Expression of EGFR family and steroid hormone receptors in ductal carcinoma in situ of the breast.

The expression of EGFR family and steroid hormone receptors was examined in a series of 40 cases of pure ductal carcinoma in situ (DCIS) of the breast by immunohistochemical staining of paraffin-embedded sections. Hematoxylin and eosin-stained sections were used to classify the tumors according to the published criteria by Holland et al. (Holland R, Peterse JL, Millis RR, et al. Semin Diagn Pathol. 1994;1 1:167-180). Of the tumors 48% were immunoreactive for EGFR, 63% for c-erbB-2, 78% for c-erbB-3, 95% for c-erbB-4, 88% for estrogen receptor (ER) and 80% for progesterone receptor (PR). Statistically significant association between histological grade (differentiation) and c-erbB-2 protein expression was seen (p <.001). In addition, expression of c-erbB-4 protein was associated with c-erbB-2 (p=.004), c-erbB-3 (p=.058), ER (p=.002) and PR (p=.004). It is concluded that c-erbB-2 expression in DCIS is associated with high-grade pathological features, and a higher c-erbB-2 expression is seen in DCIS than in invasive breast carcinomas. A possible association between extensive expression of c-erbB-4 and steroid hormone receptors in proliferative and premalignant breast epithelial cells and the c-erbB-2 expression in DCIS and invasive breast carcinomas is discussed.     

Ovarian steroids and the human breast: regulation of stem cells and cell proliferation

Ovarian steroidal control of mammary gland proliferation and differentiation is not well defined in the human. We therefore developed the athymic nude mouse model in which intact normal human breast tissue is xenografted subcutaneously and treated with human physiological serum levels of oestrogen (E) and/or progesterone (P). We showed that: (i) E, and not P, is the major steroid hormone inducing proliferation of epithelial cells in the adult non-pregnant, non-lactating breast; (ii) E induces progesterone receptor (PR) expression; and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E was required to induce proliferation. Using double label immuno-fluorescence, we demonstrated that cells expressing the oestrogen receptor- (ER) invariably contained the PR but that steroid receptor expression and cell proliferation (Ki67 antigen) were dissociated. Recently, we have demonstrated that some ER/PR-positive epithelial cells are quiescent breast stem cells suggesting that they act as “steroid hormone sensors” that secrete paracrine factors to regulate the proliferative activity of adjacent ER/PR-negative epithelial cells. The dissociation between steroid receptor expression and cell proliferation in normal epithelium was lost at an early stage in ER/PR-positive breast tumour formation perhaps indicating that they arise from deregulation of the normally quiescent breast stem cells.     

Expression of steroid hormone receptors, their regulated proteins, and bcl-2 protein in myofibroblastoma of the breast

AIMS: To investigate the possible role of steroid hormones in the pathogenesis of myofibroblastoma (MFB) of the breast, we analysed the immunohistochemical expression of oestrogen, progesterone, androgen receptors, their regulated proteins and bcl-2 protein in a series of this rare tumour. METHODS AND RESULTS: Paraffin-embedded sections from seven cases of MFB of the breast (five male; two female) were immunohistochemically tested for the expression of oestrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), oestrogen-regulated pS2 protein, androgen-regulated prostate-specific antigen (PSA) and bcl-2 protein. Rare cases of benign spindle cell tumours or tumour-like lesions of the breast (primitive fibromatosis, inflammatory pseudotumour, muscular hamartoma) which enter into the differential diagnosis with MFB, were also investigated and compared with MFB. All cases of MFB showed a diffuse (70-90% of neoplastic cells) and strong nuclear labelling with ER and PR, whereas AR was expressed only in three cases (two men and one woman) in about 60-70% of cells. Conversely, no immunostaining was detected for the pS2 protein and PSA. bcl-2 protein immunoreactivity was found in all cases of MFB, although with a variable degree of expression. No expression for steroid hormone receptors, their regulated-proteins and bcl-2 protein was observed in the rare benign spindle cell lesions of the breast included in this study. CONCLUSION: The in-situ detection of ER, PR and AR suggests that steroid hormones and their receptors are implicated in the pathogenesis of breast MFB. The consistent demonstration of bcl-2 protein, associated with a positive ER/PR status, provides evidence that bcl-2 may be an oestrogen-regulated protein also in MFB and that probably plays a role in the tumorigenesis. Finally, we postulate that the ER/PR and bcl-2 positive immunoprofile of MFB of the breast, in contrast to the negative profile of other rare primitive benign spindle cell lesions of the breast herein studied, might be exploited as an ancillary diagnostic aid in differential diagnosis of doubtful cases.     

