Preparing for the unexpected: Recommendations for returning secondary findings in late-stage cancer care.

PURPOSE: We conducted qualitative interviews with patients with cancer and providers to identify gaps in clinical care and highlight care delivery solutions for the return of secondary germline findings. METHODS: Twelve patients and 19 cancer providers from the United States were interviewed between January 2019 and May 2021. Interviews elicited feedback about patient information needs, emotional responses to secondary findings, and recommendations for improving pre-test education. RESULTS: Patients' responses ranged from gratitude to regret, depending on how much pre-test counseling they received before tumor testing. Providers cited insufficient clinic time as a major barrier to pretest education, favoring online support tools and standardized pre-test education models. Providers had differing perspectives on how pre-test education should be integrated into clinical workflows but agreed that it should include the differences between somatic and germline testing, the likelihood of medically actionable findings, and the possibility of being referred to a genetics provider. CONCLUSION: The spectrum of participants' responses to their secondary findings underscores the importance of adequate pre-test discussions before somatic sequencing. Although educational interventions could address patients' information needs and augment traditional pre-test counseling, health care systems, labs, and genetic providers may be called on to play greater roles in pre-test education.

FGF12 copy number variant associated with epileptic encephalopathy.

FGF12 related epilepsy presents with variable phenotypes. We report another patient with a duplication involving the FGF12 gene who presented similar to other published cases having normal early development and responded to phenytoin.

Rare De Novo Missense Variants in RNA Helicase DDX6 Cause Intellectual Disability and Dysmorphic Features and Lead to P-Body Defects and RNA Dysregulation.

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.

Utilizing RNA and outlier analysis to identify an intronic splice-altering variant in AP4S1 in a sibling pair with progressive spastic paraplegia.

We report a likely pathogenic splice-altering AP4S1 intronic variant in two sisters with progressive spastic paraplegia, global developmental delay, shy character, and foot deformities. Sequencing was completed on whole-blood messenger RNA (mRNA) and analyzed for gene expression outliers after exome sequencing analysis failed to identify a causative variant. AP4S1 was identified as an outlier and contained a rare homozygous variant located three bases upstream of exon 5 (NC_000014.8(NM_007077.4):c.295-3C>A). Confirmed by additional RNA-seq, reverse-transcription polymerase chain reaction, and Sanger sequencing, this variant corresponded with exon 5, including skipping, altered isoform usage, and loss of expression from the canonical isoform 2 (NM_001128126.3). Previously, loss-of-function variants within AP4S1 were associated with a quadriplegic cerebral palsy-6 phenotype, AP-4 Deficiency Syndrome. In this study, the inclusion of mRNA-seq allowed for the identification of a previously missed splice-altering variant, and thereby expands the mutational spectrum of AP-4 Deficiency Syndrome to include impacts to some tissue-dependent isoforms.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

Compound heterozygous mutations in SNAP29 is associated with Pelizaeus-Merzbacher-like disorder (PMLD).

Pelizaeus-Merzbacher-like disease (PMLD) is an autosomal recessive hypomyelinating leukodystrophy, which is clinically and radiologically similar to X-linked Pelizaeus-Merzbacher disease (PMD). PMLD is characterized by early-onset nystagmus, delayed development (motor delay, speech delay and dysarthria), dystonia, hypotonia typically evolving into spasticity, ataxia, seizures, optic atrophy, and diffuse leukodystrophy on magnetic resonance imaging (MRI). We identified a 12-year-old Caucasian/Hispanic male with the classical clinical characteristics of PMLD with lack of myelination of the subcortical white matter, and absence of the splenium of corpus callosum. Exome sequencing in the trio revealed novel compound heterozygous pathogenic mutations in SNAP29 (p.Leu119AlafsX15, c.354DupG and p.0?, c.2T > C). Quantitative analysis of the patient's blood cells through RNA sequencing identified a significant decrease in SNAP29 mRNA expression, while western blot analysis on fibroblast cells revealed a lack of protein expression compared to parental and control cells. Mutations in SNAP29 have previously been associated with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. Typical skin features described in CEDNIK syndrome, such as generalized ichthyosis and keratoderma, were absent in our patient. Moreover, the early onset nystagmus and leukodystrophy were consistent with a PMLD diagnosis. These findings suggest that loss of SNAP29 function, which was previously associated with CEDNIK syndrome, is also associated with PMLD. Overall, our study expands the genetic spectrum of PMLD.

