NF1 Variants Research Articles

Performance Evaluation of SpliceAI for the Prediction of Splicing of NF1 Variants.

Neurofibromatosis type 1, characterized by neurofibromas and café-au-lait macules, is one of the most common genetic disorders caused by pathogenic NF1 variants. Because of the high proportion of splicing mutations in NF1, identifying variants that alter splicing may be an essential issue for laboratories. Here, we investigated the sensitivity and specificity of SpliceAI, a recently introduced in silico splicing prediction algorithm in conjunction with other in silico tools. We evaluated 285 NF1 variants identified from 653 patients. The effect on variants on splicing alteration was confirmed by complementary DNA sequencing followed by genomic DNA sequencing. For in silico prediction of splicing effects, we used SpliceAI, MaxEntScan (MES), and Splice Site Finder-like (SSF). The sensitivity and specificity of SpliceAI were 94.5% and 94.3%, respectively, with a cut-off value of Δ Score > 0.22. The area under the curve of SpliceAI was 0.975 (p < 0.0001). Combined analysis of MES/SSF showed a sensitivity of 83.6% and specificity of 82.5%. The concordance rate between SpliceAI and MES/SSF was 84.2%. SpliceAI showed better performance for the prediction of splicing alteration for NF1 variants compared with MES/SSF. As a convenient web-based tool, SpliceAI may be helpful in clinical laboratories conducting DNA-based NF1 sequencing.

LGG-10. AN UNUSUAL PRESENTATION OF BILATERAL OPTIC NERVE GLIOMA IN CROUZON SYNDROME

Optic pathway gliomas (OPGs) are low grade gliomas intrinsic to the visual pathway, frequently associated with Neurofibromatosis Type 1 (NF-1). Bilateral OPGs without chiasmatic involvement are almost pathognomonic for NF-1. We report an unusual case of bilateral optic nerve glioma without chiasmatic involvement in a 17-month old male patient with craniosynostosis and Crouzon Syndrome, an autosomal dominant disorder caused by activating FGFR2 mutations associated with craniosynostosis and optic atrophy. The patient’s c.1032 G>A pathogenic variant in FGFR2 is a variant known to affect splicing and results in a protein that lacks part of an important domain involved in ligand binding. Although FGFR1 mutations have been implicated in low-grade glioma through MAPK activation, FGFR2 mutations have not yet been described in OPGs, although they have been described in epileptogenic low-grade gliomas and mixed neuronal-glial tumors. Our patient presented with worsening vision in the setting of known Crouzon Syndrome. An eye examination revealed bilateral primary optic atrophy. Brain and orbital MRIs demonstrated fusiform dilation and STIR hyperintensity of the intraorbital segments of the optic nerves bilaterally with normal pre-chiasmatic optic segments. There were no other radiographic or physical stigmata suggestive of NF-1. Next generation sequencing and copy number analysis from peripheral blood were negative for variants in NF1. RNA based studies for NF1 aberrations are pending. Although follow up MRI scans demonstrated stable size of his OPGs, the risk of further visual deficit was considered significant due to his pre-existing optic atrophy and poor baseline visual acuity. Therefore, he was started on vincristine and carboplatin chemotherapy according to A9952, and induction therapy has been well tolerated. To our knowledge, this is the first patient with Crouzon Syndrome who has developed bilateral optic pathway gliomas. Orbital MRIs should be considered for these patients with worsening visual acuity not explained by other causes.

Case report of a man with multiple paragangliomas and pathogenic germline variants in both NF1 and SDHD

We report a novel case of multiple paragangliomas in a patient who was identified with pathogenic variants in both NF1 and SDHD genes.The proband is a man with known familial NF1 disease, diagnosed clinically in childhood. Multiple head and neck paragangliomas (HNPGL) were found during investigations for acute left sided neurological symptoms, in the region of his known plexiform neurofibroma. He was referred for genetic counselling. He underwent surgery to remove a left carotid body tumor (CBT).A pheochromocytoma and paraganglioma gene panel was tested. Blood and HNPGL tumor DNA were analyzed by whole exome sequencing.In addition to the NF1 truncating variant c.5107delA, p.(Ser1703AlafsTer7), the SDHD truncating pathogenic variant c.3G > A, p.(Met1?) was found. Tumor sequencing showed no LOH of SDHD or NF1, but monoallelic loss of 11p15 and 11q12.2-q12.3 was observed.Co-occurrence of pathogenic variants in multiple cancer susceptibility genes is rare but possible, identified by the increased use of panel testing. This is the first description of a patient presenting with NF1 and SDHD dual pathology, with HNPGL development due to SDHD. This case illustrates the central role of genetic sequencing in PPGLs and the strong genotype-phenotype correlations of different genes.

