Abstract Background Central nervous system (CNS) tumors are the most common pediatric solid tumors and a major cause of childhood cancer-related mortality. With the advent of molecular profiling, the World Health Organization (WHO) has increasingly incorporated genomic data into its diagnostic framework. However, clinical grade comprehensive genomic profiling data from large cohorts of unselected pediatric CNS patients remain limited. Methods We summarize genomic data from clinically validated DNA/RNA profiling of 574 CNS tumors from 532 pediatric patients at a single institution and assess the clinical utility of these findings. Results Tier 1/2 variants were identified in 94.1% of patients with 90.8% of patients yielding clinically impactful findings. These findings resulted in a diagnosis change in 4.2% of patients and refined the histologic diagnosis in 22%. Prognostically relevant alterations were identified in 10% of patients while potential therapeutic targets were identified in 38%. Additionally, serial tumor testing in 36 patients enabled assessment of tumor evolution and differentiation of recurrent/relapsed tumors from independent primary tumors. Furthermore, 23.9% of patients were suspected to have germline pathogenic/likely pathogenic variants, with 73.2% confirmed through germline testing, representing 11.3% of the cohort. Conclusions Our study provides a large, integrated clinical dataset of genomic alterations in pediatric CNS tumors. Our findings highlight the clinical significance of genomic profiling of pediatric CNS tumors and underscore the necessity of integrating genomic results with pathologic, radiologic, and clinical features to ensure accurate tumor diagnosis and facilitate personalized, risk-adapted patient management.

Advancements in the rapidity, cost efficacy, and sensitivity of next-generation sequencing (NGS) have facilitated molecular risk stratification and precision medicine-based treatment for pediatric leukemia. The benefit of uniform cytomolecular analyses for clinical trial risk assignment is clear. However, the clinical impact of comprehensive NGS for pediatric leukemias at an institutional level is not well described. We report the genomic spectrum of one of the largest cohorts of pediatric and adolescent/young adult (AYA) acute leukemias examined to date (n = 1442) via institutional NGS testing from our large tertiary care center. We evaluated the clinical utility of genomic results for informing prognosis and treatment. We identified high utility of the comprehensive DNA-based mutational panel and RNA-fusion panel, which detected leukemia-associated variants in 99% of specimens. We observed 65% of B-cell acute lymphoblastic leukemia cases and 69% of patients with acute myeloid leukemia harbored a prognostic molecular alteration. In total, 325 of 1134 patients (29%) harbored potentially targetable molecular biomarkers, and 23% of cases with follow-up data received precision medicine-based therapy. Paired diagnostic and relapsed leukemia samples aided in differentiation between treatment-related leukemia versus lineage drift/switch. These findings demonstrate the broad utility of comprehensive molecular sequencing for pediatric/AYA leukemia at an institutional level to improve outcomes.

Abstract Introduction KP1077 is under development as an oral medication for the treatment of rare sleep disorders with Excessive Daytime Sleepiness (EDS), including idiopathic hypersomnia (IH). The active ingredient in KP1077 is serdexmethylphenidate (SDX), a prodrug of d-methylphenidate. The objectives of the study were to assess the safety (primary endpoint) and efficacy of KP1077 in patients with IH. Methods Adult patients with IH began KP1077 treatment in a 5-week open-label (OL) titration period. Possible dose levels were 80, 160, 240 and 320 mg/day SDX. Patients were randomized to receive their daily dose either once per day (just before going to sleep), or twice per day (half the daily dose just before going to sleep and half shortly after awakening). After the titration period, patients in each treatment group were randomized to placebo or continued KP1077 (optimized dose) during a 2-week double-blind (DB) withdrawal period. Assessments of safety were based on adverse events (AEs), physical examinations, clinical laboratory tests, vital signs, electrocardiograms, sleep quality, and suicidal ideation. Efficacy assessments included the Epworth Sleepiness Scale (ESS) and Idiopathic Hypersomnia Severity Scale (IHSS). Patients rated their difficulty of waking up in the morning with the Sleep Inertia Visual Analog Scale (SI-VAS) and brain fog throughout the day with an exploratory Brain Fog Scale (BFS). Results Safety and efficacy in the OL titration phase were evaluated in an interim analysis when 22 patients completed the study (target: ≥48 completers). KP1077 was well-tolerated for both treatments and all dose levels, with most frequent AEs of insomnia, headache, and nausea. Most AEs were mild, not leading to early discontinuation. Meaningful clinical improvements in scores of ESS, IHSS, SI-VAS, and BFS were observed in both treatment groups. Mean total ESS scores decreased by >9 points after 5 weeks of OL treatment. Results from all patients in the both the OL titration and DB withdrawal periods will be presented. Conclusion KP1077 was well-tolerated in patients with IH with AEs typical for a central nervous system stimulant. Meaningful clinical improvements of EDS, sleep inertia and brain fog were observed after 5 weeks of OL KP1077. Support (if any) Zevra Therapeutics

Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for w30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, >= 99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 singlenucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing -based panel testing should be a part of routine diagnostics in patients with suspected IBMFS. (J Mol Diagn 2024, 26: 191-201; https://doi.org/10.1016/j.jmoldx.2023.11.010)

Background TP53 alterations are common in certain pediatric cancers, making identification of putative germline variants through tumor genomic profiling crucial for disease management.Methods We analyzed TP53 alterations in 3123 tumors from 2788 pediatric patients sequenced using tumor-only or tumor-normal paired panels. Germline confirmatory testing was performed when indicated. Somatic and germline variants were classified based on published guidelines.Results In 248 tumors from 222 patients, 284 tier 1/2 TP53 sequence and small copy number variants were detected. Following germline classification, 86.6% of 142 unique variants were pathogenic or likely pathogenic. Confirmatory testing on 118 patients revealed germline TP53 variants in 28 of them (23 pathogenic or likely pathogenic and 5 of uncertain significance), suggesting a minimum Li-Fraumeni syndrome incidence of 0.8% (23/2788) in this cohort, 10.4% (23/222) in patients with TP53 variant-carrying tumors, and 19.5% (23/118) with available normal samples. About 25% (7/28) of patients with germline TP53 variants did not meet Li-Fraumeni syndrome diagnostic or testing criteria, while 20.9% (28/134) with confirmed or inferred somatic origins did. TP53 biallelic inactivation occurred in 75% of germline carrier tumors and was also prevalent in other groups, causing an elevated tumor-observed variant allelic fraction. Somatic evidence, however, including low variant allele fraction correctly identified only 27.8% (25/90) of patients with confirmed somatic TP53 variants.Conclusion The high incidence and variable phenotype of Li-Fraumeni syndrome in this cohort highlights the importance of assessing germline status of TP53 variants identified in all pediatric tumors. Without clear somatic evidence, distinguishing somatic from germline origins is challenging. Classifying germline and somatic variants should follow appropriate guidelines.

Introduction: There is growing recognition of germline cancer predisposition syndromes (CPS) that increase risk of hematologic malignancies including leukemias, lymphomas, bone marrow failure syndromes and myeloproliferative diseases. Somatic (tumor) testing can identify potential germline variants based on multiple factors including variant allele fraction, sequence characteristics, and other findings; however, subsequent germline testing is required to characterize the lesion. Paired tumor and germline testing (T/N testing) is more accurate to diagnose CPS and may mitigate loss to follow-up, reduce time to CPS diagnosis, and possibly alleviate unnecessary anxiety due to suspected but ultimately disproved germline conditions. Previously, our center performed tumor-only diagnostic testing for patients with newly diagnosed hematologic malignancies. In 2020 we transitioned to paired T/N testing for all new hematologic malignancy diagnoses with patient/caregiver consent. Here we report the frequency of CPS diagnoses suggested by tumor-only testing and subsequently confirmed with germline testing, as well as CPS diagnoses identified from the outset by T/N testing. Methods: Data was automatically abstracted from the Children's Hospital of Philadelphia (CHOP) Diagnostic Genomics Database (FileMaker and Nexus) for 1057 pediatric, adolescent, and young adult patients who underwent testing with the CHOP Comprehensive Hematologic Malignancy panel between 2016 and 2023. This panel interrogates 117 genes for single nucleotide variants, insertions, deletions, and copy number variants. Of those tested, 932 patients underwent tumor-only testing, and 125 patients underwent paired T/N testing. Characteristics of patients with possible pre-established genetic syndromes (e.g. Trisomy 21) were cross-checked via manual chart review. Results: Schematic representation of patient testing is summarized in Figure 1. Among 932 patients who underwent tumor-only testing, 98 were identified to have potential or previously established germline pathogenic variants. Of these, 9 had known genetic syndromes and 8 had non-cancer related genetic diagnoses (e.g. Usher syndrome). Of the 81 patients with a suspected CPS, 26 were referred from an outside hospital with unavailable follow-up data. Among suspected germline patients from our center (n=55), 34 (62%) did not undergo confirmatory testing. Of the 21 (38%) who underwent confirmatory testing, 2 (10%) were diagnosed with a CPS. For patients with T/N testing, 16 of 125 (7%) were identified to have germline conditions; 8 were pre-established diagnoses and 4 were non-cancer related genetic diagnoses. Thus, 4 (3%) of patients with T/N testing were newly diagnosed with a CPS. New CPS diagnoses included: ETV6 thrombocytopenia and leukemia predisposition syndrome, CBL syndrome, GATA2 MDS/AML predisposition syndrome, Noonan syndrome, and CEBPA AML predisposition syndrome. Conclusion: At our center, 62% of patients with suspected germline conditions on tumor-only testing had no follow up testing documented in the available medical record. These data suggest that paired T/N testing at the time of hematologic malignancy diagnosis assists in mitigating loss-to-follow up for patients with possible CPS. Additionally, T/N testing may streamline result interpretation, reduce time to diagnosis, aid in treatment decisions, and guide cascade testing for potentially affected family members. When possible, genetic testing decisions and results should be discussed with a trained genetic counselor. Over time, increasing utilization of paired T/N testing may also enhance understanding of CPS-associated genes that increase risk of hematologic malignancies.

