LZTR1 Mutations Research Articles

Abstract 3941: Wild-type KRAS dosage in mutant KRAS lung cancer: Implications for tumorigenesis and therapeutic response

Abstract Lung adenocarcinomas (LUAD) are frequently driven by activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS). Previous studies have suggested that wild-type (wt)-RAS signaling may modulate cancer progression and drug resistance in these tumors. However, the interplay between oncogenic KRAS and wt-RAS-like GTPases in lung cancer biology and treatment remains debated. Here, we investigated the impact of increased wt-KRAS dosage in LUAD harboring mutant KRAS on lung cancer phenotypes and response to anti-KRAS therapies. Increased wt-KRAS dosage occurs in LUAD due to the loss of LZTR1, a proteostatic regulator of RAS-like GTPases. LZTR1 mutations, deletions, or downregulation co-occur with oncogenic KRAS mutations in more than 40% of LUAD patients. Using a global protein stability screen, we identified LZTR1 as the main regulator of the stability of wt-KRAS, but not of the oncogenic KRAS mutants. Proteomic analysis of LUAD cells confirmed that LZTR1 loss leads to elevated expression of wt-KRAS, but not of KRAS-G12D, indicating that LZTR1 loss affects wt-RAS rather than mutant KRAS signaling. LZTR1 loss in LUAD patients is associated with poor prognosis and hypoxic expression signatures. In a mouse model of Kras-mutated LUAD, Lztr1 haploinsufficiency promoted tumor initiation and progression. Moreover, Lztr1 loss results in hypoxic tumors with inflamed, tortuous, insufficiently perfused, and leaky vessels. Integrative proteomic and phosphoproteomic analysis revealed increased RAL/TBK1 pathway activity in LZTR1-depleted lung cancer cells and Lztr1-deleted tumors. This leads to enhanced secretion of proangiogenic and proinflammatory factors, such as VEGF and TNFα, by LZTR1-deficient cells and an increased number of neutrophils in Lztr1-deleted tumors. TNF-activated neutrophils present at vascular branching points can modulate vascular remodeling by influencing the formation and function of blood vessels. Pharmacological inhibition of RAL/TBK1 signaling with amlexanox, an anti-NF-κB/TBK1 drug, reduces TNFα secretion, decreases the number of neutrophils, and normalizes the tumor-associated vasculature. We demonstrated that increased wt-KRAS dosage, but not other small GTPases, mimicked the effects of LZTR1 loss on RAL/TBK1 signaling and VEGF secretion, suggesting that LZTR1 loss alters the heterogeneity of KRAS-mutated lung cancer by modulating wt-KRAS levels. Furthermore, MRTX1133 a selective inhibitor of KRAS-G12D, showed limited effect in the Kras-G12D, Lztr1-loss LUAD model, whereas the combination of MRTX1133 and amlexanox suppress tumor growth, likely due to the restoration of the tumor vasculature by RAL/TBK1 inhibition. Our study reveals novel insights into the role of wt-KRAS dosage in mutant KRAS lung cancer and identifies potential therapeutic strategies to target patients with dysregulated KRAS proteostasis due to LZTR1 loss. Citation Format: Tonci Ivanisevic, Greetje Vande Velde, Peihua Zhao, Wout Magits, Raj N. Sewduth, Anna A. Sablina. Wild-type KRAS dosage in mutant KRAS lung cancer: Implications for tumorigenesis and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3941.

