Mutations In RAS Pathway Genes Research Articles

Characterization of myeloproliferative neoplasms based on genetics only and prognostication of transformation to blast phase.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal disorders characterized by aberrant hematopoietic proliferation and an intrinsic risk of progression to blast phase. The WHO classification 2022 identifies chronic myeloid leukemia and the BCR::ABL1 negative MPNs polycythemia vera, primary myelofibrosis and essential thrombocythemia as individual entities. However, overlaps, borderline findings or transitions between MPN subtypes occur and incomplete clinical data often complicates diagnosis. By conducting a thorough genetic analysis, we've developed a model that relies on 12 genetic markers to accurately stratify MPN patients. The model can be simplified into a decision tree for routine use. Comparing samples at chronic and blast phase revealed, that one third of patients lost their MPN driver-gene mutation, while mutations in splicing and chromatin modifying genes were stable, indicating a shared founder clone of chronic and blast phase with different driver mutations and therefore different progressing capacities. This was further supported by gain of typical de novo AML gene mutations, accompanied by gain of complex karyotypes and RAS pathway gene mutations. Our data suggest to perform a broader genetic screening at diagnosis and also at clinical progression, as driver mutations may change and the MPN-driver mutations present at diagnosis may disappear.

Error-Corrected Next-Generation Sequencing Provides a Comprehensive Overview of the Subclonal Mutation Landscape and Its Prognostic Implications in Juvenile Myelomonocytic Leukemia

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative neoplasm characterized by mutations in genes activating the RAS pathway ( PTPN11, NF1, NRAS, KRAS, and CBL). Approximately 20% of patients with JMML have secondary mutations in SETBP1 and/or JAK3, and recent droplet digital PCR (ddPCR) analyses revealed that mutations in these two genes, including very rare mutations (variant allele frequency [VAF] < 0.05) that are difficult to detect by conventional next-generation sequencing (NGS), are associated with poor prognoses. Meanwhile, the landscape of low VAF subclonal mutations of other genes in patients with JMML has not been adequately evaluated. Therefore, we utilized error-corrected NGS, which can reduce the systematic sequencing error rate of conventional NGS by incorporating molecular barcodes (MBCs). Methods: We performed error-corrected NGS in 93 patients with JMML and 11 patients with JMML-like myeloproliferative disorder with Noonan syndrome (NS/MPD) using a small panel associated with myeloid malignancies. Samples for error-corrected NGS were prepared using a SureSelect XT HS2 DNA Library Preparation Kit (Agilent Technologies), including unique dual index and duplex MBCs. A unique MBC is incorporated into each DNA fragment, and the incorporated MBC can differentiate true and false duplications, allowing for highly accurate sequencing that can detect mutations with VAF > 0.001. We used the HiSeq X platform (Illumina) and SureCall v.4.2 application (Agilent Technologies) for analysis. In addition, DNA methylation analysis was performed on the same cohort using the Infinium HumanMethylation 450K BeadChip and/or Digital Restriction Enzyme Analysis of Methylation as previously described (Murakami et al. 2018;131:1576-1586, Kitazawa et al. Blood Adv. 2021;5:5507-5518). This study was approved by the Ethics Committee of the Nagoya University Graduate School of Medicine. Results: In the cohort of 104 patients with JMML or NS/MPD, 160 mutations were identified, of which 30 (19%) had VAF < 0.05 (eight in JAK3, seven in NF1, five in ASXL1, three in NRAS, two each in PTPN11 and SOS1, and one each in SETBP1, SH2B3, and ZRSR2). Subclonal mutations in SETBP1 p.D868N and JAK3 p.R657Q identified by error-corrected NGS were confirmed using ddPCR, and consistent results were obtained for both assays (R 2 = 0.9977 and 0.9919 respectively). Of 93 patients with JMML, 30 (32%) had subclonal or clonal secondary mutations, whereas no such mutations were detected in patients with NS/MPD ( P = 0.031). Of 104 patients, 23 (22%) had secondary mutations with VAF ≥ 0.05 (Major group), 7 (7%) only had secondary mutations with VAF < 0.05 (Minor group), and the remaining 74 (71%) had no secondary mutations (Negative group). We assessed the associations of subclonal mutations detected by error-corrected NGS with the DNA methylation profile, the most important prognostic factor in JMML. Whereas 22 of 23 (96%) and 5 of 7 (71%) patients in the Major and Minor groups, respectively, had high methylation (HM) profiles, only 20 of 74 (27%) patients in the Negative group featured HM profiles ( P < 0.001). The 4-year overall survival (OS) rates were significantly lower in the Major (52.2% [95% confidence interval {CI} = 30.5%-70.0%], P = 0.01) and Minor groups (42.9% [95% CI = 9.8%-73.4%], P = 0.03) than in the Negative group (76.3% [95% CI = 64.6%-84.5%]). In a subgroup analysis of 30 patients with secondary mutations, patients with secondary mutations in RAS pathway genes (n = 18) had significantly lower 4-year OS rates than those with secondary mutations in other genes (n = 12; 32.4% [95% CI = 12.7%-54.0%] vs. 75.0% [95% CI: 40.8%-91.2%], P = 0.047). Conclusions: We successfully performed error-corrected NGS to assess comprehensive subclonal secondary mutational profiles, including very low VAF variants. The presence of subclonal mutations, particularly in RAS pathway genes, was associated with poor OS. These findings provide important information for appropriate risk stratification, which will contribute to the implementation of precision medicine for patients with JMML.

A Proposal for a Classification of Ccus, MDS and AML Primarily Based on Genetic Abnormalities Considering the Biological Continuum of These Entities