Implications of mosaicism in variant interpretation: A case of a de novo homozygous NF1 variant

Neurofibromatosis type 1 is a common multisystem autosomal dominant syndrome caused by pathogenic heterozygous variants in the neurofibromin gene (NF1). It is associated with a substantially increased cancer risk. Mosaicism for NF1 has been clinically well-established for “second hit” variants in skin lesions and tumor tissues. Here, we report on a 3-month-old boy with multiple café au lait macules (CAMs) and juvenile myelomonocytic leukemia (JMML) who was found to carry a previously established pathogenic NF1 variant (c.586+5G>A), as revealed by whole-exome sequencing. Surprisingly, however, this variant was detected in the homozygous state in the patient and was absent in the parents and siblings. Deep sequencing of this variant using blood, buccal swabs and skin samples was performed. As expected for an NF1 gene mutation promoting JMML, the variant was detected in 90.6% of the blood DNA reads, in sharp contrast to the mere 5% and 0.74% of reads in the saliva- and skin fibroblast-derived DNA, respectively. Our analysis, therefore, confirmed postzygotic origin of the variant followed by a mitotic event resulting in its homozygosity, although we could not differentiate between the possibilities of a gene conversion and mitotic crossover. Apparently de novo homozygous variants should trigger a careful investigation into mosaicism to achieve accurate interpretation.

Genomic landscape of B-other acute lymphoblastic leukemia in an adult retrospective cohort with a focus on BCR-ABL1-like subtype

Introduction BCR-ABL1-like acute lymphoblastic leukemia (ALL) is a high-risk disease with a complex genomic background. Though extensively studied, data on the frequency and mutual associations of present mutations are still incomplete in adult patients. This retrospective study aims to map the genomic landscape of B-other ALL in a cohort of adult patients with a focus on the BCR-ABL1-like ALL subtype. Methods We analyzed bone marrow and peripheral blood samples of adult B-other ALL patients treated consecutively at three major Czech teaching hospitals. Samples were analyzed by cytogenetic methods, gene expression profiling, multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS). Results Fifty-eight B-other ALL patients (not BCR-ABL1, KMT2A-rearranged, ETV6-RUNX1, TCF3-PBX1, or iAMP21) were included in the study. Median follow-up was 23.8 months. Samples from 33 patients were available for a gene expression analysis, 48.9% identified as BCR-ABL1-like ALL. Of the BCR-ABL1-like ALL cases, 18.8% harbored IGH-CRLF2 and 12.5% P2RY8-CRLF2 fusion gene. We observed a higher MRD failure rate in BCR-ABL1-like than in non-BCR-ABL1-like ALL patients after the induction treatment (50.0 vs. 13.3%, p=.05). There was a trend to worse progression-free and overall survival in the BCR-ABL1-like group, though not statistically significant. Deletions in IKZF1 gene were found in 31.3% of BCR-ABL1-like cases. Patients with concurrent IKZF1 and CDKN2A/B, PAX5 or PAR1 region deletions (IKZF1 plus profile) had significantly worse progression-free survival than those with sole IKZF1 deletion or IKZF1 wild-type (p=.02). NGS analysis was performed in 54 patients and identified 99 short variants in TP53, JAK2, NRAS, PAX5, CREBBP, NF1, FLT3, ATM, KRAS, RUNX1, and other genes. Seventy-five of these gene variants have not yet been described in B-cell precursor ALL to date. Conclusion This study widens existing knowledge of the BCR-ABL1-like and B-other ALL genomic landscape in the adult population, supports previous findings, and identifies a number of novel gene variants.

Integrated case-control and somatic-germline interaction analyses of soft-tissue sarcoma

PurposeThe contribution of rare genetic variation in the development of soft-tissue sarcoma (STS) remains underexplored. To address this gap, we conducted a whole-exome case-control and somatic-germline interaction study to identify...

NF1 Tumor Suppressor Gene Inactivation in Juvenile Myelomonocytic Leukemia: New Genetic Evidence and Consequences for Diagnostic Work-up

