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Medullary breast cancer (MBC) is a rare but enigmatic pathologic type of breast cancer. Despite features of aggressiveness, MBC is associated with a favorable prognosis. Morphologic diagnosis remains difficult in many cases. Very little is known about the molecular alterations involved in MBC. Notably, it is not clear whether MBC and ductal breast cancer (DBC) represent molecularly distinct entities and what genes/proteins might account for their differences. Using whole-genome oligonucleotide microarrays, we compared gene expression profiles of 22 MBCs and 44 grade III DBCs. We show that MBCs are less heterogeneous than DBCs. Whereas different molecular subtypes (luminal A, luminal B, basal, ERBB2-overexpressing, and normal-like) exist in DBCs, 95% MBCs display a basal profile, similar to that of basal DBCs. Supervised analysis identified gene expression signatures that discriminated MBCs from DBCs. Discriminator genes are associated with various cellular processes related to MBC features, in particular immune reaction and apoptosis. As compared with MBCs, basal DBCs overexpress genes involved in smooth muscle cell differentiation, suggesting that MBCs are a distinct subgroup of basal breast cancer with limited myoepithelial differentiation. Finally, MBCs overexpress a series of genes located on the 12p13 and 6p21 chromosomal regions known to contain pluripotency genes. Our results contribute to a better understanding of MBC and of mammary oncogenesis in general. (Cancer Res 2006; (66)9: 4634-44)

Supplementary Figure 1 from Gene Expression Profiling Shows Medullary Breast Cancer Is a Subgroup of Basal Breast Cancers

Supplementary Table 2 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Supplementary Table 4 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Supplementary Figure 2 from Gene Expression Profiling Shows Medullary Breast Cancer Is a Subgroup of Basal Breast Cancers

Supplementary Figure 1 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Supplementary Table 5 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Supplementary Table 3 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Supplementary Table 6 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Supplementary Tables 1-4 from Gene Expression Profiling Shows Medullary Breast Cancer Is a Subgroup of Basal Breast Cancers

Breast cancer is a heterogeneous disease made of various molecular subtypes with different prognosis. However, evolution remains difficult to predict within some subtypes, such as luminal A, and treatment is not as adapted as it should be. Refinement of prognostic classification and identification of new therapeutic targets are needed. Using oligonucleotide microarrays, we profiled 227 breast cancers. We focused our analysis on two major breast cancer subtypes with opposite prognosis, luminal A (n = 80) and basal (n = 58), and on genes encoding protein kinases. Whole-kinome expression separated luminal A and basal tumors. The expression (measured by a kinase score) of 16 genes encoding serine/threonine kinases involved in mitosis distinguished two subgroups of luminal A tumors: Aa, of good prognosis and Ab, of poor prognosis. This classification and its prognostic effect were validated in 276 luminal A cases from three independent series profiled across different microarray platforms. The classification outperformed the current prognostic factors in univariate and multivariate analyses in both training and validation sets. The luminal Ab subgroup, characterized by high mitotic activity compared with luminal Aa tumors, displayed clinical characteristics and a kinase score intermediate between the luminal Aa subgroup and the luminal B subtype, suggesting a continuum in luminal tumors. Some of the mitotic kinases of the signature represent therapeutic targets under investigation. The identification of luminal A cases of poor prognosis should help select appropriate treatment, whereas the identification of a relevant kinase set provides potential targets. [Cancer Res 2008;68(3):767–76]

Supplementary Section and Figure Legends from Gene Expression Profiling Shows Medullary Breast Cancer Is a Subgroup of Basal Breast Cancers

Background Immunohistochemical (IHC) signatures predictive of prognosis can be a cost effective alternative to genomic signature suitable in clinical practice for patients’ management in early stages of breast carcinoma (BrCa) in clinical. IHC procedures are very easy to handle in pathology laboratories, since require very few amount of formalin fixed tissue samples since assessed on consecutive 4 μm thick sections of paraffin embedded tumors fragments validated by pathologists’ expertise. We recently reported (C Charpin et al Int J Cancer 2009) that standardized IHC assays using high throughput densitometry on digitized microscopic images allow to predict an adverse outcome of patients with BrCa, provided that identified the IHC predictive signatures can be validated in other patients’ cohorts. In this regard we have repeated our previously reported IHC procedures on 418 series of BrCa (Marseille University) on a second series including 303 BrCa diagnosed at Yale University. Material and Methods: TMA(s) of two cohorts of patients with BrCa (418/Marseille University and 303 Yale University) were respectively investigated for immunohistochemical expression of 15 markers, including (i) those of the best signature predictive of patients outcome in N- patients in our previous studies and (ii) new markers reported in the literature as EMT cells and Stem cells. Monoclonal antibodies against HIFa, PI3, pAKT, pmTOR, moesin, P21, 4EBP-1, P27, Ker 5.6, pMAPKAPK-2, SHARP2, Claudin, ALDH, AF6, CD24, IHC was screened using standardized (Ventana Benchmark XT) on consecutive 4 μm thick sections of TMAs from both series, Quantitative measurements of immunoprecipitates were automatically assessed with TRIBVN device, and correlated to 8 year patients’ outcome. Logistic regression determined the best combination of markers to predict patients’ prognosis and results of the two cohorts were compared. Results: In node negative BrCa the best predictive combination of markers signature was HIF-1a, PI3K, Claudin, AF6, pAKT independently of ER PR and HER-2 status. When results of logistic regression with these markers was compared very similar results were obtained with 92.34 % (333/418) of well classified patients in the first set (Marseille University series) and 89.8 % (158/176) in the second set from Yale University. Discussion: The results suggest that the new optimal IHC signature predictive of prognosis identified in BrCa including HIF-1a, PI3K, Claudin, AF6 and pAKT is validated by this study with very similar results in two different cohorts of patients. This validation also suggests (1) that IHC signatures investigated in individual patients can be considered as suitable in clinical practice offering a cost effective convenient mean to predict patients’ outcome at diagnostic time (2) and to select those patients with poor prognosis, particularly in N- BrCa subset, requiring more aggressive adjuvant therapies, and to avoid useless expensive therapies and their side effects in N- patients with favourable prognosis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-12.

CK2α is a signalling molecule that participates in major events in solid tumour progression. The aim of this study was to evaluate the prognostic significance of the immunohistochemical expression of CK2α in breast carcinomas.Quantitative measurements of immunohistochemical expression of 33 biomarkers using high-throughput densitometry, assessed on digitised microscopic tissue micro-array images were correlated with clinical outcome in 1000 breast carcinomas using univariate and multivariate analyses.In univariate analysis, CK2α was a significant prognostic indicator (p0.001). Moreover, a multivariable model allowed the selection of the best combination of the 33 biomarkers to predict patients' outcome through logistic regression. A nine-marker signature highly predictive of metastatic risk, associating SHARP-2, STAT1, eIF4E, pmapKAPk-2, pAKT, caveolin, VEGF, FGF-1 and CK2α permitted to classify well 82.32% of patients (specificity 81.59%, sensitivity 92.55%, area under ROC curve 0.939). Importantly, in a node negative subset of patients an even more (86%) clinically relevant association of eleven markers was found predictive of poor outcome.A strong quantitative CK2α immunohistochemical expression in breast carcinomas is individually a significant indicator of poor prognosis. Moreover, an immunohistochemical signature of 11 markers including CK2α accurately (86%) well classifies node negative patients in good and poor outcome subsets. Our results suggest that CK2α evaluation together with key downstream CK2 targets might be a useful tool to identify patients at high risk of distant metastases and that CK2 can be considered as a relevant target for potential specific therapy.

