Minimal Residual Disease In Neuroblastoma Research Articles

Clinical application of individualized tumor-informed circulating tumor DNA for therapeutic response and relapse prediction in patients with neuroblastoma.

10053 Background: Patients with high-risk neuroblastoma (NB) generally present with widely metastatic disease and often relapse despite intensive therapy. At present, circulating tumor DNA (ctDNA)-based minimal residual disease (MRD) has been studied in various tumors and proven to be valuable in guiding treatment and predicting recurrence, but its application in NB has rarely been reported. This study aimed to assess the effectiveness of tumor-informed ctDNA monitoring for evaluating the response to induction therapy in NB patients, as well as its role in predicting the risk of relapse. Methods: We conducted a prospective cohort study of NB patients. Primary tumor samples were collected at baseline and blood samples were collected sequentially from baseline until disease progression or the last follow-up. Tumor tissue samples were subjected to whole exome sequencing (WES), and clonal mutations were selected for the individualized MRD panel. Sequencing was conducted at OrigiMed, a College of American Pathologists accredited and Clinical Laboratory Improvement Amendments certified laboratory. Results: Our cohort included 43 individuals diagnosed with NB, with a median age of 3.5 years (0.42-40 years). WES revealed that 75% (2054/2753) of the mutated genes were unique to each patient, and 98.9% (704/712) of the selected tumor-informed single nucleotide variants were variants of unknown significance, suggesting that individualized tumor-informed MRD (n = 23) is superior to fixed tumor-agnostic panel (n = 20) MRD in NB. A successful individualized MRD panel was constructed for 53.5% (23/43) of patients, with a remarkable 92.3% success rate for those older than 5 years (p < 0.01). The initial positivity rate for ctDNA detection was 68%, with the initial positivity rate being associated with the stage (p < 0.01). During treatment and follow-up, 0/6 of the patients whose ctDNA levels remained negative experienced a relapse, whereas 2/5 patients who had a negative conversion or an increase in positivity experienced a relapse. In addition, ctDNA changes matched radiological evaluations in 94% of patients. Notably, ctDNA was detected prior to radiological relapse, with a lead time of 6 months in one patient. Patients can be divided into four groups according to the mutation landscape: ALK mutation, MYCN amplification, chromosome amplification, and other groups. Among them, the rate of ctDNA clearance was the highest in the chromosome amplification group, indicating that patients in this group were the most sensitive to chemotherapy. Conclusions: Individualized tumor-informed ctDNA monitoring shows promise for evaluating therapeutic response and forecasting relapse in NB patients. This approach proves more effective than fixed tumor-agnostic panel-based MRD due to high genetic mutation uniqueness. Clinical trial information: NCT05076071 .

The expression of PHOX2B in bone marrow and peripheral blood predicts adverse clinical outcome in non-high-risk neuroblastoma

Paired-like homeobox 2B (PHOX2B) is a highly sensitive and specific biomarker for diagnosing neuroblastoma, as well as detecting minimal residual disease in neuroblastoma. The clinical significance of PHOX2B expression in bone marrow (BM) and peripheral blood (PB) samples of newly diagnosed patients with very low-, low- and intermediate-risk neuroblastoma remains unknown, to the best of our knowledge. The expression level of PHOX2B in paired BM and PB samples of patients with newly diagnosed neuroblastoma was validated using reverse transcription-quantitative polymerase chain reaction (RTqPCR). Among the 132 patients, 26 exhibited a positive PHOX2B expression BM (19.7%) and 11 in PB (8.3%) samples. PHOX2B was highly expressed in BM and PB samples from patients aged <18 months, with International Neuroblastoma Risk Group Staging System stages M and MS, 1p loss of heterozygosity, and high levels of lactate dehydrogenase, serum ferritin and neuron-specific enolase (p < 0.05). In all eligible patients, the 2-year event-free survival (EFS) and overall survival (OS) rates were 94.7 ± 2.0% and 97.7 ± 1.3%, respectively. However, the 2-year EFS rates were significantly decreased to 76.9 ± 8.3% and 63.6 ± 14.5% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). Similarly, the 2-year OS rates were also decreased to 88.5 ± 6.3% and 81.8 ± 11.6% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). In conclusion, a positive PHOX2B expression in BM and PB samples at diagnosis had a strong adverse prognostic effect on patients with non-high-risk neuroblastoma.

