NRAS Mutations Research Articles

Synergistic Anti-Tumor Effects in RAS Mut Multiple Myeloma By Targeting MAP4K2 and RAS Pathways

Mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) is an important key regulator of the stress-activated MAPK core signaling pathways. Our previous work demonstrated that MAP4K2 is highly expressed in multiple myeloma (MM) cells, and MM cells carrying a RAS mutation are more vulnerable to MAP4K2 inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins ( Li et al. Blood 2021). Targeting the aberrant RAS/MAPK signaling, MEK inhibitors have been shown to induce cytotoxicity in MM (De la Puente, P. et al. Blood Cancer J. 2016). Thus, we used a recently developed MEK inhibitor (MEKi) Trametinib to investigate the therapeutic potential of combined MEK and MAP4K2 signaling inhibition in RAS-driven MM. Tet-on sh-MAP4K2 lentivirus was introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To examine the combined effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline to silence MAP4K2 expression, followed by treatment of MEKi Trametinib at different dosages. Cell proliferation, apoptosis, and cell cycle were analyzed five days later. Data showed that MAP4K2 silencing combined with MEK inhibition significantly decreased cell proliferation to 10% compared with MEKi alone 32% ( p<0.01) by MTS assays, and enhanced cell apoptosis (MEKi alone vs. with MAP4K2 silencing: 26% vs 91%, p<0.01). Cell cycle assay demonstrated that MEKi induced G1 arrest in RAS Mut MM cells (MEKi alone:76% vs vehicle control: 54%, p<0.01), which was also significantly higher in combination with MAP4K2 silencing (92%). Western blot assay confirmed that MAP4K2 silencing combined with MEKi significantly enhanced the downregulation of IKZF1 and c-MYC compared to the MEKi treatment alone. These data suggest that the MEKi and MAP4K2 inhibition combination has synergetic anti-MM effects. To further confirm the synergetic effects, we tested the combined effects of MEKi with the MAP4K2 kinase inhibitor TL4-12. H929 (N-Ras Mut) cells were treated by vehicle control, TL4-12 (2 uM) alone, MEKi (200 nM) alone, or TL4-12 + MEKi for 4 days, followed by an apoptosis assay. The combination of TL4-12 and MEKi strongly enhanced the cytotoxicity effects compared to the single-agent treatments. Consistently, the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation assay confirmed that dual treatment significantly delayed H929 cell proliferation compared to the single-agent treatments (percentage of proliferating cells: TL4-12 alone: 86.2%; MEKi alone: 89.03%; dual treatment: 18.9%. p<0.01). Western blotting assay further confirmed that dual treatment of MEKi and MAP4K2i enhanced the decrease in MEK1/2 and ERK1/2 phosphorylation and downregulation of transcriptional factors IKZF1 and c-MYC. Taken together, our findings demonstrate that the combination of MAP4K2 inhibition with MEKi results in synergetic anti-MM effects in RAS mutation myeloma, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma.

Integrated Analysis of KIT Exon 17 Mutations and Flow-MRD Refines Risk Stratification in Pediatric Acute Myeloid Leukemia with RUNX1:: RUNX1T1

INTRODUCTION: Pediatric acute myeloid leukemia (AML) with RUNX1:: RUNX1T1 is considered a favorable risk group, with a high probability of event-free survival (EFS) in recent clinical trials. However, 20-30% of patients still experience relapse within three years and some patients have poor outcome. Several prognostic factors, including secondary genetic abnormalities and treatment response, have been previously reported. Flow cytometry-based measurable residual disease (flow-MRD) has been suggested as a poor prognostic factor in AML with RUNX1:: RUNX1T1. Additionally, KIT mutations, particularly mutations of exon 17, have been associated with poor EFS in both adult and pediatric patients. However, the combined impact of KIT exon 17 mutations and flow-MRD on prognosis has not been thoroughly investigated. This study aims to evaluate the clinical and genetic features of pediatric patients with RUNX1:: RUNX1T1-positive AML enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-12 trial and determine if integrated analysis of flow-MRD and KIT exon 17 mutations can improve risk stratification. METHOD: A total of 101 pediatric AML patients with RUNX1:: RUNX1T1 were enrolled in the study. The patients were treated in the JPLSG AML-12 trial, and their clinical and genetic characteristics were analyzed. Targeted capture sequencing was performed on bone marrow samples obtained at diagnosis. The custom gene panel was designed for mutation profiling of pediatric AML, including 504 genes. Flow-MRD was centrally monitored at the end of induction 1 (EOI1) and the end of induction 2 but was not used to guide subsequent therapies. Leukemia-associated immunophenotypes were identified in diagnostic specimens and marker combinations that allowed detection of leukemia with a sensitivity of at least 0.1% were applied to subsequent samples. RESULTS: Patients with KIT exon 17 mutations had a higher white blood cell count at diagnosis and a significantly higher rate of MRD positivity (≥0.1%) at EOI1 compared to patients without KIT exon 17 mutations. Immunophenotypic analysis showed no significant differences in CD117 or CD33 expression between patients with and without KIT exon 17 mutations; whereas CD19 expression was observed less frequently and, although not significantly, CD56 expression was observed more frequently in the patients with KIT exon 17 mutations. The genetic landscape analysis revealed that KIT mutations were the most common and tended to coexist with ARID1B mutations, while NRAS mutations were mutually exclusive. The 5-year EFS and overall survival (OS) for the entire cohort were 67.3% and 82.7%, respectively. Negative MRD at EOI1 was associated with significantly superior 5-year EFS and OS (71.6% and 85.9%) compared to positive MRD (12.5% and 37.5%)(p<0.01). Patients with KIT exon 17 mutations had significantly inferior 5-year EFS and OS (46.9% and 62.2%) compared to those without KIT exon 17 mutations (76.8% and 92.4%) (p<0.01). Furthermore, we evaluated a prognostic impact of MRD within the KIT exon 17 mutated and non-mutated cases. As for the non-mutated cases, the 5-year OS in the patients with negative MRD was 92.2%. Among the KIT-mutated cases, the 5-year OS in the patients with negative and positive MRD was 68.0% and 33.3%, respectively. CONCLUSION: Patients with both non-mutated KIT exon 17 and negative MRD have the best prognosis, while positive MRD and KIT exon 17 mutations are associated with poorer outcomes. These findings indicated that integrated analysis of flow-MRD and KIT exon 17 status enables optimal risk assignment strategies in pediatric AML with RUNX1:: RUNX1T1.

Validation and Retraining of the Stellae-123 Gene Expression Signature Improved Risk Stratification in Taiwanese Acute Myeloid Leukemia Patients

