Mutations In Signaling Pathways Research Articles

Characterization of Somatic Mosaicism and Mutational Profiling of Clonal Hematopoiesis Compared to MDS and sAML Depicts Diversities of Clonal Evolution

Background:Clonal hematopoiesis (CH) describes the presence of genetic alterations and expansion of clonal cell populations in the peripheral blood (PB) of individuals without clinical manifestation of a hematologic malignancy. CH is a common, age-related state. However, individuals carrying CH are at greater risk for hematologic cancer. It has been shown that presence of TP53- and other high-risk mutations, variant allele frequency (VAF) >10% or multiple mutations in CH carriers may confer a higher risk of preleukemic development and transformation to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Nevertheless, further insights into the role of CH in clonal evolution towards leukemia and elaborating features that are predictive of leukemic progression are needed.Aims:We have recently shown that CH is common in individuals undergoing hip replacement surgery (Hecker, Hartmann et al., Blood 2021). Here, we compared mutational spectrum in these CH individuals to MDS and secondary AML (sAML) cohorts. Additionally, we analyzed spatial and lineage distribution of CH and longitudinally monitored CH and MDS clones over time.Methods:Samples from individuals without known hematologic disease undergoing hip replacement surgery (n=288 samples from 261 individuals), patients with MDS (n=92) or sAML (n=123) were screened for variants in 68 leukemia-associated genes using a targeted sequencing approach (VAF cut off, 1%). Follow-up (FU) PB samples were available for 21 individuals with CH and 16 untreated low-risk MDS patients, 6-24 months after screening. n=5 CH bone marrow (BM) samples carrying six ASXL1-mutations were sorted for seven different cell fractions.Results:At screening, variants were detected in 127/261 (49%) healthy individuals, 84/92 (91%) MDS and 117/123 (95%) sAML patients, with median VAFs of 2.7% (ranging from 1-44%), 18.8% (1.1-87%) and 37.1% (1-99%), respectively. Individuals with CH had a median number of 1 variant per individual, whereas median detected variants per patient increased with clonal evolution with 3 variants in the MDS and 4 variants per patient in the sAML cohort.CH, MDS and sAML showed entity-specific mutation profiles (Figure 1A). Most variants in CH affected epigenetic modifiers, while mutations in splicing factors, signaling pathways and transcription factors increased with clonal progression.During FU, untreated low-risk MDS patients more frequently gained additional mutations compared to CH individuals (7/16 vs 2/21, respectively; p=0.024). However, we did not observe significant changes in clone sizes over time.In 17 hip replacement individuals both femoral heads were removed simultaneously, allowing paired analysis of two different hematopoietic sites. CH prevalence in this subgroup was 70.6% (12/17). Ten individuals showed identical mutation patterns in BM obtained from the right and left femoral head, with little differences in VAFs (Table 1). In contrast, two other individuals showed significant differences in variant detection comparing one to the other side: ASXL1-mutations were only present in one hip sample, whereas all the other variants were detectable in both sides (p<0.001), indicating possible spatial heterogeneity of CH clones in the BM compartment.To further characterize ASXL1 mosaicism across hematopoietic lineages and differentiation stages, we sorted and sequenced n=5 CH BM samples carrying six ASXL1-mutations for seven different hematopoietic cell fractions. In all cases ASXL1-mutations were identified in CD34 +CD38 -CD90 + hematopoietic stem cells (HSC). VAFs of ASXL1-mutations were differentially distributed (p=0.0049, Kruskal Wallis test): detected VAFs were significantly higher in HSC and common myeloid progenitors (CMP) compared to VAFs in T-cells (p=0.045 and p=0.011, respectively; Dunn´s multiple comparison test; Figure 1B).Conclusions:CH, MDS and sAML show characteristic mutation profiles that remained stable over a 6-24-month period. Gains of additional variants and clonal expansion associated with disease progression. Cellular distribution analysis of ASXL1 CH variants revealed characteristic repartition patterns within the hematopoietic differentiation tree. Additionally, differences in variant detection between cellular BM compartments indicates spatial heterogeneity of CH clones warranting further investigation.J.S.H. and L.H. contributed equally. [Display omitted] DisclosuresPlatzbecker: Geron: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Celgene/BMS: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Goetze: BMS/Celgene: Other: Advisory Board, Research Funding; Abbvie: Other: Advisory Board.

The Impact of Molecular and Cytogenetic Clonal Evolution on Survival in Relapsed/Refractory AML