Detection and therapy of neuroblastoma minimal residual disease using [64/67Cu]Cu-SARTATE in a preclinical model of hepatic metastases

BackgroundA major challenge to the long-term success of neuroblastoma therapy is widespread metastases that survive initial therapy as minimal residual disease (MRD). The SSTR2 receptor is expressed by most neuroblastoma tumors making it an attractive target for molecularly targeted radionuclide therapy. SARTATE consists of octreotate, which targets the SSTR2 receptor, conjugated to MeCOSar, a bifunctional chelator with high affinity for copper. Cu-SARTATE offers the potential to both detect and treat neuroblastoma MRD by using [64Cu]Cu-SARTATE to detect and monitor the disease and [67Cu]Cu-SARTATE as the companion therapeutic agent. In the present study, we tested this theranostic pair in a preclinical model of neuroblastoma MRD. An intrahepatic model of metastatic neuroblastoma was established using IMR32 cells in nude mice. The biodistribution of [64Cu]Cu-SARTATE was measured using small-animal PET and ex vivo tissue analysis. Survival studies were carried out using the same model: mice (6–8 mice/group) were given single doses of saline, or 9.25 MBq (250 µCi), or 18.5 MBq (500 µCi) of [67Cu]Cu-SARTATE at either 2 or 4 weeks after tumor cell inoculation.ResultsPET imaging and ex vivo biodistribution confirmed tumor uptake of [64Cu]Cu-SARTATE and rapid clearance from other tissues. The major clearance tissues were the kidneys (15.6 ± 5.8% IA/g at 24 h post-injection, 11.5 ± 2.8% IA/g at 48 h, n = 3/4). Autoradiography and histological analysis confirmed [64Cu]Cu-SARTATE uptake in viable, SSTR2-positive tumor regions with mean tumor uptakes of 14.1–25.0% IA/g at 24 h. [67Cu]Cu-SARTATE therapy was effective when started 2 weeks after tumor cell inoculation, extending survival by an average of 13 days (30%) compared with the untreated group (mean survival of control group 43.0 ± 8.1 days vs. 55.6 ± 9.1 days for the treated group; p = 0.012). No significant therapeutic effect was observed when [67Cu]Cu-SARTATE was started 4 weeks after tumor cell inoculation, when the tumors would have been larger (control group 14.6 ± 8.5 days; 9.25 MBq group 9.5 ± 1.6 days; 18.5 MBq group 15.6 ± 4.1 days; p = 0.064).ConclusionsClinical experiences of peptide-receptor radionuclide therapy for metastatic disease have been encouraging. This study demonstrates the potential for a theranostic approach using [64/67Cu]Cu-SARTATE for the detection and treatment of SSTR2-positive neuroblastoma MRD.

Specific and Sensitive Detection of Neuroblastoma mRNA Markers by Multiplex RT-qPCR.

