X. Trinh

The VEGF pathway and the AKT/mTOR/p70S6K1 signalling pathway in human epithelial ovarian cancer

Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples (N=86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) (R=0.29; P=0.002). Additional gene expression analysis in an independent cDNA microarray dataset (N=24) showed a negative correlation (R=−0.73, P<0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites (χ2, P=0.002) and reduced overall survival of cisplatin–taxane-based patients with serous histology (N=32, log-rank test, P=0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

Quantitative methylation profiling in tumor and matched morphologically normal tissues from breast cancer patients.

BackgroundIn the present study, we determined the gene hypermethylation profiles of normal tissues adjacent to invasive breast carcinomas and investigated whether these are associated with the gene hypermethylation profiles of the corresponding primary breast tumors.MethodsA quantitative methylation-specific PCR assay was used to analyze the DNA methylation status of 6 genes (DAPK, TWIST, HIN-1, RASSF1A, RARβ2 and APC) in 9 normal breast tissue samples from unaffected women and in 56 paired cancerous and normal tissue samples from breast cancer patients.ResultsNormal tissue adjacent to breast cancer displayed statistically significant differences to unrelated normal breast tissues regarding the aberrant methylation of the RASSF1A (P = 0.03), RARβ2 (P = 0.04) and APC (P = 0.04) genes. Although methylation ratios for all genes in normal tissues from cancer patients were significantly lower than in the cancerous tissue from the same patient (P ≤ 0.01), in general, a clear correlation was observed between methylation ratios measured in both tissue types for all genes tested (P < 0.01). When analyzed as a categorical variable, there was a significant concordance between methylation changes in normal tissues and in the corresponding tumor for all genes tested but RASSF1A. Notably, in 73% of patients, at least one gene with an identical methylation change in cancerous and normal breast tissues was observed.ConclusionsHistologically normal breast tissues adjacent to breast tumors frequently exhibit methylation changes in multiple genes. These methylation changes may play a role in the earliest stages of the development of breast neoplasia.

Comparison of molecular determinants of angiogenesis and lymphangiogenesis in lymph node metastases and in primary tumours of patients with breast cancer.

Angiogenesis and lymphangiogenesis are complex processes, driven by multiple factors. In primary breast tumours (PTs), VEGFA, ‐C and ‐D are the most important (lymph)angiogenic factors. The induction of lymphangiogenesis in axillary lymph node (LN) metastases of patients with breast cancer was described recently. To compare the molecular determinants of (lymph)angiogenesis in LN metastases and PTs of breast cancer patients, RNA was isolated from formalin‐fixed, paraffin‐embedded tissue sections of a metastatically involved and uninvolved LN and the PT from 26 lymph node‐positive patients. The expression of 12 (lymph)angiogenic markers was measured by qRT–PCR. Expression was correlated with tumour cell proliferation, angiogenesis and lymphangiogenesis, quantified by tumour cell proliferation fraction (TCP%) and (lymphatic) endothelial cell proliferation fraction [(L)ECP%]. TCP%, ECP% and LECP% were assessed on immunohistochemical double stains for CD34/Ki‐67 and D2‐40/Ki‐67, respectively. In involved LNs, the relative gene expression levels of PROX1 (p < 0.001) and FGF2 (p = 0.008) were decreased and the expression levels of VEGFA (p = 0.01) and PDGFB (p = 0.002) were increased compared to uninvolved LNs. The expression of most markers was increased in PTs compared to involved LNs. In metastatically involved LNs, the expression of VEGFA correlated with ECP% (r = 0.54, p = 0.009) and LECP% (r = 0.76, p < 0.001). In PTs, VEGFA correlated only with ECP% (r = 0.74, p < 0.001). VEGFD correlated with peritumoural LECP% (r = 0.61, p = 0.001) and with VEGFC (r = 0.78, p < 0.001). Linear regression analysis confirmed the expression of VEGFA as an independent predictor of ECP% in both PTs and LN metastases and of LECP% in LN metastases. The expression of VEGFD, but not of VEGFA, independently predicted peritumoural LECP% in PTs. Our results confirm existing data that, in PTs, angiogenesis and lymphangiogenesis are respectively driven by VEGFA and VEGFD. In contrast, in LN metastases, both processes seem to be driven by VEGFA. Lymphangiogenesis in PTs and in LN metastases might thus be driven by different factors. Copyright

A dynamic clinical pathway for the treatment of patients with early breast cancer is a tool for better cancer care: implementation and prospective analysis between 2002-2010.

