Plasmatic MMP9 released from tumor-infiltrating neutrophils is predictive for bevacizumab efficacy in glioblastoma patients: an AVAglio ancillary study.

We previously identified matrix metalloproteinase 2 (MMP2) and MMP9 plasma levels as candidate biomarkers of bevacizumab activity in patients with recurrent glioblastoma. The aim of this study was to assess the predictive value of MMP2 and MMP9 in a randomized phase III trial in patients with newly diagnosed glioblastoma and to explore their tumor source. In this post hoc analysis of the AVAglio trial (AVAGlio/NCT00943826), plasma samples from 577 patients (bevacizumab, n = 283; placebo, n = 294) were analyzed for plasma MMP9 and MMP2 levels by enzyme-linked immunosorbent assay. A prospective local cohort of 38 patients with newly diagnosed glioblastoma was developed for analysis of tumor characteristics by magnetic resonance imaging and measurement of plasma and tumor levels of MMP9 and MMP2. In this AVAglio study, MMP9, but not MMP2, was correlated with bevacizumab efficacy. Patients with low MMP9 derived a significant 5.2-month overall survival (OS) benefit with bevacizumab (HR 0.51, 95% CI 0.34-0.76, p = 0.0009; median 13.6 vs. 18.8 months). In multivariate analysis, a significant interaction was seen between treatment and MMP9 (p = 0.03) for OS. In the local cohort, we showed that preoperative MMP9 plasma levels decreased after tumor resection and were correlated with tumor levels of MMP9 mRNA (p = 0.03). However, plasma MMP9 was not correlated with tumor size, invasive pattern, or angiogenesis. Using immunohistochemistry, we showed that MMP9 was expressed by inflammatory cells but not by tumor cells. After cell sorting, we showed that MMP9 was expressed by CD45+ immune cells. Finally, using flow cytometry, we showed that MMP9 was expressed by tumor-infiltrating neutrophils. In conclusion, circulating MMP9 is predictive of bevacizumab efficacy and is released by tumor-infiltrating neutrophils.

Differential scanning calorimetry of plasma in glioblastoma: toward a new prognostic / monitoring tool.

Glioblastoma is the most frequent and aggressive primary brain tumor in adults. Recently, a growing number of studies have shown that denaturation profile of plasma samples obtained by differential scanning calorimetry (DSC) can represent a signature of a disease. In this study, we analyzed for the first time the DSC denaturation profiles of the plasma from patients with recurrent glioblastoma (n=17). Comparison to the one of healthy individuals (n=10) and to already described profiles in others cancer showed clear differences suggesting that this DSC profile may constitute a signature of glioblastoma. Parameters extracted from these profiles were used for cluster analysis which revealed the existence of glioblastoma profile subgroups which correlated with prognostic factors. Moreover, we showed that the presence of circulating bevacizumab and carmustine did not alter this calorimetric signature of the disease, indicating that an evolution of the profile could be followed without being masked by ongoing systemic treatment. Thus, our results constitute a very promising proof of principle that a specific calorimetric profile could be detected in the plasma of glioblastoma patients. Moreover, we believe that our findings point to a potential easy-to-use non-invasive monitoring tool for glioblastoma patients.

Outcome of Epstein-Barr virus-associated primary breast cancer.

The presence of the Epstein-Barr-virus (EBV) has been reported to be a pathogenic factor in breast cancer (BC). We previously demonstrated the aggressiveness of EBV-positive BC. The purpose of the present study was to evaluate the effect of EBV on the prognosis of BC according to the BC phenotype. A total of 117 patients with primary BC previously tested for the presence of EBV were evaluated. The presence of the virus was evaluated in breast specimens using quantitative PCR (qPCR). Disease-free survival (DFS) and overall survival (OS) were evaluated for 4 molecular subtypes, namely luminal A and B (lumA and lumB, respectively), human epidermal growth factor receptor 2 (HER2) and triple-negative (TN) subtypes and according to the EBV status. EBV positivity was observed in 32.5% of the cases. TN, HER2 and lumB tumours were more frequent among EBV-BC cases (P=0.02). The DFS rates were different between BC subtypes (P=0.002), but the differences were not statistically significant when the cases were stratified according to the EBV status (P=0.08 for EBV-negative and 0.06 for EBV-positive cases). The OS rates were similar for BC subtypes (P= 0.50) and when the cases were stratified according to the EBV status (P=0.16 and P=0.67 for EBV-positive and -negative cases, respectively). EBV was not associated with DFS or OS, in contrast to BC phenotypes, tumour size or nodal status. Therefore, EBV positivity was found to exert no effect on survival, despite its association with aggressive BC phenotypes.