The European Leukemia Net (ELN) recommendations provide valuable guidance in treatment decisions of patients with acute myeloid leukemia (AML). However, the genetic complexity and heterogeneity of AML are not fully covered, despite the critical role of gene expression analysis in risk stratification. Addressing this gap, the Stellae-123 score, an AI-based model that captures gene expression patterns, has demonstrated promising results in predicting survival of AML patients in four western cohorts. This study aims to assess the applicability of Stellae-123 in a Taiwanese cohort. Our cohort included 233 de novo AML patients, who were diagnosed and treated at the National Taiwan University Hospital and received standard induction therapy with 7+3 chemotherapy (or 5+2 in elder fit patients). All patients had sufficient bone marrow samples for DNA and RNA sequencing at diagnosis. The median age of the cohort was 41 years. Among the patients, ELN-2022 risk groups were distributed as follows: favorable (53.4%), intermediate (27.6%), and adverse (19%). Appling the original Stellae-123 model, which derived gene expression signatures from the BeatAML cohort RNA-seq data, our patients were categorized into low-, intermediate-, and high-risk groups, each representing 33% of the cohort. Overall, there were no differences in clinical features among these risk groups. The Stellae-123 risk groups partially aligned with the ELN risk groups, with more ELN low-risk patients in the Stellae-123 low-risk group and a higher proportion of ELN-high risk patients in the Stellae-123 high-risk group. Intriguingly, aside from mutations listed in ELN-2022, the Stellae-123 high-risk group had significantly less NRAS mutations than others. Cumulative hazards predicted by Stellae-123 model yielded a c-index of 0.612 for overall survival (OS), and the three risk groups had significantly different OS (p<0.001). We next postulated that since Stellae-123 relies on molecular features derived from the leukemic cells, it might serve as a biomarker of biological risk to predict relapse-free survival (RFS). The c-index calculated by Stellae-123 to predict RFS was 0.607, suggesting a reasonable ability to discriminate between different risk levels for RFS. Recognizing the potential variation between the original training cohort and the Taiwanese population, we addressed this concern by retraining the Stellae-123 model using Taiwanese RNAseq data. As a result, the model retraining led to significant improvements in the cross-validated c-indexes, with values of 0.656 (OS) and 0.636 (RFS), leading to an even more robust risk stratification (p<0.001, Figure 1A). As we noticed overlapping grouping between Stellae-123 and ELN-2022 in both pretrained and retrained models, we explored if Stellae-123's prognostic power was independent of ELN risk status by conducting a multivariable analysis. Remarkably, in both the pretrained and retrained Stellae-123 models, whether calculated as continuous values or categorized into three groups, they consistently demonstrated discriminative power in predicting OS and RFS, irrespective of age and ELN-2022 risk, suggesting that Stellae-123 could offer additional valuable information in predicting patient outcomes. Considering recent data showing that incorporating age helped improve the prognostication performance of ELN-2022 and that age was an independent risk factor for inferior outcome in our cohort, we examined how age could complement current risk stratification. Serial model testing revealed significant improvement of the prognostic models when age and transcriptomic data, especially the locally retrained Stellae-123 model, were combined with ELN-2022, evident by drastically declined Akaike Information Criterion (AIC) values. Time-dependent ROC curve analysis confirmed the potential of incorporating Stellae-123 and age to complement current risk stratification system (Figure 1B). In summary, we showed that the Stellae-123 predicted both OS and relapse risk in AML patients from Taiwan. Furthermore, we demonstrated that model retraining could improve prognostication by adapting the Stellae-123 to the particularities of Taiwanese patients. Given the remarkable extrapolation of the signature, it's suggested gene expression profile be incorporated in the risk stratification of AML patients who are candidates to intensive therapy.

Corelationship of Lymphocyte Subsets and Prognosis in Juvenile Myelomonocytic Leukemia

Objective: Juvenile Myelomonocytic Leukemia (JMML) is a rare hematopoietic stem cell malignant clonal disease in children and has the characteristics of MDS/MPN. Due to the poor response of JMML to chemotherapy, hematopoietic stem cell transplantation (HSCT) is currently the only cure method for JMML. Currently, there are some reports on the distribution of lymphocyte subsets in MDS. A significant lymphocytopenia was observed in MDS patients compared with similarly aged healthy people. Another study form Polish found patients with MDS have more CD3+T cells than healthy donors. But there are few related reports in JMML. The objective of this study is to analyze relationship between the distribution of lymphocyte subsets and prognosis in JMML patients, and explore new risk stratification indicatorsfor JMML personalized therapy. Method: This study retrospectively collected JMML patients clinical data from Institute of Hematology & Blood Diseases Hospital of CAMS during Jan 2008 and Nov 2022. All completed complete blood count and peripheral blood classification, fetal hemoglobin (HbF), granulocyte colony-stimulating factor (GM-CSF) high sensitivity, bone marrow morphology, immune typing, molecular biology, cytogenetics and other tests. We analyzed the relationship between distribution of lymphocyte and prognosis in these patients. Result: 96 newly diagnosed JMML patients were admitted to this study. The median age at diagnosis was 25 (2-82) months, and male to female was 3.8:1. 66 cases underwent lymphocyte subsets by flow cytometry. Median percentage of lymphocytes is 34.3%(1.8%-90.4%, normal range: 20%-40%), median percentage of CD3+T cells is 54.85%(10.3%-91%, normal range: 57%-88%), median percentage of CD3+CD4+ T cells is 30.95%(3.54%-53%, normal range: 27%-58%), median percentage of CD3+CD8+ T cells is 18.4%(2%-49.1%, normal range: 16%-45%), median percentage of NK cells is 13.95%(2.3%-50.3%, normal range: 3%-32%), and median percentage of B cells is 23.25%(4%-69.6%, normal range: 4%-18%). These cases were followed-up to Jan 2023, with a median follow-up time of 12 months (1-104 months). The overall survival (OS) rate was 52.67%, with a median OS time of 12 months (0-103 months). There were significant differences in the percentage of CD3+T cells (30.89% ± 9.46% vs 59.05% ± 10%, P<0.01) and CD3+CD4+T cells (14.87% ± 5.82% vs 35% ± 5.41%, P<0.01) between children with single NRAS mutation and compound mutations (≥2 classical five Ras pathway mutation ). In PTPN11 mutation patients, there is a significant positive correlation between OS and the percentage of lymphocytes (P=0.018). Conclusion: Our study shows differences in percentage of CD3+ T cells and CD3+CD4+ T cells between patients with NRAS mutation and compound mutations. Patients with PTPN11 mutation and higher percentage of lymphocytes will have better prognosis.

Efficacy of Demethylated Drug Combine with Low Dose Chemotherapy in Juvenile Myelomonocytic Leukemia

Objection：Juvenile myelomonocytic leukemia (JMML) is a rare pediatric leukemia with a median survival time of less than 2 years. Currently, hematopoietic stem cell transplantation (HSCT) is the only possible cure of JMML. Most of the newly diagnosed pediatric patients with JMML have high tumor burden, and rapid disease progression, and may not tolerate HSCT. In the present study, we aimed to explore the effection of Demethylated drug in combination with low-dose chemotherapy on JMML patients before HSCT. Methods：A retrospective analysis of 16 JMML patients was performed from Jul 2017 to Nov 2021. All patients were given Demethylated drug in combination with low-dose chemotherapy, 10 patients decitabine at a dose of 20 mg/m2 for 5 days, supplemented with cytarabine (50-100 mg/m2×3~5 days), and /or etoposide (50 mg/m2×3~5 days), 6 patients Azacytidine at a dose of 75mg/m2 for 7 days combined with homoharringtonine 2mg/m2 for 5-7 days. Each treatment interval was 4 weeks, bridging to HSCT after three~four courses of treatment. Results：The median age of onset of the 16 JMML patients was 36 months (range 6-51 months), the male to female ratio was 14:2, and the median volume of the spleen at diagnosis was 357cm3 (range: 175-588) by Three-dimensional ultrasound of spleen. The median white blood cell (WBC) count was 39 (range 2-128) × 109/L, the median hemoglobin 84 (range 47-110) g/l, and the median platelet 36 (range 16-75) × 109/L，the median monocyte count was 8 (range 2-26) × 109/L at diagnosis, the median RDW-CV was 19 (15-30)%, the median RDW-SD 56 (45-77) fl, the median HbF 37(1-76)%. Chromosome was performed, of which 13 cases had normal karyotype. 2 cases had tirsomy 8, 1 case had structural abnormality in chrosome 9. Next-generation sequencing results showed that 11(68.8%) cases carried PTPN11 mutations, 6 (37.5%) cases NF1 mutation, 5 (31.3%) KRAS mutation, 4 (25%) NRAS mutation, 1 case without one of the Ras pathway genes. 8 cases (50%) had more than two types of canonical JMML mutations, among them 6 cases (75%) had PTPN11 and NF1 co-mutation. 10 cases had secondary gene mutations, 3 (3/10) cases JAK3 mutation, 2 (2/10) SETBP1 mutation, 2 (2/10) SH2B3 mutation, 2(2/10) ASXL1 mutation, 2 (2/10) ARID1A mutation, 1 (1/10) SRSF2 mutation. The median treatment course was three cycles (range: 1-6), and the response rate was 68.8% (11/16) after one cycle and 81.8% (9/11) after three cycles. One case achieved complete remission (CR) after four courses of therapy. The WBC and monocyte counts were significantly decreased after treatment (P<0.001), and the spleen volume showed a decreasing tendency. Eleven patients received HSCT, 9 cases were implanted successfully and 2 cases failed, of which 1 case was relieved by secondary transplantation and 2 patients died of grade IV graft-versus-host disease (GVHD) after transplantation. The median follow-up time was 31 months. The 2-year progression-free survival rate was 75.0±10.8%. Conclusions：Demethylated drug in combination with low-dose chemotherapy could reduce JMML patients' tumor burden, improve the general condition, and obtain a clinical response rate of 81.8% after 3 cycles therapy. Therefore, such a combination regimen could be used as a therapeutic option for JMML before HSCT.