Background: According to the 5 th edition of the WHO classification (WHO 2022) and the International Consensus Classification (ICC) the main parameters used for the definition of CCUS, MDS and AML are cytopenias, dysplasia, blast count and distinct genetic abnormalities. These entities represent a disease continuum as CCUS can progress to MDS and MDS to AML. Thus, we asked the question whether this continuum can be reflected in a proposed comprehensive classification system. Methods: We evaluated 222 CCUS, 718 de novo MDS and 751 de novo AML. The diagnosis was established following WHO 2022. Bone marrow samples were analyzed by cytomorphology, chromosome banding analysis, targeted NGS panel sequencing (median coverage 1500x) and WGS (median coverage 100x). Overall survival (OS) was available for 1552/1691 patients. Median follow-up with respect to OS was CCUS: 1.7 years, MDS: 9.3 years, AML: 6.7 years. Results: In the WHO 2022 classification a subset of AML entities (AML with defining genetic abnormalities (DGA)) is defined by fusions ( PML:: RARA, RUNX1:: RUNX1T1, CBFB:: MYH11, DEK:: NUP214, RBM15:: MRTFA other defined genetic alterations/ ODGA), rearrangements ( KMT2A, NUP98, MECOM) and distinct mutations ( NPM1, CEBPA). These well-established distinct entities were kept in the classification proposal. 511 AML cases (67%) were classified as AML DGA (Figure 1). The remaining 240 AML cases classified as AML myelodysplasia-related and AML defined by differentiation according to WHO 2022 were combined with MDS and CCUS cases and categorized hierarchically into 5 distinct groups based on the presence of the following genetic abnormalities (GA group): 1. 5q deletion, 2. SF3B1 mutation, 3. other spliceosome mutations (SP: SRSF2, U2AF1, ZRSR2), 4. IDH1/2 or DNMT3A or TET2 mutations (I/D/T), 5. None of these (NO) (Figure 1). Next, within these GA groups the presence of progression markers (PM) was evaluated: PM subset A: presence of a) biallelic TP53 alterations ( TP53bi), or b) mutations in RUNX1 or ASXL1 and mutations in RAS pathway genes or KMT2A-PTD (R/A+RAS/PTD); PM subset B: presence of a) complex karyotype without TP53bi (complex), or b) RUNX1,or ASXL1 mutations (R/A), or RAS pathway mutations or KMT2A-PTD (RAS/PTD); PM subset C: absence of these PM. The assignment into GA groups and PM subsets differed substantially between CCUS, MDS and AML. With respect to GA group CCUS, MDS and AML cases were assigned into GA groups del(5q): CCUS (0%), MDS (76%), AML (24%); SF3B1: CCUS (4%), MDS (91%), AML (5%); SP: CCUS (16%), MDS (55%), AML (29%); I/D/T: CCUS (45%), MDS (30%), AML (25%); NO: CCUS (27%), MDS (62%), AML (11%). With respect to PM subsets CCUS, MDS and AML cases were assigned into A: CCUS (1%), MDS (43%), AML (56%); B: CCUS (10%), MDS (54%), AML (36%); C: CCUS (27%), MDS (69%), AML (4%) (Figure 1). Next, OS was evaluated. Median OS for PM subsets A (n=168), B (n=301) and C (n=572) was 0.7, 2.0 and 6.8 years ( p<0.001), respectively and thus were named acute myeloid neoplasm (MN), high risk MN and low risk MN. Median OS in years in each GA group in acute MN was del(5q): 0.3, SF3B1: 0.7, SP: 1.1, I/D/T: 0.9 and NO: 0.3. The respective data for high risk and low risk MN were: 1.9, 3.3, 2.0, 1.5, 1.3 and 6.4, 7.4, 6.9, 5.8, and 6.8 years (Figure 1). Conclusions: The proposed classification aims at reflecting the biological continuum of CCUS, MDS and AML and allows reclassification during the course of the disease without changing the disease entity following the concept of MPN with chronic phase and blast phase. Further, the classification reflects prognosis and also directly indicates potential therapeutic targets. It is flexible as new genetic markers can easily be added without changing the overall concept.

Suppression of KRAS and CBL Mutations and Hematological Improvement By Lenzilumab and Azacitidine Treatment in Proliferative Chronic Myelomonocytic Leukemia

Introduction: Mutations in RAS-pathway genes ( NRAS, KRAS, CBL) are amongst the most common somatic mutations in cancer and historically resistant to most therapies. Targeted therapies that impact the RAS-pathway constitute a major unmet need in oncology. The proliferative form of chronic myelomonocytic leukemia (CMML) is commonly associated with RAS-pathway mutations and has a high propensity to develop into acute myeloid leukemia. In pre-clinical models, Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ), a proprietary Humaneered ® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF, resulted in a reduction of colony numbers and viability of CMML cells, with the greatest sensitivity in cells possessing RAS-pathway ( NRAS/ KRAS/ CBL) mutations, suggesting benefit in the targeted treatment of CMML. The PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M) trial assesses the efficacy of LENZ, in addition to azacytidine (AZA), in CMML subjects with RAS-pathway mutations and high dose sodium ascorbate (ASC) and AZA in CMML subjects without RAS-pathway mutations. Interim data from 11 subjects with RAS-pathway ( NRAS/ KRAS/ CBL) mutations in PREACH-M receiving LENZ/AZA demonstrate reductions in circulating GM-CSF and CRP with 8 subjects achieving complete response or optimal marrow response. This report describes improvements in variant allele frequencies (VAF) for RAS-pathway mutations and hematologic improvements associated with LENZ/AZA treatment in CMML. Methods: PREACH-M is a Phase 2/3 non-randomized, open-label trial in 72 subjects, aged at least 18 years, with newly diagnosed CMML based on the WHO 2016 criteria; and RAS-pathway mutations at a variant allele frequency ≥3%. Subjects received 24 cycles (every 28 days) of AZA (SC; 75 mg/m 2 for 7 days) and LENZ (IV; 552 mg; d1 & d15 of cycle 1 and d1 only for all subsequent cycles). Subjects without RAS-pathway mutations received the same AZA regimen and sodium ascorbate (IV; 30g for 7 days (15g for 1st dose only; 30g thereafter if no evidence of tumor lysis syndrome); PO; 1.1g on all other days). VAF was determined from bone marrow mononuclear cells using a 41-myeloid panel using Illumina Hi-Seq with a depth of 1000x performed on bone marrow aspirates obtained at baseline and Day 1 of treatment cycles 4, 7, and 12. Results: As of July 2023, 11 subjects completed at least 3 months of LENZ/AZA treatments and follow-up sequencing data were available for 10 subjects. Five of 10 subjects showed >10% decrease in VAF for at least one mutation detected at baseline, including 5 decreased alleles in the RAS-pathway ( KRAS, CBL) out of 22 total VAF mutation responses. CBL mutations showed the largest change with a decrease in >50% VAF in three subjects. Decreases in RAS-pathway clones by LENZ/AZA were durable at 6 months. No decreases in VAF for any mutations were observed in 2 evaluable subjects following ASC/AZA treatment. Following 3 months LENZ/AZA treatment, blood monocyte count improved from a baseline of 11.0 ± 6.0 x 10 9/L to 2.0 ± 1.0 x 10 9/L (p=0.030). Blast differential improved from 10.5 ± 5.0% to 4.0 ± 3.5% (p=0.038). Platelet count increased from 90.0 ± 50.0 x 10 9/L to 150.0 ± 60.0 x 10 9/L (p=0.010). Statistical improvements remained durable for at least 6 (n=6) and 12 (n=3) months. Hemoglobin concentration increased 100.0 ± 22.0 g/L to 115.0 ± 10.0 g/L at 3 months (p=NS, n=11) and progressively increased to 122.0 ± 10.0 g/L at 12 months (p=0.024, n=3). Spleen length decreased from 15.0 ± 4.0 cm to 13.0 ± 4.0 cm (p=0.03) and decreased further to 11.0 ± 2.0 cm in subjects who received 12 months of treatment (n=3). No evidence of disease progression or relapse was observed in any subject. Conclusion: In 11 evaluable subjects with proliferative CMML and RAS-pathway mutations, GM-CSF neutralization with LENZ in addition to AZA standard of care, resulted in significant decreases in the proportion of KRAS and CBL mutant leukemic cells, accompanied by clinically significant hematologic improvements and a reduction in splenomegaly. Lenzilumab may have efficacy in preventing outgrowth of RAS-pathway mutations, specifically KRAS and CBL, in the context of CMML and other myeloid malignancies.