Background: Clonal diversity and evolution in acute myeloid leukemia (AML) are consequences of disease progression and play a pivotal role in the development of drug resistance and refractoriness to therapy. Much work has been done with characterizing clonal architecture in bulk tumors samples or single-cell DNA sequencing, but real-world data observing the impact of clonal evolution and responses to therapy remains scarce in the relapsed and refractory settings. Therefore, the purpose of this study was to analyze the impact of observed clonal evolution on survival.Patients & Methods: We retrospectively analyzed 81 patients with AML with relapsed or refractory disease from June 2018 to December 2020. Cytogenetic and molecular data were obtained at the time of diagnosis and the time of primary induction failure or relapse, when available. We identified a total of 24 patients that demonstrated evidence of clonal evolution following induction or salvage therapy. Baseline patient demographics were obtained, including cytogenetic risk and next-generation sequencing molecular profiling at diagnosis and throughout treatment alongside dates and types of induction regimens. We compared the survival of those with evidence of clonal evolution to matched groups without. The groups with and without clonal evolution were compared for baseline statistical differences using an unpaired t-test with Welch's correction and Kaplan-Meier survival analyses were computed and compared with log-rank tests using GraphPad Prism. The event for calculating the overall survival was the date of death with patients otherwise censored at the date of last contact.Results: Of the 24 patients with evidence of clonal evolution, we detected that evolution occurred in four (16.7%) patients at the time of primary induction failure (refractory to first induction), 19 (70.8%) at the time of first relapse, and 3 (12.5%) at the time of second relapse. The median age of patients with clonal evolution was 63 years (range: 23 - 74) with 17 (70.8%) males and 7 (29.2%) females. The most commonly occurring mutations were NPM1 (25.0%), FLT3-ITD (20.8%), and TP53 (20.8%). At the time of initial diagnosis, cytogenetic risk was favorable in one (4.2%), intermediate in 7 (29.2%), and adverse in 16 (66.7%). The majority (79.2%) of patients underwent first induction with 7+3 and 8 (33.3%) patients underwent allogenic SCT. At the time of clonal evolution and ignoring the adverse prognosis associated with relapse, one patient with favorable cytogenetics remained favorable, only one (14.3%) patient with intermediate cytogenetics shifted to adverse, and one (6.3%) patient in the adverse category shifted to intermediate by ELN criteria. Three (12.5%) patients out of 24 acquired FLT3-ITD and three (12.5%) acquired an additional cooperating mutation (NRAS, KRAS, or KIT) for a total of 25.0% acquiring a signaling pathway mutation. Out of 10 patients with either mutated TP53 or MLL, three (27.3%) acquired a cooperating mutation in FLT3 or RAS. We then compared the clonal evolution cohort with the 57 remaining relapsed/refractory patients without evidence of clonal evolution. There was no difference between the groups with respect to baseline characteristics, including age (p = 0.989), ECOG status at diagnosis (p = 0.4689), Charlson Comorbidity Index (CCI) score (p = 0.6454), or prior SCT (p = >0.999). The median overall survival (OS) of the clonal evolution group was 227 days and compared to 382 days in the non-evolution cohort (p = 0.843).Conclusion: We did not detect a significant OS difference between those with clonal evolution and those without. Independent of the adverse prognosis associated with relapsed or refractory disease, cytogenetic risk category appeared to remain the same with clonal evolution. Common trends during clonal evolution included acquisition of one or several cooperating mutations in FLT3 or RAS, both with and without the presence of mutated TP53 and MLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Update on the Current Genomic Landscape of Breast Implant-Associated Anaplastic Large Cell Lymphoma.

Simple SummaryBreast implant-associated lymphoma is a unique entity that arises in the setting of breast prostheses due to a complex interplay of external and internal factors. Understanding of the mechanisms of pathogenesis is yet to be fully elucidated but recurrent mutations in signalling pathways, tumour suppressors and epigenetic regulators have been reported. This article summarises the key studies to date that have described these genetic aberrancies, which have provided an insight into potential pathways to lymphogenesis.Breast implant-associated lymphoma (BIA-ALCL) is a rare subtype of anaplastic large-cell lymphoma associated with breast prostheses. Most patients present with a localised periprosthetic effusion and are managed with removal of the implant and surrounding capsule. Less commonly, the lymphoma can form a mass associated with the capsule and rarely can present with disseminated disease. Recent series characterising the genomic landscape of BIA-ALCL have led to insights into the mechanisms of lymphomagenesis. Constitutive JAK/STAT pathway activation has emerged as a likely key component while, more recently, aberrancies in epigenetic regulators have been reported. This review describes the genomic characterisation reported to date and the insight these findings have provided into this rare entity.

Oncogenic Cooperation Between IL7R-JAK-STAT Pathway Mutations.

Oncogenic Cooperation Between IL7R-JAK-STAT Pathway Mutations.

42P Analysis of canonical uveal melanoma aberrations in murine uveal melanocytes

Uveal melanoma (UM) is the most common cancer of the eye. Previous research has identified three key driver mutations in UM tumorigenesis: 1) gain of function Gq signaling pathway mutations, 2) BAP1 loss of function mutations, and 3) aberrant expression of PRAME. However, the combined impact of these aberrations is not known. The purpose of this study was to characterize the phenotype of uveal melanocytes harboring one or more of these aberrations.

A Primer on the Biology of MDS

Introduction Myelodysplastic syndromes (MDS) arise from a small population of disease-initiating hematopoietic stem cells (HSCs) that persist and expand through conventional therapies and are major contributors to disease progression and relapse.1 In the last decade, genomic technologies coupled with mouse genetic studies have greatly improved our understanding of the genetic elements driving MDS initiation and progression and the ways in which such elements functionally contribute to specific aspects of the disease’s pathobiology.2–6 These studies have revealed that MDS are typically driven by multistep processes that affect a recurrent set of genes, which leads to the clonal expansion of mutant HSCs over their normal counterparts. Disease onset can be accelerated by exposure to cytotoxic chemotherapy, the presence of germ-line predisposition mutations, or any type of stress (e.g., inflammation) that confers a fitness advantage to the mutant clone. Progression from MDS to secondary acute myeloid leukemia (sAML) is mostly associated with the expansion of HSC clones carrying preexisting or newly acquired recurrent mutations affecting hematopoietic transcription factor genes that abrogate normal differentiation or those carrying activating mutations in signaling pathways that control cellular proliferation.7

The correlation of next-generation sequencing-based genotypic profiles with clinicopathologic characteristics in NPM1-mutated acute myeloid leukemia

