KIT Mutations Research Articles

Genomic and tumour microenvironmental biomarkers of immune checkpoint inhibitor response in advanced Taiwanese melanoma.

Genomic biomarkers predicting immune checkpoint inhibitor (ICI) treatment outcomes for Asian metastatic melanoma have been rarely reported. This study presents data on next-generation sequencing (NGS) and tumour microenvironment biomarkers in 33 cases. Thirty-three patients with advanced melanoma, who underwent ICI treatment at the Chang Gung Memorial Hospital in Taiwan, were recruited. The study evaluated clinical outcomes, including response rate, disease control rate, progression-free survival (PFS) rate and overall survival (OS) rate. Archived tissue samples from 33 cases were subjected to NGS by ACTOnco, and ACTTME was employed in 25 cases. The most prevalent driver mutations were BRAF mutations (24.2%), followed by NRAS (15.2%), KIT (12.1%), KRAS (9.1%) and NF1 (9.1%) mutations. Acral/mucosal melanomas exhibited distinct mutation patterns compared to non-acral melanomas. Tumour mutational burden estimated using ACTOnco was not associated with ICI efficacy. Notably, genetic alterations in the p53 pathway (CDKNA2 loss, MDM2 gain/amplification and TP53 mutation) accounted for 36.4% and were significantly associated with unfavourable PFS (median PFS 2.7 months vs. 3.9 months, P = 0.0394). Moreover, 26 genes were identified as differentially expressed genes that were upregulated in patients with clinical benefits compared to those without benefits. Four genes, GZMH, GZMK, AIM2 and CTLA4, were found to be associated with both PFS and OS. Genetic alterations in the p53 pathway may be critical in Asian patients with melanoma undergoing ICI treatment. Further investigation is required to explore this mechanism and validate these findings.

Molecular Dynamics Simulation of KIT Mutation in a Patient wıth Piebaldism, Congenital Cataract and Aphakic Glaucoma

Background: Piebaldism (OMIM #172800) is a rare autosomal dominant genodermatosis characterized by congenital poliosis and stable patches of leucoderma. Piebaldism is caused by mutations in the KIT and SNAI2 genes. The most common mutations are detected in the KIT gene. Case presentation: A 5-year-old boy, who was followed-up for aphakic glaucoma after congenital cataract surgery, was consulted to the medical genetics and dermatology departments due to premature graying of the hair, white forelock in the frontal region of the scalp, whitening in the inner part of the eyebrows and eyelashes and patchy leukoderma with hyperpigmented islands inside on the extremities and trunk. DNA sequencing revealed a heterozygous missense c.1861G>A mutation in the KIT gene. Mutation was evaluated using in silico 3D-structure analysis and bioinformatics tools Conclusion: KIT gene has a critical role in melanoblast migration, proliferation, differentiation and survival and molecular dynamics simulation and modeling proved that this variant inhibits the migration of melanoblasts and melanocytes by reducing the enzymatic activity of the KIT protein.

The clinical experience of compassionate use program for avapritinib: implications for drug positioning in the therapeutic scenario of systemic mastocytosis

In systemic mastocytosis, cytoreductive treatment is indicated for advanced systemic mastocytosis (AdvSM) variants. The treatment scenario is rapidly diversifying especially with the introduction of KIT tyrosine kinase inhibitors. Avapritinib is a second-generation potent and selective inhibitor of the mutant KIT D816V that, based on the results of pivotal clinical trials, was approved for the treatment of adults with AdvSM by the regulatory agencies US FDA and EMA. The present article reports the experience of treating SM patients with avapritinib in an Italian compassionate use program. The data from our case series confirm the drug as being active after multiple lines of treatment allowing rapid achievement of profound responses, making it also an effective bridging strategy to allogeneic transplant in eligible patients. However, the anticipated wider use of avapritinib in the near future will require careful monitoring of side effects, especially in heavily pretreated patients.