The kinetics of blast clearance are associated with copy number alterations in childhood B-cell acute lymphoblastic leukemia

We analyzed the pattern of whole-genome copy number alterations (CNAs) and their association with the kinetics of blast clearance during the induction treatment among 195 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) who displayed intermediate or high levels of minimal residual disease (MRD). Using unsupervised hierarchical clustering of CNAs > 5 Mbp, we dissected three clusters of leukemic samples with distinct kinetics of blast clearance [A – early slow responders (n=105), B – patients with persistent leukemia (n=24), C – fast responders with the low but detectable disease at the end of induction (n=66)] that corresponded with the patients’ clinical features, the microdeletion profile,the presence of gene fusions and patients survival. Low incidence of large CNAs and chromosomal numerical aberrations occurred in cluster A which included ALL samples showing recurrent microdeletions within the genes encoding transcription factors (i.e., IKZF1, PAX5, ETV6, and ERG), DNA repair genes (XRCC3 and TOX), or harboring chromothriptic pattern of CNAs. Low hyperdiploid karyotype with trisomy 8 or hypodiploidy was predominantly observed in cluster B. Whereas cluster C included almost exclusively high-hyperdiploid ALL samples with concomitant mutations in RAS pathway genes. The pattern of CNAs influences the kinetics of leukemic cell clearance and selected aberrations affecting DNA repair genes may contribute to BCP-ALL chemoresistance.

BMX kinase mediates gilteritinib resistance in FLT3-mutated AML through microenvironmental factors

AbstractDespite the clinical benefit associated with gilteritinib in relapsed/refractory acute myeloid leukemia (AML), most patients eventually develop resistance through unknown mechanisms. To delineate the mechanistic basis of resistance to gilteritinib, we performed targeted sequencing and scRNASeq on primary FLT3-ITD-mutated AML samples. Co-occurring mutations in RAS pathway genes were the most common genetic abnormalities, and unresponsiveness to gilteritinib was associated with increased expression of bone marrow-derived hematopoietic cytokines and chemokines. In particular, we found elevated expression of the TEK-family kinase, BMX, in gilteritinib-unresponsive patients pre- and post-treatment. BMX contributed to gilteritinib resistance in FLT3-mutant cell lines in a hypoxia-dependent manner by promoting pSTAT5 signaling, and these phenotypes could be reversed with pharmacological inhibition and genetic knockout. We also observed that inhibition of BMX in primary FLT3-mutated AML samples decreased chemokine secretion and enhanced the activity of gilteritinib. Collectively, these findings indicate a crucial role for microenvironment-mediated factors modulated by BMX in the escape from targeted therapy and have implications for the development of novel therapeutic interventions to restore sensitivity to gilteritinib.

Ponatinib sensitizes myeloma cells to MEK inhibition in the high-risk VQ model

Multiple myeloma (MM) is a malignant plasma cell cancer. Mutations in RAS pathway genes are prevalent in advanced and proteasome inhibitor (PI) refractory MM. As such, we recently developed a VQ MM mouse model recapitulating human advanced/high-risk MM. Using VQ MM cell lines we conducted a repurposing screen of 147 FDA-approved anti-cancer drugs with or without trametinib (Tra), a MEK inhibitor. Consistent with its high-risk molecular feature, VQ MM displayed reduced responses to PIs and de novo resistance to the BCL2 inhibitor, venetoclax. Ponatinib (Pon) is the only tyrosine kinase inhibitor that showed moderate MM killing activity as a single agent and strong synergism with Tra in vitro. Combined Tra and Pon treatment significantly prolonged the survival of VQ MM mice regardless of treatment schemes. However, this survival benefit was moderate compared to that of Tra alone. Further testing of Tra and Pon on cytotoxic CD8+ T cells showed that Pon, but not Tra, blocked T cell function in vitro, suggesting that the negative impact of Pon on T cells may partially counteract its MM-killing synergism with Tra in vivo. Our study provides strong rational to comprehensively evaluate agents on both MM cells and anti-MM immune cells during therapy development.

High-Dimensional Analysis Identifies Mechanisms of Gilteritinib Resistance in FLT3-Mutated AML

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. DisclosuresBlachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

Primary central nervous system sarcoma with DICER1 mutation-treatment results of a novel molecular entity in pediatric Peruvian patients.

A high frequency of primary central nervous system (CNS) sarcomas was observed in Peru. This article describes the clinical characteristics, biological characteristics, and outcome of 70 pediatric patients. Data from 70 pediatric patients with primary CNS sarcomas diagnosed between January 2005 and June 2018 were analyzed. DNA methylation profiling from 28 tumors and gene panel sequencing from 27 tumors were available. The median age of the patients was 6 years (range, 2-17.5 years), and 66 of 70 patients had supratentorial tumors. DNA methylation profiling classified 28 of 28 tumors as primary CNS sarcoma, DICER1 mutant. DICER1 mutations were found in 26 of 27 cases, TP53 mutations were found in 22 of 27 cases, and RAS-pathway gene mutations (NF1, KRAS, and NRAS) were found in 19 of 27 tumors, all of which were somatic (germline control available in 19 cases). The estimated incidence in Peru was 0.19 cases per 100,000 children (<18 years old) per year, which is significantly higher than the estimated incidence in Germany (0.007 cases per 100,000 children [<18 years] per year; P < .001). Patients with nonmetastatic disease (n = 46) that were treated with a combination therapy had a 2-year progression-free survival (PFS) rate of 58% (95% CI, 44%-76%) and a 2-year overall survival rate of 71% (95% CI, 57%-87%). PFS was the highest in patients treated with chemotherapy with ifosfamide, carboplatin, and etoposide (ICE) after upfront surgery followed by radiotherapy and ICE (2-year PFS, 79% [59%-100%], n = 18). Primary CNS sarcoma with DICER1 mutation has an aggressive clinical course. A combination of surgery, chemotherapy, and radiotherapy seems beneficial. An underlying cancer predisposition syndrome explaining the increased incidence in Peruvian patients has not been identified so far. A high incidence of primary pediatric central nervous system sarcomas in the Peruvian population is described. Using sequencing technologies and DNA methylation profiling, it is confirmed that these tumors molecularly belong to the recently proposed entity "primary central nervous system sarcomas, DICER1 mutant." Unexpectedly, DICER1 mutations as well as all other defining tumor mutations (TP53 mutations and RAS-pathway mutations) were not inherited in all 19 patients where analyzation was possible. These tumors have an aggressive clinical course. Multimodal combination therapy based on surgery, ifosfamide, carboplatin, and etoposide chemotherapy, and local radiotherapy leads to superior outcomes.

