C-kit Mutation Research Articles

The Prognostic Significance of c-KIT Mutations in Core Binding Factor Acute Myeloid Leukemia

BackgroundMany recurrent mutations are encountered in core binding factor acute myeloid leukemia (CBF-AML) which may affect the prognosis. Approximately 20 to 45% of CBF-AML patients have KIT mutations which are having poor prognosis and high incidence of relapse. There is still insufficient data to categorize the patients with c-kit mutation into which risk group and there is a debate around whether Tyrosine kinase inhibitors can decrease the relapse risk and improve the prognosis of those patients. Patients and MethodsThis study was conducted throughout a period of 3 years, where 102 CBF-AML were enrolled in our study. We analyzed the incidence of c-KIT exon 8 and 17 D816V mutations in CBF-AML patients and studied the prognosis. ResultsThe prevalence of CBF-AML was 102 of 989 (10.3%), 13.7% and 8.7% in pediatrics and adults’ groups respectively. c-KIT fragment mutation analysis revealed a mutant form in 27 of 102 (26.5%) patients. Exon 8 mutation was found in 4 of 40 pediatric and 2 of 62 adult patients, while exon 17 mutation was found in 9 of 40 pediatric and 12 of 62 adult patients. The c-KIT mutations was more common in t(8;21). There was no significant relationship between c-kit mutation and CR rates, while there was a significant inferior overall, disease free as well as progression free survival in the c-KIT mutant patients as compared to the wild group (P value .045, .036 and .024 respectively) in the pediatric group, however, this significance was not evident in the adults’ group. ConclusionAccording to our study, the results may suggest c-KIT mutation as a poor risk factor in pediatric CBF-AML.

Abstract P136: Response to alpelisib in an adolescent with PIK3CA-mutated metastatic gastrointestinal stromal tumour

Abstract Introduction Gastrointestinal stromal tumours (GISTs) are mesenchymal neoplasms that arise in the gastrointestinal tract. While most GISTs are driven by mutations in cKIT or PDGFR, approximately 10% lack these variants and are referred to as ‘wild-type’. Wild-type GISTs are commonly succinate dehydrogenase (SDH)-deficient and are characterized by indolent, multi-focal disease at presentation. On occasion, patients experience symptomatic disease progression warranting treatment ; imatinib or sunitinib offer minimal benefit. PIK3CA mutations have recently been described in a small proportion of GIST. Case report A previously healthy 16-year-old female presented with a five-month history of vomiting and weight loss, compatible with gastric outlet obstruction. Computerized tomography demonstrated a large gastric mass, and multiple liver and peritoneal metastases. Biopsy showed nests of epithelioid cells with round nuclei, vesicular chromatin, and eosinophilic cytoplasm. The lesional cells were immunoreactive for CD117, DOG1, CD34, and smooth muscle actin, while negative for SDH-B immunohistochemistry, in keeping with SDH-deficient GIST. Germline and somatic panel sequencing were performed through the SickKids Cancer Sequencing Program along with RNA sequencing by Illumina TruSight RNA Pan-Cancer NGS analysis. Somatic analysis revealed a PIK3CA p.Glu545Lys (E545K) mutation at 0.12 variant allele fraction (VAF). The patient was also found to have germline heterozygous pathogenic splice variant in the SDHB gene c.75+1 G>T (p?). In the light of the molecular findings and no alternative curative options, compassionate access to alpelisib was sought. The patient started drug at the recommended adult dose of 300 mg by mouth daily, and after completing two 28-day cycles, showed marked improvement in her abdominal distension and discomfort. Her appetite improved with documented nutritional weight gain. Reimaging at this time showed a 20% reduction in the sum of maximum dimensions of target lesions (per RECIST 1.1). Repeat imaging after five months on treatment confirmed continued improvement with 25% reduction compared to baseline.The patient did not experience hypersensitivity, severe cutaneous adverse reactions, hyperglycemia or pneumonitis. She currently continues on alpelisib monotherapy. Conclusion We describe the first report of a clinically meaningful response to a PIK3CA inhibitor in an adolescent patient with advanced metastatic SDH-deficient GIST harbouring a somatic activating PIK3CA hotspot mutation. There is still much to be learned about the biology of pediatric/wild-type GIST. In these rare cases, we recommend referral to specialized centers and incorporation of comprehensive next generation sequencing into care. Sequencing should be performed both on germline and somatic DNA to evaluate for a cancer predisposition syndrome, along with possible druggable targets in PIK3CA, cKIT or PDGFRA. We believe that the lack of routine comprehensive genomic analysis may underestimate the frequency of such rare variants. Citation Format: Sarah Cohen-Gogo, Nisha Kanwar, Furqan Shaikh, Reto M. Baertschiger, Adam Shlien, David Malkin, Juan Putra, Ailish Coblentz, Anita Villani, Abha A. Gupta, Daniel A. Morgenstern. Response to alpelisib in an adolescent with PIK3CA-mutated metastatic gastrointestinal stromal tumour [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P136.