The purpose of this study was to analyze the association between next-generation sequencing (NGS) genotypic profiles and conventional clinicopathologic characteristics in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1mut). We selected 238 NPM1mut patients with available NGS information on 112 genes related to blood diseases using the χ2 and Mann-Whitney U tests and a multivariable logistic model to analyze the correlation between genomic alterations and clinicopathologic parameters. Compared with the NPM1mut/FLT3-ITD(−) group, the NPM1mut/FLT3-ITD(+) group presented borderline frequent M5 morphology [78/143 (54.5%) vs. 64/95 (67.4%); P = 0.048], higher CD34- and CD7-positive rates (CD34: 20.6% vs. 47.9%, P < 0.001; CD7: 29.9% vs. 61.5%, P < 0.001) and a lack of favorable−/adverse-risk karyotypes (6.4% vs. 0%; P = 0.031). In the entire NPM1mut cohort, 240 NPM1 mutants were identified, of which 10 (10/240, 4.2%) were missense types. When confining the analysis to the 205 cases with NPM1mut insertions/deletions-type and normal karyotype, multivariable logistic analysis showed that FLT3-ITD was positively correlated with CD34 and CD7 expressions (OR = 5.29 [95% CI 2.64–10.60], P < 0.001; OR = 3.47 [95% CI 1.79–6.73], P < 0.001, respectively). Ras-pathway mutations were positively correlated with HLA-DR expression (OR = 4.05 [95% CI 1.70–9.63], P = 0.002), and KRAS mutations were negatively correlated with MPO expression (OR = 0.18 [95% CI 0.05–0.62], P = 0.007). DNMT3A-R882 was positively correlated with CD7 and HLA-DR expressions (OR = 3.59 [95% CI 1.80–7.16], P < 0.001; OR = 13.41 [95% CI 4.56–39.45], P < 0.001, respectively). DNMT3A mutation was negatively correlated with MPO expression (OR = 0.35 [95% CI 1.48–8.38], P = 0.004). TET2/IDH1 mutations were negatively correlated with CD34 and CD7 expressions (OR = 0.26 [95% CI 0.11–0.62], P = 0.002; OR = 0.30 [95% CI 0.14–0.62], P = 0.001, respectively) and positively correlated with MPO expression (OR = 3.52 [95% CI 1.48–8.38], P = 0.004). In conclusion, NPM1mut coexisting mutations in signaling pathways (FLT3-ITD and Ras-signaling pathways) and methylation modifiers (DNMT3A and TET2/IDH1) are linked with the expressions of CD34, CD7, HLA-DR and MPO, thereby providing a mechanistic explanation for the immunophenotypic heterogeneity of this AML entity.

Abstract 2183: Genomic profiling of small bowl adenocarcinoma reveals targetable mutations in multiple signaling pathways

Abstract Background: Small bowel adenocarcinoma (SBA) is a rare malignancy of the gastrointestinal (GI) tract without approved targeted therapy. Because the treatment approaches for colorectal cancer showed limited efficacy in SBA, the NCCN guide strongly encourages SBA patients to participate in clinical trials. The identification of targetable mutations through genomic profiling will gain new insights into the etiology of SBA and enable physicians to select the matched trials for patients. Methods: To characterize actionable targets in 50 Chinese SBA patients, a 830-gene next-generation sequencing panel was applied to assess the mutational status of their tumor tissues, including SNV, insertions/deletions, CNV and re-arrangements. Also, paired genomic DNA sample was extracted and analyzed by a 139-gene NGS panel to identify pathogenic germline variants. Results: The most frequently mutated genes in this SBA cohort were TP53 (68%), KRAS (56%), APC (38%), SMAD4 (20%), CTNNB1 (16%), ARID2 (14%), FAT3 (12%), FBXW7 (12%), KMT2C (12%) and PIK3CA (12%). Potentially targetable alterations include BRAF (10%), MDM2 (10%), ERBB2/HER2 (8%), PIK3CA (4%) and MAP2K1 (4%). Except one V600E mutation, all mutations in BRAF are either type 2 (L597R) or type 3 (N581S and D594N) which were sensitive to MEK inhibitor trametinib. We also found two MAP2K1 activating mutations (K57E and K57N) which can be targeted by trametinib. ERBB2/HER2 was highly amplified in one patient (copy number 25) and ERBB2/HER2 driver mutations were seen in 3 (6%) patients including V842I (n =2) and V777L (n =1). These four patients were good candidates for HER2-targeted therapy clinical trials. We also observed two activating PIK3CA mutations (R108H and G364R) which may be sensitive to PIK3CA inhibitor alpelisib. In addition, we observed targetable gene amplification of MDM2 (n =1) and FGF3/4/19 (n =2). Interestingly, one patient carried an activating ZKSCAN1-MET fusion gene which can be targeted by FDA-approved MET inhibitor carbozantinib. Among 46 patients with available MSI status, two were designated as microsatellite instability. The median TMB of microsatellite stable patients was 3.7 mutations/Mb. Lastly, we observed five deleterious germline mutations, two for SBDS, one each for APC, ERCC5 and MLH1. Overall, at least 18 (36%) patients harbored potentially actionable genetic mutations. Conclusions: The mutational landscape of our SBA cohort provided compelling evidence that multiple signaling pathways play a role in the pathogenesis of SBA and many driver mutations in these pathways are targetable. Our findings indicated that SBA patients should participate in matched clinical trials for better management of this disease. Citation Format: Rong Liu, Xiaomo Li, Zhiming Zhao, Tonghui Ma, Fei Wang, Si Liu. Genomic profiling of small bowl adenocarcinoma reveals targetable mutations in multiple signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2183.