RRMM and Post-BCMA Treated Subjects from the CC-220-MM-001 Study Show Increased Genomic Aberrations Associated with High-Risk and Significant Dysfunction in CD4+ T-Cell Compartment Compared to NDMM Subjects

Introduction Outcomes for patients with multiple myeloma (MM) have improved significantly through development of novel therapeutics, including immunomodulatory agents (IMiDs ®), proteasome inhibitors, anti-CD38 monoclonal antibodies and new BMCA targeting T-cell redirecting therapies. While most MM patients respond well to initial therapy, over time their disease relapses, requiring several subsequent lines of treatment. However, how patient tumors and immune profiles evolve over lines of treatment and how this may impact the efficacy of subsequent treatment regimens remains poorly understood. CC-220-MM-001 is a multicohort, phase 1/2 trial (NCT02773030) studying the efficacy of iberdomide (CC-220; a cereblon [CRBN] E3 ligase modulator) as monotherapy, and in combination with other treatments in subjects with newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM), including patients who have received prior BCMA targeting agents. This study allows uniform comparison of immune and tumor biomarkers across these clinical patient segments. Methods Baseline samples collected in the CC-220-MM-001 study prior to iberdomide treatment included peripheral blood for immunophenotyping, bone marrow biopsies for immunohistochemistry and CD138+ enriched bone marrow aspirates for genomic analysis. Biomarkers were evaluated for differences between NDMM subjects and subjects refractory to IMiD agents, proteasome inhibitors, anti-CD38 antibodies, and BCMA targeting CAR-T and T-cell engager therapies. Results Analysis of immune profiles showed NDMM subjects had a less immunodeficient profile compared to RRMM subjects, particularly in the CD4+ compartment, with significantly more total and naïve CD4+ T-cells (p<0.001) and significantly lower proportion of PD-1+ CD4+ T-cells (p=0.002) and CD4+ Tregs (p=0.005). RRMM subjects had median levels of several immune cells well below the lower limit of normal (LLN) reference range for healthy individuals, including low B cells, NK-cells, and total CD3+ and CD4+ (but not CD8+) T-cells. Median number of CD4+ T-cells was approximately half of the LLN (500 cells/mL) and median ratio of CD4+ to CD8+ T-cells was ~0.75. Phenotypic assessment of immune cells in RRMM subjects showed evidence of immune stimulation with >30% T-cells expressing activation markers (HLA-DR+ and ICOS+) and exhibiting a memory phenotype (CD45RO+). In contrast, a low proportion of pre-treatment T-cells were observed to be in a proliferative state (Ki-67+; median 6.2% of CD3+) despite evidence of activation, and more than 1/3 of both CD4+ and CD8+ cells in RRMM subjects expressed PD-1, together suggesting T-cell dysfunction and/or exhaustion. These results were mostly consistent regardless of prior refractoriness to IMiDs and PIs. Conversely, subjects post anti-CD38 had lower levels of NK and CD38+ T-cells (p<0.001) and subjects post-BCMA T-cell redirecting therapies (n=22) trended to have lower absolute levels of both CD4+ and CD8+ T-cells and increased proportion of T-cells expressing activation markers than other RRMM patient segments. Analysis of the bone marrow compartment showed that while CRBN protein was quantifiably detectable in CD138+ cells in almost all samples, including in subjects coming off IMiD based regimens, 40% of RRMM subjects had evidence of molecular dysregulation of CRBN including, loss of COPS7b/COPS8, high CRBN-del-exon10 expression, CRBN LOH or CRBN mutation. A wide range of CRBN protein levels was observed, however, differences in protein levels did not correlate with refractory status to any therapy. Notably, median CRBN expression levels were 2× higher in NDMM subjects. Prevalence of translocations, hyperdiploidy, CNAs and other mutations in RRMM subjects showed 31% of subjects had KRAS mutations, 28% had Amp1q, 23% were hyperdiploid, 21% had del17p, 12% had t(11;14), 11% had NRAS mutations and 9% had t(4;14). Conclusions These data illustrate that late-line RRMM subjects have appreciable immunosuppression compared to NDMM subjects, with particular dysfunction in the CD4+ helper T-cell compartment, suggesting that the efficacy of immunotherapies in late line myeloma may benefit from combinations with agents that improve CD4+ T-cell function. These data also show enrichment of molecular high-risk segments and CRBN-related genomic aberrations in RRMM subjects in the CC-220-MM-001 study.

Practical and Flexible Genome Profiling Study Using the Halo-Shape Annealing and Defer-Ligation Enrichment (HANDLE) System: HM-Screen-JAPAN02

Background Acute myeloid leukemia (AML) is known to have a relatively small number of genomic mutations compared to solid tumors. With the development and clinical application of drugs targeting genetic mutations involved in the pathogenesis of AML, the Japanese Society of Hematology has defined “fast-track mutations” as those that should be reported promptly due to clinical necessity. A multicenter genomic profiling study (HM-SCREEN-JAPAN01: HM01 study) was conducted to comprehensively sequence 406 DNA mutations and 265 RNA aberrations in 177 AML patients, suggesting its usefulness in clinical practice, but the turnaround time (TAT) until results are reported remains an issue (Hosono, et al. Cancer Sci, 2023). Aims We have launched the HM-SCREEN-JAPAN02 (HM02) study, a practice-oriented genome profiling study that improves on the previous HM01 study and uses a newly developed targeted sequencing method. The HM02 study adopted the novel Amoy Myeloid Panel® method, which uses the HANDLE (Halo-shape ANnealing and Defer-Ligation Enrichment) system to significantly shorten TAT and facilitate specimen submission, and was designed to be completed in 7 days from sequencing to reporting. The HM02 study allows mutation analysis at any treatment timing, such as at initial presentation, during treatment, or at relapse, as well as the capability to track clonal changes through repeat analysis; the HM02 study was designed to determine whether such flexible analysis is useful in determining treatment strategy. Results The study was conducted at 23 centers in Japan, enrolling a total of 154 cases from 2020 to 2022, with 191 samples analyzed to assess the usefulness of the study. Repetitive mutation analysis was performed in 21 cases, and clonal changes could be tracked. Among the mutations detected, we selected those that were determined to be pathogenic or likely pathogenic by ClinVar. Of the 154 cases analyzed, none of the pathogenic mutations were detected in 9 cases, and 6 were of the normal karyotype. The most frequently observed mutations were TET2 (51%), FLT3 (30%), TP53 (18%), and NPM1 (17%) (Figure 1). In multivariate analysis adjusted for age, gender, and stem cell transplantation status, TP53 and NRAS mutations were associated with higher risk for death (HR=6.82 and 14.98, respectively). Of the 19 cases in which pathological mutations were repeatedly investigated, 8 were useful in determining minimal/measurable residual disease (MRD) (2 cases of MRD loss and 6 cases of MRD retained), 6 cases showed the development of new clones and 5 cases showed clonal evolution, indicating that repeated mutation analysis can be used to evaluate detailed disease status. In terms of clinical utility, HM02 mutation analysis was useful in 62% of cases (98 of 158), particularly for risk assessment and determining the indication for stem cell transplantation (Figure 2). Conclusion Flexible analysis using the novel Amoy Myeloid Panel® method suggests that it can be used to assess the risk of relapse, evaluate the need for transplantation, and select optimal therapeutic agents. NGS-based mutation analysis provides useful information at any point in AML treatment and is expected to improve outcomes.

