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Purpose: Clinical genomic sequencing of pediatric tumors is increasingly uncovering pathogenic variants in adult-onset cancer predisposition genes (aoCPG). Nevertheless, it remains poorly understood how often aoCPG variants are of germline origin and whether they influence tumor molecular profiles and/or clinical care. In this study, we examined the prevalence, spectrum, and impacts of aoCPG variants on tumor genomic features and patient management at our institution. Experimental Design: This is a retrospective study of 1,018 children with cancer who underwent clinical genomic sequencing of their tumors. Tumor genomic data were queried for pathogenic variants affecting 24 preselected aoCPGs. Available tumor whole-genome sequencing (WGS) data were evaluated for second hit mutations, loss of heterozygosity (LOH), DNA mutational signatures, and homologous recombination deficiency (HRD). Patients whose tumors harbored one or more pathogenic aoCPG variants underwent subsequent germline testing based on hereditary cancer evaluation and family or provider preference. Results: Thirty-three patients (3%) had tumors harboring pathogenic variants affecting one or more aoCPGs. Among 21 tumors with sufficient WGS sequencing data, six (29%) harbored a second hit or LOH affecting the remaining aoCPG allele with four of these six tumors (67%) also exhibiting a DNA mutational signature consistent with the altered aoCPG. Two additional tumors demonstrated HRD, of uncertain relation to the identified aoCPG variant. Twenty-one of 26 patients (81%) completing germline testing were positive for the aoCPG variant in the germline. All germline-positive patients were counseled regarding future cancer risks, surveillance, and risk-reducing measures. No patients had immediate cancer therapy changed due to aoCPG data. Conclusions: AoCPG variants are rare in pediatric tumors; however, many originate in the germline. Almost one third of tumor aoCPG variants examined exhibited a second hit and/or conferred an abnormal DNA mutational profile suggesting a role in tumor formation. aoCPG information aids in cancer risk prediction but is not commonly used to alter the treatment of pediatric cancers.

Supplementary Figure S4

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor–normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers.Significance:Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers.This article is highlighted in the In This Issue feature, p. 2945

Supplementary Methods

Purpose:Clinical genomic sequencing of pediatric tumors is increasingly uncovering pathogenic variants in adult-onset cancer predisposition genes (aoCPG). Nevertheless, it remains poorly understood how often aoCPG variants are of germline origin and whether they influence tumor molecular profiles and/or clinical care. In this study, we examined the prevalence, spectrum, and impacts of aoCPG variants on tumor genomic features and patient management at our institution.Experimental Design:This is a retrospective study of 1,018 children with cancer who underwent clinical genomic sequencing of their tumors. Tumor genomic data were queried for pathogenic variants affecting 24 preselected aoCPGs. Available tumor whole-genome sequencing (WGS) data were evaluated for second hit mutations, loss of heterozygosity (LOH), DNA mutational signatures, and homologous recombination deficiency (HRD). Patients whose tumors harbored one or more pathogenic aoCPG variants underwent subsequent germline testing based on hereditary cancer evaluation and family or provider preference.Results:Thirty-three patients (3%) had tumors harboring pathogenic variants affecting one or more aoCPGs. Among 21 tumors with sufficient WGS sequencing data, six (29%) harbored a second hit or LOH affecting the remaining aoCPG allele with four of these six tumors (67%) also exhibiting a DNA mutational signature consistent with the altered aoCPG. Two additional tumors demonstrated HRD, of uncertain relation to the identified aoCPG variant. Twenty-one of 26 patients (81%) completing germline testing were positive for the aoCPG variant in the germline. All germline-positive patients were counseled regarding future cancer risks, surveillance, and risk-reducing measures. No patients had immediate cancer therapy changed due to aoCPG data.Conclusions:AoCPG variants are rare in pediatric tumors; however, many originate in the germline. Almost one third of tumor aoCPG variants examined exhibited a second hit and/or conferred an abnormal DNA mutational profile suggesting a role in tumor formation. aoCPG information aids in cancer risk prediction but is not commonly used to alter the treatment of pediatric cancers.

Supplementary Tables 1-6

Supplementary Figure 3. HRD Sum scores from ScarHRD analysis for biallelic, monoallelic, or no loss of HR genes in aoCPG and control samples.

Supplementary Figure 1. Signature exposures in 21 samples with aoCPG variants

Supplementary Figure 2. Molecular features of tumors harboring pathogenic aoCPG variants showing the full range of additional somatic second hits.

PURPOSE Constitutional mismatch repair deficiency syndrome (CMMRD) is a lethal cancer predisposition syndrome characterized by early-onset synchronous and metachronous multiorgan tumors. We designed a surveillance protocol for early tumor detection in these individuals. PATIENTS AND METHODS Data were collected from patients with confirmed CMMRD who were registered in the International Replication Repair Deficiency Consortium. Tumor spectrum, efficacy of the surveillance protocol, and malignant transformation of low-grade lesions were examined for the entire cohort. Survival outcomes were analyzed for patients followed prospectively from the time of surveillance implementation. RESULTS A total of 193 malignant tumors in 110 patients were identified. Median age of first cancer diagnosis was 9.2 years (range: 1.7-39.5 years). For patients undergoing surveillance, all GI and other solid tumors, and 75% of brain cancers were detected asymptomatically. By contrast, only 16% of hematologic malignancies were detected asymptomatically ( P < .001). Eighty-nine patients were followed prospectively and used for survival analysis. Five-year overall survival (OS) was 90% (95% CI, 78.6 to 100) and 50% (95% CI, 39.2 to 63.7) when cancer was detected asymptomatically and symptomatically, respectively ( P = .001). Patient outcome measured by adherence to the surveillance protocol revealed 4-year OS of 79% (95% CI, 54.8 to 90.9) for patients undergoing full surveillance, 55% (95% CI, 28.5 to 74.5) for partial surveillance, and 15% (95% CI, 5.2 to 28.8) for those not under surveillance ( P < .0001). Of the 64 low-grade tumors detected, the cumulative likelihood of transformation from low-to high-grade was 81% for GI cancers within 8 years and 100% for gliomas in 6 years. CONCLUSION Surveillance and early cancer detection are associated with improved OS for individuals with CMMRD.

