Sleijfer, S.

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue (Article)

2013-07-01T00:00:00Z

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior. What's new? Circulating tumor cells (CTCs) are present in the blood stream of patients with metastatic colorectal cancer and provide the opportunity to characterize tumor cells without biopsy. The authors isolated CTCs to assess the status of KRAS and BRAF mutations, which severely limit effectiveness of anti-EGFR therapies. The analysis was challenged by the presence of more than 1,000 leukocytes in CTC-enriched fractions, but was successful in detecting mutations in as little as two CTCs when a specific, nested Allele-Specific Blocker PCR strategy was employed. These results underscore the potential of CTC analysis as an alternative to commonly used invasive approaches to test patients for mutations repeatedly during the course of the disease and treatment. Copyright

Long-term stability of T-cell activation and transduction components critical to the processing of clinical batches of gene-engineered T cells (Article)

2013-05-01T00:00:00Z

Background aims: The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). Methods: Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. Results: We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. Conclusions: High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.

Tijd om persoonlijk te worden (Inaugural Lecture)

2012-04-20T00:00:00Z

Rede, in verkorte vorm uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht Translationele Medische Oncologie aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam op 20 april 2012

Anaplastic lymphoma kinase aberrations in rhabdomyosarcoma: Clinical and prognostic implications (Article)

2012-01-20T00:00:00Z

Purpose: The aim of this study is to investigate anaplastic lymphoma kinase (ALK) protein expression and underlying genetic aberrations in rhabdomyosarcoma (RMS), with special attention to clinical and prognostic implications. Patients and Methods: A total of 189 paraffin-embedded RMS tumor specimens from 145 patients were collected on tissue microarray. ALK protein expression was evaluated by immunohistochemistry. ALK gene (2p23) copy number and translocations were determined by in situ hybridization. cDNA sequencing of the receptor tyrosine kinase domain of the ALK gene was assessed in 43 samples. Results: Strong cytoplasmic ALK protein expression was more frequently observed in alveolar RMS (ARMS) than in embryonal RMS (ERMS) (81% v 32%, respectively; P < .001). ALK gene copy number gain was detected in the vast majority of ARMS (88%), compared with 52% of ERMS (P < .001). ALK copy number correlated with protein expression in primary tumors (n = 107). We identified one point mutation (2%) and seven tumors harboring whole exon deletions (16%). In ERMS, specific ALK gain in the primary tumor correlated with metastatic disease (100% in metastatic disease v 29% in nonmetastatic disease; P = .004) and poor disease-specific survival (5-year disease-specific survival: 62% v 82% for nonspecific or no gain; P = .046). Conclusion: Because ALK aberrations on genomic and protein levels are frequently found in RMSs, in particular ARMS, and are associated with disease progression and outcome in ERMS, ALK may play a role in tumor biology and may provide a potential therapeutic target for these tumors. Future research should aim at the oncogenic role of ALK and the potential effect of ALK inhibitors in RMS.

Re: Hypertension as a biomarker of efficacy in patients with metastatic renal cell carcinoma treated with sunitinib (Article)

2011-10-19T00:00:00Z

Sunitinib-induced hypothyroidism is due to induction of type 3 deiodinase activity and thyroidal capillary regression (Article)

2011-10-01T00:00:00Z

Context: Anticancer treatment with the tyrosine kinase inhibitor sunitinib causes thyroid dysfunction. Objective: Our objective was to investigate the time course and underlying mechanisms of sunitinib-induced thyroid dysfunction. Design: Thyroid function tests of 83 patients on sunitinib were collected retrospectively for their total treatment duration between January 2006 and November 2009 and prospectively in 15 patients on sunitinib for 10 wk. Additionally, thyroid function and histology were assessed in rats on sunitinib (8 d; n = 10) and after sunitinib withdrawal (11 d; n = 7) and compared with controls (n = 7). Setting: Patients were seen at a university outpatient oncology clinic. Patients and Animals: Patients with metastatic renal cell carcinoma or gastrointestinal stromal tumors participated in the clinical study and Wistar Kyoto rats were used in the rat study. Intervention: Sunitinib was taken according to a 4 wk "on," 2 wk "off" treatment regimen. Blood samples for measurement of thyroid function were collected at baseline and at wk 4 and 10. In rats, blood, liver, and thyroid were collected to assess thyroid hormones, deiodinase activity, and thyroid histology. Main Outcome Measures: TSH and free T4levels, deiodinase activity, and thyroid histology were assessed. Results: Forty-two percent of patients in the retrospective study developed elevated TSH levels. Prospective analysis showed increased TSH levels within 10 wk of treatment, accompanied by a decreased T3/rT3ratio. In rats, serum T4and T3decreased, hepatic type 3 deiodinase activity increased, andthyroid histology showed marked capillary regression, which all but thyroid hormones reversed after sunitinib withdrawal. Conclusion: Sunitinib induces hypothyroidism due to alterations in T4/T3metabolism as well as thyroid capillary regression. Copyright

Circulating tumor cell isolation and diagnostics: Toward routine clinical use (Article)

2011-09-15T00:00:00Z

From February 7-11, 2011, the multidisciplinary Lorentz Workshop Circulating Tumor Cell (CTC) Isolation and Diagnostics: Toward Routine Clinical Use was held in Leiden (The Netherlands) to discuss progress and define challenges and potential solutions for development of clinically useful circulating tumor cell (CTC) diagnostics. CTCs, captured as "liquid biopsy" from blood, for counting and characterization using pathology and molecular assays, are expected to replace metastatic tissue biopsies to be used to predict drug response and resistance and to monitor therapy response and cancer recurrence. CTCs are highly heterogeneous; therefore, cancer type-specific isolation technologies, as well as complex clinical interpretation software, are required.

The vascular endothelial growth factor receptor inhibitor sunitinib causes a preeclampsia-like syndrome with activation of the endothelin system (Article)

2011-08-01T00:00:00Z

Angiogenesis inhibition is an established treatment for several tumor types. Unfortunately, this therapy is associated with adverse effects, including hypertension and renal toxicity, referred to as "preeclampsia." Recently, we demonstrated in patients and in rats that the multitarget tyrosine kinase inhibitor sunitinib induces a rise in blood pressure (BP), renal dysfunction, and proteinuria associated with activation of the endothelin system. In the current study we investigated the effects of sunitinib on rat renal histology, including the resemblance with preeclampsia, as well as the roles of endothelin 1, decreased nitric oxide (NO) bioavailability, and increased oxidative stress in the development of sunitinib-induced hypertension and renal toxicity. In rats on sunitinib, light and electron microscopic examination revealed marked glomerular endotheliosis, a characteristic histological feature of preeclampsia, which was partly reversible after sunitinib discontinuation. The histological abnormalities were accompanied by an increase in urinary excretion of endothelin 1 and diminished NO metabolite excretion. In rats on sunitinib alone, BP increased (ΔBP: 31.6±0.9 mm Hg). This rise could largely be prevented with the endothelin receptor antagonist macitentan (ΔBP: 12.3±1.5 mm Hg) and only mildly with Tempol, a superoxide dismutase mimetic (ΔBP: 25.9±2.3 mm Hg). Both compounds could not prevent the sunitinib-induced rise in serum creatinine or renal histological abnormalities and had no effect on urine nitrates but decreased proteinuria and urinary endothelin 1 excretion. Our findings indicate that both the endothelin system and oxidative stress play important roles in the development of sunitinib-induced proteinuria and that the endothelin system rather than oxidative stress is important for the development of sunitinib-induced hypertension.

The CYP2C19*2 genotype predicts tamoxifen treatment outcome in advanced breast cancer patients (Article)

2011-08-01T00:00:00Z

Aims: Tamoxifen is metabolized by cytochrome P450s, with an important role for CYP2D6. Recently, we demonstrated in 80 patients that CYP2C19*2 is associated with increased survival in breast cancer patients using tamoxifen. Here, we aimed to confirm this in a large group of 499 patients. Materials & methods: A total of 499 estrogen receptor-positive primary breast tumor specimens of advanced disease patients treated with first-line tamoxifen were genotyped for CYP2C19*2 and 17 variant alleles, with primary end point time-to-treatment failure (TTF). Effects of CYP2C19, independent of treatment, were analyzed in 243 primary systematic untreated patients. Results: CYP2C19*2 hetero-and homozygote patients combined showed significantly longer TTFs (hazard ratio [HR]: 0.72; 95% CI: 0.57-0.90; p = 0.004). In multivariate analysis, including CYP2D6*4 status, CYP2C19*2 remained independently associated with TTF (HR: 0.73; 95% CI: 0.58-0.91; p = 0.007). In untreated patients, the CYP2C19*17 allele was significantly associated with a longer disease-free interval (HR: 0.66; 95%CI: 0.46-0.95; p = 0.025). Conclusion: CYP2C19 genotyping is potentially important for tamoxifen therapy for advanced disease and for breast cancer prognosis.

Correlation of breast cancer susceptibility loci with patient characteristics, metastasis-free survival, and mRNA expression of the nearest genes (Article)

2011-07-12T00:00:00Z

To understand the biology of low-risk breast cancer alleles, and to investigate whether these loci also contribute to disease progression that was once established, we examined the association of SNPs tagging the low-risk breast cancer loci in or near FGFR2, LSP1, MAP3K1,H19, TOX3, POU5F1P1, MYC, and 2q35, with clinical, pathological characteristics, prognosis, and mRNA expression of the nearest genes. Tumor DNA samples of 2,480 breast cancer patients were available. Out of this cohort, 1,290 patients with lymph-node negative disease who did not receive adjuvant systemic therapy, the SNP status was associated with metastasis-free survival (MFS). In 1,401 patients, the mRNA expression levels of FGFR2, LSP1, MAP3K1,H19, TOX3, POU5F1P1, and MYC were determined and correlated with SNP genotypes. The SNP rs2981582 in FGFR2 was significantly associated with positive ER and PgR status (P < 0.001 and P = 0.003, respectively). No other significant associations with patient or tumor characteristics were observed. Only rs2107425 near H19 was significantly associated with shorter MFS in uni- and multi-variate analysis (HR: 1.53, CI: 1.12-2.08, P = 0.006 and HR: 1.59, CI: 1.16-2.20, P = 0.004, respectively), with the more aggressive minor allele displaying a recessive trait. The minor allele of SNP rs3803662 located near the TOX3 gene was associated with lower mRNA expression of this gene. In conclusion, except for the association of rs13283662 with TOX3 gene expression indicating a tumor suppressor role of TOX3, our findings suggest that breast cancer low-risk loci generally do not affect expression of the nearest gene in breast tumor tissue. Also the prognosis of patients is largely not affected by low-risk breast cancer loci except for the SNP near H19. How, this SNP affects prognosis warrants further study as it does not operate through altering H19 mRNA expression.