Abstract 2182: Evaluation of somatic and germline mutations in ampullary carcinoma reveals actionable targets in multiple signaling pathways

Abstract Background: Ampullary carcinoma (AC) is a rare gastrointestinal cancer with few effective standard treatment options. For patients with unresectable diseases, the response rates for radiotherapy, chemotherapy, and chemoradiotherapy are low. Evaluation of the actionable mutations through genomic profiling will enable patients to enroll in matched clinical trials. Methods: To characterize actionable targets in 27 Chinese AC patients, a 500-gene next-generation sequencing panel was applied to assess the mutational status of their tumor tissues, including SNV, insertions/deletions, CNV and re-arrangements. Also, paired genomic DNA sample was extracted and analyzed by a 93-gene NGS panel to characterize germline variants. Results: Recurrent somatic mutated genes were KRAS (63%), TP53 (59%), APC (26%), SMAD4 (19%), CDKN2A (15%), ELF3 (11%), ERBB2 (11%), ERBB3 (11%) and MAP2K4 (11%). Consistent with previous studies, all mutations in AC driver gene ELF3 (n =3) were inactivating (three indels and one in splicing-site). The median tumor mutation burden (TMB) of our cohort was 1.6 (0-6.1) mutations/Mb and none of our patients had high microsatellite instability (MSI-H). Overall, we observed a wide spectrum of hotspot mutations in cancer driver genes including ERBB3 (n =2), FBXW7 (n =2), U2AF1 (n =2), BRAF (n =1), ERBB2/HER2 (n =1) and PIK3CA (n =1). The HER2 R678Q mutation, located in the transmembrane domain (TMD), activated HER2 signaling by improving the active dimer interface or stabilizing an activating conformation of HER2. Interestingly, both ERBB3 mutations (G284R and D297Y) were located in the extracellular domain (ECD) and their oncogenic activity also depended on the kinase-activity of ERBB2. These three mutations, together with one case of ERBB2 amplification, can be targeted by anti-HER2 antibodies and small-molecule HER2 kinase inhibitors. The BRAF N581I mutation is resistant to BRAF inhibitor vemurafenib but sensitive to MEK inhibitor trametinib. The PIK3CA N1044K mutation is oncogenic but its sensitivity to PIK3CA inhibitor alpelisib is unknown. Additionally, one patient had high level of FGFR2 amplification which may be suitable for the clinical trials of FGFR2 inhibitors pemigatnib and erdafitnib. Furthermore, two patients carried loss-of-function germline mutations in DNA repair genes PALB2 and CHEK2 which may confer synthetic lethality with PARP inhibitors. Taken together, at least eight (29.6%) patients in our cohort had actionable therapeutic targets. Conclusions: Through comprehensive genomic characterization of Chinese AC patients, we identified multiple actionable mutations in multiple signaling pathways. Our findings indicated that molecular profiling can provide clinical benefits to a significant portion of AC patients. Citation Format: Fei Wang, Si Liu, Xiaomo Li, Zhiming Zhao, Tonghui Ma, Hongling Yuan, Rong Liu. Evaluation of somatic and germline mutations in ampullary carcinoma reveals actionable targets in multiple signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2182.

Abstract 1881: AFM24 is a novel, highly potent, tetravalent bispecific EGFR/CD16A-targeting Innate Cell Engager (ICE®) designed for the treatment of EGFR-positive malignancies

Abstract AFM24 engages CD16A (FcγRIIIa) on NK cells and macrophages with much higher affinity than monoclonal antibodies (mAbs), triggering NK cell-mediated ADCC and macrophage-mediated ADCP. AFM24's mode of action is highly differentiated from current EGFR-targeting treatment options since it does not rely on inhibition of the EGFR signaling pathway, thus rendering AFM24 insensitive to the mutational status (e.g. KRAS, BRAF) of the tumor. Indeed, the efficacy of currently marketed EGFR inhibitors is limited by i) toxicities arising from blocking of EGFR signaling in healthy tissues and ii) by the occurrence of intrinsic or acquired resistances of the tumor to EGFR-targeting inhibitors. With its distinctive mode of action profile, AFM24 is a novel treatment option for a wide range of EGFR+ patient populations including hard-to-treat patients. In preclinical assays AFM24 added up to 20% more ADCC and up to 6-fold more ADCP towards NSCLC, CRC, HNSCC and ccRCC cell lines with a wide range of EGFR densities when compared to EGFR-targeting mAbs. This superior effect of AFM24 was most pronounced towards target cell lines with low EGFR density or EGFR signaling pathway mutations, such as KRAS. AFM24's efficacy was assessed in a xenograft mouse model using different human EGFR+ cell lines such as the TNBC cell line MDA-MB-231 and an adoptively transferred, cytokine-stimulated and expanded NK cell product. This combination of AFM24 with an NK cell product demonstrated a significant dose-dependent and differentiated reduction in tumor outgrowth as compared to mice treated with NK cell product alone. This model allows for systematic screening of future IO-combination regimens, such as checkpoint inhibitors. Moreover, prevalence and activity of innate and adaptive immune cells were assessed in tumor explants of patients with EGFR+ malignancies. These data will be instrumental to guide the clinical development of AFM24 in monotherapy and combination therapies. In conclusion and considering the previously reported good safety profile in cynomolgus monkeys, the presented data shows AFM24's potential to become a novel treatment option for patients with EGFR+ cancers overcoming limitations of available EGFR-targeting therapies by fully exploiting the potential of the innate immune system. AFM24 is currently being investigated in a phase 1/2a study, NCT04259450, in EGFR+ tumors. Citation Format: Jens Pahl, Gabriele Hintzen, Uwe Reusch, Torsten Haneke, Christian Breunig, Sheena Pinto, Cassandra Choe-Juliak, Andreas Harstrick, Wolfgang Fischer, Arndt Schottelius, Joachim Koch, Erich Rajkovic. AFM24 is a novel, highly potent, tetravalent bispecific EGFR/CD16A-targeting Innate Cell Engager (ICE®) designed for the treatment of EGFR-positive malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1881.