High-Hyperdiploid Acute Lymphoblastic Leukemia in Children with LZTR1 Germline Variants

Introduction Firstly described in 1967, hyperdiploidy (HD) is the most frequent genetic abnormality in B-cell- precursor acute lymphoblastic leukemia (BCP-ALL) in children, comprising about 25% of all cases. A not yet exactly defined proportion have predisposing pathogenic germline variants in DNA repair pathway genes, chromatin remodeling factors, transcription factors regulating B-cell development (particularly ETV6) or receptor tyrosine kinases pathway genes like RAS/RAF. Among the latter, mutations have mostly been described in PTPN11 and SOS1, but not yet in other components of this central regulatory hub of cellular communication. Germline loss-of-function (LOF) LZTR1 mutations are typically linked to hereditary nerve sheath tumors. However, it remains largely unknown, if other tumor entities are associated with LZTR1 LOF germline mutations, potentially broadening the spectrum of malignancies associated with RASopathies. Aim Our aim is to understand the frequency and the impact of LZTR1 germline variants in HD BCP-ALL of childhood. Material and Methods We analyzed WES data of 283 children with BCP-ALL for the presence of pathogenic variants in the LZTR1 cancer predisposition gene. For a part of the detected variants, we performed further functional analyses to investigate the effect of the alteration on protein function. We applied a Drosophila model that is particularly suited for functional evaluation of Ras pathway activity. Results We identified LZTR1 germline variants in 10/283 (3.5%) patients (Figure 1). Interestingly, in 6 out of 283 (2.1%) children the LZTR1 variants were classified as pathogenic/likely pathogenic (P/LP). The majority of patients (5/6) harboring a P/LP germline LZTR1 variant presented with a HD BCP-ALL. Only two of our patients showed concomitant phenotypic features indicative of an underlying syndromic condition. Patient P1 presented with a HD BCP-ALL at the age of 9 years and mild psychomotor delay. Sequencing revealed a likely pathogenic variant (p.Arg283Trp) that was not yet reported in association with RASopathy syndromes. The second child P8, also diagnosed with a HD BCP-ALL, carried a well-known autosomal dominant mutation described in Noonan syndrome (NS) variant (p.Gly248Arg). The child was characterized by facial dysmorphism, as well as mild developmental delay, although a diagnosis of NS was not established prior to development of the BCP-ALL. The other three patients had no clinical peculiarities other than HD BCP-ALL. We functionally characterized patient derived LZTR1 variants in dLztr1-depleted ISC. Like wild-type hLZTR1 wt, hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg restored control-like ISC lineage production, suggesting that hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg still regulate Ras ubiquitination. In contrast, hLZTR1 p.Tyr535Ter results in even 1.8-fold higher ISC lineage production than dLztr1-RNAi. Beyond ISC production as a readout, we employed a second experimental paradigm directly addressing Ras signalling activity with a translocating modified ERK sensor. In line with Ras activity control of LZTR1, hLZTR1 wt significantly reduced ERK activity, while the putatively LOF variant hLZTR1 p.Tyr535Ter variant significantly increased Ras pathway activation by 1.4-fold over controls. We also noticed that hLZTR1 p.Tyr535Ter induced extensive membrane blebbing and nuclear fragmentation of GFP positive cells likely indicating programmed cell death. Quantification of apoptotic cells revealed an 11.1-fold increase compared to controls, which was not observed for hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg. Most notably, forced expression of hLZTR1 p.Arg283Trp andhLZTR1 p.Tyr535Ter increased mitotic recombination by 4-fold and 3.4-fold, respectively, which was not observed for the hLZTR1 p. Lys761Arg variant. Conclusion In our cohort, approximately 2% of all children with BCP-ALL harboured a P/LP LZTR1 germline variation, not necessarily linked with clinical appearance of Noonan-syndrome-like features, but to the development of a high hyperdiploid karyotype. By applying a Drosophila model, we demonstrated that patient-derived LZTR1 germline variants affect RAS pathway activation, ERK accumulation, cell proliferation, DNA recombination and apoptosis. Figure 1: Depiction of the ten LZTR1 variants detected in an unselected pediatric cohort of 283 BCP-ALL patients

P11.58.A Case of a complex neurocutaneous syndrome characterized by extensive peripheral nerve sheath tumors and somatic ERBB2 mutation