Genetic mutations in the molecular pathogenesis of gastrointestinal stromal tumor

Gastrointestinal stromal tumors are mesenchymal tumors which predominantly originate from the interstitial cells of Cajal in the intestinal lining. Around ~85% of malignant GISTs possess activating mutations in the tyrosine kinase receptors KIT or PDGFRA. The driver mutations in genes other than KIT or PDGFRA account for around 15% GISTs and belong to highly heterogeneous groups called wild-type GISTs. Around 20–40% of WT-GISTS are deficient for the succinate dehydrogenase complex (SDHA, SDHB, SDHC, SDHD). Mutations in SDH cause the accumulation of oncometabolite succinate that increases the level of HIF1α which enhances the transcription of several genes including IGF1, IGF2, and VEGF inducing the proliferation of the cancer cells. The remaining WT-GISTS are associated with mutations in BRAF, loss-of-function of NF1, hyperactivation FGFR, inactivation of tumor suppressor genes (p53 and dystrophin), and mutations in DNA damage response genes such as RAD51 and BRCA2 which results in the activation of the PI3K/mTOR or RAS/RAF/MAPK signaling cascade. Therefore, understanding the mutational status and molecular characterization of GIST plays a very crucial role in the overall management of GIST.

Prevalence of CKIT and PDGFRA Mutation in Gastrointestinal Stromal Tumors among Filipinos

Background: Gastrointestinal stromal tumors (GIST) is defined as specific, typically kit (CD117)-positive and CKIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation-driven mesenchymal tumors that can occur anywhere in the GI tract. GIST diagnosis relies heavily on immunohistomorphology. However, with the advent of molecular testing, the classification, diagnosis and targeted-therapy for gastrointestinal mesenchymal tumors have been greatly improved. In the Philippines, molecular testing is not yet readily available as in other countries. The local molecular profile of gastrointestinal stromal tumors is a point of investigation as treatment may be more tailored to the patients’ needs. Objective: This study aims to determine the prevalence of CKIT and PDGFRA mutations among formalin-fixed and paraffin embedded gastrointestinal stromal tumors and other gastrointestinal mesenchymal tumors in St. Luke’s Medical Center – Quezon City. Methods: A retrospective cross-sectional study of formalin fixed and paraffin embedded tumor samples diagnosed as Gastrointestinal Stromal Tumor from January 1, 2009 to December 31, 2017 will be analyzed for KIT and PDGFRA mutations. Result: The epidemiology of GIST remains constant in that mean age group is the 5th to 6th decade, with equal gender distribution, and stomach followed by small bowel are the most common sites. Mutational analysis of the GISTs showed predominantly KIT Exon 11 (47.83%) followed by CKIT Exon 9 (13.04%) and PDGFRA Exon 18 (10.87%). For KIT Exon 11, deletion is the most common mutations followed by point mutations. No mutation is detected in 47.83% of GISTs. Conclusion: Mutational analysis for CKIT-PDGFRA is warranted among GIST patients, as it may significantly influence treatment protocol in our patients.

Histiocytic Sarcoma Secondary to Gastrointestinal Stromal Tumors: A Literature Review.

Histiocytic sarcoma (HS) is a rare tumor that may result from the transdifferentiation of preexisting hematolymphoid neoplasms in a subset of patients. There are instances of correlation or concurrence between HS and a number of cancers, particularly B-cell-associated hematopoietic tumors. Only three cases of HS occurring subsequent to or concurrently with gastrointestinal stromal tumors (GIST) have been recorded. Our main objective was to give an overview of demographics, clinical signs and symptoms, histopathological findings, and immunohistochemical and molecular analysis when HS develops secondary to or concurrently with GIST. A search of PubMed, Google Scholar, and ScienceDirect was undertaken using Medical Subject Headings (MeSH) keywords. According to the findings of our review, there were two males (66.6%) and one female (33.3%). The average age of patients at presentation was 59.6 years. On the immunohistochemistry, three patients were positive for cluster of differentiation (CD) 68 (100%), two patients were positive for CD 163 (67%), one patient was positive for leukocyte common antigen (LCA) (33%), and only one patient was positive for CD 4, CD 10, CD 31, CD 45, human leukocyte antigen (HLA)-DR, lysozyme, and vimentin (33%). On molecular investigation, the gastric mass of only one patient (33.33%) contained a KIT mutation on exon 11. Emperipolesis was observed in one patient (33.33%) on histological examination. Our study provides an important overview of the available literature and gives insight into important diagnostic markers of HS when it occurs secondary to or concurrently with GIST.