Simple SummarySensitive detection of minimal residual disease by RT-qPCR in patients with neuroblastoma is shown to be predictive of outcome, but has not yet been introduced into clinical practice. A panel of multiple mRNA markers increases the sensitivity of minimal residual disease detection, since neuroblastoma tumors are heterogeneous tumors. Recent studies have identified two distinct phenotypes, an adrenergic and mesenchymal phenotype, that can be identified by using different mRNA markers. As generally only small volumes of bone marrow or blood are available in young neuroblastoma patients, we have developed a multiplex RT-qPCR to be able to test seven different mRNA markers, while we reduce the sample volume needed. Comparison between the multiplex RT-qPCR and RT-qPCR for the single markers showed a comparable sensitivity. This reduction in required sample volume, while saving time and resources, can assist in the introduction of minimal residual disease detection by RT-qPCR into clinical practice.mRNA RT-qPCR is shown to be a very sensitive technique to detect minimal residual disease (MRD) in patients with neuroblastoma. Multiple mRNA markers are known to detect heterogeneous neuroblastoma cells in bone marrow (BM) or blood from patients. However, the limited volumes of BM and blood available can hamper the detection of multiple markers. To make optimal use of these samples, we developed a multiplex RT-qPCR for the detection of MRD in neuroblastoma. GUSB and PHOX2B were tested as single markers. The adrenergic markers TH, GAP43, CHRNA3 and DBH and mesenchymal markers POSTN, PRRX1 and FMO3 were tested in multiplex. Using control blood and BM, we established new thresholds for positivity. Comparison of multiplex and singleplex RT-qPCR results from 21 blood and 24 BM samples from neuroblastoma patients demonstrated a comparable sensitivity. With this multiplex RT-qPCR, we are able to test seven different neuroblastoma mRNA markers, which overcomes tumor heterogeneity and improves sensitivity of MRD detection, even in those samples of low RNA quantity. With resources and time being saved, reduction in sample volume and consumables can assist in the introduction of MRD by RT-qPCR into clinical practice.

Phase I study of OKT3 x hu3F8 bispecific antibody (GD2Bi) armed T cells (GD2BATs) in GD2-positive tumors.

2533 Background: With the proven success of anti-GD2 monoclonal antibodies in eradicating minimal residual disease in neuroblastoma (NB), exploiting antibody based anti-GD2 in T cell mediated strategies has potential to combat higher disease burden and improve patient outcome. We hypothesized that arming of ex vivo expanded and activated, autologous, blood derived T cells (ATC) with chemically heteroconjugated GD2Bi should redirect them to target NB. In vitro, ATC coated (armed) with 50 ng/106 cells of GD2Bi exhibited specific killing of NB and osteosarcoma (OS) cell lines. Methods: In this phase I study (NCT02173093), patients with GD2-positive tumors received 8, biweekly infusions of GD2BATs + daily low-dose IL-2 and biweekly granulocyte-macrophage colony stimulating factor (GM-CSF). The study followed the standard 3+3 design with dose levels of 40, 80, and 160 x 106 GD2BATs/kg/infusion. Results: Twelve patients (NB = 7, OS = 3, Desmoplastic Small Round Cell Tumor = 2) were enrolled from 11/2013 to 12/2017 and 9 completed therapy. Adequate ATCs could not be grown in one patient and two patients did not complete 8 infusions because of rapid disease progression. Infusions were given in outpatient settings. All patients developed a mild, dose-independent and manageable form of cytokine release syndrome with grades 2-3 fevers/chills, headaches and occasional hypotension for up to 48 hours after infusion. No patients developed significant pain. Maximum tolerated dose was not reached. Evidence of activity was seen in several patients including one patient with OS who had a PET response, one patient with NB who had complete bone marrow response (this patient had remained progression free for 2.5 years after completion of infusions), and another NB patient who had a minor response on MIBG scan. Four patients with NB are currently alive after additional therapies at 12, 14, 18, and 47 months post BAT infusions. Conclusions: Autologous T cells from heavily pretreated patients could be expanded ex vivo to large numbers, armed with GD2Bi, cryopreserved and thawed for safe IV administration up to total dose of 1.28x109/kg. Ongoing phase II arm of the trial will focus on evaluation of clinical activity of GD2BATs in patients with NB. Clinical trial information: NCT02173093.

RT-qPCR for PHOX2B mRNA is a highly specific and sensitive method to assess neuroblastoma minimal residual disease in testicular tissue.

Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.

Highly sensitive assessment of neuroblastoma minimal residual disease in ovarian tissue using RT-qPCR-A strategy for improving the safety of fertility restoration.