Post-operative nomogram for predicting freedom from recurrence after surgery in localised breast cancer receiving adjuvant hormone therapy.

PURPOSE: To develop a prognostic nomogram to predict freedom from recurrence for patients treated with adjuvant hormonal therapy (HT) for localised breast cancer (BC). METHODS: We performed a retrospective analysis of 142 patients treated with adjuvant HT between 1996 and 2000. Clinical and pathological parameters were analysed. RESULTS: A nomogram that predicts the probability of remaining free of recurrence for 5 years after surgery with adjuvant HT was developed using a Cox proportional hazards regression model. The progesterone receptor status (p < 0.001), nodal status (p = 0.008) and cathepsin-D (p < 0.001) were retained to construct the nomogram (C-index 0.734). CONCLUSIONS: The nomogram we developed may be useful for estimating the probability of successful treatment 5 years after surgery for localised BC.

A nomogram predicting the probability of primary breast cancer survival at 2- and 5-years using pathological and biological tumor parameters.

PURPOSE: Our objective was to develop a nomogram to predict individual overall survival (OS) for primary breast cancer, based on pathological and biological tumor parameters. METHODS: A retrospective study in a cohort of 180 patients with primary breast cancer was used to build the nomogram. Pathological factors and tumor proteases measured prospectively in primary tumors were used. A multivariate Cox proportional hazards model was used to explore the relationship with OS, and regression coefficients were used to build the nomogram. The nomogram was internally validated with 200 bootstrap re-samples. RESULTS: The final variables included in the nomogram comprised tumor size (P = 0.04), nodal pathological status (P = 0.01), estrogen receptor status (P = 0.04), urokinase plasminogen activator inhibitor-1 (PAI-1; P = 0.02), thymidine kinase (P = 0.03), and cathepsin D (P = 0.004). The predictive accuracy of the nomogram at estimating the probability of OS, at both 2 and 5 years, was respectively 0.874 and 0.832 before and after calibration. CONCLUSION: A nomogram to predict 2- and 5-year OS in BC, using histological and biological parameters was successfully developed. This prognostic tool should prove useful in decision-making and therapeutic research.

Prognostic significance of tumor-related proteases as a function of the estrogen receptor status.

BACKGROUND: Tumor-related proteases such as urokinase-type plasminogen (uPA), plasminogen activator inhibitor (PAI-1), and cathepsin D (cath-D) are involved in the prognosis of breast cancer (BC) and promote the metastatic process. We investigated the relationship between these proteases and their prognostic significance according to the ER status. METHODS: uPA, PAI-1, and cath-D levels were prospectively measured in tumor from 316 patients with primary BC. The distribution and relationship between these proteases and ER subsets were analyzed as well as the prognostic significance. RESULTS: Quantitative levels of uPA, and PAI-1 were higher in ER-patients (p< 0.001) whereas no difference was observed for cath-D levels in ER subsets (p = 0.96). In patients with a positive and highly positive ER status, a high cath-D level was associated with a poorer prognosis. Patients in the highly positive ER group experienced poorer survival in the group with a high PAI-1 level compared to the group with a low PAI-1 level. In ER+ patients, cath-D (p= 0.02) and the tumor size (p= 0.03) were independent factors for OS. CONCLUSION: The prognostic significance of proteases is modulated by the ER status. Cath-D and PAI-1 could identify a high-risk group with an adverse outcome in ER+ patients.

Is quantitative oestrogen receptor expression useful in the evaluation of the clinical prognosis? Analysis of a homogeneous series of 797 patients with prospective determination of the ER status using simultaneous EIA and IHC.

