P. Huget

Platelet number and interleukin-6 correlate with VEGF but not with bFGF serum levels of advanced cancer patients

SummaryWe have compared the platelet number and the serum concentration of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6) in 80 blood samples of 50 patients with advanced cancer. We have also measured the mitogenic effect of patient sera on endothelial cells in vitro in order to estimate the biological activity of serum VEGF. Serum VEGF concentration correlated with platelet number (r = 0.61; P < 10–4). Serum IL-6 levels correlated with platelet count (r = 0.36; P < 10–3), with serum VEGF levels (r = 0.55; P < 10–4) and with the calculated load of VEGF per platelet (r = 0.4; P = 3 × 10–4). Patients with thrombocytosis had a median VEGF serum concentration which was 3.2 times higher (P < 10–4) and a median IL-6 serum level which was 5.8 times higher (P = 0.03) than in other patients. Serum bFGF did not show an association with any of the other parameters. Patient sera with high VEGF and bFGF content stimulated endothelial cell proliferation significantly more than other sera (P = 4 × 10–3). These results support the role of platelets in the storage of biologically active VEGF. Platelets seem to prevent circulating VEGF from inducing the development of new blood vessels except at sites where coagulation takes place. IL-6, besides its thrombopoietic effect, also seems to affect the amount of VEGF stored in the platelets. This is in accordance with the indirect angiogenic action of IL-6 reported previously. The interaction of IL-6 with the angiogenic pathways in cancer might explain the stimulation of tumour growth occasionally observed during IL-6 administration. It also conforms to the worse outcome associated with high IL-6 levels and with thrombocytosis in several tumour types and benign angiogenic diseases.

Prospective study of intratumoral microvessel density, p53 expression and survival in colorectal cancer

SummaryAdjuvant treatment of patients with colorectal cancer is hampered by a lack of reliable prognostic factors in addition to the clinicopathological staging system. A poorly defined but considerable fraction of Astler–Coller stage B patients will experience tumour recurrence, and some of the stage C patients will probably survive for a prolonged time after surgery without adjuvant treatment. Assessing parameters related to tumour angiogenesis has provided valuable prognostic information in different tumour types. The formation of new microvessels is part of the malignant phenotype in the majority of tumours. Alterations in tumour-suppressor genes, such as the p53 gene, or oncogenes, such as the ras gene, have been found to be responsible for changing the local balance of pro- and antiangiogenic factors in favour of the former. In this prospective study, intratumoral microvessel density (IMD) was assessed by immunostaining tissue sections for CD31 and counting individual microvessels in selected and highly vascular regions in specimens of 145 colorectal cancer patients. p53 protein overexpression was semiquantitatively determined after immunohistochemistry. In both uni- and multivariate analysis, high IMD was significantly associated with shorter survival in the patients undergoing surgery with curative intent (Astler–Coller stages A–C). p53 added prognostic power to IMD, both in Astler–Coller stage B and stage C patients. An association between IMD and mode of metastasis was also noted. High IMD was strongly associated with the incidence of haematogenous metastasis during follow-up, but not with the presence of lymphogenic metastasis observed at surgery. This study confirms the results of previous retrospective analyses of IMD and survival in colorectal cancer and warrants a clinical validation by randomizing stage B tumour patients with high IMD and p53 overexpression between adjuvant treatment or not.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer

Background:The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice.Methods:This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts.Results:As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT–PCR for CK-19 and 54% using RT–PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT–PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001).Conclusion:We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients.

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients. We simultaneously obtained whole blood, plasma and serum samples from 80 patients and 20 controls. The CellSearch System was used to enumerate CTC in blood samples. Plasma total DNA levels were determined by a QPCR method. Sera were analysed by methylation-specific QPCR for three markers: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A) and oestrogen receptor 1 (ESR1). Total DNA levels in patients were significantly increased when compared with controls (P<0.001) and correlated with the number of CTC (r=0.418, P<0.001). Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in three (16%) serum samples from controls (P=0.003). APC was hypermethylated in 29%, RASSF1A in 35% and ESR1 in 20% of breast cancer cases. Detection of a methylated gene in serum was associated with the detection of CTC in blood (P=0.03). The detection of large amounts of circulating total/methylated DNA correlated with the presence of CTC in the blood from patients with breast cancer. This can be interpreted in two ways: (a) CTC are a potential source of circulating tumour-specific DNA; (b) high numbers of CTC and circulating methylated DNA are both a phenotypic feature of more aggressive tumour biology.

