These data show that primary sphere cells isolated from high-risk neuroblastoma tumors and bone marrow aspirates contain multiple chromosomal amplifications consistent with those found in neuroblastoma. 1). Neuroblastoma histologically presents as a small round malignant tumor whose tumor cells originate from precursor cells of the sympathetic ridge. 2). This biomarker assay has been incorporated into all NANT consortium therapeutic studies in order to validate the assay and prospectively analyze disease burden assessment and to provide standard clinical disease response testing in the context of uniform therapy in large numbers of patients. An alternative in vivo assay was used to establish secondary and tertiary tumor engraftment because of latency to tumor formation in our orthotopic adrenal model of tumorigenicity.
By contrast, any level of neuroblastoma mRNA in bone marrow, even when neuroblastoma cells were not detected morphologically, predicted a poor PFS. B and C, spheres from low-risk tumors differentiated into large nestin-positive or βIII-tubulin–positive neuronal networks (B, top), whereas spheres from high-risk tumors did not (B, bottom).
30). ΔCt of 15 chosen as the median of positive ΔCt values. Your account has been temporarily locked due to incorrect sign in attempts and will be automatically unlocked in 3A–D; Table 3).
Where appropriate, standard descriptive and analytic statistical methods such as t test, ANOVA, ordinary least squares regression, and contingency table analyses were used (31). [13,14] The number of MYCN copies required to be defined as amplification is still controversial. Tumors stained positive immunohistochemically for the neuroblastoma markers NB84 and TH and for the neural progenitor cell marker nestin (arrowheads). The NB5 assay using all five genes was compared in this dataset with other analyses using other signatures (PHOX2B + TH + DCX; PHOX2B + TH + DDC; PHOX2B + TH; and TH + DCX) based on prior publications (refs.
4), pediatric brain tumors ( The bone marrow from this patient had large areas of pink neuropil on one side but no neuroblastoma cells. Patients with a high-risk neuroblastoma have a long-term survival of <40% ( The clinical characteristics of some tumors include spontaneous remission or mature differentiation.[5].
To confirm that high-risk tumor sphere lines contained chromosomal aberrations consistent with neuroblastoma, SNP analysis was performed on two primary cell lines from high-risk neuroblastoma; one from a bone marrow metastasis (NB12) and the other from a tumor (NB19). 2A–D; P < 0.0001). Fig. 1A A new approaches to neuroblastoma therapy (NANT) phase II study, Statistical methods for the analysis and presentation of the results of bone marrow transplants.
Advances in risk classification and treatment strategies for, [7]. Based on two lesions, the tumor was considered to be stage IV. Patients with relapsed neuroblastoma have a poor prognosis with a 5-year overall survival after relapse of 20% (6, 7). 30 mins. Flow cytometry and fluorescence-activated cell sorting. Because individual patients could have multiple disease evaluations performed during follow-up, and the results of these evaluation could change, implying that the patient is now potentially at higher or lower risk of relapse or progression, time-dependent covariate (TDC) Cox regression analysis was used to examine the influence of ΔCt and other variables of interest (e.g., CT/MRI, bone marrow morphology) on time to progression (32, 33). Correlation of NB5 assay ΔCt between bone marrow (BM) and blood. The primary cell lines from the spheres expressed the neural crest markers nestin, vimentin, and fibronectin and the neuroblastoma markers NB84 and TH. 4A; arrowheads). Kholodenko IV, Kalinovsky DV, Doronin II, et al. Statistical significance was obtained between these groups (P < 0.05). Neuroblastoma mRNA in bone marrow and blood, as quantified by the NB5 assay, were strongly correlated, but the amount in bone marrow was significantly greater. Once enrolled in NANT 2004-05, bone marrow and blood samples were obtained prospectively for the NB5 assay. Fig. These results indicate that neuroblastoma sphere-forming cells have a high tumorigenic potential, forming neuroblastoma tumors that metastasize with as few as 10 cells. Neuroblastoma (NB) can occur in any part of the sympathetic nervous system, and it is highly heterogeneous. B, growth curves indicated logarithmic growth of cultured primary tumors in liquid culture. These data indicate that primary cells from neuroblastoma tumors and bone marrow aspirates that express neuroblastoma and neural crest progenitor cell markers can be isolated and propagated using neural crest stem cell culture conditions. Interestingly, amplification of 7q and 11q chromosomal regions has not been described in the absence of MYCN amplification ( Approximately 105 cells were stained and analyzed on a Becton Dickinson FACSCalibur four-color analyzer. Fig.
r, accompanied by pain in both lower limbs for more than 1 month.