Abstract Schwannomatosis is a rare genetic tumor predisposition syndrome characterized by the presence of multiple non-intradermal schwannomas and the definitive absence of vestibular nerve involvement. Though considered benign, the burden of tumors can cause significant morbidity in the form of motor dysfunction and refractory neuropathic pain. Treatment is focused on mitigating these symptoms, which includes resection of offending tumors when feasible and anti-angiogenesis therapy with bevacizumab. Options for targeted medical therapies are lacking. Schwannomatosis is molecularly distinct from the other neurofibromatoses and by definition lacks constitutional mutations in NF1 and NF2. Instead, constitutional mutations in SMARCB1 or LZTR1 are often the “first hit” in tumorigenesis. Activating mutations in ERBB2 (also designated HER2) have been identified as oncogenic drivers in peripheral nerve sheath tumors in patients who lack these typical constitutional tumor suppressor gene mutations. Here we report a case of extensive peripheral nerve sheath tumors in the setting of apparent somatic mosaicism of an ERBB2 mutation. A 48-year-old man with history of chronic back pain presented with diffuse enlarging soft tissues masses primarily involving the left neck and occiput. Examination revealed areas of cutaneous heterogeneous hyperpigmentation, decreased muscle bulk and dorsiflexor weakness in the bilateral lower extremities, and steppage gait. Neuroimaging showed extensive T2 hyperintense, mildly enhancing masses within the bilateral thoracic and lumbar spine neuroforamina, intercostal nerves, paraspinal musculature, and lumbosacral plexus. Tissue from two separate tumor sites was morphologically consistent with schwannoma with some features of neurofibroma. Immunohistochemistry revealed partial loss of staining for SMARCB1. Whole exome sequencing (WES) on blood and tumor tissue did not show pathogenic germline or somatic variants for NF1, NF2, SMARCB1, LZTR1, or an array of tumor predisposition syndromes. WES on tumor tissue from both sites however did reveal a somatic ERBB2 variant (p.D769Y), suggesting mosaicism. ERBB2 D769Y has previously been classified as an activating mutation that confers sensitivity to some small molecule receptor tyrosine kinase inhibitors. Patients with ERRB2-mutated peripheral nerve sheath tumors may have broader therapeutic options in the variety of available tyrosine kinase inhibitors studied in other cancers.

A Rare Heterozygous LZTR1 Mutation in an Infant with Noonan Syndrome Running Title: LZTR1 Variant in Noonan Syndrome

A Rare Heterozygous LZTR1 Mutation in an Infant with Noonan Syndrome Running Title: LZTR1 Variant in Noonan Syndrome

Cardiovascular Abnormalities and Gene Mutations in Children With Noonan Syndrome.

Background: Common cardiac abnormalities in Noonan syndrome (NS) include congenital heart diseases (CHD), pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). Molecular diagnoses are enabling earlier and more precise diagnosis of patients who have a subtle or atypical presentation. The aims of this study were to investigate genotype-phenotype associations with respect to Noonan syndrome (NS)-associated cardiac abnormalities and catheter or surgery-based interventions conditions. Methods: From January 2019 to December 2021, 22 children with a confirmed molecular diagnosis of NS combined with cardiovascular abnormalities were consecutively enrolled into the current study. A comprehensive review was carried out of echocardiography and electrocardiogram results, second-generation whole-exome sequencing results and catheter or surgery-based interventions conditions. Results: The main manifestations of electrocardiogram abnormalities were QTc prolongation, abnormal Q wave in the precordial lead and limb lead, right ventricular hypertrophy and left or right deviation of the electrical axis. The most commonly detected abnormality was pulmonary valve dysplasia with stenosis, seen in 15 (68.2%) patients, followed by atrial septal defect in 11 (50%) patients. Seven genes (RAF1, RIT1, SOS1, PTPN11, BRAF, SOS2, and LZTR1) were found to contain disease-associated variants. The most commonly observed genetic mutations were PTPN11 (27%) and RAF1 (27%). Each genotype was associated with specific phenotypic findings. RIT1, SOS1, PTPN11, and SOS2 had common echocardiography features characterized by pulmonary valve stenosis, while RAF1 was characterized by HCM. Interestingly, patients with BRAF mutations were not only characterized by HCM, but also by pulmonary valve stenosis. In the cohort there was only one patient carrying a LZTR1 mutation characterized by left ventricle globose dilation. Ten cases underwent catheter or surgery-based interventions. All the operations had immediate results and high success rates. However, some of the cases had adverse outcomes during extended follow-up. Based on the genotype-phenotype associations observed during follow-up, BRAF and RAF1 genotypes seem to be poor prognostic factors, and multiple interventions may be required for NS patients with severe pulmonary stenosis or myectomy for HCM. Conclusions: The identification of causal genes in NS patients has enabled the evaluation of genotype-cardiac phenotype relationships and prognosis of the disease. This may be beneficial for the development of therapeutic approaches.