A Syndromic Intellectual Disability Disorder Caused by Variants in TELO2, a Gene Encoding a Component of the TTT Complex.

The proteins encoded by TELO2, TTI1, and TTI2 interact to form the TTT complex, a co-chaperone for maturation of the phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Here we report six affected individuals from four families with intellectual disability (ID) and neurological and other congenital abnormalities associated with compound heterozygous variants in TELO2. Although their fibroblasts showed reduced steady-state levels of TELO2 and the other components of the TTT complex, PIKK functions were normal in cellular assays. Our results suggest that these TELO2 missense variants result in loss of function, perturb TTT complex stability, and cause an autosomal-recessive syndromic form of ID.

A novel FBXO28 frameshift mutation in a child with developmental delay, dysmorphic features, and intractable epilepsy: A second gene that may contribute to the 1q41-q42 deletion phenotype.

Chromosome 1q41-q42 deletions have recently been associated with a recognizable neurodevelopmental syndrome of early childhood (OMIM 612530). Within this group, a predominant phenotype of developmental delay (DD), intellectual disability (ID), epilepsy, distinct dysmorphology, and brain anomalies on magnetic resonance imaging/computed tomography has emerged. Previous reports of patients with de novo deletions at 1q41-q42 have led to the identification of an evolving smallest region of overlap which has included several potentially causal genes including DISP1, TP53BP2, and FBXO28. In a recent report, a cohort of patients with de novo mutations in WDR26 was described that shared many of the clinical features originally described in the 1q41-q42 microdeletion syndrome (MDS). Here, we describe a novel germline FBXO28 frameshift mutation in a 3-year-old girl with intractable epilepsy, ID, DD, and other features which overlap those of the 1q41-q42 MDS. Through a familial whole-exome sequencing study, we identified a de novo FBXO28 c.972_973delACinsG (p.Arg325GlufsX3) frameshift mutation in the proband. The frameshift and resulting premature nonsense mutation have not been reported in any genomic database. This child does not have a large 1q41-q42 deletion, nor does she harbor a WDR26 mutation. Our case joins a previously reported patient also in whom FBXO28 was affected but WDR26 was not. These findings support the idea that FBXO28 is a monogenic disease gene and contributes to the complex neurodevelopmental phenotype of the 1q41-q42 gene deletion syndrome.

Rare variants in neuronal excitability genes influence risk for bipolar disorder.

We sequenced the genomes of 200 individuals from 41 families multiply affected with bipolar disorder (BD) to identify contributions of rare variants to genetic risk. We initially focused on 3,087 candidate genes with known synaptic functions or prior evidence from genome-wide association studies. BD pedigrees had an increased burden of rare variants in genes encoding neuronal ion channels, including subunits of GABAA receptors and voltage-gated calcium channels. Four uncommon coding and regulatory variants also showed significant association, including a missense variant in GABRA6. Targeted sequencing of 26 of these candidate genes in an additional 3,014 cases and 1,717 controls confirmed rare variant associations in ANK3, CACNA1B, CACNA1C, CACNA1D, CACNG2, CAMK2A, and NGF. Variants in promoters and 5' and 3' UTRs contributed more strongly than coding variants to risk for BD, both in pedigrees and in the case-control cohort. The genes and pathways identified in this study regulate diverse aspects of neuronal excitability. We conclude that rare variants in neuronal excitability genes contribute to risk for BD.