OBJECTIVE: Oestrogen receptor (ER) determination in breast cancer (BC) is a major yardstick for the prognosis and for response to hormonal therapy (HT). As several techniques have been proposed for ER quantification, the purpose of our study was to assess whether the qualitative or quantitative analysis of ER expression might influence the prognosis and response to treatment. MATERIALS AND METHODS: We analysed overall survival (OS) and disease-free survival (DFS) in 797 primary BC cases with ER determination by enzyme immunoassay (EIA) and immunohistochemistry (IHC). The clinical impact according to qualitative or quantitative analysis of ER expression was assessed. Response to HT was evaluated according to quantitative EIA-determined ER expression levels. RESULTS: According to the qualitative analysis of ER expression, patients with EIA-determined and IHC-determined ER-positive tumours had significantly longer OS and DFS (p<0.001). The analysis stratified on quartiles of ER levels showed significantly different outcomes according to EIA- and IHC-determined subgroups. In the group of patients who received adjuvant treatment, 5-year OS was significantly different between the groups, with a clear benefit for the highest EIA-determined ER quartiles (p<0.001). Comparatively, in terms of 5-year DFS, a clear separation was noted between groups for adjuvant treatment (p<0.001). The group with moderate ER+ values was clearly distinct from the ER-negative population. Quantitative ER expression helped to better distinguish the beneficial or detrimental effect of HT within quartiles of ER-expressing tumours. Based on the STEPP analysis which showed a trend towards an ER effect on DFS as a function of HT assignment, we confirm the benefit of HT in patients with a very high EIA-determined ER level and a detrimental impact on negative and weakly positive groups. CONCLUSION: Quantitative ER expression in BC helps to better discriminate heterogeneity in clinical outcome and response to HT.

[The consortium of Biological Resource Centres (BRC) and tumour cell and tissue banks in the Marseilles metropolitan area]. / Le réseau des Centres de Ressources Biologiques (CRB) et tumorothèques de l'agglomération marseillaise.

The goal of this presentation is to describe current and future aspects of the operations within the consortium of Biological Resource Centres (BRC) and Tumour cell and tissue banks of the Marseilles metropolitan area. The consortium was created in year 2001, through the association of several tissue and cell banks that were operating for many years in Marseilles. Existing collections are not exclusively collections of tumour cells or tissues; however, the two tumour cell and tissue banks located at the Regional Cancer Research Centre and at the University Hospital account for a very significant proportion of the collections. Our collective work leads to the recognition and funding of the consortium by Inserm, through the "Collections 2003" grant. The consortium objectives are to define a common scientific strategy, to share professional practices in the logistics and database management of the banks, to establish a quality management program, and to build a common catalogue that describes existing biological resources. Through these efforts, the ultimate goal is to adopt rules that define BRC, as defined by the Organization for Economic Co-operation and Development (OECD).

[AP-HM tumour tissue bank: molecular signature of gliomas]. / Tumorothèque de l'AP-HM: cartographie moléculaire des gliomes.

The AP-HM tissue bank is the largest one in France regarding some collections, including brain tumors. This collection was used to better characterize some gliomas. In particular because some benign gliomas such as pilocytic astrocytomas (WHO grade I) can be misclassified as malignant ones such as glioblastomas (grade IV) the first aim of our study was to find accurate diagnostic markers. This was done mainly by suppressive substractive hybridization (SSH). This study also provides a restrictive list of genes selectively involved in angiogenesis and invasion, which were highly expressed in GBM. Results were confirmed by real time quantitative RT-PCR in a large cohort of patients. In addition in order to find accurate markers which can predict GBM overall survival (OS) we selected three cohorts of GBM patients with distinctive OS (short survival < 6 months, long survival > 18 months and intermediate). Quantification of a series of markers involved in angiogenesis and invasion was done as well as cDNA array analysis.

Clinical relevance of amphiregulin and VEGF in primary breast cancers.