Validation of the Prognostic Risk Signatures for Older Patients with Acute Myeloid Leukemia Treated with Hypomethylating Agents and Venetoclax

Introduction: Hypomethylating agents (HMA) with Venetoclax (Ven) is the standard of care for patients with acute myeloid leukemia (AML) ≥ 75 yrs of age or not candidates to intensive treatment. Döhner, et al. (ASH 2022) demonstrated that the European LeukemiaNet (ELN) risk classifications do not accurately predict prognosis in HMA-Ven treated patients. A prognostic risk signature classification (PRSc) was proposed, stratifying patients by RAS, FLT3 and TP53 mutations alone. We report a validation of this PRSc in a large cohort of patients treated with HMA-Ven. Methods: This unicentric analysis included patients older than 60 years old with newly diagnosed treated with HMA-Ven in different treatment protocols. Responses were assessed with the ELN-2022 criteria. The PRSc allocated patients with [ K/N]- RAS or FLT3-ITD mutations in the intermediate benefit group, patients with TP53 in the lower benefit group, whereas patients with AML lacking these mutations were allocated in the higher benefit group. Cumulative incidence (CI) was calculated for pts achieving remission with death and relapse as competing events. Results: A total of 179 patients were included, diagnosed between 11/2014 and 12/2021. Median age was 74 yrs old (range, 61-89) and 58% were male. 31% of patients had diploid cytogenetics and 35% had complex karyotype. According to the ELN 2022 risk category, 30 (17%), 15 (8%) and 114 (64%) patients were allocated in the favorable, intermediate and adverse risk group, respectively. According to the PRSc, 75 (42%), 31 (17%) and 54 (30%) were allocated in the higher, intermediate and lower benefit group, respectively. In the PRSc intermediate benefit group, 21 patients had NRAS mutations, 9 patients had KRAS mutation and 7 patients had FLT3-ITD. Patients received treatment with decitabine 10 days with Ven (n=89, 50%), decitabine 5 days with Ven (n=69, 38%), oral decitabine 5 days with Ven (n=14, 8%) and Azacitidine 7 days with Ven (n=7, 4%). The overall response rate (ORR) was 70% (126/179), including 96 pts (54%) achieving complete remission (CR) rate and 30 (17%) having CR with incomplete hematologic recovery (CRi). The ORR for patients allocated in the ELN 2022 favorable, intermediate and adverse risk was 90% (27/30), 93% (14/15) and 63% (72/114). According to the PRSc, the ORR was 87% (65/75), 55% (17/31) and 59% (32/54) for patients of the higher, intermediate and lower benefit group, respectively. The median number of cycles to best response was 1 (1-8), and 22 patients (12%) underwent allogeneic stem cell transplantation. After a median follow-up of 35 months, the median overall survival (OS) was 11 months. According to the ELN 2022 classification, the mOS was 44, 13 and 8 months (p<0.001, C-index=0.6). When applying the PRSc, the median OS was 30, 12 and 5 months for higher, intermediate and lower benefit group (p<0.001, C-index=0.66). Median event-free survival (EFS) was 8 months, being 30, 8, and 5 months for ELN 2022 favorable, intermediate and adverse group, respectively (p<0.001, C-index=0.62). When applying the PRSc, the median EFS was 18, 8 and 4 months for higher, intermediate and lower benefit group, respectively (p<0.001, C-index=0.65). The 2-yr CI of relapse and death was 47% and 22%, respectively. By the ELN 2022 subgroups, the 2-yr CI of relapse was 29%, 43% and 55% for favorable, intermediate and adverse groups, respectively (p=0.1). The 2-yr CI of death was 12%, 21% and 25% for favorable, intermediate and adverse groups, respectively (p=0.4). By the PRSc, the 2-yr CI of relapse was 26%, 70% and 60% for higher, intermediate and lower benefit group, respectively (p=0.004). The 2-yr CI of death was 20%, 6% and 33% for higher, intermediate and lower benefit group, respectively (p=0.1). Conclusion: The PRSc classification can stratify 3 different groups of patients with distinct outcomes. In the present study the 3 categories defined by the PRSc have very similar survival than the median OS previously reported (27, 12 and 5 months for higher, intermediate and lower benefit group), with a better performance according to the C-index. Signaling mutations ( RAS or FLT3-ITD) as well as TP53 mutations represent a challenge with HMA-Ven, in which novel treatments or combinations are warranted to improve outcomes.

Deconvoluting Clonal and Cellular Architecture in IDH-Mutant Acute Myeloid Leukemia

Introduction Mutations in Isocitrate dehydrogenase 1/2 ( IDH) occur in 20% of acute myeloid leukemias (AML) and frequently co-occur with DNMT3A, NPM1, SRSF2, and NRAS. In clonal hematopoiesis (CH), IDH mutations confer high risk of AML transformation. Recently developed methods linking single cell genotype to single cell transcriptomic profiles and cell lineage offer the opportunity to define the transcriptomic consequences of each mutation across leukemic cell lineages and clonal phylogenies. Here, we develop an integrative single cell laboratory and analytical framework to study the genetic, clonal and cellular heterogeneity of IDH mutated CH and AML, across disease stages (diagnosis and relapse) and deliver a high-resolution map of IDH-mutant AML. Methods Bone marrow or peripheral blood mononuclear samples from 15 AML patients, 2 CH individuals, and 12 unaffected age-matched donors were profiled by single cell RNA-sequencing. For the AML cases, a median of 3 samples per patient were profiled under treatment with IDH inhibitor (IDHi) monotherapy (n=5 patients), IDHi in combination with intensive chemotherapy (NCT02632708, n=6) or in combination with hypomethylating agent (n=2). This dataset comprised 259,610 cells across AML (n=190,133), CH (n=16,705) and normal controls (n=52,772). Genotyping of Transcriptomes (Nam et al., 2019) (for IDH1/2, NPM1, SRSF2, and NRAS mutations) across 6 AML patients enabled genotyping 23% of cells at diagnosis for at least one clone-defining mutation. Results Through meta-analysis of 3,653 diagnostic AML samples profiled by a targeted DNA panel (Papaemmanuil et al., 2016; Tazi et al., 2022), we identified DNMT3A, SRSF2 and NPM1 as critical co-operative mutations in IDH-mutant AML. Amongst the 15 AML patients, 6 had IDH1 mutation, 8 had IDH2 mutations, and 1 had both IDH1 and IDH2. The co-occurring mutations in DNMT3A (n=11 patients), NPM1 (n=5), SRSF2 (n=5) reflected the genomic landscape of IDH-mutant AML. At diagnosis, IDH-mutant AML was enriched in immature hematopoietic stem and progenitor-like blasts. Compared to normal hematopoiesis, IDH-mutant AML blasts upregulated inflammatory response markers including TNFɑ/NFκB-signaling and downregulated proliferation-associated pathways including G2M Checkpoint, and MYC and E2F Targets, and DNA damage response. To investigate how co-mutations modify the IDH-mutant phenotype, we established an integrative single-cell profiling approach to assign single cells to distinct phylogenetic subclones and extract clone-specific transcriptional programs. SRSF2 and NPM1 co-mutations modulated the AML blast phenotype, towards erythroid ( SRSF2) or granulomonocytic lineage ( NPM1). NRAS- or SRSF2-mutant subclones defined distinct gene expression-derived clusters and upregulated proliferation and metabolic pathways. Furthermore, NPM1 co-mutated blasts upregulated KMT2A-target genes. Next, we tracked clonal responses to combination IDHi and chemotherapy using GoT and targeted DNA sequencing. In 4 cases, the ancestral clone, defined by IDH and DNMT3A mutations, retained clonal dominance in both mature and immature myeloid compartments at complete response (median 4 weeks). Conversely, subclones defined by mutations in NPM1, NRAS, or SRSF2, were either reduced or not detectable (limit of detection 2%). In two cases, mutations were not detected after therapy. In patients who relapsed after IDHi monotherapy treatment, blasts upregulated stemness, fatty acid metabolism, OxPHOS, and inflammation at relapse compared to diagnosis. Furthermore, MHC Class II was downregulated at relapse, pointing to immune evasion of AML as a potential relapse mechanism. Finally, we investigated the role of IDH2 mutations at CH. IDH-mutant CH-derived monocytes upregulated inflammation-associated pathways, mirroring the phenotypes observed in IDH-mutant AML at diagnosis. Conclusion We defined how IDH mutations and their frequent co-mutations dysregulated hematopoiesis. We leveraged state-of-the-art single cell techniques to assign leukemic cells to phylogenetic subclones and delineated the role of serially acquired co-mutations in shaping lineage bias and transcriptional programs. Furthermore, we defined genetic, clonal and transcriptional changes at three stages of the AML disease process: diagnosis, remission, and relapse under IDH inhibitor therapy.