Background With the advent of next generation sequencing, tumor and germline genomic testing are increasingly being used in the management of pediatric cancer patients. Despite this increase in testing, many pediatric hematology–oncology (PHO) providers are not confident interpreting or utilizing tumor or germline genomic results to care for their patients. Methods We developed and delivered a needs assessment survey to PHO program directors, attendings, and fellows in the United States to understand this deficiency, gather data on existing cancer genomics educational initiatives, and query preferences for creating a future curriculum. Results The survey includes 31 (41%) of 74 invited PHO program directors, 110 (11%) of 1032 invited attendings, and 79 fellows. The majority of attending physicians and fellows responding to the survey agree that understanding tumor (95% attending physicians; 95% fellows) and germline (86% attending physicians; 94% fellows) genomic information is essential for their practice. However, only 9 of 31 (29%) responding programs report that they have an existing cancer genomics curriculum. Most program directors indicated that the ideal genomics curriculum would occur during the first year of fellowship and incorporate direct patient care, online modules, and problem‐based learning. Attending physicians and fellows identified that addressing indications for ordering tumor and germline genomic testing, counseling about the risks and benefits of such testing, and interpreting and individualizing clinical management based on tumor and germline results should be included in a future curriculum. Conclusion The results of this study reveal a great need to develop a curriculum that can be offered across PHO fellowship programs to expand knowledge in the area of cancer genomics., A national needs assessment was performed to evaluate the perspectives of pediatric hematology‐oncology (PHO) fellowship program directors, attendings and fellows regarding development and deployment of a formal cancer genomics curriculum as a standard component of training. Only 29% of pediatric hematology‐oncology fellowship programs incorporate formal training in cancer genomics, yet most attending physicians and fellows report that tumor and germline genomic information is important for their clinical practice. The results of this study demonstrate the need to develop a cancer genomics curriculum for PHO fellows.

PURPOSEFor the advances of pediatric oncology next-generation sequencing (NGS) research to equitably benefit all children, a diverse and representative sample of participants is needed. However, little is known about demographic and clinical characteristics that differentiate families who decline enrollment in pediatric oncology NGS research.METHODSDemographic and clinical data were retrospectively extracted for 363 pediatric patients (0-21 years) with cancer approached for enrollment in Genomes for Kids (G4K), a study examining the feasibility of comprehensive clinical genomic analysis of tumors and paired normal samples. Demographic and clinical factors that significantly differentiated which families declined were subsequently compared, for 348 families, with enrollment in Clinical Implementation of Pharmacogenetics (PG4KDS), a pharmacogenomics study with more explicit therapeutic benefit examining genes affecting drug responses and metabolism.RESULTSFifty-three families (14.6%) declined enrollment in G4K. Race/ethnicity was the only variable that significantly differentiated study refusal according to multivariable logistic regression, with families of black children more likely to decline enrollment compared with families of non-Hispanic or Hispanic white children. Reasons for declining G4K were generally consistent with other pediatric genomics research: feeling overwhelmed and insurance discrimination fears were most frequently cited. Families of black children were also more likely to decline enrollment in PG4KDS. Thirteen (3.7%) of the 348 families approached for both studies declined PG4KDS.CONCLUSIONRace/ethnicity differentiated study declination across two different pediatric oncology genomics studies, suggesting enrollment disparities in the context of pediatric oncology genomics research. Genomics research participant samples that do not fully represent racial and ethnic minorities risk further exacerbating health disparities. Additional work is needed to understand the nuances of parental decision making in genomic research and facilitate enrollment of diverse patient populations.

Cancers arising from germline DNA mismatch-repair or polymerase-proofreading deficiencies (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion/deletion (MS-indel) burden in humans and are lethal due to inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICI) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI in these patients. ICI treatment of 45 progressive/recurrent tumours from 38 patients revealed durable objective responses in the majority, culminating in 3-year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations/Mb) enriched for combined MMRD+PPD, while MS-indels predicted response in MMRD tumours with lower mutation burden (10-100 mutations/Mb). Further, both mechanisms were associated with increased immune infiltration even in “immunologically-cold” tumours such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and associated with immune activation in both the tumour microenvironment and systemically. Further, patients with flare continuing ICI treatment achieved durable responses. Our study demonstrates improved survival for patients with tumours not previously known to respond to ICI, including CNS and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained responses to immunotherapy.

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion–deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10–100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in ‘immunologically cold’ tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy., Hypermutation and microsatellite burden determine responses and long-term survival following PD-1 blockade in children and young adults with refractory cancers resulting from germline DNA replication repair deficiency.