The characterization of novel prognostic markers in breast cancer is necessary to improve the identification of high-risk populations. In our study, the prognostic significance of VEGF and amphiregulin (AR) was investigated and compared to conventional prognostic factors in primary breast cancers. The analysis was performed using enzyme-linked immuno-assay in a series of 193 patients, and univariate and multivariate analysis were performed in the overall population as well as in pre- and post-menopausal patients subdivided in node-negative (N-) and node-positive (N+) subsets. AR (median, 44.8 pg/mg protein) appeared strongly correlated with progesterone receptors (PgR) (p = 0.0018) in the premenopausal N+ population, and with uPA (p= 0.020) and VEGF (p= 0.0053) in the postmenopausal/N+ patients. Despite these attractive data, AR expression was not significant for recurrence or survival outcome. Data revealed strong correlation between VEGF and uPA, and PAI-1, in the N+ population. Moreover, patients with high VEGF levels displayed poor outcome, with an increased risk for N+ subset. These data were confirmed by multivariate analysis that presented histologic grade (HR, 10.55, p = 0.001) and VEGF (HR, 3.89, p = 0.03) as the prominent prognostic markers for overall survival for the N+ population. Furthermore, infiltrating ductal carcinomas (IDC) were shown to express higher levels of both uPA (p < 0.0001) and VEGF (p = 0.002) than intralobular carcinomas. This retrospective study reinforces the pejorative biological role of VEGF in the progression of breast tumors. Our data also suggest that VEGF and uPA might play particular role in the biology and progression of IDC.

Pooled analysis of prognostic impact of uPA and PAI-1 in breast cancer patients.

In this report we present an extension of the pooled analysis of the prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I in breast cancer patients. We analyzed a different endpoint, metastasis-free survival (MFS). We checked the consistency of the estimates for uPA and PAI-1 for relapse-free survival (RFS) and MFS exploring possible sources of heterogeneity. Nodal status, the most important prognostic factor for breast cancer, introduced heterogeneity in the uPA/PAI-1 survival analyses, reflecting the interaction between nodal status and uPA/PAI-1. The estimates for uPA and PAI-1 were found to be consistent, even when a different transformation of their values was used. The heterogeneity of the separate data sets decreased if the levels of uPA and PAI-1 were ranked, data sets were pooled, and the analyses corrected for the base model that included all traditional prognostic factors, and stratified by data set. We conclude that uPA and PAI-1 are ready to be used in the clinic to help classify breast cancer patients into high and low risk groups.

Transmission of apoptosis in human colorectal tumor cells exposed to capecitabine, Xeloda, is mediated via Fas.

We developed an original in vitro model dedicated to the exploration of molecular pharmacology of the new oral fluoropyrimidine capecitabine (Xeloda). More specifically, in this report, we investigated whether apoptosis induced by capecitabine was mediated by the Fas/FasL system. To achieve this goal, a specific in vitro coculture model mixing hepatoma and human colorectal cell line was used. A bystander effect was observed between HepG2 and LS174T cells treated with capecitabine. Besides this, Xeloda showed a 7-fold higher cytotoxicity and markedly stronger apoptotic potential in thymidine phosphorylase (TP)-transfected LS174T-c2 cells. The striking enhancement of thymidylate synthase inhibition that we observed in cells with high TP activity was most probably at the origin of the potentiation of capecitabine antiproliferative efficacy. In addition, this increase of sensitivity was accompanied by a strong overexpression of the CD95-Fas receptor on the cell surface. Both Fas and FasL mRNA expression were triggered after exposing TP+ cells to the drug. This implication of Fas in Xeloda-induced apoptosis was next confirmed by using antagonistic anti-Fas and anti-FasL antibodies that proved to reverse capecitabine antiproliferative activity, thus highlighting the key role that Fas could play in the optimization of an antitumor response to fluoropyrimidine drugs. Our data, therefore, show that TP plays a key role in the capecitabine activity and that the Fas/FasL system could be considered as a new determinant for Xeloda efficacy.

Prognostic assessment of PTK activity in T1-T2, N0-N1, M0 breast cancer: a multicentric retrospective study.

