Plasma and tissue proteomic prognostic factors of response in primary breast cancer patients receiving neoadjuvant chemotherapy.

A pathological complete response (pCR) after neoadjuvant chemotherapy is observed in approximately 20% of breast cancer patients. A proteomic analysis was performed on plasma and tumor tissue before treatment to evaluate its potential impact on the prediction of response. One hundred and forty-nine breast cancer patients eligible for neoadjuvant chemotherapy were included in the study between February 2004 and January 2009 at three centers. The proteomic analysis was performed using SELDI Technology (ProteinChip CM10 pH4, IMAC-Cu and H50). Three acquisition protocols were used according to the mass range. Plasma and tumor proteomic signatures were generated using generalized ROC criteria and cross-validation. Twenty-eight (18.8%) patients out of 149 experienced a pCR according to Sataloff criteria. In the cytosol analysis, respectively 4, 2 and 8 proteins had significantly different levels of expression in the responders and non-responders using IMAC-Cu, H50 and CM10 pH4. Among the 8 proteins of interest on CM10 pH4, 2 (C1 and C7) were selected and were validated in 95.0 and 85.6% of the models. In the plasma analysis, respectively 12, 6 and 2 proteins had different levels of expression using the same proteinchips. Among the 12 plasma proteins of interest on IMAC-Cu, 2 (P1 and P7) were selected and were validated in 94.8 and 97.6% of the models. A combined proteomic signature was generated, which remained statistically significant when adjusted for hormone receptor status and Ki-67. Our results show that proteomic analysis can differentiate complete pathological responders in breast cancer patients after neoadjuvant chemotherapy.

Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition.

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

Measurement of mRNA of 11 biomarkers by RT-PCR to detect lymph node involvement in cervical cancer.

Lymph node metastases are a major prognostic factor in cervical carcinomas. The aim of this study was to characterize the expression of 11 markers in cervical tumors and negative lymph nodes and to determine which ones could be helpful for improving the specificity of molecular diagnosis of nodal involvement. Using TaqMan RT-PCR, we studied the expression of CK19, MUC1, HER1-HER4, VEGF, VEGF-C, uPA, MMP9, and PRAD1 in uterine cervical tumors and in histologically nonmetastatic lymph nodes of 8 patients diagnosed with locally advanced cervical cancer. We observed that CK19, MUC1, HER1-HER3, uPA, and VEGF had a significantly higher expression in cervical tumors than in the negative nodes, whereas VEGF-C expression level was higher in the negative nodes than in the tumors. PRAD1 harbored similar expression levels in the tumors and in the negative nodes. Interestingly, 1 of the 4 patients who presented a clinical recurrence, showed elevated HER1, HER2, uPA, and VEGF in the histologically negative nodes. Our results suggest that CK19, MUC1, HER1-3, uPA, and VEGF are biomarkers that have a higher expression in tumoral cervical tissues compared with the negative lymph nodes and could be useful to diagnose nodal involvement in uterine cervical carcinoma. Our results should encourage us in continue to investigate a greater number of patients, including patients with histologically involved nodes.

Real-time reverse-transcription PCR to quantify a panel of 19 genes in breast cancer: relationships with sentinel lymph node invasion.

At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0-T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2-4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERalpha, ERbeta), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35-40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.

Real-time reverse-transcription PCR to quantify a panel of 19 genes in breast cancer: relationships with sentinel lymph node invasion.

At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0-T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2-4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERalpha, ERbeta), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35-40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.

ErbB/HER ligands in human breast cancer, and relationships with their receptors, the bio-pathological features and prognosis.