The value of S-phase and DNA ploidy analysis as prognostic markers for node-negative breast cancer in the Australian setting.

This study aimed to determine the prognostic significance of DNA ploidy and S-phase fraction (SPF) measurements in our laboratory for patients with node-negative breast cancer. Frozen tumors from axillary node-negative breast cancer patients (n = 50) treated at Westmead Hospital, NSW, between 1988 and 1991 were analysed by flow cytometry. The median duration of follow-up for all patients was 8.4 years. Forty-six specimens provided evaluable DNA histograms with 43% (n = 20) diploid and 56% (n = 26) aneuploid tumors identified. Comparisons of DNA ploidy status and SPF were made with traditional prognostic variables, which included age, menopausal status, tumor size, histologic grade and hormone receptor status. Our results showed that there was no significant difference in disease-free or overall survival between patients with diploid and aneuploid tumors. Histologic grade 3 tumors were more likely to be aneuploid and had higher SPF than grade 1 or 2 tumors. Patients with grade 3 tumors and a high SPF were four times more likely to relapse than the rest of the population. These results indicate that DNA flow cytometric analysis in our laboratory provides additional prognostic data that could be utilised alongside traditional clinical and histopathologic indicators for predicting outcome for patients.     

Stage-related plasma values of transforming growth factor-beta1 are steroid receptors dependent

Transforming growth factor-beta1 (TGF-beta1) is a biomarker associated with the progression of breast cancer, characteristic by switching activity from tumor suppressor in early stages to tumor promoter at advanced disease. However, what cause this switch is still not clear. On the other hand, the relationship between steroid receptors (estrogen ER and progesterone PR) as the major discriminators of breast cancer phenotype and this paradoxical biomarker is not fully determined. In this pilot study on 52 breast cancer patients, quantitative plasma values of TGF-beta1 were determined by quantitative ELISA and steroid receptor content was measured in cytosol fraction of breast cancer tissue using dextran-coated (DCC) method. We tried to investigate the possibility that steroid receptor status of patients at different stages of disease could be the trigger that somehow causes variation of TGF-beta1 plasma levels. In nonmetastatic breast cancer patients, there was no statistically significant increase in the plasma levels of TGF-beta1, when patients are stratified by steroid receptor status (ER− vs. ER+, PR− vs. PR+). We found for the first time, that indeed in metastatic breast cancer statistically significant elevated levels of TGF-beta1 are related to negative steroid receptor status and moreover that, there is correlation between quantitative values of these parameters in this stage. This finding deserves further investigation because it could provide a new insight into more aggressive nature of steroid receptor negative tumors.     