RAS MULTI(ON) Inhibitor RMC-7977 Targets Oncogenic RAS Mutations and Overcomes RAS/MAPK-Mediated Resistance to FLT3 Inhibitors in AML Models

FLT3 inhibitors (FLT3i) such as gilteritinib are clinically active in AML, but their use is limited by resistance due to emergence of clones with RAS/MAPK mutations, both alone and in combination with FLT3 mutations, or with other drivers such as KIT mutations. RAS mutations are also commonly associated with relapse on IDH1/2 inhibitors and the BCL2 inhibitor venetoclax. Importantly, multiple heterogeneous resistant clones are identified at the time of relapse. In addition, 5-10% of all de novo patients harbor oncogenic RAS mutations. Patients with RAS mutations or other mutations that activate RAS/MAPK signaling do not benefit from clinically approved targeted therapies. Targeting oncogenic RAS has been historically challenging and inhibition of downstream effectors of the MAPK pathway, such as MEK, demonstrated modest activity and high toxicity in clinical trials. Effective inhibition of the RAS/MAPK pathway is therefore a critical unmet need in AML. RMC-7977 is a potent, oral small molecule inhibitor of both wild-type and mutant GTP-bound RAS oncoproteins (RAS MULTI) and is a preclinical tool compound representative of the clinical candidate RMC-6236, currently in clinical evaluation (NCT05379985). RMC-7977 non-covalently binds to the intracellular chaperone cyclophilin A, generating a neomorphic interface with high affinity for all isoforms of RAS. The resulting tri-complexes sterically block RAS-effector interactions required for propagating oncogenic signals. We report in vitro data supporting preclinical utility of RAS MULTI(ON) inhibition in AML models harboring RAS mutations, including those with resistance to FLT3i due to hyperactive RAS signaling. RMC-7977 suppressed cell proliferation in AML cell lines driven by FLT3-ITD (Molm-14, MV4-11), KIT N822K (Kasumi-1, SKNO-1) and RAS mutations ( NRAS Q61L -OCIAML-3, HL-60, KRAS G13D - NOMO-1) with IC 50 values between 5 and 33 nM and repressed phosphorylation of downstream effectors of the MAPK pathway MEK, ERK and RSK. We also assessed the activity of RMC-7977 in resistant Molm-14 cells that developed secondary NRAS G12C or NRAS Q61K mutations after long-term exposure to FLT3i. At concentrations as low as 5 nM, RMC-7977 restored sensitivity to gilteritinib in both NRAS-mutant resistant cell lines. Using a caspase3/7 assay, we observed that RMC-7977 induced apoptosis, but to different extents across AML cell lines. We have previously demonstrated that effective MAPK signaling inhibition increases apoptotic dependency on BCL2. Consequently, the addition of venetoclax to RMC-7977 significantly enhanced caspase activation in cell lines with FLT3, KIT and NRAS mutations. In a cell viability assay, RMC-7977 and venetoclax showed high synergistic activity as assessed by Bliss independence model. Given that intrapatient tumor heterogeneity is associated with clinical resistance to targeted therapies, we investigated the in vitro activity of RMC-7977 in models that recapitulate patterns of clonal outgrowth observed in patients treated with FLT3i. We mixed fluorescently-tagged cell lines with FLT3-ITD (Molm-14), FLT3-ITD and NRAS co-mutations (Molm-14 NRAS Q61K) and NRAS-only mutations (OCIAML-3), treated the mixtures with RMC-7977 alone and in combination (with gilteritinib or venetoclax) for 96 hours, then assessed cell viability via flow cytometry. Gilteritinib and the gilteritinib/venetoclax combination selected for survival of cells harboring NRAS mutations, but RMC-7977 inhibited outgrowth of all cell populations. The combination of RMC-7977 and gilteritinib had superior activity in mixtures containing FLT3 and FLT3- NRAS co-mutations, but no additional benefit over RMC-7977 monotherapy in cells harboring NRAS mutations alone. Strikingly, the combination of RMC-7977 and venetoclax potently suppressed cell viability equally in all cell line models and was significantly superior to RMC-7977 alone. In vivo studies investigating the tolerability and activity of RMC-7977 and RMC-7977 combinations in RAS mutant/FLT3i-resistant patient-derived xenograft models are ongoing and will be presented. Collectively, our data provide preclinical evidence that combination therapies leveraging RAS MULTI(ON) inhibition are effective in suppressing RAS-mutant AML clones, a common mechanism of resistance to currently approved targeted therapies in AML and a current area of high unmet clinical need.

Clonal Heterogeneity and Evolution during Treatment in High Hyperdiploid B-Cell Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, is caused by the accumulation of multiple genomic aberrations in developing lymphocytes. In children, high hyperdiploidy (HeH) is the most prevalent subtype of B-cell ALL (B-ALL), which is associated with a superior outcome. However, because of the high incidence of HeH B-ALL in children, this subtype is responsible for about 25% of all relapses. The prediction of relapse remains difficult, especially in these good risk subtypes. Recent studies indicate that ALL is a heterogeneous disease, with multiple leukemia clones at diagnosis, each having a distinct set of mutations. We hypothesize that these different subclones have a different sensitivity to treatment and that samples with more heterogeneity are more prone to relapse. We used single-cell sequencing to determine the clonal heterogeneity of HeH B-ALL at diagnosis and followed the clonal evolution during chemotherapy treatment. Methods: Bone marrow samples of 11 patients with HeH B-ALL were collected at diagnosis, during treatment (for 9 cases) and at relapse (for 1 case) (Table 1). Samples during treatment were enriched for leukemia cells using magnetic antibodies targeting cell membrane markers (CD10, CD19, CD34) with the MARS® gentle sorter. We performed targeted single-cell DNA sequencing with the Mission Bio Tapestri® Platform, using our custom amplicon panel of 414 amplicons across 39 commonly mutated genes. This method detected single nucleotide variants (SNVs) and small insertions/deletions (indels) based on targeted PCR amplification in about 4000 cells per sample. Results: We sequenced diagnostic samples from 11 HeH B-ALL patients. For 9 patients we also tested samples during treatment, either at day 15 of treatment (1 case) or at the end of induction (EOI) (8 cases), and for 3 cases an additional sample at the end of consolidation (EOC). Furthermore, for one patient, we analyzed samples of 3 subsequent relapses. In total, 117,148 single cells were processed, with an average of 4500 cells per sample. We detected multiple subclones in almost all diagnostic samples (9/11), ranging from 2 to 10 subclones per case. Most frequently mutated genes were KRAS, NRAS, PTPN11 and FLT3, as well as NSD2. Four cases had 2 or more different KRAS and/or NRAS subclones. Three cases had a subclone with a KRAS/ NRAS mutation co-occurring with a NSD2 mutation. For the patients with multiple subclones at diagnosis, we subsequently performed single-cell analysis on one or more follow-up samples, either during treatment or at relapse. In the samples taken at EOI, we detected residual leukemia cells in all 8 cases: multiple subclones were detected in 5 cases and only one subclone persisted in 3 cases. Residual leukemia cells were also detected in all samples collected at EOC, though in a lesser amount as compared to the previous timepoint sequenced. Interestingly, in the matched diagnosis-relapse case, we found that a minor subclone with a NSD2, PTPN11and KRAS mutation became the dominant clone at first relapse (Figure 1). Conclusions: Single-cell DNA sequencing reveals a strong genetic heterogeneity in HeH B-ALL based on a variety of mutations in KRAS, NRAS, PTPN11, FLT3 and NSD2. RAS mutations and FLT3 mutations are mutually exclusive in subclones, whereas NSD2 mutations and RAS mutations are frequently co-occurring in the same subclone. This cooperation is highlighted in our relapse patient, having a minor subclone with a KRAS and NSD2 mutation at diagnosis that became the largest subclone at relapse. Even though few leukemia cells persisted during treatment, we were able to genetically identify the residual subclones with a potentially higher therapy resistance.