BackgroundConstitutional mismatch repair deficiency syndrome (CMMRD) is the most aggressive cancer predisposition syndrome associated with multiorgan cancers, often presenting in childhood. There is variability in age and presentation of cancers and benign manifestations mimicking neurofibromatosis type 1. Genetic testing may not be informative and is complicated by pseudogenes associated with the most commonly associated gene, PMS2. To date, no diagnostic criteria exist. Since surveillance and immune-based therapies are available, establishing a CMMRD diagnosis is key to improve survival.MethodsIn order to establish a robust diagnostic path, a multidisciplinary international working group, with representation from the two largest consortia (International Replication Repair Deficiency (IRRD) consortium and European Consortium Care for CMMRD (C4CMMRD)), was formed to establish diagnostic criteria based on expertise, literature review and consensus.ResultsThe working group established seven diagnostic criteria for the diagnosis of CMMRD, including four definitive criteria (strong evidence) and three likely diagnostic criteria (moderate evidence). All criteria warrant CMMRD surveillance. The criteria incorporate germline mismatch repair results, ancillary tests and clinical manifestation to determine a diagnosis. Hallmark cancers for CMMRD were defined by the working group after extensive literature review and consultation with the IRRD and C4CMMRD consortia.ConclusionsThis position paper summarises the evidence and rationale to provide specific guidelines for CMMRD diagnosis, which necessitates appropriate surveillance and treatment.

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor–normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. Significance: Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers. This article is highlighted in the In This Issue feature, p. 2945

NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development.

To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97–99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing., Clinical oncology is rapidly adopting next-generation sequencing technology for nucleotide variant and indel detection. Here the authors present a three-platform approach (whole-genome, whole-exome, and whole-transcriptome) in pediatric patients for the detection of diverse types of germline and somatic variants.

PTEN hamartoma tumor syndrome (PHTS), DICER1 syndrome, and hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome are pleiotropic tumor predisposition syndromes that include benign and malignant neoplasms affecting adults and children. PHTS includes several disorders with shared and distinct clinical features. These are associated with elevated lifetime risk of breast, thyroid, endometrial, colorectal, and renal cancers as well as melanoma. Thyroid cancer represents the predominant cancer risk under age 20 years. DICER1 syndrome includes risk for pleuropulmonary blastoma, cystic nephroma, ovarian sex cord–stromal tumors, and multinodular goiter and thyroid carcinoma as well as brain tumors including pineoblastoma and pituitary blastoma. Individuals with HLRCC may develop multiple cutaneous and uterine leiomyomas, and they have an elevated risk of renal cell carcinoma. For each of these syndromes, a summary of the key syndromic features is provided, the underlying genetic events are discussed, and specific screening is recommended. Clin Cancer Res; 23(12); e76–e82. ©2017 AACR. See all articles in the online-only CCR Pediatric Oncology Series.

Li-Fraumeni syndrome (LFS) is an autosomal dominantly inherited condition caused by germline mutations of the TP53 tumor suppressor gene encoding p53, a transcription factor triggered as a protective cellular mechanism against different stressors. Loss of p53 function renders affected individuals highly susceptible to a broad range of solid and hematologic cancers. It has recently become evident that children and adults with LFS benefit from intensive surveillance aimed at early tumor detection. In October 2016, the American Association for Cancer Research held a meeting of international LFS experts to evaluate the current knowledge on LFS and propose consensus surveillance recommendations. Herein, we briefly summarize clinical and genetic aspects of this aggressive cancer predisposition syndrome. In addition, the expert panel concludes that there are sufficient existing data to recommend that all patients with LFS be offered cancer surveillance as soon as the clinical or molecular LFS diagnosis is established. Specifically, the panel recommends adoption of a modified version of the “Toronto protocol” that includes a combination of physical exams, blood tests, and imaging. The panel also recommends that further research be promoted to explore the feasibility and effectiveness of these risk-adapted surveillance and cancer prevention strategies while addressing the psychosocial needs of individuals and families with LFS. Clin Cancer Res; 23(11); e38–e45. ©2017 AACR. See all articles in the online-only CCR Pediatric Oncology Series.

BACKGROUND The incorporation of genomic testing to identify targetable somatic alterations and predisposing germline mutations into the clinical setting is becoming increasingly more common. Despite its potential usefulness, to the authors' knowledge physician confidence with regard to understanding and applying genomic testing remains unclear, particularly within the realm of pediatric oncology. METHODS Before initiating an institutional feasibility study regarding the integration of clinical genomic testing, the authors surveyed pediatric oncologists regarding their confidence around understanding of genomic testing, perceived usefulness of test results, preferences around the disclosure of germline test results, and possible risks and benefits of testing. RESULTS Among survey respondents (52 of 88 contacted; response rate of 59%), only a minority were confident in interpreting, using, and discussing somatic (35%) or germline (27%) genomic test results. Providers who were confident in interpreting somatic results were significantly more likely to anticipate using the results to plan the treatment of patients with relapsed or refractory cancers (P = .009). Similarly, providers who reported confidence in interpreting germline results were significantly more likely to discuss and use these results as part of clinical care (P