The Pharmacogenomics of Bipolar Disorder study (PGBD): identification of genes for lithium response in a prospective sample.

BACKGROUND: Bipolar disorder is a serious and common psychiatric disorder characterized by manic and depressive mood switches and a relapsing and remitting course. The cornerstone of clinical management is stabilization and prophylaxis using mood-stabilizing medications to reduce both manic and depressive symptoms. Lithium remains the gold standard of treatment with the strongest data for both efficacy and suicide prevention. However, many patients do not respond to this medication, and clinically there is a great need for tools to aid the clinician in selecting the correct treatment. Large genome wide association studies (GWAS) investigating retrospectively the effect of lithium response are in the pipeline; however, few large prospective studies on genetic predictors to of lithium response have yet been conducted. The purpose of this project is to identify genes that are associated with lithium response in a large prospective cohort of bipolar patients and to better understand the mechanism of action of lithium and the variation in the genome that influences clinical response. METHODS/DESIGN: This study is an 11-site prospective non-randomized open trial of lithium designed to ascertain a cohort of 700 subjects with bipolar I disorder who experience protocol-defined relapse prevention as a result of treatment with lithium monotherapy. All patients will be diagnosed using the Diagnostic Interview for Genetic Studies (DIGS) and will then enter a 2-year follow-up period on lithium monotherapy if and when they exhibit a score of 1 (normal, not ill), 2 (minimally ill) or 3 (mildly ill) on the Clinical Global Impressions of Severity Scale for Bipolar Disorder (CGI-S-BP Overall Bipolar Illness) for 4 of the 5 preceding weeks. Lithium will be titrated as clinically appropriate, not to exceed serum levels of 1.2 mEq/L. The sample will be evaluated longitudinally using a wide range of clinical scales, cognitive assessments and laboratory tests. On relapse, patients will be discontinued or crossed-over to treatment with valproic acid (VPA) or treatment as usual (TAU). Relapse is defined as a DSM-IV manic, major depressive or mixed episode or if the treating physician decides a change in medication is clinically necessary. The sample will be genotyped for GWAS. The outcome for lithium response will be analyzed as a time to event, where the event is defined as clinical relapse, using a Cox Proportional Hazards model. Positive single nucleotide polymorphisms (SNPs) from past genetic retrospective studies of lithium response, the Consortium on Lithium Genetics (ConLiGen), will be tested in this prospective study sample; a meta-analysis of these samples will then be performed. Finally, neurons will be derived from pluripotent stem cells from lithium responders and non-responders and tested in vivo for response to lithium by gene expression studies. SNPs in genes identified in these cellular studies will also be tested for association to response. DISCUSSION: Lithium is an extraordinarily important therapeutic drug in the clinical management of patients suffering from bipolar disorder. However, a significant proportion of patients, 30-40 %, fail to respond, and there is currently no method to identify the good lithium responders before initiation of treatment. Converging evidence suggests that genetic factors play a strong role in the variation of response to lithium, but only a few genes have been tested and the samples have largely been retrospective or quite small. The current study will collect an entirely unique sample of 700 patients with bipolar disorder to be stabilized on lithium monotherapy and followed for up to 2 years. This study will produce useful information to improve the understanding of the mechanism of action of lithium and will add to the development of a method to predict individual response to lithium, thereby accelerating recovery and reducing suffering and cost. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01272531 Registered: January 6, 2011.

In vitro-differentiated neural cell cultures progress towards donor-identical brain tissue.