Tumors from low-risk neuroblastoma tumors acquired adherent growth characteristics upon passaging. Conception and design: A. Marachelian, J.G.
The primary sphere line NB12, passage 3, showed amplification of chromosomal regions in 1p, 7q, 10q, 11q, 15q, and 17q when using a copy number cutoff of >8 (Supplementary Table S1), as previously observed in neuroblastoma tumors ( Moreover, tumor cells stained positive for the neuroblastoma markers NB84, TH, and the progenitor marker nestin ( They were isolated as sphere-forming cells in medium that supports stem cell growth, without going through the crisis stage that typically accompanies the generation of immortalized cell lines.
Villablanca, H.A. 10; 5A, top). We observed extensive neuronal differentiation in low-risk patient sphere cells and little or no differentiation in high-risk patient sphere cells, suggesting that the capacity of neuroblastoma tumor sphere cells to differentiate reflects the clinical phenotype of neuroblastoma tumors. This is the first prospective study to quantify expression of five neuroblastoma-associated genes in bone marrow and blood of patients with relapsed/refractory neuroblastoma and to correlate expression with concurrent CT/MRI and MIBG imaging and morphologic bone marrow evaluations. Seeger, Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc. 2C). (A) Small round tumor cells expressing CD56, CgA, NSE, and S100 in. Fig. Although all cellular fractions formed tumors in immunocompromised mice, CD24+/CD34+ cells formed tumors in half the time of all other cellular fractions (19.0 ± 0.0 days compared with 34.0 ± 0.72 days; To determine whether the tumors could be serially passaged, we followed secondary and tertiary tumor formation in NOD/SCID mice. Histopathology of bone marrow showed positive expression of CD56, CgA, NSE, and S100 (Fig. Fig. Primary culture of tumor spheres from tumors and bone marrow aspirates. ): J.A. A subpopulation of cells within tumor spheres from high-risk tumors expressed CD24 and CD34 ( Our data provide the first example of primary human solid tumor TICs forming tumors with as few as 10 unsorted cells, and suggest that relative to other solid tumor cells, a high proportion of cells in high-risk neuroblastoma bone marrow metastases are tumorigenic. Medicine. http://creativecommons.org/licenses/by/4.0. Sorting was performed on cells stained with purified monoclonal CD34 and Alexa 488–conjugated goat anti-mouse secondary antibody followed by phycoerythrin-conjugated monoclonal CD24 antibody. MYCN examined in bone marrow cells by FISH was detected with negative amplification. C and D, Correlation of bone marrow and blood NB5 ΔCt and MIBG Curie score. 38– Importantly, quantifying neuroblastoma mRNA in both bone marrow and blood provides an independent predictor of PFS. A small fraction (∼0.24%) of neuroblastoma tumor-derived sphere cells from two different patients (NB12 and NB25) stained positive for the metastatic marker CD24 (middle). When comparing the baseline characteristics of the undetectable versus detectable patients by NB5 assay, there were no statistically significant differences in MYCN status, age at diagnosis, or history of prior relapse. Diagnose: Bone marrow examination revealed NB cell invasion. In this regard, undifferentiated and differentiated neuroblastoma TICs and SKPs share expression of many neural and progenitor markers, including vimentin, fibronectin, TH, βIII-tubulin, NFM, NB84, nestin, S100β, GFAP, and GalC. As a … Liu, F. Goodarzian, H.C. Tran, S. Groshen, R. Sposto, R.C. The glycoprotein CD24 was investigated as a candidate unique identifier for the TIC in our neuroblastoma tumor spheres as this antigen has been shown to be expressed on renal cell carcinomas, small-cell lung carcinomas, and neuroblastomas ( Cytogenetic analysis of tumor spheres. Isolation of neuroblastoma cells using neural crest stem cell conditions provides for the first time an expandable source of cells from low-risk tumors and from bone marrow metastases from high-risk tumors. Fig. Copy number loss was defined as <1.2 copies. In fact, the absence of NB5 detectable mRNA in bone marrow, which is almost always associated with a negative bone marrow morphologically, predicts a high probability of PFS.