Neurofibromatosis type 1 (NF-1) predisposes to juvenile myelomonocytic leukemia (JMML) via loss of function of the NF1 tumor suppressor gene and consecutive deregulation of Ras signal transduction. Affected individuals usually carry one defective NF1 allele in the germline; somatic inactivation of the second NF1 allele in hematopoietic cells is associated with transformation to leukemia. We previously demonstrated that a major mechanism for biallelic loss of NF1 function in patients with JMML/NF-1 is mitotic recombination leading to uniparental disomy (UPD) of the 17q chromosome arm (Flotho, 2007; Steinemann, 2010). Using contemporary resequencing and microarray technology, we have now revisited the genetics of NF1 inactivation in JMML. Specifically, we addressed two questions: 1) Are genetic findings in leukemic cells of JMML/NF-1 patients consistent with the clinical diagnosis and the two-hit concept? 2) Does the quintuple-negative (QN) group of JMML (patients without clinical evidence of NF-1 and negative for mutations in PTPN11, KRAS, NRAS, or CBL) contain unrecognized cases likely driven by NF1? We investigated 156 children with JMML registered in studies EWOG-MDS 98 or 2006 and tested for mutations in PTPN11, KRAS, NRAS, and CBL. Twenty-five children (16%) were clinically diagnosed as NF-1 based on &amp;gt;=6 café-au-lait spots (CALS) or family history plus CALS. Family history was positive in 9 JMML/NF-1 patients; &amp;gt;=6, 4, and 1 CALS were described in 23, 1, and 1 patients, respectively. The median age at diagnosis of JMML in the NF-1 group was 35.9 months (range, 4.2 to 71.4). Sixteen children (10%) were grouped as JMML-QN. Granulocyte DNA from bone marrow or peripheral blood collected at time of diagnosis was used for next-generation sequencing of the entire NF1 coding sequence (custom Ampliseq enrichment and Miseq, Illumina). Pathogenicity of NF1 variants was assessed according to ACMG criteria. Affymetrix Cytoscan HD array analysis was applied to detect segmental deletions or copy number-neutral loss of heterozygosity (LOH). Among 25 JMML/NF-1 cases, 8 exhibited an NF1 loss-of-function mutation at near-100% variant allelic frequency (VAF) in combination with UPD involving almost the entire 17q arm, suggesting single mitotic recombination as the leukemic driver. One case had an NF1 mutation at near-100% VAF and segmental 17q UPD, suggesting the unusual occurrence of double mitotic recombination. Nine cases carried two independent pathogenic NF1 mutations at near-50% VAF each; here, germline and somatic events could not be distinguished due to unavailability of non-hematopoietic or parental DNA. Four cases exhibited an NF1 microdeletion in combination with a pathogenic NF1 mutation at near-100% VAF; non-hematopoietic tissue available in 2 of these 4 cases failed to display the mutation, indicating the microdeletion as the constitutional event. A deleterious mutation at 71% VAF but no LOH was revealed in one sample. Only monoallelic evidence of NF1 deficiency was found in 2 cases. In the JMML-QN group, 9/16 cases had previously unrecognized NF1 alterations. Compound-heterozygous pathogenic NF1 mutations were found in 2 and homozygous pathogenic NF1 mutations combined with focal LOH in 3 patients, strongly suggesting NF1 as the JMML driver. In the absence of clinical NF-1 features, the findings in these 5 children may be explained by postzygotic NF1 mosaicism, onset of JMML before clinical manifestation of NF-1, or double somatic NF1 hits in the hematopoietic lineage. Four cases carried monoallelic pathogenic or likely pathogenic NF1 mutations with VAF at ~50% or less, providing inconclusive evidence of driver function as it is also possible that these are secondary hits in a myeloproliferative neoplasm (MPN) driven by an unidentified event. There was no genetic evidence of NF1 involvement in the remaining 7 JMML-QN cases. Several important conclusions can be drawn from our study: The clinical diagnosis is reliable in children with JMML/NF-1; we propose that it can be made on the basis of CALS and JMML alone as the patients are generally too young to display the conventional spectrum of NF-1 features. Children without features of NF-1 should only be assigned to JMML-QN after genetic work-up of NF1 because this will unmask involvement of NF1 in a significant number of cases. In suspected JMML-QN without identifiable NF1 lesion, other forms of MPN should be considered. Disclosures Locatelli: Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemeyer:Celgene: Consultancy; Novartis: Consultancy.

A child with juvenile myelomonocytic leukemia possessing a concurrent germline CBL mutation and a NF1 variant of uncertain significance: A rare case with a common problem in the era of high-throughput sequencing

Genetic changes in juvenile myelomonocytic leukemia (JMML) determine distinct subtypes, treatments, and outcomes. JMML with germline CBL mutation and somatic NRAS mutation possibly achieves spontaneous remission, but hematopoietic stem cell transplantation is indicated for other subtypes of JMML. We hereby report a child with JMML harboring a germline CBL mutation (c.1111T>C) and an NF1 variant (c.3352A>G) concurrently. After evaluation, we considered that the NF1 variant was not the major contributor. After one year of observation, this case had no signs of disease progression. This case highlights the importance of combining available evidence and clinical findings in caring for patients with unusual genomic variations.