Multiple research groups have observed neuropathological phenotypes and molecular symptoms in vitro using induced pluripotent stem cell (iPSC)-derived neural cell cultures (i.e. patient-specific neurons and glia). However, the global differences/similarities that may exist between in vitro neural cells and their tissue-derived counterparts remain largely unknown. In this study, we compared temporal series of iPSC-derived in vitro neural cell cultures to endogenous brain tissue from the same autopsy donor. Specifically, we utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, and the following three results support this conclusion: (i) there was a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain; (ii) there was an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue; and (iii) there was a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. Taken together, these results are consistent with in vitro neural development and physiological progression occurring predominantly by transcriptional activation of downregulated genes rather than deactivation of upregulated genes.

Genome-wide association of bipolar disorder suggests an enrichment of replicable associations in regions near genes.

Although a highly heritable and disabling disease, bipolar disorder's (BD) genetic variants have been challenging to identify. We present new genotype data for 1,190 cases and 401 controls and perform a genome-wide association study including additional samples for a total of 2,191 cases and 1,434 controls. We do not detect genome-wide significant associations for individual loci; however, across all SNPs, we show an association between the power to detect effects calculated from a previous genome-wide association study and evidence for replication (P = 1.5×10(-7)). To demonstrate that this result is not likely to be a false positive, we analyze replication rates in a large meta-analysis of height and show that, in a large enough study, associations replicate as a function of power, approaching a linear relationship. Within BD, SNPs near exons exhibit a greater probability of replication, supporting an enrichment of reproducible associations near functional regions of genes. These results indicate that there is likely common genetic variation associated with BD near exons (±10 kb) that could be identified in larger studies and, further, provide a framework for assessing the potential for replication when combining results from multiple studies.

Plasma cytokine profiling in sibling pairs discordant for autism spectrum disorder.

OBJECTIVE: Converging lines of evidence point to the existence of immune dysfunction in autism spectrum disorder (ASD), which could directly affect several key neurodevelopmental processes. Previous studies have shown higher cytokine levels in patients with autism compared with matched controls or subjects with other developmental disorders. In the current study, we used plasma-cytokine profiling for 25 discordant sibling pairs to evaluate whether these alterations occur within families with ASD. METHODS: Plasma-cytokine profiling was conducted using an array-based multiplex sandwich ELISA for simultaneous quantitative measurement of 40 unique targets. We also analyzed the correlations between cytokine levels and clinically relevant quantitative traits (Vineland Adaptive Behavior Scale in Autism (VABS) composite score, Social Responsiveness Scale (SRS) total T score, head circumference, and full intelligence quotient (IQ)). In addition, because of the high phenotypic heterogeneity of ASD, we defined four subgroups of subjects (those who were non-verbal, those with gastrointestinal issues, those with regressive autism, and those with a history of allergies), which encompass common and/or recurrent endophenotypes in ASD, and tested the cytokine levels in each group. RESULTS: None of the measured parameters showed significant differences between children with ASD and their related typically developing siblings. However, specific target levels did correlate with quantitative clinical traits, and these were significantly different when the ASD subgroups were analyzed. It is notable that these differences seem to be attributable to a predisposing immunogenetic background, as no other significant differences were noticed between discordant sibling pairs. Interleukin-1ß appears to be the cytokine most involved in quantitative traits and clinical subgroups of ASD. CONCLUSIONS: In the present study, we found a lack of significant differences in plasma-cytokine levels between children with ASD and in their related non-autistic siblings. Thus, our results support the evidence that the immune profiles of children with autism do not differ from their typically developing siblings. However, the significant association of cytokine levels with the quantitative traits and the clinical subgroups analyzed suggests that altered immune responses may affect core feature of ASD.

Underreporting of SMARCB1 alteration by clinical sequencing: Integrative patho-genomic analysis captured SMARCB1/INI-1 deficiency in a vulvar yolk sac tumor.