Patients harboring germline pathogenic biallelic variants in genes involved in the recognition and repair of DNA damage are known to have a substantially increased cancer risk. Emerging evidence suggests that individuals harboring heterozygous variants in these same genes may also be at heightened, albeit lesser, risk for cancer. Herein, we sought to determine whether heterozygous variants in RECQL4, the gene encoding an essential DNA helicase that is defective in children with the autosomal recessive cancer-predisposing condition Rothmund–Thomson syndrome (RTS), are associated with increased risk for childhood cancer. To address this question, we interrogated germline sequence data from 4435 pediatric cancer patients at St. Jude Children's Research Hospital and 1127 from the National Cancer Institute Therapeutically Applicable Research to Generate Effective Treatment (TARGET) database and identified 24 (0.43%) who harbored loss-of-function (LOF) RECQL4 variants, including five of 249 (2.0%) with osteosarcoma (OS). These RECQL4 variants were significantly overrepresented in children with OS, the cancer most frequently observed in patients with RTS, as compared to 134,187 noncancer controls in the Genome Aggregation Database (gnomAD v2.1; P = 0.00087, odds ratio [OR] = 7.1, 95% CI, 2.9–17). Nine of the 24 (38%) individuals possessed the same c.1573delT (p.Cys525Alafs) variant located in the highly conserved DNA helicase domain, suggesting that disruption of this domain is central to oncogenesis. Altogether these data expand our understanding of the genetic factors predisposing to childhood cancer and reveal a novel association between heterozygous RECQL4 LOF variants and development of pediatric OS.

Dear Editor,Retinoblastoma and Hermansky-Pudlak syndrome (HPS) are two rare ophthalmic disorders which arise from sporadic or germline mutations in specific genes. Retinoblastoma occurs in 1 in 15,...

Clinical genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. In the Genomes for Kids study (NCT02530658) we used a three-platform sequencing approach, including whole genome (WGS), whole exome (WES) and RNA sequencing, to examine tumor and paired germline genomes from prospectively identified children with cancer. The goal of the study was to assess the potential of comprehensive next generation sequencing to elucidate the molecular mechanisms underlying tumor formation and investigate the potential of this information to influence clinical decision-making.The cohort, with a median age of 6 yrs, range 0 - 26 yrs, included 301 patients with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type or stage. Patients with hematologic malignancies accounted for 41% of cases, 31% had CNS tumors, and 28% had other non-CNS solid tumors. This cohort also included 18 patients with very rare tumor types, defined here as occurring in less than 2 cases per million person per year.Two hundred fifty three patients (84%) had sufficient tumor for three-platform sequencing and all 301 had adequate paired germline samples. Following analysis, 86% of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer predisposing (18%) variants. The inclusion of WGS enabled detection of oncogenic gene fusions, as well as 22 cases in which oncogenes were activated through enhancer hijacking, a particularly frequent occurrence in hematologic malignancies. In addition, WGS effectively detected clinically relevant small intragenic deletions (15% of tumors) and a variety of mutational signatures, which were not detectable through analysis of whole exome data. Evaluation of 56 pathogenic germline variants in the context of paired tumor sequence data helped establish the disease relevance of several genes that are not typically associated with the cancer type in question, providing critical insights on a case-by-case basis. Examples include a pathogenic germline variant in MUTYH in a patient with retinoblastoma whose tumor exhibited a mutation signature associated with reactive oxygen species indicative of loss of MUTYH function; and conversely, a likely pathogenic variant in PMS2 in a rare brain cancer, which did not exhibit a mutation signature associated with microsatellite instability. This study successfully demonstrated the power of this three-platform approach to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. As a result of these findings, we have incorporated this three-platform approach into our routine real-time clinical service at St. Jude Children's Hospital. Citation Format: David A. Wheeler, Scott Newman, Joy Nakitandwe, Chimene A. Kesserwan, Elizabeth M. Azzato, Michael C. Rusch, Sheila Shurtleff, Armita Bahrami, Brent Orr, Jeffery M. Klco, Dale J. Hedges, Kayla V. Hamilton, Scott G. Foy, Michael N. Edmonson, Andrew Thrasher, Jiali Gu, Lynn W. Harrison, Lu Wang, Roya Mostafavi, Manish Kubal, Jamie Maciaszek, Michael Clay, Annastasia Ouma, Antonina Silkov, Yanling Liu, Zhaojie Zhang, Yu Liu, Samuel W. Brady, Xin Zhou, Mark Wilkinson, Delaram Rahbarinia, Jay Knight, Jian Wang, Charles G. Mullighan, Rose B. McGee, Emily A. Quinn, Elsie L. Gerhardt, Leslie M. Taylor, Regina Nuccio, Jessica M. Valdez, Stacy J. Hines-Dowell, Alberto Pappo, Giles Robinson, Liza-Marie Johnson, Ching-Hon Pui, David W. Ellison, James R. Downing, Jinghui Zhang, Kim E. Nichols. Genomes for Kids: Comprehensive DNA and RNA sequencing defining the scope of actionable mutations in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 642.