Risk Stratified Treatment for Patients with Newly Diagnosed Juvenile Myelomonocytic Leukemia: A Phase 1/2 Non-Randomized Study of Trametinib and Azacitidine with or without Chemotherapy

Background: Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of childhood. The biochemical hallmark of JMML is aberrant signaling through the Ras pathway caused by initiating mutations in NF1, NRAS, KRAS, RRAS, RRAS2, SH2B3, PTPN11,or CBL. While hematopoietic stem cell transplantation (HSCT) can be curative, 5-year event-free survival of children with JMML after HSCT is only ~50%. Recently, several studies have identified mutational burden and DNA methylation as predictive of clinical outcomes in patients with JMML. In the T2020-004 phase 1/2 clinical trial, we will define lower-risk patients as those with one somatic alteration and low DNA methylation and high-risk patients as those with more than one somatic alteration or intermediate/high DNA methylation. Trametinib is an orally bioavailable, reversible, highly selective allosteric inhibitor of MEK1/2 in the Ras/MAPK signaling pathway. Trametinib is FDA-approved for the treatment of adults with advanced melanoma with BRAF V600E or V600K mutations and in children with Ras-mutant solid tumors in combination with dabrafenib. Clinical investigation of trametinib monotherapy via the Children's Oncology Group ADVL1521 phase 2 trial (NCT03190915) showed efficacy in children with relapsed/refractory JMML. Phase 2 clinical trial evaluation of azacitidine in children with newly-diagnosed JMML was also efficacious and led to its FDA approval. In this trial, trametinib will be tested in combination with azacitidine in lower-risk patients and in combination with azacitidine and chemotherapy for high-risk patients. Study Design and Methods: This is a non-randomized, risk stratified, Phase 1/2 study for patients with newly-diagnosed JMML (NCT05849662) conducted via the Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) Consortium with safety and efficacy phases for both lower-risk and high-risk cohorts. Lower-risk patients will receive trametinib once-daily for 28 days in combination with azacitidine administered daily for five days per cycle. Lower-risk patients can receive up to 12 cycles and will proceed to HSCT only if they experience progressive disease or relapse. High-risk patients will receive trametinib administered once-daily for 28 days in combination with azacitidine, fludarabine, and cytarabine (aza/FLA) administered daily for five days per cycle. High-risk patients will receive up to two cycles of therapy before proceeding to HSCT off protocol. Key Eligibility: Patients aged 1 month to 21 years who meet World Health Organization criteria for JMML will be eligible. Objectives: The primary safety objectives are to determine the safety of combining trametinib with azacitidine for lower-risk patients and combining trametinib with aza/FLA for high-risk patients. The secondary efficacy objectives are to determine the event-free survival for lower-risk patients in the absence of HSCT and the molecular response rates pre-HSCT for high-risk patients. Sample Size and Statistical Design: A rolling 6 trial design will be used during Phase 1 of the study to determine the recommended phase II dose (RP2D) of trametinib for lower-risk and high-risk patients in combination with their respective therapies. Starting trametinib dose is 0.032mg/kg daily if less than 6 years of age, or 0.025mg/kg daily if 6 years or older, with one dose level de-escalation if indicated. Phase II of the study involves lower-risk and high-risk cohort expansions at the RP2D of trametinib. Target accrual is 22 patients in the lower-risk arm and 42 patients in the high-risk arm. The study is open to accrual at all TACL consortium sites. We acknowledge the TACL Consortium's scientific contribution to and participation in this study, including participating member institutions, investigators, research teams, and the TACL Operations Center. Novartis Pharmaceuticals Corporation provided the investigative drug in support of this trial. This study is supported by the National Institute of Health, National Cancer Institute (R37CA266550), the Pediatric Cancer Research Foundation, and the Cannonball Kids Cancer Foundation.

Clinical Outcomes and Mutational Profile Associated with Dysmyelopoiesis in Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is defined by absolute (≥ 0.5 x10 9/L) and relative (≥ 10%) monocytosis in peripheral blood. Presence of significant dysplasia in any of the three myeloid series, in particular dysgranulopoiesis, is an almost constant finding in CMML. In the absence of a clonal marker, a scenario very unlikely with the advent of next-generation sequencing (NGS), significant myeloid dysplasia becomes a crucial criterion for establishing the diagnosis. Although bone marrow dysplasia has demonstrated independent prognostic value in MDS, its significance in relation to CMML has never been assessed. The main interest of our work was to evaluate the genomic determinants of dysplasia and to assess its prognostic impact. We analyzed a series of 240 patients with CMML with clinical, morphological, immunophenotypic, cytogenetic and molecular information. Patients were diagnosed following WHO and ICC 2022 recommendations. As a novelty with respect to previous WHO classifications, the new definition of CMML includes those patients previously categorized as oligomonocytic CMML (OM-CMML). That is, myelodysplastic neoplasms (MDS) or unclassifiable myelodysplastic/myeloproliferative neoplasms with relative monocytosis and a monocyte count of 0.5 × 10 9/L to <1 × 10 9/L. A total of 167 (71%) patients were diagnosed with CMML-1 and 67 (29%) with CMML-2. Likewise, 193 (80%) patients were diagnosed with dysplastic CMML, 55 of whom would meet criteria for OM-CMML, and 47 (20%) were diagnosed with proliferative CMML. We assessed associations between dysmyelopoiesis and gene mutations by Spearman's rank correlation coefficient. Dysgranulopoiesis showed a significant positive correlation with NRAS, TET2 and ZRSR2 mutations; while a significant negative correlation with SETBP1 was observed. Dysmegakaryopoiesis showed a significant positive correlation with ASXL1 and TP53. Dyserythropoiesis showed a significant positive correlation with DNMT3A and SF3B1. The heatmap and matrix showing these correlations, as well as the differences in the degree of dysmyelopoiesis among these specific mutations are depicted in Figure 1-A & 1-B. Later, we wanted to assess the prognostic impact of dysgranulopoiesis, dyserythropoiesis and dysmegakaryopoiesis in terms of overall survival (OS) and leukemia-free survival (LFS) in our series. By applying the MaxStat package in R, it allowed us to detect the best cut-off points for dysgranulopoiesis (>75%) and dysmegakaryopoiesis (>20%) to separate our series into two groups with significantly different outcomes (Figure 1-C). We were unable to detect a significant cut-off point for dyserythropoiesis and the presence of multilineage dysplasia also showed no prognostic impact. CMML patients with dysgranulopoiesis above 75% showed a significant shorter median OS (34.4 vs 67.8 months; Hazard Ratio (HR): 1,99 [1.24-3.19]; P=0.0036) and LFS (33.4 vs 66.6 months; HR: 1,87 [1.17-3]; P=0.008). Likewise, patients with dysmegakaryopoiesis above 20% also showed a significant shorter median OS (30.2 vs 67.1 months; HR: 2.52 [1.63-3.88]; P<0.0001) and LFS (30.4 vs 66.6 months; HR: 2.76 [1.78-4.28]; P<0.0001). Remarkably, dysmegakaryopoiesis above 20% and dysgranulopoiesis above 75% were identified as independent prognostic factors for both OS and LFS after adjustment by multivariable Cox regression analyses by CMML-specific prognostic scoring system (CPSS) and Mayo Prognostic Model. Furthermore, the addition of dysmegakaryopoiesis above 20% and dysgranulopoiesis above 75% to these prognostic indices improved their prognostic accuracy in terms of OS as assessed by a C-index for right-censored data (Figure 1-C). In conclusion, our study has allowed us to detect mutations in genes related to dysmyelopoiesis. In addition, both dysgranulopoiesis and dysmegakaryopoiesis have demonstrated a negative prognostic impact on the survival of patients in our series that was not captured by the CPSS and Mayo prognostic model. Their worse prognosis could be partially justified by their association with poor prognostic mutations (e.g. NRAS, ASXL1, TP53), which allows us to visualize them as a good surrogate of poor prognostic molecular profiles.