Circulating tumour cells and lung microvascular tumour cell retention in patients with metastatic breast and cervical cancer

We have shown that in up to half of the patients with metastatic breast cancer (MBC), higher numbers of circulating tumour cells (CTCs) are present in the central venous blood (CVB) compared to the peripheral venous blood (PVB), suggesting that the lungs might retain a substantial number of CTCs. Here we report the presence of tumour cell emboli (TCE) in the microvasculature of the lungs in three out of eight patients with MBC and one patient with metastatic cervical carcinoma who had markedly elevated numbers of CTCs in the blood. All these patients suffered from symptomatic dyspnoea not easily attributable to other causes. No TCE were observed in five patients with MBC and elevated CTC counts and three patients with MBC who had low CTC counts (<5/7.5 ml). To investigate whether CTCs derived from CVB or PVB exhibit different transcriptional characteristics that might explain selective CTC retention, paired CTC samples from CVB and PVB of 12 patients with advanced breast cancer were subjected to gene expression analysis of 105 genes. No significant differences in CTC gene expression were observed. Together, these data suggest that potentially clinically relevant CTC retention in the microvasculature of the lung can occur in a subset of patients with advanced metastatic breast and cervical cancer, which seems to be transcriptionally non-selectively.

Trastuzumab emtansine in breast cancer

Introduction: Trastuzumab emtansine (T-DM1) is a human epidermal growth factor receptor 2 (HER2)–targeted antibody–drug conjugate (ADC) composed of trastuzumab, a stable linker (MCC), and the cytotoxic agent DM1 (derivative of maytansine). Administration of T-DM1 leads to limited systemic exposure of free DM1, with no evidence of DM1 accumulation after repeated dosing. Areas covered: Phase I and Phase II clinical trials with T-DM1 as a single agent and in combination with paclitaxel, docetaxel, and pertuzumab have shown substantial clinical activity and a favorable safety profile. A randomized, open-label, first-line trial comparing trastuzumab and docetaxel with single agent T-DM1 showed a significant improved progression-free survival for T-DM1. Expert opinion: T-DM1 has successfully completed second-line Phase III development for advanced HER2-positive breast cancer. The Phase III EMILIA study demonstrated an overall survival benefit for T-DM1 compared to the combination of lapatinib and capecitabine in taxane-trastuzumab pretreated patients. T-DM1 may offer delivery on a personalized basis of very potent cytotoxic agents in a cellular selective manner.

Proposal for magnetic resonance imaging-guided salvage radiotherapy for prostate cancer

Abstract Background: A subset of patients experience a biochemical recurrence following radical prostatectomy. Radiotherapy can salvage those patients, provided that all disease is encompassed within the target volume. We hypothesized that this can be achieved more adequately with magnetic resonance imaging (MRI)-guided treatment planning. Material and methods: From January 2009 to April 2014, 183 patients were referred to our department for salvage radiotherapy (SRT). According to protocol, patients received a planning computed tomography (CT) as well as an MRI in treatment position. All MRI scans were retrospectively reviewed by an experienced uro-radiologist. Results: Median prostate-specific antigen (PSA) value at time of referral was 0.3 ng/ml (range 0.02–4.7 ng/ml). MRI did not show any suspected macroscopic disease in 137 patients (75%). In 46 (25%) patients, MRI did indicate a pelvic recurrence. The mean PSA level was significantly higher in patients with a suspected recurrence on MRI (0.4 vs. 1.4 ng/ml, p < .001) on a Student’s t-test. The mean follow-up was 33 months (range 5–69 months). Biochemical disease-free survival (bDFS) was significantly worse in patients with suspected disease on MRI [hazard ratio (HR) 2.9, p < .0001]. bDFS was significantly worse in the subgroup where the macroscopic recurrences on MRI received a lower radiation dose (HR 3.4, p = .01). Conclusion: MRI detects loco-regional disease in a substantial subset of patients with a biochemical recurrence after prostatectomy, especially in a PSA above 0.5 μg/l. Lack of MRI-based dose escalation on these macroscopic recurrences could explain some of the biochemical progression observed after SRT.