Analysis of risk factors of relapse after allogeneic hematopoietic stem cell transplantation in patients with t (8;21) acute myeloid leukemia

目的探讨t(8;21)急性髓系白血病（AML）异基因造血干细胞移植（allo-HSCT）后复发的影响因素。方法对2008年1月至2020年10月期间在中国医学科学院血液病医院接受allo-HSCT的t(8;21) AML患者进行回顾性分析。结果73例t(8;21) AML患者纳入研究，男39例，女34例，中位年龄36（13～54）岁。73例患者中10例出现血液学复发，3年累积复发率（CIR）为15.7％（95％CI 7.3％～26.8％），中位复发时间为9.2（2.0～47.6）个月。73例患者中19例死亡，移植后3年总生存（OS）率为68.9％（95％CI 56.4％～81.4％）。移植后3个月内融合基因下降≥3个对数级组移植后3年CIR明显低于下降<3个对数级组［13.3％（95％CI 4.5％～26.8％）对57.1％（95％CI 13.2％～85.6％），P<0.001］；移植后12个月内融合基因水平下降≥4个对数级组移植后3年CIR明显低于下降<4个对数级组［5.1％（95％CI 0.9％～15.4％）对25.0％（95％CI 0.3％～71.3％），P<0.001］。Cox多因素分析显示移植当天造血干细胞回输前（0 d）RUNX1-RUNX1T1融合基因高定量（≥1.58％）［P＝0.006，HR＝28.849（95％CI 2.680～310.524）］及初诊时骨髓流式细胞术原始细胞比例≥60％［P＝0.015，HR＝6.640（95％CI 1.448～30.457）］是血液学复发的独立危险因素；c-Kit及Flt3基因突变对移植后血液学复发无明显影响（P＝0.877，P＝0.773）。初诊时骨髓流式细胞术原始细胞比例≥60％［P<0.001，HR＝8.925（95％CI 2.702～29.476）］、达到完全缓解所需的疗程数≥2个［P＝0.013，HR＝4.495（95％CI 1.379～14.649）］是影响OS的独立危险因素。结论0 d RUNX1-RUNX1T1融合基因定量水平≥1.58％及初诊时骨髓流式细胞术原始细胞比例≥60％是影响t(8;21) AML患者allo-HSCT后血液学复发的独立危险因素。移植后RUNX1-RUNX1T1融合基因监测有助于评估复发的危险性。

Compromised Host Stem Cell Competitiveness Affords Fanconi Stem Cell Engraftment in C-Kit Mutant Humanized Mice

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment.In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation.FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice.In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. DisclosuresNo relevant conflicts of interest to declare.

Results of the 6-Year Follow-up of the Gimema AML1310 Trial: A Risk-Adapted, MRD-Directed Therapy for Young Adults with Newly Diagnosed Acute Myeloid Leukemia

