MHC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/neu decamer LIAHNQVRQV spanning residues 85-94 (HER-2(10(85))). HER-2(10(85)) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(10(85)) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(10(85))-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the rapid expansion of HLA-A2.1/HER-2(10(85))pentamer+/CD8+ cells (PENT+/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(10(85))-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-gamma intracellular staining and in the high sensitivity CD107alpha degranulation assay. Finally, HER-2(10(85)) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(10(85)) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(10(85)) as a possible target for anticancer immunotherapy.

Increased frequency of CD4+ cells expressing CD161 in cancer patients.

PURPOSE: Although the function of natural killer receptors on T cells infiltrating tumors and their potential effect on antitumor immunity has been investigated, little is known about T cells expressing NKR-P1A (CD161) in cancer patients. In the present study, we examined T cells expressing CD161 in the peripheral blood, the tumor tissue and in malignant effusions of patients with several types of malignancies. EXPERIMENTAL DESIGN: Expression of CD161 in CD4(+) or CD8(+) (lacking CD56) T cells isolated from peripheral blood (n = 61), tumor specimens (n = 8), and malignant effusions (n = 37) of cancer patients was examined using four-color flow cytometry. Proliferative capacity and cytokine production of purified CD4(+)CD161(+)CD56(-) cells were studied after weak or strong stimulation, with or without costimulation, in the presence or absence of interleukin 2. The possible regulatory function of activated CD4(+)CD161(+)CD56(-) cells on T-cell alloresponses was also investigated. RESULTS: CD4(+) cells expressing CD161 were increased in cancer patients, compared with healthy individuals. This increase in the peripheral blood of cancer patients positively correlated with disease stage and was augmented at the tumor site. Phenotypic analysis revealed that CD4(+)CD161(+) cells are memory T cells, with low expression of activation markers. CD4(+)CD161(+) cells play an immunoregulatory role through cytokine production, because upon receiving costimulatory signals via CD28, they exert suppressive activity on autologous peripheral blood mononuclear cell alloresponses. CONCLUSIONS: CD4(+)CD161(+)CD56(-) cells represent a distinct memory T-cell population significantly increased in cancer patients. Depending on the type of signals provided by the tumor microenvironment, CD4(+)CD161(+) cells may regulate the immune response.

Multidisciplinary therapy of locally far-advanced or inflammatory breast cancer with fixed perioperative sequence of epirubicin, vinorelbine, and Fluorouracil chemotherapy, surgery, and radiotherapy: long-term results.

BACKGROUND: Based on phase II data in advanced breast cancer (BC), the fluorouracil, epirubicin, and vinorelbine (FEN) combination was assessed as perioperative chemotherapy, integrated in a multidisciplinary treatment for locally advanced BC. PATIENTS AND METHODS: Patients with newly diagnosed inoperable (stage IIIB or inflammatory) BC. Multimodality treatment protocol consisted of four preoperative courses of fluorouracil (600 mg/m(2) day 1), epirubicin (75 mg/m(2) day 1), and vinorelbine (25 mg/m(2) day1 and day 8), all i.v. every 21 days, followed by radical or conservative surgery according to clinical response and four postoperative identical chemotherapy courses aimed to eradicate micrometastatic disease. Locoregional radiotherapy was offered to all patients after the completion of chemotherapy followed by hormonotherapy according to hormone receptor status. The primary end points of the study were: (a) clinical and pathological response, (b) downstaging and conversion to operable disease, and (c) recurrence-free survival (RFS) and overall survival (OS). RESULTS: Forty-eight women, one stage IIIA, 27 (56.2%) stage IIIB, two stage IIIC (4.1%), and 12 (25%) with inflammatory BC, aged 34-75 years (median, 52), were accrued. Thirty-eight and 34 patients completed the planned pre- and postoperative chemotherapy, respectively. Totals of 175 and 135 cycles were administered pre- and postoperatively, respectively. Toxicity of both phases, mainly hematologic, was in general acceptable without treatment-related death. Venous reactions were a frequent problem. All but three tumors were converted to operable, 31.3% with breast conservation. The clinical response rate (RR) was 77.7% (22.2% complete) and pathological RR was 73.3% (complete, 20% in both primary and axilla). After a median follow-up of 72 months, 62.5% and 16.7% of patients remain relapse free at 3 and 5 years, respectively, while 83% and 58.3% were alive 3 and 5 years, respectively, after the start of chemotherapy. Median RFS and OS have not yet been reached, and are currently 37+ and 62+ months, respectively. CONCLUSION: This fixed number of FEN perioperative courses schedule followed by radiotherapy is safe and highly active in inducing both local and distant control of locally far-advanced BC. This strategy is at least not inferior to other established regimens or strategies for locally far-advanced BC, while the integration of taxanes or new targeted agents may help show its true value for this challenging clinical entity.