Breast cancer outcomes by steroid hormone receptor status assessed visually and by computer image analysis

Mohammed Z M A, Edwards J, Orange C, Mallon E, Doughty J C, McMillan D C & Going J J
(2012) Histopathology  61, 283–292 Breast cancer outcomes by steroid hormone receptor status assessed visually and by computer image analysis Aims: To compare the assessment of steroid hormone receptor immunohistochemistry by eye and by computer‐aided image analysis, and to examine their relationships with survival in breast cancer. Methods and results: Allred scores and weighted histoscores for oestrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry were determined by eye (visual histoscore) for 459 primary invasive ductal breast carcinomas in triplicate tissue microarrays. Histoscores were also determined by computerized image analysis (automated histoscore). ER and PR status determined by these different methods were compared with each other and in their ability to predict survival over at least 142 months of follow‐up. Allred and visual histoscore were highly associated for ER and PR (both P < 0.001). By univariate analysis, Allred score and visual histoscore for ER and PR were highly associated with recurrence‐free and cancer‐specific survival (both P < 0.001) in patients with invasive ductal breast cancer overall, in those who received tamoxifen, and in those with recurrence on tamoxifen. Visual and automated histoscores were in excellent agreement for ER and PR (both P < 0.001), and were equally effective in predicting recurrence and survival for patients with invasive breast cancer who received tamoxifen. Conclusions: Automated histoscore appears to be a valid alternative to visual histoscore or Allred score for determining ER and PR status.     

Correlation of steroid hormone receptors (SR) with clinical and pathological features of human breast cancer

Paraffin-embedded materials from 104 patients with breast cancer were assayed for the expression of estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) with avidin-biotin complex method. A significant positive correlation was found between SR status and cell differentiation or lymph node status. Tumors with elastosis tend to contain ER, PR and AR more frequently. Patients with positive ER, PR or AR tumors were 1.2 to 2.6 times more likely to survive than those with negative ones. The five-year survival rate increased with the increase of number and amount of positive SR. These results indicate that ER, PR and AR detected by ABC method may be used as biomarkers for tumor differentiation and have prognostic values in breast cancer.     

Comparison of monoclonal immunocytochemical and immunoenzymatic methods for steroid receptor evaluation in breast cancer

The production of monoclonal antibodies against estrogen receptor (ER) and progesterone receptor (PR) has permitted the development of the enzyme immunoassay (EIA) and immunocytochemical assay (ICA) for steroid receptor determination. The results obtained with these two techniques, using the same monoclonal antibodies, were compared in a large series of breast carcinomas (187 for ER and 100 for PR). The correlation between these methods was significant for ER (rs = 0.54) and PR (rs = 0.55) (P less than 0.001) but was lost when the receptor concentrations determined by EIA were less than or equal to 15 and less than or equal to 30 fmol/mg protein for ER and PR, respectively. When these values are considered as cutoffs, the concordance between the two methods was 84.5% for ER and 73% for PR. An analysis of discordant results revealed that low epithelial cellularity generally was present in ICA-positive, EIA-negative specimens, whereas only focal positivity with ICA, or positivity of only normal peripheral mammary ducts and lobules, frequently was found in ICA-negative, EIA-positive tumors. In conclusion, there is good correlation between the results obtained by EIA and ICA methods for detection of ER and PR. The authors suggest that biochemical and histochemical methods for steroid receptors could be considered complementary and used together for the analysis of breast cancer.     

Quantitative DNA analysis and proliferation in breast carcinomas. A comparison between image analysis and flow cytometry.

The DNA content and proliferation in 100 invasive breast carcinomas were evaluated by computerized image analysis (IA) and flow cytometry (FCM). For DNA content, image analysis of Feulgen-stained slides of fresh tumor imprints were compared with flow cytometry of propidium iodide-stained disaggregated fresh tumor tissue. The DNA indices obtained by the two methods showed close correlation by linear regression analysis (r = 0.89, p less than .001). There were 44 (44%) diploid and 56 (56%) aneuploid tumors. There was agreement between the two methods in detection of aneuploidy in 81% of tumors. Image analysis required smaller tissue samples, permitted direct visualization and selection of tumor cells, and was more sensitive in detecting tetraploid and highly aneuploid cell populations. In contrast, flow cytometry histograms provided better resolution, and were more effective in detecting multiploid tumors and near-diploid aneuploid tumors. Aneuploidy was significantly related to various adverse prognostic parameters, namely, negative estrogen receptor, high mitotic rate, high histologic and nuclear grades. Proliferation was evaluated by measuring the FCM S phase fraction (SPF), and by image analysis quantitation of immunohistochemical staining using Ki-67 monoclonal antibody. SPF and Ki-67 count showed modest correlation (r = 0.42). Both SPF and Ki-67 count were significantly related to the mitotic rate, histologic and nuclear grades. Our results indicate that the two methods provide comparable results, but offer individual advantages and are complementary techniques in analyzing DNA ploidy and proliferation in breast carcinomas.     