Background: In the AML1310 trial, we applied a comprehensive AML risk assessment, based on the integration of cytogenetic/genetic data and measurable residual disease (MRD) status, to optimize patients' (pts) therapeutic post-remission allocation. By doing so and using the NCCN2009 risk-stratification, favorable-risk (FR) pts (NPM1 mut/FLT3-ITD wt or CBF positive without c-Kit mutations) were to receive an autologous stem cell transplant (AuSCT); poor-risk (PR) pts (adverse karyotype or FLT3-ITD mut) were to receive an allogeneic stem cell transplant (ASCT); intermediate-risk (IR) pts (intermediate karyotype or FLT3-TKD mut or CBF positive with c-Kit mut) were to receive AuSCT or ASCT depending on the levels of MRD, measured by flow cytometry after consolidation therapy. Allocation to ASCT required the procedure to be performed whatever the source of stem cells (identical sibling, unrelated, cord blood, haploidentical). At that stage of analysis, 2-year overall (OS) and disease-free survival (DFS) of the whole series was 56% and 54%, respectively. Two-year OS and DFS were 74% and 61% in the FR category, 42% and 45% in the PR category, 79% and 61% in the IR MRD-negative category, 70% and 67% in the IR MRD-positive category (Venditti, Blood 2019:134(12);935-945) .Aims: With an extended median follow-up of 6 years, we wanted to evaluate the long-term impact on outcome of the strategy explored in the AML1310 trial. Specifically, we meant to analyze long-term duration of OS and DFS according to the category of risk, and the corresponding post-remission therapy delivered.Results: Three hundred-61/500 patients (72%) achieved a CR, 342/361 completed the consolidation phase and were treatment allocated: 165 (48%) to ASCT (122 PR, 43 IR MRD-positive) plus 23 rescued after salvage therapy, for a total of 188 candidates; 150 (44%) to AuSCT (115 FR, 35 IR MRD-negative) plus 27 IR patients (8%) with no leukemia-associated phenotype, for a total of 177 candidates. Overall, 110/177 (62%) and 130/188 (71%) AuSCT or ASCT candidates received it, respectively. Median OS and DFS was 3.1 and 2.9 years, respectively. Probability of 6-years OS and DFS of the whole series was 43.6% and 43.1%, respectively; cumulative incidence of relapse (CIR) was 39.7%. Probability of 6-years OS and DFS were 58.5% and 50.1% in the FR category; 35.4% and 38.0% in the PR category; 43.1% and 45.7% in the IR category. Probability of 6-year OS and DFS of patients with no LAIP detected was 32.5% and 29.1% (Fig.1 A-B). We did not observe difference in OS and DFS between the IR-MRD negative and IR-MRD positive category: 56.6% vs 62.3% and 51.6% vs 48.6%, respectively. Six-year CIR was 42.2%, 37.6% and 35.8% in the FR, PR and IR category, respectively (Fig. 1 C). Six-year CIR of patients with no LAIP detected was 50% (Fig.1 C). Six-year CIR and non relapse mortality (NRM) of transplanted patients was 35% and 15.8%, respectively (Fig.1 D).Conclusions: With an extended follow-up of 6 years, the present analysis confirms the long-term advantage of adopting a risk-adapted, MRD-directed strategy to implement the post-remission therapeutic decision. For patients belonging to the FR or IR-MRD negative category, an excess of toxicity was prevented by delivering an AuSCT. This is also in keeping with the ELN2017 recommendations, suggesting that AuSCT still has a role in selected situations. On the other hand, the vast majority of PR and IR-MRD positive patients received an ASCT, with a remarkable 6-year OS and DFS benefit. The effectiveness of such an approach translated in a low NRM in transplanted patients. Six-year OS, DFS and CIR of no LAIP patients also confirm that the choice of AuSCT was suboptimal and that in this situation ASCT should have been preferred. Based on the present knowledge, an MRD-directed approach is being explored also in FR patients, in the GIMEMA AML1819 trial. [Display omitted] DisclosuresVenditti: Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding. Luppi: Abbvie: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; MSD: Honoraria; Gilead Science: Honoraria, Other: Travel grant; Daiichi-Sankyo: Honoraria; Jazz Pharma: Honoraria. Voso: Celgene/BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz: Consultancy, Honoraria, Speakers Bureau; Abbvie: Speakers Bureau; Novartis: Speakers Bureau. Vignetti: Novartis: Honoraria; Incyte: Honoraria; Amgen: Consultancy, Honoraria. Buccisano: Novartis: Honoraria, Speakers Bureau.

Co-Targeting E-Selectin/CXCR4 with GMI-1359 Facilitates AML Stem Cell Mobilization and Protects BM Niches from Anti-Leukemia Therapy

Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA).Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p<0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively).In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts.Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. [Display omitted] DisclosuresFogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

Rh-endostatin combined with chemotherapy in patients with advanced or recurrent mucosal melanoma: retrospective analysis of real-world data.

Mucosal melanoma is rare and has distinct clinical and genetic features. Even with advances in targeted and immune therapies, the survival of patients with advanced or recurrent mucosal melanomas remains poor. The standard treatment remains controversial and we conducted this real-world study aimed to explore continuous intravenous recombinant human endostatin (Rh-endostatin) infusion plus chemotherapy in this population in the first-line setting. Overall, 43 patients with advanced or recurrent mucosal melanoma treated at Fudan University Shanghai Cancer Center between April 2017 and August 2020 were retrospectively included. Patients received dacarbazine plus cisplatin or temozolomide plus cisplatin per the investigators' preference. Rh-endostatin (105mg/m2) was administered with continuous infusion for 168h (Civ 168h). Of the 43 patients, 72.1% had metastatic disease, and the most common primary site was the gastrointestinal tract (51.2%). The most commonly observed mutations were NRAS (23.1%), BRAF (7.7%) and CKIT mutations (5.1%). An objective response was observed in 12 (30.0%) of the 40 evaluable patients, and disease control was achieved in 31 (77.5%) patients. With a median follow-up of 17.6months, the median progression-free survival (PFS) and overall survival (OS) were 4.9 and 15.3months, respectively. Additionally, high lymphocyte-to-monocyte ratio (LMR) (p = 0.023, HR 0.29, 95% CI: 0.10-0.84) and BRAF/KIT/RAS mutation (p = 0.028, HR 0.24, 95% CI: 0.07-0.86) were independently correlated with prolonged OS. Toxicity was manageable overall. Continuous Rh-endostatin infusion plus chemotherapy was effective and safe for the treatment of advanced or recurrent mucosal melanoma. High LMR was correlated with favorable PFS and OS in this patient population.

Molecular evaluation of mutations in acute myeloid leukemia patients from Turkey: A single-center study.

