Segmental Chromosomal Aberrations Research Articles

17q Gain in Neuroblastoma: A Review of Clinical and Biological Implications.

Neuroblastoma (NB) is the most frequent extracranial solid childhood tumor. Despite advances in the understanding and treatment of this disease, the prognosis in cases of high-risk NB is still poor. 17q gain has been shown to be the most frequent genomic alteration in NB. However, the significance of this remains unclear because of its high frequency and association with other genetic modifications, particularly segmental chromosomal aberrations, 1p and 11q deletions, and MYCN amplification, all of which are also associated with a poor clinical prognosis. This work reviewed the evidence on the clinical and biological significance of 17q gain. It strongly supports the significance of 17q gain in the development of NB and its importance as a clinically relevant marker. However, it is crucial to distinguish between whole and partial chromosome 17q gains. The most important breakpoints appear to be at 17q12 and 17q21. The former distinguishes between whole and partial chromosome 17q gain; the latter is a site of IGF2BP1 and NME1 genes that appear to be the main oncogenes responsible for the functional effects of 17q gain.

Computed Tomography-Based Radiomics Signature for Predicting Segmental Chromosomal Aberrations at 1p36 and 11q23 in Pediatric Neuroblastoma.

This study aimed to develop and assess the precision of a radiomics signature based on computed tomography imaging for predicting segmental chromosomal aberrations (SCAs) status at 1p36 and 11q23 in neuroblastoma. Eighty-seven pediatric patients diagnosed with neuroblastoma and with confirmed genetic testing for SCAs status at 1p36 and 11q23 were enrolled and randomly stratified into a training set and a test set. Radiomics features were extracted from 3-phase computed tomography images and analyzed using various statistical methods. An optimal set of radiomics features was selected using a least absolute shrinkage and selection operator regression model to calculate the radiomics score for each patient. The radiomics signature was validated using receiver operating characteristic curves to obtain the area under the curve and 95% confidence interval (CI). Eight radiomics features were carefully selected and used to compute the radiomics score, which demonstrated a statistically significant distinction between the SCAs and non-SCAs groups in both sets. The radiomics signature achieved an area under the curve of 0.869 (95% CI, 0.788-0.943) and 0.883 (95% CI, 0.753-0.978) in the training and test sets, respectively. The accuracy of the radiomics signature was 0.817 and 0.778 in the training and test sets, respectively. The Hosmer-Lemeshow test confirmed that the radiomics signature was well calibrated. Computed tomography-based radiomics signature has the potential to predict SCAs at 1p36 and 11q23 in neuroblastoma.

Neuroblastoma Patients' Outcome and Chromosomal Instability.

Chromosomal instability (CIN) induces a high rate of losses or gains of whole chromosomes or parts of chromosomes. It is a hallmark of most human cancers and one of the causes of aneuploidy and intra-tumor heterogeneity. The present study aimed to evaluate the potential prognostic role of CIN in NB patients at diagnosis. We performed array comparative genomic hybridization analyses on 451 primary NB patients at the onset of the disease. To assess global chromosomal instability with high precision, we focused on the total number of DNA breakpoints of gains or losses of chromosome arms. For each tumor, an array-CGH-based breakpoint instability index (BPI) was assigned which defined the total number of chromosomal breakpoints per genome. This approach allowed us to quantify CIN related to whole genome disruption in all NB cases analyzed. We found differences in chromosomal breakages among the NB clinical risk groups. High BPI values are negatively associated with survival of NB patients. This association remains significant when correcting for stage, age, and MYCN status in the Cox model. Stratified analysis confirms the prognostic effect of BPI index in low-risk NB patients with non-amplified MYCN and with segmental chromosome aberrations.

Stage 4N neuroblastoma before and during the era of anti-GD2 immunotherapy.