BACKGROUND Patients with cancer are increasingly offered genomic sequencing, including germline testing for cancer predisposition or other disorders. Such testing is unfamiliar to patients and families, and clear communication is needed to introduce genomic concepts and convey risk and benefit information. METHODS Parents of children with cancer were offered the opportunity to have their children's tumor and germline examined with clinical genomic sequencing. Families were introduced to the study with a 2-visit informed consent model. Baseline genetic knowledge and self-reported literacy/numeracy were collected before a study introduction visit, during which basic concepts related to genomic sequencing were discussed. Information was reinforced during a second visit, during which informed consent was obtained and a posttest was administered. RESULTS As reflected by the percentage of correct answers on the pretest and posttest assessments, this model increased genetic knowledge by 11.1% (from 77.8% to 88.9%; P

Background/aims Non-irradiative local therapies have shown promise in delaying or supplanting external beam radiotherapy (EBRT) and enucleation in patients with retinoblastoma. We hypothesised that prior focal therapy does not compromise the efficacy of delayed episcleral plaque brachytherapy (epBRT).Methods We performed an institutional review board-approved medical record review of patients with retinoblastoma who were treated with I-125 epBRT prior to (primary) or following chemoreduction (delayed), alone and in combination with non-irradiative focal therapy. Clinical and treatment characteristics were retrieved. Treatment failure was defined as the need for subsequent EBRT and/or enucleation. Event-free and ocular survival rates were calculated from the date of plaque placement. The cumulative incidences (CIs) of treatment failure and enucleation were compared across strata using Gray’s test.Results We identified 50 patients with retinoblastoma (54 eyes), who received a total of 56 plaques between January 1986 and December 2010, with a median follow-up of 8.3 years (range, 0.8–21.2 years). The median time from diagnosis to plaque placement was 12.7 months (range, 0.1–128 months). The CI and 95% CI of treatment failure and enucleation following epBRT at 5 years was 37%±7.2% and42.2%±7.3%, respectively. The lack of prior diode or green laser therapy was predictive of increased risk for treatment failure (p=0.02 and 0.03). International Classification group C or D was predictive of decreased time to enucleation (p=0.004). The use of any focal therapy was not predictive of time to treatment failure (p=0.33).Conclusions The use of non-irradiative focal therapies prior to or following epBRT does not decrease the time to enucleation or treatment failure.

Malignant progression of a benign or low-grade tumor in individuals with germline alteration of SMARCB1 gene is not well characterized. In a family in which two carrier children had germline SMARCB1 mutations and atypical teratoid rhabdoid tumor, we report malignant progression of a nerve sheath tumor over a 7-year period in an affected adult family member. Prompt identification of the germline SMARCB1 alteration and the resultant rhabdoid tumor predisposition syndrome can help guide genetic counseling and surveillance in affected family members.

Context Parathyroid carcinoma is exceedingly rare in children. We describe a case of parathyroid cancer in a young female who was originally classified as benign and managed surgically. Upon her diagnosis with malignancy, concurrent with metastatic lung involvement, she was referred for medical and surgical palliation to control her symptomatic hypercalcemia. We briefly review published childhood cases, consider the challenges in differentiating malignant from benign hyperparathyroidism in this age group, and discuss the association of CDC73 mutations with parathyroid carcinoma. Case presentation A 13-year-old African American girl with a history of parathyroid adenoma, diagnosed at 8 years of age with multiple recurrences, presented with hypercalcemia and elevated parathyroid hormone when her disease had been reclassified as malignant. Germline gene analysis revealed a heterozygous partial deletion of CDC73. The patient underwent palliative surgery for disease metastatic to her lungs. She continues with medical management of her hypercalcemia. Conclusions A case of pediatric parathyroid carcinoma associated with haploinsufficiency of CDC73 is discussed. We review all published cases of pediatric parathyroid carcinoma and offer diagnostic considerations for a parathyroid mass in a child.

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with a high risk of developing early-onset malignancies of the blood, brain, and intestinal tract. We present the case of a patient with T-lymphoblastic lymphoma at the age of 3 years, followed by Burkitt lymphoma 10 years later. This patient also exhibited numerous nonmalignant findings including cafe au lait spots, lipomas, bilateral renal nodules, a nonossifying fibroma, multiple colonic adenomas, and a rapidly enlarging pilomatrixoma. The spectrum of malignant and nonmalignant neoplasms in this patient highlights the remarkable diversity, and early onset, of lesions seen in children with CMMRD.

As the understanding of the genetic etiology of childhood cancers increases, the need for the involvement of experts familiar with the provision of genetic counseling for this population is paramount. In October 2016, the American Association for Cancer Research organized the AACR Childhood Cancer Predisposition Workshop in which international experts in pediatric cancer predisposition met to establish surveillance guidelines for children with cancer predisposition. Identifying for whom, when, why, and how these cancer predisposition surveillance guidelines should be implemented is essential. Genetic counselors invited to this workshop provide a genetic counseling framework for oncology professionals in this article. Points of entry and recommendations regarding the provision and timing of the initial and subsequent genetic counseling sessions are addressed. The genetic counseling and testing processes are reviewed, and the psychologic impact related to surveillance is explored. Pediatric cancer genetics will continue to grow and evolve as a field, and genetic counseling services will be vital to ensure appropriate identification and management of at-risk children moving forward. Clin Cancer Res; 23(13); e91–e97. ©2017 AACR. See all articles in the online-only CCR Pediatric Oncology Series.