Angiogenic escape and tumour progression in two patients with metastatic breast cancer receiving bevacizumab treatment.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1029 Background: Vascular endothelial growth factor A (VEGF-A) has an important role in tumour progression by promoting angiogenesis. VEGF-A inhibitors, such as bevacizumab (Bev) and VEGF-Trap are being introduced into the treatment of breast cancer in order to target angiogenesis by inhibiting VEGF-A. Material and Methods: Two patients with metastatic breast cancer are described having tumour progression while being treated with single agent Bev. Both patients participated in the AVADO clinical phase III study, where shown to have received Bev in combination with docetaxel (D) as first line treatment for metastatic breast cancer. The first patient (A) had received 6 cycles of D and Bev and was for 4 months on single agent Bev (15mg/kg/3wk) before progressing. Pt B had received 9 cycles of D and Bev and was on 7 months of single agent Bev (15mg/kg/3wk) before disease progression. Tumour biopsies of progressing lesions were obtained after informed consent. Routine histological assessment and a CD34/Ki67 double staining were performed on their primary tumour as well as on the newly developed metastasis (A+B). Chalkley counts (CC) and endothelial cell proliferation fractions (ECP) were assessed by two independent observers. RT-PCR Taqman low density arrays with a gene panel of 94 angiogenesis related genes were performed in triplicate on both metastasis and compared to 10 other primary breast tumours. Results: Both lesions showed a high CC, respectively 7.5±0.62 (A) and 4.8±0.2 (B). Both lesions had elevated ECP values of 14% (A) and 8% (B). Using the 2 (-__CT) method and 18S as an internal control, the VEGFR1 mRNA was highly overexpressed in both A (25.18±0.12 fold change) and B (38.60±0.07 fold change) compared to the mean of 10 unselected primary breast tumours serving as controls (p<10-7). Similarly, in metastasis B, VEGF-B, TGFB1 and PDGFRA were found to be overexpressed, i.e. out of range [min-max] of the 10 primary breast tumours. A had out of range overexpression of VEGF-C. The gene expression of VEGF-A, VEGF-D, VEGFR2, VEGFR3, PDGFB and PDGFRB in both A and B were found to be in the range of the 10 controls. Conclusion: We describe two patients with progressive disease while being treated with Bev after an initial response on the combination of D and Bev. These new sites of disease showed a highly angiogenic and apparently vascular dependent growth pattern, in spite of high dosed anti-VEGF-A regimen. This suggests the existence of an important VEGF-A independent alternative modality of the tumour to promote angiogenesis. VEGFR1 was remarkably overexpressed in both metastases compared to controls. The expression of placental growth factor, a VEGFR1 specific ligand is further being explored. Knowledge of the biology of Bev resistance is essential since it could be useful in designing well considered combinations of targeted therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1029.

A phase I dose-escalation trial of stereotactic ablative body radiotherapy for non-spine bone and lymph node metastases (DESTROY-trial)