10037 Background: Patients with stage 4N neuroblastoma (distant metastases limited to lymph nodes) stand out as virtually the only survivors of high-risk neuroblastoma (HR-NB) before myeloablative therapy (MAT) and immunotherapy with anti-GD2 monoclonal antibodies (mAb) became standard. No report presents results with contemporary treatment programs for 4N. The only large study to date (from the International Neuroblastoma Risk Group) analyzed patients diagnosed 1990-2002, found 4N to have significantly better event-free (EFS) and overall survival (OS) compared to other stage 4 patients, and recommended considering less intensive therapy for 4N. Methods: We conducted a retrospective analysis of patients treated at our center from 1985-2021 to analyze our large experience to fill the gap in the literature on treatment of 4N, namely, results with contemporary multi-modality therapy including MAT and anti-GD2 mAbs. Results: We found and reviewed 48 pediatric 4N patients ( < 18 years old) treated 1985-2021 at our center. Biological features included segmental chromosomal aberrations in 21/21 MYCN-non-amplified patients, mutations of ALK in 4/22 (18%), ATRX in 4/16 (25%), and TERT in 4/12 (33%) patients. Among 33 MYCN-non-amplified high-risk patients ( > 18 months old), 19 are relapse-free 1.6+-to-36.9+ (median 12.2+) years post-diagnosis, including 13 without prior MAT and 3 with no mAb, while 14 patients (7 without prior immunotherapy) relapsed (all in soft tissue initially). Eleven of the 14 died, including 3 who developed late osteomedullary metastases after 3-5 relapses in soft tissue alone. Of 13 MYCN-amplified 4N patients, 6 are relapse-free 4.5+-to-25.8+ (median 11.7+) post-diagnosis (all received mAbs; 3 underwent MAT) and 3 are in 2nd remission 4.4+-to-21.1+ years post-relapse (all soft tissue). For all high-risk 4N patients, 5/10-year EFS was 55%/52%, and OS was 76%/64%. MAT was not prognostic. The 2 patients with intermediate-risk 4N (14 months old) are relapse-free 7+ years post-resection of primary tumors; distant disease spontaneously regressed. Conclusions: The natural history of 4N is marked by NB confined to soft tissue without early relapse in bones or bone marrow, sites where mAbs have proven efficacy. These findings plus the possibility of survival without MAT and/or mAb immunotherapy, as seen elsewhere and at our center, support consideration of treatment reduction for selected MYCN-non-amplified 4N patients.

Multiple Genes with Potential Tumor Suppressive Activity Are Present on Chromosome 10q Loss in Neuroblastoma and Are Associated with Poor Prognosis

Neuroblastoma (NB) is a tumor affecting the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. Despite recent advances in understanding the complexity of NB, the mechanisms determining its progression are still largely unknown. Some recurrent segmental chromosome aberrations (SCA) have been associated with poor survival. However, the prognostic role of most SCA has not yet been investigated. We examined a cohort of 260 NB primary tumors at disease onset for the loss of chromosome 10q, by array-comparative genomic hybridization (a-CGH) and Single Nucleotide Polymorphism (SNP) array and we found that 26 showed 10q loss, while the others 234 displayed different SCA. We observed a lower event-free survival for NB patients displaying 10q loss compared to patients with tumors carrying other SCA. Furthermore, analyzing the region of 10q loss, we identified a cluster of 75 deleted genes associated with poorer outcome. Low expression of six of these genes, above all CCSER2, was significantly correlated to worse survival using in silico data from 786 NB patients. These potential tumor suppressor genes can be partly responsible for the poor prognosis of NB patients with 10q loss.

Segmental chromosomal aberrations as the poor prognostic factor in children over 18 months with stage 3 neuroblastoma without MYCN amplification.

Patients with stage 3 neuroblastoma (NBL) according to International Neuroblastoma Staging System (INSS) without MYCN amplification represent a heterogenous group with respect to disease presentation and prognosis. Retrospective analysis of 40 stage 3 patients with NBL without MYCN amplification was performed. The prognostic value of age at diagnosis (under 18 vs over 18 months), International Neuroblastoma Pathology Classification (INPC) diagnostic category and presence of segmental or numerical chromosomes aberrations were evaluated, as well as biochemical markers. Array comparative genomic hybridization (aCGH) for analyzing copy number variations and Sanger sequencing for ALK point mutations were done. In 12 patients (two patients under 18 months), segmental chromosomal aberrations (SCA) were found and numerical chromosomal aberrations (NCA) were found in 16 patients (14 patients under 18 months). In children over 18 months SCA were more common (p=0.0001). Unfavorable pathology was significantly correlated with SCA genomic profile (p=0.04) and age over 18 months (p=0.008). No therapy failures occurred in children with NCA profile over or under 18 months or in children under 18 months, irrespective of pathology and CGH results. Three treatment failures occurred in the SCA group, in one patient CGH profile was not available. For the whole group at 3, 5 and 10-year OS and DFS were 0.95 (95% CI 0.81-0.99), 0.91 (95% CI 0.77-0.97) and 0.91 (95% CI 0.77-0.97), and 0.95 (95% CI 0.90-0.99), 0.92 (95% CI 0.85-0.98) and 0.86 (95% CI 0.78-0.97), respectively. DFS was significantly lower in the SCA group than in the NCA group (3-years, 5-years, and 10-years DFS 0.92 (95% CI 0.53-0.95), 0.80 (95% CI 0.40-0.95) and 0.60 (95% CI 0.16-0.87) vs 1.0, 1.0 and 1.0, respectively, p=0.005). The risk of treatment failure was higher in patients with SCA profile, but only in patients over 18 months. All relapses occurred in children having obtained the complete remission, with no previous radiotherapy. In patients over 18 months, SCA profile should be taken into consideration for therapy stratification as it increases the risk of relapse and this group may require more intensive treatment.