Myoepithelial carcinoma (MEC) is a rare type of breast cancer composed purely of myoepithelial cells. Most often it presents with a spindle cell morphology that can mimic several benign and malignant lesions and may be misdiagnosed by the pathologist. We report 15 cases of MEC, which were sent to our consultation practice: Five of them were initially diagnosed as benign. The patients, all female, ranged from 45 to 86 years in age (mean 69.5) and-with one exception-presented with a breast mass. The tumor size measured between 1 and 4.8 cm (mean 2.6 cm). Microscopically, the tumors had infiltrative growth pattern most frequently with thin anastomosing cords of tumor cells associated with an intimately admixed reactive spindle cell stroma. The neoplastic myoepithelial cells were emanating from the myoepithelial cell layer of entrapped ductules in every case. The nuclei showed mild to moderate pleomorphism, and the mitotic activity ranged from 0 to 9/10 high power field. Immunohistochemical stains for p63, CD10, CK903, and CK5/6 reacted strongly and diffusely with the tumor cells, and mainly the reactive stroma had weak positivity for calponin, S-100, and smooth muscle actin. Estrogen receptor, progesteron receptor, and Her2 immunostains were negative, but strong epidermal growth factor receptor expression was observed. Follow-up was available for seven patients: All of them were alive at last contact; one patient had local recurrence, and one developed pulmonary metastases. MEC is a potentially aggressive malignant neoplasm sharing many features with metaplastic carcinomas. Morphologically, it is often difficult to distinguish it from benign spindle cell proliferations.

Background: c Kit (CD117) expression in tissues has been reported as a relevant target for specific therapy in some human malignancies, but has been poorly documented in breast carcinomas Methods: The prognostic significance of c Kit in a series of 924 breast carcinomas (mean follow-up, 79 months) was investigated using standardised high-throughput quantitative densitometry of immunohistochemical precipitates in tissue microarrays. Results: c Kit was expressed in 14.7% breast carcinomas (and in 42 out of 586 node-negative tumours). In univariate analysis, (log-rank test) the score of c Kit expression correlated with poor patient outcome P=0.02 and particularly in node-negative cases (P=0.002). In multivariate Cox analysis, c Kit was an indicator of metastasis independent of 25 other concomitantly evaluated markers of prognosis. Logistic regression showed that c Kit ranked 10 out of 25 (P=0.041), and was included in a 10-marker signature that allowed 79.2% of the patients to be correctly classified in the metastatic or metastasis-free categories independently of hormone receptors and HER-2 status. Interestingly, c Kit was also a significant predictor of metastasis in node-negative tumours (2 out of 25 ranking, P

Quantitative immunocytochemical assays of 1,200 breast carcinomas were assessed after construction of tissue microarrays. A total of 42 markers were evaluated for prognostic significance by univariate log rank test (mean follow-up, 79 months), using quantitative scoring by an image analysis device and specific software. Complete data were obtained for 924 patients, for whom 27 of the 42 markers proved to be significant prognostic indicators. Analysis of these 27 markers by logistic regression showed that 18 (cMet, CD44v6, FAK, moesin, caveolin, c-Kit, CK14, CD10, P21, P27, pMAPK, pSTAT3, STAT1, SHARP2, FYN, ER, PgR and c-erb B2), and 15 when ER, PgR and c-erb B2 were excluded, were 80.52% and 78.9% predictive of disease outcome, respectively. The immunocytochemical assays on 4 micron thick sections of fixed tissue are easy to handle in current practice and are cost-effective. Quantitative densitometric measurement of immunoprecipitates by computer-assisted devices from digitized microscopic images allows standardized high-throughput "in situ" molecular profiling within tumors. It is concluded that this 15 marker immunohistochemical signature is suitable for current practice, since performed on paraffin sections of fixed tumor samples, and can be used to select patients needing more aggressive therapy, since this signature is about 80% predictive of poor clinical outcome. Also, the markers included in the signature may be indicative of tumor responsiveness to current chemotherapy or suggest new targets for specific therapies.

Genomic studies have led to new taxonomic classifications of breast carcinomas. Proteomic investigations using tissue microarrays have yielded complementary results and are useful in identifying potential molecular targets for specific therapies. Searching for new drug targets is particularly important for tumors of poor prognosis, such as breast tumors that lack estrogen receptors and HER2 amplification; in these tumors, certain molecules probably play a significant role in tumor spreading through the stromal microvasculature. We investigated 930 breast carcinomas categorized according to patients' survival (range of follow-up = 4-10 years; median follow-up = 6.5 years) using (1) automated immunohistochemical procedures (Ventana, Cedex, France) with tissue microarrays (Alphelys, Plaisir, France) and (2) quantification of immunoprecipitates assessed by automated image analysis densitometry (SAMBA, Meylan, France). Expression of c-Met and CD146 and that of signaling transducers PI3K, FAK, and FYN were compared in living and deceased patients. Expression of some proteins recently reported to be characteristic of basal cell carcinomas was also assessed, namely, CK5-6, caveolin-1, carbonic anhydrase IX, p63, and CD117; these also constitute potential targets for therapies for aggressive tumors. Overexpression of these proteins was observed in deceased or metastatic patients (P < .01 to P < .00001), particularly node-negative patients (except for FYN, p63, and CD146). c-Met and CD146 are involved in tumor spreading, and our results suggest that they probably play an important role in patients' death, along with other proteins involved in hypoxia (carbonic anhydrase IX) and other cell functions or structures (caveolin-1, CD117, CK5-6, and p63) that are expressed in an aggressive subtype of basal cell carcinoma for which no specific therapy is available.