BACKGROUND: The aim of this study is to provide an expression profile of ErbB/HER ligands in breast cancer. We analysed the relationships with their receptors, the bio-pathological features and prognosis. PATIENTS AND METHODS: Epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), amphiregulin (AREG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EREG) and neuregulins1-4 (NRG1-4) were quantified in 363 tumours by real-time reverse transcription-polymerase chain reaction using TaqMan probes. RESULTS: Ligands were detected in 80%-96% of the cases, except NRG3 (42%) and EREG (45.5%). At least one ligand was expressed in 304 cases (cut-off: upper quartile). Almost all combinations of receptor and ligand co-expressions were observed, but TGFalpha is preferentially expressed in tumours co-expressing EGFR/HER3, NRG3 in those co-expressing EGFR/HER4, AREG and EREG in those co-expressing HER2/HER4. EGF and AREG were associated with estradiol receptors, small tumour size, low histoprognostic grading, high HER4 levels. TGFalpha, HB-EGF and NRG2 were negatively related to these parameters. In Cox univariate analyses, EGF was a prognostic factor. CONCLUSION: Our study demonstrates that (i) ErbB/HER ligands, including BTC and EREG, are expressed in most breast cancers; and (ii) TGFalpha, HB-EGF and NRG2 high expressions are related to the biological aggressiveness of the tumours.

Prognostic value of tumoral sialyltransferase expression and circulating E-selectin concentrations in node-negative breast cancer patients.

AIMS AND BACKGROUND: A crucial step in the metastatic process is the interaction between the endothelial molecule E-selectin and its tumoral ligands sialyl-Lewis- and sialyl-Lewis. Sialyltranferases are involved in the biosynthesis of these ligands. The aim of this study was to assess the prognostic value of tumoral sialyltransferase expression and of circulating soluble E-selectin (sE-selectin) in node-negative breast cancer patients. METHODS: Using a multiplex RT-PCR method, we measured the expression of five sialyltransferases (ST3Gal III, ST6Gal I, ST3Gal IV, ST3Gal I and ST3Gal II) in tumors of 135 surgically treated node-negative breast cancer patients. Circulating sE-selectin concentrations were measured by an ELISA method prior to surgery. We also analyzed tumor size, histoprognostic grading and steroid hormone receptor status. RESULTS: The median follow-up was 7.5 years. Expression of estrogen receptors was associated with a good prognosis for relapse-free survival in univariate analysis. A high ST3Gal III/ST6Gal I ratio and a high sE-selectin concentration were associated with a bad prognosis for relapse-free survival and overall survival in univariate and multivariate analysis. CONCLUSION: In the present study, tumoral sialyltransferase expression and circulating sE-selectin concentrations had prognostic value in patients with node-negative breast cancer. This result provides further evidence for the important role of these agents in the metastatic process.

Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line.

To evaluate whether leptin plays a putative role in breast tumorigenesis, we studied the expression of its long and short receptor isoforms in various tumoral breast tissues. We applied semiquantitative RT-PCR method to RNA extracted from 20 breast cancer biopsies and two human breast cancer cell lines (T47-D and MCF-7). Our results showed the expression of both leptin receptor transcripts in all tumoral tissues examined. By in situ hybridization experiments, we localized leptin receptors in proliferating epithelial cells. Study of leptin effects on human breast cancer cells growth was performed by [3H]-thymidine incorporation method and colorimetric MTT assay. We demonstrated that leptin (50-100 ng/ml) significantly stimulates proliferation of the human breast cancer cell line T47-D (P<0.05). Western blot analysis indicated that leptin induces a time-dependent activation of mitogen-activated protein kinases (MAPKinase) 1 and 2 in T47-D cell line. Moreover, the specific MAPK-inhibitor PD 98059 blocked cell proliferation induced by leptin. In conclusion, we demonstrate that leptin receptors are expressed in breast cancer and that leptin induces proliferation in the T47-D cell line via activation of the MAPKinases pathway. These data suggest that leptin and its receptors may be implicated in mammary cell proliferation and in breast cancer pathogenesis.

Proteomics of breast cancer for marker discovery and signal pathway profiling.

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

Expression of BRCA1 gene in ewe mammary epithelial cells during pregnancy: regulation by growth hormone and steroid hormones.

