The Isolation of Ecdysterone Inducible Genes by Hybridization Subtraction Chromatography

We have developed a procedure for selectively enriching a mRNA population for inducible sequences. Other than the induced mRNA species, the population of mRNA in control cells is approximately the same as the mRNA population in induced cells. Cytoplasmic mRNA from control cells is bound to oligo (dT)-cellulose and used as a template for reverse transcriptase, the oligo (dT) serving as a primer. After removing the template mRNAs, the cDNA-cellulose column is used to hybridize a population of mRNAs from induced cells. The non-hybridized poly A + RNAs are greatly enriched in the inducible sequences. We have used this technique of hybridization subtraction chromotography to select a mRNA population enriched for the mRNAs inducible by ecdysterone in Schneider\u27s Line 2 Drosophila cells. This population of RNAs was used to screen a recombinant library. Preliminary results indicate that approximately 10% of the RNA in the probe population represents ecdysterone inducible sequences. Methods are described for optimizing the cDNA synthesis reaction (we obtain ≥ 30% efficiency) and hybridizing RNA to the cDNA-cellulose resin. This method can be used to select induced mRNAs regardless of the way in which the induction is brought about

MIBG avidity correlates with clinical features, tumor biology, and outcomes in neuroblastoma: A report from the Children’s Oncology Group

BackgroundPrior studies suggest that neuroblastomas that do not accumulate metaiodobenzylguanidine (MIBG) on diagnostic imaging (MIBG non‐avid) may have more favorable features compared with MIBG avid tumors. We compared clinical features, biologic features, and clinical outcomes between patients with MIBG nonavid and MIBG avid neuroblastoma.ProcedurePatients had metastatic high‐ or intermediate‐risk neuroblastoma and were treated on Children’s Oncology Group protocols A3973 or A3961. Comparisons of clinical and biologic features according to MIBG avidity were made with chi‐squared or Fisher exact tests. Event‐free (EFS) and overall (OS) survival compared using log–rank tests and modeled using Cox models.ResultsThirty of 343 patients (8.7%) had MIBG nonavid disease. Patients with nonavid tumors were less likely to have adrenal primary tumors (34.5 vs. 57.2%; P = 0.019), bone metastases (36.7 vs. 61.7%; P = 0.008), or positive urine catecholamines (66.7 vs. 91.0%; P < 0.001) compared with patients with MIBG avid tumors. Nonavid tumors were more likely to be MYCN amplified (53.8 vs. 32.6%; P = 0.030) and had lower norepinephrine transporter expression. Patients with MIBG nonavid disease had a 5‐year EFS of 50.0% compared with 38.7% for patients with MIBG avid disease (P = 0.028). On multivariate testing in high‐risk patients, MIBG avidity was the sole adverse prognostic factor for EFS identified (hazard ratio 1.77; 95% confidence interval 1.04–2.99; P = 0.034).ConclusionsPatients with MIBG nonavid neuroblastoma have lower rates of adrenal primary tumors, bone metastasis, and catecholamine secretion. Despite being more likely to have MYCN‐amplified tumors, these patients have superior outcomes compared with patients with MIBG avid disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138438/1/pbc26545_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138438/2/pbc26545.pd

Evaluation of Norepinephrine Transporter Expression and Metaiodobenzylguanidine Avidity in Neuroblastoma: A Report from the Children's Oncology Group

Purpose. (123)I-metaiodobenzylguanidine (MIBG) is used for the diagnostic evaluation of neuroblastoma. We evaluated the relationship between norepinephrine transporter (NET) expression and clinical MIBG uptake. Methods. Quantitative reverse transcription PCR (N = 82) and immunohistochemistry (IHC; N = 61) were performed for neuroblastoma NET mRNA and protein expression and correlated with MIBG avidity on diagnostic scans. The correlation of NET expression with clinical features was also performed. Results. Median NET mRNA expression level for the 19 MIBG avid patients was 12.9% (range 1.6–73.7%) versus 5.9% (range 0.6–110.0%) for the 8 nonavid patients (P = 0.31). Median percent NET protein expression was 50% (range 0–100%) in MIBG avid patients compared to 10% (range 0–80%) in nonavid patients (P = 0.027). MYCN amplified tumors had lower NET protein expression compared to nonamplified tumors (10% versus 50%; P = 0.0002). Conclusions. NET protein expression in neuroblastoma correlates with MIBG avidity. MYCN amplified tumors have lower NET protein expression

