Isolation of disseminated neuroblastoma cells from bone marrow aspirates for pretreatment risk assessment by array comparative genomic hybridization.

In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior.

Predicting outcomes for children with neuroblastoma using a multigene-expression signature: a retrospective SIOPEN/COG/GPOH study.

BACKGROUND: More accurate prognostic assessment of patients with neuroblastoma is required to better inform the choice of risk-related therapy. The aim of this study is to develop and validate a gene-expression signature to improve outcome prediction. METHODS: 59 genes were selected using an innovative data-mining strategy, and were profiled in the largest neuroblastoma patient series (n=579) to date using real-time quantitative PCR starting from only 20 ng of RNA. A multigene-expression signature was built using 30 training samples, tested on 313 test samples, and subsequently validated in a blind study on an independent set of 236 tumours. FINDINGS: The signature has a performance, sensitivity, and specificity of 85.4% (95% CI 77.7-93.2), 84.4% (66.5-94.1), and 86.5% (81.1-90.6), respectively, to predict patient outcome. Multivariate analysis indicates that the signature is a significant independent predictor of overall survival and progression-free survival after controlling for currently used risk factors: patients with high molecular risk have a higher risk of death from disease and higher risk of relapse or progression than patients with low molecular risk (odds ratio 19.32 [95% CI 6.50-57.43] and 3.96 [1.97-7.97] for overall survival and progression-free survival, respectively, both p<0.0001). Patients at an increased risk of an adverse outcome can also be identified in the current treatment groups, showing the potential of this signature for improved clinical management. These results were confirmed in the validation study, in which the signature was also independently statistically significant in a model adjusted for MYCN status, age, International Neuroblastoma Staging System stage, ploidy, International Neuroblastoma Pathology Classification grade of differentiation, and mitosis karyorrhexis index (odds ratios between 4.81 and 10.53 depending on the model for overall survival and 3.68 [95% CI 2.01-6.71] for progression-free survival). INTERPRETATION: The 59-gene expression signature is an accurate predictor of outcome in patients with neuroblastoma. The signature is an independent risk predictor, identifying patients with an increased risk of poor outcome in the current clinical-risk groups. The method and signature is suitable for routine laboratory testing, and should be evaluated in prospective studies. FUNDING: The Belgian Foundation Against Cancer, the Children Cancer Fund Ghent, the Belgian Society of Paediatric Haematology and Oncology, the Belgian Kid's Fund and the Fondation Nuovo-Soldati (JV), the Fund for Scientific Research Flanders (KDP, JH), the Fund for Scientific Research Flanders, the Institute for the Promotion of Innovation by Science and Technology in Flanders, Strategisch basisonderzoek, the Fondation Fournier Majoie pour l'Innovation, the Instituto Carlos III, the Italian Neuroblastoma Foundation, the European Community under the FP6, and the Belgian programme of Interuniversity Poles of Attraction.

Array comparative genomic hybridization and flow cytometry analysis of spontaneous abortions and mors in utero samples.

BACKGROUND: It is estimated that 10-15% of all clinically recognised pregnancies result in a spontaneous abortion or miscarriage. Previous studies have indicated that in up to 50% of first trimester miscarriages, chromosomal abnormalities can be identified. For several decades chromosome analysis has been the golden standard to detect these genomic imbalances. A major drawback of this method is the requirement of short term cultures of fetal cells. In this study we evaluated the combined use of array CGH and flow cytometry (FCM), for detection of chromosomal abnormalities, as an alternative for karyotyping. METHODS: In total 100 spontaneous abortions and mors in utero samples were investigated by karyotyping and array CGH in combination with FCM in order to compare the results for both methods. RESULTS: Chromosome analysis revealed 17 abnormal karyotypes whereas array CGH in combination with FCM identified 26 aberrations due to the increased test success rate. Karyotyping was unsuccessful in 28% of cases as compared to only two out of hundred samples with inconclusive results for combined array CGH and FCM analysis. CONCLUSION: This study convincingly shows that array CGH analysis for detection of numerical and segmental imbalances in combination with flow cytometry for detection of ploidy status has a significant higher detection rate for chromosomal abnormalities as compared to karyotyping of miscarriages samples.

Influence of neuroblastoma stage on serum-based detection of MYCN amplification.