Abstract 5895: Whole-genome characterization of lung adenocarcinomas lacking alterations in RTK/RAS/RAF/MAPK pathway

Abstract Lung adenocarcinomas (LUAD) are typically characterized by genetic activation of the receptor tyrosine kinase (RTK)/RAS/RAF/MAP kinase (MAPK) pathway. A minority of LUAD cases (20-25%) lack apparent genetic alterations in this pathway, and thus are ineligible for most targeted therapies. These candidate “oncogene negative” LUADs may harbor novel classes of oncogenic drivers or represent a biologically distinct class of tumors. To characterize the genomic landscape of oncogene-negative LUADs, we nominated 98 cases that were found to lack an activating RTK/RAS/RAF/MAPK pathway alteration in a TCGA study utilizing whole exome sequencing, microarray, and transcriptome data. We profiled these tumors with high-depth whole genome sequencing (WGS), with the goal of identifying noncoding and structural variant driver DNA alterations in both known and novel loci. Of the 98 cases, 20 harbored somatic KRAS mutations that had been missed in the prior WES and transcriptome studies because of insufficient coverage, including 8 cases with the recently targetable p.G12C mutation. 16 samples harbored oncogenic or loss-of-function structural variants in FGFR1, MAPK1, EGFR, NF1, RASA1, ARAF, NTRK2 and NRG1. 5 other samples with SNV or indels in EGFR, ERBB2 and SOS1 were reclassified as oncogene positive. Thus via comprehensive genomic analysis, we confirmed that 57 of the 98 WGS cases did not harbor any detectable alterations in genes encoding any known RTK/RAS/RAF/MAPK members, representing 13% cases chosen as “lung adenocarcinomas” for the TCGA study. Among the 57 confirmed oncogene-negative LUADs, we identified focal deletions targeting the promoter and transcription start site of tumor suppressor genes STK11, KEAP1 and SMARCA4 in 10 samples. Expression and methylation profiling suggested an enrichment of the TP53-deficient phenotype, including cell cycle and FOXM1 deregulation, among the oncogene-negative samples. Moreover, novel promoter mutations associated with increased expression were identified in ILF2, which regulates DNA damage response pathways. Finally, a subset of confirmed oncogene-negative LUADs harbored increased expression of neuroendocrine markers, suggesting that these oncogene-negative samples may either be mis-diagnosed as LUAD or represent LUAD with mixed features of other subtypes of lung cancer; indeed, 14 of the 57 confirmed oncogene-negative cases show histological features of large cell neuroendocrine lung carcinoma. This would suggest that 10% of the cases in this study are both lung adenocarcinoma and “oncogene-negative” to date. Our results provide some of the first comprehensive genomic characterization of oncogene-negative LUADs, implicating TP53 and structural variants in the pathogenesis of this common and difficult to treat entity. Citation Format: Jian Carrot-Zhang, Siddhartha Devarakonda, Nicolas Robine, Xiaotong Yao, Tiago C. Silva, Jeff Damrauer, Aditya Deshpande, Ming-Sound Tsao, Christina Yao, Chris Wong, Lisui Bao, Hyo Young Choi, Ina Felau, Jean C. Zenklusen, Gordon Robertson, Tuan Trieua, Wei-Wei Liang, Meng Zhou, Esther Rheinbay, Neil Hayes, Ekta Khurana, Li Ding, Peter Laird, Olivier Elemento, John Weinstein, David Kwiatkowski, Chris Benz, Josh Stuart, Lixing Yang, Mauro Castro, William Travis, Katherine Hoadley, Ben Berman, TCGA Analysis Network, Matthew Meyerson, Ramaswamy Govindan, Marcin Imielinski. Whole-genome characterization of lung adenocarcinomas lacking alterations in RTK/RAS/RAF/MAPK pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5895.

Genetics of renovascular hypertension in children.

In most cases of renovascular hypertension in children, the cause is unclear. The aim of this study was to investigate genetic variation as a factor in the development of renovascular hypertension in children. In a cohort of 37 unrelated children from a single tertiary referral center, exome sequencing was performed. We assessed variants in recognized and suspected disease genes and searched for novel ones with a gene-based variant-burden analysis. In the majority of patients, exome sequencing could not identify causative variants. We found a pathogenic variant in a recognized associated disease gene in five patients (three pathogenic variants in NF1, one in ELN and a deletion of chromosome 7q11.23, consistent with Williams syndrome). In two other patients, (likely) pathogenic variants were found in putative renovascular hypertension genes (SMAD6 and GLA), with clinical implications for both. Ten additional patients carried variants of uncertain significance (VUS) in known (n = 4) or putative (n = 6) renovascular hypertension disease genes. Rare variant burden analysis yielded no further candidate genes. Genetic contributors, such as germline mutations in NF1, ELN, 7q11.23del were present in only 5 out of 37 (14%) children with renovascular hypertension. Twelve other children (32%) had potentially causal variants identified, including a pathogenic variant in SMAD6; a vasculopathy gene hitherto unknown to link with renovascular hypertension. Most importantly, our data show that exome sequencing can rarely identify the cause of renovascular hypertension in nonsyndromic children. We suggest that nongenetic factors or somatic genetic variation will play a more important role.