The KRAS-G12D Mutation Induces Metabolic Vulnerability in B-Cell Acute Lymphoblastic Leukemia

Mutations in RAS pathway genes are highly prevalent in acute lymphoblastic leukemia (ALL). However, the effects of RAS mutations on ALL cell growth have not been experimentally characterized, and effective RAS-targeting therapies are being sought after. Here, we found that Reh ALL cells bearing the KRAS-G12D mutation showed increased proliferation rates in vitro but displayed severely compromised growth in mice. Exploring this divergence, proliferation assays with multiple ALL cell lines revealed that the KRAS-G12D mutation drives rewiring of methionine and arginine metabolism. Our isotope tracing results showed that mutant KRAS promotes catabolism of methionine and arginine to support anabolism of polyamines and proline, respectively. Chemical inhibition of polyamine biosynthesis selectively killed KRAS-G12D B-ALL cells, implicating the ornithine decarboxylase 1 (ODC1) enzyme as a potential vulnerability for RAS mutant B-ALL. Finally, chemically inhibiting AKT/mTOR signaling abrogated the altered amino acid metabolism and strongly promoted the in vivo growth of KRAS-G12D cells in B-ALL xenograft model. Our study thus illustrates how hyperactivated AKT/mTOR signaling exerts distinct impacts on hematological malignancies vs. solid tumors.

Mutation-Specific Signaling Profiles and Therapeutic Vulnerabilities of Induced Pluripotent Stem Cell Models of Juvenile Myelomonocytic Leukemia

Background: Juvenile myelomonocytic leukemia (JMML) is an uncommon, frequently fatal clonal myeloproliferative neoplasm of early childhood driven by mutations in Ras pathway genes and characterized by hyperactive Ras/MAPK signaling. Clinical outcomes for many children with JMML remain dismal despite use of cytoreductive chemotherapy and hematopoietic stem cell transplantation, indicating continued need for new therapeutic approaches. Others and we have demonstrated promising preclinical activity of MEK inhibition in PTPN 11 -mutant (PTPN11) JMML, but the specific signaling characteristics and potential therapeutic vulnerabilities of the more recently-discovered CBL -mutant (CBL) JMML are not known. We hypothesized that genetic subtypes of JMML have distinct biochemical signaling profiles that may be differentially targeted with specific kinase inhibitors.Methods: To test this hypothesis, we created induced pluripotent stem cell (iPSC) lines from children with JMML harboring somatic PTPN11E76K or germline CBLY371 mutations and from a normal healthy donor. We measured hypersensitivity of iPSCs to granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose-based colony-forming unit assays (MCAs). We then assessed the ability of MEK, JAK, and PI3K pathway inhibitors (Figure 1) to inhibit colony formation and signaling phosphoproteins using MCAs and phosphoflow cytometry (PC) analysis, respectively.Results: We observed similar in vitro GM-CSF hypersensitivity and colony formation of PTPN11 and CBL iPSCs in MCAs, recapitulating the phenotype of primary JMML cells. We detected constitutive Ras/MAPK (phosphorylated ERK; pERK) and PI3K pathway signaling (pS6 and pAkt) by PC analysis in both PTPN11 and CBL iPSCs versus control iPSCs (p<0.001 and p<0.05 by ANOVA, respectively) with greater pERK levels in PTPN11 cells (p<0.05 by Tukey's post-test). As predicted, activated signaling and colony formation of PTPN11 iPSCs were preferentially inhibited by the MEK inhibitors PD0325901 (PD901) and trametinib in PC analyses and in MCAs. Signaling and colony growth were abrogated in both JMML iPSC subtypes incubated with the PI3Kd inhibitor idelalisib or the mTOR inhibitor rapamycin. In contrast, we detected increased pJAK2 and pSTAT5 levels in CBL iPSCs (p<0.05), but not in PTPN11 iPSCs, with inhibition of colony growth and JAK/STAT signaling in CBL iPSCs incubated with the JAK inhibitors momelotinib and ruxolitinib. These data suggest preferential sensitivity of PTPN11 iPSCs to MEK inhibitors and of CBL iPSCs to JAK inhibitors, as well as similar sensitivity of both JMML iPSC subtypes to PI3K and mTOR inhibition.Conclusions: Our studies demonstrate mutation-specific signaling profiles of JMML iPSCs and therapeutic potential of Ras/MAPK, JAK/STAT, and PI3K/Akt/mTOR pathway kinase inhibitors in genetic subsets of JMML. Creation of additional iPSCs from patients with other JMML-associated mutations will facilitate assessment of additional drugs, alone or as combination therapy. Clinical investigation of mutation-specific kinase inhibitor therapies in children with JMML is warranted. [Display omitted] DisclosuresTasian:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Research Funding. Gandre-Babbe:GlaxoSmithKline: Employment.