RESUME L’importance de l’angiogenese dans les tumeurs solides est un indicateur pronostique et la vascularisation tumorale constitue une cible therapeutique pour les therapies antiangiogeniques. Le CD105 (endogline) est significativement exprime dans les cellules endotheliales activees en culture et dans les microvaisseaux du stroma tumoral. De ce fait, la quantification de l’expression immunohistochimique du CD105 peut donc etre un parametre potentiellement utile pour la prise en charge des malades, ce qui a ete montre dans une seule etude preliminaire. Neanmoins, ce marqueur n’a pas encore ete precisement evalue sur des series consequentes. Notre etude a porte sur l’expression immunohistochimique du CD105 sur coupes de tissu congele dans une serie de 929 cancers du sein. Les resultats ont ete correles a l’evolution a long terme des malades (mediane de suivi = 11.3 ans). Les resultats montrent qu’un nombre eleve de microvaisseaux CD105 positifs est significativement (p = 0.001, Kaplan Meier) correle avec une survie globale plus courte. Cette correlation persiste, bien que moins forte lorsque les malades sans metastase ganglionnaire sont consideres (p = 0.035). De plus, une forte expression du CD105 est correlee avec un fort risque de metastases a distance (p = 0.006, Kaplan Meier) et notamment chez les patientes sans metastase ganglionnaire (p = 0.001). L’etude multifactorielle (modele de Cox) montre que l’immuno-expression CD105 est un marqueur pronostique independant. Nos resultats montrent que, dans les cancers du sein, l’expression immunohistochimique du CD105 dans les vaisseaux du stroma tumoral permet d’identifier un sous-groupe de patientes depourvues de metastase ganglionnaire axillaire, mais ayant un plus mauvais pronostic. De plus, l’immunodetection du CD105 peut etre consideree comme un parametre utile pour selectionner les patientes qui pourraient, dans l’avenir, beneficier d’une therapie antiangiogenique specifique, notamment les conjugues a l’anti CD105.

CD105 (endoglin) is expressed significantly in activated endothelial cells in culture and in tumor microvessels. Quantification of CD105 immunocytochemical expression that may be clinically relevant has not been accurately evaluated. We studied CD105 expression on frozen tissue sections by using immunohistochemical assays in a series of 929 patients and correlated the findings with long-term follow-up (median, 11.3 years). Univariate (Kaplan-Meier) analysis showed that the number of CD105+ microvessels (cutoff, 15 vessels) correlated significantly with poor overall survival among all patients ( P = .001). This correlation was less significant in node-negative patients ( P = .035). Marked CD105 expression also correlated with a high risk for metastasis among all patients ( P = .006) and among node-negative patients ( P = .001). Multivariate analysis (Cox model) identified CD105 immunodetection as an independent prognostic indicator. Our results suggest that immunohistochemical expression of CD105 has practical clinical relevance for identifying node-negative patients with a poor prognosis. Moreover, immunodetection of CD105 also may be considered a potential tool for selecting patients who could benefit from specific antiangiogenic therapy, using anti-CD105 conjugates.

RESUME Dans l’objectif d’une meilleure evaluation de la proliferation epitheliale de l’endometre, nous avons voulu utiliser un modele in vitro d’evaluation des effets de traitements hormonaux, comme d’un « hormonogramme », afin de mesurer les effets des steroides sexuels composant les traitements hormonaux substitutifs de la menopause et en consequence de pouvoir prevenir l’apparition de lesions precancereuses de l’endometre. La methode utilisee a consiste a tester les effets de differents types de progestatifs couramment utilises en therapeutique, la progesterone, l’acetate de medroxyprogesterone (MPA), l’acetate de nomegestrol (TX) et l’acetate de norethindrone (NOR) sur l’endometre humain proliferatif en culture d’explants, en utilisant deux moyens : la production de prostaglandine F2α (PGF2α) dans les milieux de culture et l’immunoexpression des recepteurs a l’estradiol (RE), des recepteurs a la progesterone (RP) et des antigenes de proliferation Ki67 sur les tissus. Les resultats ont montre (a) qu’apres culture, des etudes quantitatives n’ont pu etre realisees que pour l’evaluation de l’immunoexpression en RE ou RP mais non pour l’immunoexpression en Ki67. Le marquage immunohistochimique en Ki67 s’est revele en effet trop faible et insuffisant dans l’endometre non tumoral pour etre quantifie ; (b) en ce qui concerne la production de PGF2α, elle est diminuee par la progesterone, le TX et le MPA dans les deux types d’endometre proliferatif, homogene ou heterogene ; (c) en ce qui concerne l’immunoexpression des recepteurs, la progesterone diminue uniquement l’expression des RP dans les endometres proliferatifs. L’immunoexpression des RP dans les cellules stromales est diminuee par tous les progestatifs dans les explants d’endometre proliferatif homogene. Le TX diminue l’expression en RP et RE au sein des glandes et du stroma des endometres proliferatifs homogenes ou heterogenes. Le MPA agit de la meme maniere que TX sur les endometres heterogenes. En bref, ces resultats montrent que la majorite des effets observes in vitro par les traitements progestatifs sur l’endometre sont des diminutions de production de PGF2α et des diminutions d’immunoexpression en RP et/ou RE, bien que les progestatifs n’aient pas les memes effets in vitro sur les differents types d’endometre proliferatif (homogene et heterogene). Cette etude ouvre la perspective de nouvelles possibilites d’exploration de l’endometre humain sur culture d’explants, notamment dans le cadre de l’etude de l’activite de proliferation endometriale.

The objective of this study was to evaluate the endometrial response to sexual steroids in organ culture using two means: prostaglandin (PG) F2α output in medium culture and steroid receptor immunoexpression in tissue. Human endometrium samples were classified in homogeneous and heterogeneous proliferative or secretory subsets. In proliferative endometrium explant culture, progesterone (10–7 M) induced a significant decrease in PGF2α output, but this was not the case in secretory endometrium, whereas no significant effect of estradiol (10–8 M) was observed. Before culture, homogeneous and heterogeneous proliferative endometrium presented the same pattern of estradiol receptor (ER) and progesterone receptor (PR) expression evaluated by quantitative immunocytochemistry. After culture, immunoreactive ER and PR were detected on the explant. PR immunoexpression rates after culture were lower than before culture in glands on homogeneous proliferative and in stroma on heterogeneous proliferative endometrium explants without in vitro steroid addition. In secretory endometrium, no significant difference was observed between ER or PR immunoexpression rates before culture and after culture. These results provided the hormonal receptivity status of endometrium after culture and will thus serve as a reference for evaluating in vitro steroid effects on endometrium explants. Our preliminary results suggest that cultures of endometrium explants are a valid model for studying the effects of hormonal treatment on homogeneous as well as heterogeneous endometrium. These data could be particularly relevant for evaluating the potential response to hormone stimulation and treatment of endometria sampled in perimenopausal patients.

Objective. Conservative treatment for ductal carcinoma in situ of the breast exposes patients to the risk of infiltrating recurrence which can lead to metastasis. The primary purposes of this retrospective study were to evaluate diagnostic and therapeutic methods over a 10-year period and to validate prognostic factors. This information should greatly improve patient selection for conservative treatment or mastectomy. Study design. A multi-institutional data base including 575 patients treated between 1983 and 1993 was established by combining data from 16 French institutions. Survival at 5 and 7 years was studied as a function of various prognostic factors. Results. Recurrence-free survival at 7 years was 0.96 after modified radical mastectomy and 0.83 after breast-conserving treatment and radiotherapy (P=0.003). Metastasis-free survival at 7 years was 0.99 after modified radical mastectomy and 0.94 after breast-conserving treatment and radiotherapy (not significant). No factor was predictive of local recurrence after mastectomy. Clinical stage was the only factor significantly correlated with metastasis after mastectomy. Recurrence-free survival after breast-conserving treatment with radiotherapy was significantly lower for patients with comedo carcinoma, multifocal lesions, or unclear resection margins, regardless of whether the histological type was comedo or non-comedo carcinoma. Metastasis-free survival was significantly lower for patients with multifocal lesions and for patients with unclear margins after excision of comedo carcinoma. Conclusions. Breast-conserving treatment with radiotherapy is a valid alternative to mastectomy. Patients must be selected carefully on the basis of morphological criteria. Swift gains in therapeutic outcome can be obtained by stressing quality control at each stage of diagnosis and treatment.