By contrast, any level of neuroblastoma mRNA in bone marrow, even when neuroblastoma cells were not detected morphologically, predicted a poor PFS. B and C, spheres from low-risk tumors differentiated into large nestin-positive or βIII-tubulin–positive neuronal networks (B, top), whereas spheres from high-risk tumors did not (B, bottom).
30). ΔCt of 15 chosen as the median of positive ΔCt values. Your account has been temporarily locked due to incorrect sign in attempts and will be automatically unlocked in 3A–D; Table 3).
Where appropriate, standard descriptive and analytic statistical methods such as t test, ANOVA, ordinary least squares regression, and contingency table analyses were used (31). [13,14] The number of MYCN copies required to be defined as amplification is still controversial. Tumors stained positive immunohistochemically for the neuroblastoma markers NB84 and TH and for the neural progenitor cell marker nestin (arrowheads). The NB5 assay using all five genes was compared in this dataset with other analyses using other signatures (PHOX2B + TH + DCX; PHOX2B + TH + DDC; PHOX2B + TH; and TH + DCX) based on prior publications (refs.
4), pediatric brain tumors ( The bone marrow from this patient had large areas of pink neuropil on one side but no neuroblastoma cells. Patients with a high-risk neuroblastoma have a long-term survival of <40% ( The clinical characteristics of some tumors include spontaneous remission or mature differentiation.[5].
To confirm that high-risk tumor sphere lines contained chromosomal aberrations consistent with neuroblastoma, SNP analysis was performed on two primary cell lines from high-risk neuroblastoma; one from a bone marrow metastasis (NB12) and the other from a tumor (NB19). 2A–D; P < 0.0001). Fig. 1A A new approaches to neuroblastoma therapy (NANT) phase II study, Statistical methods for the analysis and presentation of the results of bone marrow transplants.
Advances in risk classification and treatment strategies for, [7]. Based on two lesions, the tumor was considered to be stage IV. Patients with relapsed neuroblastoma have a poor prognosis with a 5-year overall survival after relapse of 20% (6, 7). 30 mins. Flow cytometry and fluorescence-activated cell sorting. Because individual patients could have multiple disease evaluations performed during follow-up, and the results of these evaluation could change, implying that the patient is now potentially at higher or lower risk of relapse or progression, time-dependent covariate (TDC) Cox regression analysis was used to examine the influence of ΔCt and other variables of interest (e.g., CT/MRI, bone marrow morphology) on time to progression (32, 33). Correlation of NB5 assay ΔCt between bone marrow (BM) and blood. The primary cell lines from the spheres expressed the neural crest markers nestin, vimentin, and fibronectin and the neuroblastoma markers NB84 and TH. 4A; arrowheads). Kholodenko IV, Kalinovsky DV, Doronin II, et al. Statistical significance was obtained between these groups (P < 0.05). Neuroblastoma mRNA in bone marrow and blood, as quantified by the NB5 assay, were strongly correlated, but the amount in bone marrow was significantly greater. Once enrolled in NANT 2004-05, bone marrow and blood samples were obtained prospectively for the NB5 assay. Fig. These results indicate that neuroblastoma sphere-forming cells have a high tumorigenic potential, forming neuroblastoma tumors that metastasize with as few as 10 cells. Neuroblastoma (NB) can occur in any part of the sympathetic nervous system, and it is highly heterogeneous. B, growth curves indicated logarithmic growth of cultured primary tumors in liquid culture. These data indicate that primary cells from neuroblastoma tumors and bone marrow aspirates that express neuroblastoma and neural crest progenitor cell markers can be isolated and propagated using neural crest stem cell culture conditions. Interestingly, amplification of 7q and 11q chromosomal regions has not been described in the absence of MYCN amplification ( Approximately 105 cells were stained and analyzed on a Becton Dickinson FACSCalibur four-color analyzer. Fig.
r, accompanied by pain in both lower limbs for more than 1 month.