Impact of Genomic and Clinical Factors on Outcome of Children ≥18 Months of Age with Stage 3 Neuroblastoma with Unfavorable Histology and without MYCN Amplification: A Children's Oncology Group (COG) Report.

Patients ≥18 months of age with International Neuroblastoma Staging System (INSS) stage 3 unfavorable histology (UH), MYCN-nonamplified (MYCN-NA) tumors have favorable survival rates compared with other high-risk neuroblastoma populations. The impact of select clinical and biological factors on overall survival (OS) and event-free survival (EFS) were evaluated. Patients enrolled on Children's Oncology Group (COG) A3973 (n = 34), ANBL0532 (n = 27), and/or biology protocol ANBL00B1 (n = 72) were analyzed. Tumors with available DNA (n = 65) and RNA (n = 42) were subjected to whole-exome sequencing (WES) and RNA sequencing. WES analyses and gene expression profiling were evaluated for their impact on survival. Multivariate analyses of EFS/OS using significant factors from univariate analyses were performed. 5-year EFS/OS for patients treated with high-risk therapy on A3973 and ANBL0532 were 73.0% ± 8.1%/87.9% ± 5.9% and 61.4% ± 10.2%/73.0% ± 9.2%, respectively (P = 0.1286 and P = 0.2180). In the A3973/ANBL0532 cohort, patients with less than partial response (PR; n = 5) at end-induction had poor outcomes (5-year EFS/OS: 0%/20.0% ± 17.9%. Univariate analyses of WES data revealed that subjects whose tumors had chromosome 1p or 11q loss/LOH and chromosome 5 or 9 segmental chromosomal aberrations had inferior EFS compared with those with tumors without these aberrations. Multivariate analysis revealed that 11q loss/LOH was an independent predictor of inferior OS [HR, 3.116 (95% confidence interval, 1.034-9.389), P = 0.0435]. Patients ≥18 months of age at diagnosis who had tumors with UH and MYCN-NA INSS stage 3 neuroblastoma assigned to high-risk therapy had an 81.6% ± 5.3% 5-year OS. Less than PR to induction therapy and chromosome 11q loss/LOH are independent predictors of inferior outcome and identify patients who should be eligible for future high-risk clinical trials.

Loss of ALK hotspot mutations in relapsed neuroblastoma.

ALK is the most commonly mutated oncogene in neuroblastoma with increased mutation frequency reported at relapse. Here we report the loss of an ALK mutation in two patients at relapse and a paired neuroblastoma cell line at relapse. ALK detection methods including Sanger sequencing, targeted next-generation sequencing and a new ALK Agena MassARRAY technique were used to detect common hotspot ALK variants in tumors at diagnosis and relapse from two high-risk neuroblastoma patients. Copy number analysis including single nucleotide polymorphism array and array comparative genomic hybridization confirmed adequate tumor cell content in DNA used for mutation testing. Case 1 presented with an ALK F1174L mutation at diagnosis with a variant allele frequency (VAF) ranging between 23.5% and 28.5%, but the mutation was undetectable at relapse. Case 2 presented with an ALK R1257Q mutation at diagnosis (VAF=39%-47.4%) which decreased to <0.01% at relapse. Segmental chromosomal aberrations were maintained between diagnosis and relapse confirming sufficient tumor cell content for mutation detection. The diagnostic SKNBE1n cell line harbors an ALK F1174S mutation, which was lost in the relapsed SKNBE2c cell line. To our knowledge, these are the first reported cases of loss of ALK mutations at relapse in neuroblastoma in the absence of ALK inhibitor therapy, reflecting intra-tumoral spatial and temporal heterogeneity. As ALK inhibitors are increasingly used in the treatment of refractory/relapsed neuroblastoma, our study highlights the importance of confirming whether an ALK mutation detected at diagnosis is still present in clones leading to relapse.