Skull Base Tumors: Neuropathology and Clinical Implications.

Tumors that arise in and around the skull base comprise a wide range of common and rare entities. Recent studies have advanced our understanding of their pathogenesis, which in some cases, have significantly influenced clinical practice. The genotype of meningiomas is strongly associated with their phenotype, including histologic subtype and tumor location, and clinical outcome. A single molecular alteration, NAB2-STAT6 fusion, has redefined the category of solitary fibrous tumors to include the previous entity hemangiopericytomas. Schwannomas, both sporadic and familial, are characterized by near ubiquitous alterations in NF2 , with additional mutations in SMARCB1 or LZTR1 in schwannomatosis. In pituitary adenohypophyseal tumors, cell lineage transcription factors such as SF-1, T-PIT, and PIT-1 are now essential for classification, providing a more rigorous taxonomy for tumors that were previously considered null cell adenomas. The pituicyte lineage transcription factor TTF-1 defines neurohypophyseal tumors, which may represent a single nosological entity with a spectrum of morphologic manifestations (ie, granular cell tumor, pituicytoma, and spindle cell oncocytoma). Likewise, the notochord cell lineage transcription factor brachyury defines chordoma, discriminating them from chondrosarcomas. The identification of nonoverlapping genetic drivers of adamantinomatous craniopharyngiomas and papillary craniopharyngiomas indicates that these are distinct tumor entities and has led to successful targeted treatment of papillary craniopharyngiomas using BRAF and/or mitogen-activated protein kinase inhibitors. Similarly, dramatic therapeutic responses have been achieved in patients with Langerhans cell histiocytosis, both with BRAF -mutant and BRAF -wildtype tumors. Familiarity with the pathology of skull base tumors, their natural history, and molecular features is essential for optimizing patient care.

175 Concomitant LZTR1 and NF1 mutations contribute to the diversity of the Neurofibromatosis 1 phenotypic spectrum

Neurofibromatosis 1 (NF1) is a genetic disorder caused by germline mutations in the NF1 gene, manifested by various tissues proliferation, including plexiform neurofibromas. Even though somatic loss of NF1 is a common molecular mechanism, plexiform neurofibroma phenotype, varies from indolent to locally aggressive, suggesting contribution of additional modifiers. We investigated the development of a life-threatening, rapidly progressive plexiform neurofibroma in a 9-month-old female infant with NF1. Germline and somatic genetic analysis was conducted through whole exome sequencing and chromosomal microarray analysis. Site-directed mutagenesis, Lentiviral transduction, cell culture and western blot were used for functional studies. In addition to a de-novo pathogenic variant in the NF1 gene, the proband harbored a known pathogenic variant in another Rasopathy gene, LZTR1 which was inherited from the patient’s mother. Further somatic analysis revealed NF1 loss of heterozygosity and a variant in the gene encoding a G Protein Coupled Receptor, GNAZ. Cells expressing the mutant GNAZ exhibited increased ERK activation compared to those expressing the wild type GNAZ. We suggest that variants in NF1, LZRT1, and GNAZ act synergistically in our patient, leading to activation of the MAPK pathway and contributing to the severity of her plexiform neurofibromatosis. This case study demonstrates the power of next generation sequencing in understanding the patho-mechanisms of complex cases, and contributes to the knowledge of germline and somatic non-NF1 variants affecting the clinical phenotype.

Abstract NG08: Oncoprotein duality in cancer and developmental syndromes: Lessons learned from RAS GTPases

Abstract NG08: Oncoprotein duality in cancer and developmental syndromes: Lessons learned from RAS GTPases

Generation of a Mouse Model to Study the Noonan Syndrome Gene Lztr1 in the Telencephalon.

The leucine zipper-like transcriptional regulator 1 (Lztr1) is a BTB-Kelch domain protein involved in RAS/MAPK pathway regulation. Mutations in LZTR1 are associated with cancers and Noonan syndrome, the most common RASopathy. The expression and function of Lztr1 in the developing brain remains poorly understood. Here we show that Lztr1 is expressed in distinct regions of the telencephalon, the most anterior region of the forebrain. Lztr1 expression was robust in the cortex, amygdala, hippocampus, and oligodendrocytes in the white matter. To gain insight into the impact of Lztr1 deficiency, we generated a conditional knockout (cKO) restricted to the telencephalon using Foxg1IREScre/+. Lztr1 cKOs are viable to postnatal stages and show reduced Lztr1 expression in the telencephalon. Interestingly, Lztr1 cKOs exhibit an increase in MAPK pathway activation in white matter regions and subsequently show an altered expression of stage-specific markers in the oligodendrocyte lineage with increased oligodendrocyte progenitor cells (OPCs) and decreased markers of oligodendrocyte differentiation. Moreover, Lztr1 cKOs also exhibit an increased expression of the astrocyte marker GFAP. These results highlight the generation of a new mouse model to study Lztr1 deficiency in the brain and reveal a novel role for Lztr1 in normal oligodendrocyte and astrocyte development in the telencephalon.

EPCO-04. GENOMIC AND EPIGENOMIC HALLMARKS OF SCHWANNOMATOSIS SCHWANNOMAS

Abstract Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often, painful neuronal tumors called schwannomas (SWNs). Very little is known about the epigenomic and genomic alterations in SWNTS related SWNs (SWNTS-SWNs) other than germline mutations in SMARCB1 and LZTR1 plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q. Herein, we have comprehensively established the specific molecular signatures of SWNTS-SWNs. We found that tumor anatomic location was associated with pain and distinct DNA methylation and transcriptional signatures. DNA sequencing revealed several novel non-22q deletions, specifically in LZTR1-mutant cases. Whole-genome sequencing identified novel recurrent structural rearrangements. Further, chromosomal aberrations in SWNTS-SWNs were accompanied by increased transcription of mismatch repair genes. Our transcriptome analysis detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, more commonly in LZTR1-mutant tumors. In addition, we identified the specific genetic, epigenetic, and transcriptional hallmarks of painful SWNs that may be harnessed to develop new treatments for this debilitating syndrome.

Epigenomic, genomic, and transcriptomic landscape of schwannomatosis

Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Abstract A14: Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling

Abstract Genetic evidence explicitly points out a role for LZTR1, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, in human disease. Mutations in LZTR1 concurrent with loss of heterozygosity have been associated with glioblastoma and schwannomatosis. LZTR1 emerges as a predisposition gene for paediatric neoplasms. LZTR1 mutations are significantly higher than the background in liver cancers and testicular germ cell tumours. LZTR1 has been also recently added to the list of genes causing Noonan Syndrome, a RASopathy disorder. However, how LZTR1 contributes to human disease and whether it regulates the RAS/MAPK pathway are not known. We found that whereas Lztr1 knockout in mice is embryonically lethal, Lztr1 haploinsufficiency partially recapitulates Noonan syndrome phenotypes, including growth delay, craniofacial dysmorphism, and congenital heart defects. On the other hand, LZTR1 loss in primary Schwann cells drives their dedifferentiation into proliferating cells. By trapping LZTR1 complexes from intact mammalian cells, we identified RAS as a substrate for the LZTR1/CUL3 ubiquitin ligase complex. Immunoprecipitation experiments confirmed that endogenous LZTR1 is associated with endogenous RAS. Ubiquitome and proximity ligation analyses showed that Lztr1 loss abrogates ubiquitination of endogenous RAS specifically at lysine 170. Molecular simulations together with the analysis of Hras-K170R knock-in cells demonstrated that LZTR1-mediated ubiquitination at lysine 170 inhibits RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations abolish RAS ubiquitination due to disruption of either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. In line with the work of Bigenzahn et al., Giulio Superti-Furga lab, CeMM (see separate abstract), our study implies LZTR1-mediated RAS ubiquitination as a key step in the control of the RAS GTPases. The discovered molecular mechanism provides a fundamental explanation for the role of LZTR1 in human disease. Citation Format: Mikhail Steklov, Silvia Pandolfi, Maria Francesca Baietti, Paolo Carai, Mingzhen Zhang, Hyunbum Jang, Yanyan Cai, Eric Legius, Jan Tavernier, Kris Gevaert, Francis Impens, Stephane Heymans, Ludwine Messiaen, Ruth Nussimov, Sven Eyckerman, Anna Sablina. Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A14.

COMP-10. THE MUTATIONAL PROFILE OF GLIOBLASTOMA-DERIVED CELL-FREE DNA IN PLASMA REPRESENTS A DISTINCT SUBSET OF THE SOMATIC MUTATIONS PRESENT IN GLIOBLASTOMA SOLID TUMOR DNA

Abstract Glioblastoma’s mutational landscape varies widely in the same tumor. Using conventional criteria to identify mutations from a focal tissue specimen (e.g., variant allele frequency > 5%) undersamples glioblastoma’s broad clonal diversity, which may limit detection of glioblastoma-derived circulating cell-free DNA in plasma (i.e., circulating tumor DNA; ctDNA). Here, we sought to enhance somatic variant identification in solid tumor DNA to improve detection and characterize glioblastoma-derived ctDNA. Tumor DNA and plasma cell-free DNA (collected < 24 hours prior to the surgical procedure) were isolated from eight glioblastoma patients. DNA was capture-enriched using a custom-designed, glioblastoma-targeted, next-generation sequencing panel (124 genes, 118 kb) followed by paired-end sequencing. Samples were prepared in duplicate with molecular barcodes to enable detection of very-low frequency variants. Somatic mutations in tumor DNA were identified using variable allele frequency thresholds and were considered positive in ctDNA if present in both duplicate samples. Using a lower allele frequency threshold to identify mutations in tumor DNA significantly increased detection of ctDNA (F(1.04,7.29)=14.81, P=0.006). At a solid tumor allele frequency threshold of ≥ 5%, only a single patient (12.5%) had tumor mutations detected in ctDNA. However, at a threshold ≥ 1%, all patients (100%) had at least one tumor mutation detected in ctDNA. Moreover, at a threshold ≥ 0.5%, 7 out of 8 patients (87.5%) had > 12 tumor mutations present in ctDNA. The increased detection of ctDNA enabled the subsequent discovery that somatic mutations in APC, KIT, MSH6, and NF1 were more likely to be present in ctDNA compared to somatic mutations in ATRX, LZTR1, SLC26A3, and TERT which were absent in ctDNA (χ 2=8.0, P=0.005). Thus, stronger sampling of glioblastoma’s genetic heterogeneity in tumor DNA improves detection of ctDNA allowing comparisons between mutational profiles that may lead to the identification of similarities and differences with key biologic and/or clinical implications.