Differential expression of estrogen, progesterone, and epidermal growth factor receptors in normal, benign, and malignant human breast tissues using dual staining immunohistochemistry.

Distribution of estrogen (ER), progesterone (PR) receptors, and epidermal growth factor (EGF) receptors was assayed by dual staining immunohistochemistry on 28 selected cytosolic ER-positive breast carcinomas and 22 nonmalignant breast tissues. ER-positive tumor cells were detected in 26 (93%) and EGF receptor positive tumor cells were detected in 7 (25%) carcinomas. In five tumors both ER and EGF receptors were detected but localized in distinct tumor cells. Only in one case of ductal carcinoma in situ co-expression was observed in a subset of tumor cells. In contrast, simultaneous expression of ER/PR and EGF receptors was observed in non-neoplastic ductal remnants in the majority of the carcinomas and the fibroadenomas. In addition, double-positive cells were occasionally detected in luminal epithelial cells of normal breast tissue and mastopathies. This study shows that ER/PR and EGF receptors in breast tumor cells are inversely related at the single cell level. However, demonstration of ER/PR and EGF receptors in individual normal luminal cells shows that expression is not mutually exclusive.     

The flow-cytometric analysis of DNA content and S-phase fraction (SPF) of human breast cancer

The DNA ploidy of breast cancer tissue from paraffin blocks was measured by flow cytometry in 122 patients. In this material there was a difference in lymph node involvement and in the presence of distant metastases between diploid and aneuploid tumors. Diploid tumors were smaller than aneuploid tumors. Aneuploid tumors were more common in postmenopausal than in premenopausal women. Near tetraploid tumors were found in older patients rather than tumors with other ploidy patterns. The ploidy pattern was not associated with survival during the mean follow-up of 4.1 years. We specially studied the S-phase fraction (SPF) which was distinctly higher in aneuploid tumors than in diploid tumors. Also near tetraploid, hypertetraploid and multiploid tumors showed higher SPF than diploid tumors. The median of SPF in our material was 8.5%. Positive axillary lymph nodes were found in 32% of the patients who had tumors with an SPF below the median and in 46% of those with tumors above the median (SPF greater than 8.5%). The difference, however, was not statistically significant. In our material the SPF of the tumor did not show a significant association with survival. However, longer follow up time is needed for firm conclusions on the predictive value of cell DNA on survival.     

Endometrial carcinoma: association of steroid hormone receptor expression with low angiogenesis and bcl-2 expression

In endometrial tissues, malignant change may be accompanied by a loss of hormone dependence which is, usually, reflected in a parallel loss of oestrogen and progesterone receptors (ER and PR). In this study, the steroid receptor status of 164 endometrial carcinomas was related to intratumoural angiogenesis and the apoptotic proteins bcl-2 and p53. Relationships to conventional histopathological features and patient survival were also sought. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues. The mean follow-up was 55 months (range 19-167 months). Specific nuclear staining for ER and PR was detected in 35% and 32% of endometrial carcinomas, respectively, and was very commonly co-expressed (P<0.0001). The failure of demonstrating a steroid receptor complement in endometrial neoplasms was, in general terms, an adverse prognostic sign. Thus, ER or PR loss was significantly associated with non-endometrioid carcinomas (ER P=0.01; PR P=0.004) and with deep myometrial invasion (ER P<0.0002), high intratumoural angiogenesis (PR P<0.01) and the absence of bcl-2 expression (PR P<0.005). There was a trend for patients with ER or simultaneous ER/PR expression to have an improved survival, but this association did not reach the level of statistical significance. In multivariate analysis (all stages), tumour cell type (endometrioid versus non-endometrioid carcinomas) and stage of disease were the only variables associated with prognosis (P=0.01 and P<0.0001, respectively), with tumour cell type retaining its independent prognostic value and within stage-I endometrial carcinomas (P=0.02). It is suggested that the loss of steroid hormone receptors in endometrial carcinomas is associated with a more aggressive phenotype and the switching-on of angiogenic pathways.     