Impact of PTPN11 Mutations in De Novo, Secondary and Relapsed/Refractory Acute Myeloid Leukemia

Background Somatic mutations in the protein phosphatase non-receptor type 11 ( PTPN11) gene occur frequently in children with juvenile myelomonocytic leukemia and less commonly in adults with AML, being present in less than 5% of reported cases. Uncertainty exists regarding the prognostic impact of PTPN11 mutations in AML, largely owing to their observed rarity. Some retrospective studies have identified PTPN11 mutations as deleterious prognostic markers in AML, though others have noted that their negative impact is mitigated by concurrent NPM1 mutations, which frequently co-occur. We analyzed the outcomes of patients at our institution with AML and PTPN11 mutations in order to add to the literature regarding the relevance of this mutation. Methods We conducted a retrospective case series of patients at a single-center, tertiary-care institution who were diagnosed with AML between January 1 st, 2020 and June 1 st, 2023. Inclusion criteria included: 18 years of age or older, primary follow-up at the study institution, documented diagnosis of AML and identification of a PTPN11 mutation by NGS. Outcomes of interest included cytogenetic and molecular profiles, response to induction or salvage therapy, progression-free survival (PFS) and overall survival (OS). Results Twenty-two (12 men and 10 women) patients with PTPN11 mutant AML were identified. The median age at diagnosis was 60 years (range, 31-89 years). Nineteen patients had a PTPN11 mutation identified at diagnosis and 3 had a PTPN11 mutation appear at the time of relapse. Nine patients with de novo AML had PTPN11 mutations identified at diagnosis. Six were female and the average age at diagnosis was 60 years (range, 44-89 years). The most common co-occurring mutations were NPM1 (8 patients), DNMT3A (3 patients), TET2 (2 patients) and FLT3-TKD (2 patients). Cytogenetics were normal or non-adverse in 8 patients, and complex/adverse-risk in 1 patient. Seven patients received treatment - 4 with intensive chemotherapy and 3 with a hypomethylating agent (HMA) + venetoclax. Treatment response was CR/CRi in 6 patients (85.7%). At a median follow-up of 195 days, median PFS and OS were not reached. Two patients relapsed at 76 and 139 days after response. No patients received an allogeneic transplant in first remission. Ten patients with therapy-related or secondary-AML (t/s-AML) were identified, all with PTPN11 mutations noted at the time of AML diagnosis. Two patients had preceding MDS and 3 had preceding MDS/MPN. Median age was 63 years (range, 31-70 years). The most common co-occurring mutations were RUNX1 (5 patients), FLT3-ITD (2 patients) and NRAS/KRAS (3 patients). Chromosome 7 abnormalities were present in 4 of 5 patients with RUNX1 mutations. Cytogenetics were complex or adverse-risk in 9 patients. Eight patients received treatment - 6 with intensive chemotherapy and 2 with HMA + venetoclax. Treatment response was CR/CRi in 3 patients (37.5%). Median PFS was 183 days and median OS in treated patients was 307 days. The only patient with an ongoing response at the time of data cut-off received an allogeneic transplantation in CR1. Three patients with AML had emergence of a PTPN11 mutation at the time of relapse, at 245, 343 and 2262 days after initial diagnosis. PTPN11 was the only new mutation identified in 2 patients, and emerged in conjunction with an NRAS and KDM6A mutation in 1 patient. One patient received treatment, attaining a CRi after HMA + venetoclax, but relapsed 112 days later. The other 2 patients passed at 10 and 21 days after relapse. Conclusions PTPN11 mutations were relatively rare, occurring in only 22 patients with AML during a 3.5-year time span. Stark differences were noted in patients with de novo AML versus t/s-AML and PTPN11 mutations. De novo AML patients frequently demonstrated NPM1 mutations and a normal karyotype, while t/s-AML patients frequently had RUNX1 and FLT3 mutations, as well as adverse cytogenetics. Although the impact of PTPN11 mutations in AML may be influenced by concurrent NPM1 mutations or cytogenetic characteristics, the context of AML may ultimately have the largest impact.

Mutational and Cytogenetic Dynamics at Diagnosis and Relapse in Acute Myeloid Leukemia: Influence of Intensity and Type of Treatment

Introduction: Cytogenetic and molecular aberrations are common at diagnosis in patients with acute myeloid leukemia (AML) and may have significant prognostic impact. Relapse after prior response is associated with a poor outcome. The dynamics of cytogenetic and molecular abnormalities at relapse and their impact on outcome is less well studied. Methods: This is a retrospective single center study including patients with AML diagnosed from April 2017 to October 2022 who relapsed after achieving a prior response. Mutations were detected using a targeted 81-gene NGS panel and cytogenetics were assessed using conventional karyotyping and FISH testing. To assess disappearance, maintenance or appearance of cytogenetic and molecular aberrations, they were evaluated at diagnosis and relapse and differences in incidence were evaluated with the Chi-square and Fisher test. Results: The cohort includes164 patients who had relapsed after achieving remission. The median age at diagnosis and relapse was 67 years (range, 20-94) and 68 years (range, 21-95). 53% were male. Patients received intensive treatment (IT; 40% of patients, 41% of whom with the addition of venetoclax) or low intensity therapy (LIT; 60% of patients, 72% with venetoclax). The median time from diagnosis to relapse was 8.9 months (range, 2.2-52). According to the ELN 2022 risk classification, 15%, 15% and 70% had favorable, intermediate and adverse risk, respectively. At diagnosis, the most frequent mutations were DNMT3A (32%), TP53 (31%), RUNX1 (19%), TET2 (18%), SRSF2 (17%) and NPM1 (17%). At relapse, the most frequent mutations were DNMT3A (35%), TP53 (35%), TET2 (24%), RUNX1 (20%) and SRSF2 (17%). The median number of mutations per patient at diagnosis and relapse was 3 (0-12) and 3 (0-14), respectively (p=0.07). Most frequent karyotypes at diagnosis were diploid (34%), complex (32%), chromosome 5 abnormalities (29%) and chromosome 7 abnormalities (26%). At relapse, the most frequent karyotypes included complex (36%), chromosome 7 abnormalities (31%), chromosome 5 abnormalities (30%) and diploid karyotype (27%). When analyzing each mutation individually, the rate of mutation clearance from diagnosis to relapse was higher for FLT3-ITD (58%, p=0.01), FLT3-TKD (83%, p<0.001), KIT (88%, p=0.003), NF1 (71%, p=0.004) and WT1 mutations (71%, p=0.004), compared to other remaining mutations (32%). Conversely, ASXL1 (95%, p=0.01), DNMT3A (84%, p=0.006), SRSF2 (89%, p=0.01), TET2 (84%, p=0.02) and TP53 mutations (86%, p=0.002) persisted more frequently at relapse compared to other mutations (66%). At relapse, WT1 mutations were more frequently acquired (77%, p=0.001), compared to other mutations (36%). Diploid karyotype changed more frequently than other cytogenetic abnormalities (33% vs 5%, p <0.001), whereas complex karyotype persisted at relapse (98% vs 87%, p=0.04) more frequently. Chromosome 7 abnormalities were more frequently acquired at relapse (23%), compared to other cytogenetic abnormalities acquired at relapse (11%, p=0.04). We then compared mutational dynamics from diagnosis to relapse between patients receiving IT and LIT. NRAS mutations disappeared more frequently from diagnosis to relapse in patients treated with IT (73%), compared with patients treated with LIT (27%, p=0.01) whereas TP53 mutations were acquired more frequently in IT treated patients (67% vs 10%). Patients treated with IT acquired chromosome 7 abnormalities more frequently than patient treated with LIT (60% vs 14%, p=0.007). No significant differences were found regarding disappearance, maintenance or appearance of mutations when comparing patients treated with or without Venetoclax. Conclusion: The number of mutations does not vary significantly from diagnosis to relapse, and most mutations persist, suggesting maintenance of leukemic clones as the cause of relapse. Overall, genes involved in signaling pathways tend to disappear more frequently at relapse whereas chromatin and histone modifiers as well as TP53 mutations tend to persist. NRAS mutations disappear more frequently at relapse with IT, whereas TP53 mutations are acquired more frequently.