GISTs with NTRK Gene Fusions: A Clinicopathological, Immunophenotypic, and Molecular Study.

The most common mutations in gastrointestinal stromal tumors (GISTs) are KIT or PDGFRA mutations. Recently, neurotrophic tyrosine receptor kinase (NTRK) fusions have been reported in WT GISTs, which increased interest in introducing tropomyosin receptor kinase (TRK) inhibitors as treatments for GISTs with NTRK fusions. Hence, we aimed to screen NTRK fusions in WT GISTs; we used fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and immunohistochemistry (IHC) to screen NTRK fusions in 46 WT GISTs and evaluate each method. We further reviewed NTRK fusion-positive GISTs from the literature and performed clinical and pathological analyses; two GISTs with an ETV6-NTRK3 fusion (5%) were identified, while only one (50%) was positive for Pan-TRK expression. On the other hand, among the six GISTs with Pan-TRK-positive expression, only one (17%) harbored NTRK fusion. The literature review revealed the strong consistency between FISH and NGS and the limited value of Pan-TRK IHC in screening NTRK fusions in GISTs. In addition, the clinical and pathological analysis showed that GISTs with NTRK rearrangement occurred less frequently in the stomach, were more frequently larger in size, and the epithelioid type presented with a higher risk of recurrence. The NTRK3 fusion has been more common than the NTRK1 fusion in GISTs to date; our study identified two ETV6-NTRK3 fusions in 46 WT GISTs. Compared with FISH and IHC, NGS is preferred for screening WT GISTs, including NTRK rearrangements. However, since GISTs with NTRK fusions are rare, further studies including more samples and mechanistic investigations should be conducted in the future.

Mast Cell Activation Syndromes: Comparison Between Two Scoring Models to Predict for Mast Cell Clonality

The Red Española de Mastocitosis (Spanish Network on Mastocytosis) score (REMAs) and the National Institutes of Health idiopathic clonal anaphylaxis score (NICAS) were developed for more efficient screening of mast cell (MC) clonality in MC activation syndromes. In a limited idiopathic anaphylaxis case series, the NICAS showed higher accuracy compared with the REMAs. To compare the performance of the REMAs against the NICAS in the diagnosis of MC clonality. We compared the diagnostic value of the REMAs against the NICAS in 182 patients (63% men, median age 56 years) who presented with anaphylaxis triggered by Hymenoptera venom allergy (45%), drugs (15%), food (11%), idiopathic anaphylaxis (20%), and mixed causes (10%). KIT mutation was assessed in parallel in whole blood and bone marrow (BM) and, when negative, in highly purified BM MC. TPSAB1 was genotyped in a subset of 71 patients. We found higher accuracy and rates of correctly classified patients for the REMAs (82% and 84%) compared with the NICAS (75% and 75%; P= .02 and P= .03, respectively), particularly among men (P= .05), patients with systemic mastocytosis (P= .05), those presenting anaphylaxis owing to any cause featuring urticaria (P= .04), cardiovascular symptoms (P= .02), and/or presyncope (P= .02) and those with a blood-negative/BM-positive KIT mutational profile (P= .002), but not hereditary α-tryptasemia-associated genotypes. Combined assessment of the REMAs and KITD816V in blood yielded an overall improved classification efficiency of 86% versus 84% for REMAs. The combined use of the REMAs and blood detection of KITD816V is recommended, but more sensitive blood-based molecular assays to detect KITD816V are needed.