Integrated Molecular Profiling of Juvenile Myelomonocytic Leukemia

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that occurs in infancy and early childhood, characterized by excessive myelomonocytic cell proliferation and granulocyte-macrophage colony-stimulating factor hypersensitivity. Over 80% of JMML patients have been reported to harbor mutually exclusive somatic and/or germline mutations in canonical RAS pathway genes (e.g., PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several poor prognostic factors, including the presence of two or more mutations, acute myeloid leukemia-like (AML-like) gene expression profiles, and selected gene promoter methylation profiles. However, the molecular pathogenesis in 10%-20% of patients and the relationships among biomarkers have not been well defined. This study aimed to assess the molecular pathogenesis in patients and the relationships among several biomarkers and outcomes.Patients and Methods: We performed comprehensive genetic analyses in 150 JMML patients, including 15 with Noonan syndrome-associated myeloproliferative disorder (NS/MPD), using whole-exome sequencing (n = 69, 46%), targeted deep sequencing (n = 92, 61%), RNA sequencing (n = 129, 86%), and genome-wide methylation analysis (n = 106, 71%).Results: We identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in three of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro . Therefore, we administered crizotinib to a patient with the RANBP2-ALK fusion gene. This patient achieved complete molecular remission after the administration of crizotinib for 51 days and successfully bridged to allogeneic stem cell transplantation. This patient survived without disease recurrence for 15 months after transplantation.We performed a genome-wide methylation analysis in 106 patients and combined the data with repository data from normal CD34+ samples (n = 5) and AML CD34+CD38− samples (n = 14) for an unsupervised consensus clustering of CpG methylation profiles, which yielded two distinct subgroups, hypermethylation profile (n = 45, 42%) and hypomethylation profile (n = 61, 58%). All AML samples had a hypermethylation profile, whereas all normal CD34+ and NS/MPD samples had a hypomethylation profile. A gene set enrichment analysis revealed marked enrichment of AML-associated genes in the hypermethylation cluster. The 5-year OS rate was 46.0% [95% confidence interval (CI), 31.0%-59.8%] among patients with a hypermethylation profile and 73.4% (95% CI, 57.6%-84.1%) among those with a hypomethylation profile (p = 0.0028). The 5-year TFS was significantly poorer in the hypermethylation group than in the hypomethylation group [2.2% (95% CI, 0.2%-10.1%) vs. 41.2% (95% CI, 27.1%-54.8%), p = 3.1 × 10−10]. Differential gene expression analysis for the hypermethylation and hypomethylation profiles revealed overexpression of LIN28B and its downstream target HMGA2 in the hypermethylation profile.Lastly, we integrated the molecular analyses of JMML, including genome-wide methylation profiling, RNA sequencing, and resequencing data and found that the hypermethylation profile was highly correlated with the most established risk factors for JMML, including older age, higher HbF level, lower platelet count, PTPN11/NF1 mutations, presence of two or more mutations, LIN28B overexpression, and AML-like expression profiles, as well as poor survival.Discussion: We identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations. Crizotinib represents a promising candidate drug for treating JMML in patients with ALK/ROS1 fusions. In addition, we established a risk profiling system based on genome-wide methylation analysis and showed that the hypermethylation profile is highly associated with poor survival and most established poor prognostic biomarkers for JMML. These finding will contribute to the development of precision treatments for JMML patients. DisclosuresNo relevant conflicts of interest to declare.

Morphologic and Immunophenotypic Differences in Juvenile Myelomonocytic Leukemias With CBL and Other Canonical RAS-pathway Gene Mutations: A Single Institutional Experience.

The diagnostic criteria for juvenile myelomonocytic leukemia have recently been revised to include clinical findings and RAS-pathway gene mutations per the 2016 World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues. Differing clinical behaviors have been observed in cases with CBL versus other RAS-pathway gene (RAS-p) mutations, notably the patients with CBL mutations can be self-limiting with spontaneous resolution. Additional clinical characteristics and histopathologic findings between these subsets are less well-described. We performed a retrospective search and identified cases with either CBL or RAS-p mutations, as per targeted and/or massively parallel sequencing. Eight patients had sufficient material for review, including cytogenetic studies and peripheral blood, bone marrow aspirate, and/or biopsy with flow cytometry analyses. Three patients showed CBL mutations and lower percentages of hemoglobin F and peripheral blood absolute monocyte counts, lesser degrees of leukocytosis compared with the RAS-p cohort, and normal megakaryocyte morphology and myeloblast immunophenotypes. Two of these patients were managed with observation only and experienced resolution of their disease. The patients with RAS-p mutations had severe thrombocytopenia, moderate to severe anemia, and experienced variable clinical outcomes. Abnormal megakaryocyte morphology and decreased numbers of megakaryocytes were seen in cases with RAS-p mutations. In addition, 3 of 4 cases with flow cytometry data demonstrated aberrant CD7 expression in myeloblasts. Our study is the first to identify morphologic and immunophenotypic differences between juvenile myelomonocytic leukemia cases with CBL or RAS-p mutations, and further supports previous reports of significantly different clinical behaviors between these subsets of patients.

RAS pathway mutation is an added-value biomarker in pediatric Philadelphia-negative B-cell acute lymphoblastic leukemia with IKZF1 deletions.

IKZF1deletion is an unfavorable factor in Philadelphia negative (Ph -) B-cell acute lymphoblastic leukemia. However, the effects ofIKZF1deletions co-existing genetic alterations in Ph (-) ALL have not been extensively studied. Bone marrow samples from 368 children with Ph (-) ALL were analyzed by using multiplex ligation-dependent probe amplification kit for detection of gene deletions and Sanger sequencing for mutational analysis of RAS pathway genes. The outcome was analyzed on 215 patients treated with Taiwan Pediatric Oncology Group-ALL-2002 protocol. IKZF1 deletions were present in 12.8% and IKZF1plus in 6.3% of patients. Mutations of RAS pathway genes were detected in 25.0% of IKZF1-deleted patients. The 10-year event-free survival (EFS) of IKZF1-undeleted patients was significantly better compared with IKZF1-deleted patients (80.0%vs. 47.8%, p=0.001). Compared with outcome of patients harboring IKZF1 deletion alone, no difference in EFS was observed in patients with IKZF1plus , whereas three patients carried both IKZF1 and ERG deletions had a superior 10-year EFS (100%). The 10-year EFS of patients with any gene mutation of RAS pathway was worse than that of patients with wild-type genes (79.1%vs. 61.6%, p=0.033). In multivariate analysis, RAS pathway mutations and IKZF1 deletion were independent predictors of inferior EFS. Co-existence of IKZF1 deletion with RAS pathway mutations had a worst 10-year EFS (11.1 ± 10.5%) and 10-year OS (53.3 ± 17.6%). Our results showed that RAS pathway mutation is an added-value biomarker in pediatric IKZF1-deleted Ph (-) ALL patients.