Expression of vascular cell adhesion molecules (VCAM) in tumors is associated with endothelial cell activation and may facilitate adherence of carcinomatous cells to the vessel wall, promoting bloodborne metastases. Expression of VCAM was investigated in 202 breast carcinomas using automated (Ventana System) and quantitative (SAMBA image analyzer) immunoperoxidase staining of frozen sections. Positive VCAM immunoreactivity was observed in 83 tumors (41%) (mean immunostained surface, 12.4%; SD, 10.5). The mean area of immunostaining was correlated with clinical and pathologic prognostic indicators and with the immunohistochemical expression in tissue sections of various indicators of cell proliferation, metastatic potential, and drug resistance or sensitivity, evaluated according to the same method. There was no correlation of VCAM immunoreactivity with tumor size, type, or grade or with nodal status. Also, no significant correlation was observed between VCAM and MIB1/Ki67, p53, Bcl-2, E cadherin, CD44v, cathepsin D, CD31, P-gp, ER, PR, or pS2. However, VCAM immunoreactivity was significantly correlated with ELAM and VLA2 (P = .001) and VLAs (P = .008) expression. The results suggest that VCAM expression in breast carcinoma tissue sections is likely not a prognostic indicator. Its practical clinical relevance, if any, must be established by correlation with patients' outcomes and tumor sensitivity to drugs.

PURPOSE bcl-2 protein is detectable in human cancers and may be involved in the response to antineoplastic drugs or endocrine therapy in breast carcinomas. In a previous study, we had developed optimal technical conditions for bcl-2 immunodetection. The aim of the present report was to determine the prognostic significance of bcl-2 expression in breast carinomas by the use of a similar immunocytochemical procedure. METHODS bcl-2 immunocytochemical assays were performed on frozen sections by automated immunoperoxidase technique (Ventana) and computer-assisted analysis of digitized colored microscopic images (SAMBA) in a series of 170 breast carcinomas. The results of automated quantitative immunocytochemical assays were correlated with patient follow-up (120 months). RESULTS Intense bcl-2 immunocytochemical expression in tumors (cutpoint, 15%) significantly correlated with longer disease-free survival and longer recurrence-free survival in the entire cohort of patients (P = .028 and P = .035, respectively) and also in node-negative subgroups of patients (P = .028 and P = .01; Kaplan-Meier long-rank test; NCSS 6.0.1 software). But bcl-2 immunostained surfaces (cutpoint, 15%) did not correlate with overall survival. In multivariate analysis (proportional hazards regression, Cox model), bcl-2 prognostic significance in terms of disease-free survival was only independent of the tumor size and grade and histoprognostic index (Nottingham prognostic index [NPI]). CONCLUSION bcl-2 immunohistochemical expression is a significant indicator of favorable outcome only in terms of disease-free and local recurrence-free survival. However, bcl-2 expression in tumors is an independent weakly prognostic indicator in breast carcinomas. bcl-2 immunodetection assessed in optimal technical conditions (frozen samples, automation, quantitative analysis, scatter diagram cutoffs) may have some limited practical clinical relevance for the management of patients with breast carcinomas.

VLA2 is thought to be involved in the metastatic process in malignant tumours, in particular in carcinomatous cell adhesion to vessel basement membrane. VLA2 expression was immunohistochemically investigated in 204 breast carcinomas. Frozen tissue sections were probed with monoclonal anti-VLA2 using automated (Ventana ES 320 System) and quantitative (SAMBA 2005 image processor) immunoperoxidase. A positive anti-VLA2 immunoreaction was observed in 48 tumours (23.5%), within epithelial carcinomatous cells. The VLA2-positive surface in tumours varied from 3% to 20% (mean 8.75, S.D. 7.17) and was correlated with histoprognostic indicators and tumour expression of various antigens detected using the same method as that for VLA2. The results show that VLA2 immunoexpression was independent of the tumour size, grade, type and aneuploidy, and of the nodal status. VLA2 significantly correlated with ELAM, VCAM, VLA3 and P-glycoprotein (P-gp) (P < 0.01) and inversely correlated with cathepsin D (P < 0.001), but was independent of Ki67/MIB1, p53, bcl-2, c-erbB-2, E cadherin, CD44v, CD31, oestrogen and progesterone receptors' (ER, PR) antigenic sites and pS2. The exact role, if any, of VLA2 in tumour cell dissemination remains to be elucidated and the clinical relevance of VLA2 immunodetection in breast carcinomas requires further investigation of the correlation between VLA2 immunocytochemical expression and patients' outcome and response to chemotherapy. Images Figure 1 Figure 2 Figure 3

The relationship between pregnancy and the outcome of breast cancer remains controversial. The purpose of this study was to determine the prognostic value of pregnancy at the time of diagnosis of primary infiltrating breast cancer. In a retrospective multi-center study we compared a group of 154 patients presenting pregnancy-associated (PA) breast cancer with a control group of 308 patients presenting non-pregnancy-associated (non-PA) breast cancer. Classic prognostic factors, treatment modalities, disease-free survival and overall survival were compared in the 2 groups. The relative importance of pregnancy was assessed by Cox multivariate analysis. There was a significantly higher proportion of inflammatory breast cancer, large tumors and negative receptor status in the PA group. Five-year recurrence-free survival, metastasis-free survival and overall survival were lower both in the whole PA group and in the PA sub-group excluding patients with inflammatory breast cancer than in the corresponding non-PA groups. According to clinical stage, histoprognostic grade and microscopic lymph-node involvement, probability of 5-year metastasis-free survival and overall survival was lower in the PA group. Outcome was significantly poorer after chemotherapy for patients in the PA sub-group than in the non-PA sub-group. Multivariate analysis demonstrated that pregnancy was an independent and significant prognostic factor. Pregnancy has an adverse effect on the outcome of breast cancer. Concurrent or recent pregnancy should be taken into account in the development of new systemic therapies. Our findings have important implications for further research into the basic mechanisms of cancer.