P-564 Advanced paternal age is associated with higher rate of segmental chromosomal aberrations in preimplantation embryos

Abstract Study question Is there a correlation between paternal age and the frequency of chromosomal aberrations in embryos created by in vitro fertilization (IVF)? Summary answer There is an association between paternal age and segmental aberration rates in embryos derived from young oocyte providers after adjusting for age,sperm,and IVF-cycle characteristics. What is known already Paternal reproductive aging(APA) is less understood than maternal reproductive aging(AMA). Embryo aneuploidy presents one of the most important determinants of transfer cycle success. The association of AMA with increased rate of aneuploidy is well established. The systematic review and meta-analysis published by our group did not identify an association of APA with aneuploidy, based on ∼10,000 pooled embryos, however, the power to detect subchromosomal aberrations was limited by the sensitivity of the PGT-A platform used by each center. Our aim was to study the impact of APA on chromosomal aberrations in a large sample size analyzed by high resolution PGT-A. Study design, size, duration This was a retrospective cohort study performed at the CReATe Fertility Centre Genetics Laboratory, Toronto, Canada, between January 2019-December 2021. A total of 17731 blastocysts from 3506 cycles (2502 patients) were analyzed using high resolution NGS PGT-A (10Mb resolution; 30%-70% reported as mosaicism). Clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: ≤33years and ≥34years. Each group was divided into four sub-groups by paternal age: Group A 20-29(n = 484); GroupB 30-39(n = 9704); GroupC 40-49(n = 6341); GroupD≥50(n = 1202). Multiple logistic/ordinal regressions were conducted to evaluate euploidy, mosaicism and aneuploidy rates between age groups, as appropriate. Each was further sub-analyzed by type of abnormality A-single-whole-chromosome, S-segmental, C-complex (≥2 full-chr-aberrations). Segmental aberrations were divided into subgroups of S + (segmental gain), S-(segmental loss) and CS(complex segmental). Main results and the role of chance High resolution NGS PGT-A analysis of 17731 embryos showed 54.9% were euploid, 31.5% were aneuploid and 13.6% mosaic. In the oocyte provider’s age of ≤ 33 group, significant differences between paternal age group A vs. B, C and D were found in euploidy rates (68.8%, 62.5%, 61.7%, 59.0% respectively; p = 0.00559). There were no significant differences in aneuploidy rates (19.1%, 22.7%, 22.5%, 24.6% respectively; p = 0.07184) or mosaicism rates (12.1%, 14.8%, 15.8%, 16.3%; respectively, p = 0.23754). In the oocyte provider’s age of ≥ 34 group, decreasing euploidy rates and increasing aneuploidy rates were observed between all paternal age groups (p = 0.00001 and p &amp;lt; 0.00001,respectively), and no significant differences in mosaicism rates (14%, 12%, 10.2%, 11.6% respectively) reflecting the maternal age effect on euploidy/aneuploidy. In the oocyte provider’s age of ≤ 33 group, paternal group A (2.3%) had significantly lower segmental (S) rates compared to groups B (4.3%, p = 0.027599), C (3.3%, p = 0.039924) and D (4.6%, p = 0.018478). In addition, segmental losses (S-) were lower in group A (1.3%) compared to groups B (3.4%, p = 0.020431), C (3.1%, p = 0.043166) and D (3.7%, p = 0.016385). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The results of this study provide important information to assist fertility practitioners when providing pre-conception counseling to patients, particularly for couples in which the male partner belongs to an older age group. Trial registration number na

Abstract 3755: DNA methylation patterns, but not genetic sequence alterations, are concordant across multiply sampled sites within individuals with unilateral and bilateral Wilms tumors