RECQL4 encodes an essential helicase that repairs DNA damage and maintains genomic stability. Biallelic pathogenic germline mutations in RECQL4 cause the autosomal recessive (AR) Rothmund-Thomson, Baller-Gerold, and RAPADILINO syndromes. Predisposition to cancer has been observed in all three syndromes, with osteosarcoma (OS) representing the greatest risk. Monoallelic pathogenic variants in DNA damage response genes (e.g., ATM, NBN) are associated with a moderate increase in cancer risk. Building upon this notion, we sought to determine whether monoallelic RECQL4 loss of function (LOF) variants contribute to childhood cancer, particularly OS. Here, LOF variants were defined as nonsense, frameshift, or canonical splice altering variants that are classified as pathogenic or likely pathogenic based on the 2015 ACMG Guidelines. Among 4,436 pediatric cancer patients at St. Jude and 1,127 in the National Cancer Institute TARGET database (total: 5,563 patients), we identified 20 individuals (0.36%; tumor types in Table) harboring germline monoallelic RECQL4 LOF variants. Compared to reference controls in the Genome Aggregation Database (gnomAD), we observed an enrichment of RECQL4 LOF variants in pediatric cancer patients (P = 0.046, prevalence ratio [PR] = 1.51). We next assessed for enrichment of RECQL4 LOF variants across tumor types (Table). This examination revealed a significant association between monoallelic RECQL4 mutations and leukemia (P = 0.032, PR = 1.91) and more notably, with OS (P = 0.0028, PR = 7.03) where 1.7% of pediatric OS patients carried monoallelic RECQL4 LOF variants. No evidence of association was observed for the other tumor types examined. Our data provide the first evidence of an association linking germline monoallelic RECQL4 LOF variants to childhood cancer, especially OS. Examination of larger cohorts are warranted to elucidate the extent to which these mutations increase the risk for leukemia and OS and the mechanisms by which they promote tumor formation. Pediatric Cancer PatientsgnomAD ControlsCancer Risk (Fisher’s Exact Test)Average age at cancer diagnosis (range)Carriers, RECQL4mut/wtTotal Patients, RECQL4wt/wtCarriers, RECQL4mut/wtTotal Controls, RECQL4wt/wtPrevalence Ratio (95% CI)P ValueAll cancers7 (0.3 - 18)2055632811206591.51 (0.99, 2.30)0.046OS10 (6 - 16)42432811206597.03 (2.65, 18.69)0.0028CNS tumor515792811206590.74 (0.10, 5.26)0.74GCT0.31752811206595.74 (0.81, 40.88)0.16Leukemia4 (3 - 11)1124312811206591.91 (1.07, 3.41)0.032Lymphoma10 (7 - 18)35862811206592.19 (0.71, 6.76)0.16OS = osteosarcoma; CNS tumor = central nervous system tumor; GCT = germ cell tumor Citation Format: Jamie L. Maciaszek, Gang Wu, Kayla Hamilton, Rose B. McGee, Zhaoming Wang, Regina Nuccio, Stacy Hines-Dowell, Lynn Harrison, Elsie L. Gerhardt, Annastasia Ouma, Scott Newman, Aman Patel, Joy Nakitandwe, Elizabeth Azzato, Alberto S. Pappo, Sheila A. Shurtleff, David W. Ellison, James R. Downing, Melissa M. Hudson, Leslie L. Robison, Victor Santana, Jinghui Zhang, Kim E. Nichols, Chimene A. Kesserwan. Increased prevalence of germline monoallelic RECQL4 mutations in children with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3651.

The last 30 years have witnessed tremendous advances in our understanding of the cancer genetic susceptibility syndromes, including those that predispose to hematopoietic malignancies. The identification and characterization of families affected by these syndromes is enhancing our knowledge of the oncologic and nononcologic manifestations associated with predisposing germ line mutations and providing insights into the underlying disease mechanisms. Here, we provide an overview of the cancer genetic susceptibility syndromes, focusing on aspects relevant to the evaluation of patients with leukemia and lymphoma. Guidance is provided to facilitate recognition of these syndromes by hematologists/oncologists, including descriptions of the family history features, tumor genotype, and physical or developmental findings that should raise concern for an underlying cancer genetic syndrome. The clinical implications and management challenges associated with cancer susceptibility syndromes are also discussed.

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with a high risk of developing early-onset malignancies of the blood, brain, and intestinal tract. We present the case of a patient with T-lymphoblastic lymphoma at the age of 3 years, followed by Burkitt lymphoma 10 years later. This patient also exhibited numerous nonmalignant findings including café au lait spots, lipomas, bilateral renal nodules, a nonossifying fibroma, multiple colonic adenomas, and a rapidly enlarging pilomatrixoma. The spectrum of malignant and nonmalignant neoplasms in this patient highlights the remarkable diversity, and early onset, of lesions seen in children with CMMRD.

BackgroundThe prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. MethodsIn 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data...

Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition., Author Summary Inherited mutations of transcription factors have recently been associated with susceptibility to acute leukemia. Here we report two unrelated kindreds with inherited mutations in ETV6, the gene encoding the transcription factor ETS variant 6. These families were characterized by a low platelet count (thrombocytopenia) and acute lymphoblastic leukemia (ALL). Sequencing a panel of genes identified germline ETV6 mutations associated with leukemia and thrombocytopenia in multiple individuals tested. In one family, there was a substitution within the DNA binding domain of ETV6, termed L349P, and in the second there were five base pairs missing in ETV6 (N385fs), causing an abnormally truncated protein. We overexpressed the ETV6 mutants in the HeLa cell line and measured protein levels and localization within the cells. Instead of localizing to the nucleus, as expected for a transcription factor, the mutant proteins were found in the cytoplasm. The mutant proteins also showed decreased ability to regulate the expression of other genes typically suppressed by ETV6. These findings suggest that germline ETV6 mutations cause a new type of heritable leukemia. This discovery makes possible the pre-symptomatic diagnosis of leukemia susceptibility in families with germline ETV6 mutations, and also provides new information on the causes of leukemia.