Sporadic and Familial Variants in NF1: An Explanation of the Wide Variability in Neurocognitive Phenotype?

Background: Cognitive impairment is the most common neurological manifestation in NF1 and occurs in 30–70% of NF1 cases. The onset and severity of each specific cognitive deficit varies greatly from child to child, with no apparent external causes. The wide variability of phenotype is the most complex aspect in terms of management and care. Despite multiple research, the mechanism underlying the high heterogeneity in NF1 has not yet been elucidated. While many studies have focused on the effects of specific and precise genetic mutations on the NF1 phenotype, little has been done on the impact of NF1 transmission (sporadic vs. familial cases). We used a complete neuropsychological evaluation designed to assess five large cognitive areas: general cognitive functions (WISC-IV and EVIP); reading skills (“L'Alouette,” ODEDYS-2 and Lobrot French reading tests); phonological process (ODEDYS-2 test); visual perceptual skills (JLO, Thurstone and Corsi block tests) and attention (CPT-II), as well as psychosocial adjustments (CBCL) to explore the impact of NF1 transmission on cognitive disease manifestation in 96 children affected by NF1 [55 sporadic cases (29♀, 26♂); 41 familial cases (24♀, 17♂)].Results: Familial and Sporadic form of NF1 only differ in IQ expression. The families' socioeconomic status (SES) impacts IQ performance but not differently between sporadic and familial variants. However, SES is lower in familial variants than in the sporadic variant of NF1. No other cognitive differences emerge between sporadic and familial NF1.Conclusions: Inheritance in NF1 failed to explain the phenotype variability in its entirety. IQ differences between groups seems in part linked to the environment where the child grows up. Children with NF1, and especially those that have early diagnoses (most often in inherited cases), must obtain careful monitoring from their early childhood, at home to strengthen investment in education and in school to early detect emerging academic problems and to quickly place them into care.Trial Registration: IDRCB, IDRCB2008-A01444-51. Registered 19 January 2009.

Examination of the genetic factors underlying the cognitive variability associated with neurofibromatosis type 1

PurposeNeurofibromatosis type 1 (NF1) is an autosomal dominant disorder associated with cognitive deficits. The NF1 cognitive phenotype is generally considered to be highly variable, possibly due to the observed T2-weighted hyperintensities, loss of heterozygosity, NF1-specific genetic modifiers, or allelic imbalance. MethodsWe investigated cognitive variability and assessed the contribution of genetic factors by performing a retrospective cohort study and a monozygotic twin case series. We included data of 497 children with genetically confirmed NF1 and an IQ assessment, including 12 monozygotic twin and 17 sibling sets. ResultsIndividuals carrying an NF1 chromosomal microdeletion showed significant lower full-scale IQ (FSIQ) scores than individuals carrying intragenic pathogenic NF1 variants. For the intragenic subgroup, the variability in cognitive ability and the correlation of IQ between monozygotic NF1 twin pairs or between NF1 siblings is similar to the general population. ConclusionsThe variance and heritability of IQ in individuals with NF1 are similar to that of the general population, and hence mostly driven by genetic background differences. The only factor that significantly attenuates IQ in NF1 individuals is the NF1 chromosomal microdeletion genotype. Implications for clinical management are that individuals with intragenic NF1 variants that score <1.5–2 SD below the mean of the NF1 population should be screened for additional causes of cognitive disability.

Synchronous Periampullary Tumors in a Patient With Pancreas Divisum and Neurofibromatosis Type 1.

IntroductionIn this study, we describe for the first time a Neurofibromatosis type 1 patient with pancreas divisum, multiple periampullary tumors and germline pathogenic variants in NF1 and CFTR genes.Case reportA 62-year-old female NF1 patient presented with weakness, choluria, nausea, and diffuse abdominal pain to an emergency room service. Magnetic resonance imaging revealed an abdominal mass involving the periampullary region and pancreas divisum. After surgical resection, three synchronous neoplasms were detected including two ampullary tumors (adenocarcinoma of the major ampulla and a neuroendocrine tumor of the minor ampulla) and a gastrointestinal stromal tumor (GIST). Germline multigene panel testing (MGPT) identified two pathogenic heterozygous germline variants: NF1 c.838del and CFTR c.1210-34TG[12]T[5].ConclusionThis is the first report of a Neurofibromatosis type 1 patient with pancreas divisum and multiple periampullary tumors harboring pathogenic germline variants in NF1 and CFTR genes. The identification of two germline variants and a developmental anomaly in this patient may explain the unusual and more severe findings and underscores the importance of comprehensive molecular analyses in patients with complex phenotypes.