BACKGROUND: MYCN oncogene amplification has been defined as the most important prognostic factor for neuroblastoma (NB), the most common solid extracranial neoplasm in children. High copy numbers are strongly associated with rapid tumor progression and poor outcome, independently of tumor stage or patient age, and this has become an important factor in treatment stratification. PROCEDURE: By real-time quantitative PCR analysis, we evaluated the clinical relevance of circulating MYCN DNA of 267 patients with locoregional or metastatic NB in children less than 18 months of age. RESULTS: For patients in this age group with INSS stage 4 or 4S NB and stage 3 patients, serum-based determination of MYCN DNA sequences had good sensitivity (85%, 83%, and 75% respectively) and high specificity (100%) when compared to direct tumor gene determination. In contrast, the approach showed low sensitivity patients with stages 1 and 2 disease. CONCLUSION: Our results show that the sensitivity of the serum-based MYCN DNA sequence determination depends on the stage of the disease. However, this simple, reproducible assay may represent a reasonably sensitive and very specific tool to assess tumor MYCN status in cases with stage 3 and metastatic disease for whom a wait and see strategy is often recommended.

Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma: quality assurance on behalf of SIOPEN-R-NET.

The clinical utility of detecting minimal residual disease (MRD) in children with neuroblastoma (NB) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) is not clear. This in part reflects the lack of uniform methodology for analysis and reporting. Reference laboratories across Europe have therefore established standard operating procedures (SOPs) for the detection of NB cells by QRT-PCR. Haemopoietic samples are collected into PAXgene blood RNA tubes, which stabilise mRNA for 48 h at room temperature and more than 6 months at -80 degrees C. Tyrosine hydroxylase (TH) was selected as the target for NB cell detection, expression is normalised to beta2-microglobulin and reported using the DeltaDeltaCt method. The sensitivity of QRT-PCR increased from 58% to 90% following the development of SOPs. A robust, transferable, objective method for the detection of NB cells by QRT-PCR has been defined to improve the power and consistency of studies on MRD in children with NB.

DRAQ5: improved flow cytometric DNA content analysis and minimal residual disease detection in childhood malignancies.

BACKGROUND: The majority of flow cytometric DNA content analyses are performed on whole peripheral blood, bone marrow or tumor samples containing significant numbers of non-malignant cells which hamper specific DNA content analysis. Simultaneous analysis of DNA content and immunophenotype greatly improves the specificity of DNA-ploidy measurements. Therefore, a three-color flow cytometric assay using DRAQ5 was developed and validated. METHODS: The results of DNA content analysis using DRAQ5 and propidium iodide were compared using peripheral blood samples of 15 healthy volunteers. The reproducibility of the multiparameter DRAQ5 assay was assayed in 10 analytical runs and the sensitivity was evaluated with addition experiments. RESULT: Using DRAQ5, slightly wider CVs of the G0/G1 peak were obtained (average CV=3.29%). When DRAQ5 staining and immunophenotyping were combined, the within- and between-run imprecision was 1.98% and 1.67%, respectively and the DNA content of 25 DNA-hyperdiploid tumor cells (DNA-index=1.16) per 4.10(4) mononuclear cells (0.06%) could be determined. CONCLUSIONS: The multiparameter DRAQ5 assay has a superior sensitivity and specificity compared to the propidium iodide based method. Since at least 25 DNA-hyperdiploid cells per 10(4) DNA-diploid mononuclear cells could be detected, multiparameter DRAQ5 DNA content analysis could be used to study minimal residual disease.

Prognostic significance of multidrug resistance-related proteins in childhood acute lymphoblastic leukaemia.

An important problem in the treatment of children with acute lymphoblastic leukaemia (ALL) is pre-existent or acquired resistance to structurally and functionally unrelated chemotherapeutic compounds. Various cellular mechanisms can give rise to multidrug resistance (MDR). Best studied is the transmembrane protein-mediated efflux of cytotoxic compounds that leads to decreased cellular drug accumulation and toxicity. Several MDR-related efflux pumps have been characterised, including P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP) and lung resistance protein (LRP). P-gp expression and/or activity has been associated with unfavourable outcome in paediatric ALL patients, whereas MRP1 and BCRP do not seem to play a major role. LRP might contribute to drug resistance in B-lineage ALL, but larger studies are needed to confirm these results. The present review summarises the current knowledge concerning multidrug resistance-related proteins and focuses on the clinical relevance and prognostic value of these efflux pumps in childhood ALL.

Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow.

Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.

Detecting minimal residual disease in neuroblastoma patients-the present state of the art.

