BIRC5 (survivin): a pejorative prognostic marker in stage II/III breast cancer with no response to neoadjuvant chemotherapy.

PURPOSE: Neoadjuvant systemic therapy (NAC) is currently used in the treatment of stage II/III breast cancer. Pathological complete response as a surrogate endpoint for clinical outcomes is not completely validated for all subgroups of breast cancers. Therefore, there is a need for reliable predictive tests of the most effective treatment. METHODS: We used a combination of predictive clinical, pathological, and gene expression-based markers of response to NAC in a prospective phase II multicentre randomized clinical trial in breast cancer patients, with a long follow-up (8 years). This study concerned the subpopulation of 188 patients with similar levels of pathological response rates to sequential epirubicin/cyclophosphamide and docetaxel to determine predictive marker of pCR and DFS. We used a set of 45 genes selected from high throughput analysis and a standardized RT-qPCR. We analyzed the predictive markers of pathological complete response (pCR) and DFS in the overall population and DFS the subpopulation of 159 patients with no pCR. RESULTS: In the overall population, combining both clinical and genomic variables, large tumor size, low TFF1, and MYBL2 overexpression were significantly associated with pCR. T4 Stage, lymphovascular invasion, negative PR status, histological type, and high values of CCNB1 were associated with DFS. In the no pCR population, only lymphovascular invasion and high values of BIRC5 were associated with DFS. CONCLUSIONS: We confirm the importance of ER-related and proliferation genes in the prediction of pCR in NAC-treated breast cancer patients. Furthermore, we identified BIRC5 (survivin) as a main pejorative prognostic factor in patients with breast cancers with no pCR. These results also open perspective for predictive markers of new targeted therapies.

Challenging single- and multi-probesets gene expression signatures of pathological complete response to neoadjuvant chemotherapy in breast cancer: experience of the REMAGUS 02 phase II trial.

This study was designed to identify predictive signatures of pathological complete response (pCR) in breast cancer treated by taxane-based regimen, using clinicopathological variables and transcriptomic data (Affymetrix Hgu133 Plus 2.0 devices). The REMAGUS 02 trial (n = 153,training set) and the publicly available M.D. Anderson data set (n = 133, validation set) were used. A re-sampling method was applied. All predictive models were defined using logistic regression and their classification performances were tested through Area Under the Curve (AUC) estimation. A stable set of 42 probesets (31 genes) differentiate pCR or no pCR samples. Single-or 2-probesets signatures, mainly related to ER pathway, were equally predictive of pCR with AUC greater then 0.80. Models including probesets associated with ESR1, MAPT, CA12 or PIGH presented good classification performances. When clinical variables were entered into the model, only CA12 and PIGH, remained informative (p = 0.05 and p = 0.005) showing that a combination of a few genes provided robust and reliable prediction of pCR.

Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab.

BACKGROUND: Phosphatidylinositol 3-kinase (PI3K) pathway activation has been suggested to negatively influence response to anti-HER2 therapy in breast cancer patients. The present study focused on mutations of the PIK3CA gene, encoding one of the two PI3K subunits. METHODS: PIK3CA mutations were assessed by direct sequencing in 80 HER2-positive patients treated with 1 year of trastuzumab. All patients preoperatively received four cycles of anthracycline-based chemotherapy, followed by four cycles of docetaxel and 1 year of trastuzumab, starting either before surgery with the first cycle of docetaxel and continuing after surgery (neoadjuvant trastuzumab arm, n=43), or only after surgery (adjuvant trastuzumab arm, n=37). RESULTS: PIK3CA mutations were found in 17 tumours (21.3%). Better disease-free survival (DFS) was observed in patients with PIK3CA wild-type compared with mutated tumours (P=0.0063). By combining PIK3CA status and treatment arms, four separate prognostic groups with significantly different DFS (P=0.0013) were identified. CONCLUSION: These results confirm that the outcome of HER2-positive patients treated with trastuzumab is significantly worse in patients with PIK3CA-mutated compared with wild-type tumours.

A nomogram to predict individual prognosis in node-negative breast carcinoma.