Ovarian tissue cryopreservation (OTC) is the only option available to preserve fertility in prepubertal females with neuroblastoma (NB), a childhood solid tumor that can spread to the ovaries, with a risk of reintroducing malignant cells after an ovarian graft. We set out to determine whether the analysis of TH (tyrosine hydroxylase), PHOX2B (paired-like homeobox 2b), and DCX (doublecortin) transcripts using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) could be used to detect NB contamination in ovarian tissue. Analyses were performed on benign ovarian tissue from 20 healthy women between November 2014 and September 2015 at the University Hospital of Clermont-Ferrand. Pericystic benign ovarian tissues were collected and contaminated with increasing numbers of human NB cells (cell lines IMR-32 and SK-N-SH) before detection using RT-qPCR. TH and DCX transcripts were detected in uncontaminated ovarian tissue from all the donors, hampering the detection of small numbers of tumor cells. By contrast, PHOX2B was not detected in any uncontaminated ovarian fragment. PHOX2B levels were significantly increased from 10 NB cells. Our study is the first to evaluate minimal residual disease detection using NB mRNAs in human ovarian tissue. Only PHOX2B was a reliable marker of NB cells contaminating ovarian tissue. These results are encouraging and offer hope in the near future for grafting ovarian tissue in women who survive cancer, whose fertility has been jeopardized by treatment, and who could benefit from OTC without oncological risk.

Abstract LB-87: A novel epigenetic blood test to monitor minimal residual disease in high-risk neuroblastoma.

Abstract Background: Cells present as minimal residual disease (MRD) following frontline therapy drive chemoresistant tumour relapse, thus their detection and targeting is a major priority in cancer therapy. Neuroblastoma (NB) is an aggressive neural crest-derived malignancy of infants and young children. A hallmark of NB is its clinical heterogeneity and the majority of infants with NB have a unique biology that results in spontaneous remission through differentiation in the absence of therapy, despite disease that may be disseminated to the liver, skin, and bone marrow at diagnosis. These infants (with stage 4S disease) have survival rates that exceed 95%. Children with high-risk NB, however, largely characterized by amplification of the MYCN oncogene, often have a disease that is therapy resistant. Although modest improvements in outcome have been achieved as a result of therapy intensification, 5-year event free survival in high-risk patients remains approximately 40-50%. Typically, high-risk neuroblastoma recurs after near complete remission is achieved and the development of effective strategies directed at MRD remains an unmet clinical need. Detection of MRD in neuroblastoma is usually performed using real-time quantitative (RQ)-PCR of neuroblastoma-specific transcripts. Tumor-selective mRNA markers levels are highly dependent on gene expression, which can vary between patients (by a factor of up 1000) and change during treatment. Furthermore, the applicability of a PCR target for MRD detection is also determined by its background expression in hematologic cells. Chromatin conformation patterns integrate the spatial arrangement of the chromatin and build a signature for the physiological status of the cell representing early changes in the genetic and epigenetic regulation. These signatures form the basis for a new technology, Episwitch™, which can identify epigenetic patterns linked with cancer progression and aggressiveness. Results: Based on a screen of human NB cell lines containing amplification of MYCN or expression of high levels of MYCN protein versus NB cell lines lacking expression of MYCN, we have identified a panel of four NB-associated genetic markers: HDM2, PHOX2B, TERT and TH. HDM2 and TERT are direct transcriptional targets of MYCN. This epigenetic signature is currently being validated in primary NB samples. An in vivo proof of concept study utilizing the equivalent mouse epigenetic signature is being performed in a relapse model of MYCN-driven NB. Conclusion: In this study we have developed and tested a novel blood test based on Episwitch™ technology that discriminates MYCN-associated NB. Our data suggest this non-invasive test can be used to monitor remission/relapse status quantitatively with high sensitivity in high-risk MYCN-associated neuroblastoma, thus informing treatment decisions. Citation Format: Kevin Petrie, Megan Field, Zai Ahmad, Mehrnoush Dezfouli, Karen Barker, Magdalena Jeznach, Laura Glass, Albert Hallsworth, Yordan Sbirkov, Howard Womersley, Arthur Zelent, Philip Jordan, Alexandre Akoulitchev, Louis Chesler. A novel epigenetic blood test to monitor minimal residual disease in high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2013-LB-87