While cyto- and histological screening of bone marrow samples are still accepted as the gold standard for initial staging of neuroblastoma patients, these applications are insufficient during or after therapy because it is not always possible to detect tumour cell infiltration below the level of 1% by morphology alone. For monitoring of minimal residual disease, techniques offering a considerably higher sensitivity have been developed. Immunocytology, RT-PCR and flow cytometry are most frequently used, but differ with regard to targets (single cells, RNA transcripts), measured parameters (tumour cell number, antigen expression, cytomorphology, cytogenetic aberrations, level/number of RNA transcripts), specificity (uni-/multi-parameter analysis) and sensitivity (number of investigated cells). The pros and cons of these methods are reviewed. Precise quantification of residual tumour cells in bone marrow and blood may show a future impact on risk grouping and therapeutic strategies for patients with disseminated disease, but the potential clinical application of these techniques has to be preceded by thorough standardisation and validation in multi-centre studies.

Comparison of two functional flow cytometric assays to assess P-gp activity in acute leukemia.

One of the possible causes of treatment failure in acute leukemia is the emergence of multidrug resistance caused by P-glycoprotein (P-gp) overexpression. We compared a flow cytometric assay using JC-1 with a technique using rhodamine 123 (rho123) to evaluate the P-gp function in acute leukemia. Samples from 50 acute leukemia patients were analyzed by both functional assays. The P-gp expression was assessed by an immunological flow cytometric test and the association between the P-gp status and the clinical outcome was evaluated. Of all samples, 28% showed a reversible JC-1 efflux and 36% scored positive for the rho123 assay. In two cases, the leukemic blasts showed a reversible JC-1 efflux whereas they were negative for rho123. These patients had blast cells with a very low P-gp activity. Six samples scored positive for the rho123 assay but were negative for the JC-1 test. Five of these samples did not express P-glycoprotein and were considered false positive. We found a strong correlation between the JC-1 and the rho123 test (R(s)=0.59, p<0.0001) and the JC-1 and the immunological assay (R(s)=0.29, P=0.05). There was also an association between the JC-1 status and the clinical outcome of adult patients (chi2=6.30, P=0.04). In conclusion, we recommend the JC-1 assay to study the P-gp activity in acute leukemia because it is more specific and less labor intensive than conventional functional flow cytometric tests using rhodamine 123. In addition, the JC-1 assay can be used to identify adult patients with an increased risk for adverse clinical outcome.

Detection of residual neuroblastoma cells in bone marrow: comparison of flow cytometry with immunocytochemistry.

BACKGROUND: Because the cytomorphologic examination of bone marrow (BM) aspirates appears not sensitive enough to detect residual neuroblastoma cells, two four-color flow cytometric assays using different combinations of CD9, CD81, CD56, CD45, and anti-GD2 were evaluated. METHODS: The sensitivity of the flow cytometric assays was assessed by spiking experiments in normal peripheral blood samples. Twenty-eight BM samples, 12 biopsies, and 3 peripheral blood stem cell (PBSC) preparations from 22 patients with neuroblastoma were analyzed. The results were compared with those of an anti-GD2 immunocytochemical reference assay. RESULTS: Flow cytometric and immunocytochemical analyses showed residual neuroblastoma cells in four BM samples. One PBSC preparation and 20 BM samples were negative for both assays. Four BM and two PBSC samples scored positive for the immunocytochemical assay but were negative for the flow cytometric tests. This was due to the limited number of cells that were flow cytometrically analyzed. A strong correlation between the flow cytometric and immunocytochemical tests was found (chi2 = 6.4, P = 0.011). CONCLUSIONS: When an equal amount of cells is analyzed, the sensitivity of the flow cytometric assays is to be about 10 times lower than that of the immunocytochemical test. However, the flow cytometric assays can be used to screen for residual cells in clinical samples with a sensitivity of one neuroblastoma cell in 10(4) to 10(5) normal mononuclear cells. Flow cytometry is simple, quick, and cost effective compared with immunocytochemistry. In addition, the flow cytometric assays can be used to screen for residual neuroblastoma cells in case of a GD2-negative primary tumor. Therefore we recommend flow cytometry for the detection of residual neuroblastoma cells.

Neuroblastoma mRNAs predict outcome in children with stage 4 neuroblastoma: a European HR-NBL1/SIOPEN study.

PURPOSE: To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. METHODS: RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. RESULTS: High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. CONCLUSION: High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

N-cadherin in neuroblastoma disease: expression and clinical significance.

