Residual Disease In Neuroblastoma Research Articles

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.

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.

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.

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 …

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.

The synthetic retinoid RO 13-6307 induces neuroblastoma differentiation in vitro and inhibits neuroblastoma tumour growth in vivo.

Retinoids modulate cell proliferation, differentiation and apoptosis in a variety of tumour cells including leukaemia and neuroblastoma, a childhood tumour of the sympathetic nervous system. 13-cis retinoic acid is in clinical use against minimal residual disease in neuroblastoma, where the effect seems to depend on dose, scheduling and tumour mass. Novel retinoids are searched for, to improve potency and lower toxicity. We investigated the effect of the synthetic retinoid Ro 13-6307 on neuroblastoma growth in vitro on SK-N-BE(2) and SH-SY5Y cells. Furthermore, effects on tumour growth and the toxicity profile were investigated in a rat xenograft model. Effects of Ro 13-6307 were compared to 13-cis RA (retinoic acid) in vitro and in vivo. Neuroblastoma cells treated with 1 microM Ro 13-6307 exhibited neuronal differentiation, decreased proliferation and accumulation of cells in G1 phase in at least the same magnitude as 5 microM 13-cis RA. No apoptosis was detected in vitro. Treatment of nude rats with neuroblastoma using Ro 13-6307, 0.12 mg p.o. daily, decreased neuroblastoma growth in vivo, in terms of tumour volume during treatment and tumour weight at sacrifice (p < 0.05). In contrast, Ro 13-6307, 0.08 mg p.o. daily, resulted in no significant reduction in tumour growth. All rats treated with Ro 13-6307 gained less weight than control rats, but they exhibited no other signs of toxicity. The toxicity profile of Ro 13-6307 was similar to what we found with 13-cis RA. Our preclinical results suggest that Ro 13-6307 may be a candidate retinoid for clinical oral therapy of neuroblastoma in children.

Radiochemotherapy of postoperative minimal residual disease in neuroblastoma.

Two-year survival of neuroblastoma patients has not been substantially improved in the last 20 years, either in localized and regional disease or in metastatic tumors (Tab. 1) [1, 16, 19, 23]. As in other tumors of childhood the anatomic extent of the disease is the main prognostic factor (Fig. 1), but host-tumor interrelations seem to be much more decisive in neuroblastoma [7] than the ability of chemotherapeutic agents to eradicate microscopic foci of metastatic deposits as they do in Wilm’s tumor and soft-tissue sarcoma. Thus, guidelines for treatment of postoperative minimal residual disease have been found in supervision of the urinary level of the biochemical markers of neuroblastoma by serial assays [14] and in the reappraisal of the various factors influencing survival [1, 4, 6, 9, 12, 23]. Age at diagnosis is the most prominent of these factors (Fig. 2), and infants under 1 year of age have a surprisingly good prognosis compared to children of 1 year or more, whether disease is localized or widespread.KeywordsMinimal Residual DiseaseActuarial SurvivalNeuroblastoma PatientAnatomic ExtentMain Prognostic FactorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.