BACKGROUND: Currently, the benefit of chemotherapy (CT) in node-negative breast carcinoma (NNBC) is discussed. The evaluation of classical clinical and histological factors is limited to assess individual outcome. A statistical model was developed to improve the prognostic accuracy of NNBC. METHODS: A total of 305 node-negative breast carcinomas who underwent surgery (+/- radiotherapy) but no adjuvant treatment were selected. Putative prognosis factors including age, tumour size, oestrogen receptor (ER), progesterone receptor (PgR), Scarff-Bloom-Richardon (SBR) grading, urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and thymidine kinase (TK) were evaluated. The developed model was internally validated using Harrell's concordance index. A prognosis index (PI) was proposed and compared with Adjuvant! Online program. RESULTS: Age (p < 0.001), pathological tumour size (pT) (p < 0.001), PgR (p = 0.02), and PAI-1 (p ≤ 0.001) were included in the Cox regression model predicting Breast cancer specific survival (BCSS) at 5-years. Internal validation revealed a concordance index of 0.71. A PI score was derived from our nomogram. The PI score was significantly associated with BCSS (hazard ratio (HR): 4.1 for intermediate, p=0.02, HR: 8.8, p < 0.001 for high group) as compared to Adjuvant! Online score (HR: 1.4, p=0.14). CONCLUSION: A nomogram can be used to predict probability survival curves for individual breast cancer patients.

Psychological distress and delusional memories after critical care: a literature review.

BACKGROUND: A considerable number of intensive care unit (ICU) survivors report delusional memories, which refer to dreams, nightmares, paranoid delusions and hallucinations experienced in the ICU. These memories often have a strong vividness, long duration and high emotional impact. AIM: The aim of this review was to investigate and synthesize published literature about psychological distress associated with delusional memories of adult ICU survivors. METHODS: Using key terms, a search was conducted in major health care electronic databases [Cumulative Index for Nursing and Allied Health Literature (CINAHL), PubMed, Web of Science and PsycInfo] focusing on articles published between 1990 and 2009 in English-language journals. FINDINGS: Ten articles met the inclusion criteria. Recall of delusional memories at various intervals after ICU discharge was associated with post-traumatic stress disorder (PTSD)-related symptoms in many studies, while associations with other aspects of psychological distress, mainly feelings of fear, anxiety and depression, were also reported. Recent studies did not seem to confirm the protective role of factual memories. CONCLUSIONS: The findings support the association between delusional memories and PTSD-related symptoms, but further research is needed to confirm their association with other psychological disorders. Development of a safety sense in the ICU can protect patients against the emotional impact of both delusional and stressful factual ICU memories. Appropriate follow-up of high-risk patients could improve their long-term psychological recovery.

Prolonged tamoxifen treatment increases relapse-free survival for patients with primary breast cancer expressing high levels of VEGF.

Previous retrospective studies have shown that high intratumoural levels of vascular endothelial growth factor (VEGF) correlate with an inferior outcome for patients treated with adjuvant tamoxifen. Our objectives were to validate the impact of VEGF on survival after adjuvant tamoxifen and to investigate the interaction between VEGF and treatment duration. For this purpose tumour homogenates from 402 patients with operable oestrogen receptor positive breast cancer (BC), treated with tamoxifen for 2 (n=149) or 5 years (n=253) as the only systemic adjuvant therapy were included. The median follow-up time for surviving patients was 9.8 years (range 0.5-14.8 years). Expression of VEGF was assessed by an enzyme-linked immunosorbent assay and investigated in relation to the standard BC parameters and survival. In the total population, higher VEGF was significantly correlated with shorter recurrence-free survival (RFS) (HR=1.63, 95%CI=1.11-2.39, p=0.010), breast cancer corrected survival (BCCS) (HR=1.82, 95%CI=1.13-2.93, p=0.014) and overall survival (OS) (HR=1.51, 95%CI=1.11-2.05, p=0.009). High VEGF was significantly associated with reduced RFS (HR=2.61, 95%CI=1.45-4.70, p=0.001) after two years of tamoxifen, whilst no difference was seen in patients treated for five years (HR=1.09, 95%CI=0.64-1.84, p=0.760). A statistically significant interaction was observed between high VEGF expression and improved RFS after 5-year tamoxifen (p=0.034). In concordance with previous studies, high VEGF was significantly correlated with shorter survival. We present data not reported previously revealing that patients expressing high levels of VEGF display a better outcome provided that tamoxifen is given for five years. Further studies on the impact of VEGF on a 5-year regimen are motivated.

Methylated genes as new cancer biomarkers.

Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

Harmonisation of multi-centre real-time reverse-transcribed PCR results of a candidate prognostic marker in breast cancer: an EU-FP6 supported study of members of the EORTC - PathoBiology Group.