The Role of p75NTR and Its Signaling Pathways in Fenretinide (4HPR)-Induced Apoptosis in Neuroblastoma Cells (P06.003)

Objective: The overall goal is to understand the role of p75NTR and its signaling pathways in Fenretinide (4-hydroxyphenyl retinamide - 4HPR) induced apoptosis in neuroblastoma cells and determine some of the signaling pathways that mediate this effect. Understanding the role of p75NTR in mediating 4HPR-induced apoptosis will facilitate development of targeted therapies for the treatment of neuroblastoma by providing insight into pathways that could be targeted for combination chemotherapy. Background 4HPR is well tolerated in clinical trials and has been shown to cause apoptosis by increasing ROS generation in cancer cells. Targeting minimal residual disease in neuroblastoma involves sensitizing these tumors to chemotherapeutics by manipulating receptor signaling. p75NTR has a pro/anti-apoptotic role depending on the intracellular conditions. Design/Methods: S-type neuroblastoma cells (SH-EP1, SK-N-AS, SK-N-MC) with varying amounts of p75NTR are used to determine the effect of p75NTR signaling on the 4HPR-concentration curve. Changes in oxidative stress as a result of 4HPR treatment are recorded using western blotting and fluorescent microscopy. Results: 4HPR reduces cell viability in a variety of neuroblastoma cell lines when they are treated for 24(15%)-72 hrs (40%). p75NTR expression is pro-apoptotic and sensitizes SH-EP1 cells by decreasing viability, metabolic health and upregulating 4HPR-induced apoptosis. Treatment of SH-EP1 cells with 4HPR for 72 hours revealed no change in production of cellular OH- or cytoslic O2- but a sharp increase was observed in mitochondrial O2- production(Scr>p75shRNA transfected SH-EP1). Scavenging oxygen radicals in the mitochondria or inhibiting Complex II inhibits the pro-apoptotic effect of Fenretinide. Akt signaling is upregulated in SH-EP1 cells with p75shRNA compared to native SH-EP1 cells. Conclusions: This will facilitate the sensitization of minimal residual disease and preventing recurrence using combination chemotherapeutics for treatment by creating conditions of increased oxidative stress and enhance response by tumors to conventional chemotherapeutics causing apoptosis. Disclosure: Dr. Ganeshan has nothing to disclose. Dr. Schor has nothing to disclose.

Stability of PCR Targets for Monitoring Minimal Residual Disease in Neuroblastoma

In neuroblastoma (NB) patients, minimal residual disease (MRD) can be detected by real-time quantitative PCR (qPCR) using NB-specific target genes, such as PHOX2B and TH. However, it is unknown whether the mRNA levels of these targets vary either during treatment or at relapse. If marker genes are not stably expressed, estimation of MRD levels in bone marrow (BM) or peripheral blood will be hampered. We studied the stability of a panel of qPCR markers in primary tumors at diagnosis compared with i) paired metastasis (n = 7), ii) treated (n = 10), and iii) relapse (n = 6) tumors. We also compared relative expression of the targets in iv) primary tumors and BM at diagnosis (n = 17), v) BM and peripheral blood at diagnosis (n = 20), vi) BM at diagnosis and during treatment (n = 26), and vii) BM from different puncture sides (n = 110). Especially at diagnosis, PCR target expression is quite stable. Accurate quantification is possible when expression level can be related to the primary tumor; however, PCR target expression can alter on treatment and at relapse. If the median value of relative expression of a panel of PCR targets is used, most variations due to treatment and outgrowth of subclones level out, allowing for reliable application and quantification of MRD-PCR targets in NB patients.