OBJECTIVE: Steroid hormones (estradiol and progesterone) in association with prolactin and growth hormone are involved in lobulo alveolar development of the mammary gland during pregnancy. We hypothesized that the BRCA1 gene may be induced by these different hormones. METHODS AND RESULTS: In this study, we have demonstrated by Northern blot and in situ hybridization, that the expression of ovine (o) BRCA1 mRNA in mammary epithelial cells increased dramatically during a short period in the second half of pregnancy (days 70 to 112) and decreased at the end of pregnancy. The increase in oBRCA1 mRNA expression is concomitant with rapid lobulo alveolar growth. Using an in vivo protocol to artificially induce mammary gland development, we demonstrated by the real-time RT-PCR method that growth hormone in association with estrogen, progesterone and hydrocortisone induces an increase of BRCA1 mRNA expression in the ewe mammary gland. Moreover, we showed that estradiol and progesterone induce oBRCA1 expression in primary cultures of ewe mammary gland. CONCLUSIONS: These results suggest that BRCA1 is a potential regulator of the effects of steroid hormones and growth hormone in the induction of mammary epithelial cell proliferation.

[Proteomic analysis: why and how ?]. / Analyse protéomique: pourquoi-comment?

The proteome, first formalized in 1995, designs all the proteins expressed by the genome of a cell, tissu, or organ at a defined time. Proteomic analysis leads to a description of the regulation of gene expression by the study of proteins and of their post-translational modifications. Proteomic analysis is based on three technologies: 1) Two-dimensional electrophoresis allowing the separation of thousands of proteins from a single mixture; 2) mass spectrometry allowing the characterization of picoquantities of polypeptides and providing data on post-translational modifications; 3) Bioinformatic which is required for the quantification of protein level and for the constitution of databases of protein expression profiles. Complementing the methods of the genomics, the use of proteomic analysis is widely spreading in the fields of fundamental biology, biomedicine and pharmacology for the identification of new biological markers and therapeutic targets.

Expression of nerve growth factor receptors and their prognostic value in human breast cancer.

Nerve growth factor (NGF) has been shown recently to be mitogenic for human breast cancer cells. In the present study, we have assayed the expression of NGF receptors (NGFRs: TrkA and p75) mRNAs in 363 human primary breast cancers, using real-time quantitative reverse transcription-PCR. NGFRs were found in all of the tumor biopsies. TrkA and p75 were positively correlated and were respectively associated with the histoprognostic grading and the tumor type. NGFRs were both related to progesterone receptors. In univariate analyses, TrkA (>upper quartile) was associated with longer overall survival. Histoprognostic grading, tumor size, node involvement, and steroid receptors were also prognostic factors. In Cox multivariate analyses, TrkA was not a prognostic parameter. This study demonstrates the expression of NGFRs in breast cancer and points out that patients with high levels of TrkA have a more favorable overall survival prognosis.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways.

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-kappaB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

Proteomic analysis reveals that 14-3-3sigma is down-regulated in human breast cancer cells.

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, we have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 were the same in both normal and transformed cells. The data support the idea that 14-3-3sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

Proteomic detection of changes in protein synthesis induced by fibroblast growth factor-2 in MCF-7 human breast cancer cells.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of breast cancer cell growth through stimulation of tyrosine kinase receptors and activation of the mitogen-activated protein kinase cascade. In the present study, we have investigated changes in protein synthesis induced by FGF-2 stimulation of the prototypic human breast cancer cell line MCF-7. Using high-resolution two-dimensional electrophoresis of (35)S amino acid metabolically labeled proteins and computerized analysis of 2D autoradiograms, we found that four proteins were up-regulated within the first 12 h of FGF-2 stimulation. Mass spectrometry analysis (MALDI-TOF and MS-MS) of tryptic fragments and database searches allowed the identification of these FGF-2-regulated proteins as the heat shock proteins HSP90 and HSP70, the proliferating cell nuclear antigen (PCNA), and the transcriptionaly controlled tumor protein (TCTP). We then analyzed the distribution of these proteins in various cancerous and normal breast epithelial cells. Interestingly, the four FGF-2-regulated proteins were found to be constitutively up-regulated in ras-transfected MCF-7 cells, indicating their relevance to the up-regulation of cellular proliferation. Moreover, HSP90 and PCNA were found at higher levels in cancerous cells than in normal cells. The role of HSP90 was further investigated using the specific inhibitor geldanamycin. We showed that the functionality of HSP90 is strictly required in order to obtain FGF-2 mitogenic stimulation in MCF-7 cells, indicating the crucial role played by this molecular chaperone in the control of breast cancer cell growth. Finally, these results show that proteomic analysis is a valuable method for identifying potential markers or therapeutic targets related to cancer growth.