The capability to assay for the status of individual genes or proteins in patient blood or tissues has grown exponentially over the last two decades. Molecular diagnostics has had a particularly important impact on the practice of neurology, both due to the keen interest of the practicing neurologist and the fact that many of the first identified disease genes have been for inherited neurological conditions. With the dramatically expanding knowledge of the primary biochemical defect for hundreds of disorders comes the anticipation that pathophysiological cascades can be slowed or halted, or primary biochemical deficiencies replaced. While this hope has yet to be realized, molecular human genetics has transformed the way in which the inherited and noninherited neurological diseases are researched, and experimental therapeutics are pursued. Finally, molecular diagnostics can provide a definitive diagnosis to a patient (both symptomatic and presymptomatic) while also permitting the genetic counseling of the patient’s family.

Next-generation sequencing (NGS) of the whole genome, whole exome, and transcriptome has enabled characterization of genetic landscapes of multiple cancers. By analyzing over 2,000 pediatric cancer patients, we have developed a comprehensive database for recurrent somatic alterations and pathogenic germline mutations as part of the St. Jude/Washington University Pediatric Cancer Genome Project. However, there is no systematic evaluation on whether NGS is able to identify germline and somatic lesions reported by existing molecular diagnostic assays and what combination of NGS platforms is best suited for clinical sequencing. Here we report the first comprehensive study that employs whole-genome sequencing at 30-45X coverage, whole-exome sequencing at 100X coverage and transcriptome sequencing using matched tumor/normal samples from cancer patients. A pilot study was carried out to perform NGS analysis on 78 children of leukemia, solid tumor or brain tumor with a total of 112 diagnostic or prognostic biomarkers previously characterized by multiple molecular diagnostic assays. We implemented an analysis pipeline that integrates the genetic lesions detected by all three NGS platforms to characterize somatic and germline single nucleotide variations (SNVs), short insertions and deletions (indels), structural variations including fusions, karyotypes, copy number alterations, loss of heterozygosity, tumor purity and tumor-in-normal contamination. The turn-around time for data analysis is 2 weeks with an overall sensitivity of 99% on detecting known biomarkers. Extensive validation of >3,000 somatic sequence mutations or structural variations from 38 cases shows that the specificity for somatic SNV, indel and structural variation is at 98%, 95% and 84% across the genome. We demonstrate that in addition to providing cross-validation, multi-platform NGS is required for detecting all genetic lesions of pathological significance including complex re-arrangements such as chromothripsis. In addition to known pathogenic or likely pathogenic mutations, our analysis has also unveiled novel pathogenic mutations (e.g. a germline deletion in TP53 in one patient with medulloblastoma) and identified multiple variants of unknown significance that may be worth further exploration (e.g. an in-frame deletion of exons 3-9 of DNMT3A in one neuroblastoma). Our study demonstrates that NGS is able to detect a wide range of genetic lesions currently characterized by multiple molecular diagnostic assays, providing critical insight into the design of clinical sequencing for ongoing studies. Citation Format: Jinghui Zhang, Michael Rusch, Joy Nakitandwe, Zhaojie Zhang, Michael N. Edmonson, Matthew Parker, Xiaotu Ma, Jared Becksfort, Andrew Thrasher, Jiali Gu, Yongjin Li, Erin Hedlund, Aman Patel, John Easton, Donald Yergeau, Bhavin Vadodaria, Xiang Chen, Tanja A. Gruber, Rose McGee, David Ellison, Sheila Shurtleff, James R. Downing. Molecular diagnosis for pediatric cancer through integrative analysis of whole-genome, whole-exome and transcriptome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2628.

Germline mutations of transcription factors (e.g. PAX5, CEBPA, GATA2, RUNX1) have been associated with an inherited susceptibility to acute leukemia. Here we report 2 unrelated kindreds harboring germline mutations in ETV6, the gene encoding the transcription factor ETS variants 6. These kindreds were primarily characterized by thrombocytopenia and acute lymphoblastic leukemia (ALL). The first kindred, identified at MSKCC and HMC, includes 9 individuals with thrombocytopenia, and 3 individuals with pre-B ALL. Sequencing a subset of common and somatically altered leukemia genes in this family identified a rare heterozygous non-synonymous missense variation (T>C) in 6 family members with thrombocytopenia and 2 with ALL. Notably, this variant did not segregate in 9 individuals in the kindred without these phenotypes. The amino acid alteration is predicted to lead to an L349P substitution within the DNA binding domain of ETV6 (L349P, NPP_001978). In silico analyses using SIFT and Polyphen assigned pathogenic variant classifications to the L349P variant. The proband of the second family presented to SJCRH with pre-B ALL, a history of thrombocytopenia, and demonstrated hypersensitivity to methotrexate (grade 3 bone marrow suppression). His leukemia transformated to myelodsyplastic syndrome and acute myeloid leukemia, which was treated by unrelated donor transplantation. The proband's mother, maternal aunt and maternal grandfather had thrombocytopenia, and the maternal great grandfather was diagnosed with chronic myeloid leukemia. The proband and his mother were found to carry a heterozygous 5 bp deletion in ETV6, N385fs (c.1153-5_1153_1delAACAG), which is predicted to produce a truncated protein of 389 amino acids with the last 4 residues different from the canonical ETV6 sequence. This variant has not been described in public sequencing data repositories. Finally, a review of 1,120 cases from the SJCRH/Washington University Pediatric Cancer Genome Project, including 588 cases of leukemia (among which 472 were ALL), revealed 3 rare germline ETV6 variants: L442P (n = 1), R181H (n = 1), and V37M (n = 1), with 2 of these variants L442P and R181H predicted to be pathogenic based on prediction and/or genomic location. Notably, these 2 variants each occurred in patients with ALL. Collectively, these findings suggest that germline ETV6 mutations are associated with a novel syndrome of thrombocytopenia with susceptibility to leukemia. Furthermore, these mutations may account for approximately 0.4% (2/472) of pediatric ALL cases. Further phenotypic characterization of affected patients and functional assessment of ETV6 germline variants, in progress, will be required to reveal the clinical effects of these mutations, their incidence in patients with ALL and their role in leukemogenesis. Citation Format: Vijai Joseph, Michael F. Walsh, Sabine Topka, Gang Wu, Rose B. McGee, Emily Quinn, Hiroto Inaba, Christine Hartford, Ching-Hon Pui, Alberto S. Pappo, Michael Edmonson, Lauren Jacobs, Villano Danylo, Kasmintan Schrader, Pragna Gaddam, Zsofia Stadler, Michael Zhang, Polina Stepensky, Peter Steinherz, James Bussel, M Harit, Michael Weintraub, Akiko Shimamura, Jinghui Zhang, James R. Downing, Kenneth Offit, Kim E. Nichols. Germline mutations in ETV6 confer risk of thrombocytopenia and acute lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2033. doi:10.1158/1538-7445.AM2015-2033