Abstract Wilms tumor, the most common renal malignancy of childhood, is a disease marked by heterogeneity. One tumor can harbor multiple histologic subtypes and multifocal or bilateral disease is common occurring in up to 10% of patients. Several molecular features have been associated with Wilms tumors and some are used for risk stratification. However, it is unclear which, if any, features are consistent throughout a single tumor or shared between tumors in cases of bilateral disease. To answer this question, we generated genomic and epigenomic data from multiply sampled Wilms tumors from 10 patients - 3 with unilateral disease and 7 with bilateral disease. Multiple samples from each tumor as well as matched normal tissues were included when available. All tumors and matched normal tissues had DNA methylation array data available. Segmental chromosomal aberrations (SCAs) were inferred from methylation array data. Sequencing data were available for all bilateral patients but not for unilateral patients. We found that SCAs were discordant both between pairs of bilateral tumors and within multiply sampled unilateral tumors. This discordance included SCAs used for clinical risk stratification (chromosome 1q gain, 1p loss). Sequence variants were also rarely shared between pairs of bilateral tumors. On the other hand, DNA methylation patterns were concordant across all samples for each individual. We previously described 3 distinct DNA methylation patterns in Wilms tumors and found that all samples from each patient classified into the same subgroup. Furthermore, DNA methylation at Wilms tumor-specific and subgroup-specific differentially methylated regions had high (&amp;gt;0.8) correlation in all samples from all cases. Our data demonstrate that, in contrast to genomic changes such as SCAs and sequence alterations, DNA methylation patterns in Wilms tumors are frequently shared between paired tumors in bilateral disease and in multiple regions within individual tumors. This is consistent with earlier studies demonstrating that gain of methylation at the H19 imprinted region is often the earliest event in Wilms tumors and in some cases likely occurs in the embryonic intermediate mesoderm prior to the development of individual kidneys. Our findings demonstrate the utility of Wilms tumor DNA methylation patterns in overcoming tumor heterogeneity for risk stratification in cases of bilateral or multifocal Wilms tumors. Citation Format: Jack Brzezinski, Sanaa Choufani, Cheryl Shuman, Rodrigo Romao, Armando Lorenzo, Rosanna Weksberg. DNA methylation patterns, but not genetic sequence alterations, are concordant across multiply sampled sites within individuals with unilateral and bilateral Wilms tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3755.

Retrospective Analysis of INRG Clinical and Genomic Factors for 605 Neuroblastomas in Japan: A Report from the Japan Children's Cancer Group Neuroblastoma Committee (JCCG-JNBSG).

Neuroblastomas (NBs) exhibit broad and divergent clinical behaviors and tumor risk classification at diagnosis is crucial for the selection of an appropriate therapeutic strategy for each patient. The present study aimed to validate the clinical relevance of International Neuroblastoma Risk Group (INRG) prognostic and genomic markers in a Japanese NB cohort using a retrospective analysis. Follow-up data based on 30 common INRG queries in 605 NB cases diagnosed in Japan between 1990 and 2014 were collected and the genome signature of each tumor sample was integrated. As previously indicated, age, tumor stage, MYCN, DNA ploidy, the adrenals as the primary tumor site, serum ferritin and lactate dehydrogenase (LDH) levels, segmental chromosome aberrations, and the number of chromosome breakpoints (BP) correlated with lower survival rates, while the thorax as the primary tumor site and numerical chromosome aberrations correlated with a favorable prognosis. In the patient group with stage 4, MYCN non-amplified tumors (n = 225), one of the challenging subsets for risk stratification, age ≥ 18 months, LDH ≥ 1400 U/L, and BP ≥ 7 correlated with lower overall and event-free survival rates (p < 0.05). The genome subgroup GG-P2s (partial chromosome gain/loss type with 1p/11q losses and 17q gain, n = 30) was strongly associated with a lower overall survival rate (5-year survival rate: 34%, p < 0.05). Therefore, the combination of the tumor genomic pattern (GG-P2s and BP ≥ 7) with age at diagnosis and LDH will be a promising predictor for MYCN-non-amplified high-risk NBs in patient subsets, in accordance with previous findings from the INRG project.

Comparison of Next-Generation Sequencing and Fluorescence In Situ Hybridization for Detection of Segmental Chromosomal Aberrations in Neuroblastoma.