•SMARCB1/INI1-deficient gynecologic tumors are rare and clinically aggressive. A subset shows primitive yolk sac tumor features.•Due to technical limitation of next generation sequencing (NGS) and interlaboratory variability in sequencing methodologies and analytical pipelines, SMARCB1 deficiency caused by somatic copy number variations (SCNV) may be underreported by NGS.•To improve identification of SMARCB1/INI1-deficient neoplasm, we propose the following strategy: First, careful pathology slide review and detection of rhabdoid cells should raise the possibility of SMARCB1/INI1 deficiency. Second, INI1 IHC is a useful complementary test to exclude clinical suspicion of SMARCB1 deficiency in the context of negative molecular reporting. Third, knowledge of potential underreporting of SMARCB1 mutation would avoid underdiagnosis.

Implementation, Evolution, and Laboratory Performance of Methods-Based Proficiency Testing for Next-Generation Sequencing Detection of Germline Sequence Variants.

CONTEXT.­: Next-generation sequencing (NGS)-based assays are used for diagnosis of diverse inherited disorders. Limited data are available pertaining to interlaboratory analytical performance of these assays. OBJECTIVE.­: To report on the College of American Pathologists (CAP) NGS Germline Program, which is methods based, and explore the evolution in laboratory testing practices. DESIGN.­: Results from the NGS Germline Program from 2016-2020 were analyzed for interlaboratory analytical performance. Self-reported laboratory testing practices were also evaluated. RESULTS.­: From 2016-2020, a total of 297 laboratories participated in at least 1 program mailing. Of the 289 laboratories that provided information on tests offered, 138 (47.8%) offered only panel testing throughout their enrollment, while 35 (12.1%) offered panels and exome testing, 30 (10.4%) offered only exomes, 9 (3.1%) offered only genomes, and 15 (5.2%) offered panels, exomes, and genomes. The remainder (62 laboratories, 21.4%) changed their test offerings during the 2016-2020 timeframe. Considering each genomic position/interval, the median detection percentage at variant positions across the 2016-2020 mailings ranged from 94.3% to 100%, while at reference positions (no variant detected), the median correct response percentage was 100% across all mailings. When considering performance of individual laboratories, 89.5% (136 of 152) to 98.0% (149 of 152) of laboratories successfully met the detection threshold (≥90% of the variants present), while 94.6% (87 of 92) to 100% (163 of 163) of laboratories met the 95% specificity threshold across mailings. CONCLUSIONS.­: Since the inception of this program, laboratories have consistently performed well. The median sensitivity and specificity of detection of sequence variants included in this program (eg, single nucleotide variants, insertions, and deletions) were 100.0%.

Genetic and Protein Network Underlying the Convergence of Rett-Syndrome-like (RTT-L) Phenotype in Neurodevelopmental Disorders.

Mutations of the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2) cause classical forms of Rett syndrome (RTT) in girls. A subset of patients who are recognized to have an overlapping neurological phenotype with RTT but are lacking a mutation in a gene that causes classical or atypical RTT can be described as having a 'Rett-syndrome-like phenotype (RTT-L). Here, we report eight patients from our cohort diagnosed as having RTT-L who carry mutations in genes unrelated to RTT. We annotated the list of genes associated with RTT-L from our patient cohort, considered them in the light of peer-reviewed articles on the genetics of RTT-L, and constructed an integrated protein-protein interaction network (PPIN) consisting of 2871 interactions connecting 2192 neighboring proteins among RTT- and RTT-L-associated genes. Functional enrichment analysis of RTT and RTT-L genes identified a number of intuitive biological processes. We also identified transcription factors (TFs) whose binding sites are common across the set of RTT and RTT-L genes and appear as important regulatory motifs for them. Investigation of the most significant over-represented pathway analysis suggests that HDAC1 and CHD4 likely play a central role in the interactome between RTT and RTT-L genes.

A Case Report of Immunotherapy-Resistant MSI-H Gastric Cancer with Significant Intrapatient Tumoral Heterogeneity Characterized by Histologic Dedifferentiation.