BackgroundIn an oligometastatic setting, metastasis-directed treatment could render patients disease free, possibly for a protracted interval. Stereotactic ablative radiotherapy (SABR) is one of the treatment modalities that can be offered to these patients. In addition, the radiobiological qualities of SABR are promising for the use in perceived radioresistant tumours. There is also emerging evidence that SABR can stimulate the immune response, and a specific therapeutic window may exist for the optimal use of radiotherapy as an immune adjuvant. However, when SABR is considered for non-spine bone or lymph node metastases, the optimal fractionation schedule is not yet known.MethodsThe DESTROY-trial is a non-randomized prospective phase I trial determining a regimen of choice for patients with non-spine bone and lymph node metastases. A total of 90 patients will be included in three different treatment regimens. They will be offered stereotactic ablative radiotherapy in 5, 3 or 1 fractions. Dose-limiting toxicity will be recorded as primary endpoint. Acute and late toxicity, local response and local recurrence, and progression-free survival are secondary endpoints. Liquid biopsies will be collected throughout the course of this study from the second fractionation schedule on.DiscussionDespite its almost universal use in (oligo-)metastatic patients, the level of evidence supporting radical local treatment in general, and stereotactic radiotherapy in particular, is low. This prospective phase I trial will evaluate different SABR regimens for metastases and the differences in immune-stimulatory effects.Trial registrationThe Ethics committee of the GZA Hospitals (B099201732915) approved this study on 05/07/2017. Amendment for translational research was approved on 06/02/2018. Trial registered on Clinicaltrials.gov (NCT03486431) on 03/04/2018 – Retrospectively registered.

Abstract 4072: Molecular characterization of single tumor cells isolated from blood samples using immunomagnetic enrichment and dielectrophoretic cell sorting: A feasibility study

Introduction Molecular characterization of CTC holds considerable promise for the identification and monitoring of therapeutic targets in cancer patients under systemic treatment. Molecular profiling of CTC is however frustrated by white blood cell (WBC) signatures overwhelming those emanating from the CTC minority. In this study, we investigated the feasibility to isolate and molecularly characterize single tumor cells (TC) and small pools of up to 10 pure TC from immunomagnetically enriched blood samples using a semi-automated platform for dielectrophoretic cell sorting (DEPArray, Silicon Biosystems, Bologna, IT). Methods MDA-MB-231 human breast cancer cells were spiked into healthy donor blood at a concentration of 1000 cells/7.5 ml blood. Clinical patient samples were obtained from patients with metastatic breast cancer. Blood samples were subjected to EpCAM based immunomagnetic enrichment using the CellSearch Profile Kit (CellSearch, Veridex, Raritan, NJ, USA). Enriched samples were immunofluorescently stained for EpCAM-PE, CD45-APC and Hoechst and subsequently further purified using a DEPArray cell sorter. Single TC, pools of 5 TC, pools of 10 TC and pools of 20 WBC were recovered into individual reaction tubes. Molecular analyses consisting of whole genome amplification followed by K-ras mutation analysis and RT-qPCR for an in-house selected panel of breast cancer related transcripts were performed. Results Genomic DNA (gDNA) was succesfully amplified and detected in 3/5 (60%) single MDA-MB-231 TC and 4/4 (100%) pools of 5-10 TC and 20 WBC. K-ras mutation analysis revealed the G13D mutation - heterozygously present in the MDA-MB-231 cell line - in all TC samples and in none of two WBC samples, indicating 100% purity of the sorted cell samples. In addition, gDNA was succesfully detected and amplified in 5/9 (55%) single TC and 5/6 (83%) pools of 5-10 TC and 20 WBC from a clinical patient sample. Transcriptional profiles of pools of 5-10 MDA-MB-231 TC samples, were consistently correlated with publicly available gene expression profiles of MDA-MB-231 cells (Spearman R 2 =0.36±0.03) and inversely correlated with gene expression profiles of MCF-7 cells (Spearman R 2 =−0.09±0.03), indicating correct classification according to molecular subtypes. In a clinical patient sample, transcriptional profiles of pools of CTC could be correctly distinguished from WBC. Discussion We show the feasibility of an integrated workflow for the molecular characterization of single TC and small pools of up to 10 pure TC isolated from immunomagnetically enriched blood samples using a semi-automated dielectrophoretic cell sorting technique. Further optimization is being undertaken to allow for single cell transcriptional profiling and results will be expanded in clinical patient samples in order to gain insight into in vivo CTC heterogeneity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4072. doi:1538-7445.AM2012-4072