The Role of p14ARF Methylation in Neuroblastoma Minimal Residual Disease

Objectives: The aim of the present study is to investigate the clinical significance of epigenetic changes in neuroblastoma, we evaluated the relationship between therapeutic variables and the pattern of gene methylation in neuroblastoma cell lines. Methods: In this minimal residual disease model, cytotoxic effects of blocked p14ARF by a demethylating agent on KELLY and SHSY-5Y human neuroblastoma cell lines were assessed following addition of the drugs used in the Turkish Pediatric Oncology Group (TPOG) chemotherapy protocol. Drug induced effects on cell viability, cell damage and apoptotic cell death ratio were assessed with trypan blue dying. Cytotoxic effects differed among the used neuroblastoma cell lines. For the investigation of mechanisms of this effect, p14 ARF gen methylation and expression levels, and MYCN expression levels were targeted. Expressions of mRNA and protein were determined with real-time PCR and ELISA, respectively. Results: The p14 ARF gene expression levels in KELLY 5-aza-CdR, Vincristin, Vincristin+5aza-CdR, Dacarbazine, Ifosfamide+5-aza-CdR, Doxorubicine+5-aza-CdR, Etoposide+5aza-CdR groups were found to be significantly higher than those measured in corresponding groups of SHSY-5Y (p<0.05). The percentages of p14ARF gene promoter unmethylation were higher in MYCN (-) groups than MYCN (+) groups (p=0.034). Conclusion: In this pioneering study we suggest not only a new therapeutic approach for early or late relapses following the standard treatment protocols, but also demonstrate the effect of chemoterapeutic agents on p14 ARF gene. The present study suggests that clinically aggressive neuroblastoma cell lines have aberrant methylation of p14ARF genes and provides a

Multiple target molecular monitoring of bone marrow and peripheral blood samples from patients with localized neuroblastoma and healthy donors

Multiple target molecular monitoring of minimal residual disease in neuroblastoma (NB) patients may increase sensitivity and overcome tumor heterogeneity. However, multiple target analysis is costly and time consuming, thus improvement with respect to single target monitoring needs to be achieved. Italian patients with localized NB were evaluated at diagnosis for TH, GD2-s, DDC, DCX, ELAV-4, STX, and Phox2b mRNA expressions. Patients with metastatic NB were tested as positive controls, together with NB primary tumors and cell lines, while healthy donors were tested as negative controls. All NB-related markers but Phox2b were expressed in healthy donors, and in a high percentage of patients with localized NB without association with clinical events. The introduction of cut-off levels increased marker specificity, although the percentage of positive results was only slightly modified. While TH positivity in PB samples significantly associated with a worse prognosis, a paradox association was found for GD2-s mRNA expression. No correlation and agreement between quantitative and qualitative results obtained with the two assays were found. In the set of samples tested for all markers, no pattern of expression was found to be associated with a specific clinical situation. These findings suggest that positive molecular results may not reflect the presence of disease, and that correlation among different markers is small in condition of low tumor burden. Thus, to reduce cost and amount of precious samples, in addition to TH, whose prognostic value was confirmed, only Phox2b warrants further evaluation in multi-center, prospective studies for high risk patients.

Antagonism of Cytotoxic Chemotherapy in Neuroblastoma Cell Lines by 13-cis-Retinoic Acid Is Mediated by the Antiapoptotic Bcl-2 Family Proteins

13-cis-Retinoic acid (13-cis-RA) is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immunoblotting. Pretreatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC(99)) increased from clinically achievable levels to nonachievable levels, greater than 5-fold (cisplatin) to greater than 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxicity for neuroblastoma in vitro, mediated at least in part via the antiapoptotic Bcl-2 family of proteins.

Detecting Minimal Residual Disease in Neuroblastoma: The Superiority of a Panel of Real-Time Quantitative PCR Markers