Sex steroid receptors in lymphoid cells of human endometrium

Sex steroid hormone action on target tissues is mediated through binding of estrogen and progesterone to specific intranuclear proteins, the estrogen and progesterone receptors (ER and PR). Therefore, in the present report the authors investigated for the presence of ER and PR in lymphoid cells of endometrial stroma that may serve as potential targets for estrogen- and progesterone-mediated effects in endometrium. The presence of ER was shown in nine proliferative and ten secretory endometria and the presence of PR in three secretory and one proliferative endometria. ER and PR were localized by monoclonal antibodies, to the nuclei of cells, with the use of, respectively, peroxidase-antiperoxidase (PAP) and avidin-biotin-peroxidase complex (ABC) methods. Lymphoid cells were then delineated by decoration of their plasma membranes with the use of monoclonal antibodies to HLA-DR, leukocyte common antigen (LCA), Leu-4 (CD3), and IL-2 receptor molecules with the use of an ABC staining procedure. A group of cells in the lymphoid aggregates in endometrial stroma showing membranous staining for HLA-DR, LCA, and Leu-4 molecules had nuclear ER. IL-2 receptor-positive cells were rare in endometrium, and no PR-positive cells were found in lymphoid aggregates. Furthermore, HLA-DR and ER were expressed in the glandular and surface epithelium in the proliferative phase and in occasional glands in the basalis in the late secretory phase. The presence of an ER-positive lymphoid cell population in endometrial lymphoid aggregates suggests that these cells may serve as target cells for estrogen.     

Expression of EGFR Family and Steroid Hormone Receptors in Ductal Carcinoma in situ of the Breast

The expression of EGFR family and steroid hormone receptors was examined in a series of 40 cases of pure ductal carcinoma in situ (DCIS) of the breast by immunohistochemical staining of paraffin-embedded sections. Hematoxylin and eosin-stained sections were used to classify the tumors according to the published criteria by Holland et al. (Holland R, Peterse JL, Millis RR, et al. Semin Diagn Pathol. 1994;11:167-180). Of the tumors 48% were immunoreactive for EGFR, 63% for c-erbB-2, 78% for c-erbB-3, 95% for c-erbB-4, 88%for estrogen receptor (ER) and 80% for progesterone receptor (PR). Statistically significant association between histological grade (differentiation) and c-erbB-2 protein expression was seen ( p <.001). In addition, expression of c-erbB-4 protein was associated with c-erbB-2 ( p =.004), c-erbB-3 ( p =.058), ER ( p =.002) and PR ( p =.004). It is concluded that c-erbB-2 expression in DCIS is associated with high-grade pathological features, and a higher c-erbB-2 expression is seen in DCIS than in invasive breast carcinomas. A possible association between extensive expression of c-erbB-4 and steroid hormone receptors in proliferative and premalignant breast epithelial cells and the c-erbB-2 expression in DCIS and invasive breast carcinomas is discussed.     