RARE-42. PRIMARY INTRACRANIAL SARCOMA WITH DICER1-MUTATION - TREATMENT RESULTS OF A NEW MOLECULAR ENTITY

OBJECTIVEAn unexpectedly high incidence of sarcomas of the Central Nervous System (SCNS) was recently observed in Peru. We describe clinical and biological characteristics of the disease.METHODSSeventy pediatric patients with primary SCNS diagnosed between January 2005 and June 2018 were analyzed. DNA methylation profiling and gene panel sequencing was available from 28 and 27 tumors, respectively.RESULTSMedian age was 6 years (range 2–17.5), 66/70 patients had supratentorial tumors, 56 patients intratumoral hemorrhage at diagnosis. Three patients fulfilled clinical criteria of NF1; 35 had café-au-lait spots and/or freckling. DNA-methylation profiling classified 28/28 as “intracranial spindle cell sarcoma with rhabdomyosarcoma-like features and DICER1 mutations”. DICER1 mutations were found in 26/27, TP53 mutations in 22/27, and RAS-pathway gene mutations (NF1, KRAS, NRAS) in 19/27 tumors, all of which were somatic (germline control available in n=19 cases). Survival was analyzed in 57 patients with non-metastatic disease who received adjuvant therapy. Two patients had metastatic disease, eleven did not receive or abandoned treatment. Two-year OS was 66.3% (95%-CI: 54–81%), 2-year PFS 51% (38–67%). PFS was highest in patients treated with postoperative ICE chemotherapy followed by radiotherapy and ICE (2y-EFS 79% [59–100%], n=18) and worse after upfront radiotherapy followed by ICE (40% [19–85%]; n=10) or VAC (50% [28–88%], n=12) and radiotherapy only (21% [6–71%], n=11; p=0.008).CONCLUSIONPrimary SCNS with DICER1 mutation have an aggressive clinical course. A combination of chemotherapy and radiotherapy seems beneficial. A link to a cancer predisposition syndrome could not be established so far.

Abstract IA33: Development of effective therapies for neurofibromatosis type 1 and other pediatric RAS-driven tumors

Abstract The RAS pathway is critical for cell survival and development. More than 20% of all adult cancers and many of the most aggressive pediatric cancers are driven by sporadic mutations of the RAS family of genes. In spite of substantial efforts by academia and the pharmaceutical industry, no effective therapies targeting RAS-mutated cancers have been identified to date. RASopathies are genetic disorders characterized by germline mutations in RAS pathway genes. These syndromes, such as neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome, and Costello syndrome (CS), have a widely variable incidence (1:1000 NS -1:300,000 CS), are usually first diagnosed in children, and exhibit a wide variety of manifestations including an increased risk of development of pediatric cancers such as sarcomas, leukemias, and CNS tumors (up to 42-fold increased risk compared to the general population). No approved medical therapies exist for RASopathies. Through the conduct of a longitudinal natural history study, preclinical studies in collaboration with extramural investigators, and early-phase trials, the NCI Pediatric Oncology Branch recently identified the MEK inhibitor selumetinib as the first active therapy for debilitating peripheral nerve sheath tumors called plexiform neurofibromas (PN) in NF1. The POB has identified and clinically characterized atypical neurofibromas (ANF) in NF1 as precursors to aggressive sarcomas called malignant peripheral nerve sheath tumors (MPNST). This has allowed for the development of targeted therapies and prevention strategies for these tumors. This example raises the possibility of developing effective therapies for manifestations of other RASopathies. Recent comprehensive molecular, epigenetic, and genomic studies have demonstrated the mechanisms of RAS pathway activation in several pediatric malignancies including rhabdomyosarcoma, relapsed neuroblastoma, gliomas, and leukemias. These studies will enable the development of clinical trials targeting these tumors more effectively. Citation Format: Brigitte Widemann. Development of effective therapies for neurofibromatosis type 1 and other pediatric RAS-driven tumors [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 IA33.

Abstract IA30: Dissecting the playbook of cancer: Genomic analysis of 100,000 human tumors reveals elaborate patterns of activation of the RTK-RAS-MAPK pathway

Abstract Massive tumor sequencing allows us to progress beyond cataloging driver mutations, to discovery of the intricate patterns of genomic alterations that are required to enable a successful tumor. We used patterns of mutation co-occurrence to map activation of the ras pathway in over 16,000 lung adenocarcinomas. This revealed a network of 40 highly mutually exclusive mutations in 19 genes, 18 of which are known ras pathway genes (the RTKs EGFR, ERBB2, MET, ALK, RET, ROS1, KIT, FGFR2; NRAS and KRAS, BRAF and CRAF, MEK1/2, CBL, NF1, SOS1, and RIT1), indicating that each mutation is a strong pathway driver and is redundant with other pathway mutations. Together, these mutations cover 71% of all lung adenocarcinomas and include several previously uncharacterized mutations. Analysis of the remaining tumors uncovers additional patterns of ras pathway activation. Some have additional mutations in ras pathway genes that have the opposite pattern—they are co-occurrent with each other, either in an additive manner where several mutations are enriched with each other, such as rare raf and ras family mutations, or synergistic, where a particular pair of mutations is highly co-occurrent, often in the same gene. These pairs are exclusive of other pathway mutations, suggesting that they are also strong pathway activators. We also see partially exclusive mutations, including amplifications of several genes and loss-of-function RAF family mutations that are likely to be weak activators of the ras pathway in additional tumors. Finally, this approach also identifies recurrent mutations that are independent of other pathway mutations, giving clinical evidence that their function in human tumors is distinct from the common function of other ras pathway mutations. We validated these patterns of exclusivity, additivity, and synergy in 80,000 genomic profiles from 400 other tumor types, and identified additional exclusive ras pathway alterations in several other tissues. Across all cancers, 30% of all tumors had exclusive mutations indicating that they were ras pathway driven. The individual mutations varied greatly between tissues, suggesting that there exist extensive tissue-specific enhancers and inhibitors of each oncogenic mutation, which may be of great relevance to drug and biomarker development. This initial foray into the future of massive tumor genomic datasets shows that we can transform genomic data from individual mutations into patterns of pathway activation, and distinguish pathway-driving mutations from other hotspots, providing a better understanding of the oncogenic strategies used by each human tumor. The degree of mutational exclusivity seen can predict the degree of oncogene addiction, and patterns of co-mutation can suggest strategies for combination therapies, as well as allow reverse translation to enable better matching of preclinical and basic research models to oncogenic strategies that are most relevant to human tumors. Citation Format: Gerard Manning, Nick Lounsbury, Ryan Hartmaier, Sally Trabucco, Ethan Sokol. Dissecting the playbook of cancer: Genomic analysis of 100,000 human tumors reveals elaborate patterns of activation of the RTK-RAS-MAPK pathway [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 IA30.

Frequency and Prognostic Impact of IKZF1 and Co-Existed Gene Alterations in Childhood B-ALL in Taiwan

Introduction: IKZF1 deletion was firstly reported to be very frequent in Philadelphia (Ph) positive B-cell acute lymphoblastic leukemia (B-ALL), and continuously found in other B-ALL subtypes. IKZF1plus defined as the presence of any of CDKN2B, CDKN2A,PAX5 or PAR1 in the absence of ERG deletion and was reported to describe a new minimal residual disease (MRD)-dependent poor prognostic profile in B-ALL.1 We aimed to analyze the frequency and prognostic relevance of IKZF1 deletion with or without co-occurring gene alterations in Taiwanese children with B-ALL treated with TPOG-ALL-2002 protocol or MRD-directed TPOG-ALL-2013 protocol. Methods: Bone marrow samples at diagnosis from 561 children excluding Ph+ and infant ALL were analyzed. Detection of gene deletion was carried out with multiplex ligation dependent probe amplification (MLPA) kit (SALSA MLPA P335 and P327) and mutations of RAS pathway genes (NRAS, KRAS, and PTPN11) were assessed by Sanger sequencing. The outcome was analyzed on 259 patients treated with TPOG-ALL-2002 protocol and 158 patients treated with TPOG-ALL-2013 protocol. Results: IKZF1 deletions were present in 11.2% of 561 patients. DEL 4-7, DEL 1-8, and other variants were present in 50.8%, 14.3%, and 34.9%, respectively of 63 IKZF1-deleted patients. Co-existence of additional deletions with IKZF1 deletion was detected in 34.0% for CDKN2B, 37.3% for CDKN2A, 27.7% for PAX5, 2.1% forPAR1, and 8.0% for ERG. IKZF1plus comprised of 4.4% of 546 patients who had all the genes examined. Co-existed mutations of RAS pathway genes were detected in 5.0% for PTPN11, 13.3% for NRAS, and 10.0% for KRASin the IKZF1-deleted patients. The 5-year event-free survival (EFS) of IKZF1-undeleted patients was significantly better compared with IKZF1-deleted patients in TPOG-2002 cohort (82.5 ± 2.6% vs. 56.9 ± 9.1%, P = 0.002) as did TPOG- 2013 cohort (89.4 ± 3.9% vs. 59.1 ± 19.8%, P = 0.012). The 5-year overall survival (OS) of IKZF1-deleted patients was worse than that of patients with undeleted IKZF1 in TPOG-2013 cohort (58.3 ± 19.8% vs. 89.2 ± 4.2%, P = 0.004) but not in TPOG-2002 cohort (90.9 ± 5.0% vs. 86.9 ± 2.3%, P = 0.846). DEL 1-8 patients had an inferior 5-year EFS (33.3 ± 19.2%) compared with DEL 4-7 (63.3 ± 12.0%,) or DEL-others (60.0 ± 18.4 %) in TPOG- 2002 cohort (P = 0.003). No significant difference in OS was observed between patients among different IKZF1-deleted subtypes in TPOG-2002 cohort (P = 0.283) (Fig. 1A) but there was different in TPOG-2013 cohort (P = 0.003) (Fig. 1B). Patients with IKZF1 deletion alone had comparable 5-year EFS and 5-year OS compared with patients with IKZF1plus in TPOG-2002 cohort whereas patients with IKZF1 deletion alone had worse 5-year EFS (33.3 ± 27.2% vs. 80.0 ± 17.9%, P = 0.284) and 5-year OS (33.3 ± 27.2% vs. 80.0 ± 17.9%, P = 0.415) than that of KZF1plus in TPOG-2013 cohort. The prognostic impact of IKZF1 deletion with co-existed gene alterations in TPOG-2002 cohort was further analyzed as follows: IKZF1plus with ≧ 2 co-existed deletions had an inferior 10-year EFS (23.8 ± 20.3%) compared with patients with IKZF1 deletion alone (36.7 ± 17.5%) or IKZF1-undeleted patients (81.5 ± 2.7%) (P = 0.005) (Fig. 1C). Three patients carried both IKZF1 and ERG deletions had a superior 5-year EFS (100%) compared with IKZF1-deleted alone or IKZF1-undeleted patients (P = 0.005) (Fig. 1D). The 10-year EFS of patients with any gene mutation of RAS pathway was worse than that of patients with RAS wild-type genes (66.6% ± 6.4% vs. 83.7% ± 2.6%, P = 0.011). In multivariate analysis, in addition to the initial white blood count &gt; 50 x 109/L and KMT2A-rearranged, IKZF1 deletion (HR=2.609, 95% CI: 1.363-5.034; P = 0.004) and RAS pathway gene mutations (HR=2.360, 95% CI: 1.336-4.168; P = 0.003) were independent genetic predictors for inferior EFS. Co-existence of IKZF1 deletion with RAS pathway mutations had worst 5-year EFS (20.0% ± 12.6%, P &lt; 0.0001, Fig. 1E), and 10-year OS (53.3% ± 17.6%, P = 0.042, Fig. 1F). Conclusions: Our results showed that IKZF1 deletion alone was associated with an inferior OS in MRD-directed TPOG-2013 treated cohort but not in TPOG-2002 cohort in which it became significant when co-existed with RAS pathway mutations. Reference: 1. IKZF1plus defines a new minimal residual disease-dependent very-poor prognostic profile in pediatric B-cell precursor acute lymphoblastic leukemia. J Clin Oncol. 2018, 1240-1249. Figure 1 Disclosures No relevant conflicts of interest to declare.

Detection of Subclonal SETBP1 and JAK3 Mutations in Patients with Juvenile Myelomonocytic Leukemia Using Droplet Digital PCR