PCR-based detection of minimal residual disease (MRD) in neuroblastoma (NB) patients can be used for initial staging and monitoring therapy response in bone marrow (BM) and peripheral blood (PB). PHOX2B has been identified as a sensitive and specific MRD marker; however, its expression varies between tumors. Therefore, a panel of markers could increase sensitivity. To identify additional MRD markers for NB, we selected genes by comparing SAGE (serial analysis of gene expression) libraries of healthy and NB tissues followed by extensive real-time quantitative PCR (RQ-PCR) testing in samples of tumors (n = 56), control BM (n = 51), PB (n = 37), and cell subsets. The additional value of a panel was determined in 222 NB samples from 82 Dutch stage 4 NB patients (54 diagnosis BM samples, 143 BM samples during/after treatment, and 25 PB samples). We identified 2 panels of specific RQ-PCR markers for MRD detection in NB patients: 1 for analysis of BM samples (PHOX2B, TH, DDC, CHRNA3, and GAP43) and 1 for analysis of PB samples (PHOX2B, TH, DDC, DBH, and CHRNA3). These markers all showed high expression in NB tumors and no or low expression in control BM or PB samples. In patients' samples, the PHOX2B marker detected most positive samples. In PB samples, however, 3 of 7 PHOX2B-negative samples were positive for 1 or more markers, and in BM examinations during treatment, 7% (6 of 86) of the PHOX2B-negative samples were positive for another marker. Because of differences in the sensitivities of the markers in BM and PB, we advise the use of 2 different panels to detect MRD in these compartments.

Detecting Minimal Residual Disease in Neuroblastoma: Still a Ways to Go

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for 10% of all cancers in children. The development of neuroblastoma involves both embryonic and tumorigenic factors, making the tumor very heterogeneous both biologically and clinically. Neuroblastoma tumors originate from embryonic neural crest cells committed to the development of the sympathetic nervous system, and their malignancy varies from totally benign with spontaneous regression to highly malignant (1). Prognostic factors include age of the patient at diagnosis, INSS (International Neuroblastoma Staging System) stage, tumor histopathology, and ploidy status of DNA content (2). Despite the availability of many clinical and biologic markers proposed as predictive of disease outcome and the use of advanced multimodal therapy, treatment results are still suboptimal for patients with these tumors. Thus, how to best estimate patient prognosis and choose optimal individual therapy remain fundamental challenges. Since the discovery by Moss et al. (3) 20 years ago of neuroblastoma cells in the blood of a majority of patients with known disseminated disease, much work has been devoted to the development and improvement of methods for detection of minimal residual disease (MRD).1 In this issue of Clinical Chemistry Stutterheim et al. (4) address the question of how to best identify the presence of neuroblastoma cells in blood and bone marrow by use of reverse transcription quantitative PCR (RQ-PCR). The authors recently investigated the use of paired-like homeobox 2b ( PHOX2B )2 as a marker for MRD in neuroblastoma and claimed reported that this transcript was the most specific single marker for this purpose (5). In the current study they selected 28 genes from SAGE libraries that showed high expression in neuroblastoma tumors and little or no expression in normal tissues. Preliminary studies of these 28 genes and 5 previously described markers (identified by using RQ-PCR and …

Prognostic influence of minimal residual disease detected by flow cytometry and peripheral blood stem cell transplantation by CD34+ selection in childhood advanced neuroblastoma

To determine whether neuroblastoma (NB) minimal residual disease (MRD) in bone marrow (BM) detected by flow cytometry could predict prognosis and whether tumor cell purging by CD34(+) cell selection prior to transplantation will impact on disease-free survival. NB MRD in BM was evaluated by flow cytometry with CD45-FITC-/CD81-PE+/CD56-PECy5+ monoclonal antibodies cocktail. Peripheral blood stem cell (PBSC) was enriched via positive CD34(+) cell selection by magnetic-activated cell separation system (MACS). Eleven of 31 patients with CD45(-)/CD81+/CD56+ cells by flow cytometry at diagnosis became negative after an average of four courses of chemotherapy. All 11 patients remained alive without evidence of disease. Thirteen of the 20 patients with positive MRD relapsed and 1 patient died from disease (mean 25.8 months). There was a significant difference between these two groups. MRD in BM was tested before PBSC transplantation (PBSCT) for 19 NB patients. Fourteen was negative, 4 of them relapsed and 10 patients remained alive without evidence of disease. Another 5 patients with positive MRD, all of them relapsed (mean 17 months after PBSCT) with a significant difference between these two groups. Fourteen of 19 PBSC were purged with CD34(+) selection procedure. Six of 14 relapsed (mean 18.43 months after PBSCT). Five patients did not purge for CD34(+) selection, and 3 of them relapsed with no significant difference between these two groups. Positive MRD in BM after an average of four courses of chemotherapy and before PBSCT is an unfavorable factor for stage IV NB. CD34(+) selection purging for PBSCT may not improve the prognosis for children with neuroblastoma in advanced stage.