Both somatic and germline alterations in TP53 are common in cancers. Identification of potential germline variants through tumor sequencing is important for patient care. We investigated TP53 sequence and copy number variants in 2,810 tumors from 2,197 pediatric patients by targeted NGS panels with germline confirmation when indicated. Somatic and germline variant classification was performed using published guidelines. In total, 228 TP53 sequence variants were detected in 199 tumors from 173 individuals. Seventy-seven percent of 199 tumors harbored two TP53 alterations indicating biallelic loss of TP53. Germline testing performed on 79 patients revealed that 24 carried a germline TP53 variant, three of whom had a second germline variant in another cancer gene too. For the remaining 55 patients, four had only a germline alteration in a different cancer gene. No significant difference was found regarding variant allele fraction, tumor type, or presence of additional TP53 somatic changes between the confirmed germline and somatic variants. Of the 115 unique Tier 1/2 TP53 variants, 78 are missense, and 86 would be classified as P/LP and 29 as VUS if germline. Although most VUS are located at somatic mutation hotspots, twelve have never been reported as germline. Our study demonstrates the necessity of routinely assessing germline status of TP53 variants found in pediatric cancers. Variant classification should be performed using appropriate somatic or germline criteria as a variant may be significant somatically, yet of uncertain germline significance. While somatic data may support germline classification, germline variants cannot be interpreted based on somatic information.

Bone marrow failure syndromes (BMFS) are a group of heterogeneous disorders often associated with germline changes in pediatric patients, known as inherited BMFS (IBMFS). We developed an NGS panel interrogating 159 genes associated with IBMFS, which evaluates sequence and copy number variations. Testing can be ordered as a comprehensive panel or several sub-panels with reflex to a larger panel. A total of 236 patients were tested and a definitive or possible molecular diagnosis was found in 39 cases (16.5%) including 2 patients carrying two potential genetic etiologies. Inconclusive findings were identified in 50 cases (21.1%) due to partial phenotypic overlap or limited variant evidence. The reported variants in 89 cases involve 44 genes with pathogenic variants most commonly seen in ELANE, followed by GATA2. The remaining 146 cases (61.9%) were negative with no variants, or only one variant in a recessive disorder gene. Our study affirms that targeted panels can facilitate the diagnosis of IBMFS; however, challenges remain. Most samples tested were blood, which may complicate variant interpretation if a latent/active myelodysplasia or leukemia is present, as was observed in two of these cases. Another challenge is secondary findings, such as a heterozygous pathogenic BRCA1 variant detected in a child with aplastic anemia. These challenges may be partially resolved by collecting additional familial and phenotypic information to help distinguishing acquired from inherited BMFS, obtaining appropriate tissue for testing, and refining the panel gene list or expanding to exome/genome sequencing to capture variants that may be missed by targeted analysis.