Single-Cell Proteogenomics Analysis of AML Samples Treated with Decitabine and Venetoclax Reveals Divergent Treatment Escape Mechanisms

Background: The combination therapy of hypomethylating agents and venetoclax (HMA+VEN) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist, including relapse and treatment resistance. Previous studies have revealed genetic ( RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms in the context of HMA+VEN therapy. Despite these advancements, our comprehensive understanding of the HMA+VEN resistance mechanisms remains incomplete. To gain further insights into the resistance mechanism in HMA+VEN therapy, we implemented single-cell proteogenomics analysis on longitudinally collected samples from AML patients treated with decitabine+venetoclax (DAC+VEN) trial. Methods: We performed a high-throughput, single-cell targeted DNA-antibody sequencing (DAb-seq) using Tapestri platform from Mission Bio Inc. Sixty-three AML samples sequentially collected from 33 unique patients who participated in DAC+VEN clinical trial (NCT03404193) were analyzed. Our custom DNA panel covered 37 recurrently mutated genes in AML, whereas the custom antibody panel included either 15 cell surface proteins (N = 20 patients) or 48 proteins (N = 13 patients). Cellular phenotypes were inferred based on the patterns of cell surface protein expression. Median 2,767 cells were sequenced per sample, with total 196,719 cells sequenced for this study. Results: The median age of the cohort was 70 years (range 21-84). Twenty-four (73%) patients had primary AML and 9 (27%) had secondary or therapy-related AML. Based on the FAB classification, 13 patients had M0/M1/M2, 6 had M4/M5, one had M7, and the rest had unknown subtypes. The most frequent gene mutation at baseline was DNMT3A (45%), followed by NPM1 (39%), TET2 (30%), and TP53 (24%). Among the 33 patients, 32 had evaluable treatment response; 27 (82%) were defined as responders by achieving CR or CRi, whereas 5 (15%) were non-responders. Six patients experienced relapse after responding to the therapy, and their baseline-relapse pairs were analyzed by the single-cell platform. Using the composition of cellular phenotype data, AML samples were classified into either stem-like (N = 5), progenitor-like (N = 1), predominantly monocytic (N = 4), predominantly erythroid (N = 2) and mixed population AML (mixed population of stem, progenitor, monocyte or erythroid-like population, N = 21). Genotype-phenotype analysis showed a significant correlation between the KRAS mutation and the mixed phenotype (p= 0.0403), as well as between the U2AF1 mutation and the mixed phenotype (p= 0.0471). There was no significant association between treatment response and AML phenotype. Analysis of six baseline-relapse pairs showed that relapse was associated with phenotypic shift of AML cells. Stem-like cells were largely eradicated at relapse and replaced by more differentiated cells. Among the six baseline-relapse pairs, three showed monocytic shift, one showed erythroid shift and two showed mixed monocytic/erythroid shift. Phenotypic shifts at relapse were not always associated with expansion of new genetic mutations, except for in two cases: one had monocytic shift accompanied with expansion of KRAS mutation, and another had erythroid shift accompanied with expansion of NRAS mutation. Conclusions: Phenotypic transition from stem-like cells to more differentiated cells (either monocytic or erythroid) was frequently observed in AML relapse following DAC+VEN therapy. These findings are consistent with the known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. Close monitoring of these phenotypic alterations throughout the course of HMA+VEN treatment may potentially facilitate the precise identification of cellular populations predisposed to relapse.

Exploring the In Vitro and In Vivo Therapeutic Potential of BRAF and MEK Inhibitor Combination in NRAS-Mutated Melanoma.

Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genes ATF4, CHOP, and NUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.

Mucinous cystadenocarcinoma of the breast harbours TRPS1 expressions and PIK3CA alterations.

Breast mucinous cystadenocarcinoma (BMCA) is a rare tumour recently recognised as a distinct entity by the World Health Organisation Tumour Classification Series. BMCA is a triple-negative tumour that lacks specific immunohistochemical markers; therefore, distinguishing it from mimickers such as ovarian and pancreatic cystadenocarcinomas requires careful clinicopathological correlation. Due to its rarity, little is known about the molecular alterations that underlie BMCA. In this study, we used immunohistochemical staining methods to investigate TRPS1 (trichorhinophalangeal syndrome type 1) expression in BMCA and compare it to expression in ovarian and pancreatic mucinous cystadenocarcinomas. We also collected tumour samples from three BMCA patients for molecular analysis by MALDI-TOF mass spectrometry, real-time polymerase chain reaction, whole exome sequencing and fluorescence in-situ hybridisation. TRPS1 immunoreactivity was found only in BMCA tumour cells and not in the ovarian and pancreatic counterparts. One of the three BMCA tumours also showed a PIK3CA hot-spot mutation, which was confirmed by whole genome next-generation sequencing (NGS). No KRAS, NRAS, BRAF or AKT mutations were found. To our knowledge, this is the first demonstration of TRPS1 expression in BMCA patients and the first identification of a PIK3CA hotspot mutation in these tumours. These findings provide insights into the molecular mechanisms underlying BMCA tumorigenesis and suggest a potential drug target for this rare and poorly understood cancer.

Targeted next-generation sequencing of Japanese patients with sinonasal mucosal melanomas identifies frequent NRAS and CTNNB1 mutations

ObjectiveMucosal melanoma is a rare malignancy; however, the reported incidence rate of mucosal melanoma is higher in Asians than in Caucasians. Sinonasal mucosal melanoma (SNMM) is an aggressive malignancy with a poor prognosis due to distant metastasis. Systemic therapy with BRAF inhibitor and MEK inhibitor is one of the standards of care for cutaneous melanoma patients with BRAF V600 mutations. However, no molecular targeted therapy for patients with mucosal melanoma has been established. Relatively few studies have described the genetic mutations associated with mucosal melanoma because of its low frequency. Furthermore, to the best of our knowledge, the genetic mutations among Japanese patients have not been reported. Therefore, in the current study, we evaluated the genetic and clinicopathological characteristics of patients with SNMM. MethodsA total of 18 tissue samples obtained from patients with SNMM were analyzed for genetic mutations based on targeted next-generation sequencing to investigate the driver of tumorigenesis and/or candidate genes for predicting clinical outcomes in SNMM. We also performed immunohistochemistry for patients identified with CTNNB1 mutations. ResultsEight of the 18 (44 %) patients had genetic mutations. The most frequent mutation was NRAS (6/18, 33 %), followed by CTNNB1 (2/18, 11 %) and BRAF (1/18, 5.6 %). One patient had both NRAS and CTNNB1 mutations. Clinical outcomes did not differ significantly between those with and without genetic mutations. NRAS mutations were associated with relatively higher T classification and worse survival rates, although the differences were not significant. The nuclear translocation of β-catenin was detected in both tumors with CTNNB1 mutations. The amino acid change in the BRAF mutation was K601R in exon 15. In the current study, no BRAF V600 mutations were detected. ConclusionGenetic mutations were not significantly associated with clinical outcomes. However, NRAS mutations may be a prognostic predictor and CTNNB1 mutation may be a treatment effector for immune check inhibitors. A larger prospective study is required to clarify the clinical importance of genetic mutations in patients with SNMM.

EPCO-28. MULTIPLATFORM MOLECULAR AND FUNCTIONAL GENOMIC ANALYSES OF NF1 MUTANT GLIOBLASTOMA IDENTIFY DISTINCT CO-MUTATION PATTERNS AND DRUGGABLE DEPENDENCIES UNDERLYING SELUMETINIB RESPONSE

Abstract The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.