Prognostic value of the type I growth factor receptors in a large series of human primary breast cancers quantified with a real-time reverse transcription-polymerase chain reaction assay.

We measured the expression of the type I growth factor receptor gene family [epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3 and c-erbB-4] in a series of 365 unselected primary breast cancers. The expression was quantified with a real-time one-step reverse transcriptase-PCR (RT-PCR) assay, based upon the 5' nuclease activity of the Taq polymerase and using an Abi Prism 7700 Sequence Detector System (Perkin-Elmer, Courtaboeuf, France). c-erbB-3 and c-erbB-4 were positively correlated to each other (Spearman test) and negatively correlated to EGFR. EGFR and c-erbB-2 were inversely correlated to the presence of estradiol receptors (ER) and progesterone receptors (PgR), and positively correlated to the histoprognostic grading (HPG). Conversely, c-erbB-3 and c-erbB-4 were positively correlated to the presence of ER and PgR, and inversely correlated to the grading HPG. EGFR was inversely related (chi2 test) to the presence of ER and PgR, and positively associated with HPG. In contrast, both c-erbB-3 and c-erbB-4 were inversely related to HPG, and positively associated with the presence of ER and PgR. The expression level of EGFR and c-erbB-2 was significantly higher in ER- and PgR-negative tumors compared with ER- and PgR-positive tumors (Student's t test), and in tumors with higher grade compared with tumors with lower grade. The expression level of c-erbB-3 and c-erbB-4 was significantly higher in ER- and PgR-positive tumors compared with ER- and PgR-negative tumors and in tumors with lower grade compared with tumors with higher grade. In overall survival studies, Cox univariate analyses showed prognostic values of EGFR [> or = median; P = 0.026; risk ratio (RR), 1.6], c-erbB-3 (> or = median; P = 0.0093; RR, 0.58), c-erbB-4 (> or = median; P = 0.0024; RR, 0.52), HPG, node involvement, tumor diameter, ER, and PgR. In Cox multivariate analyses, tumor diameter, ER, and PgR had a prognostic value. In relapse-free survival studies, univariate analyses demonstrated prognostic values of tumor diameter, node involvement, and c-erbB-4 (P = 0.015; RR, 0.65). These three parameters maintained their prognostic value in multivariate analyses (c-erbB-4, P = 0.035; RR, 0.67). This study confirms that EGFR expression and c-erbB-2 expression are markers of tumor aggressiveness in breast cancer. Conversely, we demonstrate that c-erbB-3 and c-erbB-4 elevated expressions are associated with a better prognosis.

Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status.

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC(50)values ranging from 0.2-22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r = 0.880, P = 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity.

A real-time one-step reverse transcriptase-polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer.

We developed a real-time one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method for the routine quantification of c-erbB-2 oncogene expression in breast cancer, using a 7700 ABI PRISM Sequence Detector System (Perkin Elmer-Applied Biosystems, Courtaboeuf, France). The real-time quantification of the polymerase chain reaction products is based on the TaqMan 5' nuclease assay. The optimal experimental conditions we determined were as follows: 6 mM MgCl2, 200 nM of fluorogenic probe, 200 nM of each primer, and 12.5 units MuLV reverse transcriptase. The GAPDH housekeeping gene was used for normalization of c-erbB-2 expression. In human breast cancer cell lines, the normalized expression of c-erbB-2 ranged from 8 x 10(-6) to 2,600 x 10(-6), the two highest values corresponding to the c-erbB-2 overexpressing cells MDA-MB-453 and SK-BR-3. In a series of 100 breast cancer samples, c-erbB-2 normalized expression was found to range from 0.4 x 10(-6) to 350 x 10(-6). A close correlation was observed between this real-time one-step quantitative RT-PCR method and both semiquantitative conventional RT-PCR (N = 22; r = 0.8543; P < .0001) and c-erbB-2 protein expression (p185) quantified by an enzyme immunoassay (EIA) (N = 27; r = 0.71; P < .0001). The current realtime RT-PCR assay is rapid, sensitive, and reproducible and appears particularly suitable to quantify gene expression in large series of samples.