BackgroundDue to increasing the complexity of breast cancer treatment it is of paramount importance to develop structured care in order to avoid a chaotic and non-consistent management of patients. Clinical pathways, a result of the adaptation of the documents used in industrial quality management namely the Standard Operating Procedures, can be used to improve efficiency and quality of care. They also aim to re-centre the focus on the patient’s overall journey, rather than the contribution of each specialty or caring function independently.MethodsThe effect of the implementation and prospective systematic evaluation of a clinical care pathway for the management of patients with early breast cancer in a single breast unit is evaluated over a long time interval (between 2002 and 2010). Annual analysis of predefined clinical outcome measures, service indicators, team indicators, process indicators and financial indicators was performed. Pathway quality control meetings were organized at least once a year. Systematic feedback was given to the team members, and if necessary the pathway was adapted according to evidence based literature data and in house pathway related data in order to improve quality.ResultsThe annual number of patients included in the pathway (289 vs. 390, P <0.01), proportion of patients with Tis-T1 tumors (42% vs. 58%, P <0.01), negative lymph nodes (44% vs. 58%, P <0.01) and no metastases at diagnosis (91.5% vs. 95.9%) has risen significantly between 2002 and 2010. Evolution of mandatory quality indicators defined by EUSOMA shows a significant improvement of quality of cancer care. Particularly, the proportion of patients having anti-hormonal therapy (84.8% vs. 97.4%, P = 0.002) and adjuvant chemotherapy according to the guidelines (72% vs. 95.6%, P = 0.028) increased dramatically. Patient satisfaction improved significantly (P <0.05). Progression free 4-year survival was significantly higher for all patients, for T1 tumors only and for T2-T4 tumors only, treated between 2006 to 2008 compared to between 1999 to 2002 and 2003 to 2005 (P = 0.006, P = 0.05, P = 0.06, respectively). Overall 4-year survival of the entire population treated between 2006 and 2008 was significantly better (P = 0.05).ConclusionsAlthough the patient characteristics changed over the years due to better screening, this clinical pathway and regular audit of quality indicators for the treatment of patients with operable breast cancer proved to be important tools to improve the quality of care, patient satisfaction and outcome.

Quantitative assessment of DNA hypermethylation in the inflammatory and non-inflammatory breast cancer phenotypes

In this study, a comparative quantitative methylation profiling of inflammatory breast cancer (IBC) and non-IBC was set up for the identification of tumor-specific methylation patterns. Methylation ratios of six genes measured in benign breast tissues (n=9) and in tumor samples from non-IBC (n=81) and IBC (n=19) patients using quantitative methylation-specific PCR. Median methylation ratios observed in breast cancer (n=100) were significantly higher than those observed in benign breast tissues for 5 of 6 genes (TWIST, HIN-1, RASSF1A, RARβ2 and APC). Only one of the individual genes studied, RARβ2, showed differential methylation ratios in IBC and non-IBC (P=0.016). Using the maximal methylation ratio observed in benign breast tissue as a threshold, the methylation frequency of two genes, RARβ2 and APC, was significantly increased in IBC (n=19) when compared to non-IBC (n=81): 53% vs. 23% for RARβ2 (P=0.012) and 84% vs. 54% for APC (P=0.017). Using hierarchical clustering, methylation patterns could not classify breast cancers according to their phenotype. The finding of differential frequencies of methylation in IBC and non-IBC for 2 out of 6 genes suggests that gene-specific patterns of methylation could provide a basis for molecular classification of IBC Testing for additional genes could help to define the IBC phenotype based on patterns of aberrant gene promoter methylation.

Abstract P3-02-10: Comparison of Circulating Tumour Cells in Peripheral and Central Venous Blood of Patients with Metastatic Breast Cancer: A Pilot Study