OS2.3 Relevance of Neurofibromatosistype 1 and schwannomotosis in extramedullary spine tumors

Abstract BACKGROUND Extramedullary spine tumors represent two-thirds of all primary spine neoplasms. Approximately half of these are peripheral nerve sheath tumors, mainly neurofibromas and schwannomas and neurofibromatosis or schwannomatosis can be suspected. Given the rarity of this condition the clinical genetic and radiological features remains to be better define. The aim of this study was to characterize the clinical, radiologic presentation of patients with widespread spinal disease and to identify gene mutation. MATERIAL AND METHODS We selected patient with a at least: intradural extramedullary, or extradural intraspinal (tumor within the spinal canal), or extradural paraspinal (tumor at the neural foramenor extending outward into adjacent tissues) neoplasms and no other tumors such as meningiomas in the spine at spine MRI. Patients’ DNA were analyzed by Targeted NGS by means a custom gene panel including NF1, NF2, LZTR1, SMARCB1 genes. RESULTS 63 patients were identified31 had few isolated tumors, involving spinal roots (Multiple Neurofibromas Few Spinal Root, MNFSR), 18 had bilateral neurofibromas involving all spinal roots. 14 had a single lesion; 10 cases were familiar and 53 sporadic. Genetic analysis showed NF1 gene mutations (in prevalence splicing or missense) in 49 cases LZTR1 mutations in 3 and in the others 11 no mutation or deletion was detected. Pain was the hallmark symptom in patients with LZTR1 mutations, while all familial cases all had NF1 diagnosis. About 50% of them had few cutaneous manifestations. CONCLUSION In patients with extramedullary spine tumors is important to look for signs of neurofibromatosis or schawannomatosis and if there are present genetic testing should be performed.

Oligo-astrocytoma in LZTR1-related Noonan syndrome

Mutations in LZTR1, already known to be causal in familial schwannomatosis type 2, have been recently involved in a small proportion of patients with autosomal dominant and autosomal recessive Noonan syndrome. LZTR1 is also a driver gene in non syndromal glioblastoma. We report a 26-year-old patient with typical Noonan syndrome, and the dominantly transmitted c.850C > T (p.(Arg284Cys)) variant in LZTR1. An oligoastrocytoma was diagnosed in the patient at the age of 22 years; recurrence of the tumor occurred at age 26, as a ganglioblastoma. The patient had been transiently treated with growth hormone between ages 15 and 17. Considering the implication of LZTR1 in sporadic tumors of the nervous system, we hypothesize that gliomas are a possible complication of LZTR1-related Noonan syndrome. This report also supports a possible link between occurrence of a cerebral tumor in Noonan syndrome and a previous treatment with growth hormone.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination.

The leucine zipper-like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants

PurposeTo characterize the molecular genetics of autosomal recessive Noonan syndrome. MethodsFamilies underwent phenotyping for features of Noonan syndrome in children and their parents. Two multiplex families underwent linkage analysis. Exome, genome, or multigene panel sequencing was used to identify variants. The molecular consequences of observed splice variants were evaluated by reverse-transcription polymerase chain reaction. ResultsTwelve families with a total of 23 affected children with features of Noonan syndrome were evaluated. The phenotypic range included mildly affected patients, but it was lethal in some, with cardiac disease and leukemia. All of the parents were unaffected. Linkage analysis using a recessive model supported a candidate region in chromosome 22q11, which includes LZTR1, previously shown to harbor mutations in patients with Noonan syndrome inherited in a dominant pattern. Sequencing analyses of 21 live-born patients and a stillbirth identified biallelic pathogenic variants in LZTR1, including putative loss-of-function, missense, and canonical and noncanonical splicing variants in the affected children, with heterozygous, clinically unaffected parents and heterozygous or normal genotypes in unaffected siblings. ConclusionThese clinical and genetic data confirm the existence of a form of Noonan syndrome that is inherited in an autosomal recessive pattern and identify biallelic mutations in LZTR1.

LZTR1 mutations associated with greater pain among patients with schwannomatosis (P6.142)

LZTR1 mutations associated with greater pain among patients with schwannomatosis (P6.142)