Protein tyrosine kinases (PTKs) play a major role in the transduction of intracellular mitogenic signal. PTKs are also involved in the process of cellular transformation. A number of studies have reported increased PTK activities in cytosolic fractions from human breast carcinoma. However, the possible pronostic value of these activities is difficult to establish from these studies, mostly conducted on limited numbers of patients. In order to clear up the issue, we have investigated a large series of patients with a long follow-up, using a retrospective multicentric study (894 breast cancers T1-T2, N0-N1, M0; median follow-up: 67 months). PTKs were measured using a radioenzymatic assay as described in our previously report. We confirmed the already observed correlation between PTK activities and Scarff-Bloom grading (p < 10(-5)), negative estrogen receptor (ER), and progesterone receptor (PR) status. By contrast, we found in this study a correlation between PTK values and clinical nodal status (p = 0.00027) not showed in our precedent analysis. In Cox multivariate analysis, PTK activity does not emerge as a significant pronostic parameter. On the other hand, tumor PTK activity assay may prove of great interest in clinical research using newly developed tyrosine kinase inhibitors in order to assess their biological impact and eventually to predict the responsiveness to these new therapeutic agents.

Flow cytometric S-phase fraction measurement in breast carcinoma: Influence of software and histogram resolution.

BACKGROUND: S-phase fraction (SPF) measurement by flow cytometry is a clinically useful prognostic factor in patients with breast carcinoma. Standardized SPF determination is essential. As part of a multicenter study, we evaluated the influence of the choice of software and histogram resolution (256, 512, or 1,024 channels) on SPF quantification. METHODS: One hundred thirty-three DNA histograms were analyzed in three laboratories with Modfit 5.2, Modfit LT, and Multicycle AV software. Strict rules for histogram interpretation and software management were applied. The following five options were compared: MF 5.2 1024, MF 5.2 256, MF LT 256, MC AV 256, and MC AV 512. RESULTS: In the DNA diploid and aneuploid groups, SPF distributions were not statistically different among the five options. Excellent quantitative correlations were obtained between pairs of options. When using tertiles as cutpoints for SPF classification, concordance rates ranged from 79.7% to 93.2% for DNA diploid samples and from 87.8% to 95.9% for DNA aneuploid samples, the best results being obtained with software working with a similar histogram resolution. CONCLUSIONS: Standardized use of commercially available software, including the choice of histogram resolution, provides comparable SPF results.

PAI-1 and EGFR expression in adult glioma tumors: toward a molecular prognostic classification.

PURPOSE: Molecular classification of gliomas is a major challenge in the effort to improve therapeutic decisions. The plasminogen activator system, including plasminogen activator inhibitor type 1 (PAI-1), plays a key role in tumor invasion and neoangiogenesis. Epidermal growth factor receptor (EGFR) is involved in the control of proliferation. The contribution of PAI-1 and EGFR to the survival of gliomas was retrospectively investigated. METHODS AND MATERIALS: Fifty-nine adult gliomas treated by neurosurgery and conventional irradiation were analyzed, including 9 low-grade (2) and 50 high-grade (3-4) tumors (WHO classification). PAI-1 was measured on cytosols and EGFR on solubilized membranes using ELISA methods. RESULTS: High PAI-1 levels were strongly associated with high histologic grade (p < 0.001) and histologic necrosis (p < 0.001). PAI-1 also correlated positively with patient age (p = 0.05) and negatively with Karnofsky index (p = 0.01). By univariate analysis of the high-grade population, higher PAI-1 (p < 0.0001) and EGFR values (p = 0.02) were associated with shorter overall survival. Only PAI-1 was an independent factor in multivariate analysis. Grade 3 tumors with low PAI-1 (100% 3-year overall survival rate) presented the same clinical outcome as the low-grade tumors. CONCLUSIONS: In this prognostic study, PAI-1 and EGFR expression revealed similarities and differences between high-grade gliomas that were not apparent by traditional clinical criteria. These data strongly support that biologic factors should be included in glioma classification and the design of clinical trials to treat more homogeneous populations.

Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). METHODS: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. RESULTS: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). CONCLUSIONS: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategies.