Kraan, J.

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

Elevated numbers of regulatory T cells, central memory T cells and class-switched B cells in cerebrospinal fluid of patients with anti-Hu antibody associated paraneoplastic neurological syndromes (Article)

2013-04-08T00:00:00Z

Multi-parametric flow cytometry was used to study lymphocyte subsets and dendritic cells in paired blood and CSF samples from 11 newly diagnosed patients with progressive anti-Hu antibody associated paraneoplastic neurological syndromes (Hu-PNS), 9 patients with other inflammatory neurologic disorders (IND), and 12 patients with other non-inflammatory neurologic disorders (OND). Hu-PNS patients had elevated numbers of regulatory T cells, central memory T cells, class-switched B cells and dendritic cells in their CSF. These findings support the hypothesis that the immune system is locally activated in Hu-PNS, and suggests common etiological pathways between Hu-PNS and other inflammatory central nervous system disorders.

Flow cytometric characterization of cerebrospinal fluid cells (Article)

2011-09-01T00:00:00Z

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately.

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.

Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time (Article)

2011-03-12T00:00:00Z

Immediately after sampling, leukocyte counts in native cerebrospinal fluid (CSF) start to decrease rapidly. As the time lapse between CSF collection to analysis is not routinely registered, the clinical significance of decreasing cell counts in native CSF is not known. Earlier data suggest that addition of serum-containing medium to CSF directly after sampling prevents this rapid decrease in leukocyte counts and, thus, may improve the accuracy of CSF cell counting and cell characterization. Here, we prospectively examined the effect of storage time after lumbar puncture on counts of leukocytes and their major subsets in both native CSF and after immediate addition of serum-containing medium, measured by flow cytometry and microscopy. We collected CSF samples of 69 patients in tubes with and tubes without serum-containing medium and determined counts of leukocytes and subsets at 30 minutes, 1 hour, and 5 hours after sampling. Compared to cell counts at 30 minutes, no significant decrease in cell number was observed in CSF with serum-containing medium 1 and 5 hours after sampling, except for the granulocytes at 1 hour. In native CSF, approximately 50% of leukocytes and all their subsets were lost after 1 hour, both in flow cytometric and microscopic counting. In 6/7 (86%) samples with mild pleocytosis (5-15 × 10(6) leukocytes/l), native CSF at 1 hour was incorrectly diagnosed as normocellular. In conclusion, addition of serum-containing medium to CSF directly after sampling prevents cell loss and allows longer preservation of CSF cells prior to analysis, both for microscopic and flow cytometric enumeration. We suggest that this protocol results in more accurate CSF cell counts and may prevent incorrect conclusions based on underestimated CSF cell counts.

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.

Central memory CD4+ T cells dominate the normal cerebrospinal fluid (Article)

2011-01-01T00:00:00Z

Background: To use cerebrospinal fluid (CSF) immune phenotyping as a diagnostic and research tool, we have set out to establish reference values of white blood cell (WBC) subsets in CSF. Methods: We assessed the absolute numbers and percentages of WBC subsets by 6 color flow cytometry in paired CSF and blood samples of 84 individuals without neurological disease who underwent spinal anaesthesia for surgery. Leukocyte (i.e., lymphocytes, granulocytes, and monocytes), lymphocyte (i.e., T [CD4 + and CD8 +], NK, NKT and B cells), T cell (i.e., naïve, central memory, effector memory, and regulatory) and dendritic cell subsets (i.e., myeloid and plasmacytoid) were studied. Results: CSF showed a predominance of T cells, while granulocytes, B and NK cells were relatively rare compared to blood. The majority of T cells in CSF consisted of CD4 + T cells (∼70%), most of them (∼90%) with a central memory phenotype, while B cells were almost absent (<1%). Among the small population of dendritic cells in CSF, those of the myeloid subtype were more frequent than plasmacytoid dendritic cells (medians: 1.7% and 0.4% of leukocytes, respectively), whilst both subsets made up 0.2% of leukocytes in blood. Conclusions: This study reports reference values of absolute numbers and percentages of WBC subsets in CSF, which are essential for further investigation of the immunopathogenesis of neuro inflammatory diseases. Furthermore, the relative abundance of CD4 + T cells, mainly with a central memory phenotype, and the presence of dendritic cells in CSF suggests an active adaptive immune response under normal conditions in the central nervous system (CNS).

Levels of circulating endothelial cells in normotensive and severe preeclamptic pregnancies (Article)

2010-11-01T00:00:00Z

Background: Preeclampsia is a disease hypothesized to originate from widespread endothelial dysfunction or damage. This study investigated whether circulating endothelial cells (CEC) can serve as a surrogate marker for disease severity in patients with preeclampsia, and if their number correlates to serum endothelial biomarkers for activation, dysfunction, or damage of those cells. Methods: Blood was drawn consecutively from 30 patients admitted with a diagnosis of severe preeclampsia. Thirty healthy, normotensive, patients matched for age, body mass index, and gestational age served as a control group. We determined the number of CEC and serum concentrations of biomarkers indicative of endothelial damage (thrombomodulin) and activation (E-selectin), and the antiangiogenic protein (endoglin), which reflects endothelial dysfunction. Results: Median CEC counts did not differ significantly between preeclamptic patients and the control group (median 5.3 vs. 3.5 CEC/mL, respectively) and were mostly within the normal range (i.e., <20 CEC/mL). However, serum concentrations of thrombomodulin (median 3.6 vs. 5.2 ng/mL; P = 0.006), E-selectin (median 32.0 vs. 42.9 ng/mL; P = 0.02), and especially endoglin (median 5.0 vs. 76.2 ng/mL; P < 0.0001) were significantly increased in severe preeclamptic patients. CEC counts did not correlate with any of the clinical parameters or routinely determined laboratory indices. Conclusion: Preeclampsia is characterized by endothelial dysfunction and activation rather than actual endothelial damage as characterized by increased CEC counts.

Levels of circulating endothelial cells in normotensive and severe preeclamptic pregnancies (Article)

2010-11-01T00:00:00Z

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

2010-06-10T00:00:00Z

Combination of IL-21 and IL-15 enhances tumour-specific cytotoxicity and cytokine production of TCR-transduced primary T cells (Article)

2010-06-01T00:00:00Z

IL-21, and to a lesser extent IL-15, inhibits differentiation of antigen-primed CD8 T cells and promotes their homeostasis and anti-tumour activity. Here, we investigated molecular mechanisms behind tumour-specific responses of primary murine T lymphocytes engineered to express a TCR directed against human gp100/HLA-A2 following short-term exposure to IL-15 and/or IL-21. We demonstrated that IL-15 + IL-21, and to a lesser extent IL-21, enhanced antigen-specific T-cell cytotoxicity, which was related to enhanced expression of granzymes A and B, and perforin 1. Furthermore, IL-15 + IL-21 synergistically enhanced release levels and kinetics of T-cell IFNγ and IL-2, but not IL-10. Enhanced secretion of IFNγ was accompanied by increased gene expression and cytosolic protein content, and was restricted to effector memory T cells. To summarize, we show that IL-15 + IL-21 improves antigen-specific responses of TCR-transduced effector T cells at multiple levels, which provides a rationale to treat T cells with a combination of these cytokines prior to their use in adoptive TCR gene therapy.

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.

Quantification of circulating endothelial cells by flow cytometry (Article)

2009-05-15T00:00:00Z

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.

Contamination of synthetic HuD protein spanning peptide pools with a CMV-encoded peptide (Article)

2008-11-01T00:00:00Z

To detect HuD-specific T cells in patients with Hu-antibody associated paraneoplastic neurological syndromes (Hu-PNS), we used short-term stimulation assays with HuD protein spanning peptide pools (PSPP) with purities of at least 70% and found reproducible false-positive CD8+ T-cell responses in three of 127 individuals (two healthy controls and one Hu-PNS patient), which all shared HLA-A*2402 and HLA-B*1801. After testing the 15-mer peptides of the HuD antigen separately, we discovered that the same three 15-mers yielded the CD8+ T cell response in those three individuals. This highly unusual result could not be reproduced when using new batches of peptides with a higher level of purity (>82% and >95%). Therefore, we assumed this response was not directed against the HuD peptides and analyzed the HuD 15-mers by Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS), which showed the presence of a cytomegalovirus (CMV)-encoded peptide (AIAEESDEEEAIVAY) as a contaminant. The three responding individuals all were CMV-seropositive and the contaminating peptide appeared to fit in the binding groove of HLAB* 18. Our data reveal that synthetic PSPP may contain immunogenic contaminations which may cause false positive results in T-cell stimulation assays.

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.

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.

B and T cell imbalances in CSF of patients with Hu-antibody associated PNS (Article)

2008-03-01T00:00:00Z

In paraneoplastic neurological syndromes associated with Hu-antibodies (Hu-PNS) an important role for cellular immunity is hypothesized. We characterized the cerebrospinal fluid (CSF) pleocytosis in Hu-PNS patients by assessing the major lymphocyte subsets by flow cytometry. The B cell subset in the CSF of Hu-PNS patients showed a significant absolute (~ 20×) and relative (~ 3×) expansion, while the numbers of CD4+ T cells, CD8+ T cells and NK cells only showed an absolute expansion (~ 4-7×) compared to the controls. On the other hand, the NKT cell subset showed a significant relative reduction in CSF and in blood of Hu-PNS patients. The relative B cell expansion is consistent with the intrathecal synthesis of Hu-antibodies, while the increased number of T and NK cells supports an additional role for cellular immunity in the pathogenesis of Hu-PNS. In addition, the autoimmune hypothesis of Hu-PNS is supported by the relative NKT cell deficiency.

Flow cytometric lymphocyte subset enumeration: 10 Years of external quality assessment in the benelux countries (Article)

2008-03-01T00:00:00Z

A biannual external quality assessment (EQA) scheme for flow cytometric lymphocyte immunophenotyping is operational in the Benelux countries since 1996. We studied the effects of the methods used on assay outcome, and whether or not this EQA exercise was effective in reducing between-laboratory variation. Eighty test samples were distributed in 20 biannual send-outs. Per send-out, 50-71 participants were requested to enumerate CD3+, CD4+, and CD8+T cells, B cells, and NK cells, and to provide methodological details. Participants received written debriefings with personalized recommendations after each send-out. For this report, data were analyzed using robust multivariate regression. Five variables were associated with significant positive or negative bias of absolute lymphocyte subset counts: (i) platform methodology (i.e., single-platform assays yielded lower CD4+and CD8+T-cell counts than did dual-platform assays); (ii) sample preparation technique (i.e., assays based on mononuclear cells isolation yielded lower T-cell counts than those based on red cell lysis); (iii) gating strategies based on CD45 and sideward scatter gating of lymphocytes yielded higher CD4+T-cell counts than those based on "backgating" of lymphocytes guided by CD45 and CD14); (iv) stabilized samples were generally associated with higher lymphocyte subset counts than nonstabilized samples; and (v) laboratory. Platform methodology, sample stabilization, and laboratory also affected assay variability. With time, assay variability tended to decline; this trend was significant for B-cell counts only. In addition, significant bias and variability of results, independent of the variables tested for in this analysis, were also associated with individual laboratories. In spite of our recommendations, participants tended to standardize their techniques mainly with respect to sample preparation and gating strategies, but less with absolute counting techniques. Failure to fully standardize protocols may have led to only modest reductions in variability of results between laboratories.

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.

Imbalances in circulating lymphocyte subsets in Hu antibody associated paraneoplastic neurological syndromes (Article)

2007-12-01T00:00:00Z

In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies, neuron-specific Hu antigens expressed by the tumour hypothetically trigger an immune response that cross-reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized cell-mediated immune pathogenesis of these syndromes, we analysed the circulating lymphocyte subsets in untreated patients with SCLC, PNS and Hu antibodies (n = 18), SCLC without PNS (n = 19) and controls (n = 29) using flow cytometry. SCLC patients with PNS had a variety of imbalances within their circulating lymphocyte subsets as compared with SCLC patients without PNS and healthy controls: (i) a lymphopenia of the major subsets (i.e. B, CD4+and CD8+T lymphocytes); (ii) increased proportions of activated CD4+and CD8+T cells; (iii) reduced numbers of terminally differentiated effector CD8+T cells and cells with a cytotoxic T-cell phenotype (CD56+and CD57+). Although indirect, our data provide further support for the involvement of T cells in the pathogenesis of Hu antibody associated PNS.

Potential biomarkers in metastatic gastrointestinal stromal tumors (Article)

2007-10-15T00:00:00Z

Response to Goon et al. (Article)

2007-09-01T00:00:00Z

No evidence for circulating HuD-specific CD8+ T cells in patients with paraneoplastic neurological syndromes and Hu antibodies (Article)

2007-07-01T00:00:00Z

Aim: In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+T cells in a large cohort of Hu-PNS patients and controls. Patients and methods: Blood was tested from 43 Hu-PNS patients, 31 Hu antibody negative SCLC patients without PNS and 54 healthy controls. Peripheral blood mononuclear cells (PBMC) were stimulated with HuD protein-spanning peptide pools (15-mers) and individual HuD-derived peptides (9-mers) and analysed by cytokine flow cytometry and interferon-γ ELISPOT-assays. Additionally, HuD-based Class I HLA multimers were used to visualize HuD-specific CD8+T cells. Results: No HuD-specific CD8+T cells could be detected in the blood of Hu-PNS patients or controls. Conclusions: Our results do not support a role for HuD-specific CD8+T cells in Hu-PNS. Further studies should focus on the detection of circulating HuD-specific CD4+T cells and examine the antigen specificity of T cells in affected tissues.

Discriminant function analysis as decision support system for the diagnosis of acute leukemia with a minimal four color screening panel and multiparameter flow cytometry immunophenotyping (Article)

2007-06-01T00:00:00Z

Despite several recommendations for standardization of multiparameter flow cytometry (MFC) the number, specificity and combinations of reagents used by diagnostic laboratories for the diagnosis and classification of acute leukemias (AL) are still very diverse. Furthermore, the current diagnostic interpretation of flow cytometry readouts is influenced arbitrarily by individual experience and knowledge. We determined the potential value of a minimal four-color combination panel of 13 monoclonal antibodies (mAbs) with a CD45/sideward light scatter-gating strategy for a standardized MFC immunophenotyping of the clinically most relevant subgroups of AL. Bone marrow samples from 155 patients with acute myeloid leukemia (AML, n=79), B-cell precursor acute lymphoblastic leukemia (BCP-ALL, n=29), T-cell precursor acute lymphoblastic leukemia (T-ALL, n=12) and normal bone marrow donors (NBMD, n=35) were analyzed. A knowledge-based learning algorithm was generated by comparing the results of the minimal panel with the actual diagnosis, using discriminative function analysis. Correct classification of the test sample according to lineage, that is, BCP-ALL, T-ALL, AML and differentiation of NBMD was achieved in 97.2% of all cases with only six of the originally applied 13 mAbs of the panel. This provides evidence that discriminant function analysis can be utilized as a decision support system for interpretation of flow cytometry readouts.

Flow cytometric CD34+ stem cell enumeration: Lessons from nine years' external quality assessment within the Benelux countries (Article)

2007-05-15T00:00:00Z

Background: A biannual external quality assurance (EQA) scheme for flow cytometric CD34+ haematopoietic stem cell enumeration has been operational in the Benelux countries since 1996. In an evaluation of the results of 16 send-outs, we studied the effects of the methods used on assay outcome and whether or not this exercise was effective in reducing between-laboratory variation. Methods: Data were analyzed using robust multivariate regression. This approach is relatively insensitive to outliers and is used to assess the effect of methodological aspects of CD34+ cell counting on the bias and variability. Results: Five variables were associated with significant bias of absolute CD34+ cell counts: (i) unique laboratory number (ULN), (ii) gating strategy; (iii) CD34 mAb fluorochrome; (iv) type of flow cytometer, and (v) method of sample preparation. In addition, ULN and platform methodology (i.e., single vs. dual) contributed significantly to the variability of this assay. Overall, the variability in results of CD34+ cell enumeration has declined with time; in particular, after a practical workshop in which participants were trained to use the "single platform ISHAGE protocol." Conclusions: Between-laboratory variation in CD34+ cell enumeration can be reduced by standardization of methodologies between centres. Our approach, i.e., EQA with targeted training and feedback in response to reported results, has been successful in reducing the variability of CD34+ cell enumeration between participants.

The impact of impurities in synthetic peptides on the outcome of T-cell stimulation assays (Article)

2007-04-04T00:00:00Z

Protein-spanning peptide pools have proven valuable as a screening tool for detecting T-lymphocyte responses against a wide range of proteins. We have used this approach in our search for T cells reactive to the onconeural protein HuD. We found positive responses in only 3 of 127 individuals; however, these were highly unusual in that the same class I HLA alleles and peptides were involved. These T-cell responses were not confirmed when peptides re-synthesized by the same manufacturer with similar and with higher purity levels were used. Our observations indicated that these T-cell responses were not directed against the designed HuD peptides. Here, we report on (i) comparisons of the peptide batches analyzed by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) that did -and did not - elicit T-cell responses and (ii) a detailed analysis of the various by-products of peptides, irrespective of T-cell assay outcome. We found numerous differences between the peptide batches, such as omissions of amino acids in the primary structure of the peptides. Furthermore, some batches revealed strong interactions with calcium ions or contained sulfated peptides. Our data reveal that different batches from the same peptide may contain artefacts that influence the outcome of HLA-restricted T-cell response assays. Copyright

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.

Parallel detection of transduced T lymphocytes after immunogene therapy of renal cell cancer by flow cytometry and real-time polymerase chain reaction: implications for loss of transgene expression (Article)

2005-12-01T00:00:00Z

Abstract We have started a phase I/II immunogene therapy study of metastatic renal cell cancer (RCC), using autologous T lymphocytes transduced ex vivo with a gene encoding a single-chain receptor based on the monoclonal antibody (mAb) G250 [scFv(G250)]. G250 recognizes carbonic anhydrase IX, which is overexpressed by RCC cells. We have developed and validated flow cytometric and real-time polymerase chain reaction (PCR) assays to quantitatively detect transduced T cells in patient blood. The flow assay was based on staining with the anti-G250 idiotype mAb NuH82 and showed a sensitivity of 0.06% scFv(G250)(1) cells within CD3(1) T cells. The real-time PCR method showed a sensitivity of 14 copies of scFv(G250) DNA per 100 ng of total DNA, which enabled detection of 0.008% scFv(G250)(1) T cells within leukocytes. Both assays were further validated for their specificity and reproducibility. When applied to blood samples from three RCC patients treated with intravenous infusions of scFv(G250)(1) T cells, the kinetics of scFv(G250)(1) T cell counts as detected by flow cytometry were similar to those detected by real-time PCR, although PCR allowed detection of transduced T cells over a longer period of time (i.e., for patient 3, 7 versus 32 days, respectively). Interestingly, follow-up studies of patient 3 demonstrated that the number of circulating scFv(G250)(1) T cells remained fairly constant during the first 7 days posttreatment, whereas the number of gene copies increased during the same period of time. These results suggest loss of scFv(G250) membrane expression on adoptive transfer, which would have important implications for the antitumor efficacy of this form of immunogene therapy.