The aim of this study was to compare next-generation sequencing (NGS) with the traditional fluorescence in situ hybridization (FISH) for detecting segmental chromosomal aberrations (SCAs) such as 1p deletion, 11q deletion and 17q gain, which are well-known predictive markers for adverse outcome in neuroblastoma. The tumor tissue obtained from 35 patients with neuroblastoma was tested by FISH and targeted NGS, which is specially designed to detect copy number alterations across the entire chromosomal region in addition to mutations in 353 cancer-related genes. All chromosomal copy number alterations were analyzed using the copy number variation plot derived from targeted NGS. FISH was performed to detect 1p deletion, 11q deletion and 17q gain. The copy numbers of 1p, 11q, and 17q obtained via NGS were correlated with those acquired via FISH. The SCAs determined by NGS were matched with those by FISH. Most 17q gain of mismatched cases detected by NGS alone showed a subsegmental gain of 17q. FISH revealed 11q deletion and 17q gain in a few tumor cells of two cases, which were not detected by NGS. NGS can be a sensitive complementary and alternative method to the conventional FISH for detecting SCAs.

Revised Neuroblastoma Risk Classification System: A Report From the Children's Oncology Group.

Treatment planning for children with neuroblastoma requires accurate assessment of prognosis. The most recent Children's Oncology Group (COG) risk classification system used tumor stage as defined by the International Neuroblastoma Staging System. Here, we validate a revised classifier using the International Neuroblastoma Risk Group Staging System (INRGSS) and incorporate segmental chromosome aberrations (SCA) as an additional genomic biomarker. Newly diagnosed patients enrolled on the COG neuroblastoma biology study ANBL00B1 between 2007 and 2017 with known age, International Neuroblastoma Staging System, and INRGSS stage were identified (N = 4,832). Tumor MYCN status, ploidy, SCA status (1p and 11q), and International Neuroblastoma Pathology Classification histology were determined centrally. Survival analyses were performed for combinations of prognostic factors used in COG risk classification according to the prior version 1, and to validate a revised algorithm (version 2). Most patients with locoregional tumors had excellent outcomes except for those with image-defined risk factors (INRGSS L2) with MYCN amplification (5-year event-free survival and overall survival: 76.3% ± 5.8% and 79.9% ± 5.5%, respectively) or patients age ≥ 18 months with L2 MYCN nonamplified tumors with unfavorable International Neuroblastoma Pathology Classification histology (72.7% ± 5.4% and 82.4% ± 4.6%), which includes the majority of L2 patients with SCA. For patients with stage M (metastatic) and MS (metastatic, special) disease, genomic biomarkers affected risk group assignment for those < 12 months (MYCN) or 12-18 months (MYCN, histology, ploidy, and SCA) of age. In a retrospective analysis of patient outcome, the 5-year event-free survival and overall survival using COG version 1 were low-risk: 89.4% ± 1.1% and 97.9% ± 0.5%; intermediate-risk: 86.1% ± 1.3% and 94.9% ± 0.8%; high-risk: 50.8% ± 1.4% and 61.9% ± 1.3%; and using COG version 2 were low-risk: 90.7% ± 1.1% and 97.9% ± 0.5%; intermediate-risk: 85.1% ± 1.4% and 95.8% ± 0.8%; high-risk: 51.2% ± 1.4% and 62.5% ± 1.3%, respectively. A revised 2021 COG neuroblastoma risk classifier (version 2) that uses the INRGSS and incorporates SCAs has been adopted to prospectively define COG clinical trial eligibility and treatment assignment.

Neuroblastoma Molecular Risk-Stratification of DNA Copy Number and ALK Genotyping via Cell-Free Circulating Tumor DNA Profiling.

Simple SummaryNeuroblastoma is composed of several molecular subtypes that are associated with distinct outcomes. Risk classification into these subtypes is routinely assessed at diagnosis and is based on the evaluation of well-defined chromosomal changes found in the tumor DNA which requires a tissue biopsy. However, current clinical practice limits the amount of tumor tissue available for molecular diagnostics. Tumors often shed cells and subcellular components into body fluids. Liquid biopsy is an emerging method to detect such cancer biomolecules in the bloodstream and holds great promise for personalized medicine. Here, we present the use of liquid biopsy to provide molecular classification for neuroblastoma that is independent to tissue biopsy and can potentially be used as a companion diagnostic test to reduce tissue requirements. We will also discuss the pre-analytical considerations for the successful application of this approach in the clinical setting.Background: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation. Methods: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR. Results: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6–8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment. Conclusions: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.

Long-Term Outcome and Role of Biology within Risk-Adapted Treatment Strategies: The Austrian Neuroblastoma Trial A-NB94.