Pathologic germ line mutations that predispose patients to cancer are estimated to occur in 4-30% of all pediatric oncology cases. In addition to leukemia specific familial predisposition syndromes, children with rare constitutional syndromes, heterogeneous dysmorphic syndromes, and multiple-cancer hereditary predisposition syndromes are all at an increased risk for hematologic malignancies. However, to date no genome-wide analysis has been done to define the range of germ line mutations that occur in pediatric patients with hematological malignancies. To determine the frequency of pediatric cancer patients that have germ line variants of pathological significance in genes that predisposed to cancer, we analyzed the germ line and tumor DNA from 1120 pediatric cancer patients that were enrolled in the St. Jude – Washington University Pediatric Cancer Genome Project (PCGP). Samples were analyzed by whole-genome sequencing (n = 595), whole-exome sequencing (n = 456), or both (n = 69). Single nucleotide variants (SNVs), insertions/deletions (indels), structural variations (SV) and copy number alterations (CNAs) were detected using our analytical pipeline and all single nucleotide polymorphisms (SNPs) previously identified in non-cancer populations were filtered out. Our analysis then focused on the 23 cancer predisposition genes recently recommended for germ line analysis by the American College of Genetics and Genomics, along with an additional 8 genes that have been previously shown to predispose to pediatric cancer at a high penetrance. All variants in these 31 genes were classified as pathologic, likely pathologic, uncertain significance, likely benign, and benign based on literature review and in-silico predictions on the effect of novel mutations. An expanded analysis including a total of 565 genes known to play a role in oncogenesis was also evaluated. Pathologic or likely pathologic germ line variants in one of the 31 genes were detected in 8% (90/1120) of patients, including: 16% (46/287) of patients with solid tumors, 8.6% (21/245) with brain tumors, and 3.9% (23/588) with leukemia. Expanding this analysis to 565 cancer gene resulted in only a slight increase, with a pathologic or likely pathologic variant being detected in 8.6% (97/1120) of patients. The most frequently effected genes included TP53 (n=48), APC (n=7) and BRCA2(n=6). Importantly, in >50% of these patients, analysis of their tumor DNA revealed the absence of a wild type allele for the cancer predisposition gene that was altered in the germ line. The 588 pediatric patients with leukemia included 116 acute myeloid leukemias (AMLs: FAB M7 n=20; Core Binding Factor leukemias n=86; MLL-R n=10) and 472 acute lymphoblastic leukemias (ALLs: E2A-PBX1 n=53; ERG-R n=39; TEL-AML1 n=53; Hyperdiploid n=69; Hypodiploid n=47; BCR-ABL1 n=40; T-ALL n=32; MLL-R n=40; BCR-ABL-like n=31; and Other n=68). Across this cohort, 3.9% (23/588) of leukemia patients harbored a pathologic germ line mutations in one of the 31 cancer pre-disposing genes. This number increased to 4.6% (27/588; 28 mutations) when the expanded gene list was evaluated. TP53 (n=10) was the most frequently altered germ line gene in pediatric leukemia patients and was found predominantly in low-hypodiploid ALL, as previously reported. Germ line pathologic variants were also identified in KRAS, RUNX1, APC, BRCA2, and RET (2 cases each), and NRAS, SH2B3, BRCA1, MUTYH, PTCH1, SDHA,VHL, and NF2 (1 case each). Although germ line mutations in RUNX1 and SH2B3are typically associated with myeloid neoplasms, we identified these lesions in 3 cases of B lineage ALL suggesting an association with a wider spectrum of leukemia. In conclusion, a small but significant proportion of pediatric patients with leukemia carry a germ line variant of pathologic significance in a cancer predisposition gene. These results suggest that these germ line lesions likely play a direct role in the pathogenesis of the patient’s presenting leukemia. Moreover, our results suggest that these patients would benefit from future clinical surveillance for the development of a second cancer. Lastly, these data demonstrate the power of comprehensive next generation DNA/RNA sequencing for the identification of pediatric patients who carry a germ line pathologic variant in a cancer predisposition gene. Disclosures No relevant conflicts of interest to declare.