We describe a patient with both gastric adenocarcinoma and metastatic squamous cell carcinoma (SCC) of unknown primary site. The possibility of a single malignant clonal process as opposed to differing primaries was supported by the finding of both histologies exhibiting high microsatellite instability. Despite evidence of tumor microsatellite instability, the patient's disease process did not respond to immune checkpoint inhibition. Our pursuit of whole-exome sequencing and comparing the single-nucleotide variant profiles of both tumors supported a single clonal process with the development of significant intratumoral heterogeneity. High intratumoral heterogeneity has posed a challenge to precision medicine approaches, but we also provide a review of the literature of this phenomenon mediating resistance to immunotherapy strategies.

Next-Generation Sequencing Somatic and Germline Assay Troubleshooting Guide Derived From Proficiency Testing Data.

CONTEXT.­: Next-generation sequencing-based assays are increasingly used in clinical molecular laboratories to detect somatic variants in solid tumors and hematologic malignancies and to detect constitutional variants. Proficiency testing data are potential sources of information about challenges in performing these assays. OBJECTIVE.­: To examine the most common sources of unacceptable results from the College of American Pathologists Next-Generation Sequencing Bioinformatics, Hematological Malignancies, Solid Tumor, and Germline surveys and provide recommendations on how to avoid these pitfalls and improve performance. DESIGN.­: The College of American Pathologists next-generation sequencing somatic and germline proficiency testing survey results from 2016 to 2019 were analyzed to identify the most common causes of unacceptable results. RESULTS.­: On somatic and germline proficiency testing surveys, 95.9% (18 815/19 623) and 97.8% (33 890/34 641) of all variants were correctly identified, respectively. The most common causes of unacceptable results related to sequencing were false-negative errors in genomic regions that were difficult to sequence because of high GC content. False-positive errors occurred in the context of homopolymers and pseudogenes. Recurrent errors in variant annotation were seen for dinucleotide and duplication variants and included unacceptable transcript selection and outdated variant nomenclature. A small percentage of preanalytic or postanalytic errors were attributed to specimen swaps and transcription errors. CONCLUSIONS.­: Laboratories demonstrate overall excellent performance for detecting variants in both somatic and germline proficiency testing surveys. Proficiency testing survey results highlight infrequent, but recurrent, analytic and nonanalytic challenges in performing next- generation sequencing-based assays and point to remedies to help laboratories improve performance.

Comprehensive Genomic Profiling of High-Risk Pediatric Cancer Patients Has a Measurable Impact on Clinical Care.

PURPOSE: Profiling of pediatric cancers through deep sequencing of large gene panels and whole exomes is rapidly being adopted in many clinical settings. However, the most impactful approach to genomic profiling of pediatric cancers remains to be defined. METHODS: We conducted a prospective precision medicine trial, using whole-exome sequencing of tumor and germline tissue and whole-transcriptome sequencing (RNA Seq) of tumor tissue to characterize the mutational landscape of 127 tumors from 126 unique patients across the spectrum of pediatric brain tumors, hematologic malignancies, and extracranial solid tumors. RESULTS: We identified somatic tumor alterations in 121/127 (95.3%) tumor samples and identified cancer predisposition syndromes on the basis of known pathogenic or likely pathogenic germline mutations in cancer predisposition genes in 9/126 patients (7.1%). Additionally, we developed a novel scoring system for measuring the impact of tumor and germline sequencing, encompassing therapeutically relevant genomic alterations, cancer-related germline findings, recommendations for treatment, and refinement of risk stratification or prognosis. At least one impactful finding from the genomic results was identified in 108/127 (85%) samples sequenced. A recommendation to consider a targeted agent was provided for 82/126 (65.1%) patients. Twenty patients ultimately received therapy with a molecularly targeted agent, representing 24% of those who received a targeted agent recommendation and 16% of the total cohort. CONCLUSION: Paired tumor/normal whole-exome sequencing and tumor RNA Seq of de novo or relapsed/refractory tumors was feasible and clinically impactful in high-risk pediatric cancer patients.