Certain genetic mutations could have a role in the etiology of acute myeloid leukemia (AML). Hereby, in this study, we primarily aimed to investigate the distribution of genetic mutations in AML patients. We also attempted to analyze the incidence of genetic mutations in AML patients from Turkey.This retrospective study included a total of 126 patients diagnosed with AML, who had molecular mutation test results or records in their patient files. The patients who were not citizens of the Republic of Turkey were not included in the study.It was observed that analyses for at least 1 c-kit exon mutation had been carried out on 76 patients, which detected no c-kit mutation among the types of genetic mutations investigated in all of those 76 patients. We found the frequency of FMS-like tyrosine kinase 3-internal tandem duplication mutation as 25%. The prevalence of translocation(15;17) was approximately 11% and the prevalence of translocation(8;21) was % 6.25. In addition, we also showed that the frequency of inversion16 was nearly 3.7%.Lastly, the possibility of c-kit mutation in AML patients from Turkey might actually be low.

616 Rare KIT gene mutation in a recurrent GIST: Case Report

Abstract Introduction The diagnosis and treatment of Gastro-intestinal stromal tumours (GISTs) has been revolutionized by molecular pathology and targeted therapy. Description This patient was diagnosed with locally advanced gastric GIST in 2009. He was initially treated neoadjuvantly with imatinib from 2009- 2010. He underwent laparoscopic resection in 2010. Pathology showed almost complete response with only 1.5mm focus of viable tumour. He did not receive adjuvant imatinib as this was not established practice in 2010. Recurrent disease was resected in 2011. Mitotic count was 200/50hpf. Adjuvant imatinib was given for 5 years then discontinued in 2016. Imaging showed no recurrence over this time period. Molecular testing showed Kit Exon 11 mutation- this is common in GISTs and associated with response to imatinib. Recurrent disease was diagnosed 2018 with a 10x9cm mass between residual stomach and liver– he recommenced imatinib with partial response (maximal response was reached in 2020, but a new 3cm lesion was noted) He underwent further resection of the residual stomach and liver segmentectomy in 2020. Histology showed acellular areas of myxoid degeneration, indicating treatment response however viable tumour remained. Sequencing was performed. This showed the expected mutation in exon 11 but also a mutation in exon 13 of KIT- this has been shown recently to confer resistance to imatinib. Discussion Over 90% of GISTs harbour mutations in c-KIT. Recent work has demonstrated that some tumours acquire secondary mutations conferring resistance, following prolonged TKI therapy. Radiological and histopathological features correlate with such events and assist in deciding surgical management.

S3071 Humongous Refractory Gastrointestinal Stromal Tumor With an Unusual Mutation

Introduction: Gastrointestinal stromal tumors (GIST) are rare mesenchymal neoplasms that arise from any part of the gastrointestinal tract. Its incidence varies between 7 and 8 percent per million inhabitants per year with a and median age of diagnosis of 63 years old. Extragastrointestinal stromal tumors make up less than 5 percent of cases, and most cases are secondary to advanced disease. Here we present a case of a catastrophic progression GIST with few or no previously documented mutation refractory to available therapies. Case Description/Methods: A 46-year-old man with a history of Omental GIST in remission after 5 years of treatment after imatinib attended the emergency room due to abdominal pain. Symptoms began suddenly with constipation, early satiety, and changes in stool size. Physical examination with a distended abdomen and positive liquid wave. laboratory analysis with 9.4 g / dL of hemoglobin. Abdominal CT reveals a large mesenteric mass measuring 15.6 x 10.2 x 24.6 cm in the lower abdomen extending into the pelvic region. Biopsy of the lesion reported a high-risk recurrent GIST with high Ki67 proliferation and a very high mitotic range. High-throughput and pathological sequences reported negative mutations in C-kit, CD117 and CD34, CDX-2, Betacatenin, LCA, CD3, CD20, Vimentin, and EXON 13,14 positive. Treatment was started with unanswered imatinib and then switched to sunitinib with disease progression and worsening disease burden. Treatment with regorafenib was started but, unfortunately, aggressive progression of the disease with a mass effect that affects multiple organs, so the patient did not tolerate the therapy. During the prolonged stay, the patient was complicated by spontaneous bacterial peritonitis, massive pulmonary embolism, and multi-organ failure, resulting in his death. Discussion: Our patient presented mutations in exon 13 and 14 rarely described in the literature that makes up less than 1% of GIST cases and does not have definitive treatment. It is presumed that the mutation in exon 13 responds to imatinib although in our patient it did not slow it down. progression of the disease. KIT-negative GISTs that progress during or after treatment with sunitinib and imatinib can be challenging for pathologists and clinicians. The implementation of next-generation gene sequences in clinical practice provides a major step forward in the implementation of target therapies and further studies. Tumor markers need to be developed for early detection, monitoring, and treatment.