Background Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative disease. Approximately 85% of patients with JMML harbor germline and/or somatic mutations in RAS pathway genes, such as PTPN11, NF1, CBL, NRAS, and KRAS. In a subset of patients with JMML, SETBP1 and JAK3 mutations were identified as secondary mutations in addition to primary RAS mutations. These secondary mutations are associated with the disease progression and poor clinical outcome. Recently, it has been reported that subclonal SETBP1 mutation also correlates with a dismal prognosis. Therefore, we hypothesized that subclonal JAK3 mutation is present in a higher than expected number of patients with JMML and associated with poor prognosis. The aim of this study is to identify patients with subclonal SETBP1 and/or JAK3 mutations at the diagnosis using droplet digital PCR (ddPCR) and to elucidate their clinical outcomes. Patients and Methods We enrolled 128 patients with JMML and 15 with Noonan syndrome-associated myeloproliferative disorder (NS/MPD). Using bone marrow (BM) or peripheral blood derived genomic DNA, ddPCR was performed in the 143 patients to detect SETBP1 p.D868N and JAK3 p.R657Q hotspot mutations with low variant allele frequencies (VAF). The study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results To assess the false-positive rate of the ddPCR assay for each mutation, the assay was also performed in 30 healthy volunteers. Among these, the false-positive rate (mean ± standard deviation) for SETBP1 and JAK3 mutations was 0.010% ± 0.010% and 0.013% ± 0.012%, respectively. Due to the presence of false-positive droplets, the sensitivity and the quantitative linearity was evaluated for &gt;0.01% VAF. The significant correlation between the expected and the observed VAF in SETBP1 and JAK3 was observed (R-squared, 0.9923 and 0.9922, respectively). Therefore, 0.05% VAF was defined as the cut-off value in this assay. Among the 143 patients, ddPCR detected SETBP1 and JAK3 mutations in nine (6.3%) and fifteen (10.5%), respectively. SETBP1 and JAK3 mutations, including variants with low allele frequencies, were not detected in NS/MPD. Among patients with SETBP1 and/or JAK3 mutations, two and six patients harbored less than 1.0% VAF. Patients with less than 1.0% VAF in SETBP1 or JAK3 mutation exhibited a significantly poorer 2-year transplantation-free survival than those without SETBP1 and JAK3 mutations (P = 3.05 × 10-3). JMML is genetically characterized by an extremely small number of somatic mutations (an average of 0.8 mutations/exome/patient). However, we demonstrated that among 19 patients with SETBP1 and/or JAK3 mutations, five patients (26.3%) harbored both the mutations. This finding suggested a statistically significant co-occurrence of SETBP1 and JAK3 mutations in JMML. In order to determine whether SETBP1 and JAK3 mutations were present in the same clone or not, we performed colony formation assays using BM cells in one of the five patients with both SETBP1 and JAK3 mutations. This case harbored 0.9% VAF in JAK3 and 41.9% in SETBP1 in addition to 46.0% in PTPN11 mutation (c.227A&gt;C, p.E76A), respectively. In total, 93 colonies were collected and individually analyzed by Sanger sequencing, of which two colonies (2.1%) were identified with both SETBP1 and JAK3 mutations. Conclusions ddPCR is a useful tool to assess subclonal SETBP1 and JAK3 hotspot mutations and to estimate the prognosis. It would be better to start preparing for hematopoietic stem cell transplantation when patients with JMML harbored subclonal SETBP1 and/or JAK3 mutations at the diagnosis. While JMML is characterized by a paucity of somatic mutations, clones harboring SETBP1 and JAK3 mutations were identified. This finding suggests that SETBP1 and JAK3 mutation are susceptible to each other. Furthermore, the serial acquisition of SETBP1 and JAK3 mutations might correlate with the disease progression. Disclosures No relevant conflicts of interest to declare.

Integrated Genomic-Transcriptomic Study Highlights Accumulation of Genetic Variants and Activation of Inflammatory Pathways

INTRODUCTION: The molecular basis of Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) is unclear. So-called "driver" mutations in JAK2, CALR, or MPL are present in the vast majority of cases, but there is no compelling evidence to explain how each mutant gene can lead to phenotypically distinct and/or overlapping disease phenotypes. In an attempt to understand the molecular events that underlay clinical characteristics of MPNs, we studied gene expression profiles and sequenced hematopoietic cells of MPN patients with a focus on myelofibrosis (MF) landscape. METHODS: Patients were consented to MI-ONCOSEQ study approved by the University of Michigan IRB. Peripheral blood or bone marrow aspirates were either enriched for CD34 expressing cells or peripheral blood or bone marrow mononuclear cells (PBMC/BMMC) were analyzed. MI-ONCOSEQ is a Next Generation Sequencing platform to identify genetic aberrations in 1,711 genes and gene expression and fusion analysis of 24,774 capture targets by transcriptome sequencing. Gene expression data was analyzed between various sub-groups of MF based upon clinical (spleen size) or molecular (mutations in Ras pathway genes) characteristics. Gene set enrichment analysis was conducted for the MF cohorts (23 CD34 enriched, 76 PBMC/BMMC) and ET/PV/PrePMF cohort (12 CD34, 35 PBMC/BMMC). RESULTS: We analyzed the genetic landscape of 163 patients with MPNs: 113 with overt MF and 50 with ET (18), PV (23), or prePMF (9). In addition to driver genes, 183 other gene variants were observed. The number of gene variants was higher in older patients (median 4 and 5 variants in those aged 70-80 and &gt;80 yrs respectively) with MF as compared to ET/PV/PrePMF where the median number of variants did not exceed 3. Mutations in ASXL1, TET2, RAS and SRSF2 increased in frequency with age (Fig 1). Gene expression profiles of sub-groups of MF were further analyzed to understand aberrantly regulated molecular pathways. Hierarchical clustering of all MF patients showed that CD34 enriched samples to be distinct from the PBMC/BMMC cohort and therefore these cohorts were analyzed separately. Moreover, hierarchical clustering suggested differences in patients with large spleens. In the comparison of MF to ET/PV/PrePMF within the CD34 population gene enrichment highlights hemopoiesis, leukemia related pathways as well as endoplasmic reticulum and Golgi transport pathways. Previously, we saw that RAS pathway mutations predicted proliferative disease with high WBC counts. Therefore, we focused on RAS pathway mutated cohorts versus RAS wild type cohorts and identified dysregulated pathways by gene set enrichment analysis (Fig 2). The RAS mutated MF cohort (PBMC/BMMC fraction) showed up-regulation of cytokines IL6 (p-value 9.39E-06), IL8 (p-value 1.16E-04), and IL1beta (p-value 5.24E-04) and down-regulation of the TNF superfamily (p-value 9.98E-04). Most notably, there was up regulation of NFkB transport to the nucleus (p-value 8.69E-05) and transcription factor activity. In general, several metabolic pathways were affected and inflammatory pathways were up-regulated. Since spleen size is an indicator of disease severity, progression and response to therapy, gene set enrichment between cohorts of patients with different spleen size (&gt;6cm by physical exam versus &lt;6cm) was analyzed (Fig 3). The data suggest dysregulation of megakaryocyte differentiation (p-value 2.05E-05), cytokine production (p-value 9.09E-05) and signaling, JAK-STAT pathway (p-value 5.10E-05), RAS signaling (p-value 7.67E-05) and NFkB pathway (p-value 1.31E-04) in patients with larger spleens. CONCLUSIONS: Age is a high risk for many hematological malignancies. We analyzed the number of genetic variants by age and determined that accumulation of higher number of mutations might indicate why disease progresses rapidly in older patients. Not only genetic variants, but gene expression changes also contribute to the pathogenesis of MF. Analysis of gene expression changes show enrichment of genes regulating JAK-STAT pathway activity and cytokine production as anticipated, but also implicates epigenetic regulators and the RAS signaling pathway in disease biology. Moreover, enriched pathways in gene expression analysis underscore the dysregulation of NFkB, perhaps as a result of inflammatory response. Thus these pathways are promising candidates for intervention in patients with MF. Disclosures Pettit: Samus Therapeutics: Research Funding. Talpaz:Imago BioSciences: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; CTI BioPharma: Research Funding; Constellation: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Samus Therapeutics: Research Funding.