NF1 Deletions Research Articles

Cooperative genomic lesions in HRAS-mutant cancers predict resistance to farnesyltransferase inhibitors.

In the clinical development of farnesyltransferase inhibitors (FTIs) for HRAS-mutant tumors, responses varied by cancer type. Co-occurring mutations may affect responses. We aimed to uncover cooperative genetic events specific to HRAS-mutant tumors and to study their effect on sensitivity to FTIs. Using targeted sequencing data from the MSK-IMPACT and Dana-Farber Cancer Institute Genomic Evidence Neoplasia Information Exchange databases, we identified comutations that were observed predominantly in HRAS-mutant versus KRAS-mutant or NRAS-mutant cancers. HRAS-mutant cancers had a higher frequency of coaltered mutations (48.8%) in the MAPK, PI3K, or RTK pathway genes, compared with KRAS-mutant (41.4%) and NRAS-mutant (38.4%) cancers (p < 0.05). Class 3 BRAF, NF1, PTEN, and PIK3CA mutations were more prevalent in HRAS-mutant lineages. To study the effects of comutations on sensitivity to FTIs, HrasG13R was transfected into "RASless" (Kraslox/lox/Hras-/-/Nras-/-/RERTert/ert) mouse embryonic fibroblasts (MEFs), which sensitized nontransfected MEFs to tipifarnib. Comutation in the form of Pten or Nf1 deletion and Pik3caH1047R transduction led to resistance to tipifarnib in HrasG13R-transfected MEFs in the presence or absence of KrasWT, whereas BrafG466E transduction led to resistance to tipifarnib only in the presence of KrasWT. Combined treatment with tipifarnib and MEK inhibition sensitized cells to tipifarnib in all settings, including in MEFs with PI3K pathway comutations. HRAS-mutant tumors demonstrate lineage-dependent MAPK or PI3K pathway alterations, which confer resistance to tipifarnib. The combined use of FTIs and MEK inhibition is a promising strategy for HRAS-mutant tumors.

Genetic/epigenetic effects in NF1 microdeletion syndrome: beyond the haploinsufficiency, looking at the contribution of not deleted genes

NF1 microdeletion syndrome, accounting for 5–11% of NF1 patients, is caused by a deletion in the NF1 region and it is generally characterized by a severe phenotype. Although 70% of NF1 microdeletion patients presents the same 1.4 Mb type-I deletion, some patients may show additional clinical features. Therefore, the contribution of several pathogenic mechanisms, besides haploinsufficiency of some genes within the deletion interval, is expected and needs to be defined. We investigated an altered expression of deletion flanking genes by qPCR in patients with type-1 NF1 deletion, compared to healthy donors, possibly contributing to the clinical traits of NF1 microdeletion syndrome. In addition, the 1.4-Mb deletion leads to changes in the 3D chromatin structure in the 17q11.2 region. Specifically, this deletion alters DNA-DNA interactions in the regions flanking the breakpoints, as demonstrated by our 4C-seq analysis. This alteration likely causes position effect on the expression of deletion flanking genes.Interestingly, 4C-seq analysis revealed that in microdeletion patients, an interaction was established between the RHOT1 promoter and the SLC6A4 gene, which showed increased expression. We performed NGS on putative modifier genes, and identified two “likely pathogenic” rare variants in RAS pathway, possibly contributing to incidental phenotypic features.This study provides new insights into understanding the pathogenesis of NF1 microdeletion syndrome and suggests a novel pathomechanism that contributes to the expression phenotype in addition to haploinsufficiency of genes located within the deletion.This is a pivotal approach that can be applied to unravel microdeletion syndromes, improving precision medicine, prognosis and patients’ follow-up.

Abstract PO1-23-09: A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC)