MIBG scans in patients with stage 4 neuroblastoma reveal two metastatic patterns, one is associated with MYCN amplification and in MYCN-amplified tumours correlates with a better prognosis

Methods: Diagnostic 123I-MIBG scans from 249 patients (123 from a European and 126 from the COG cohort) were assessed for metastatic spread in 14 body segments and the form of the lesions: “focal” (clear margins distinguishable from adjacent background) or “diffuse” (indistinct margins, dispersed throughout the body segment). The total numbers of diffuse and focal lesions were recorded. Patients were then categorized as having lesions exclusively focal, lesions more focal than diffuse, lesions more diffuse than focal, or lesions exclusively diffuse.Results: Diffuse lesions affected a median of seven body segments and focal lesions a median of two body segments (P < 0.001, both cohorts). Patients with a focal pattern had a median of 2 affected body segments and those with a diffuse pattern a median of 11 affected body segments (P < 0.001, both cohorts). Thus, two MIBG-avid metastatic patterns emerged: “limited-focal” and “extensive-diffuse”. The median numbers of affected body segments in MYCN-amplified (MNA) tumours were 5 (European cohort) and 4 (COG cohort) compared to 9 and 11, respectively, in single-copy MYCN (MYCNsc) tumours (P < 0.001). Patients with exclusively focal metastases were more likely to have a MNA tumour (60 % and 70 %, respectively) than patients with the other types of metastases (23 % and 28 %, respectively; P < 0.001). In a multivariate Cox regression analysis, focal metastases were associated with a better event-free and overall survival than the other types of metastases in patients with MNA tumours in the COG cohort (P < 0.01).Conclusion: Two metastatic patterns were found: a “limited and focal” pattern found mainly in patients with MNA neuroblastoma that correlated with prognosis, and an “extensive and diffuse” pattern found mainly in patients with MYCNsc neuroblastoma.Purpose: The aim of this study was to find clinically relevant MIBG-avid metastatic patterns in patients with newly diagnosed stage 4 neuroblastoma

Semiquantitative mIBG scoring as a prognostic indicator in patients with stage 4 neuroblastoma: a report from the Children\u27s oncology group

Radiolabeled metaiodobenzylguanidine (mIBG) is a highly sensitive and specific marker for detecting neuroblastoma. A semiquantitative mIBG score (Curie score [CS]) was assessed for utility as a prognostic indicator for a cohort of patients with high-risk metastatic disease. METHODS: mIBG scans from 280 patients with mIBG-avid, stage 4 neuroblastoma enrolled on the Children\u27s Oncology Group (COG) protocol A3973 were evaluated at diagnosis (n = 280), after induction chemotherapy (n = 237), and after an autologous stem cell transplantation (n = 178). Individual mIBG scans were evaluated at 10 different anatomic regions, with the scoring of each site (0-3) based on the extent of disease at that anatomic region. RESULTS: There was no correlation between CS at diagnosis and subsequent treatment outcome. Patients with a CS \u3e 2 after induction therapy had a significantly worse event-free survival (EFS) than those with scores 2 identified a cohort of patients at greater risk for an event, independent of other known neuroblastoma factors, including age, MYCN status, ploidy, mitosis-karyorrhexis index, and histologic grade. For MYCN-amplified tumors, the presence (CS \u3e 0) versus absence (CS = 0) of residual mIBG avidity after induction was associated with a significantly worse outcome (3-y EFS: 11.8% +/- 7.8% vs. 49.6% +/- 7.7%, respectively; P = 0.003). After transplantation, patients with a CS \u3e 0 had an EFS inferior to that of patients with a CS of 0 (3-y EFS: 28.9% +/- 6.8% vs. 49.3% +/- 4.9%, respectively [n = 133]; P = 0.009). CONCLUSION: Curie scoring carries prognostic significance in the management of patients with high-risk neuroblastoma. In particular, patients with CSs \u3e 2 after induction have extremely poor outcomes and should be considered for alternative therapeutic strategies