Simple SummaryNeuroblastoma, the most common extracranial malignancy of childhood, shows a highly variable course of disease ranging from spontaneous regression or maturation into a benign tumor to an aggressive and intractable cancer in up to 60% of patients. To adapt treatment intensity, risk staging at diagnosis is of utmost importance. The A-NB94 trial was the first in Austria to stratify therapy intensity according to tumor staging, patient’s age, and MYCN amplification status, the latter being a biologic marker turning otherwise low-risk tumors into high-risk disease. Recent publications showed a prognostic impact of various genomic features including segmental chromosomal aberrations (SCAs). We retrospectively investigated the relevance of SCAs within this risk-adapted treatment strategy. The A-NB94 approach resulted in an excellent long-term survival for the majority of patients with acceptable long-term morbidity. An age- and stage-dependent frequency of SCAs was confirmed and SCAs should always be considered in future treatment decision making processes.We evaluated long-term outcome and genomic profiles in the Austrian Neuroblastoma Trial A-NB94 which applied a risk-adapted strategy of treatment (RAST) using stage, age and MYCN amplification (MNA) status for stratification. RAST ranged from surgery only to intensity-adjusted chemotherapy, single or multiple courses of high-dose chemotherapy (HDT) followed by autologous stem cell rescue depending on response to induction chemotherapy, and irradiation to the primary tumor site. Segmental chromosomal alterations (SCAs) were investigated retrospectively using multi- and pan-genomic techniques. The A-NB94 trial enrolled 163 patients. Patients with localized disease had an excellent ten-year (10y) event free survival (EFS) and overall survival (OS) of 99 ± 1% and 93 ± 2% whilst it was 80 ± 13% and 90 ± 9% for infants with stage 4S and for infants with stage 4 non-MNA disease both 83 ± 15%. Stage 4 patients either >12 months or ≤12 months but with MNA had a 10y-EFS and OS of 45 ± 8% and 47 ± 8%, respectively. SCAs were present in increasing frequencies according to stage and age: in 29% of localized tumors but in 92% of stage 4 tumors (p < 0.001), and in 39% of patients ≤ 12 months but in 63% of patients > 12 months (p < 0.001). RAST successfully reduced chemotherapy exposure in low- and intermediate-risk patients with excellent long-term results while the outcome of high-risk disease met contemporary trials.

Typical numerical alterations in genome identified by array CGH analysis in neuroblastoma tumors

&lt;abstract&gt;&lt;sec&gt; &lt;title&gt;Introduction&lt;/title&gt; &lt;p&gt;The clinical variability in the course of neuroblastoma (NB) is closely linked to diverse genetic changes acquired by tumor cells. Rapid NB progression is associated with oncogene MYCN amplification (MNA) and segmental chromosomal aberrations (SCA). Alternatively, numerical chromosomal alterations (NCA) have positive impact on treatment. So far, no studies have been undertaken to identify NCA that may group NB patients. Therefore, the aim of the study was to identify NCA typical for NB.&lt;/p&gt; &lt;/sec&gt;&lt;sec&gt; &lt;title&gt;Materials and methods&lt;/title&gt; &lt;p&gt;Copy number alterations in NB tumor genome (fresh samples N = 94; formalin-fixed paraffin-embedded specimens N = 66) were analyzed with a pangenomic array CGH technique.&lt;/p&gt; &lt;/sec&gt;&lt;sec&gt; &lt;title&gt;Results&lt;/title&gt; &lt;p&gt;The profile with NCA was observed in 72 (45%) cases, NCA+SCA in 37 (23%), normal in 35 (22%) and MNA in 16 (10%). Samples with NCA were characterized by whole chromosome gains: 17, 7, 6 (78%, 65%, 51%, respectively) and copy loss of chromosome 14 (57%). Similarly to NCA, patients with a combined NCA and SCA profile were also characterized by gain of whole chromosome 17 and 7 (35% both) and loss of chromosome 14 (38%), but with lower frequency. In the combined NCA and SCA profiles, typical NB changes such as deletion 1p36 (27%) and gain 17q (41%) were observed, as well as deletion 11q (24%). The same alterations were detected in MNA samples (44%, 44%, 19%, respectively). A difference was found in spanning 11q deletion between MNA and NCA+SCA subgroup, which may suggest new prognostic markers in NB. In MNA subgroup specific NCA was not indicated.&lt;/p&gt; &lt;/sec&gt;&lt;sec&gt; &lt;title&gt;Conclusions&lt;/title&gt; &lt;p&gt;The hypothesis that NCA in NB tumors are more frequent in younger children with good prognosis was confirmed. To gain new insights into the pathogenesis of NB and to establish molecular targets for diagnosis and therapy, candidate genes in the altered chromosomal regions must be investigated.&lt;/p&gt; &lt;/sec&gt;&lt;/abstract&gt;