High EVI1 Expression Predicts Adverse Outcomes in Children With De Novo Acute Myeloid Leukemia

BackgroundA high ecotropic viral integration site 1 (EVI1) expression (EVI1 high) is an independent prognostic factor in adult acute myeloid leukemia (AML). However, little is known of the prognostic value of EVI1 high in pediatric AML. This study aimed to examine the biological and prognostic significance of EVI1 high in uniformly treated pediatric patients with AML from a large cohort of seven centers in China.MethodsA diagnostic assay was developed to determine the relative EVI1 expression using a single real-time quantitative polymerase chain reaction in 421 newly diagnosed pediatric AML patients younger than 14 years from seven centers in southern China. All patients were treated with a uniform protocol, but only 383 patients were evaluated for their treatment response. The survival data were included in the subsequent analysis (n = 35 for EVI1 high, n = 348 for EVI1 low).ResultsEVI1high was found in 9.0% of all 421 pediatric patients with de novo AML. EVI1 high was predominantly found in acute megakaryoblastic leukemia (FAB M7), MLL rearrangements, and unfavorable cytogenetic aberrance, whereas it was mutually exclusive with t (8; 21), inv (16)/t (16; 16), CEBPA, NPM1, or C-KIT mutations. In the univariate Cox regression analysis, EVI1 high had a significantly adverse 5-year event-free survival (EFS) and overall survival (OS) [hazard ratio (HR) = 1.821 and 2.401, p = 0.036 and 0.005, respectively]. In the multivariate Cox regression analysis, EVI1 high was an independent prognostic factor for the OS (HR = 2.447, p = 0.015) but not EFS (HR = 1.556, p = 0.174). Furthermore, EVI1 high was an independent adverse predictor of the OS and EFS of patients with MLL rearrangements (univariate analysis: HR = 9.921 and 7.253, both p < 0.001; multivariate analysis: HR = 7.186 and 7.315, p = 0.005 and 0.001, respectively). Hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) provided EVI1 high patients with a tendential survival benefit when compared with chemotherapy as a consolidation (5-year EFS: 68.4% vs. 50.8%, p = 0.26; 5-year OS: 65.9% vs. 54.8%, p = 0.45).ConclusionIt could be concluded that EVI1 high can be detected in approximately 10% of pediatric AML cases. It is predominantly present in unfavorable cytogenetic subtypes and predicts adverse outcomes. Whether pediatric patients with EVI1 high AML can benefit from HSCT in CR1 needs to be researched further.

Molecular profiling and identification of prognostic factors in Chinese patients with small bowel adenocarcinoma.

Small bowel adenocarcinoma (SBA) is a rare malignancy with a poor prognosis and limited treatment options. Despite prior studies, molecular characterization of this disease is not well defined, and little is known regarding Chinese SBA patients. In this study, we conducted multigene next‐generation sequencing and 16S ribosomal RNA gene sequencing on samples from 76 Chinese patients with surgically resected primary SBA. Compared with colorectal cancer and Western SBA cohorts, a distinctive genomic profile was revealed in Chinese SBA cohorts. According to the levels of clinical actionability to targetable alterations stratified by OncoKB system, 75% of patients harbored targetable alterations, of which ERBB2, BRCA1/2, and C‐KIT mutations were the most common targets of highest‐level actionable alterations. In DNA mismatch repair–proficient (pMMR) patients, significant associations between high tumor mutational burden and specific genetic alterations were identified. Moreover, KRAS mutations/TP53 wild‐type/nondisruptive mutations (KRASmut/TP53wt/non‐dis ) were independently associated with an inferior recurrence‐free survival (hazard ratio [HR] = 4.21, 95% confidence interval [CI] = 1.94‐9.14, P < .001). The bacterial profile revealed Proteobacteia, Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Cyanobacteria were the most common phyla in SBA. Furthermore, patients were clustered into three subgroups based on the relative abundance of bacterial phyla, and the distributions of the subgroups were significantly associated with the risk of recurrence stratified by TP53 and KRAS mutations. In conclusion, these findings provided a comprehensive molecular basis for understanding SBA, which will be of great significance in improving the treatment strategies and clinical management of this population.

Gastrointestinal Stromal Tumours.

To analyse the gastrointestinal stromal tumours (GIST) patients' inter-demographics, histological type and association with secondary tumours. A case series. Place and Duration of the Study: Department of General Surgery, Haydarpasa Numune Training and Research Hospital, Istanbul, Turkey, between January 2010 and December 2018. Fifty-eight patients diagnosed with GIST and operated at the study place were analysed retrospectively. The demographics, symptoms, diagnosis, treatment, tumour location, histopathology, risk classification, and prognosis were recorded. The mean age was 60.62 ± 10.63 (37-83) years and the male to female ratio was 1:1. The most common symptom was abdominal pain (51.7%). Tumour site was the stomach in the majority (86.2%), followed by the small intestine and colon. One patient also had a secondary malignancy. The most common histologic type was spindle cell, followed by mixed cell type. C-kit (CD117) and CD34 mutations were positive in 87.9% and 75.9% of the cases. One patient had liver metastasis on diagnosis and another had peritoneal implants per-operatively, who died after 36 months due to midgut volvulus. The mean follow-up period was 32.03 ± 13.67 months. Two patients developed liver metastasis in the early postoperative period. Surgical resection and imatinib treatment have been provided with good prognosis. The most common histology is spindle cell type. GISTs might be associated with other cancers which should be searched and analysed. Key Words: Gastrointestinal stromal tumour, Secondary malignancy, Treatment, Prognosis.