Spontaneous remission of acute myeloid leukemia with NF1 alteration

Acute myeloid leukemia (AML) is defined by the presence of ≥ 20% myeloblasts in the blood or bone marrow. Spontaneous remission (SR) of AML is a rare event, with few cases described in the literature. SR is generally associated with recovery from an infectious or immunologic process, and more recently possibly with clonal hematopoiesis. We review the literature and assess the trends associated with SR, and report a new case of a 58-year-old man with a morphologic diagnosis of AML associated with a severe gastrointestinal (GI) tract infection. The patient had an NF1 variant that was previously unreported in AML as the only clonal abnormality. After treatment of the infection, the increased blast population subsided with no leukemia-directed therapy, and the patient has remained in a continuous, spontaneous complete remission for > 2 years.

Detection of the Genomic Variants in the Paired De Novo-Relapsed Samples with Pan-Cancer Panel and Whole Exome-Seq in Adult B-Cell Acute Lymphoblastic Leukemia

Background: The relapse rate is around 20-40% in adult B-cell acute lymphoblastic leukemia (B-ALL). The genetic defects are the major reasons for the relapse and poor outcome. We screened the genomic variants with Pan-cancer panel in B-ALL patients and whole exome-seq (WES) in the paired de novo-relapsed B-ALL samples. Methods: The xGen Pan-Cancer Panel (IDT), which has been designed with the probes targeting 127 significantly mutated genes from the TCGA database across 12 tumor tissue types (Nature, 502:333-339) was used. Agilent SureSelect Human All Exon V4+UTRs (Agilent) was used to target coding exons plus UTRs for the WES analysis. The genomic DNA from 81 Philadelphia chromosome positive (Ph+) B-ALL patient samples (71 de novo and 10 relapsed, 14 to 77 years old) collected between June 2008 and June 2016 at Zhongda Hospital Southeast University were used for the Pan-cancer panel screening. All DNA samples were sheared and generated approximately 260bp DNA fragments. The fragmented DNA was processed into Illumina compatible sequencing libraries using the Kapa Hyper Prep Kit. Each library was uniquely barcoded and captured by either the Pan-cancer panel or WES probes, followed by PCR amplification and sequencing on a HiSeq 2500 (Illumina) with 2x100 bp reads. The sequencing reads were aligned to the human genome (hg19) by following Broad Institute's GATK best practice pipeline to call germline short variants (SNPs and Indels). Called variants were annotated using ANNOVAR (version 2.3). Variants with exonic, nonsynonymous, stopgain, or stoploss, novel SNPs (not covered by dbSNP138), and with predicted deleterious/damaging functions were manually surveyed by IGV to confirm. Two paired de novo-relapse samples from Ph(+) B-ALL patients were performed the WES analysis. Results: We identified a total of 3945 single nucleotide variants (SNVs), 2222 insertions and deletions (INDELs) in the Pan-cancel panel analysis in 81 Ph(+) B-ALL patients. Among these, 101 genomic variants are with amino acid change, 8 are with stopgain, and 5944 have not been previously reported. We evaluated the frequency and distribution of likely pathogenic variants (PVs) detected in the cohort. Likely PVs were defined by SIFT algorithm which predicts whether an amino acid substitution is likely to affect protein function. Defined by the SIFT's qualitative score 'deleterious', we detected 46 PVs. Among these, PVs were commonly detected in KMT2C, APC, CDKN1A, NSD, BRCA1, EPHA3, and PIK3CG. The PVs were also validated with the Sanger DNA sequencing in the patients. The patients with the likely PVs have significantly higher WBC count (61*10^9/L vs. 24.45*10^9/L, P=0.004). Survival analysis showed that the patients with likely PVs had a worse event-free survival (EFS) and overall survival (OS), the difference was statistically significant (8 months vs. 15 months, P=0.017 and 14 months vs. 25 months, P=0.021). In order to gain an insight to the gene mutations contributing to the disease relapse, we compared the mutants spectrum between the de novo and relapsed samples. We found the genomic variants in NF1, CDK12, mTOR, and USP9X genes appeared mostly in relapsed samples, indicating their roles in the relapse. Using the WES, we further analyzed the genomic variants in two paired de novo and relapsed samples. We detected totally 40354 (de novo) and 16822 (relapsed) genomic variants, and among these 10415 (de novo) and 3082 (relapsed) are with amino acid change in patient 1; likewise, 30130 (de novo) and 14003 (relapse), and among these 7200 (de novo) 2534 (relapsed) with amino acid changes in patient 2. Totally 216 genomic variants with amino acid changes in 162 genes appeared only in the two relapsed samples, among which 10 genomic variants in ADAMTS8, CDK11B, EFCAB4B, FBXL21, HYDIN, IRF2BPL, MIR205HG, PRIM2, ZNF717, ZNF880 appeared in both relapsed samples, revealing their driver roles in relapse. Also, 110 of the 216 genomic variants are not previously reported. Conclusion: Genomic variants in common human cancer driver genes were also detected in B-ALL patients. The new genomic variants detected in the relapse samples may be involved in the oncogenesis of the relapse, which will be further verified with functional analysis. Our data also suggested the significance of developing more efficient new therapies to prevent the relapse in hematological malignancies. Disclosures No relevant conflicts of interest to declare.