AIM: Assessment of intra- and inter-laboratory variation in multi-centre real-time reverse-transcribed PCR (qRT-PCR)-based mRNA quantification of a prognostic marker in breast cancer using external quality assurance (EQA). METHODS: A questionnaire on the methodologies used and EQA calibrators were sent to 5 participating laboratories from 4 European countries, which measured mRNA levels of PITX2 splice variants and reference genes by qRT-PCR. RESULTS: Differences in the methodology included PCR quantification methodology and equipment, RNA extraction and cDNA synthesis procedures. The intra-laboratory coefficient of variation (CV) ranged from 5 to 23%, and the inter-laboratory CV ranged from 17 to 30%. The inter-laboratory CV was reduced to 13% by using prediluted calibrators and by harmonising the data in the central QA laboratory. Additional normalisation using reference genes did not decrease the variation further. CONCLUSIONS: Both externally provided calibrators and centralised harmonisation are required to reduce the intra-laboratory variation in multi-centre qRT-PCR results to an acceptable level.

Oligonucleotide microarray analysis of estrogen receptor alpha-positive postmenopausal breast carcinomas: identification of HRPAP20 and TIMELESS as outstanding candidate markers to predict the response to tamoxifen.

The estrogen receptor alpha (ER alpha) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER alpha status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. In this aim, we used a two-step strategy. We first screened genes of interest by a pangenomic 44 K oligonucleotide microarray in a series of ten ER alpha-positive tumors from five tamoxifen-treated postmenopausal patients who relapsed (distant metastasis) and five tamoxifen-treated postmenopausal patients who did not relapse, matched with respect to age, Scarff-Bloom-Richardson grade, lymph node status, and macroscopic tumor size. Genes of interest (n=24) were then investigated in an independent well-characterized series of ER alpha-positive unilateral invasive primary breast tumors from postmenopausal women who received tamoxifen alone as adjuvant hormone therapy after primary surgery. We identified four genes (HRPAP20, TIMELESS, PTPLB, and MGC29814) for which high mRNA levels were significantly associated with shorter relapse-free survival (log-rank test). We also showed that hormone-regulated proliferation-associated 20 kDa protein (HRPAP20) and TIMELESS are 17beta-estradiol-regulated in vitro and are ectopically expressed in OH-Tam-resistant cell lines. In conclusion, these findings point to HRPAP20 and TIMELESS as promising markers of tamoxifen resistance in women with ER alpha-positive breast tumors.

Identification of novel genes that co-cluster with estrogen receptor alpha in breast tumor biopsy specimens, using a large-scale real-time reverse transcription-PCR approach.

The estrogen receptor alpha (ERalpha) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERalpha-positive and ERalpha-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERalpha-positive breast tumors compared with ERalpha-negative breast tumors. In addition to well-known ERalpha-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERalpha-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERalpha in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

Gene transcript assay by real-time RT-PCR in epithelial breast cancer cells selected by laser microdissection.

The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERalpha, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor homogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.

Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma.

In a retrospective study of 488 women with primary breast cancer, after a median follow-up of 10 years, we sought interactions between disease-free survival (DFS) and overall survival (OS) and tumor antigen levels of two components of the plasminogen system, urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and the transmembrane growth factor receptor c-erbB-2. We used ELISAs (American Diagnostica, Greenwich, CT, USA) to quantify uPA and PAI-1 antigen levels in cytosols, and a double monoclonal antibody-based assay (EIA) (Ciba Corning Diagnostics, Alameda, CA, USA) to quantify c-erbB-2 in membrane extracts of the same tissues. Weak positive correlations were found between uPA and c-erbB-2 (r(s) = 0.146; p = 0.001) and between PAI-1 and c-erbB-2 (r(s) = 0.154; p < 0.001). In the overall population, using univariate analyses, c-erbB-2 overexpression and high uPA and PAI-1 antigen levels (> 300 IU/mg, > 1.40 ng/mg and > 5.53 ng/mg, respectively) were significantly associated with shorter DFS (p = 0.003, p < 0.001 and p < 0.001, respectively) and OS (p < 0.001 in all cases). Using multivariate analyses, PAI-1, node status and tumor size were independent predictors of DFS and c-erbB-2 was retained in the model only for OS. In the node-negative subgroup, PAI-1 was the strongest significant survival predictor both for OS (p = 0.003; HR 2.52) and DFS (p < 0.001; HR 2.39). This study shows that in primary breast cancer c-erbB-2 offers no additional prognostic information when uPA and/or PAI-1 are candidates in the multivariate analyses.