Coexisting Oncogenic Signaling Pathway Mutations in Cnl/Acml/CMML/MPN-U

Purpose: Chronic neutrophilic leukemia (CNL), chronic myelomonocytic leukemia (CMML), atypical myeloid leukemia (aCML), and unclassified myeloproliferative neoplasms (unMPN) are rare entities of myeloproliferative or myelodysplastic/myeloproliferative (MDS/MPN) syndromes. Due to poorly defined molecular mechanisms, these diseases have been, until now, diagnosed based on morphology and exclusionary criteria and treated empirically, resulting in dismal outcomes. Recent application of next generation sequencing has shed light on the genetic mechanisms of CNL/aCML/CMML/MPN-U. In particular, Maxson et al. and others have shown that CSF3R membrane proximal and transmembrane mutations are present in 83-89% of CNL and 3.3-44% of aCML patients, and expression of CSF3R membrane proximal mutants is sufficient to recapitulate the clinical features (mature granulocyte accumulation) of CNL in a mouse model. This has led to revision of the WHO diagnostic criteria for CNL, enabling a more accurate diagnosis and facilitating the development of targeted treatment for this disease. Aside from the CSF3R driver mutations, few recurrent signaling pathway mutations have been characterized in this group of diseases that could serve as the basis for such targeted therapeutic strategies. The aim of the current study is to characterize the frequency and distribution of signaling pathway driver mutations in a large cohort of CNL/aCML/CMML/MPN-U patients with the hope of informing diagnosis, prognosis and treatment.Methods and results: We performed whole exome sequencing on a large cohort of patients (n=160) with plausible diagnosis of CNL/aCML/MPN-U/CMML. We screened hotspot variants in genes representing kinases, non-kinase growth factor and cytokine receptors. We observed that 66.9% of patients harbor at least one signaling pathway driver mutations (CSF3R 20.6%, NRAS 17.5%, JAK2 9.4%, CBL 6.5%, PTPTN11 5.6%, ETNK1 5.6, KRAS 4.4%, NF1 3.1%, FLT3 3.1%, SH3B2 3.1%, etc.), largely involving JAK/STAT and RAS signaling pathways. Interestingly, MPL mutations and CALR indels were rare in our cohort. The variants of signaling pathway driver mutations were observed with high allele frequencies: CSF3R (52.1%), JAK2 (48.9%), NRAS (46.3%), PTPN11 (40.7%), CBL (34.4%) and KRAS (29.5%), indicating the majority of these driver mutations are present in the dominant leukemic clones.Interestingly, 21.5% and 3.7% patients harbor two and three different active signaling pathway mutations, respectively. Clonality analysis demonstrated a co-occurrence pattern in more than half of the cases where more than two signaling pathway mutations were identified, underscoring a complex genetic pathogenesis. CSF3R was shown to co-occur with NRAS, SH2B3, PTPN11, NTRK2, CBL, ETNK1 and ABL1 mutations; NRAS mutations were present in combination with CSF3R, KRAS, JAK2, CBL, STAT3, NTRK2, FLT3, and GNB1 mutation. Since CSF3R and RAS mutation co-occur at high frequencies (n=5, 22%) in this cohort. We performed drug sensitivity assays, and we observed reduced drug sensitivity to either JAK or MEK inhibitors in cells transformed by co-existing mutations, whereas drug combinations targeting the co-existing mutations demonstrated synergistic cytotoxicity .Conclusions: In sum, CNL/aCML/MPN-U/CMML is a group of heterogeneous diseases associated with high frequencies of signaling pathway mutations in the major clone. More than 25% of patients demonstrate multiple different pathway mutations in a co-existing pattern, which warrants comprehensive screening of molecular targets and high-efficacy drug combination strategies. DisclosuresTyner:AstraZeneca: Research Funding; Incyte Corporation: Research Funding; Leap Oncology: Consultancy; Syros: Research Funding; Seattle Genetics: Research Funding; Agios Pharmaceuticals: Research Funding; Takeda Pharmaceutical Company: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceutica: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding.

Characteristics, Mutation Profiles and Outcomes of Patients with Acute Myeloid Leukemia and a History of Autoimmune Disease