Targeting KIT by frameshifting mRNA transcripts as a therapeutic strategy for aggressive mast cell neoplasms

Activating mutations in c-KIT are associated with the mast cell (MC) clonal disorders cutaneous mastocytosis and systemic mastocytosis and its variants, including aggressive systemic mastocytosis, MC leukemia, and MC sarcoma. Currently, therapies inhibiting KIT signaling are a leading strategy to treat MC proliferative disorders. However, these approaches may have off-target effects, and in some patients, complete remission or improved survival time cannot be achieved. These limitations led us to develop an approach using chemically stable exon skipping oligonucleotides (ESOs) that induce exon skipping of precursor (pre-)mRNA to alter gene splicing and introduce a frameshift into mature KIT mRNA transcripts. The result of this alternate approach results in marked downregulation of KIT expression, diminished KIT signaling, inhibition ofMC proliferation, and rapid induction of apoptosis in neoplastic HMC-1.2 MCs. We demonstrate that invivo administration of KIT targeting ESOs significantly inhibits tumor growth and systemic organ infiltration using both an allograft mastocytosis model and a humanized xenograft MC tumor model. We propose that our innovative approach, which employs well-tolerated, chemically stable oligonucleotides to target KIT expression through unconventional pathways, has potential as a KIT-targeted therapeutic alone, or in combination with agents that target KIT signaling, in the treatment of KIT-associated malignancies.

Gastrointestinal Stromal Tumors-A Mini Review.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. They are potentially malignant, and have an unpredictable evolution. The origin of these tumors is in the interstitial cells of Cajal, which are cells that are interposed between the intramural neurons and the smooth muscle cells of the digestive tract. GISTs are characterized by mutations in the gene c-Kit, but also other mutations, such as those of the platelet-derived growth factor receptor alpha. The most common locations of these tumors are the stomach and small intestine, although they can occur at any level of the digestive tract and occasionally in the omentum, mesentery and peritoneum. Most cases of GISTs are sporadic, and about 5% of cases are part of family genetic syndromes. The correct diagnosis of GIST is determined by histopathological examination and immunohistochemistry. According to histopathology, there are three main types of GISTs: spindle cell type, epithelioid type and mixed type. The therapeutic management of GIST includes surgery, endoscopic treatment and chemotherapy. The prognosis of patients with GIST varies depending on a number of factors, such as risk category, GIST stage, treatment applied and recurrence after treatment.

Advancement of precision oncology by integration of highly sensitive protein profiling technologies and patient-derived cell models for functional efficacy testing

Precision oncology is defined as the individualized treatment of a patient´s tumor based on molecular, cellular and functional analyses of tumor tissue specimen (National Center for Tumor Diseases Heidelberg. Precision oncology, 2020). To date, mostly genomic analyses of tumor biopsy tissue are used to identify aberrations present in the patient’s tumor and in result matching drug treatments addressing these alterations. Based on such analyses and considering available clinical and histopathological data, oncologists discuss and select tailored therapies in molecular tumor boards established at clinical centers. A prominent example of a precision therapeutic is Imatinib, which was approved amongst others for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST) harboring activating BCR-ABL gene fusion and c-KIT mutations, respectively. Another early example of precision oncology is the use of tyrosine kinase inhibitors (e.g. Gefitinib, Erlotinib) for treatment of non-small-cell lung cancer (NSCLC) harboring activating mutations in the epidermal growth factor receptor (EGFR). More recently, tissue-agnostic, targeted treatment approaches for tumors harboring TRK gene fusions received clinical approval. Promising results of this novel approach to tumor therapy have led to significant changes and restructuring in the treatment of tumor patients at clinical centers worldwide in recent years. Necessary infrastructures with molecular genomic core facilities and molecular tumor boards have been established and centers for personalized medicine for patient treatment have been founded in many places. Here, we provide an overview on the status of practical implementation of precision oncology at clinical centers with a focus on existing challenges of this approach as well as future opportunities with regard to the integration of additional technologies with emphasis on protein profiling technologies and patient-derived cellular model systems.