Expression of the bcl-2 gene was investigated in 218 human breast carcinomas by immunohistochemical analysis. Immunodetections were assessed using (1) frozen sections, (2) documented commercially available monoclonal antibody (bcl-2/124, Dako), (3) automation of immunoperoxidase technique (Ventana) and (4) quantitative evaluation of results by image analysis (SAMBA) and statistical analysis of quantitative data (BMDP software). Bcl-2 protein expression was correlated with current prognostic indicators and with molecular markers detected by the same procedure as for Bcl-2. It was shown that Bcl-2 expression is not related to patients' age, tumour size and type or lymph node status, but an inverse relationship was observed between Bcl-2 and tumour grade (P < 0.0001). An inverse relationship was also observed between Bcl-2 expression and p53 (P < 0.0001), Ki67/MIB1 antigen- (P = 0.0012), and P-gp- (P = 0.002) positive immunoreactions. In contrast, anti-Bcl-2 positive reaction was significantly associated with ER-positive (P < 0.001) and with ER/PR-positive or ER/PR/pS2-positive immunoreactions (P < or = 0.005). Bcl-2 expression was independent of CD31 and cathepsin D expression. Thus, Bcl-2 protein, thought to be antiapoptotic, exhibits parodoxical expression in human breast carcinomas. It is strongly detected in low-grade tumours (well-differentiated) with low (MIB1) growth fraction, but is independent of the tumour progression (size, node status, CD31, and cathepsin D). Bcl-2 acting on apoptosis is related to p53 gene abnormalities in breast carcinomas. Bcl-2 protein expression may also be involved in response to endocrine therapy (associated to ER/PR/pS2 positive immunoreactions) and probably with chemoresistance mechanisms (inverse relationship with P-gp). Images Figure 1 Figure 2

AIM: To determine the prognostic significance and clinical relevance of cathepsin D detected by immunocytochemical assays (ICAs) in breast carcinomas. METHODS: 151 patients presenting with palpable or impalpable breast carcinomas and who had not received any kind of adjuvant chemotherapy or endocrine therapy who were operated from January 1986 to May 1987 were studied. ICAs of tumour specimens were performed in optimal technical conditions (frozen sections, automated immunoperoxidase technique (Ventana), and computer assisted analysis of digitised coloured microscopic images (SAMBA)) to determine cathepsin D immunocytochemical expression. Results of quantitative ICAs were correlated with overall and disease free survival over 8.4 years of follow up in axillary lymph node positive and negative patients. RESULTS: Cathepsin D immunocytochemical expression in tumours of 15% or more was significantly associated with poor overall survival in the whole group and in node positive patients (Kaplan Meier, log rank test p = 0.003 and p = 0.007); however, it was not correlated with survival in node negative patients. Cathepsin D immunocytochemical expression (> 15%) correlated with short disease free (p = 0.015) and short recurrence free survival (p = 0.021) in the group as a whole but not when node positive and negative patients were evaluated separately. CONCLUSIONS: In optimal conditions (automated and quantitative ICAs on frozen sections) cathepsin D immunohistochemical expression is a significant prognostic indicator in terms of overall, disease free, and recurrence free survival; however, there is no correlation when node negative patients are considered separately.

The prognostic significance of c-erbB-2 oncoprotein overexpression detected in tumours by immunocytochemical assays (ICAs) was investigated in 148 breast carcinomas. ICAs were performed under optimal technical conditions with frozen tissue sections and included automated immunoperoxidase technique and computer-assisted analysis (densitometry) of digitized coloured microscopic images. Results of quantitative ICAs (expressed in percentages of c-erbB-2-positive surfaces and mean optical densities) were correlated with the patients' follow-up in axillary lymph node-positive (N+) and node-negative (N-) subgroups of patients. Patients' follow-up ranged from 9 months (for the first death) to 101 months (for the 121 alive patients) with a 62.5 months mean overall follow-up. It was shown that marked c-erbB-2 immunocytochemical expression in tumours (cut-off point 35%) significantly correlated with the patients' poor overall survival in N+ and in N- patients (Kaplan-Meier, log-rank test, P = 0.045 and P = 0.015). Also, marked c-erbB-2 immunohistochemical expression correlates with short disease-free (P = 0.005), recurrence-free (P = 0.048) and metastasis-free survival (P = 0.05) (Kaplan-Meier, log-rank test) in N+, but not in N- subgroups. It is concluded that in optimal conditions (automated and quantitative ICAs on frozen sections) c-erbB immunohistochemical expression is a significant prognostic indicator in terms of overall and disease-free survival. The c-erbB-2 protein prognostic significance is independent of node status in terms of overall survival, but not of disease-free survival. Images Figure 1

The reduction of E-cadherin expression, which is involved in the initial step of invasion and metastasis of cancer, was investigated in 218 human breast carcinomas. Quantitative immunohistochemical assays (ICAs) were performed on frozen sections. Quantitation was assessed by processing digitized microscopic images of immunoreactions using a computerized system of image analysis (SAMBA). The results were correlated with clinicopathological data and quantitative immunodetection of other molecules. E-cadherin expression was significantly (P < 0.001) stronger in ductal carcinomas than in lobular carcinomas and stronger (P < 0.01) in low grades than in high grades, but E-cadherin was independent of lymph node status and tumour size. Also an inverse significant (P < 0.01) relationship was observed between E-cadherin expression on tissue sections and positive immunoreactions with anti-P53, MIB1 (growth fraction), and anti-c-erb-B2 product. Conversely, strong positive and anti-E-cadherin immunoreactions correlated with strong positive anti-ER and anti-PR immunoreactions (P < 0.01). No relationship was observed between E-cadherin and the results of quantitative ICAs of cathepsin D, CD31, and P-glycoprotein, assessed on consecutive sections from the same frozen tissue samples. The results show that preserved E-cadherin expression correlates with high degree of tumour differentiation, low proliferative activity, and low expression of prognostic markers. The deregulation of E-cadherin is independent of other steps of tumour invasion, such as protease digestion of extracellular matrix and angiogenesis.

CD44 variants carrying sequences encoded by exon v6 are preferentially expressed in metastatic animal cancer cell lines. CD44v6 overexpression correlates tumor dedifferentiation and progression in some human carcinomas, but the relationship of CD44v6 overexpression with metastatic behavior of tumor observed in animal models is controversial, particularly in breast carcinomas. The discrepancies probably result from analytical bias. We investigated CD44v6 and CD44s expression in 218 frozen samples of primary breast carcinomas. Immunocytochemical procedure was performed under optimal technical conditions using commercially available 2F-10 monoclonal antibody (MAb), a microprocessor-controlled automated device (Ventana Medical Systems, Tucson, AZ), and quantitative evaluation of results by processing digitized-colored microscopic images (SAMBA, Grenoble, France). CD44v6 expression in tissue sections was shown to be independent of the patient age, tumor size, histological types and grades, and the lymph node status. CD44v6 expression was also independent of the expression of molecules endowed with poor prognostic significance detected by MAbs (anti-p53, anti-c-erb B-2 protein, MIB1) on consecutive sections. No significant relationship could be evidenced either between CD44v6 expression, and CD31 involved stromal angiogenesis and cathepsin D. Finally, CD44v6 was independent of markers of hormone dependence (estrogen and progesterone receptors, pS2) and of multidrug resistance (P-glycoprotein). Similar results were observed with anti-CD44s. We conclude that the true prognostic significance of CD44v6 overexpression still remains to be shown under rigorous technical conditions (frozen samples, well-documented MAbs, and optimal standardization of procedure using automation and quantitative analysis) providing data appropriate for further correlation with long-term patient follow-up.

Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours into invasive malignancies. Many signalling pathways are involved in EMT, but the key regulatory kinases in this important process have not been clearly identified. Protein kinase CK2 is a multi-subunit protein kinase, which, when overexpressed, has been linked to disease progression and poor prognosis in various cancers. Specifically, overexpression of CK2α in human breast cancers is correlated with metastatic risk. In this article, we show that an imbalance of CK2 subunits reflected by a decrease in the CK2β regulatory subunit in a subset of breast tumour samples is correlated with induction of EMT-related markers. CK2β-depleted epithelial cells displayed EMT-like morphological changes, enhanced migration, and anchorage-independent growth, all of which require Snail1 induction. In epithelial cells, Snail1 stability is negatively regulated by CK2 and GSK3β through synergistic hierarchal phosphorylation. This process depends strongly on CK2β, thus confirming that CK2 functions upstream of Snail1. In primary breast tumours, CK2β underexpression also correlates strongly with expression of EMT markers, emphasizing the link between asymmetric expression of CK2 subunits and EMT in vivo. Our results therefore highlight the importance of CK2β in controlling epithelial cell plasticity. They show that CK2 holoenzyme activity is essential to suppress EMT, and that it contributes to maintaining a normal epithelial morphology. This study also suggests that unbalanced expression of CK2 subunits may drive EMT, thereby contributing to tumour progression.

To assess the value of direct colposcopic vision (DCV) for optimizing large loop excision of the transformation zone (LLETZ) for the treatment of cervical intraepithelial neoplasia (CIN). Data from 648 patients who underwent excisional procedures for CIN and were included in two previously published cohort studies were retrospectively reviewed. Women who had a LLETZ were included for analysis (n = 436). Margin status, surgical specimen dimensions and volume were analysed according to the use of colposcopy during procedure. Compared to LLETZ guided by previous colposcopy report only, and to LLETZ performed immediately after colposcopy, DCV allowed for a significantly higher rate of clear margins: 33 (52.4 %), 104 (68.0 %) and 142 (84.5 %), respectively (p

The relationship of age with prognostic factors and outcome of breast cancer has long been controversial due to numerous confounding factors. In order to clarify the prognostic value of age, we analyzed a homogeneous population of 1,266 patients treated for breast cancer at the same institution (mean follow-up: 62 months). Three groups were compared: patients under 35 years of age, non-menopausal patients over 35 years of age, and post-menopausal patients under the age of 70 years. A higher frequency of undifferentiated tumors, histoprognostic grade-3 cancer, microscopic lymph-node involvement and negative hormonal receptor status was observed in patients under 35 years. In addition, clinical but not anatomical tumor size was greater in young patients, suggesting higher stromal activity. Metastasis-free survival and overall survival were significantly poorer before 35 years. Differences were observed when patients were matched with regard to stage, anatomic size, histoprognostic grade, microscopic lymph-node involvement and receptor status. Multivariate analysis of both overall and metastasis-free survival demonstrated that age younger than 35 years was an independent risk factor. Younger women had a higher risk of local recurrence but, unlike older women, they did not experience any worsening of the already unfavorable outcome due to recurrence.

The distribution of PECAM-1/CD31 molecule was investigated in 133 breast carcinomas using monoclonal antibody and frozen sections. Anti-CD31 labels endothelial cells and reflects stromal angiogenesis. The CD31 immunoreactivity was evaluated by computer-assisted analysis of digitized microscopic images. The automatic screening of the whole preparation and the measurements of the mean CD31 immunostained surface was performed in each case. A similar procedure was achieved for p53, cathepsin D, P-gp, pHER-2/neu, Ki67, pS2 estrogen and progesterone antigenic sites immunodetection. The image analysis of positive CD31 surface was variable, ranging from 4% to 33% (mean 14.7%, SD = 5.43). The CD31 positive surface correlated (P < .01) with the Nottingham prognostic index, but not with the tumor size, the node status, the tumor grade, nor with the patient age. Also the CD31 immunoreactivity was independent of the pHER-2/neu, Ki67 antigen, p53, ER, PR and pS2 immunodetectable expression in tumors, but correlates with that of cathepsin D (P = .024) and P-gp (P = .028), which reflects the multi-drug resistance capacity of tumor cells. In conclusion, CD31 positive vessels assessed on frozen sections by image analysis constitute an excellent method of evaluating tumor stromal angiogenesis, and can be further used for clinical purposes. The results also suggest that the CD31/PECAM molecule may be involved in the spread of tumor by interacting with extracellular matrix lysis that results from the tumor cell proteasic activity and with multidrug resistance. (Key words : PECAM/CD31 ; Immunohistochemical assays ; Image analysis) Am J Clin Pathol 1995 ;103 :443-448.

A series of 200 breast carcinomas was investigated on frozen sections using PAb 1801 p53 monoclonal antibody and streptavidin biotin peroxidase complex. Densitometric analysis of the immunoprecipitates was assessed by processing digitized microscopic images. p53 was observed in the nucleus of 48% of the tumors. Some tumors (14 of 91) tested in parallel on paraffin sections were negative, although positive on frozen sections. Image analysis showed that the surfaces positive with anti-p53 and the staining intensity were decreased (P < .01) on paraffin sections. The p53 tumor expression was independent of patient age, tumor size, axillary lymph node status, HER-2/neu and cathepsin D expression, and nuclear morphometric parameters. However, p53 correlated with high histological grade (P < .01), lack of estrogen receptor (ER) (P = .0015) and progesterone (PR) (P = .0065) antigenic sites, pS2 detection (P = .03), high Ki-67 immunoreactivity (P = .018), large silver-stained nucleolar organizer region (AgNOR) nuclear surface ratio (P < .02), and degree of hyperploidy (P < .03), and was more often observed in the comedocarcinomas. The results suggest that p53 expression in breast carcinomas is not a totally independent prognostic indicator and that the clinical relevance and prognostic significance of p53 expression in breast carcinomas can be reliably assessed provided that the procedures are standardized, particularly with regard to the use of frozen sections and image analysis processing of the immunodetection.