BackgroundSomatic genetic testing is rapidly becoming the standard of care in many adult and pediatric cancers. Previously, the standard approach was single-gene or focused multigene testing, but many centers have moved towards broad-based next-generation sequencing (NGS) panels. Here, we report the laboratory validation and clinical utility of a large cohort of clinical NGS somatic sequencing results in diagnosis, prognosis, and treatment of a wide range of pediatric cancers.MethodsSubjects were accrued retrospectively at a single pediatric quaternary-care hospital. Sequence analyses were performed on 367 pediatric cancer samples using custom-designed NGS panels over a 15-month period. Cases were profiled for mutations, copy number variations, and fusions identified through sequencing, and their clinical impact on diagnosis, prognosis, and therapy was assessed.ResultsNGS panel testing was incorporated meaningfully into clinical care in 88.7% of leukemia/lymphomas, 90.6% of central nervous system (CNS) tumors, and 62.6% of non-CNS solid tumors included in this cohort. A change in diagnosis as a result of testing occurred in 3.3% of cases. Additionally, 19.4% of all patients had variants requiring further evaluation for potential germline alteration.ConclusionsUse of somatic NGS panel testing resulted in a significant impact on clinical care, including diagnosis, prognosis, and treatment planning in 78.7% of pediatric patients tested in our institution. Somatic NGS tumor testing should be implemented as part of the routine diagnostic workup of newly diagnosed and relapsed pediatric cancer patients.

Introduction: Inherited bone marrow failure syndromes (IBMFSs) are a group of heterogeneous disorders characterized by failure to generate enough blood cells in bone marrow. IBMFSs account for ∼30% of pediatric bone marrow failure and are associated with developmental abnormalities and increased cancer risk. We have developed and clinically validated a large NGS-based panel to facilitate the diagnosis of IBMFSs.

PURPOSE The diagnosis of cancer predisposition in pediatric patients with cancer is vital for treatment decisions, surveillance, and management of at-risk family members. Somatic tumor testing can identify potential underlying constitutional variants that confer increased cancer risk. Here, we report the characteristics of constitutional variants identified through tumor testing. MATERIALS AND METHODS Data were abstracted from medical record review of 1,023 patients who received inhouse somatic tumor testing over a 28-month period. Patients were identified for testing using referral criteria developed as a collaboration between genomic diagnostics, pathology, and oncology. Characteristics of patients who underwent constitutional testing, including family history and variant loss of heterozygosity, were tracked. RESULTS From 1,023 patients who underwent somatic tumor sequencing in a 28-month period, 210 variants were identified in 141 patients (13.8%) that were concerning for cancer predisposition syndromes requiring intervention. A total of 73 variants in 41 patients have undergone clinical confirmatory testing thus far. Of these, 26 variants were confirmed to be constitutionally present (35.6%). Among patients tested, 23 (56.1%) of 41 total patients were diagnosed with a cancer predisposition syndrome. CONCLUSION Our data demonstrate that more than one third of variants in tumor somatic sequencing that were concerning for underlying cancer predisposition were constitutionally confirmed. Overall, somatic tumor testing identified potential cancer predisposition syndromes in pediatric patients, and some would not have been identified on the basis of clinical history alone.

Purpose Hereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes. Methods A tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes. Results ES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis. Conclusion The tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Genomic alterations in B-ALL are essential for disease diagnosis, prognosis, and treatment. We performed integrated genomic analysis on 136 pediatric B-ALL patients using the CHOP Comprehensive Hematological Cancer NGS Panel along with conventional cytogenetic studies. The panel interrogates 118 cancer genes for sequence and copy number variants (CNV) and 110 genes for known and novel fusions. Clinically significant genomic changes were identified in 89.7% of patients. Fusion genes or gene rearrangements were detected in 52.9% of the patients, 20% of them were never reported before. ETV6-RUNX1 (27.8%) was the most common fusion, followed by Ph+/Ph-like fusions (26.4%), KMT2A-rearrangements (15.3%), TCF3-PBX1 (8.3%), and TCF3-HLF (2.8%). Many fusions were cryptic and were missed by conventional cytogenetics or even by FISH. One hundred thirty-three mutations were observed in these patients with most of the mutations in genes involving RAS-pathway (31.9%), followed by PTPN11 (8.2%), PAX5 (7.4%), CREBBP (6.7%), TP53 (5.2%), and JAK2 (4.4%). RAS mutations are common in both primary and relapsed cases, while TP53 mutations were only observed in relapsed cases. CNVs were detected in 86.2% of the patients including hyperdiploidy (18.5%), iAMP21 (3.8%), hypodipolidy/near-haploidy (3.1%), and cryptic deletions involving IKZF1, CDKN2A/B, or ETV6 (73.1%). Three patients with normal cytogenetics were found to carry Ph-like ALL-associated alterations by NGS assay. These genomic alterations have significantly impacted patient care, especially in cases of Ph-like ALL. Our comprehensive NGS testing has provided critical evidence for both precision diagnoses and therapeutic decisions.