Imbalance between genomic gain and loss identifies high-risk neuroblastoma patients with worse outcomes

Survival in high-risk neuroblastoma (HR-NB) patients remains poor despite multimodal treatment. We aimed to identify HR-NB patients with worse outcomes by analyzing the genomic instability derived from segmental chromosomal aberrations. We calculated 3 genomic instability indexes for primary tumor SNP array profiles from 127 HR-NB patients: (1) Copy number aberration burden (%gainslength+%losseslength), (2) copy number load (CNL) (%gainslength-%losseslength) and (3) net genomic load (NGL) (%gainsamount-%lossesamount). Tumors were classified according to positive or negative CNL and NGL genomic subtypes. The impact of the genomic instability indexes on overall survival (OS) was assessed with Cox regression. We identified 38% of HR-NB patients with poor 5-year OS. A negative CNL genomic background was related to poor prognosis in patients ≥18 months showing tumors with homogeneous MYCN amplification (9.5% survival probability, P < 0.05) and patients with non-MYCN amplified NB (18.8% survival probability related to >2.4% CNL, P < 0.01). A positive CNL genomic background was associated with worse outcome in patients with heterogeneous MYCN amplification (22.5% survival probability, P < 0.05). We conclude that characterizing a tumor genomic background according to predominance of genome gained or lost contributes toward improved outcome prediction and brings greater insight into the tumor biology of HR-NB patients.

Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line

BackgroundIncreased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible.MethodsWe applied high density SNPa and NGS techniques to in vivo and in vitro models (orthotropic xenograft vitronectin knock-out mice and 3D bioprinted hydrogels with different stiffness) using two representative neuroblastoma cell lines (the MYCN-amplified SK-N-BE(2) and the ALK-mutated SH-SY5Y), to discern how tumor genomics patterns and clonal heterogeneity of the two cell lines are affected.ResultsWe describe a remarkable subclonal selection of genomic aberrations in SK-N-BE(2) cells grown in knock-out vitronectin xenograft mice that also emerged when cultured for long times in stiff hydrogels. In particular, we detected an enlarged subclonal cell population with chromosome 9 aberrations in both models. Similar abnormalities were found in human high-risk neuroblastoma with MYCN amplification. The genomics of the SH-SY5Y cell line remained stable when cultured in both models.ConclusionsFocus on heterogeneous intratumor segmental chromosome aberrations and mutations, as a mirror image of tumor microenvironment, is a vital area of future research.

Clinically and biologically relevant subgroups of Wilms tumour defined by genomic and epigenomic analyses

BackgroundAlthough cure rates for Wilms tumours (WT) are high, many patients receive therapy with attendant long-term complications. Our goal was to stratify WT using genome-wide analyses to identify candidate molecular features for patients who would benefit from a reduction in therapy.MethodsWe generated DNA methylation and exome sequencing data on WT–kidney pairs (n = 57) and unpaired tumours (n = 27) collected either at our centre or by the Children’s Oncology Group. Samples were divided into a discovery set (n = 32) and validation set (n = 52).ResultsAnalysis of DNA methylation revealed two subgroups of WT with distinct features. Subgroup A has a similar DNA methylation profile to mature kidney, while Subgroup B has genome-wide dysregulation of DNA methylation. The rate of non-synonymous missense mutations and segmental chromosomal aberrations was higher in Subgroup B tumours, suggesting that this group has genome instability related to its epigenetic state. Subgroup A had a higher proportion of cases of bilateral disease. Tumours with high-risk histology or from patients who relapsed were only found in Subgroup B.ConclusionWe have identified subgroup-specific molecular events that could inform future work supporting more targeted therapeutic approaches and patient stratification. We propose a novel developmental tumour model based on these findings.

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

PURPOSEFor localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome.PATIENTS AND METHODSDiagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children’s Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group.RESULTSPatients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038).CONCLUSIONGenomic aberrations of resectable non–MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.