Background: Enumeration of circulating tumour cells (CTC), as performed by the CellSearch System (Veridex, Raritan, NJ, USA), has prognostic significance in patients with metastatic breast cancer (MBC). Questions remain on how these cells - if all - are capable to survive in the circulation and ultimately give rise to distant metastases. Experiments in mouse models have shown that CTC only appear transiently in the circulation after injection in the tail vein or left ventricle. Data on the discrete localisation of CTC in the blood stream and CTC kinetics in human breast cancer is still limited. The aim of this pilot study was to directly compare the amount of CTC measured in peripheral and central venous blood samples of patients with MBC in order to reveal possible differences. Methods: So far, we have included 18 patients with MBC presenting at our hospital with either primary metastatic (N=2) or progressive disease (N=16). Most patients suffered from diffuse metastatic involvement and were extensively pretreated. Clinically evident lung metastases were documented in 4/18 patients. Peripheral venous blood (PVB) - obtained from an antecubital vein - and central venous blood (CVB) samples - obtained from the subcutaneous port catheter - were drawn into CellSave Preservative tubes (Immunicon, Huntingdon Valley, PA) and processed in parallel using the CellSearch Circulating Tumor Cell Test (Veridex, Raritan, NJ, USA). With this test, CTC captured from 7.5 mL whole blood, are defined as EpCAM+, nuclear cells (DAPI+), expressing cytokeratins 8/18/19 and lacking CD45 expression. Results: CTC were detected in both PVB and CVB samples of 16/18 patients. In 2 patients no CTC were detected, neither in PVB nor in CVB. In 16 patients with detectable CTC, the number of CTC was found to be significantly higher in CVB (median: 65; range: 3-4036) than in PVB (median: 45; range: 1-4013) samples (Wilcoxon Signed Ranks Test, P=0.001). When analyzing samples parewise, the number of CTC in CVB was higher than the number of CTC in PVB in 15/16 patients, although marginally in 4 of 15 patients (median ratio CVB/PVB: 1.8; range: 1.0-3.1). Only 1 patient showed a higher number of CTC in PVB (absolute value: 49 CTC/7.5 mL blood) as compared to CVB (absolute value: 41 CTC/7.5 mL blood). Conclusions: In this study, a substantial decrease in the number of CTC was observed between CVB and PVB of patients with MBC. This difference might be relevant in the clinical setting as a quantitative cut off in the number of CTC is used to distinguish between patients with good and worse prognosis. As hand metastases are most uncommon, the lungs are suggested to function as the main sieve, even in the absence of clinically overt lung metastases. However, further research in larger patient populations and - where feasible - including radial arterial blood sampling, is required in order to confirm these results and to investigate human CTC kinetics in depth. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-10.

A Phase II Study of the Combination of Endocrine Treatment and Bortezomib in Patients with Endocrine Resistant Metastatic Breast Cancer.

e11072 Background: The majority of patients with hormone receptor-positive metastatic breast cancer die from disease progression despite different types of anti-hormonal treatments. Preclinical studies have indicated that resistance to anti-hormonal therapies may be the result of an activated NF-κB signalling pathway in breast cancer. Bortezomib is a proteasome inhibitor that blocks the NF-κB pathway. Recent pharmacodynamic and pharmacokinetic xenograft studies have shown that drug exposure may be a crucial factor for the efficacy of bortezomib in solid tumours. METHODS The aim was to investigate whether the addition of bortezomib to anti-hormonal therapy would result in antitumour activity in patients with both progressive and measurable disease on the identical endocrine agent. Clinical benefit was defined as patients obtaining SD, PR or CR after 2 cycles, lasting for at least another five weeks. Bortezomib was administered on days 1, 8, 15, 22 of a 5 week regimen (1.6 mg/sqm). RESULTS Eight patients received an aromatase inhibitor and bortezomib, while one received tamoxifen and bortezomib. There were 3 grade III gastro-intestinal toxicities. Median time to treatment failure was 69 days [35-140]. Two out of the 9 patients had stable disease for more than 10 weeks. Despite an effective target inhibition, suggested in peripheral blood mononuclear cells and available tumour samples, no objective antitumour responses were observed. CONCLUSIONS Addition of a proteasome inhibitor to anti-hormonal therapy resulted in a clinical benefit rate of 22% in a limited number of patients with endocrine resistant and progressive metastatic breast cancer. The demonstrated proteasome inhibition in tumour tissue provides evidence that the lack of clinical responses is not attributed to deficient drug exposure. Eudract-N°2006-004144-23.