Abstract 1482: M4645 is a potent and highly selective inhibitor of disease relevant cKIT mutations for the treatment of metastatic or recurrent GIST

Abstract Oncogenic mutations in cKIT in exons 9 and 11 are the main driver of gastrointestinal stromal tumors (GIST). Imatinib is standard of care in 1L in unresectable metastatic or recurrent GIST, yet secondary resistance mutations in exons 13/14 and 17/18 of the cKIT kinase domain frequently arise limiting efficacy. Currently approved 2L and 3L therapies sunitinib and regorafenib target these mutations only to some extent and show limited clinical benefit. Even with the recent approval of ripretinib as 4L treatment, a high unmet medical need remains for patients with imatinib resistance mutations in exon13/14. M4645 was designed to particularly inhibit the frequent V654A resistance mutation in exon 13 of cKIT. Based on cKIT inhibitor scaffolds identified within the Merck compound collection, a 3,5,6-substituted indazole-lead was discovered. Substituent optimization in the 5- and 6-positions of the core scaffold resulted in highly selective kinase inhibitors. Through variation at the 3-position, potency and physicochemical properties were optimized, resulting in the candidate molecule M4645 which combined potency, kinase selectivity, and solubility with a good safety profile. The excellent kinase selectivity could be rationalized by the crystal structure of cKIT V654A mutant kinase domain in complex with M4645. M4645 showed potent inhibition of cKIT autophosphorylation and of downstream P-ERK1/2 and P-AKT signaling in cell lines bearing activating and resistance mutations of cKIT. Dose-dependent inhibition of cKIT activation was demonstrated in a GIST xenograft model expressing the cKIT resistance mutation V654A in exon 13. Strong antitumor activity was observed in cell line and patient-derived GIST xenograft models with this imatinib resistance mutation. Compared to imatinib, M4645 induced prolonged anti-tumor response in a GIST model with a primary activating mutation of cKIT. In summary, the favorable profile and strong antitumor activity, particularly in GIST models expressing the V654A resistance mutation, suggests superiority of M4645 compared to currently available &amp;gt; 2L treatment options for unresectable metastatic or recurrent GIST patients. Citation Format: Nina Linde, Andreas Blum, Dieter Dorsch, Ulrich Graedler, Michael Busch, Nina Glaser, Carl Petersson, Aaron Ruff, Eva Sherbetjian, Christina Esdar. M4645 is a potent and highly selective inhibitor of disease relevant cKIT mutations for the treatment of metastatic or recurrent GIST [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 1482.

Abstract 1133: Combined inhibition of cKIT and Lysine-Specific Demethylase 1 as a therapeutic strategy in acute myeloid leukemia

Abstract Responses to kinase inhibitors in AML are short-lived with the inevitable development of resistance. Combination therapy with agents from distinct functional classes is one strategy to overcome this resistance. Using AML cell lines and primary patient samples harboring oncogenic cKIT mutations we demonstrate that inhibition of the epigenetic regulator Lysine-Specific Demethylase 1 (LSD1) markedly augments the cytotoxic effect of the KIT inhibitor avapritinib. Transcriptomic and epigenetic profiling revealed synergistic suppression of Myc activity accompanied by a global loss of acetylation and decreased expression of key leukemia cell cycle genes. This epigenetic reorganization results from disruption of key transcription factor networks responsible for maintaining leukemia cell self-renewal and survival. These findings demonstrate that combined KIT and LSD1 inhibition is an exciting new therapeutic avenue for KIT mutant AML. Previously, we have shown targeting the leukemic epigenome as a possible mechanism to augment the efficacy of kinase inhibitors. Thus, we hypothesize that targeting both the cKIT and CBF mutations with combination therapy will result in a deeper molecular response. Kasumi-1 cells harbor mutant cKIT, making an ideal model to study this combination. We observed a synergistic response to a clinically available KIT inhibitor, avapritinib, and two different LSD1 inhibitors, ORY-1001 and GSK-LSD1. Colony formation of healthy CD34+ cells and cell growth of cKIT WT cells were not significantly altered by the combination, thus showing the specificity of this combination by targeting mutant KIT. Bulk RNA-seq and master regulator analysis revealed greater suppression of Myc target genes with dual inhibition of KIT and LSD1. Using Cleavage Under Targets and Tagmentation (CUT&amp;Tag), we observed synergistic loss of H3K27Ac at the promoters of genes necessary for cell proliferation. Additionally, we used Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) to interrogate Myc localization, and found a greater loss of Myc binding with dual inhibition, overlapping with the loss of acetylation at these loci. This study demonstrates the synergistic efficacy of combination therapy and identifies key biomarkers for drug activity, namely loss of histone acetylation and Myc binding. Collectively, combined KIT and LSD1 inhibition may be an effective therapeutic approach for cKIT mutant AML. Citation Format: Brittany M. Smith, Daniel J. Coleman, Lauren Maloney, Cody Coblentz, Zachary Schonrock, Joseph Estabrook, Theresa Lusardi, Brian J. Druker, Julia E. Maxson, Theodore P. Braun. Combined inhibition of cKIT and Lysine-Specific Demethylase 1 as a therapeutic strategy in acute myeloid leukemia [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 1133.