BACKGROUND Chemotherapy failure that is due to cellular drug resistance remains a major problem in most cancer patients. One type of drug resistance that has been characterized is the multidrug resistance phenomenon, which demonstrates a reduced ability of cancer cells to accumulate drugs as a result of the effects of an energy-dependent unidirectional drug efflux pump with a broad substrate specificity. This drug pump is composed of a 170-kd transmembrane glycoprotein referred to as the P-glycoprotein (P-gp) that uses energy in the form of adenosine triphosphate to transport drugs through a channel formed by transmembrane segments. PURPOSE Our purpose was to detect the levels of P-gp expression in frozen untreated breast carcinomas by immunocytochemical assays and to correlate these levels to current prognostic indicators and, in a few cases, to MDR1 (also known as PGY1) mRNA expression by polymerase chain reaction (PCR). METHODS The immunocytochemical expression of the multidrug resistance gene, P-gp, was investigated using a specific monoclonal antibody (JSB1) against P-gp in 5-microns frozen sequential sections of breast carcinomas obtained from 213 patients. Microscopic images of immunostained preparations were evaluated by image analysis and were compared with MDR1 transcription (mRNA) assessed by PCR in 16 patients. Quantitative P-gp immunocytochemical assays were correlated to histoprognostic factors and immunocytochemical indicators. RESULTS Among the 213 breast carcinomas tested, 113 (53%) were P-gp positive, but in 28% of the tumors, the immunostained surface accounted for less than 5% of the total area stained. Quantitative immunocytochemistry reflecting the amount of intracellular P-gp antigen strongly correlated (r = 0.865; two-sided, P < .0001; Pearson's test) with the quantitative evaluation of the scanner analysis of mRNA transcripts. The P-gp expression was significantly (two-sided, P < .001) correlated with p53 expression in tumors, to cathepsin D and Ki67 (two-sided, P < .01) immunoreactivity, and to a lesser extent, the detection of estrogen receptor antigenic sites (two-sided, P = .019). P-gp expression was found to be independent of expression of progesterone receptor and pS2, pHER-2/neu, and CD31 in tumors and from patient age, tumor size, histologic types, grades and Nottingham prognostic index, and nodal status. CONCLUSIONS The quantitative immunocytochemical assays of P-gp are correlated to PCR analysis of MDR1 expression, and such correlations can be useful in evaluating potential multidrug resistance in breast cancer. However, the clinical significance of P-gp immunodetections remains to be further determined.

Mammary hamartomas are breast disorders currently underestimated and not well recognized. Forty-one hamartomas diagnosed among 5,834 breast biopsies, histologically examined during the last 7 years, are reported. Hamartomas accounted for 1.2% of benign lesions and 4.8% of benign breast tumors. Clinically, hamartomas were revealed by breast palpable lump, usually painless. Typically, but inconsistently, mammography showed sharply circumscribed density, separated from adjacent normal breast by a thin radiolucent zone. Macroscopically, hamartomas were slightly larger and softer than common adenofibromas, were well limited, whitish, pinkish and fleshy, with yellow islands of fat tissue. Histologically, hamartomas exhibited pushing borders with a pseudoencapsulation, and consisted of a combination of variable amount of stromal and epithelial components. Stromal components mainly consisted in a prominent fibrohyalin feature usually associated to small islands of adipose tissue and edematous changes. Epithelial structures showed variable features of benign breast disease. The overall architecture was lobulated but not nodular. The histological diagnosis was mainly a diagnosis of exclusion and hamartomas diagnosis relies on clinical, radiological and pathological criteria. Hamartomas result more from breast dysgenesis than from tumorous process.

We recently reported that standardized quantitative immunohistochemical (IHC) assays allowed prediction of an adverse outcome among 572 node negative (N-) patients with breast carcinoma (BrCa). To further validate our prior findings, we repeated the IHC stains including a second series of BrCa diagnosed at Yale University. Tissue microarrays (TMAs) of two cohorts of patients with BrCa (418 Marseille University and 303 Yale University) were respectively investigated for IHC expression of 15 markers (HIF-1α, PI3K, pAKT, pmTOR, moesin, P21, 4(E) BP-1, P27, Ker5-6, pMAPKAPK-2, SHARP2, claudin-1, ALDH, AF6 and CD24). Quantitative measurements of immunoprecipitates densitometry assessed with an image analyzer were correlated with 8-year patients' outcome and compared in the two cohorts. The best predictive signature consisted of a combination of five markers that included HIF-1α, PI3K, claudin-1, AF6 and pAKT in N- BrCa. This combination permitted an accurate prediction of outcome in 92.34% (386/418) of N- patients in the first set (Marseille) and 89.8% (158/176) in the second set (Yale). The close results in both cohorts confirmed the validity of this original IHC signature predictive of prognosis in node negative BrCa. This validation suggests that in clinical practice, it would be possible with standardized kits (i) to identify patients with poor prognosis at diagnosis time, particularly in the N- BrCa subset, who would require more aggressive adjuvant therapy and (ii) to avoid useless expensive therapies and their side effects in N- patients with favorable prognosis.

pS2 expression was studied in 212 breast carcinomas. Immunocytochemical assays (ICAs) were performed on frozen sections and evaluated by digital image analysis. pS2 immunostaining was compared in frozen sections and paraffin sections. The pS2 positivity was observed in 45% of the cases on frozen sections. But in pS2 positive tumors, the tumor surface which was immunostained was small (m=14.3%). In 22% of immunoreactive tumors the positive surface was 5% or less. In contrast to frozen sections, the pS2 positivity on paraffin sections could not be evaluated by image analysis system because of the background. No significant correlation was observed between pS2 expression and patient age, tumor size, histological type and grade, nor with lymph node status. The pS2 positivity was significantly correlated to ER and PR positive immunodetection on frozen sections. But pS2 was independent from pHER-2/neu and cathepsin expression whereas there was a significant inverse relationship between pS2 and Ki67. This study shows that pS2-ICAs on frozen sections and image analysis are optimal and standardized conditions for the evaluation of pS2 expression in breast carcinomas, and suitable for selecting patients for hormone therapy.

A series (n = 322) of breast carcinomas was investigated from 1991 to 1993 using digital image analysis. Nuclear morphometry and DNA content, and AgNORs were evaluated on cell imprints from fresh tissue samples which were further stored frozen (-80-degrees-C). Data were correlated to morphological prognostic factors and immunocytochemical expression of cell markers assessed on frozen sections and evaluated by densitometry after image analysis processing. Nuclear morphometric parameters, DNA nuclear content and AgNORs were independent from the tumor size, histological grades, and the tumor content of immunodetectable pHER-2/neu, Cathepsin D, ER, PR, pS2, and p53. But, DNA index and hyperploidy degree correlated with the mitosis index (p < 0.01) and Ki67 immunostaining (p = 0.003, p < 0.0001) whereas the shape factor and nucleus large diameter correlated with the degree of cell anisocytosis (p < 0.01). Nuclear surface and large diameter were greater in ductal carcinomas (p = 0.028) and in comedocarcinomas (p < 0.001) than in lobular carcinomas. These results suggest that image analysis processing provides accurate data to refine histoprognostic grading and additional parameters to evaluate tumor proliferative activity.

Immunocytochemical assays of cathepsin D were assessed in a series of breast carcinomas (n = 257) using monoclonal M1G8 anti-total cathepsin D and the avidin-biotin-peroxidase complex. Cathepsin immunoreactivity was compared in frozen and paraffin sections. All tumours were anti-cathepsin-positive. Positive staining was observed in carcinoma and stromal cells and in the extracellular matrix. The amount of immunodetectable cathepsin in tissue was measured by computer-assisted image analysis (SAMBA 2005). Both the percentage of immunostained tumour surface and the mean optical densities were processed as continuous variables for statistical analysis and correlated with prognostic factors. It was shown that cathepsin D was independent of the tumour size, the lymph node status, hormone receptors, and pHER-2/neu overexpression. Cathepsin was significantly correlated with anti-EGFR (P = 0.012) and Ki67 (P = 0.002) immunoreactivity, tumour grade (P = 0.032), vascular invasion (P = 0.0081), proliferation index (P = 0.0045), and, to a lesser extent with AgNORs (P = 0.0504) and the degree of hyperploidy (P = 0.057). Tissue fixation and paraffin embedding significantly decreased cathepsin immunoreactivity. These results show that cathepsin D is not a totally independent prognostic factor in breast carcinomas.