A microRNA Expression Profile Consisting of 15 microRNAs, Including miR205, Is Associated with Poor Prognosis in Breast Cancer

Introduction: MicroRNAs (miRNA) are a class of non-coding RNAs able to regulate gene expression at the post-transcriptional level. In breast cancer, levels of specific miRNAs differ between malignant and normal breast tissue and are able to classify tumors according to clinicopathologic variables. This highlights the potential of miRNAs as novel prognostic and/or predictive indicators. In this study we sought for miRNAs denoting poor prognosis in breast cancer.Materials and methods: 377 miRNAs were profiled in 70 breast tumor samples using the human MicroRNA A Array Set version 2.0 (Applied Biosystems). All miRNAs with Ct-values less than 35 in 25% of the samples were included for analysis. Data normalization was performed relative to the median miRNA expression level per sample and expression values were log2-transformed. Principal component analysis (PCA) was performed to identify metagenes of miRNAs associated with clinicopathological variables (TNM-status, tumor stage, histological grade, ER-, PR-, ErbB2- and P53-status) and prognostic/predictive gene signature-classifications (wound healing response, invasiveness gene signature, 70-gene prognostic signature, genomic grade index, recurrence score, HOXB13/IL17RB-expression ratio and the molecular breast cancer subtypes). Results were validated by investigating relationships between the expression of a relevant miRNA target gene signature and clinicopathological variables in 5 publicly available breast cancer gene expression data sets.Results: Using PCA-analysis we identified a metagene significantly associated with the Luminal B phenotype, an elevated genomic grade index, an elevated recurrence score, an activated wound healing response, the invasiveness gene signature and poor prognosis according to the 70-gene prognostic profile (range Rs: 0.325-0.372; P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4062.

Gene Promoter Hypermethylation in Tumors and Matched Affected Lymph Nodes of Breast Cancer Patients

Gene promoter hypermethylation plays a pivotal role in breast carcinogenesis and breast cancer progression. In this study, we investigated 6 gene promoters (RASSF1A, HIN-1, RARβ2, TWIST, CCND2 and APC) for aberrant DNA methylation by real-time methylation-specific PCR in primary tumors and matched affected lymph nodes from 30 patients with breast cancer. These data were used to explore associations between gene promoter hypermethylation and clinicopathological features, including tumor cell proliferation, angiogenesis and hypoxia marker expression.Overall, gene promoter methylation levels (ratios of DNA copy numbers for methylated and reference genes) were higher in primary tumors than in matched lymph nodes. Notably, we found significant correlations between methylation levels in primary tumors and matched lymph nodes for 5 of 6 genes (P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1149.

Prognostic Significance of Real-Time RT-PCR Detection of Disseminated Tumour Cells in Bone Marrow and Circulating Tumour Cells in Patients with Breast Cancer.

Background: Despite optimal staging and treatment, up to 40% of stage I and II breast cancer (BC) patients will develop recurrent disease over time. These patients are believed to have disseminated tumour cells (DTC) at the time of diagnosis. Detection of minimal disease in bone marrow (BM) has been suggested to be a more direct approach to select metastasis-prone patients among this 9good prognosis group9. Peripheral blood (PB) sampling however is more convenient. The aim of this study was to evaluate whether the detection of DTC in either PB or BM predicts overall survival (OS). The initial analysis was published with a mean follow up time of 786 days (Benoy et al., BrJC 2006). We now report on these data after a mean follow up of nearly 5 years.Material and methods: PB and BM samples were collected from 148 patients with primary (M0, n=116) and metastatic (M+, n=32) BC before the initiation of any local or systemic treatment. PB of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as control group. DTC were detected by measuring relative gene expression (RGE) for cytokeratin 19 (CK19) and mammaglobin (MAM), using a quantitative RT-PCR detection method. The 95 percentile of the RGE for CK19 and MAM of the control group was used as cutoff to determine elevation in BC patients. Kaplan-Meier analysis was used to predict OS.Results: Mean follow up time was 1518 days (+/- 719). Elevated CK19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. MAM expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK19 or MAM expression in BM had a worse OS than patients without elevated expression levels (p=0.002 (CK19) and p=0.001 (MAM)). For PB, no statistical significant difference in OS was observed between patients with or without elevated CK19 (p=0.227), but a strong trend for predicting OS was observed according to MAM status (p=0.054). Separate analyses of M0 and M+ patients revealed only a marked difference in OS according to BM CK19 in the M+ patient group. In M0 patients disease free survival (DFS) was not significantly predicted by CK19 and/or MAM status in BM alone (p=0.173 (CK19), p=0.219 (MAM)), but the presence of the double positive phenotype showed a trend for predicting DFS (p=0.059).Conclusions: DTC measured as elevated CK19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DTC in BM was highly predictive for OS. However, with longer follow up differences in OS between patients with or without elevated CK19 and particularly MAM expression in PB also tend to become more significant. Furthermore, on the long term, double positivity for CK19/MAM in BM seems to predict DFS in patients with localized BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3019.