Strategy for Identification of an Inherited Leukemia Predisposition in a 299 Patients Cohort with Tumor-Only Sequencing Data

Myeloid neoplasms (MN) are usually sporadic late-onset cancers; nevertheless, growing evidence suggests that ~5% of the cases could emerge as a consequence of inherited predisposition. Indeed, the 2016 revision of the World Health Organization (WHO) includes hereditary myeloid malignancies (HMMs) as a separate disease entity. Distinguishing somatic from germline variants is of vital importance, in order to establish an appropriate individualized management and counsel the patients and their relatives. Germline tissues are required to discriminate HMMs alterations, but they are not always available for testing at the time of diagnosis; therefore additional strategies should be put in place in order to identify variants potentially conferring genetic predisposition to MN. The aim of the present work is to develop a tool to identify HMMs cases from tumour-only sequencing data. To this end, we reviewed tumor-only NGS reports from 299 cases of patients with myeloid malignancies sequenced with a custom Pan Myeloid Panel (PMP) that targets 48 genes, of which 21 are described in literature as HMM-associated genes. All patients signed a written informed consent form for genetic testing, research and tissue banking provided by the Biobank of the University of Navarra (UN) and were approved by the Ethical and Scientific Committees of the UN. Variant calling files (VCF) of the 299 cases were run through an algorithm aiming at identifying those variants suggestive of being of germline nature (Figure 1). We considered as indicative of potential inherited origin, variants detected in bone marrow samples at a ~50% VAF classified as pathogenic, likely pathogenic or of unknown significance. This first filter yielded 90 "likely HMMs" patients harboring a total of 104 suspicious of being germline variants affecting at least one of the 21 HMM-associated genes. Our algorithm included three additional filters: mutational patterns suggestive of germline nature of the variants, analysis of variant allele frequency (VAF) on follow up data, and sequencing of non-myeloid tissues (Figure 1). Firstly, we found 10 "likely HMMs" patients presenting double hit mutations in one of three genes: DDX41 (n=7), CEBPA (n=2) and RUNX1 (n=1). Additionally, there was available follow up data for 8 cases included in our cohort, harboring 11 suspicious variants. From them, VAFs in 4 variants affecting ASXL1, CBL, IKZF1 and GATA2 genes, drastically changed over time, indicating that those variants were somatic, whereas the other 7 variants affecting ASXL1 (n=2), DDX41 (n=2), IKZF1 (n=1), and RUNX1 (n=1) genes suspected to be germline maintained VAF ~50% with evident change of the accompanying variants over time. Finally, we collected non-myeloid tissue samples from 8 patients, harboring a total of 9 variants in ASXL1 (n=2), DDX41 (n=1), GATA2 (n=1), IKZF1 (n=1), NF1 (n=3), and SH2B3 (n=1) genes. Sequencing data confirmed germline nature of 6 of the 9 tested variants; 3 of the 6 germline variants have previously been shown to be linked to MN (DDX41 p.Asp140Glyfs*2 in UPN1, ASXL1 p.Gly967del in UPN11, and NF1 p.Met992del in UPN19). Of note, two of these bona-fide pathogenic germline mutations had been previously highlighted as potential germline variants when applying our criteria of mutational patterns and/or follow up data indicative of germline nature, in so showing the effectiveness of our algorithm (DDX41 in UPN1, and ASXL1 in UPN11). Regarding the usefulness of the different tissues, we found that skin fibroblasts, hair follicles and CD3+ T cells helped discriminating the nature of the variants; on the contrary, DNA from hair follicles showed poor DIN values and scarce DNA concentration. Similarly, DNA isolated from buccal swab showed in general poor quality metrics, and high level of contamination with tumour DNA in one case, proving it unsuitable for discrimination of the nature of the variants in some instances. Our data supports the importance of considering variants found upon tumor-only sequencing as potentially of germline origin, and we offer a pipeline to define the nature of the variants. We hope to provide insights for genetic laboratories facing this relatively new challenge of discriminating somatic from germline variants in tumor sequencing data. Disclosures No relevant conflicts of interest to declare.