Natural CD8+ T-cell responses against MHC class I epitopes of the HER-2/ neu oncoprotein in patients with epithelial tumors.

HER-2/ neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/ neu-positive ((+)) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/ neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2-binding nona-peptide 369-377 (HER-2(9(369))). In the present study, we examined patients with HER-2/ neu(+) breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/ neu-derived and HLA-A2-restricted "cytotoxic" peptides and to a novel one spanning amino acids 777-785 also with HLA-A2-binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon gamma (IFN-gamma) ELISpot assay detecting T cells at the single cell level secreting IFN-gamma. CTLp were defined as peptide-specific precursors per 10(6) peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/ neu-negative ((-)) tumors and healthy individuals. Of the HER-2/neu(+) patients examined, 31% had increased CTLp to HER-2(9(952)), 19% to HER-2(9(665)), 16% to HER-2(9(689)), and 12.5% HER-2(9(435)), whereas only 2 of 32 patients (6%) responded to HER-2(9(777)). The CTLp recognizing HER-2(9(952)) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu(-) patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN-gamma production, preexisting CTL immunity to all five HER-2/ neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9(435)), HER-2(9(952)), HER-2(9(689)), HER-2(9(665)), and HER-2(9(777)) is present in patients with HER-2/ neu(+) tumors of distinct histology, (2) HER-2(9(777)) is a naturally processed peptide expressed on the surface of HER-2/ neu(+) tumors, as are the other four peptides, and (3) HER-2/ neu(+) prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/ neu-based immunotherapy.

Quantitative fluorescence cytometric measurement of estrogen and progesterone receptors: correlation with the hormone binding assay.

We describe, here, a rapid flow cytometry technique for the detection and quantification of estrogen (ER) and progesterone (PgR) receptors in several human cell lines and in clinical samples obtained from breast cancer tumors. ER and PgR quantitation can be very useful in patients with breast cancer as their role in diagnosis and prognosis is well established. However ligand binding assays and immunohistochemical assays are difficult to measure heterogeneity in individual cells. On the other hand, flow cytometry is a convenient tool for quantification in individual cells. Flow cytometric results with breast cancer cell lines and clinical samples were compared to those obtained by quantitative biochemical ER and PgR performed by the standard dextran-coated charcoal biochemical assay. The latter assay is affected by the level of endogenous steroids. This is also the case in the routine measurement of ER/PgR in patient's tumor cells whereby estradiol molecules in patient's serum produced negative or low values in the biochemical assay. The mAbs used in our flow cytometric method bind to their specific ER or PgR independently of whether they are preoccupied by their ligands and they produce reliable results. With the use of beads calibrated in MESF (Molecules of Equivalent Soluble Fluorochrome) units, the ER and PgR can be measured on a per cell basis. The flow cytometric method showed a strong correlation with biochemical receptor assessments of either ER alpha (ER alphaDCC, r = 0.918, p = 0.073) or PgR (PgRDCC, r = 0.75, p = 0.001). This study demonstrates that ER alpha and PgR can be detected by flow cytometry on a per cell basis in intact cells, and can be quantitated reliably in terms of MESF without the limitations of competition with serum's estradiol molecules.