Introduction: Therapy-related acute myeloid leukemia (t-AML) covers a poorly defined group (grp) of patients (pts) who develop AML following cytotoxic or radiation therapy. Most pts have a prior malignancy but 5-20% have exposure to cytotoxic therapy for benign disorders, such as autoimmune diseases (AD). The aim of this study was to define the characteristics, mutational profiles and outcomes of pts with AD and AML.Methods: Pts with AML diagnosed from 1999-2017 were identified and divided into 4 grps: de novo AML, AML with a prior history of AD (AD-AML), t-AML excluding AD-AML, and AML evolving from antecedent myelodysplastic syndrome (MDS) or other myeloid stem cell disorders without prior history of AD (s-AML). Molecular data were obtained using different platforms of next generation sequencing (NGS) of genes that are known to be commonly mutated in AML (number of covered genes varied from 37 - 60). Categorical data were analyzed using Fisher's exact test for 2-grp comparisons of categorical data with 2 levels; chi-square tests for comparisons of categorical data involving >2 grps and/or factors with >2 levels; t-tests and ANOVA were used for comparisons of continuous factors, and the log rank test was used for comparisons of overall (OS) and relapse-free survival (RFS).Results: A total of 910 pts were included: 13% (n=118) with AD-AML, 60.7% (n=553) de novo AML, 16% sAML (n=146) and 10.2% t-AML (n=93). Median age at diagnosis was 63 years; 55% were male; 66% received intensive induction therapy. Cytogenetic data were available for 94% of pts and, according to MRC 2010 classification, were favorable in 16%, intermediate in 56% and poor in 29% of the pts. Most prevalent AD were Crohn's/ulcerative colitis (n=20, 17%), rheumatoid arthritis (n=18, 15%), psoriasis (n=12, 10%), and lupus (n=11, 9%); 11% of pts had 2 disorders (Table). Median age at diagnosis of first AD was 54 years (range 16-94) and median interval from AD diagnosis to AML was 81 months. The majority of pts (53%) did not receive treatment (not including steroids) for their AD. 27% of pts were treated with immunomodulating agents, 26% with cytotoxic agents and 14% with anti-inflammatory agents; 19% received ≥ 2 drug classes. Poor risk cytogenetics were more common in AD-AML than in de novo AML (37% vs. 22%, p=.0003), which was driven mainly by chromosome 7 (19% vs. 9%, p=.004) and 17 (11% vs. 5%, p=.01) abnormalities. NGS data were available for 338 pts, of whom 40 had AD-AML. Mutations in SRSF2 were more common in AD-AML (23%) than in de novo (10%, p=.03) and sAML (7%, p=.03). In pts with sAML arising from MDS/myeloproliferative disorders (MPN) and available NGS data (n=82), SRSF2 mutations were more common in pts with AD (5/16, 31%) versus non-AD (4/66, 6%; p=.01). Compared with t-AML, pts with AD-AML had more frequent mutations in NPM1 (18% vs. 0%, p=.03) and NRAS (15% vs. 0%, p=.03). Overall, mutations in signaling pathways were more common in pts with AD-AML (55% vs. 36%, p=.03), while mutations in genes involved with histone and chromatin modification were less common (7% vs. 20%, p=.04). Rate of complete remission and RFS were similar among de novo (71%, 15 months), AD-AML (68%, 13 months) and t-AML (68%, 13.5 months) groups. For the AD-AML grp, the median OS was 16 months; significantly shorter compared with de novo AML (25 months, p=.02), and similar to t-AML (10 months, p=.71) and sAML (10 months, p=.14) (Figure).Conclusion: Patients with AML and a prior history of AD have cytogenetic profile and outcomes similar to those with t-AML but with distinct molecular features. In pts with MDS/MPN transformed into AML, SRSF2 mutations are more common in those with history of AD. These findings need to be validated in large series; however, they suggest that spliceosome inhibitors might be a future focus of investigation in pts with AD-AML. [Display omitted] DisclosuresGerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Advani:Takeda/ Millenium: Research Funding; Pfizer: Consultancy.

Genetic Basis and Therapies for Vascular Anomalies.

Vascular and lymphatic malformations represent a challenge for clinicians. The identification of inherited and somatic mutations in important signaling pathways, including the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin), RAS (rat sarcoma)/RAF (rapidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinases), HGF (hepatocyte growth factor)/c-Met (hepatocyte growth factor receptor), and VEGF (vascular endothelial growth factor) A/VEGFR (vascular endothelial growth factor receptor) 2 cascades has led to the evaluation of tailored strategies with preexisting cancer drugs that interfere with these signaling pathways. The era of theranostics has started for the treatment of vascular anomalies. Registration: URL: https://www.clinicaltrialsregister.eu; Unique identifier: 2015-001703-32.

Next Generation Sequencing Identifies Smoldering Multiple Myeloma Patients with a High Risk of Disease Progression

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that arises from the precursor states: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Some patients rapidly progress from MGUS/SMM to MM, while others remain indolent with minimal progression over their lifetime. The genetic and molecular factors that underlie disease evolution and distinguish progressors from non-progressors are not well elucidated. To address these questions, we examined clinically-annotated samples from SMM patients, including sequential samples at the SMM stage and at time of progression.Methods: We utilized next generation sequencing methods to study 186 SMM patient samples. We performed whole exome sequencing (WES) of 70 tumor-germline matched samples (mean target coverage 50X/100X for germline/tumor) and targeted deep sequencing on 116 samples of progressor and non-progressor SMM (mean target coverage ~500X). We also performed ultra-low pass whole-genome sequencing (ULP-WGS) of 116 SMM samples as well as 20 available cell-free DNA (cfDNA) samples to detect the tumor fraction before proceeding with targeted sequencing. For WES, libraries were constructed with Agilent SureSelect XT2 library prep kit, and hybridized to Agilent's whole exome V5+UTR capture probes and then sequenced on HiSeq 4000 (Illumina). For targeted sequencing, libraries were enriched by hybridizing to the customized target bait (60 MM related genes and 56 pan-cancer genes). For ULP-WGS, libraries were prepared with Kapa Hyper Prep kit and custom adapters. Sequencing data were analyzed using previously established analytic pipelines including ichorCNA, MuTect, RecapSeg, MutSig, GISTIC, ABSOLUTE, and PHYLOGIC. High-risk SMM (HRSMM) and low-risk SMM (LRSMM) were defined according to the criteria proposed by Rajkumar et al. in Blood 2014.Results: Higher mutation load (average 1.44 mutations/Mb) was observed in HRSMM patients, which is close to the average of 1.6 mutations/Mb in symptomatic MM, compared to LRSMM patients (average 0.73 mutations/Mb, p -value = 0.001). Somatic mutations in known signaling pathways were observed four times more frequently (43.8% vs. 9.5%, p- value = 0.015) in HRSMM patients compared to LRSMM patients. Mutations in MAPK pathway genes (KRAS, NRAS, BRAF) were detected in 21.7% of HRSMM vs. 0% in LRSMM (p- value = 0.004). Mutations in NFkB pathway were present in 10% of patients and were also associated with HRSMM (17.4% vs. 0%, p- value = 0.018). MM somatic copy number aberrations (SCNAs) were detected in 64% of all SMM samples, and were two times more frequently in HRSMM than LRSMM patients (78.1% vs. 42.9%, p- value = 0.020). Phylogenetic studies of sequential patient samples, who progressed from SMM to MM, revealed different patterns of clonal evolution. Furthermore, we have performed ULP-WGS of available cfDNA samples from 20 patients of this cohort. Interestingly, tumor fraction of cfDNA was higher in HRSMM compared to LRSMM patients (p- value = 0.03). Together, this data suggests that specific genomic alterations are associated with HRSMM and could potentially be predictive of the risk to progression in SMM patients.Conclusion: This study demonstrates that next generation sequencing (WES, targeted-sequencing, and ULP-WGS) of SMM patients can identify genomic alterations associated with HRSMM vs LRSMM through different parameters such as mutation load, somatic mutations, and SCNAs. DisclosuresLaubach:Novartis, Takeda, Celgene: Consultancy; Novartis, Takeda, Celgene, Onyx: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Research Funding. Anderson:C4 Therapeutics: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder. Kumar:Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Skyline: Honoraria.