One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions, might play a central role in the metastasis of neuroblastoma (NB), a solid tumor of neuroectodermal origin. Using Reverse Transcription Quantitative PCR (RT-qPCR), Western blot, immunocytochemistry and Tissue MicroArrays (TMA) we demonstrate the expression of N-cadherin in neuroblastoma tumors and cell lines. All neuroblastic tumors (n = 356) and cell lines (n = 10) expressed various levels of the adhesion protein. The N-cadherin mRNA expression was significantly lower in tumor samples from patients suffering metastatic disease. Treatment of NB cell lines with the N-cadherin blocking peptide ADH-1 (Exherin, Adherex Technologies Inc.), strongly inhibited tumor cell proliferation in vitro by inducing apoptosis. Our results suggest that N-cadherin signaling may play a role in neuroblastoma disease, marking involvement of metastasis and determining neuroblastoma cell viability.

Hop bitter acids efficiently block inflammation independent of GRalpha, PPARalpha, or PPARgamma.

Hop (Humulus lupulus L.) is an essential ingredient of beer, where it provides the typical bitter taste, but is also applied in traditional folk medicine for sedative and antibacterial purposes. In this study, we demonstrate and compare the anti-inflammatory effect of various classes of hop bitter acids (HBA), including alpha-acids (AA), beta-acids (BA), and iso-alpha-acids (IAA), in fibroblasts, which are important players in the inflammatory response. All three studied classes of HBA blocked the tumor necrosis factor alpha (TNF)-induced production of the cytokine IL6, and inhibited the transactivation of the pro-inflammatory transcription factors nuclear factor kappa B (NF-kappaB), activator protein-1 (AP-1), and cAMP-response element-binding protein (CREB). In this respect, the six-membered ring compounds AA and BA showed equal potency, whereas the five-membered ring compounds, IAA, were effective only when used at higher concentrations. Furthermore, with regard to the mechanism of NF-kappaB suppression, we excluded a possible role for glucocorticoid receptor alpha (GRalpha), peroxisome proliferators-activated receptor alpha/gamma (PPARalpha or PPARgamma), nuclear receptors (NRs) that are also known to inhibit inflammation by directly interfering with the activity of pro-inflammatory transcription factors. Interestingly, combining hop acids and selective agonists for GRalpha, PPARalpha, or PPARgamma resulted in additive inhibition of NF-kappaB activity after TNF treatment, which may open up new avenues for combinatorial anti-inflammatory strategies with fewer side effects. Finally, systemic administration of HBA efficiently inhibited acute local inflammation in vivo.

Potential application of ELAVL4 real-time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells.

BACKGROUND: Reliable detection of neuroblastoma cells in bone marrow (BM) is critical because BM involvement influences staging, risk assessment, and evaluation of therapeutic response in neuroblastoma patients. Standard cytomorphologic examination of BM aspirates is sensitive enough to detect single tumor cells. Consequently, more sensitive and specific detection methods are indispensable. METHODS: We used real-time quantitative reverse transcription-PCR (QPCR) of the tyrosine hydroxylase (TH), GD2 synthetase (GALGT), and embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) genes to detect disseminated neuroblastoma cells. We assessed assay sensitivity by addition experiments and then analyzed 97 neuroblastic tumor, BM, peripheral blood (PB), or peripheral blood stem cell (PBSC) samples from 30 patients. The QPCR results were compared with those of a standardized immunocytochemical assay. RESULTS: The molecular markers were highly expressed in all evaluated tumor samples. In addition, 32%, 11%, and 38% of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively. The TH and ELAVL4 assays could detect 1 neuroblastoma cell in 10(6) mononuclear cells. By contrast, the GALGT QPCR assay could detect 1 neuroblastoma cell in 10(4) mononuclear cells. We assessed the potential prognostic value of TH, GALGT, and ELAVL4 QPCR by analyzing subsequent samples from 3 patients with stage 4 disease. Preliminary results indicated that persistence of high ELAVL4 expression has prognostic value. CONCLUSIONS: ELAVL4 QPCR can be used to detect residual neuroblastoma cells in clinical samples. However, combination of several molecular markers and screening techniques should be considered to ensure reliable detection of rare neuroblastoma cells.

The combined analysis of P-glycoprotein expression and activity predicts outcome in childhood acute lymphoblastic leukemia.

The link between drug resistance and relapse was often suggested, but rarely demonstrated in long-range clinical studies. Since it is nowadays recommended to validate immunocytochemical results, the authors studied prospectively 52 acute lymphoblastic leukemia (ALL) patients with an immunocytochemical test and a functional flow cytometric test. The 4-year EFS and OS were 79.3% and 85.2%, respectively. Patients scoring positive in both tests had a significantly higher relapse rate and worse survival (log rank p = 0.007 and 0.047 for event-free survival and overall survival, respectively). Among the different prognostic variables evaluated, only the combination of P-gp expression and activity was a statistically significant parameter predicting relapse in childhood ALL.