Abstract Background: CDK4/6i are part of the standard management of HR+ MBC. The MAPK pathway has been implicated in mediating resistance to CDK4/6 blockade (Wander 2020). A variety of molecular alterations dysregulate MAPK signaling, including NF1 mutations (NF1-MUT) (Wallace 2012), though NF1 has not been implicated in CDK4/6i resistance. To assess this, we analyzed retrospective clinical data from multiple institutions, real-world data (RWD) from the GuardantINFORM database, and preclinical models of HR+ breast tumors. The primary objective of this study was to characterize clinical outcomes of CDK4/6i in patients (pts) with NF1-MUT MBC, and to validate those findings in laboratory models. Methods: Pts with pathogenic (p)NF1-MUT were identified via sequencing of circulating tumor DNA (ctDNA) or tumor tissue. Pathogenic was defined as nonsense, insertion/deletion, frameshift, or oncogenic missense mutations. Progression free survival (PFS) on CDK4/6i was analyzed, and intrinsic resistance was defined as progression &amp;lt; 6 months on 1st line (1L) CDK4/6i or &amp;lt; 3 mo on 2nd line and beyond (2L+). Acquired resistance was defined as PFS &amp;gt;6 mo (1L) or &amp;gt;3 mo (2L+) with a NF1 alteration detected post-progression. RWD from the GuardantINFORM dataset had Guardant360 (G360) ctDNA testing linked with claims data on &amp;gt;37,000 pts with advanced or MBC tested between June 2014 and March 2023. Pts on CDK4/6i within 90 days pre-G360 were stratified as NF1-MUT (n = 28) vs. NF1-non-mutant (NF1-nonMUT) (n = 1133) and analyzed for real-world time-to-next-treatment (rwTTNT) and real-world overall survival (rwOS). Propensity score weighting for age, sex, line of therapy, and year of G360 were used to create a balanced control group. To determine whether NF1 loss-of-function alterations were causal of CDK4/6i resistance, we used CRISPR/Cas9 to delete NF1 in MCF7 breast cancer cells. MCF7 wild-type (WT) and two NF1-knockout (KO) clones were treated with palbociclib ± estrogen deprivation, followed by cell viability assays. MAPK and PI3K/AKT pathway activation were measured by immunoblot analysis. Results: Across 4 institutions, 40 pts with HR+, HER2- MBC expressing pNF1-MUT and prior CDK4/6i were included. The mean age at MBC diagnosis was 56 years, and the majority of pts received no prior treatment in the metastatic setting before CDK4/6i (range 0 - 5 prior lines). Intrinsic or acquired resistance to CDK4/6i was seen in 29/40 (73%) tumors harboring pNF1-MUT. We analyzed pts with baseline pNF1-MUT who received 1L CDK4/6i plus endocrine therapy (n = 13) and found that the median PFS was 7.7 months (range 2 - 18 mo). We next examined GuardantINFORM RWD and discovered a significant difference in rwTTNT and rwOS on CDK4/6i for NF1-MUT (n = 28) vs. weighted NF1-nonMUT (n = 28) tumors. Mean age (62 years) and level of pretreatment (majority 2L+) were similar between the groups. Compared to NF1-nonMUT, NF1-MUT pts had a shorter median rwTTNT on CDK4/6i regimens (4.2 vs. 12.4 mo, weighted log-rank p&amp;lt; 0.0001) and worse rwOS (15.8 vs 45.2 mo, weighted log-rank p=0.016). In MCF7 cells, NF1-KO exhibited a palbociclib IC50 11- to 13-fold higher than NF1-WT controls. In estrogen-deprived media, treatment with 250 nM palbociclib for 6 days reduced MCF7 NF1-WT cell viability 75% but only 5-20% in NF1-KO cells. As expected, phosphorylation of ERK and AKT were strongly induced in NF1-KO cells relative to NF1-WT. Conclusions: Using multicenter clinical and real-world evidence, we demonstrate that NF1-MUT is associated with inferior duration and worse outcomes on CDK4/6i in MBC. Pts with baseline pNF1-MUT on 1L CDK4/6i and endocrine therapy had a median PFS below typical responses expected in clinical practice. A link between NF1 loss and CDK4/6i resistance was supported by in vitro experiments in HR+ breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, therefore, future directions include investigating alternative or combination treatment strategies targeting the MAPK and/or PI3K/AKT pathways in these tumors. *MRL and RCP contributed equally. ABH and SAW are co-senior authors. Citation Format: Maxwell Lloyd, M Rosario Chica-Parrado, Todd Knepper, Chang-Ching Lin, Emily Podany, Christine Walko, Fabiana Napolitano, Caroline Weipert, Jiemin Liao, Arielle Medford, Lianne Ryan, Jennifer Keenan, Nicole Zhang, Shiyuan Liu, Gerburg Wulf, Katherine Clifton, Cynthia Ma, Hyo Han, Leif Ellisen, Aditya Bardia, Carlos Arteaga, Ariella Hanker, Seth Wander. A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-09.

Droplet Digital PCR for Fast and Accurate Characterization of NF1 Locus Deletions: Confirmation of the Predominant Maternal Origin of Type-1 Deletions

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) setalong the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.

Deregulated expression of polycomb repressive complex 2 target genes in a NF1 patient with microdeletion generating the RNF135-SUZ12 chimeric gene

Neurofibromatosis type I (NF1) microdeletion syndrome, accounting for 5–11% of NF1 patients, is caused by the heterozygous deletion of NF1 and a variable number of flanking genes in the 17q11.2 region. This syndrome is characterized by more severe symptoms than those shown by patients with intragenic NF1 mutation and by variable expressivity, which is not fully explained by the haploinsufficiency of the genes included in the deletions. We here reevaluate an 8-year-old NF1 patient, who carries an atypical deletion generating the RNF135-SUZ12 chimeric gene, previously described when he was 3 years old. As the patient has developed multiple cutaneous/subcutaneous neurofibromas over the past 5 years, we hypothesized a role of RNF135-SUZ12 chimeric gene in the onset of the patient’s tumor phenotype. Interestingly, SUZ12 is generally lost or disrupted in NF1 microdeletion syndrome and frequently associated to cancer as RNF135. Expression analysis confirmed the presence of the chimeric gene transcript and revealed hypo-expression of five out of the seven analyzed target genes of the polycomb repressive complex 2 (PRC2), to which SUZ12 belongs, in the patient’s peripheral blood, indicating a higher transcriptional repression activity mediated by PRC2. Furthermore, decreased expression of tumor suppressor gene TP53, which is targeted by RNF135, was detected. These results suggest that RNF135-SUZ12 chimera may acquire a gain of function, compared with SUZ12 wild type in the PRC2 complex, and a loss of function relative to RNF135 wild type. Both events may have a role in the early onset of the patient’s neurofibromas.

Abstract 6011: Methylation profiling reveals the role of PRC2 in regulating DNA methylome changes in malignant peripheral nerve sheath tumors

Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer predisposition syndrome defined by germline deletion of one NF1 allele. Somatic loss of heterozygosity in the remaining NF1 allele in Schwann lineage cells gives rise to benign tumors known as plexiform neurofibromas. These tumors have a high risk of developing into cancerous lesions known as malignant peripheral nerve sheath tumors (MPNST) through the sequential deletion of CDKN2A/B followed by recurrent mutations in SUZ12 or EED, constituting the inactivation of polycomb repressor complex 2 (PRC2). PRC2 is a transcriptional repressor complex involved in the silencing of several genes throughout cell development and differentiation. Loss of this complex leads to a depletion in trimethylation marks on Histone H3 Lysine 27 (H3K27me3) and the subsequent gain of acetylation marks on the same lysine residue (H3K27Ac). However, full range of epigenetic consequences of these events are currently not well characterized, especially with respect to MPNST formation and development. The Largaespada lab has developed Schwann cell lines engineered to harbor deletions in NF1 and either SUZ12 or EED and assayed the methylation status of 850,000 CpG sites using the EPIC array. The resulting methylation profiles reveal a large degree of change in promoter methylation between cell lines with and without PRC2 inactivation. These changes, however, appear to be inconsistent. To further understand this phenomenon, I have analyzed differential methylation patterns between lines with SUZ12/EED deletions on top of NF1 deletion compared to those with NF1 deletion alone. Pathway analysis of genes whose promoters are differentially methylated upon PRC2 reveals a bias towards developmental modules such as morphogenesis and neuronal differentiation, suggesting that DNA methylation is a mechanism by which PRC2 loss promotes stemness in Schwann lineage cells prior to MPNST formation. Additionally, differentially methylated regions were overlapped with functional non-coding elements in the genome to understand potential ripple effects in the epigenome. These findings underline the importance of DNA methylation in contributing to tumor formation and define unique functions by which PRC2 specifically leverages diverse methylation states to drive malignant transformation in Schwann lineage cells. Citation Format: Christopher M. Stehn, Minu Bhunia, Kyle B. Williams, Alex T. Larsson, David A. Largaespada. Methylation profiling reveals the role of PRC2 in regulating DNA methylome changes in malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6011.

Abstract 3552: YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma

Abstract Background/Objectives: Despite intensive multimodal therapy, greater than 50% of children with high-risk neuroblastoma (HR NB) relapse with incurable disease. Next generation sequencing of primary HR NB tumors identified an increase in activating mutations in the RAS/RAF/MAPK pathway. Moreover, gene set enrichment analyses showed a significant decrease in expression of genes suppressed by the Yes-Associated Protein (YAP) at relapse, suggesting increased YAP transcriptional repression. YAP binds with TEAD family transcription factors to regulate gene expression. We have shown that YAP promotes chemotherapy and MEK inhibitor resistance in RAS-mutated NB tumors in vivo by suppressing the expression of Harakiri (HRK), a BH3-only pro-apoptotic protein activated in response to tumor environmental stress such as serum starvation. Our overall objective is to elucidate how YAP represses HRK and tumor suppressor genes globally, and to enhance MEK inhibitor potency by combining MEK inhibition with agents that inhibit YAP or induce HRK to restore the tumor environmental stress response and apoptosis in RAS-mutated NB. Design/Methods: We used publicly available databases to identify TEAD binding sites on the HRK gene locus in NB. To assess the global state of methylation, we treated NB cells, SK-N-AS (NRASQ61K mutation, MYCN non-amplified) and NLF (NF1 deletion, MYCN amplified), with demethylating agent azacitidine and evaluated HRK expression. To identify the specific TEAD (1-4) binding partner to YAP, we performed siRNA and co-immunoprecipitation studies. We further tested novel YAP-TEAD small molecule inhibitors with varying TEAD1-4 inhibition specificity in SK-N-AS and NLF cells in vitro. Results: We observed that TEAD binds near cis-regulatory regions on the HRK gene locus in NB. We found that HRK expression is restored when SK-N-AS and NLF cells are treated with azacitidine despite YAP expression increasing. We also identified TEAD2 as the specific binding partner to YAP in NB and found that TEAD2 is necessary for HRK regulation. Novel YAP-TEAD small molecule inhibitors affect NB cell viability under serum-deprived conditions in vitro, especially the inhibitor with highest specificity against TEAD2, and affect YAP-TEAD downstream targets. Conclusions: YAP-TEAD2 binding is essential for HRK regulation in RAS-mutated NB and thus is a logical therapeutic target to restore therapy response. Further studies are ongoing to test YAP-TEAD small molecule inhibitors in combination with MEK inhibitors and in vivo. Citation Format: Jenny Shim, Andrew Ho, Hunter C. Jonus, Adeiye A. Pilgrim, Benjamin G. Barwick, Tracy T. Tang, Lawrence H. Boise, Kelly C. Goldsmith. YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3552.

Imagawa-Matsumoto syndrome: SUZ12-related overgrowth disorder.

The SUZ12 gene encodes a subunit of polycomb repressive complex 2 (PRC2) that is essential for development by silencing the expression of multiple genes. Germline heterozygous variants in SUZ12 have been found in Imagawa-Matsumoto syndrome (IMMAS) characterized by overgrowth and multiple dysmorphic features. Similarly, both EZH2 and EED also encode a subunit of PRC2 each and their pathogenic variants cause Weaver syndrome and Cohen-Gibson syndrome, respectively. Clinical manifestations of these syndromes significantly overlap, although their different prevalence rates have recently been noted: generalized overgrowth, intellectual disability, scoliosis, and excessive loose skin appear to be less prevalent in IMMAS than in the other two syndromes. We could not determine any apparent genotype-phenotype correlation in IMMAS. The phenotype of neurofibromatosis type 1 arising from NF1 deletion was also shown to be modified by the deletion of SUZ12, 560 kb away. This review deepens our understanding of the clinical and genetic characteristics of IMMAS together with other overgrowth syndromes related to PRC2. We also report on a novel IMMAS patient carrying a splicing variant (c.1023+1G>C) in SUZ12. This patient had a milder phenotype than other previously reported IMMAS cases, with no macrocephaly or overgrowth phenotypes, highlighting the clinical variation in IMMAS.

TB-1 ADDITIONAL GENETIC ALTERATIONS DIFFERENTIALLY ALTER THE TRANSCRIPTOMIC LANDSCAPE OF H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA

Abstract Histone H3 K27M mutation is a hallmark mutation for H3 K27M-mutant diffuse midline glioma (DMG), but targeting this mutation has yet to achieve a significant survival benefit in clinical trials. Recent analyses revealed alterations in several genes, such as NF1 and PDGFRA, are observed in substantial subpopulations of H3 K27M-mutant DMG patients in addition to H3 mutation and recurrent TP53 mutations, indicating patient-to-patient tumor heterogeneity and the potential necessity of tailored target therapy for the treatment of this disease. Here, using our human induced pluripotent stem cells (iPSC)-derived glioma avatar platform, we designed DMG models by introducing TP53R248Q with or without heterozygous H3 K27M mutation in combination with further genetic modifications of either NF1 knockout or PDGFRAD842V overexpression to recapitulate DMG subpopulations. Mice with TP53R248Q; H3F3AK27M (QM) tumors survived significantly longer than those harboring QM;NF1-/- (QMN) tumors and QM; PDGFRAD842Voe (QMP) tumors. RNA-sequencing of those induced DMG (iDMG) neurospheres revealed altered patterns of upregulation of MAPK pathway genes both in QMN and QMP-iDMG neurospheres compared to their H3 wildtype counterparts with the same combinations of genetic alterations, suggesting that those additional mutations modifies the oncogenic signaling associated with H3 K27M mutation. Further, differential expression analysis comparing QMN and QMP-iDMG neurospheres revealed 405 differentially expressed genes. Gene set enrichment analysis showed upregulation of transcriptional programs related to mesenchymal signature in QMN-iDMG neurospheres and proneural signature in QMP-iDMG neurosphere as expected. These data show that NF1 deletion and PDGFRAD842V overexpression significantly alter gene expression in H3 K27M-mutant iDMG tumors, potentially opening up a new therapeutic avenue in these devastating tumors with patient-to-patient heterogeneity. Further work using these models will shed light on the development of tailored therapy based on detailed genetic information on each patient sample, such as combining targeted kinase inhibition with HDAC inhibitors that have shown promise in the clinic.

Differentiation Scoring (DS) Derived from Cellworks Computational Omics Biology Model (CBM) Predicts Response to Hypomethylating Agents (HMA) and Patient Survival in Myelodysplastic Syndrome (MDS)

Background: Differentiation arrest resulting from aberrant methylation represents a key pathogenic and phenotypic feature of MDS. Hypomethylating agents (HMA) can reverse epigenetic dysregulation, but as single agents fail to control the disease in approximately 50% of patients, many of whom progress to AML and carry a dismal prognosis. We hypothesized that Differentiation Scoring (DS) derived from Cellworks Computational Omics Biology Model (CBM) of each patient's genomic aberrations could serve as a biomarker to predict response to HMA. Methods: We studied 169 MDS patients treated with azacytidine (n = 86) or decitabine (n = 83) whose genomic aberrations and clinical outcomes were available from public databases. Biosimulation of the disease state was computationally derived from pathways that were up- or down-regulated by individual genomic aberrations. DS was defined as the ratio of the quantified impact of key differentiation biomarkers in myeloid malignancies (CEBPA, GATA1, SPI1 and ITGAM) divided by the impact of HOXA9 transcription, a known repressor of differentiation, and normalized on a log2 scale. We also used CBM to determine the change in DS attributable to HMA to define an efficacy score (ES) as a parameter of drug response. A Pearson correlation coefficient was obtained using the two variables. DS in clinical responders (R) and non-responders (NR) was evaluated with the Student t-test. We defined DS > 1.5 as DS-high and < 1.5 as DS-low. Survival data were available for 101 patients and correlated with DS-high (n=49) and DS-low (n=52) groups using a KM-curve. Results: Median age of this cohort was 69 years (range, 20-87 years), including 56 females, 111 males, and 2 of unknown sex. Regarding HMA response, 62 patients were R while 107 were NR. Prior to therapy, 86 patients had DS-high and 83 had DS-low scores. Post-treatment DS were correlated with HMA ES (R = 0.58, p < 2.2e-16), and showed a favorable HMA response for DS-high patients. Biosimulation showed the incremental benefit on DS of HMA was greater for people with DS-high than DS-low scores (44% vs 16%, p = 7.51 e-17). Median DS was higher in R than NR (p=0.00232). Patients with DS-high scores had superior survival compared to those with DS-low scores (log rank p = 0.0016). (Figure 1a) Different genomic aberrations contributed to the DS scores. DS-high patients had more TP53 mutations, 17p del, 8q amp, and 5q del. On the other hand, DS-low patients more often had mutations in ASXL1, SRSF2, RUNX1, DNMT3A, EZH2, NF1, NRAS, CBL, or 7q deletion. (Figure 1b) Loss of TP53 due to mutation or deletion upregulates DNMT1 and silences HOXA9 transcription, leading to DS-high scores. KAT6A (8q) amp also upregulates EZH2 and CpG methylation, while 5q del causes GATA1 transcription, increasing CpG methylation via SALL4 to produce high ß-catenin signaling that led to DS-high scores. By contrast, 7q loss, ASXL1, SRSF2, and EZH2 mutations downregulated EZH2, diminished CpG methylation and unsilenced HOXA9 transcription. As a result, HOXA9 arrested differentiation results in DS-low scores. Additionally, NF1 or NRAS mutations upregulate ERK signaling which inhibits CEBPA, and also caused DS-low scores. Conclusions: Computational biosimulation based on patients’ individual genomic aberrations reveals a spectrum of DS among patients with MDS. DS strongly predicted HMA response. In general, NR had lower DS and survival than DS-high patients. While patients with DS-low scores might have fared even worse without HMA therapy, we propose that DS-low patients should be considered for novel combination therapy at the time of diagnosis given their unsatisfactory outcomes with HMA therapy as a single agent. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unmatched Whole-Genome Sequencing As a Clinical Tool for Hematological Neoplasms with Significant Utility in Cases with Tumor-in-Normal Contamination

Introduction For patients with acute leukemias, mapping of clinical biomarkers is critical for disease classification, risk stratification and treatment decisions. At diagnosis, molecular profiling is primarily based on cytogenetic analysis, SNP-arrays and increasingly complemented with targeted assays to include Fluorescent In Situ Hybridization (FISH), and DNA/RNA panel assays. Recently, Duncavage et al. showed that for acute myeloid leukemia, whole genome sequencing (WGS) can replace cytogenetics and detect the hotspot mutations in the ELN2017 guidelines (Duncavage et al., 2021). However, the 2022 revision of disease classification and risk stratification led to the incorporation of additional events characterized by a diverse spectrum of non-hotspot mutations (Khoury et al. 2022, Bataller et al. 2022). To this end, the diagnostic utility of WGS for comprehensive characterization of relevant biomarkers across hematological neoplasms is not established. A further limitation for matched WGS (mWGS) is the requirement of a normal sample at diagnosis, as sources of control tissue can be contaminated with tumor cells. Methods To resolve this unmet clinical need, we developed a computational framework for the analysis of WGS data without the use of a normal germline control (uWGS). We applied this uWGS in 2 independent cohorts of patients (Table 1). In cohort 1 of 49 patients, representative of a broad range of hematological neoplasms, uWGS findings were compared against standard-of-care molecular assays to include cytogenetics, FISH, SNP-arrays, targeted-gene panels and RNA sequencing for findings of clinical relevance (disease classification, risk stratification, therapy informing) as annotated by OncoKB (Chakravarty et al. 2017). To further evaluate whether an uWGS can detect the same spectrum of acquired mutations that a matched WGS workflow (mWGS), we compared findings for 52 B-ALL patients in cohort 2 with mWGS data, and we estimated the tumor-in-normal contamination (TiN) with the algorithm deTiN (Taylor-Weiner et al., 2018). Results In Cohort 1, uWGS detected 100% (24/24) of highly confident findings (cancer cell fraction >20) of clinical relevance described by standard-of-care assays (0.49 average, 0-3 range) (Fig.1). Moreover, uWGS identified 9 mutations in 8 patients that were not detected by standard-of-care. These include 2 disease-defining P2RY8::CRLF2 in B-ALL, 2 canonical t(7;9)(q34;q34.3) NOTCH1-intergenic in T-ALL, a TCF3 D561V point mutation and a IGH::MYC event in Burkitt lymphoma, 2 focal deletions in CDKN2A and IKZF1 in B-ALL, and a focal NF1 deletion in JMML. Importantly, 5/8 of these cases did not have a clinically relevant alteration identified by standard-of-care. In Cohort 2, we evaluated what proportion of relevant events identified by mWGS can be detected by uWGS. Putative biomarkers had been identified by mWGS in 88% of patients (46/52). These included delineations of aberrant karyotypes where chromosome banding failed (5/52), newly described fusion genes (i.e. UBTF::ATXN7L3, EP300::ZNF384, in 27/52) and recurrent gene mutations (i.e. PAX5 P80R, ZEB2 H1038R, in 14/52). As a result, uWGS workflow captured 100% of biomarkers identified in the mWGS (5/5 ploidy, 27/27 fusion, 14/14 coding). Remarkably, evaluation of TiN levels reveals that 4 driver events had been missed in the mWGS workflow, affecting genetic alterations across variant classes. For example, a focal BTG1 loss detected by uWGS was not called by mWGS. Overall, 25% of B-ALL cases had evidence of TiN >2% (14/55, ranges 2-57%), resulting in lower sensitivity of detection of copy number variants (CNVs) in mWGS. When looking into the whole spectrum of CNVs detected only by uWGS, we found lower levels of precision in mWGS that were associated with evidence of TiN (median 0.45 vs 0.64, p-value 0.03). Additionally, the other 3 drivers missed by mWGS were indels in CHEK2, JAK1 and TCF3. Conclusions We have developed and validated an uWGS workflow for the detection of clinically relevant alterations in leukemia across variant classes to include CNVs, translocations and point mutations. We demonstrated that for hematological neoplasms, uWGS rescues events that can be missed by mWGS workflows owing to TiN, which shows how uWGS enables the detection of clinically relevant biomarkers, and the opportunity to discover new clinical findings using a single test and a single biopsy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

123. Chromosomal microarray assists interpretation of cytogenetic abnormalities in hematopoietic malignancies

Chromosome analysis is a first-line ancillary test performed on bone marrows to evaluate for hematopoietic malignancy. While characteristic cytogenetic findings correlate with pathology in most cases, the chromosomal abnormalities are sometimes too complex for complete characterization, limiting interpretation of the results. Additionally, chromosomal abnormalities may lack correlation with the hematopathology findings. Here, we present two cases where chromosomal microarray was performed, which aided interpretation of chromosome analysis. Case 1: A patient with a history of acute myeloid leukemia (AML) with NPM1 mutation had a bone marrow evaluated for residual disease. Although chromosome analysis identified a mosaic balanced translocation between chromosomes 7 and 17, concurrent ancillary studies were unrevealing. The significance of this finding was unclear given the unremarkable morphology and flow cytometry. Chromosomal microarray identified deletions of NF1 and SUZ12 on chromosome 17 as well as deletion of ASXL1 on chromosome 20, which may be a driver for leukemogenesis in this case. Case 2: A patient presented with circulating blasts and was diagnosed with AML. Chromosome analysis demonstrated deletion of chromosome 5q and loss of chromosome 7. However, additional complex chromosomal abnormalities were also identified, including a jumping translocation. Chromosomal microarray showed complex abnormalities of chromosome 1p, gain of chromosome 5p including TERT and IL7R, deletion of IKZF1 on chromosome 7, and deletion of TP53 on chromosome 17. These chromosomal microarray findings provide greater insight toward AML pathogenesis. These case reports demonstrate the utility of chromosomal microarray in further defining chromosomal findings with unclear significance to further understand disease pathogenesis.

Elucidating the diversity of malignant mesenchymal states in glioblastoma by integrative analysis

BackgroundMultiple glioblastoma studies have described a mesenchymal (MES) state, with each study defining the MES program by distinct sets of genes and highlighting distinct functional associations, including both immune activation and suppression. These variable descriptions complicate our understanding of the MES state and its implications. Here, we hypothesize that there is a range of glioma MES states, possibly reflecting distinct prior states in which a MES program can be induced, and/or distinct mechanisms that induce the MES states in those cells.MethodsWe integrated multiple published single-cell and bulk RNA sequencing datasets and MES signatures to define a core MES program that recurs across studies, as well as multiple function-specific MES signatures that vary across MES cells. We then examined the co-occurrence of these signatures and their associations with genetic and microenvironmental features.ResultsBased on co-occurrence of MES signatures, we found three main variants of MES states: hypoxia-related (MES-Hyp), astrocyte-related (MES-Ast), and an intermediate state. Notably, the MES states are differentially associated with genetic and microenvironmental features. MES-Hyp is preferentially associated with NF1 deletion, overall macrophage abundance, a high macrophage/microglia ratio, and M2-related macrophages, consistent with previous studies that associated MES with immune suppression. In contrast, MES-Ast is associated with T cell abundance and cytotoxicity, consistent with immune activation through expression of MHC-I/II.ConclusionsDiverse MES states occur in glioblastoma. These states share a subset of core genes but differ primarily in their association with hypoxia vs. astrocytic expression programs, and with immune suppression vs. activation, respectively.

Abstract LB067: NF1 deletion potentiates tumorigenesis and activates expression of cancer-related kinases in an iPSC-based model of H3.3K27M diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). However, H3.3K27M alone is insufficient for tumorigenesis in existing DIPG models, suggesting that it interacts with co-occurring oncogenic mutations. NF1 deletion co-occurs with TP53 mutation and H3.3K27M in DIPG, but the impact of these mutations individually and together are understudied. To address this gap, we designed a model of DIPG based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express either wild-type or deleted NF1 in conjunction with heterozygous H3.3K27M and a loss of function mutation in TP53. Edited iPSC were chemically differentiated into neural progenitor cells (iNPC), which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Mice with TP53 mut;H3.3K27M tumors survived significantly (p&amp;lt;0.05) longer than those harboring TP53 mut;NF1 -/-;H3.3K27M tumors. In vitro proliferation assays of neurospheres generated from these tumors (termed iDIPG) confirmed this result, suggesting that NF1 deletion synergizes with the other two mutations to accelerate tumor growth. To determine which transcriptional pathways could be involved in this faster tumorigenesis, we performed RNA-sequencing TP53 mut;H3.3K27M (TK) and TP53 mut;NF1 -/-;H3.3WT (TNK) iDIPG neurospheres as well as their iNPC precursors. iDIPG and iNPC clustered by NF1 status. Differential expression analysis comparing TNK and TK iDIPG neurospheres revealed 7226 differentially expressed genes (p&amp;lt;0.05). Gene set enrichment analysis showed upregulation of transcriptional programs related to cell cycle and kinase signaling cascades in TNK iDIPG neurospheres; therefore, we focused our analysis on differentially expressed kinases to determine whether NF1 deletion significantly impacts expression of the kinome. We found 141 kinases upregulated in TNK iDIPG neurospheres, including several members of the MAP kinase family and EGFR, which is amplified or activated in a large number of high-grade gliomas and therefore represents an attractive therapeutic target. Taken together, these data show that NF1 deletion is associated with a significant alteration of kinase expression in H3.3K27M iDIPG, potentially opening up a new therapeutic avenue in these devastating tumors. Further work using this model will focus on screening for kinases necessary for TNK iDIPG neurospheres survival in culture and investigating synergy between targeted kinase inhibition and HDAC inhibitors, which have shown promise in H3.3K27M DIPG. Citation Format: Kasey Skinner, Tomoyuki Koga, Shunichiro Miki, Robert F. Gruener, R. Stephanie Huang, Frank Furnari, Ryan Miller. NF1 deletion potentiates tumorigenesis and activates expression of cancer-related kinases in an iPSC-based model of H3.3K27M diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB067.

Abstract 1168: From clinical mutational data to a novel lung cancer mouse model -NF1inactivation cooperates withRASA1inactivation to drive non-small cell lung cancer (NSCLC)

Abstract 11% of non-small cell lung cancer (NSCLC) patients harbor NF1 mutations. The enrichment of NF1 mutations in lung adenocarcinoma (LUAD) cases that otherwise lack activated oncogene mutations indicates that NF1 loss may play a necessary role in the activation of the RAS pathway. However, conditional, biallelic loss of Nf1 in the mouse lung is insufficient for LUAD initiation over 2 years, indicating additional genetic or epigenetic events cooperate with Nf1 loss in lung tumorigenesis. To better understand NF1 mutant lung cancers, we analyzed a lung cancer dataset with 47227 patients’ genomic mutation data from Foundation Medicine (FMI) and found that NF1 mutations (5270 patients) are associated with a higher mutational burden compared to NF1 wild type lung cancer patients in the lung cancer patients’ clinical database. We then analyzed the AACR GENIE non-small cell lung cancer datasets for genes mutated concurrently with NF1 mutation. We found that NF1 mutation statistically significantly cooccurs with RASA1 (p &amp;lt;0.001 for concurrency) and with TP53 mutations in the non-small lung cancer dataset. NF1-mutated samples lack other key driver oncogene mutations such as EGFR (p &amp;lt;0.001 for mutual exclusivity). To functionally validate our findings of genetic cooperativity, we conditionally deleted the two key GAPs: Nf1 and Rasa1 (with Trp53) in vivo. Deletion of both Nf1 and Rasa1 but neither the Nf1 or Rasa1 deletion alone lead to LUAD when combined with Trp53 deletion. Our new mouse model provides a unique tool for our ongoing effort to understand the mechanism of Nf1 &amp; Rasa1 tumor initiation in the lung and will aid preclinical investigations of targeted therapies. Citation Format: Lin Song, Thaidy Y. Rodriguez, Adam Olshen, Eric Collisson. From clinical mutational data to a novel lung cancer mouse model -NF1inactivation cooperates withRASA1inactivation to drive non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1168.

The Role of Schwann Cells in the Onset of Neuropathic Pain in Neurofibromatosis-1

Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes individuals to benign tumors in the peripheral nervous system (PNS). Pain is a primary cause of decreased quality of life in NF1 patients. Specific loss of Nf1 in SCs and SC precursors is responsible for neurofibroma formation, but the role of SCs in nociception and pain development in NF1 is not known. Here, we wanted to determine whether Schwann cells (SCs), sensory neurons, or both contributed to neuropathic pain-like behaviors in an animal model of NF1. We performed behavioral assessments of mechanical sensitivity, and single unit electrophysiological recordings of sensory neurons using our ex vivo preparation from mice with SC specific (DhhCre;Nf1f/f) or sensory neuron specific (PirtCre;Nf1+/f) deletion of Nf1 and compared results to Nf1+/- mice and littermate controls. We then performed similar assessments in animals' wildtype for Nf1 with chemogenetic manipulation of SC calcium activity. Data was correlated with DRG gene expression using realtime PCR and nerve structure using electron microscopy. Findings show that Nf1 deletion in SCs produced mechanical hypersensitivity, while Nf1 deletion in sensory neurons did not. Behavioral hypersensitivity in the SC specific Nf1 mutants, correlated with sensitization of myelinated A-fiber nociceptors and unmyelinated polymodal C-fibers (CPM) to mechanical stimuli. This occurred with Remak bundle disruption but not tumor formation. Chemo-genetic manipulation of SC calcium activity induced mechanical hypersensitivity at the behavioral level and produced sensitization of CPM neurons to mechanical stimuli compared to CNO treated controls. The observed peripheral sensitization correlated with elevation of cytokines and growth factors in the L2/L3 DRGs. This suggests that alterations in SCs are directly responsible for changes in specific sensory neuron subtypes that possibly modulate neuropathic pain development in NF1. This study may help to identify novel cell-specific treatment strategies to ameliorate pain in NF1 patients.

Analysis of NF1 gene variants among thirteen patients with neurofibromatosis type 1

To detect variants of NF1 gene among thirteen patients with neurofibromatosis type 1. Genomic DNA was extracted from peripheral blood samples of the patients. High-throughput sequencing was employed to detect potential variants of the NF1 and NF2 genes. Thirteen pathogenic variants were identified among the patients, which included one NF1 deletion, three missense variants, three nonsense variants and six frameshifting variants. Among these, 10 variants have been associated with neurofibromatosis type 1. c.4180A>T (p.Asn1394Tyr), c.4217dupT (p.Leu1406fs) and c.1753dupT(p.Leu585Phefs*3) were unreported previously. Based on the guidelines of the American College of Medical Genetics and Genomics, c.4180A>T (p.Asn1394Tyr) was predicted to be likely pathogenic (PS2+PM1+PM2+PP2), while c.4217dupT (p.Leu1406fs) and c.1753dupT (p.Leu585Phefs*3) were predicted to be pathogenic (PVS1+PS2+PM2). Variants of the NF1 gene probably underlay the disease among these children. Above findings have enriched the the spectrum of NF1 gene variants.

Nf1 and Sh2b3 mutations cooperate in vivo in a mouse model of juvenile myelomonocytic leukemia

AbstractJuvenile myelomonocytic leukemia (JMML) is initiated in early childhood by somatic mutations that activate Ras signaling. Although some patients have only a single identifiable oncogenic mutation, others have 1 or more additional alterations. Such secondary mutations, as a group, are associated with an increased risk of relapse after hematopoietic stem cell transplantation or transformation to acute myeloid leukemia. These clinical observations suggest a cooperative effect between initiating and secondary mutations. However, the roles of specific genes in the prognosis or clinical presentation of JMML have not been described. In this study, we investigate the impact of secondary SH2B3 mutations in JMML. We find that patients with SH2B3 mutations have adverse outcomes, as well as higher white blood cell counts and hemoglobin F levels in the peripheral blood. We further demonstrate this interaction in genetically engineered mice. Deletion of Sh2b3 cooperates with conditional Nf1 deletion in a dose-dependent fashion. These studies illustrate that haploinsufficiency for Sh2b3 contributes to the severity of myeloproliferative disease and provide an experimental system for testing treatments for a high-risk cohort of JMML patients.

Expanding the clinical phenotype of RASopathies in 38 Turkish patients, including the rare LZTR1, RAF1, RIT1 variants, and large deletion in NF1.

RASopathies are a group of disorders caused by pathogenic variants in the genes encoding Ras/mitogen-activated protein kinase pathway and share overlapping clinical and molecular features. This study is aimed to describe the clinical and molecular features of 38 patients with RASopathies. Sanger or targeted next-generation sequencing of related genes and multiplex ligation-dependent-probe amplification analysis for NF1 were performed. The pathogenic variant detection rate was 94.4%. While PTPN11 was responsible for 50% of 18 patients with Noonan syndrome (NS), SOS1, LZTR1, RIT1, and RAF1 were responsible for the remaining 27.8%, 11.1%, 5.5%, and 5.5%, respectively. Three variants in LZTR1 were novel, of which two were identified in the compound heterozygous state in a patient with intellectual disability and hypertrophic cardiomyopathy, whereas the third variant was found in the heterozygous state in a patient with pulmonary stenosis and normal intelligence. We described pyloric stenosis, knee dislocation, and cleft palate in patients with SOS1, RIT1, and RAF1 variants, respectively, that was not previously reported. We detected a PTPN11 variant in three patients from same family with NS with multiple lentigines. BRAF and MAP2K2 variants were found in eight patients with Cardiofaciocutaneous syndrome. Two variants in HRAS were detected in two Costello syndrome patients, one with a mild and the other with a severe phenotype. While large NF1 deletions were identified in four Neurofibromatosis-NS patients with intellectual disability, intelligence was normal in one patient with missense variant. In conclusion, this study provided three novel variants in LZTR1 and expanded the clinical phenotype of rare RASopathies.

Genomic Analysis of Hairy Cell Leukemia Identifies Novel Recurrent Genetic Alterations

Hairy cell leukemia (HCL) comprises the clonal malignancies classical and variant hairy cell leukemia (vHCL). Classical HCL (cHCL) is characterized by a near 100% frequency of the BRAFV600E mutations while ~50% of vHCL have MAP2K1 mutations. However, recurrent genetic alterations cooperating with BRAFV600E or MAP2K1 mutations in HCL, as well as those in MAP2K1 - wild type vHCL are not well defined. Therefore, we performed deep targeted mutational and copy number (CN) analysis of cHCL (n=53) and vHCL (n=8) to gain additional insights into the pathogenesis and mechanisms of therapeutic resistance in HCL.Diagnostic bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs) were obtained from 53 cHCL and 8 vHCL patients from multiple centers. Mutational and CN analysis of MNCs (and FACS-purified HCL cells for some cases) was performed using a targeted next-generation sequencing assay, which sequences all coding regions of 585 genes recurrently mutated in leukemias, lymphomas, and solid tumors.Of the 53 cHCL patients, 100% were marked by BRAFV600E mutations, and the next most commonly mutated genes were the histone methyltransferase KMT2C (MLL3) and CDKN1B occurring in 15% (8/53) and 11% (6/53) of patients, respectively. Other recurrent mutations in cHCL affected genes involved in transcriptional regulation (BRD4, CEBPA, CREBBP, RUNX1, EP300, and MED12), Notch signaling (NOTCH1 and NOTCH2), and DNA repair (RAD50) (Fig. A). The most recurrent copy alterations in cHCL were deletions of chromosome 7q and 13q and gains of chromosome 5. Chromosome 7q and 13q deletions were confirmed by FISH. While recurrent 7q deletions have previously been reported in cHCL, genes in the minimally deleted region of 7q were not known and identified here to include SMO (7q32) and BRAF (7q34). Recurrent 13q deletions in cHCL include the tumor suppressor RB1 and the miR-15a and miR-16-1 microRNA cluster at 13q14.3 (Fig. B-D).Sequencing across 8 additional vHCL patients identified change-of-function mutations in both CCND3 and U2AF1, each in 13% (1/8) of vHCL patients and hotspot mutations in TP53 (38%; 3/8) (Fig. E). The CCND3 and U2AF1 mutations were absent in cHCL suggesting additional genetic differences between cHCL and vHCL. These findings may have therapeutic relevance as CCND3 mutations are thought to confer sensitivity to CDK4/CDK6 inhibitors while those in U2AF1 confer sensitivity to spliceosome inhibitors. Other mutations affecting genes involved in transcriptional regulation (CEBPA, CREBBP, DDX3X, and PBRM1) and chromatin remodeling (KMT2C, KDM6A, and KDM5C) were also identified (Fig. E). As with cHCL, chromosome 7q deletions were also present in vHCL and always included BRAF (7q34). Additionally, we identified recurrent 3p deletions in vHCL, which include a critical tumor suppressor locus encoding VHL, SETD2, BAP1, and PBRM1 (Fig. F).Prior studies have identified acquired KRAS mutations as driving vemurafenib resistance in HCL. Here we identified an activating mutation in NRAS in a treatment naïve cHCL patient. In addition, genomic analysis of the pre-treatment sample from a patient that developed de novo vemurafenib resistance uncovered a clonal hemizygous BRAFV600E mutation, as well as heterozygous deletions of BRAF, NF1, NF2, and TP53 (Fig. G). Consistent with the heterozygous deletion of NF1 and NF2, the de novo vemurafenib-resistant patient showed decreased expression of both NF1 and NF2 while vemurafenib-sensitive patients without NF1 or NF2 copy loss did not demonstrate decreased expression of NF1/NF2 by qRT-PCR. To understand the functional role of Nf1 and Nf2 loss and the potential contribution to RAF inhibitor resistance in the hematological system, we performed shRNA-mediated downregulation of Nf1 or Nf2 in Ba/F3 cells stably expressing BRAFV600E. Silencing of either Nf1 or Nf2 alone or concomitant downregulation of Nf1 and Nf2 simultaneously conferred vemurafenib resistance in vitro (Fig. H).Combined, these data identify several novel drivers of HCL. While activating MAPK mutations are critical for both cHCL and vHCL, our data suggest additional shared cooperating alterations, as well as disease-specific alterations targeting BRAF, KMT2C, and CDKN1B in cHCL and MAP2K1, CCND3, U2AF1, TP53, and KMT2C in vHCL. Finally, these data nominate several novel potential therapeutic approaches for vHCL and identify a novel mechanism of vemurafenib resistance seen clinically. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.