Tumors from low-risk neuroblastoma tumors acquired adherent growth characteristics upon passaging. Conception and design: A. Marachelian, J.G.
The primary sphere line NB12, passage 3, showed amplification of chromosomal regions in 1p, 7q, 10q, 11q, 15q, and 17q when using a copy number cutoff of >8 (Supplementary Table S1), as previously observed in neuroblastoma tumors ( Moreover, tumor cells stained positive for the neuroblastoma markers NB84, TH, and the progenitor marker nestin ( They were isolated as sphere-forming cells in medium that supports stem cell growth, without going through the crisis stage that typically accompanies the generation of immortalized cell lines.
Villablanca, H.A. 10; 5A, top). We observed extensive neuronal differentiation in low-risk patient sphere cells and little or no differentiation in high-risk patient sphere cells, suggesting that the capacity of neuroblastoma tumor sphere cells to differentiate reflects the clinical phenotype of neuroblastoma tumors. This is the first prospective study to quantify expression of five neuroblastoma-associated genes in bone marrow and blood of patients with relapsed/refractory neuroblastoma and to correlate expression with concurrent CT/MRI and MIBG imaging and morphologic bone marrow evaluations. Seeger, Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc. 2C). (A) Small round tumor cells expressing CD56, CgA, NSE, and S100 in. Fig. Although all cellular fractions formed tumors in immunocompromised mice, CD24+/CD34+ cells formed tumors in half the time of all other cellular fractions (19.0 ± 0.0 days compared with 34.0 ± 0.72 days; To determine whether the tumors could be serially passaged, we followed secondary and tertiary tumor formation in NOD/SCID mice. Histopathology of bone marrow showed positive expression of CD56, CgA, NSE, and S100 (Fig. Fig. Primary culture of tumor spheres from tumors and bone marrow aspirates. ): J.A. A subpopulation of cells within tumor spheres from high-risk tumors expressed CD24 and CD34 ( Our data provide the first example of primary human solid tumor TICs forming tumors with as few as 10 unsorted cells, and suggest that relative to other solid tumor cells, a high proportion of cells in high-risk neuroblastoma bone marrow metastases are tumorigenic. Medicine. http://creativecommons.org/licenses/by/4.0. Sorting was performed on cells stained with purified monoclonal CD34 and Alexa 488–conjugated goat anti-mouse secondary antibody followed by phycoerythrin-conjugated monoclonal CD24 antibody. MYCN examined in bone marrow cells by FISH was detected with negative amplification. C and D, Correlation of bone marrow and blood NB5 ΔCt and MIBG Curie score. 38– Importantly, quantifying neuroblastoma mRNA in both bone marrow and blood provides an independent predictor of PFS. A small fraction (∼0.24%) of neuroblastoma tumor-derived sphere cells from two different patients (NB12 and NB25) stained positive for the metastatic marker CD24 (middle). When comparing the baseline characteristics of the undetectable versus detectable patients by NB5 assay, there were no statistically significant differences in MYCN status, age at diagnosis, or history of prior relapse. Diagnose: Bone marrow examination revealed NB cell invasion. In this regard, undifferentiated and differentiated neuroblastoma TICs and SKPs share expression of many neural and progenitor markers, including vimentin, fibronectin, TH, βIII-tubulin, NFM, NB84, nestin, S100β, GFAP, and GalC. As a … Liu, F. Goodarzian, H.C. Tran, S. Groshen, R. Sposto, R.C. The glycoprotein CD24 was investigated as a candidate unique identifier for the TIC in our neuroblastoma tumor spheres as this antigen has been shown to be expressed on renal cell carcinomas, small-cell lung carcinomas, and neuroblastomas ( Cytogenetic analysis of tumor spheres. Isolation of neuroblastoma cells using neural crest stem cell conditions provides for the first time an expandable source of cells from low-risk tumors and from bone marrow metastases from high-risk tumors. Fig. Copy number loss was defined as <1.2 copies. In fact, the absence of NB5 detectable mRNA in bone marrow, which is almost always associated with a negative bone marrow morphologically, predicts a high probability of PFS.