Abstract 2252: Genome profiling of thymic carcinoma identifies putative driver mutations in the NF-κB signaling pathway

Abstract Background: Thymic epithelial tumors (TETs), including thymoma and thymic carcinoma (TC), are one of the rarest cancers. Unlike thymoma, TC is clinically agressive and responds poorly to first-line chemotherapy and second-line systemic therapy. Currently, sunitinib is the only receptor tyrosine kinase inhibitor approved for TC treatment. However, most TC patients do not have the corresponding c-Kit mutations. Because the molecular drivers of TC are almost unknown, characterization of the mutational landscape of TC will broaden our understanding of TC etiology and facilitate the development of effective targeted therapies. Methods: Comprehensive genomic profiling, including SNV, indels, copy number alterations and re-arrangement analysis, was performed with a 830-gene next-generation sequencing panel on tumor tissues from a cohort of 32 TET patients (16 TCs and 16 thymomas). Results: Recurrent mutated genes in this TET cohort were TP53, CYLD, KMT2C, BRCA1, CHD2, PBRM1, ARID2, ASXL1, BRD7, CDKN2A, EGFR, KIT, MYH9, NFKB1A, RB1 and TET2. TP53 is the most frequently mutated gene, seen in 31% (5/16) of TCs and 6% (1/16) of thymomas. We found two actionable mutations in TCs (KIT D579del and IDH1 R132C) and one in thymomas (KIT V560D). Consistent with previous reports, we observed recurrent mutations in multiple genes encoding epigenetic regulator proteins (ASXL1, TET2 KMT2C, CHD2, PBRM1, ARID2 and BRD7), all in TCs but not thymomas. Most importantly, we found four CYLD mutations (two stop gain, one frameshift and one missense) and two NFKBIA mutations (one frameshift and one in splicing-site) in 6 TC samples. Both CYLD and NFKBIA genes encode negative regulators of NF-κB signaling pathway. CYLD is a Ub-specific protease (USP)-type deubiquitinase that preferentially disassembles Lys63-Ub, a post-translational modification required for the activation of NF-κB, while NFKBIA causes trapping REL dimers induced inhibitory activity of dimeric NF-κB/REL protein complexes. Furthermore, genomic alterations in NFKBIA and CYLD were mutually exclusive. Our results indicated that loss-of-function mutations in CYLD and NFKBIA may function as the molecular drivers of TC through constitutive activation of NF-κB signaling pathway. Conclusion: Our findings provide an enhanced road map for the etiology of TC. Further investigation is warranted into the roles of NF-κB pathway and epigenetic regulators dysfunction in TC pathogenesis and their potential for clinical trials. Citation Format: Naixin Liang, Xiaomo Li, Tonghui Ma, Chao Gao, Zhili Cao, Lei Cao, Zhongxing Bing, Zhenhuan Tian, Chao Guo, Shanqing Li, Yadong Wang, Jianchao Xue, Ji Li, Xiaoguang Li, Peng Liu, Shuyang Zhang. Genome profiling of thymic carcinoma identifies putative driver mutations in the NF-κB signaling pathway [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 2252.

Abstract PL03-02: (Re)emerging principles of cancer therapy

Abstract It is likely that some of the mutations that occur late during evolution, whether passenger mutations or passenger mutations, are likely only tolerated (let alone advantageous to the cancer cell) because of the mutations that preceded them. If so, targeting mutations that occur early during tumor evolution should selectively kill cancer cells by unmasking the otherwise deleterious effects of subsequent mutations. There are now many precision cancer drugs that target such early mutations, including EGFR inhibitors for lung cancer, c-Kit mutations for GI stromal tumors, and B-Raf inhibitors for B-Raf mutant melanomas. Inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL) is an early event in kidney cancer. Inactivated proteins can sometimes be tackled by exploiting epistatic relationships or synthetic lethality. For example, pVHL suppresses the HIF2 transcription factor, which in turn regulates VEGF. VEGF inhibitors are now mainstays of kidney cancer treatment. HIF2 was viewed as “undruggable”, but undruggable proteins can sometimes be targeted by allosteric modulators or degraders. A first-in-class allosteric HIF2 inhibitor has advanced to phase 3 trials based on promising early data. Most cancer drugs work better in the frontline setting, and a recent study of previously untreated von Hippel-Lindau disease patients strongly suggests this will be true for the HIF2 inhibitor. Combination therapy will be required to cure most cancer. It is tempting to combine drugs that have interdependent actions in an attempt to achieve synergy, but this increases the risk of cross-resistance. The classical way to prevent resistance is to combine drugs that have distinct mechanisms of action (to minimize toxicity and cross-resistance). In this regard, immunotherapy has emerged as a powerful modality to treat cancer cells, and complements therapies aimed at inducing cancer cell death. Both kidney cancer and melanomas are highly immunogenic and can respond to immune checkpoint blockade, yet kidney cancer has a much lower mutational burden than melanoma. Recent work suggests that kidney cancers promiscuously express endogenous retroviruses, which might serve as a source of neoantigens. If true, it might be possible to pharmacologically enhance ERV expression to augment immunotherapy. Citation Format: William George Kaelin. (Re)emerging principles of cancer therapy [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 PL03-02.