Integrating collection of biospecimens in clinical trials: The approach of the european organization for research and treatment of cancer (Article)

2011-06-01T00:00:00Z

Access to biospecimens and their derivatives, that is, human biological materials (HBM), for translational research (TR) is considered a major bottleneck hindering successful bench to bedside translation. Clinical trials offer a unique opportunity to collect HBM in a specialized setting that allows prospectively designed, high-quality TR that would be difficult to fulfill from community- or population-based HBM collections alone. Increasingly, as the field advances toward personalized treatment of cancer patients, access to HBM is becoming a necessity for patient enrollment in a new generation of clinical studies that are designed and driven by molecular hypotheses. The European Organization for Research and Treatment of Cancer (EORTC) is one of the largest networks for clinical trials in oncology. The EORTC is re-focusing its strategy, building on experiences and expertise gained over the years from specific initiatives such as EORTC Group activities and the EORTC Virtual Tumour Bank, by developing new mechanisms to support investigators with the practical aspects of HBM collection as part of EORTC clinical studies. Due to the complex, multidisciplinary nature of HBM collection and TR, integration of HBM collection into clinical trials warrants careful upfront planning and input from a range of expertise. To simplify HBM collection in clinical studies, the EORTC has developed a simple checklist containing the key elements of HBM collection setup and combines these into a simple tool for practical use. Through identifying and managing key risk areas, this can maximize the HBM collection success while achieving efficient clinical trial development. This article focuses on the key elements of HBM collection and the approaches of the EORTC for efficiently integrating this collection into clinical trial development.

mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients (Article)

2011-06-01T00:00:00Z

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer. Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule-based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors. Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels. Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs.

Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146 (Article)

2011-05-01T00:00:00Z

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch™ technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients (Article)

2011-04-01T00:00:00Z

Summary: Circulating tumor cells (CTCs) have rapidly developed as important cancer biomarkers after their enumeration proved to be prognostic in metastatic breast, colorectal and prostate cancer, and their rise or decline after the first cycle of therapy showed to predict therapy response. Besides mere counting, CTCs can be isolated and subsequently analyzed using various molecular applications, including miRNA expression analysis. Recently, miRNA expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation likely has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Additionally to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Since determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs, this review focuses on measuring miRNAs in the circulation of cancer patients.

External quality assurance of circulating tumor cell enumeration using the CellSearch® system: A feasibility study (Article)

2011-03-01T00:00:00Z

Background: Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi-automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories. Methods: Blood samples from six cancer patients and controls were distributed to 14 independent laboratories to test between-laboratory, between-assay, and between-instrument variation. Additionally, between-operator variability was assessed through the interpretation of blinded images of all blood samples on a website. Results: Shipment and storage of samples had no influence on CTC values. Between-instrument (coefficient of variation (CV) < 12%) and between-assay variation was low (CV ≤ 20%), indicating high reproducibility. However, between-laboratory CV ranged from 45 to 64%. Although inter-operator agreement on image interpretation (Fleiss' I° statistics) ranged from " to "almost perfect," image interpretation, particularly of samples containing high numbers of apoptotic cells, was the main contributor to between-laboratory variation. Conclusions: This multicenter study shows the feasibility of an EQA program for CTC detection in patient samples, and the importance of continuation of such a program for the harmonization of CTC enumeration.

Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells (Article)

2011-01-06T00:00:00Z

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted with a chimeric antibody receptor (CAR) directed toward carbonic anhydrase IX (CAIX), an antigen highly expressed in renal cell carcinoma. In the majority of patients, we observed distinct humoral and/or cellular anti-CAIX-CAR T-cell immune responses in combination with a limited peripheral persistence of transferred CAIX-CAR T cells in the majority of patients. Humoral immune responses were anti-idiotypic in nature and neutralized CAIX-CAR-mediated T-cell function. Cellular anti-CAIX-CAR immune responses were directed to the complementarity-determining and framework regions of the CAR variable domains. In addition, 2 patients developed immunity directed against presumed retroviral vector epitopes. Here, we document the novel feature that therapeutic cells, which were ex vivo engineered by means of transduction with a minimal γ-retroviral vector, do express immunogenic vector-encoded epitopes, which might compromise persistence of these cells. These observations may constitute a critical concern for clinical ex vivo γ-retroviral gene transduction in general and CAR-retargeted T-cell therapy in particular, and underscore the need to attenuate the immunogenicity of both transgene and vector.

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients (Article)

2011-01-01T00:00:00Z

Two-protein signature of novel serological markers apolipoprotein-A2 and serum amyloid alpha predicts prognosis in patients with metastatic renal cell cancer and improves the currently used prognostic survival models (Article)

2010-12-01T00:00:00Z

Background: In metastatic renal cell cancer (mRCC), the Memorial Sloan-Kettering Cancer Center (MSKCC) risk model is widely used for clinical trial design and patient management. To improve prognostication, we applied proteomics to identify novel serological proteins associated with overall survival (OS). Patients and methods: Sera from 114 mRCC patients were screened by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Identified proteins were related to OS. Three proteins were subsequently validated with enzyme-linked immunosorbent assays and immunoturbidimetry. Prognostic models were statistically bootstrapped to correct for overestimation. Results: SELDI-TOF MS detected 10 proteins associated with OS. Of these, apolipoprotein A2 (ApoA2), serum amyloid alpha (SAA) and transthyretin were validated for their association with OS (P = 5.5×10-9, P = 1.1×0-7 and P = 0.0004, respectively). Combining ApoA2 and SAA yielded a prognostic two-protein signature [Akaike's Information Criteria (AIC) = 732, P = 5.2×10-7]. Including previously identified prognostic factors, multivariable Cox regression analysis revealed ApoA2, SAA, lactate dehydrogenase, performance status and number of metastasis sites as independent factors for survival. Using these five factors, categorization of patients into three risk groups generated a novel protein-based model predicting patient prognosis (AIC = 713, P = 4.3×10-11) more robustly than the MSKCC model (AIC = 729, P = 1.3×10-7). Applying this protein-based model instead of the MSKCC model would have changed the risk group in 38% of the patients. Conclusions: Proteomics and subsequent validation yielded two novel prognostic markers and survival models which improved prediction of OS in mRCC patients over commonly used risk models. Implementation of these models has the potential to improve current risk stratification, although prospective validation will still be necessary.

Effects of mannose-binding lectin polymorphisms on irinotecan-induced febrile neutropenia (Article)

2010-10-01T00:00:00Z

Objective. Mannose-binding lectin (MBL) is important in the innate immune response. MBL2 gene polymorphisms affect MBL expression, and genotypes yielding low MBL levels have been associated with an elevated risk for infections in hematological cancer patients undergoing chemotherapy. However, these reported associations are inconsistent, and data on patients with solid tumors are lacking. Here, we investigated the effects of MBL2 genotypes on irinotecan-induced febrile neutropenia in patients with solid tumors. Patients and Methods. Irinotecan-treated patients were genotyped for the MBL2 gene. Two promoter (550 H/L and 221 X/Y) and three exon polymorphisms (52 A/D, 54 A/B, and 57 A/C) were determined, together with known risk factors for irinotecan-induced toxicity. Neutropenia and febrile neutropenia were recorded during the first course. Results. Of the 133 patients, 28% experienced severe neutropenia and 10% experienced febrile neutropenia. No associations were found between exon polymorphisms and febrile neutropenia. However, patients with the H/H promoter genotype, associated with high MBL levels, experienced significantly more febrile neutropenia than patients with the H/L and L/L genotypes (20% versus 13% versus 5%). Moreover, patients with the HYA haplotype encountered significantly more febrile neutropenia than patients without this high MBL-producing haplotype (16% versus 4%). In the subgroup with wild-type exon polymorphisms (A/A), patients with the high MBL promoter phenotype had the highest incidence of febrile neutropenia, regardless of known risk factors. Conclusion. Patients with high MBL2 promoter genotypes and haplotypes seem more at risk for developing febrile neutropenia. If confirmed, these preliminary findings may contribute to more individualized approaches of irinotecan treatment.

The addition of pravastatin to chemotherapy in advanced gastric carcinoma: A randomised phase II trial (Article)

2010-08-19T00:00:00Z

Purpose: Statins have for long been considered to play a potential role in anticancer treatment based upon their ability to inhibit the mevalonate synthesis pathway. This randomised phase II trial compared the efficacy and safety of pravastatin added to epirubicin, cisplatin and capecitabine (ECC versus ECC + P) in patients with advanced gastric carcinoma. Methods: Patients were randomised to receive up to six cycles of 3-weekly ECC with or without pravastatin (40 mg, once daily from day 1 of the first cycle until day 21 of the last cycle). Primary end-point was progression-free rate at 6 months (PFR6months). Secondary end-points were response rate (RR), progression-free survival (PFS), overall survival (OS) and safety. For early termination in case of futility, a two-stage design was applied (P0 = 50%; P1 = 70%; α = 0.05; β = 0.10). Results: Thirty patients were enrolled. PFR6months was 6/14 patients (42.8%) in the ECC + P arm, and 7/15 patients (46.7%) in the control arm, and therefore the study was terminated after the first stage. In the ECC and ECC + P arm, RR was 7/15 (46.7%) and 5/15 (33.3%), median PFS was 5 and 6 months and median OS was 6 and 8 months, respectively. Toxicity data showed no significant differences, although there was a trend towards more gastrointestinal side-effects such as diarrhoea and stomatitis in the ECC + P arm. Conclusion: In this randomised phase II trial the addition of pravastatin to ECC did not improve outcome in patients with advanced gastric cancer. Therefore, further testing of this combination in a randomised phase III trial cannot be recommended.

(Pre-)Clinical pharmacology and activity of pazopanib, a novel multikinase angiogenesis inhibitor (Article)

2010-08-02T00:00:00Z

Pazopanib is a recently approved, novel tyrosine kinase inhibitor specifically designed to impair angiogenesis by abrogating vascular endothelial growth factor receptor 2 (VEGFR-2) to exert its function. Pazopanib inhibits VEGF-induced endothelial cell proliferation in vitro and angiogenesis in vivo and demonstrates antitumor activity in mouse models. Furthermore, the pazopanib concentration resulting in maximal inhibition of VEGFR-2 phosphorylation in vivo was in line with the steady-state concentration required to inhibit growth of tumor xenografts, suggesting that pazopanib's mechanism of action is indeed through VEGFR-2 inhibition. In a phase I trial, a generally well-tolerated dose was identified at which the majority of patients achieved pazopanib plasma concentrations above the concentration required for maximal in vivo inhibition of VEGFR-2 phosphorylation in preclinical models. Administered as monotherapy, evidence of antitumor activity was observed in phase II studies in several tumor types, including soft tissue sarcoma, renal cell cancer (RCC), ovarian cancer, and non-small cell lung cancer. Recently, the U.S. Food and Drug Administration granted approval for treatment with pazopanib in patients with RCC based on the longer progression-free survival time observed with this agent in a placebo-controlled, randomized trial. This review summarizes the preclinical and clinical pharmacokinetics and pharmacodynamics of pazopanib, as well as data on clinical activity, that ultimately resulted in its recent approval.

mRNA levels of CD31, CD144, CD146 and von Willebrand factor do not serve as surrogate markers for circulating endothelial cells (Article)

2010-08-01T00:00:00Z

Circulating endothelial cells (CEC) are considered a promising marker to determine the extent of vascular damage. However, currently available and validated CEC enumeration assays are laborious, time consuming and costly, which limits their clinical utility. Here, we evaluated the feasibility of quantifying mRNA levels of the endothelium-associated markers CD31, CD144, CD146 and von Willebrand factor (vWf) in peripheral blood (PB) of healthy donors, patients, and human umbilical veins by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and their use as surrogate markers for CEC. Whole blood samples and CD146+ cell-enriched fractions were assessed for mRNA and protein expression of CD31, CD144, CD146 and vWf by RT-PCR and flow cytometry, respectively. We showed the feasibility to detect endothelial mRNA isolated from HUVEC numbers as low as 10. However, no endothelial mRNA could be measure in whole blood samples, and only low levels of CD31 and CD146 mRNA were detected in suspensions of isolated CEC with numbers up to 4,450 CEC per sample. We conclude that mRNA levels of CD31, CD144, CD146 and vWf in whole blood as detected by real time RT-PCR cannot be used as biomarkers for end-stage endothelial cells such as CEC.

Circulating endothelial cells, circulating tumour cells, tissue factor, endothelin-1 and overall survival in prostate cancer patients treated with docetaxel (Article)

2010-07-01T00:00:00Z

Purpose: We investigated whether serum markers of angiogenesis endothelin-1 (ET-1) and tissue factor (TF), and/or markers of vascular damage such as circulating endothelial cells (CECs), or their relative changes during treatment, were prognostic for overall survival (OS) in castration resistant prostate cancer (CRPC) patients. Additionally, we combined these markers with circulating tumour cells (CTCs) to construct a predictive nomogram for treatment outcome. Patients and methods: One hundred and sixty two CRPC patients treated with a docetaxel containing regimen had blood drawn before and at 2-5 weeks and 6-8 weeks after treatment start. Prospectively determined CTC and CEC levels, and retrospectively measured serum concentrations of ET-1 (pg/mL) and TF (pg/mL) were evaluated to determine their prognostic value for OS. Results: Baseline CEC, TF and ET-1 were not prognostic for OS. A ≥3.8-fold increase in CEC 2-5 weeks after treatment initiation was associated with decreased OS (median 10.9 versus 16.8 months; P = 0.015), as was any decrease in TF levels compared to baseline levels (median 11.9 versus 21.5 months; P = 0.0005). As previously published, baseline and CTC counts ≥5 at 2-5 weeks were also predictive of decreased OS. Combining CTC with changes in TF and CEC 2-5 weeks after treatment initiation yielded four groups differing in OS (median OS 24.2 versus 16.0 versus 11.4 versus 6.1 months; P < 0.0001). Conclusion: CEC, CTC and TF levels alone and combined can predict early on OS in CRPC patients treated with docetaxel-based therapy. A prospective study to confirm the use of these markers for patient management is needed.

Circulating tumor cells and sample size: The more, the better (Article)

2010-06-10T00:00:00Z

Decreased exposure to sunitinib due to concomitant administration of ifosfamide: results of a phase I and pharmacokinetic study on the combination of sunitinib and ifosfamide in patients with advanced solid malignancies (Article)

2010-06-08T00:00:00Z

Background:This study aimed to define the maximally tolerated dose (MTD) of sunitinib combined with two different infusion schedules of ifosfamide. Methods:Patients with advanced solid tumours, good performance score, good organ function, and no standard therapy available were eligible. Continuous once daily sunitinib, in escalating doses per cohort, was combined with ifosfamide, 9 g m-2 for 3 days or 6 g m-2 for 5 days, administered every 3 weeks. Pharmacokinetic (PK) and pharmacodynamic (PD) assessments were performed. Results:With growth-factor support, the MTD of sunitinib combined with either ifosfamide schedule was 12.5 mg in 32 patients enrolled. Neutropenia-related adverse events were dose-limiting toxicities. Sunitinib did not affect ifosfamide PK. Ifosfamide significantly decreased exposure to sunitinib and increased exposure to its metabolite, SU12662. No consistent changes in PD parameters were observed. Conclusion:With growth-factor support, the MTD of sunitinib with both ifosfamide schedules was 12.5 mg. Ifosfamide produced decreased sunitinib blood levels because of CYP3A induction. As PK interactions cannot explain the relatively low sunitinib doses that can be combined with ifosfamide, synergy in toxicity is likely. Whether this also holds true for anti-tumour activity needs to be further explored.British Journal of Cancer advance online publication, 18 May 2010; doi:10.1038/sj.bjc.6605696 www.bjcancer.com.

Multicentric parallel phase II trial of the polo-like kinase 1 inhibitor BI 2536 in patients with advanced head and neck cancer, breast cancer, ovarian cancer, soft tissue sarcoma and melanoma. The first protocol of the European Organization for Research and Treatment of Cancer (EORTC) Network Of Core Institutes (NOCI) (Article)

2010-05-14T00:00:00Z

Aims: BI 2536 is a selective and potent small-molecule inhibitor of polo-like kinase 1. We performed a multi-centre, multi-tumour phase II trial to investigate the efficacy, safety and pharmacokinetics of BI 2536 in five solid tumour types. Patients and methods: Patients with advanced head and neck, breast and ovarian cancer, soft tissue sarcoma and melanoma were selected according to protocol-defined general and tumour-specific criteria. They were ≥18 years old, had a good performance status, adequate bone marrow, renal and liver function, measurable progressive disease and had completed other relevant systemic treatments >4 weeks ago. BI 2536 200-250 mg was given intravenously on day 1 every 3 weeks until intolerance, progression or refusal. The study was based on a Simon two-stage design, with 12 patients entering in stage 1 and additional 25 patients to be entered in case of at least one response in the first stage. The rate of objective responses (RECIST criteria) was chosen as primary end-point. Results: Seventy six patients were included, 71 started treatment and received a median number of two cycles (four in ovarian cancer). Frequent grade 3-4 adverse events were neutropaenia (81.6%), thrombocytopaenia (19.7%), febrile neutropaenia (19.7%), anaemia (15.5%) and pain (9.9%). We did not observe confirmed objective responses. All cohorts were closed after the entry of 14-15 eligible non-responding patients. Pharmacokinetic analyses revealed multi-compartmental behaviour and a rapid distribution of BI 2536. Conclusions: BI 2536 showed limited antitumour activity according to the design of this trial in five different tumour types. Derivatives of BI 2536 with a more favourable pharmacological profile are currently explored further in prospective studies.

T cell receptor (TCR) gene therapy to treat melanoma: Lessons from clinical and preclinical studies (Article)

2010-04-01T00:00:00Z

Importance of the field: Adoptive T cell therapy (ACT) with tumour infiltrating lymphocytes is currently the best treatment option for metastatic melanoma. Despite its clinical successes, ACT has limitations in availability and generation of therapeutic T cells for a larger group of patients. Introduction of tumour-specific T cell receptors into T cells, termed TCR gene therapy, can provide an alternative for ACT that is more widely applicable and might be extended to other types of cancer. Areas covered in this review: The current status of TCR gene therapy studies including clinical challenges, such as on-target toxicity, compromised anti-tumour T cell responses, compromised T cell persistence and potential immunogenicity of receptor transgenes. Strategies to address these challenges are covered. What the reader will gain: A listing and discussion of strategies that aim at improving the efficacy and safety of TCR gene therapy. Such strategies address antigen choice, TCR mis-pairing, functional avidity and persistence of T cells, immune responses towards receptor transgenes, and combination of ACT with other therapies. Take home message: To ensure further clinical development of TCR gene therapy, it is necessary to choose safe T cell target antigens, and implement (combinations of) strategies that enhance the correct pairing of TCR transgenes and the functional avidity and persistence of T cells.

Bioanalytical method for the quantification of sunitinib and its n-desethyl metabolite SU12662 in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry (Article)

2010-03-11T00:00:00Z

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the quantitative determination of sunitinib and its n-desethyl metabolite SU12662, in 100 μl aliquots of human potassium EDTA plasma with deuterated sunitinib as internal standard. As sunitinib was found to be extremely sensitive to light causing rapid conversion of the Z (cis)-isomer to the E (trans)-isomer, the sample extraction and cleaning-up were performed under sodium-light and in amber vials. The extraction involved a simple liquid-liquid extraction with tert-butyl methyl ether. Chromatographic separations were achieved on an Aquity UPLC®BEH C181.7 μm, 2.1 mm × 50 mm column eluted at a flow rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 4 min, with elution times of 1.05, 1.43, 0.95, and 1.34 min, for the E (trans)- and Z (cis)-isomers of sunitinib and the E (trans)- and Z (cis)-isomers of SU12662, respectively. The multiple reaction monitoring transitions were set at 399 > 326 (m/z), at 371 > 283 (m/z) and at 409 > 326 (m/z) for sunitinib, SU12662 and the internal standard, respectively. The calibration curves were linear over the range of 0.200 to 50.0 ng/ml with the lower limit of quantitation validated at 0.200 ng/ml for both sunitinib and SU12662. The within-run and between-run precisions were within 11.7%, while the accuracy ranged from 90.5 to 106.8%.

Increasing tumoral 5-fluorouracil concentrations during a 5-day continuous infusion: a microdialysis study (Article)

2010-01-01T00:00:00Z

Purpose: Response to anticancer therapy is believed to be directly related to the concentration of the anticancer drug in the tumor itself. Assessment of intra-tumor drug pharmacokinetics can be helpful to gain more insight into mechanisms involved in the (in)sensitivity of tumors to anticancer therapy. We explored the pharmacokinetics of 5-fluorouracil in both plasma and tumor tissue during a 5-day continuous infusion of 5-fluorouracil in patients with cancer. Sampling for measurement of 5-fluorouracil in tumor tissue was performed using microdialysis. Experimental design: In seven patients with an accessible (sub)cutaneous tumor treated with a continuous 5-fluorouracil infusion, plasma and microdialysate samples from tumor and normal adipose tissue were collected over a period of 5 days. Results: For six patients, drug concentrations in both tumor tissue and plasma were available. Concentration-time curves of unbound 5-fluorouracil were lower in tumor tissue compared to the curves in plasma, but exposure ratios of tumor tissue versus plasma increased during the 5-day infusion period. The presence of circadian rhythmicity of 5-fluorouracil pharmacokinetics in the tumor itself was demonstrated as 5-fluorouracil concentrations in tumor extracellular fluid were higher during the night than during daytime. Conclusion: Microdialysis was successfully employed in patients with cancer during a continuous 5-day 5-fluorouracil infusion. Plasma and tumor pharmacokinetics of 5-fluorouracil differed substantially with increasing 5-fluorouracil concentrations in tumor over time, possibly resulting from a lowered interstitial fluid pressure by 5-fluorouracil itself. This microdialysis 5-fluorouracil model might be useful to monitor the effect of drug delivery modulating strategies in future studies.

Patterns and incidence of chromosomal instability and their prognostic relevance in breast cancer subtypes (Article)

2010-01-01T00:00:00Z

One of the hallmarks of human solid tumors is chromosomal instability (CIN). We studied global patterns as well as individual levels of CIN and determined the prognostic relevance among breast cancer subtypes. For this, we used single nucleotide polymorphism copy number data of 313 primary lymph-node negative breast cancers. The level of CIN for individual samples was determined by counting the total number of chromosomal segments showing a gain or loss per specimen. Hierarchical clustering resulted in four groups showing distinct patterns of abnormalities, predominantly characterized by 1q gain, 8q gain, 1q&8q gain, or no gain of these loci. Estrogen receptor (ER)-positive and ER-negative samples showed an uneven distribution (statistically significant) across the cluster-groups, as did the molecular subtypes and triple-negative tumors (negative for estrogen-, progesterone-, and her2/neu-receptor). The CIN-score was significantly higher in ER-negative and triple-negative samples. Among luminal cancers, luminal B had a higher CIN-score than luminal A. The CIN-score was significantly associated with prognosis, measured by the time to distant metastasis, in ER-positive, luminal B, and her2/neu subtypes, but not in ER-negative patients. Our study points to a multifaceted role for CIN in breast cancer. Within ER-negative samples, CIN is likely related to the onset but other factors govern the progression of the disease. In contrast, CIN is clearly associated with progression in ER-positive, luminal B, and her2/neu subtypes; thus, assessing CIN in these subtypes may contribute to personalized patient management.

Decreased expression of EZH2 is associated with upregulation of ER and favorable outcome to tamoxifen in advanced breast cancer (Article)

2010-01-01T00:00:00Z

Abstract The purpose of this study is to investigate EZH2 in a large series of breast cancer patients for its prognostic and predictive value, and to evaluate its functional role in treatment response in vitro. EZH2 levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. EZH2 expression was downregulated with siRNAs in MCF7, to assess expression alterations of putative EZH2 downstream genes and to determine cell numbers after treatment with the anti-estrogen ICI 164384. In 688 lymph node-negative patients who did not receive adjuvant systemic therapy, EZH2 was not significantly correlated with metastasis-free survival (MFS). In 278 patients with advanced disease treated with first-line tamoxifen monotherapy, the tertile with highest EZH2 levels was associated with the lowest clinical benefit (OR = 0.48; P = 0.02) and with a shorter progression-free survival (PFS) in both univariate (HR = 1.80; P < 0.001) and multivariate analysis, including traditional factors (HR = 1.61; P = 0.004). In vitro, EZH2 silencing in MCF7 caused a 38% decrease in cell numbers (P < 0.001) whereas ICI 164384 treatment resulted in a 25% decrease (P < 0.001) compared to controls. Combining EZH2 silencing with ICI treatment reduced cell numbers with 67% (P < 0.001) compared to control conditions. EZH2 downregulation was associated with an almost two-fold upregulation of the estrogen receptor alpha (ER) (P = 0.001). In conclusion, EZH2 has no prognostic value in breast cancer. High levels of EZH2 are associated with poor outcome to tamoxifen therapy in advanced breast cancer. Downregulated EZH2 leads to upregulation of the ER and better response to anti-estrogens.

Prognostic and predictive factors for outcome to first-line ifosfamide-containing chemotherapy for adult patients with advanced soft tissue sarcomas. An exploratory, retrospective analysis on large series from the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC-STBSG) (Article)

2010-01-01T00:00:00Z

Background: Adult patients with advanced soft tissue sarcomas (STS) are generally treated similarly, regardless of great differences between STS subtypes, disease presentation and patients' characteristics. As ifosfamide is frequently applied in first line systemic therapy, we aimed to establish prognostic and predictive factors for outcome to ifosfamide-based therapy. Methods: A retrospective, exploratory analysis was performed on data from 1337 advanced STS patients who received first-line ifosfamide-containing chemotherapy. For predictive factor analysis, 660 patients treated with doxorubicin monotherapy served as comparators. Results: Independent favourable prognostic factors for overall survival (OS) were good performance status, female gender, low histological grade, extremity primary tumour site and locally advanced disease; for progression-free survival (PFS), the combination of doxorubicin and ifosfamide, locally advanced disease, and tumour entity with a lower risk to progress for synovial sarcoma patients compared to leiomyosarcoma. For response, independent favourable prognostic factors were doxorubicin combined with ifosfamide, higher histological grade, and histology with synovial sarcoma patients having the highest chance to respond. Predictive factor analysis showed that compared to doxorubicin monotherapy, patients who benefited less from ifosfamide-based therapies were leiomyosarcoma patients in terms of OS, and patients with liposarcoma for response. No predictive factors were found for PFS. Conclusion: In this study, we established an independent set of prognostic and predictive factors for outcome to ifosfamide-based chemotherapy in advanced STS patients. This study provides important information for the interpretation and design of clinical trials for specific STS entities and may contribute to further treatment individualisation of advanced STS patients.

It takes two to tango: Combinations of conventional cytotoxics with compounds targeting the vascular endothelial growth factor-vascular endothelial growth factor receptor pathway in patients with solid malignancies (Article)

2010-01-01T00:00:00Z

Through advances in molecular biology, insight into the mechanisms driving malignancies has improved immensely and as a result, various factors playing an essential role in the biology of numerous tumor types have been revealed. By using compounds that specifically block the function of a single factor being crucial for tumor pathogenesis, it was hoped to exert antitumor activity while avoiding toxicities characteristic for conventional chemotherapy. One of the processes of crucial importance in the development of cancer, and consequently an attractive target, is angiogenesis. In recent years, several key factors for angiogenesis have been identified, including ligands, receptors, and transduction signaling factors. Of these, the vascular endothelial growth factor (VEGF) pathway has been found to be activated in numerous tumor types and considered one of the main drivers of angiogenesis. Roughly, VEGF-mediated angiogenesis can be inhibited by two approaches: either by monoclonal antibodies directed towards VEGF or its corresponding receptors, or by kinase inhibitors targeting the signal transduction of the VEGF receptors. As monotherapy, several kinase inhibitors exert antitumor activity in tumor types such as renal cell carcinoma. However, in most tumor types, the antitumor activity of compounds targeting the VEGF pathway is limited. In recent years, evidence is mounting that the paradigm of one single factor that drives malignant behavior applies rarely and is an oversimplification for most tumors in which there are multiple driving pathways. Consequently, multitargeting rather than single-targeting approaches are required. One of the means is by combining targeted agents with conventional cytotoxics. As the VEGF pathway also affects the sensitivity of tumor cells to chemotherapeutics, combinations of compounds targeting this pathway and conventional cytotoxics have been explored. This review addresses such combinations.

Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR (Article)

2009-12-01T00:00:00Z

Detection of circulating tumor cells (CTCs) in whole blood from metastatic cancer patients by the CellSearch™ CTC Test (Veridex LLC, Warren, NJ, USA) has been shown to have clinical relevance. In addition to enumeration, there is great interest in molecular characterization of these CTCs. We aimed to establish a robust method to perform mRNA expression analysis of multiple genes by a real-time reverse transcriptase (RT)-PCR on small numbers of CTCs enriched from whole blood by the CellSearch™ system. Despite the 4 log depletion of leukocytes after CellSearch enrichment, the CTC-enriched fractions still contained leukocytes, in particular B-lymphocytes, which severely interfered with our CTC-specific gene expression profiling. After extensive washing and leukocyte-specific depletion by anti-CD45 coated magnetic beads prior to CellSearch™ enrichment, the number of leukocytes present in the enriched fraction was still high (range 60-929). However, by using a set of genes with no or minor expression by leukocytes, we succeeded to perform quantitative gene expression profiling specific for as little as one breast cancer CTC present in a CTC-enriched environment typically containing over 800 contaminating leukocytes. Our method allows molecular characterization specific for as little as one CTC, and can be used to expand the understanding of the biology of metastasis and, potentially, to improve patient management.

CITED2 and NCOR2 in anti-oestrogen resistance and progression of breast cancer (Article)

2009-12-01T00:00:00Z

Background:Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance.Methods:Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT-PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer.Results:mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease.Conclusions: Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.

Outcomes of first-line chemotherapy in patients with advanced or metastatic leiomyosarcoma of uterine and non-uterine origin (Article)

2009-12-01T00:00:00Z

Although leiomyosarcomas (LMSs) form the largest subgroup of soft tissue sarcomas (STSs), the efficacy of chemotherapy in this group is largely unclear, partly because older studies are contaminated with gastrointestinal stromal tumors (GISTs). In this retrospective study we investigated the outcome of first line chemotherapy in 65 patients with unresectable or metastatic LMS. The overall response rate (ORR) was 18%; and the median progression-free (PFS) and overall survival (OS) were 3.8 and 9.7 months respectively. No statistically significant differences in outcomes for uterine and non-uterine LMS were found. In non-uterine LMS, however, the PFS and OS seemed to be longer for females than for males, potentially negatively affecting outcomes in this group. If our observations are confirmed in other series, they would suggest that studies performed in STS patients should not only stratify for histological subtype but also for uterine versus non-uterine LMS and for gender. Copyright

Cardiovascular and renal toxicity during angiogenesis inhibition: Clinical and mechanistic aspects (Article)

2009-12-01T00:00:00Z

Inhibition of angiogenesis with humanized monoclonal antibodies to vascular endothelial growth factor (VEGF) or with tyrosine kinase inhibitors targeting VEGF receptors has become an established treatment for various tumor types. Contrary to expectations, angiogenesis inhibition by blocking VEGF-mediated signaling is associated with serious side effects including hypertension and renal and cardiac toxicity in a substantial proportion of patients. Fortunately, most of these side effects as discussed in this paper seem to be manageable, but likely become more problematic when survival increases. Although several hypotheses regarding the etiology of angiogenesis inhibition-related cardiovascular and renal side effects have been postulated, many of the underlying pathophysiological mechanisms remain to be elucidated. This may lead to the development of more specific angiogenesis inhibitors, better management of their side effects and may potentially provide new insights into the pathogenesis of cardiovascular disease in general.

Management of aggressive fibromatosis: Can we unravel the maze of treatment options? (Article)

2009-11-01T00:00:00Z

Chemotherapy: Adjuvant chemotherapy in older patients with breast cancer (Article)

2009-10-14T00:00:00Z

Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-a therapy (Article)

2009-10-01T00:00:00Z

Abstract Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-alpha (IFN-alpha). Animal studies showed that IFN-alpha administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases (MAOs), and finally in a change in NO production due to the H(2)O(2) generated by MAOs. We examined the potential relationship between NO production in plasma and MAO-B activity in platelets of 43 cancer patients during 8 weeks of treatment with IFN-alpha. NO synthesis was quantitated by measuring both the ratio of citrulline and arginine (CIT/ARG-ratio) and total nitrite/nitrate (NOx) levels. Compared to baseline, MAO activity and NOx increased, while the CIT/ARG-ratio decreased. No associations were found between NOx, MAO and CIT/ARG-ratio. Only few associations were observed between changes in the biochemical parameters and changes in psychopathology induced by IFN-alpha, of which the association between changes in CIT and lassitude was the most consistent. The results suggest that peripheral NO production and MAO activity are unrelated to each other, and that peripheral changes in these biochemical parameters induced by IFN-alpha are unlikely to contribute to definite psychiatric disturbance.

Mapatumumab, a fully human agonistic monoclonal antibody that targets TRAIL-R1, in combination with gemcitabine and cisplatin: A phase I study (Article)

2009-09-01T00:00:00Z

Purpose: To evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of mapatumumab, a fully human monoclonal antibody targeting tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), in combination with gemcitabine and cisplatin. Experimental Design: Patients with advanced solid tumors received gemcitabine 1,250 mg/m2 i.v. on days 1 and 8 and cisplatin 80 mg/m2 i.v. on day1 of each 21-day cycle. Escalating mapatumumab doses were administered i.v. every 21 days. Toxicity was evaluated and pharmacokinetic analysis of plasma mapatumumab, gemcitabine, 2-difluoro-2-deoxyuridine, and unbound and total platinum was done. TRAIL-R1 tumor expression was determined immunohistochemically. Results: Forty-nine patients received mapatumumab (1 mg/kg, n = 4; 3 mg/kg, n = 7; 10 mg/kg, n = 12; 20 mg/kg, n = 13; or 30 mg/kg, n = 13). A median of six cycles (range, 1-48) was administered. The adverse events most commonly observed reflect the toxicity profile of gemcitabine and cisplatin. Dose-limiting toxicities were seen in 3 of 12 patients at 10 mg/kg, consisting of grade 3 transaminitis, neutropenic fever, and grade 4 thrombocytopenia. At 20 mg/kg, 2 of 12 patients had dose-limiting toxicities, including grade 4 thrombocytopenia and grade 4 fatigue. The maximum tolerated dose was not reached. Pharmacokinetic interactions have not been observed. Twelve patients had a partial response, and 25 patients showed stable disease with a median duration of 6 months. Conclusions: Mapatumumab in combination with gemcitabine and cisplatin is safe and well tolerated at doses up to 30 mg/kg. Further studies on this combination are warranted.

Gastrointestinal Stromal Tumors II: Medical Oncology and Tumor Response Assessment (Article)

2009-08-01T00:00:00Z

The finding of mutations of KIT in gastrointestinal stromal tumors (GISTs) and subsequent development of kinase-directed therapy in metastatic GIST serve as a touchstone for the translation of laboratory research into clinical therapeutics. A variety of novel developments have followed the discovery of clinical activity of kinase-directed therapy against GIST. Radiological assessment of GIST challenges the standard of care for assessing tumor responses, ie, Response Evaluation Criteria in Solid Tumors (RECIST). Furthermore, the determination of the relationship of specific KIT mutations and sensitivity and resistance to kinase-directed agents and the assessment of inhibitor levels and the quality of response to those agents have implications beyond the treatment of sarcomas. These discoveries and the next chapters in this developing story are discussed in this review.

On the origin of (CD105+) circulating endothelial cells (Article)

2009-08-01T00:00:00Z

Cells designated by the CellSearch™ assay as circulating endothelial cells (CEC) (CD146+/CD105+/ CD45- nuclear cells) are thought to derive from damaged vasculature. As CD105 has been suggested to be expressed by endothelial cells from malignant vasculature particularly, it is currently unknown whether this assay is suitable to determine CECs in non-malignant diseases. Also, more insight is needed whether CECs as detected by this assay predominantly measures CECs or also endothelial progenitor cells (EPCs), which originate from the bone marrow and reflect angiogenesis rather than vascular damage. CEC counts were determined in nine patients treated with isolated limb perfusion with tumour necrosis factor (TNF) a and melphalan, and in 10 healthy donors. Given the severe vascular damage caused by venesection and cannulation of the main vessels, we expected a significant increase in CEC counts in case CEC were of vascular rather than of bone marrow origin. Additionally, this finding, as well as the presence of CD105+CEC in the blood of healthy controls, would confirm that healthy endothelial cells express CD105. Numbers of CD146+/CD105+/CD45-nuclear CEC increased significantly after venesection and cannulation. After administration of TNF, a large fraction of non-intact, possibly apoptotic CEC appeared. This study shows that the Cell-Search™ assay detects CECs originating from damaged vasculature. Furthermore, CD105 expression is found on CEC from damaged normal vasculature rendering further exploration of the value of CEC determined by this assay worthwhile not only in malignant diseases but also in non malignant disorders characterised by vascular damage.

Application of prolonged microdialysis sampling in carboplatin-treated cancer patients (Article)

2009-07-01T00:00:00Z

Purpose: To better understand the mechanisms underlying (in)sensitivity of tumors to anticancer drugs, assessing intra-tumor drug pharmacokinetics (PKs) could be important. We explored the feasibility of microdialysis in tumor tissue for multiple days in a clinical setting, using carboplatin as model drug. Methods: Plasma and microdialysate samples from tumor and adipose normal tissues were collected up to 47 h after dosing in eight carboplatin-treated patients with an accessible (sub)cutaneous tumor. Results: Pharmacokinetics were evaluable in tumor tissue in 6/8 patients and in adipose normal tissue in 3/8 patients. Concentration-time curves of unbound platinum in both the tissues followed the pattern of the curves in plasma, with exposure ratios of tissue versus plasma ranging from 0.64 to 1.46. Conclusions: Microdialysis can be successfully employed in ambulant patients for multiple days, which enables one to study tissue PK of anticancer drugs in normal and malignant tissues in more detail.

Circulating endothelial cells: A potential parameter of organ damage in sickle cell anemia? (Article)

2009-07-01T00:00:00Z

Objective laboratory tools are needed to monitor developing organ damage in sickle cell disease (SCD). Circulating endothelial cells (CECs) are indicative of vascular injury. We determined whether elevated CEC can be detected in asymptomatic SCD with the CellSearch system and whether the CEC count is related to clinical and blood-based biomarkers of disease severity. Fifteen consecutive clinically asymptomatic HbSS patients and 15 matched HbAA controls were analyzed for CEC counts, laboratory parameters of disease severity (Hb, leukocyte counts, HbF%), plasma levels of markers for endothelial activation (sVCAM-1, VWF:Ag) and of endogenous inhibitors of nitric oxide synthase (asymmetrical dimethylarginine [ADMA]). CEC counts were significantly higher in patients (12 cells/mL, IQR 8-29) as compared to controls (4 cells/mL, 3-10) (P = 0.007). CEC counts were significantly higher in patients with pulmonary hypertension (PHT) (P = 0.015), and increased with increasing number of affected organs (0-4 involved organs, P = 0.002). No significant correlations between CEC and any other laboratory parameter were detected. In conclusion, CECs could prove to be an important new tool for assessing developing vasculopathy and organ damage in SCD.

Soft tissue sarcomas: ESMO clinical recommendations for diagnosis, treatment and follow-up (Article)

2009-06-11T00:00:00Z

Quantification of circulating endothelial cells by flow cytometry (Article)

2009-05-15T00:00:00Z

The applicability of mTOR inhibition in solid tumors (Article)

2009-05-01T00:00:00Z

The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin-pathway (PI3K/AKT/mTOR-pathway) plays a role in the regulation of cell proliferation, cell survival, angiogenesis and resistance to anti-tumor treatments. In many tumor types the PI3K/AKT/mTOR-pathway is found activated through several different underlying mechanisms. Since this pathway is believed to largely drive the malignant behavior of several of these tumors, mTOR-inhibition is considered an attractive means to apply as anti-tumor treatment. Currently, four mTOR-inhibitors are explored for clinical use: rapamycin, temsirolimus (CCI-779), everolimus (RAD001) and deforolimus (AP23573). As monotherapy, mTOR-inhibitors yield interesting anti-tumor activity against various tumor types at the expense of relatively mild toxicities. This recently resulted in the registration of two mTOR-inhibitors for patients with metastatic renal cell carcinoma (RCC) while randomized studies in other tumors are currently in progress. Furthermore, mTOR-inhibitors are well-suited drugs to combine with other anti-tumor drugs as in preclinical models mTOR-inhibition overcomes chemoresistance. Consequently, mTOR-inhibitor-containing multidrug regimens are subject to clinical studies. As holds true for all anti-tumor therapies, identification of patients who are likely to respond to mTOR-inhibitor-containing therapies is of utmost importance to avoid over- or undertreatment. Preliminary results suggest that several factors reflecting activation of mTOR in tumors may be used for this purpose. This review addresses the mechanism of action and current clinical experience with mTOR-inhibitors as well as their role in overcoming resistance to conventional therapies. Additionally, potential predictors of outcome to mTOR-inhibition are discussed.

Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer (Article)

2009-03-01T00:00:00Z

Abstract The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool, as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However, implementation of this assay into clinical routine has been cumbersome, as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers, and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature, including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients.

The price of success: Cost-effectiveness of molecularly targeted agents (Article)

2009-02-01T00:00:00Z

The advent of molecularly targeted agents has brought major breakthroughs in oncology. However, because these drugs are expensive and financial resources are limited, attention to their cost-effectiveness is rapidly increasing. It seems that establishment of cost-effectiveness is complex but is determined for the most part by the cost of drug acquisition and the drug's efficacy. This article examines the cost-effectiveness of molecularly targeted drugs.

Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells (Article)

2009-01-01T00:00:00Z

Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling - normal-like, basal, HER2-positive, and luminal A and B - were identified by CellSearch, a US Food and Drug Administration-approved test that uses antibodies against the cell surface-expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed.

GIST as the model of paradigm shift towards targeted therapy of solid tumors: Update and perspective on trial design (Article)

2008-12-01T00:00:00Z

Angiogenesis inhibition in non-GIST soft tissue sarcomas (Article)

2008-11-01T00:00:00Z

Because angiogenesis is of crucial importance in the pathogenesis of cancer, blocking the function of proangiogenic factors has been shown to improve the outcomes of patients with several cancer types. Given the poor survival durations of patients with advanced soft-tissue sarcomas (STSs), which has remained stable at a median of 12 months over the last 20 year, there is an unmet need for novel agents active against these tumors. Like in other tumors, accumulating evidence points at an important role for angiogenic factors in STSs, rendering these factors attractive treatment targets. This review discusses the currently available evidence supporting a role for angiogenic factors in the pathogenesis of STSs and the first preliminary study results obtained with angiogenesis inhibitors.

The use of bevacizumab in colorectal, lung, breast, renal and ovarian cancer: Where does it fit? (Article)

2008-11-01T00:00:00Z

Bevacizumab is approved for the treatment of colorectal cancer, breast cancer, non-small cell lung cancer and renal cell cancer. Before embracing this expensive agent for many other indications, it remains critical to be aware of the evidence upon which oncologists base their day-to-day clinical practice. In this review, we address the results of clinical studies upon which bevacizumab's current use is based and discuss some future perspectives.

Correlation between circulating endothelial cell counts and plasma thrombomodulin levels as markers for endothelial damage (Article)

2008-10-01T00:00:00Z

Increased numbers of circulating endothelial cells (CEC) in peripheral blood have been observed in diseases with vascular involvement, and are considered a promising surrogate marker for vascular damage. It was the objective of this study to evaluate the correlation between putative soluble markers of endothelial injury, activation, and endothelial proliferation, and absolute numbers of CEC. CEC were evaluated in 125 healthy donors and 40 patients with metastatic carcinoma by automated CD146 driven immunomagnetic isolation. Plasma concentrations of E-selectin, endoglin, and thrombomodulin were assessed by ELISA in plasma obtained from 40 healthy donors and 40 patients. CEC numbers in blood were positively correlated with plasma thrombomodulin levels, but not with levels of E-selectin and endoglin. Multivariate analysis demonstrated a significant increase in CEC numbers with age. The levels of plasma biomarkers were not influenced by age. Higher levels of thrombomodulin and E-selectin were observed in males when compared to females. In conclusion, CEC numbers correlate positively with plasma levels of thrombomodulin.

Neurotoxic and neuroprotective metabolites of kynurenine in patients with renal cell carcinoma treated with interferon-α: Course and relationship with psychiatric status (Article)

2008-10-01T00:00:00Z

Aims: Immunotherapy with interferon-α (IFN-α) is associated with psychiatric side-effects, including depression. One of the putative pathways underlying these psychiatric side-effects involves tryptophan (TRP) metabolism. Cytokines including IFN-α induce the enzyme indoleamine 2,3-dioxygenase (IDO), which converts TRP to kynurenine (KYN), leading to a shortage of serotonin (5-HT). In addition, the production of neurotoxic metabolites of KYN such as 3-hydroxykynurenine and quinolinic acid (QA) might increase and contribute to IFN-α-induced psychopathology. In contrast, other catabolites of KYN, such as kynurenic acid (KA), are thought to have neuroprotective properties. Methods: In a group of 24 patients treated with standard IFN-α for metastatic renal cell carcinoma (RCC), combined psychiatric and laboratory assessments were performed at baseline, 4 and 8 weeks, and at 6 months. Results: No psychopathology was observed, despite an increase in neurotoxic challenge as reflected in indices for the balance between neurotoxic and neuroprotective metabolites of KYN. Conclusions: The present hypothesis that a shift in the balance between neurotoxic and neuroprotective metabolites of KYN underlies the neuropsychiatric side-effects of IFN-α-based immunotherapy, is neither supported nor rejected.

Dose selection in phase I studies: Why we should always go for the top (Article)

2008-09-15T00:00:00Z

In reply (Article)

2008-09-15T00:00:00Z

Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response (Article)

2008-09-01T00:00:00Z

Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative realtime PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. Results: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P < 0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of ∼40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P = 0.001), and (b) no clinical benefit (odds ratio, 0.81; P = 0.035) and shorter progression-free survival (hazard ratio, 1.19; P = 0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. Conclusion: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereasTNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

Retronectin-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield (Article)

2008-07-21T00:00:00Z

BACKGROUND: For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin in combination with Lifecell X-foldtrade mark cell culture bags. METHODS: In our current study, we evaluated two alternative types of bags for the Retronectin-mediated retroviral transduction of human T cells: the Miltenyi DC-generation bag and the Takara CultiLife Spin bag. RESULTS: In static transductions, but not in spinoculation, the DC-generation bags and CultiLife Spin bags performed as well as Lifecell X-foldtrade mark bags in Retronectin-assisted retroviral transduction of human T cells with respect to transduction efficiency, lymphocyte subset composition and lymphocyte function. However, both types of bags performed less well than Lifecell X-foldtrade mark cell culture bags in terms of cell yield. DISCUSSION: Adjusted numbers of cells at the start of transduction should be used when using the Miltenyi or Takara bags in order to compensate for the lower cell yield following transduction.

Irinotecan-induced central nervous system toxicity. Report on two cases and review of the literature (Article)

2008-07-01T00:00:00Z

Decrease of CD117 expression as possible prognostic marker for recurrence in the resected specimen after imatinib treatment in patients with initially unresectable gastrointestinal stromal tumors: A clinicopathological analysis (Article)

2008-07-01T00:00:00Z

Gastrointestinal stromal tumors (GIST) are the most common malignant mesenchymal tumors of the gastrointestinal tract. The principal treatment modality for primary GIST is surgery whereas for metastatic GIST, imatinib has an established role. In patients with locally advanced and metastatic GIST, the role of surgery in the imatinib era is still unclear. Fifteen patients with locally advanced (n≤9) and/or metastatic GIST (n≤6) were treated with imatinib followed by resection. Detailed histopathological examination was performed before and after treatment with imatinib, which was given for a median of 11 months before surgery. Ten patients showed a radiographic partial response, four patients had stable disease, and one patient progressed. At the time of surgery, the median tumor diameter was 6.5g cm. In all the nine patients with locally advanced GIST, a R0 resection could be performed. Histopathological examination showed imatinib effects in all tumors, including the case with progressive disease. All patients with locally advanced disease (n≤9) were alive after a median follow-up of 40 months (range: 18g-59), of which seven patients were free of disease. Four of the six patients treated for metastatic GIST died of disease after 30, 45, 50, and 74 months of follow-up. Remarkably, in five of six patients in whom CD117 expression was diminished or lost in the resection specimen, disease recurrence was observed. In patients with retained CD117 expression, one of the nine patients had recurrent disease. In conclusion, preoperative imatinib treatment in patients with locally advanced GIST resulted in a decrease of tumor load in most patients, enabling complete surgical resection. For patients with metastatic GIST, the role of surgery remains less clear. Loss or decrease of CD117 expression in the resected specimen after imatinib treatment may be associated with disease recurrence.

Circulating endothelial cells in oncology: Pitfalls and promises (Article)

2008-06-03T00:00:00Z

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

Soft tissue sarcomas: ESMO clinical recommendations for diagnosis, treatment and follow-up (Article)

2008-05-01T00:00:00Z

Editorial: The long and winding road to better cancer cell-specific therapies (Article)

2008-05-01T00:00:00Z

Plasma activity of prolyl endopeptidase in relation to psychopathology during immunotherapy with IFN-α in patients with renal cell carcinoma (Article)

2008-05-01T00:00:00Z

Abnormal activity in peripheral blood of the cytosolic enzyme prolyl endopeptidase (PEP, EC 3.4.21.26, post prolyl cleaving enzyme, prolyl oligopeptidase) has been found in patients with a variety of psychiatric disorders, most consistently in mood disorders. Mood disturbance is a well-known side effect of immunotherapy with interferon-α (IFN-α). Earlier, we documented a decrease in serum PEP activity in the first 4 weeks of treatment with IFN-α. In 24 patients (16 men, 8 women, median age 60.5 years, range 47-72 years) with metastatic renal cell carcinoma (RCC), psychiatric assessment and blood sampling were performed before and at 4 and 8 weeks and at 6 months after initiation of treatment with IFN-α. No episodes of depression were observed, and the sum score and the scores on the subscales for depression and hostility of the Symptom Check List-90 (SCL-90) did not change during follow-up, whereas the anxiety scores were somewhat lower at 4 and 8 weeks compared with baseline. No change in plasma PEP activity and no relationships between change in psychiatric parameters and change in plasma PEP activity were found. As more subtle relationships between PEP activity and psychiatric status could have easily been obscured, a role for PEP in the pathophysiology of IFN-α-induced mood disturbance can neither be confirmed nor excluded.

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years (Article)

2008-04-01T00:00:00Z

Abstract. Recombinant retroviruses are one of the most commonly used gene transfer vehicles for therapeutic gene delivery. The stability of viral vectors upon long-term storage, anticipated to be short lived, is expected to impact timeline and financial course of clinical immunogene therapy. However, to date little is known about vector stability. Therefore, we analyzed the stability of retroviral vectors produced in culture supernatants (RTVsup) for ex vivo gene therapy upon long-term storage. We have generated RTVsups derived from two packaging cell lines, PG13 and Phoenix(Ampho). Both lines produced murine leukemia virus-derived SFG-scFv(G250)-CD4gamma vector, which were pseudotyped with the gibbon ape leukemia virus envelope and amphotropic envelope, respectively. The supernatants were stored at -80 or -196 degrees C. To date, the PG13-derived RTVsups have been evaluated over a period of 9 years (1998-2007). In addition, a clinical batch of Phoenix(Ampho)-derived RTVsup has been evaluated over a period of 5 years (2002-2007). Here, we show that both RTVsups, when stored up to 9 and 5 years, respectively, do not show any sign of decay in their capacity to functionally transduce primary human T cells. These data provide evidence that in terms of 'life expectancy' the production and storage of clinical batches of RTVsup for gene therapy warrants the corresponding professional and financial risks.

Drug Insight: Gastrointestinal stromal tumors (GIST) - The solid tumor model for cancer-specific treatment (Article)

2008-02-01T00:00:00Z

We are living in an exciting era in the treatment of cancer, using drugs that target specific proteins rather than agents that cause more general cytotoxic effects. The identification of proteins and signal transduction pathways that play crucial roles in the pathogenesis of cancer has allowed treatments to be designed that target these tumor-driven events. Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors and were among the first solid tumor types for which such a novel treatment (in this case imatinib) became available. The tyrosine kinase inhibitor imatinib targets the human KIT receptor and the platelet-derived growth factor receptor-α. This drug exhibits impressive antitumor effects against GIST and has become the first-line therapy for advanced disease. Major insights into the mechanism of action of this drug, drug resistance, and patient management issues have been gleaned. Additionally, new drugs developed for the treatment of GIST have been identified. As a consequence, lessons learned from GIST are widely applicable to other tumor entities, thereby rendering GIST the paradigm of solid tumors treated with tyrosine kinase inhibitors. This Review discusses the pathogenesis of GIST, treatment strategies, mechanisms accounting for drug resistance, and potential future perspectives.

Interferon-alpha in oncology patients: fewer psychiatric side effects than anticipated (Article)

2008-01-01T00:00:00Z

Interferon-alpha (IFN-alpha) treatment in both oncological and hepatological settings is associated with depression. If IFN-alpha treatment induces depression in high numbers, it could serve as a model for studying the pathophysiology of depression, in general. The authors therefore studied 43 oncology patients treated with standard or pegylated IFN-alpha with baseline psychiatric assessment and at regular time-points in the first 6 months of treatment. Apart from a severe depression because of brain metastases, authors observed only two clinically relevant depressive states. Contrary to findings in most of the literature, most depressive episodes in this study were self-limiting and short-lasting and were associated with either episodes of flu-like symptoms common at the start of the treatment or with concurrent psychosocial events. In the group as a whole, scores on both observer-based and self-report rating scales did not show clinically relevant changes. The results of this study indicate that IFN-alpha treatment is not suitable as a study model for depression in general.

Trial methodology in soft tissue sarcomas; the 'one plus one is three or zero' phenomenon (Article)

2008-01-01T00:00:00Z

The pathogenesis of cancer related fatigue: Could increased activity of pro-inflammatory cytokines be the common denominator? (Article)

2008-01-01T00:00:00Z

Cancer related fatigue (CRF), defined as a persistent subjective sense of tiredness related to cancer or cancer treatment that interferes with daily functioning, is highly prevalent and probably the most underestimated and insufficiently treated complication in cancer patients. Therefore, unravelling the pathogenesis of CRF is of great importance allowing the identification of novel therapeutic targets. It is generally believed that the pathogenesis is multicausal, explaining why most therapeutic interventions directed towards only one potential causative factor are unsuccessful. In this regard, it is of interest that increased activity of pro-inflammatory cytokines might be a common denominator causing CRF. Detailed insight in the central role of increased activity of pro-inflammatory cytokines in CRF will hopefully offer an effective approach in the treatment of CRF by affecting a broad array of proposed causative factors such as anaemia, disturbances of the hypothalamic-pituitary-adrenal axis and altered brain serotonin metabolism.

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo (Article)

2007-12-01T00:00:00Z

Abstract BACKGROUND: We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)(+) T cells is reflected by changes of selected immune parameters measured in peripheral blood. METHODS: ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)(+) T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. RESULTS: Prior to infusion, scFv(G250)(+) T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX(+) RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-gamma were produced, whilst production of TNF-alpha, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-gamma was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-gamma increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. CONCLUSION: ScFv(G250)-receptor-mediated functions of the scFv(G250)(+) T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-gamma levels.

Circulating tumour cell detection on its way to routine diagnostic implementation? (Article)

2007-12-01T00:00:00Z

Circulating tumour cells (CTCs) have been considered for a long time in reflecting the aggressiveness of tumours. As a result, many attempts have been made to develop assays that reliably detect and enumerate CTCs, but only recently have such assays been available. The first clinical results obtained with such assays strongly suggest that in some tumour types, CTC detection and enumeration can be used to estimate prognosis and may serve as an early marker to assess anti-tumour activity of a treatment. Furthermore, through technical advances, CTCs can be characterised for several features, which may shortly yield better prognostic and predictive classification systems and may also provide improved insight into biological processes including dissemination, drug resistance and treatment-induced cell death. This review addresses CTCs, and in particular, technical issues concerning their detection, clinical results obtained so far, and future perspectives.

The pharmacologic basis of ifosfamide use in adult patients with advanced soft tissue sarcomas (Article)

2007-11-01T00:00:00Z

The treatment outcome of patients with locally advanced and metastatic soft tissue sarcomas is poor. Doxorubicin is regarded as standard treatment, but its use is featured by the occurrence of cardiotoxicity. This hinders the administration of this drug at high doses or in combination with, in theory, attractive newly developed targeted drugs, such as vascular endothelial growth factor (VEGF) pathway inhibitors. The combination of doxorubicin and VEGF pathway inhibitors has been shown to yield an unacceptable high rate of cardiomyopathy. Ifosfamide is the only drug that consistently shows response rates comparable to those of doxorubicin. The lack of cardiotoxicity renders this drug a much more attractive alternative than doxorubicin to be explored at high doses or as part of new drug combinations. This review addresses the clinical pharmacology, metabolism, and present role of ifosfamide in the treatment of locally advanced and/or metastatic soft tissue sarcomas, excluding gastrointestinal stromal tumors, the Ewing-like sarcomas, and other small blue round cell tumors. Furthermore, this review focuses on the anticipated growing role of ifosfamide in the development of new treatment strategies.

Bleomycin and scuba diving: where is the harm? (Article)

2007-11-01T00:00:00Z

Potential biomarkers in metastatic gastrointestinal stromal tumors (Article)

2007-10-15T00:00:00Z

Interferon-α influences tryptophan metabolism without inducing psychiatric side effects (Article)

2007-10-01T00:00:00Z

Background: Interferon-α (IFN-α) treatment is often associated with psychiatric side effects and has been found to lower the amount of tryptophan (TRP) available to the brain. The alterations in tryptophan metabolism might underlie the psychiatric side effects during treatment with IFN-α. Methods: In this study, 43 oncology patients treated with IFN-α were included. In order to study de novo depressions, depressed patients at baseline were excluded. Psychiatric evaluation comprising clinical judgment combined with a structured psychiatric interview and observer-based and self-report rating scales was performed at baseline and at 4 weeks, 8 weeks and 6 months after the start of treatment with IFN-α, and in the case of emerging psychopathology. Blood samples were drawn at the same evaluation times and assessed for concentrations of TRP, large neutral amino acids, kynurenine, 5-hydroxyindole acetic acid, neopterin and biopterin. Results: During treatment with IFN-α, several alterations in laboratory parameters occurred that were consistent with an increased degradation of peripheral TRP. Psychometric ratings revealed hardly any psychiatric changes. No consistent associations were found between changes in the laboratory assessments determined and the diverse psychiatric measures. Conclusion: In this study, IFN-α was found to alter TRP metabolism without inducing psychiatric side effects. Therefore, a possible relationship between TRP metabolism and depression was not substantiated by this study. Copyright

Gynaecological sarcomas (Article)

2007-09-01T00:00:00Z

PURPOSE OF REVIEW: To review gynaecological sarcomas, their management and future perspectives. RECENT FINDINGS: Soft tissue sarcomas are a group of tumours consisting of a wide variety of subtypes. The most common subtypes encountered in the female tract are uterine leiomyosarcomas and endometrial stromal sarcomas. Other subtypes do occur but at a much lower frequency. Mixed Müllerian tumours were previously considered to be a subtype of sarcoma, but recent evidence has revealed that they should in fact be regarded as carcinomas. Given the different approaches for different subtypes of sarcomas, their rarity and the complexity of treatment, the management of patients with gynaecological sarcomas should be delivered by multidisciplinary teams experienced in the treatment of these entities. SUMMARY: Gynaecological sarcomas are rare tumours and are treated differently from gynaecological carcinomas. Hopefully, improved insight into the pathogenesis of gynaecological sarcomas will yield novel therapies in the near future.

Response to Goon et al. (Article)

2007-09-01T00:00:00Z

Pulmonary alterations during BEP? Always be aware of BIP (Article)

2007-06-01T00:00:00Z

Improved insight into resistance mechanisms to imatinib in gastrointestinal stromal tumors: A basis for novel approaches and individualization of treatment (Article)

2007-06-01T00:00:00Z

Gastrointestinal stromal tumor (GIST) is one of the first solid tumor types in which a tyrosine kinase inhibitor, imatinib, has become standard of care for patients with advanced disease. Although imatinib yields antitumor activity in the vast majority of patients, it is likely that all patients eventually experience progressive disease given enough time. In recent years, major progress has been made in the elucidation of mechanisms conferring resistance to imatinib that result in progressive disease. Insight into these resistance mechanisms has already resulted in the availability of strategies that can be applied in cases of progressive disease and it is likely that more approaches will be defined in the next years. Additionally, it can be anticipated that in the near future treatment will be guided according to factors determining sensitivity to imatinib. This review focuses on the factors inducing imatinib resistance that have been elucidated so far, the currently available and potential novel treatment options for patients with progressive disease, and how insight into resistance mechanisms may allow individualized treatment in the near future for patients with advanced GISTs.

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets (Article)

2007-03-15T00:00:00Z

Background: Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low-to-intermedate, sideward light scatter (SSC)low, CD45-, CD31++and CD146+is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658-3661). Here, we set out to confirm the endothelial nature of these cells. Methods: We isolated cells with a FSClow-to-intermediate, SSClow, CD31++, CD45dimimmunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel-Palade bodies in the sorted cells. Results: CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel-Palade bodies, and revealed CMOIC to be large platelets. Conclusion: The vast majority of cells with the immunophenotype FSClow-to-intermediate, SSClow, CD45-, CD31++do not express CD146 and are large platelets rather than endothelial cells.

Influence of pegylated interferon-α therapy on plasma levels of citrulline and arginine in melanoma patients (Article)

2007-01-01T00:00:00Z

The aim of this study was to evaluate the effect of pegylated interferon-alpha (PEG-IFN-α) on the plasma citrulline/arginine ratio, regarded as an index of nitric oxide (NO) synthesis, in patients with high-risk melanoma. Forty patients were randomly assigned to either PEG-IFN-α treatment (n = 22) or to observation only (control group, n = 18). The treatment group received 6 μg PEG-IFN-α/kg once a week during 8 weeks, followed by a maintenance dose of 3 μg/kg/wk. Blood was collected at different time points, plasma concentrations of citrulline and arginine were measured and the ratio of citrulline/arginine was calculated. Patients treated with PEG-IFN-α showed a significant decrease in the concentrations of citrulline and in the citrulline/arginine ratio during the whole study period, both compared to baseline values and to the control group. The data suggest that therapy with PEG-IFN-α results in a marked decrease in the synthesis of NO in melanoma patients.

Process validation and clinical evaluation of a protocol to generate gene-modified T lymphocytes for imunogene therapy for metastatic renal cell carcinoma: GMP-controlled transduction and expansion of patient’s T lymphocytes using a carboxy anhydrase IX-specific scFv transgene (Article)

2006-12-01T00:00:00Z

Abstract BackgroundAdoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes.MethodsA batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients.ResultsThe clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses.DiscussionWe have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment

Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience (Letter To Editor)

2006-05-01T00:00:00Z

Using single-agent therapy in adult patients with advanced soft tissue sarcoma can still be considered standard care. (Article)

2005-11-01T00:00:00Z

The group of soft tissue sarcomas in adult patients is a heterogeneous group with more than 40 different subtypes. While local treatment remains the mainstay for localized disease, systemic chemotherapy can importantly contribute in the treatment of advanced soft tissue sarcoma. For patients with metastatic disease, chemotherapy is a palliative treatment in the vast majority of the cases. In this setting, toxicity should not outweigh the potential benefits resulting from chemotherapy. In patients with locally advanced disease too extensive for local treatment, systemic chemotherapy can contribute to cure, provided that tumor shrinkage renders subsequent optimal local treatment possible. In these cases, chemotherapeutic regimens yielding the highest response rates achievable should be used. In the last decades, several randomized studies have aimed to determine whether combination regimens yield benefit over single-agent treatment in terms of response rate and overall survival. This review addresses the current available data on chemotherapy for adult patients with soft tissue sarcoma, excluding gastrointestinal stromal tumor, the Ewing-like sarcomas, and other small blue round cell tumors. In addition, it is increasingly recognized that future research in soft tissue sarcoma should focus on the identification of tumor factors that can serve as targets for treatment and that the diverse tumor subtypes should be analyzed separately for their sensitivity to systemic treatment. This review also focuses on these and other strategies that will hopefully lead to better out comes in this disease entity in the near future.

Adoptive immuno-gene therapy of cancer with single chain antibody [scFv(Ig)] gene modified T lymphocytes (Article)

2004-04-01T00:00:00Z

Abstract Adoptive transfer of antigen-specific T cells has recently shown therapeutic successes in the treatment of viral infections and tumors. T cells specific for the antigen of interest can be generated in vitro, and adoptively transferred back to provide patients with large numbers of immune-competent T cells. Adoptive T cell therapy, however, is a patient-tailored treatment that unfortunately is not universally applicable to treat viral infections and tumors. We and others have demonstrated that the transfer of genes encoding antigen-specific receptors into T cells (i.e., genetic retargeting) represents an attractive alternative to induce antigen-specific immunity. Currently, we evaluate this concept in a clinical protocol to treat patients with metastatic renal cell cancer (RCC) using autologous RCC-specific gene-modified T lymphocytes

Aplastic anaemia: a review (Article)

2003-01-01T00:00:00Z

Aplastic anaemia is featured by bone marrow hypocellularity and peripheral pancytopenia and is a potentially fatal disease. In recent years, insight in it pathogenesis has increased. It appears that activated autoreactive T lymphocytes induce apoptosis of haematopoietic stem cells resulting in a hypocellular bone marrow. Nowadays, it can be treated by stem cell transplantation or immunosuppressive therapy. This review focuses on the pathophysiology and treatment of aplastic anaemia.

Bleomycin-induced pneumonitis (Article)

2001-01-01T00:00:00Z

The cytotoxic agent bleomycin is feared for its induction of sometimes fatal pulmonary toxicity, also known as bleomycin-induced pneumonitis (BIP). The central event in the development of BIP is endothelial damage of the lung vasculature due to bleomycin-induced cytokines and free radicals. Ultimately, BIP can progress in lung fibrosis. The diagnosis is established by a combination of clinical symptoms, radiographic alterations, and pulmonary function test results, while other disorders resembling BIP have to be excluded. Pulmonary function assessments most suitable for detecting BIP are those reflecting lung volumes. The widely used transfer capacity of the lungs for carbon monoxide appeared recently not to be specific when bleomycin is used in a polychemotherapeutic regimen. There are no proven effective treatments for BIP in humans, although corticosteroids are widely applied. When patients survive BIP, they almost always recover completely with normalization of radiographic and pulmonary function abnormalities. This review focuses on BIP, especially on the pathogenesis, risk factors, and its detection.