Semiquantitative mIBG Scoring as a Prognostic Indicator in Patients with Stage 4 Neuroblastoma: A Report from the Children’s Oncology Group

UnlabelledRadiolabeled metaiodobenzylguanidine (mIBG) is a highly sensitive and specific marker for detecting neuroblastoma. A semiquantitative mIBG score (Curie score [CS]) was assessed for utility as a prognostic indicator for a cohort of patients with high-risk metastatic disease.MethodsmIBG scans from 280 patients with mIBG-avid, stage 4 neuroblastoma enrolled on the Children's Oncology Group (COG) protocol A3973 were evaluated at diagnosis (n = 280), after induction chemotherapy (n = 237), and after an autologous stem cell transplantation (n = 178). Individual mIBG scans were evaluated at 10 different anatomic regions, with the scoring of each site (0-3) based on the extent of disease at that anatomic region.ResultsThere was no correlation between CS at diagnosis and subsequent treatment outcome. Patients with a CS &gt; 2 after induction therapy had a significantly worse event-free survival (EFS) than those with scores ≤ 2 (3-y EFS: 15.4% ± 5.3% vs. 44.9% ± 3.9%, respectively; P &lt; 0.001). A postinduction CS &gt; 2 identified a cohort of patients at greater risk for an event, independent of other known neuroblastoma factors, including age, MYCN status, ploidy, mitosis-karyorrhexis index, and histologic grade. For MYCN-amplified tumors, the presence (CS &gt; 0) versus absence (CS = 0) of residual mIBG avidity after induction was associated with a significantly worse outcome (3-y EFS: 11.8% ± 7.8% vs. 49.6% ± 7.7%, respectively; P = 0.003). After transplantation, patients with a CS &gt; 0 had an EFS inferior to that of patients with a CS of 0 (3-y EFS: 28.9% ± 6.8% vs. 49.3% ± 4.9%, respectively [n = 133]; P = 0.009).ConclusionCurie scoring carries prognostic significance in the management of patients with high-risk neuroblastoma. In particular, patients with CSs &gt; 2 after induction have extremely poor outcomes and should be considered for alternative therapeutic strategies

Ototoxicity in children with high-risk neuroblastoma: Prevalence, risk factors, and concordance of grading scales - A report from the Children\u27s Oncology Group

Purpose: Platinum-based therapy is the mainstay for management of high-risk neuroblastoma. Prevalence of platinum-related ototoxicity has ranged from 13% to 95% in previous reports; variability is attributable to small samples and disparate grading scales. There is no consensus regarding optimal ototoxicity grading. Furthermore, prevalence and predictors of hearing loss in a large uniformly treated high-risk neuroblastoma population are unknown. We address these gaps in our study. Patients and Methods: Audiologic testing was completed after administration of cisplatin alone (\u3c 400 mg/m2; exposure one) or after cisplatin (400 mg/m2) plus carboplatin (1,700 mg/m 2; exposure two). Hearing loss was graded using four scales (American Speech-Language-Hearing Association; Brock; Chang; and Common Terminology Criteria for Adverse Events, version 3 [CTCAEv3]). Results: Of 489 eligible patients, 333 had evaluable audiologic data. Median age at diagnosis was 3.3 years. Prevalence of severe hearing loss differed by scale. For those in the exposure-one group, prevalence ranged from 8% per Brock to 47% per CTCAEv3 (Brock v CTCAEv3 and Chang, P \u3c .01; CTCAEv3 v Chang, P = .16); for those in the exposure-two group, prevalence ranged from 30% per Brock to 71% per CTCAEv3 (all pair-wise comparisons, P \u3c .01). In patients requiring hearing aids, hearing loss was graded as severe in 49% (Brock), 91% (Chang), and 100% (CTCAEv3). Risk factors for severe hearing loss included exposure to cisplatin and carboplatin compared with cisplatin alone and hospitalization for infection. Conclusion: Severe hearing loss is prevalent among children with high-risk neuroblastoma. Exposure to cisplatin combined with myeloablative carboplatin significantly increases risk. The Brock scale underestimates severe hearing loss and should be used with caution in this setting. © 2014 by American Society of Clinical Oncology