Abstract 3105: Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma

Abstract Background: Analysis of circulating tumor DNA (ctDNA) provides a noninvasive method to profile tumor-associated genomic aberrations, heterogeneity and evolution. Methods: Peripheral blood from 5 newly diagnosed and 33 relapsed high-risk neuroblastoma patients was serially profiled for ctDNA (n=1-7 samples/patient; total n=122 samples) with the FoundationACT assay that utilizes hybrid capture-based genomic profiling of 62 genes. Samples were sequenced to a median unique coverage depth of at least 3168x and variants were evaluated and compared with temporally-matched tumor sequencing and imaging evaluations. Results: Ninety-three percent (114/122) of peripheral blood samples yielded suitable cell-free DNA for sequencing. ctDNA was detected in 68% (78/114) of samples (maximum somatic allele frequency [MSAF] &amp;gt;0; median MSAF=0.53%; range MSAF 0-79%). At least 1 pathogenic genomic alteration (genomic short-variant or amplification) was found in 54% (61/114) of samples (range 1-6 alterations). Fifty-four percent (13/24) of detected ctDNA genomic-short variants were not present in temporally matched tumor samples at diagnosis or relapse (collected within 3 months of each other; n=23 patients), including pathogenic variants in ALK, TP53, TERT, NF1, FLT3, PTPN11, and PIK3CA. There was 100% concordance between the detection of MYCN (6/6) and ALK (3/3) amplification in paired ctDNA/tissue samples. For example, a newly diagnosed patient with stage 4 neuroblastoma had MYCN amplification noted on both tumor and ctDNA sequencing, however had 3 separate ALKmutations (R1275Q, F1245L, and F1174L) uniquely identified in ctDNA. Twenty-eight patients had multiple ctDNA samples sequenced (range 2-7 samples) and 50% (14/28) had alterations emerge or regress across serial samples. For example, pathogenic variants in ALK, BRCA2, NRAS, PTEN, TP53, CDKN2A, PTPN11, ABL1,CDH1, MET, ERRFl1 and ERBB2 appeared in subsequent ctDNA sequencing that were not present in the initial ctDNA or tumor sequencing. Overall, serial ctDNA profiling identified additional pathogenic variants in driver cancer genes beyond that derived from tumor sequencing in 45% (17/38) of cases, including identifying targetable ALK-RAS-MAPK pathway alterations uniquely in ctDNA in 21% (8/38) of cases. For the 18 patients that had 3 or more ctDNA samples, 33% (6/18) developed ctDNA unique ALK-RAS-MAPK pathway mutations during therapy. Finally, in most cases ctDNA profiling was complementary to standard imaging surveillance, however in 3 cases rising ctDNA allele frequencies occurred prior to clinical or imaging signs of disease relapse; additional correlation of ctDNA data and disease evaluations is ongoing. Conclusions: Sequencing of ctDNA from neuroblastoma patients identified clinically actionable tumor-associated genetic aberrations emerging under the selective pressure of standard and targeted therapies. Citation Format: Kristopher R. Bosse, Samantha Buongervino, Maria Lane, Adam Hyman, Maria Gemino-borromeo, Jennifer Saggio, Alana Fitzsimmons, Brady Forcier, Anne Murphy, John Wick, Matthew Cooke, Jennifer Webster, Russell Madison, Alley Welsh, Vincent A. Miller, Siraj M. Ali, John M. Maris, Yael P. Mosse. Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma [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 3105.

NF1 Patients Receiving Breast Cancer Screening: Insights from The Ontario High Risk Breast Screening Program.

Neurofibromatosis Type I (NF1) is caused by variants in neurofibromin (NF1). NF1 predisposes to a variety of benign and malignant tumor types, including breast cancer. Women with NF1 <50 years of age possess an up to five-fold increased risk of developing breast cancer compared with the general population. Impaired emotional functioning is reported as a comorbidity that may influence the participation of NF1 patients in regular clinical surveillance despite their increased risk of breast and other cancers. Despite emphasis on breast cancer surveillance in women with NF1, the uptake and feasibility of high-risk screening programs in this population remains unclear. A retrospective chart review between 2014–2018 of female NF1 patients seen at the Elizabeth Raab Neurofibromatosis Clinic (ERNC) in Ontario was conducted to examine the uptake of high-risk breast cancer screening, radiologic findings, and breast cancer characteristics. 61 women with pathogenic variants in NF1 enrolled in the high-risk Ontario breast screening program (HR-OBSP); 95% completed at least one high-risk breast screening modality, and four were diagnosed with invasive breast cancer. Our findings support the integration of a formal breast screening programs in clinical management of NF1 patients.

Breast cancer risk in neurofibromatosis type 1 is a function of the type of NF1 gene mutation: a new genotype-phenotype correlation

BackgroundNeurofibromatosis type 1 (NF1) predisposes to breast cancer (BC), but no genotype-phenotype correlations have been described.MethodsConstitutional NF1 mutations in 78 patients with NF1 with BC (NF1-BC) were compared with the...

P1.03-10 The Molecular Spectrum of NF1 Variants in Chinese Non-Small-Cell Lung Cancer Patients

P1.03-10 The Molecular Spectrum of NF1 Variants in Chinese Non-Small-Cell Lung Cancer Patients