Genetic Characterization and Clinical Features of Helicobacter pylori Negative Gastric Mucosa-Associated Lymphoid Tissue Lymphoma.

Simple SummaryThe pathogenesis of H. pylori-associated gastric MALT lymphoma has been well characterized, but the genetic basis and clinical features of H. pylori negative gastric cases remain elusive. In the present study, we investigated the genetic profiles of a large series of H. pylori negative gastric MALT lymphoma by targeted sequencing for a panel of genes specifically designed for marginal zone lymphoma, together with assessment of common translocations and comprehensive clinical data. Targeted sequencing confirmed that NF-κB activation is a major driver in the pathogenesis of H. pylori negative MALT lymphoma, as shown by frequent TNFAIP3 inactivating mutations and also by translocations of MALT1/IGH. This study adds new insights into the genetic background of H. pylori negative MALT lymphoma and will potentially allow us to more specifically target the underlying molecular pathways in future therapeutic concepts.Background: In Western countries, the prevalence of gastric mucosa-associated lymphoid tissue (MALT) lymphoma has declined over the last three decades. Contemporaneously, H. pylori negative gastric MALT lymphoma is increasingly encountered, and their genetic basis and clinical features remain elusive. Methods: A total of 57 cases of H. pylori negative gastric MALT lymphoma were reviewed and investigated for chromosome translocation by fluorescence in-situ hybridization and for somatic mutations by the targeted sequencing of 93 genes. Results: MALT1 translocation, most likely t(11;18)(q21;q21)/BIRC3-MALT1, was detected in 39% (22/57) cases, and IGH translocation was further seen in 12 MALT1-negative cases, together accounting for 60% of the cohort. Targeted sequencing was successful in 35 cases, and showed frequent mutations in NF-κB signaling pathways (TNFAIP3 = 23%, CARD11 = 9%, MAP3K14 = 9%), together affecting 14 cases (40%). The NF-κB pathway mutations were mutually exclusive from MALT1, albeit not IGH translocation, altogether occurring in 86% of cases. There was no significant correlation between the genetic changes and clinicopathological parameters. The patients showed a median of progression-free survival (PFS) of 66.3 months, and a significant superior PFS when treated with systemic versus antibiotic therapy (p = 0.004). Conclusion: H. pylori negative gastric MALT lymphoma is characterized by highly frequent genetic changes in the NF-κB signaling pathways.

Profiling of 520 Candidate Genes in 50 Surgically Treated Chinese Small Cell Lung Cancer Patients.

Small cell lung cancer (SCLC) is one of the severe malignancies with high mortality. Surgically resected tumor tissues from 50 Chinese SCLC patients were collected for next-generation sequencing to detect 520 cancer‐related genes. The most frequently altered genes were TP53 (94.0%), RB1 (86.0%), LRP1B (44.0%), SPTA1 (26.0%) and KMT2D (24.0%). We detected that NOTCH2, JAK2 and CDK12 (P<0.05) had a significantly higher mutation frequency in Chinese SCLC compared to the Cologne and MSKCC. The single nucleotide variation (SNV) is dominated by C>A (34.1%). We found a significant association between TMB-H (≥10.3muts/Mb) and ATM (P=0.023), CREBBP (P=0.010), KMT2D(P=0.050) and LRP1B (P=0.005) gene mutations in Chinese SCLC patients. Immunostaining was performed using the following antibodies: TTF-1, CgA, CD56, Syn, and Ki-67. Correlation analysis between the expression of 6 markers and mutations in signaling pathways showed that Syn and CgA expression were associated with 4 (cGMP-PKG, Chemokine, TGF-β and Phospholipase D) and 2 (cGMP-PKG and Phosphatidylinositol) signaling pathway mutations. Kaplan-Meier curve showed that age<55 years, mutant ARID2 and high TMB (≥7muts/Mb) were associated with a better prognosis, while the prognosis of patients with mutations in the Ras pathway was significantly improved. High TMB is an important prognostic factor for SCLC patients showed by multivariate analysis. In the combined cohort composed of current and two previous studies, survival analysis showed that SCLC patients with mutant LRP1B demonstrated better OS (P=0.0017). Patients with a high TMB (≥7muts/Mb) have a better prognosis (P=0.0053), consistent with our results in the Chinese cohort. We characterized the genomic alterations profile of Chinese SCLC patients and analyzed the correlation between genomic changes and immunohistochemical phenotypes at the signaling pathway level. Our data might provide useful information in the diagnosis and treatment for Chinese SCLC patients.