Comparison of immunocytochemistry, real-time quantitative RT-PCR and flow cytometry for detection of minimal residual disease in neuroblastoma

A quantitative and precise measure of treatment response is warranted in neuroblastoma patients. We compared three quantitative methods often used for detection of minimal residual disease in such patients. Specificity, sensitivity and concordance of immunocytochemistry, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry were compared using experimental cell suspensions (n = 8) and clinical samples (n = 126). Neuroblastoma cells were identified by immunocytochemistry and flow cytometry using anti-GD2 (14.G2a) and anti-NCAM (5.1H11) antibodies, whereas tyrosine hydroxylase mRNA was the molecular target for quantitative RT-PCR. The sensitivity using flow cytometry was 1-2 logs less than using immunocytochemistry or quantitative RT-PCR. All control samples (n = 35) tested negative by immunocytochemistry, whereas 2/34 (6%) and 1/14 (7%) were false positive by quantitative RT-PCR and flow cytometry respectively. Concordant results were obtained in 85% of patient samples (n = 116) analyzed in parallel by quantitative RT-PCR and immunocytochemistry, whereas 71% of samples analyzed by flow cytometry and immunocytochemistry were concordant (n = 35). The correlation between tumor cell levels analyzed by quantitative RT-PCR and immunocytochemistry was high (r = 0.78, p < 0.001). Quantitative RT-PCR and immunocytochemistry both reliably detected very low levels of neuroblastoma cells in clinical samples. The agreement and correlation between these methods were high. In comparison, flow cytometry was less sensitive.

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.

The Doublecortin Gene, A New Molecular Marker to Detect Minimal Residual Disease in Neuroblastoma

Neuroblastoma (NB) is a pediatric cancer of highly variable clinical outcome. Much effort is devoted to detection of minimal residual (MRD) disease through RT-PCR or immunology of tissue-specific markers. Tyrosine hyrdroxylase (TH) has demonstrated a high utility to assess disease dissemination, although this marker can be lost due to clonal variability. Here we propose the use of the doublecortin (DCX) gene as a new molecular marker of neuroblastoma cells. DCX specifically appears in migrating neurons of the central and peripheral nervous system and interacts with and regulates the microtobule cytoskeleton. We have studied this gene by real-time quantitative RT-PCR in a total of 47 primary tumors and 202 samples of bone marrow or peripheral blood from 34 high-risk neuroblastoma patients as well as in 41 normal controls. The expression of DCX demonstrated a good specificity and concordance with TH, showing a higher expression rate in all the sample types studied as well as at different time points from diagnosis. We conclude that DCX would be a more efficient marker of minimal disease in neuroblastoma and perhaps other tumors of neuronal lineage.

Comparison of five colour- flowcytometry and PCR in the detection of minimal residual disease in neuroblastoma

In contrast to acute lymphatic leukemia, the clinical impact of minimal residual disease (MRD) in patients with neuroblastoma (NB) is unclear. In order to detect and monitor residual NB cells, we established a new five colour-flowcytometry panel and assays using RT-PCR and Real-time PCR for tyrosine hydroxylase (TH). All methods were able to detect one NB cell in 105 normal cells. We analyzed a total of 322 samples (bone marrow and autologous peripheral blood stem cells which were collected for autologous transplantation) from 44 NB patients. Seventy-three percent of the 322 samples were positive, 15% were negative for NB cells. We found a highly significant correlation between flowcytometry, Real-time PCR and RT-PCR (P≤.0001). We conclude that both PCR for TH and five colour-flowcytometry are valuable tools to monitor minimal residual disease in patients with NB. Currently, we perform a prospective study to evaluate a potential correlation between MRD and survival.