[RNA isolation and purification methods]. / Les méthodes d'extraction et de purification des ARN.

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.

["Standards, Options and Recommendations 2001" for radiotherapy in patients with non-metastatic infiltrating breast cancer. Update. National Federation of Cancer Campaign Centers (FNCLCC)]. / Standards, Options et Recommandations 2001 pour la radiothérapie des patientes atteintes d'un cancer du sein infiltrant non métastatique, mise à jour. Fédération nationale des centres de lutte contre le cancer (FNCLCC).

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of french cancer centers (FNCLCC), the 20 french cancer centers, and specialists from french public universities, general hospitals and private clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and the outcome of cancer patients. The methodology is based on a literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To develop clinical practice guidelines for non metastatic breast cancer patients according to the definitions of the Standards, Options and Recommendations project. METHODS: Data were identified by searching Medline, web sites, and using the personal reference lists of members of the expert groups. Once the guidelines were defined, the document was submitted for review to 148 independent reviewers. RESULTS: This article presents the chapter radiotherapy resulting from the 2001 update of the version first published in 1996. The modified 2001 version of the standards, options and recommendations takes into account new information published. The main recommendations are: (1) Breast irradiation after conservative surgery significantly decrease the risk of local recurrence (level of evidence A) and the decrease in the risk of local recidive after chest wall irradiation is greater as the number of risk factors for local recurrence increases (level of evidence A). (2) After conservative surgery, a whole breast irradiation should be performed at a minimum dose of 50 Gy in 25 fractions (standard, level of evidence A). (3) A boost in the tumour bed should be performed in women under 50 years, even if the surgical margins are free (standard, level of evidence B). (4) Internal mammary chain irradiation is indicated for internal or central tumours in the absence of axillary lymph node involvement (expert agreement) and in the presence of lymph node involvement (standard, level of evidence B1). (5) Sub- and supra-claviculr lymph node irradiation is indicated in patients with axillary node involvement (standard, level of evidence B1).

Real-time reverse transcription PCR assay of CYP19 expression: application to a well-defined series of post-menopausal breast carcinomas.

Aromatase, the product of the CYP19 gene, plays a key role in androgenic steroids transformation into estrogens from various hormonal sensitive tissues. Thus, in situ expression of CYP19 has been suggested to be involved in breast tumor growth especially in post-menopausal patients.We developed a real-time quantitative RT-PCR assay based on fluorescent TaqMan methodology to quantify total CYP19 gene expression at the mRNA level in breast tumors. This method, based on nucleic acid quantification in homogeneous solutions, has the potential to become a standard in terms of its sensitivity, wide dynamic range and high-throughput capacity. In a well-defined series of 107 post-menopausal breast tumor samples, relative CYP19 mRNA levels ranged from 1 to 131. Among the four major CYP19 exon I-spliced transcripts, designated I.a, I.b, I.c and I.d, mRNA levels of the latter three correlated positively with total CYP19 mRNA levels. In ER alpha-positive breast tumors, CYP19 and ER alpha mRNA levels correlated negatively with each other (P=0.0078, r=-0.266), while CYP19 and ER beta mRNA levels correlated positively (P=0.00012, r=+0.388). Patients with high CYP19 mRNA levels did not relapse more frequently or have shorter relapse-free survival than other patients. Finally, mRNA levels of IL6, a major CYP19 regulatory factor, were significantly higher in tumors strongly expressing CYP19 than in tumors weakly expressing CYP19 (P=0.018). In conclusion, CYP19 expression did not influence the outcome of post-menopausal patients with breast cancer.

The CGA gene as new predictor of the response to endocrine therapy in ER alpha-positive postmenopausal breast cancer patients.

We recently identified CGA (coding for the alpha subunit of glycoprotein hormones) as a new estrogen receptor alpha (ER alpha)-responsive gene in human breast tumors. Here, we assessed the relationship between CGA status (as determined by real-time quantitative RT-PCR) and the response to tamoxifen therapy in a well-defined cohort of 125 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. CGA overexpression, observed in 37.6% of patients, was associated with good relapse-free survival (P=0.037; univariate analysis). CGA status, combined with ERBB2 status (a marker of poor outcome), was an independent predictor of the response to tamoxifen (P=0.020; multivariate analysis). CGA status, especially when combined with ERBB2 status, may thus provide useful predictive information on tamoxifen responsiveness in breast cancer.