Immunohistochemical detection of the estrogen receptor‐α and progesterone receptor in the canine pregnant uterus and placental labyrinth

The presence of hormone receptors is as important as the amount of hormone to predict hormone action. Therefore, the presence of estrogen receptors of the alpha subtype (ER‐α) and progesterone receptors (PR) was evaluated in six pregnant uteri including the placenta and in three postpartum uteri of dogs. This preliminary study is part of our immunohistochemical research project on steroid hormone receptor distribution in the canine female genital tract. Specific staining for ER‐α or PR was found only in cell nuclei. Staining for ER‐α was rare in the various cell types of pregnant and postpartum uteri. Staining for PR was absent or weak in epithelial cells. Moderate staining for PR was observed in endometrial stromal cells and myometrial smooth muscle cells, two cell types playing an important role in the maintenance of pregnancy. Stromal cells stained more frequently positive for ER‐α and PR than epithelial cells, indicating that both hormones may act on epithelial cells indirectly via stromal cells. In the placental labyrinth, fetal cells showed no evidence of ER‐α or PR. In contrast, both receptors were present in maternal mesenchymal cells that were located around the basement membrane of the maternal blood vessels. These cells showed signs of decidualization. No difference in PR distribution was seen between pregnant and postpartum uterine tissue, suggesting that during parturition the decrease in serum progesterone levels and the concomitant increase in the estrogen/progesterone ratio are probably more important than the decline in receptor availability. Anat Rec 260:42–50, 2000. © 2000 Wiley‐Liss, Inc.     

Androgen receptor expression in archival human breast tumors

Hormone receptors play a major role in growth and hormonal therapy of breast and prostate tumors. Quantitative results from the ligand binding assays cannot determine heterogeneity in receptor expression nor can they discriminate between expression of the stromal and the tumor cells. Availability of antibodies to hormone receptors has led to the development of immunohistochemistry as a standard method for monitoring of hormone receptor expression under a microscope. However, this method is based on examination of a small number of cells. Laser flow cytometry has been extensively used for monitoring of receptor expression in human liquid tumors. As most of the hormone receptor expression is nuclear, we have developed methods for flow cytometric analysis of receptor expression in nuclei isolated from enzyme treated paraffin sections. The present report based on gated analysis of androgen receptor expression in nuclei isolated from archival formalin fixed/paraffin embedded breast tumors shows that receptor expression in aneuploid sub-populations is greater than that of the diploid cells.     

Estrogen receptor-negative,progesterone receptor-positive breast carcinoma: poor clinical outcome

OBJECTIVE: The biological significance of breast carcinomas negative (-) for estrogen receptor (ER), but positive (+) for progesterone receptor (PR) is unclear. It has been proposed that these tumors contain ER whose presence is masked in binding assays by endogenous estrogen. We analyzed the clinical outcome of 17 patients with ER-PR+ tumors. METHODS: The disease-free and overall survival of a series of 300 women with invasive breast carcinoma was followed for 7 to 79 (median 41) months. RESULTS: The recurrence rate was significantly greater in ER-PR+ tumors (8/17 [47%]) than in ER+PR+ tumors (27/177 [15%]), and it was similar to the high recurrence rate of ER-PR- tumors (21/57 [37%]). The cancer-related death rate was 3 1/2 times higher in the ER-PR+ group than in the ER+PR+ group. A significant association between ER-PR+ tumors and tumor recurrence or cancer-related death persisted even after correction for other variables associated with poor outcome (eg, tumor size and lymph node involvement). CONCLUSIONS: Estrogen receptor-negative, progesterone receptor-positive breast carcinomas are biologically different from ER+PR+ tumors and have a poor clinical outcome.     