Glyceraldehyde-3-phosphate dehydrogenase gene expression in human breast cancer.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been widely used as a control RNA in Northern blotting and in reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We investigated the expression of GAPDH in a large series of primary breast cancers and in MCF7 human mammary epithelial breast cancer cells treated with oestradiol. The expression of GAPDH was quantified by a real-time one-step RT-PCR assay, based upon the 5' nuclease activity of Taq polymerase using an Abi Prism 7700 Sequence Detector System (Perkin Elmer, France). Using the Spearman test, GAPDH expression was found to correlate inversely with the age of the patients at diagnosis (P = 0.003; r = -0.147), oestradiol receptors (ER) (P<0.0001; r = -0.327) and progesterone receptors (PgR) (P < 0.0001; r = -0.206). A positive correlation was observed between GAPDH expression and the histo-prognostic grading (HPG) (P < 0.0001; r = 0.344). Moreover, the overall survival (OS) and the relapse-free survival (RFS) were significantly reduced in patients whose tumours showed an enhanced level of GAPDH expression (OS, P = 0.046; RFS, P = 0.021). Multivariate analyses demonstrated that GAPDH was not an independent prognostic factor. Finally, in MCF7 cells treated with oestradiol. a statistically significant dose-dependent increase in GAPDH expression was observed. These results show that GAPDH expression is associated with breast cancer cell proliferation and with the aggressiveness of tumours. The present study demonstrates that, in cancer, the use of GAPDH gene expression should not be used as a control RNA.

Increased concentrations of the circulating angiogenesis inhibitor endostatin in patients with systemic sclerosis.

OBJECTIVE: Endostatin is an angiogenesis inhibitor derived from type XVIII collagen. The aim of this study was to determine the concentrations of circulating endostatin in patients with systemic sclerosis (SSc), and to assess the relationship between these concentrations, extension of tissular sclerosis, and presence of cutaneous scars or ulcers. METHODS: The study involved 50 patients with SSc and 30 healthy subjects. Cutaneous extension of sclerosis was graded according to Barnett's classification system: 33 patients had grade I SSc and 17 patients had grades II or III SSc. The results of pulmonary function tests were abnormal in 31 of 50 patients, 8 of whom also had abnormalities on chest radiograms. Cutaneous scars or ulcers were found in 22 of 50 patients. Endostatin concentrations were determined using a competitive enzyme immunoassay method. RESULTS: The mean circulating endostatin concentration was significantly higher in the SSc group than in the healthy subjects group (mean +/- SD 53.2 +/- 22.4 ng/ml versus 9.9 +/- 9.7 ng/ml; P < 10(-4)), in patients with grade II or grade III SSc than in patients with grade I SSc (63.2 +/- 20.2 ng/ml versus 45.1 +/- 15.6 ng/ml; P < 10(-2)), in patients with abnormal findings on chest radiograms than in patients with normal findings on chest radiograms (67.6 +/- 22.4 ng/ml versus 50.4 +/- 21.6 ng/ml; P < 0.05), and in patients with cutaneous scars or ulcers than in patients without these manifestations (60.9 +/- 25.9 ng/ml versus 47.2 +/- 13.3 ng/ml; P < 10(-2)). CONCLUSION: Circulating endostatin concentrations are significantly increased in patients with SSc. Production of endostatin may result from tissular sclerosis and could contribute to the development of ischemic manifestations.