Actionable Tumor Alterations and Treatment Protocol Enrollment of Pediatric and Young Adult Patients With Refractory Cancers in the National Cancer Institute-Children's Oncology Group Pediatric MATCH Trial.

PURPOSE: The National Cancer Institute-Children's Oncology Group Pediatric MATCH trial aimed to facilitate evaluation of molecular-targeted therapies in biomarker-selected cohorts of childhood and young adult patients with cancer by screening tumors for actionable alterations. PATIENTS AND METHODS: Tumors from patients age 1-21 years with refractory solid tumors, lymphomas, or histiocytic disorders were subjected to cancer gene panel sequencing and limited immunohistochemistry to identify actionable alterations for assignment to phase II treatment arms. The rates of treatment arm assignment and enrollment were compared between clinical and demographic groups. RESULTS: Testing was completed for 94.7% of tumors submitted. Actionable alterations were detected in 31.5% of the first 1,000 tumors screened, with treatment arm assignment and enrollment occurring in 28.4% and 13.1% of patients, respectively. Assignment rates varied by tumor histology and were higher for patients with CNS tumors or enrolled at Pediatric Early Phase Clinical Trials Network sites. A reported history of prior clinical molecular testing was associated with higher assignment and enrollment rates. Actionable alterations in the mitogen-activated protein kinase signaling pathway were most frequent (11.2%). The most common reasons provided for not enrolling on treatment arms were patients receiving other treatment or poor clinical status. CONCLUSION: The Pediatric MATCH trial has proven the feasibility of a nationwide screening Protocol for identification of actionable genetic alterations and assignment of pediatric and young adult patients with refractory cancers to trials of molecularly targeted therapies. These data support the early use of tumor molecular screening for childhood patients with cancer whose tumors have not responded to standard treatments.

Detection of single-copy chromosome 17q gain in human neuroblastomas using real-time quantitative polymerase chain reaction.

Regional genomic alterations resulting from single-copy allelic loss or gain have been well characterized in many human cancers and are often of prognostic relevance. Unbalanced gain of 17q material is common in malignant human neuroblastomas and typically results from unbalanced translocations. Unbalanced 17q gain may be an independent predictor of disease outcome, but technical difficulties with quantifying such gain using fluorescent in situ hybridization gives this method limited clinical applicability. We now describe a duplex genomic DNA-based quantitative polymerase chain reaction assay to determine the presence or absence of unbalanced gain of chromosome 17q in primary neuroblastoma specimens. The technique was first refined and validated in a panel of nine human neuroblastoma-derived cell lines by direct comparison with dual-color fluorescent in situ hybridization. Prospective blinded comparison of quantitative polymerase chain reaction and fluorescence in situ hybridization in 40 human neuroblastoma primary tumor samples showed a sensitivity of 96% and 100% specificity for detecting unbalanced 17q gain when a relative 17q copy number ratio of 1.3 was used to define unbalanced gain. Tumors with ratios >1.3 were highly associated with malignant tumor phenotypic features such as metastatic disease (P <.0001) and tumor MYCN amplification (P =.008). These data suggest that quantitative polymerase chain reaction determination of 17q status is feasible and highly specific in primary tumor samples. Sensitivity may be limited because of the inherent complexity of both the chromosomal rearrangements and heterogeneity of some tumor samples. Taken together, quantitative polymerase chain reaction can be used as a high-throughput screening tool for 17q aberrations, but a subset of samples may also require fluorescence in situ hybridization analysis in an attempt to conclusively determine 17q allelic status.

ID2 expression is not associated with MYCN amplification or expression in human neuroblastomas.

MYCN is a biologically and clinically important oncogene in human neuroblastoma as genomic amplification reliably predicts for aggressive tumor behavior and a poor prognosis. However, the mechanism by which MYCN amplification and overexpression contributes to a highly malignant phenotype remains obscure. ID2 is a dominant inhibitor of the RB1 tumor suppressor gene product and recently was suggested to be a direct transcriptional target of MYCN. Overexpression of Id2 protein has thus been postulated to result in functional inactivation of retinoblastoma in MYCN-amplified neuroblastomas, offering a potential explanation for the undifferentiated and highly proliferative nature of most MYCN-amplified neuroblastomas, as well as the paucity of retinoblastoma pathway mutations observed in clinical samples. We therefore sought to determine the likelihood that ID2 overexpression is associated with MYCN amplification and overexpression in human neuroblastoma. ID2 was not differentially expressed in 39 primary neuroblastoma specimens analyzed by oligonucleotide array-based expression analysis, and there was no correlation with MYCN expression levels. ID2 mRNA and protein expression was highly variable and independent of MYCN amplification status and mRNA expression in 10 human-derived neuroblastoma cell lines. In addition, ID2 mRNA expression was not associated with MYCN gene amplification status (P = 0.15) or MYCN expression (r = 0.22) in 131 separate diagnostic primary neuroblastoma samples analyzed by real-time quantitative RT-PCR. These data suggest that transcriptional regulation of ID2 by the MycN oncoprotein is unlikely to be a seminal molecular event resulting in a highly malignant neuroblastoma phenotype.

No evidence for the presence of an imprinted neuroblastoma suppressor gene within chromosome sub-band 1p36.3.

Deletion of the distal short arm of chromosome 1 occurs in 35% of primary neuroblastomas (NBs). These deletions tend to be large and extend to the telomere, but a common region within sub-band 1p36.3 is consistently lost. Despite intensive investigation, no candidate tumor suppressor gene within this region has been shown to undergo tumor-specific mutation consistent with biallelic inactivation. In addition, initial studies demonstrated preferential loss of the maternally inherited 1p homologue in NBs with 1p loss of heterozygosity (LOH) without MYCN amplification. This has led to the widely accepted hypothesis that a genomically imprinted NB suppressor gene is the target of 1p deletion in this subset. To test this hypothesis we have studied 293 primary NBs for LOH within 1p36.3 and determined the parental origin of the deleted 1p homologue. LOH within 1p36.3 was demonstrated in 55 NBs (19%). Of these, 29 occurred in tumors without MYCN amplification: 13 had deletion of the maternally inherited 1p, whereas 16 had deletion of the paternally inherited 1p (P = 0.58). These data strongly refute a parent-of-origin effect for 1p deletions in NB and exclude the existence of an imprinted NB suppressor locus in this region.