A comparison of human breast cancer cell kinetics measured by flow cytometry and thymidine labeling

Flow cytometric determination of tumor ploidy and S-phase fraction following collagenase dissociation and thymidine labeling was performed on 75 consecutive breast cancers. Estrogen and progesterone receptor levels and routine histologic examination also were obtained on each tumor. Cell viability following collagenase dissociation varied from 13 to 95% with a mean of 71%. Thirty-six tumors were diploid, four tetraploid, and four hypertetraploid, and the remainder had DNA indices between 1.1 and 1.9. There was no significant correlation between tumor ploidy and tumor size or estrogen receptor positivity or negativity. The percentage of cells in S-phase varied from 1.2 to 20.0% with a mean of 6.0% utilizing a rectilinear model for histogram analysis that integrated a 10-contiguous channel sample containing the lowest number of cells in S-phase (S-pFL). The mean S-pFL of diploid carcinomas (3.43%) was significantly lower than that of hyperdiploid carcinomas (8.38%). There was good correlation between S-phase fraction determined by thymidine-labeling index (TLI) and S-pFL (r = 0.772, p = 0.0001). S-pFL predicted whether a tumor would be above or below median TLI with an accuracy of 90.5%. Estrogen receptor-negative cancers tended to have higher TLIs and S-pFLs than estrogen receptor-positive cancers; however, there was no correlation between progesterone receptor positivity or negativity and TLI and S-pFL.     

Comparative assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry.

Although tumor DNA content and proliferation are usually determined by flow cytometry (FCM), quantitative microscopic image analysis is a viable alternative technique that also provides important histologic correlations. To compare these methods, we measured DNA content and proliferation in 54 consecutive breast cancers and 15 benign breast lesions by FCM and IA. DNA content determination was concordant in 49 of 54 cancers measured by FCM and IA. Four of the discordant cases were aneuploid by IA and diploid by FCM. There was good correlation between the DNA index (DI) measured by FCM and IA (r = 0.89, P less than 0.0001). Proliferation was assessed by IA quantitation of Ki-67 and PCNA/Cyclin antibody staining, as well as by flow cytometric S-phase fraction (SPF). Ki-67 positivity was greater in breast cancer than in benign controls (21.6% +/- 13.1% vs. 7.9% +/- 5.6% [P less than 0.0001]), as was PCNA/Cyclin positivity (10.2 +/- 6.7% vs. 2.7 +/- 2.5% [P less than 0.0001]). S-phase fraction measured by FCM was 7.9% +/- 5.7% for carcinomas and 3.17% +/- 2.1% for benign controls (P less than 0.003). Ki-67 and Cyclin staining, as well as SPF, were significantly increased in aneuploid compared to diploid tumors, and increased staining was associated with worsening nuclear grade. There were significant correlations between SPF and Ki-67 staining (r = 0.48, P less than 0.0001) and SPF and Cyclin staining (r = 0.48, P less than 0.0001). We conclude that FCM and IA provide comparable measurements of DNA content, although occasional discrepancies occur. Image analysis provides a valuable alternative method for assessing tumor cell proliferation and may offer certain advantages over FCM.     

Prognostic implications of HER-2 status in steroid receptor-positive, lymph node-negative breast carcinoma

Patients with lymph node-negative breast carcinoma (LNNBC) and positive hormone receptor (HR) status during estrogen-based endocrine therapy have different prognoses. The contribution of HER-2 (human epidermal growth factor receptor-2) has not been extensively evaluated. We stained 230 LNNBCs for estrogen and progesterone receptors (ER and PR) and HER-2. HER-2 gene status was studied in 150 randomly selected tumors by chromogenic in situ hybridization and cases with discordant or nondefinitive results by fluorescence in situ hybridization. Patients with ER+ and/or PR+ tumors were treated with tamoxifen. We found positive expression of ER, PR, and HER-2 in 73.7%, 67%, and 27.8%, respectively, and HER-2 amplification in 18.0%. Poorer outcome was seen for patients with ER+ and/or PR+/HER-2 overexpressing tumors and as a trend for patients with HER-2 amplification. ER/PR and HER-2 expression showed an independent prognostic value. In LNNBCs with positive HR status, HER-2 overexpression and/or amplification confer an aggressive tumor phenotype, and this might be related to tamoxifen unresponsiveness.