Genomic analysis of cellular hierarchy in acute myeloid leukemia using ultrasensitive LC-FACSeq

Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

The prognostic value of cytoskeletal regulation and vesicle trafficking signaling pathway mutation in breast cancer.

e13068 Background: Breast cancer (BC) is the most commonly diagnosed cancer in 2020, with an estimated 2.3 million new cases and the leading cause of cancer death in women. The cytoskeleton is a dynamic system of protein filaments that is regulated by dozens of proteins which are, in turn, regulated by phosphoinositides. Cytoskeletal regulation and vesicle trafficking (CRVT) signaling pathway is associated with many changes in the structure and movements of cells. Few studies addressing the association between genetic mutation of the CRVT pathway and the disease prognosis have been reported up to now. Here we investigate the genetic status of CRVT pathway as a biomarker for predicting overall survival (OS) and relapse-free survival (RFS) for breast cancer patients. Methods: We used cBioPortal platform to analyze a cohort of 10646 breast cancer samples. Mutations in 7 genes ( APC, BAP1, KEAP1, PAK1, PTEN, SRC, STAT3 ) are the most common indication in the CRVT signaling pathway. We define overall survival (OS) as the time between the procedure date when the tumor specimen was collected and the date of death or last follow-up visit. And we define relapse-free survival (RFS) as the time from surgical resection to relapse of disease. We estimated and compared the survival curves of these two groups using Kaplan-Meier method and log-rank tests. The statistical significance level was set to 5% ( p &lt; 0.05). Results: Genomic alterations of the CRVT-indicator genes were found in 2582 (n = 2582/10646, 24.25%) samples with sorts of alterations identified including variants of non-synonymous mutations, splicing mutations, short in-frame insertion and deletion, short frame shift insertion and deletion, and copy number gain and loss. PTEN was most commonly altered (n = 2329), followed by PAK1 (n = 863), APC (n = 718), STAT3 (n = 341), KEAP1 (n = 317), BAP1(n = 292), and SRC (n = 278). Based on the result, patients were divided into the CRVT and non-CRVT group. The median OS of the CRVT group was 141.57 moth, which was significantly shorter than the OS of the non-CRVT group (log-rank test p= 0.000662). The median RFS of the CRVT group was 167.76 moth, which was also significantly shorter than the RFS of the non-CRVT group (log-rank test p= 0.0169). Conclusions: To the best of our knowledge, this study is the first investigation to evaluate the prognostic value of CRVT pathway mutation in patients with breast cancer. Our investigation shows that CRVT pathway mutation could significantly predict poor prognosis in breast cancer patients. The result of our analysis should be better confirmed with additional relevant research in the future using updated analyses.

Roles of the Notch Signaling Pathway in Ovarian Functioning.

The Notch signaling pathway regulates cell invasion, adhesion, proliferation, apoptosis, and differentiation via cell-to-cell interactions and plays important physiological roles in the ovary. This review summarizes current knowledge about the Notch signaling pathway in relation to ovarian functions and reveals the potential underlying mechanisms. We conducted an in-depth review of relevant literature to determine the current status of research into the Notch signaling pathway in relation to ovarian functioning and reveal potential underlying mechanisms. The activation of different Notch receptors promotes the formation of primordial follicles and proliferation of granulosa cells and inhibits steroid secretion. Abnormal regulation of the Notch signaling pathway or direct mutations might lead to over-activation or under-activation of the receptors, resulting in Notch upregulation or downregulation. It can also disrupt the normal physiological functions of the ovary. The lncRNA HOTAIR and growth hormones improved premature ovarian failure (POF) and promoted follicle maturation in a mouse model of POF by upregulating Notch1 expression. They also stimulated the Notch1 signaling pathway, increased the level of plasma estradiol, and decreased the level of plasma follicle-stimulating hormone. Thus, Notch1 could serve as a novel therapeutic target for POF. Several studies have reported multiple roles of Notch in regulating female primordial follicle formation and follicle maturation. Direct mutations in Notch-related molecules or abnormal gene regulation in the signaling pathway can lead to ovarian dysfunction. However, the underlying mechanisms are not fully understood.

Genomic correlates of response and resistance to immune checkpoint inhibitors in carcinomas of unknown primary.

Cancers of unknown primary (CUP) are highly aggressive tumours with limited molecular characterization. These tumours can be particularly sensitive to immune checkpoint inhibitors (ICI) by mounting a seemingly more effective anti-tumour immune response. Unlike other tumour lineages, the biological basis and clinical efficacy of ICI in CUP remain largely unknown. The cBioPortal database was accessed to select eligible cases from the MSK-IMPACTTM Clinical Sequencing Cohort. The tumour cell genomic correlates of response and resistance to ICI in patients with CUP were compared to those with ICI-eligible tumours: cervical cancer, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, non-small-cell lung cancer, melanoma, Merkel cell carcinoma and urothelial bladder cancer. Among a total of 234 patients with CUP, the identified genomic alterations were mainly mutation correlates of resistance to ICI, notably mutations in oncogenic signalling pathways including KRAS, STK11 and EGFR (24.7%, 10.9% and 4.2%, respectively). Compared to other tumours considered eligible for ICI, CUP presents a higher prevalence of alterations in the oncogenic signalling pathways KRAS and STK11. CUP patients treated with ICI had similar median overall survival with and without genomic correlates of response and resistance to ICI. An exploratory analysis showed that patients with TMB >10 mutations had a trend for better outcomes. A tumour mutation burden >10 mutations per megabase can provide a potential genomic correlate of response to ICI in patients with CUP. Further research is warranted to validate these findings.