A series of 160 impalpable breast carcinomas was collected from 1979 to 1991. Mammographs showed microcalcifications (64%), or opaque images (36%). Surgical specimens were X-rayed during the intervention in order (i) to ascertain that the lesions detected on mammographs were removed, and (ii) to guide the pathologist in sampling tissue fragment for an appropriate microscopic evaluation of the lesions. During the intervention, the peroperation histological diagnosis was correct in 63% of the cases, whereas malignancies were underscored in 37%. No false positive diagnosis was recorded. A large majority (92%) of false diagnoses stated during surgery were in situ carcinomas diagnosed as epitheliosis and invasive carcinomas diagnosed as in situ carcinomas. In 63% of the cases the axillary lymph node could be removed during the first intervention. In 91% of the cases "in sano" margins of resection were evaluated as such during the intervention. The size of tumors ranged from 1 to 60 mm (m = 10 mm - SD = 8.45), 70% measuring less than 10 mm. Carcinomas were in situ (23.75%), microinvasive (13.75%) and invasive (62.5%). Carcinomas were ductal (78.1%), lobular (18.7%) and of other types (14.2%). A majority of intraductal carcinoma (68%) were comedocarcinomas. Invasive carcinomas accounted for grade I in 37% of the cases, grade II in 56%, grade III in 7%, ductal carcinomas and for tubular carcinomas in 15%. Immunodetection could be performed on frozen sections in 78 cases. Tumors were receptor positive in 58% of the cases. The greater growth fraction (Ki-67) and higher detection of HER-2/neu oncogene product were observed in comedocarcinomas. Diploid tumors accounted for 52% of those evaluated (n = 48).

Immunocytochemical assays for EGFR were performed on frozen sections from breast carcinomas (n = 209). Results were evaluated by computer assisted image analysis to accurately define the percentage of immunostained surface and the mean optical densities. Thirty seven percent (n = 77/209) of the tumors were EGFR positive, but about one third of them were faintly reactive (35%). No significant relationship was observed between EGFR tumor content and patient age, tumor size, histological type, histoprognostic grade, or axillary lymph node status. A negative correlation was observed with the results of estrogen receptor immunocytochemical assays and a positive correlation with immunodetectable cathepsin D and Ki 67 antigen evaluated according the same method. No correlation was found with HER-2/neu protein, aneuploidy, nucleolar organizor region distribution, and nuclear morphometry, also assessed by image analysis. These results suggest that immunocytochemical assays assessed on frozen sections and evaluated by image analysis are suitable for current and standardized evaluation of EGFR which has been previously documented as a prognostic indicator in breast carcinomas.

Background Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known. Methods Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival. Results HIF-1α isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1αTAG) and HIF-1α736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1αTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1αTAG (P = 0.048), and total HIF-1α (P = 0.048) mRNA levels. HIF-1αTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1αTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01). Conclusions Our results show for the first time that mRNA expression of a HIF-1αTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis. See commentary: http://www.biomedcentral.com/1741-7015/8/45

The aim of this study was to identify a prognostic immunohistochemical signature indicative of risk of early metastasis in node-negative breast carcinomas that would also be relevant to the development of new tailored therapy. Quantitative measurements of the immunohistochemical expression of 64 markers (selected from literature data) using high-throughput densitometry (as a continuous variable) of digitised microscopic micro-array images were correlated with clinical outcome in 667 node-negative breast carcinomas (mean follow-up 102 months). Multivariable fractional polynomials model of logistic regression allowed the selection of the best combination of markers (in terms of sensitivity and specificity) to predict patient outcome without any categorisation using predefined cut-points for individual marker measurements. A highly predictive ten-marker (out of 64) signature was identified comprising PI3K, pmTOR, pMAPKAPK-2, SHARP-2, P21, HIF-1alpha, Moesin, p4EBP-1, pAKT and P27 that well classified 91.4% of node-negative patients (specificity 90.9%, sensitivity 93.7%, area under ROC curve 0.958) independently of estrogen receptors (ER), and progesterone receptors (PR) and HER-2 status (91.6% well classified patients when ER, PR, HER-2 excluded). It is concluded that quantitative immunoprofiling of node-negative breast carcinomas is helpful in selecting patients who should not receive aggressive adjuvant chemotherapy and provides data for the development of tailored therapy.

Silver-stained nucleolar organizer regions (AgNORs) in human breast carcinoma were studied using a computer-assisted system of image analysis. Standardized, automatic measurements of 7 morphometric parameters (area, perimeter, shape factor, bend energy, angle, and small and large diameters) performed on paraffin sections and cell imprint were compared and correlated with nuclear morphometry, histopathological grading, tumor growth fraction, (monoclonal Ki67-immunostaining), DNA nuclear content (stoechiometric Feulgen staining) and axillary lymph node invasion. The major findings were as follows: (i) variations in AgNORs and nuclear parameters were correlated, (ii) the ratio of AgNOR area/nuclear area was significantly different in low and high grade tumors, (iii) mean AgNOR parameter values increased significantly with the tumor growth fraction and tumor hyperploidy and were significantly higher in patients with axillary lymph node metastases and (iv) AgNOR evaluation was more accurate for cell preparations than for tissue sections.

We aimed in this study at identifying prognostic immunohistochemical molecular signatures indicative of disease outcome, also relevant for development of new specific therapies, in triple-negative (ER, PR, c-erbB2- negative) breast carcinoma subtypes. We evaluated 42 markers in tissue micro-arrays from a series of 924 breast carcinomas including 184 triple-negative tumors using standardized quantitative immunocytochemical assays and correlated the data with patients' outcome (mean follow-up of 79 months). When 27/42 markers including basal-like markers first found to be individually significant for prognosis in a univariate analysis (log-rank test) in 924 tumors, were secondly evaluated in the triple-negative tumor subtype (184/924), eleven including maspin, P21, P27, PTEN, caveolin, EGFR, FAK, P38, pMAPK, STAT1 and CD10 were 89.2% predictive of disease outcome in logistic regression. When markers reported in the literature as expressed in basal-like subtype were evaluated in the 924 series, only eight (EGFR, CK14, moesin, caveolin, cMet, ckit, CD44v6, C10) were prognosis predictive in univariate analysis (log-rank test) and in logistic regression were predictive of disease outcome in 66.3% independently of ER, PR and c-erbB2 expression and in 72% in triple-negative tumor subset. The results suggest that the category of 'triple-negative' breast carcinomas does not exactly overlap the basal-like subtype, and that immunoprofiling of triple-negative tumors (not similar to that of basal-like tumors) may be helpful to select patients for more aggressive treatment and provides a basis for development of tailored therapy.