Highlights from Recent Cancer Literature.

Highlights from Recent Cancer Literature.

Small Gastric Stromal Tumors: An Underestimated Risk

Small gastrointestinal stromal tumors (GISTs) are defined as tumors less than 2 cm in diameter, which are often found incidentally during gastroscopy. There is controversy regarding the management of small GISTs, and a certain percentage of small GISTs become malignant during follow-up. Previous studies which used Sanger targeted sequencing have shown that the mutation rate of small GISTs is significantly lower than that of large tumors. The aim of this study was to investigate the overall mutational profile of small GISTs, including those of wild-type tumors, using whole-exome sequencing (WES) and Sanger sequencing. Thirty-six paired small GIST specimens, which were resected by endoscopy, were analyzed by WES. Somatic mutations identified by WES were confirmed by Sanger sequencing. Sanger sequencing was performed in an additional 38 small gastric stromal tumor samples for examining hotspot mutations in KIT, PDGFRA, and BRAF. Somatic C-KIT/PDGFRA mutations accounted for 81% of the mutations, including three novel mutation sites in C-KIT at exon 11, across the entire small gastric stromal tumor cohort (n = 74). In addition, 15% of small GISTs harbored previously undescribed BRAF-V600E hotspot mutations. No significant correlation was observed among the genotype, pathological features, and clinical classification. Our data revealed a high overall mutation rate (~96%) in small GISTs, indicating that genetic alterations are common events in early GIST generation. We also identified a high frequency of oncogenic BRAF-V600E mutations (15%) in small GISTs, which has not been previously reported.

Primary mucosal melanomas of the head and neck are characterised by overexpression of the DNA mutating enzyme APOBEC3B.

Primary head/neck mucosal melanomas (MMs) are rare and exhibit aggressive biologic behaviour and elevated mutational loads. The molecular mechanisms responsible for high genomic instability observed in head/neck MMs remain elusive. The DNA cytosine deaminase APOBEC3B (A3B) constitutes a major endogenous source of mutation in human cancer. A3B-related mutations are identified through C-to-T/-G base substitutions in 5'-TCA/T motifs. Herein, we present immunohistochemical and genomic data supportive of a role for A3B in head/neck MMs. A3B protein levels were assessed in oral (n =13) and sinonasal (n =13) melanomas, and oral melanocytic nevi (n =13) by immunohistochemistry using a custom rabbit α-A3B mAb (5210-87-13). Heterogeneous, selective-to-diffuse, nuclear only, A3B immunopositivity was observed in 12 of 13 (92.3%) oral melanomas (H-score range=9-72, median=40) and 8 of 13 (62%) sinonasal melanomas (H-score range=1-110, median=24). Two cases negative for A3B showed prominent cytoplasmic staining consistent with A3G. A3B protein levels were significantly higher in oral and sinonasal MMs than intraoral melanocytic nevi (P < 0.0001 and P=0.0022, respectively), which were A3B-negative (H-score range=1-8, median=4). A3B levels, however, did not differ significantly between oral and sinonasal tumours (P > 0.99). NGS performed in 10 sinonasal MMs revealed missense NRAS mutations in 50% of the studied cases and one each KIT and HRAS mutations. Publicly available whole-genome sequencing (WGS) data disclosed that the number of C-to-T mutations and APOBEC3 enrichment score were markedly elevated in head/neck MMs (n=2). The above data strongly indicate a possible role for the mutagenic enzyme A3B in head/neck melanomagenesis, but not benign melanocytic neoplasms.

Molecular and immunohistochemical comparison between primary gastrointestinal mucosal melanomas and atypical gastrointestinal stromal tumors.

It is sometimes challenging to differentiate between gastrointestinal (GI) mucosal melanoma and gastrointestinal stromal tumors (GISTs) because they share some similarities in histological and molecular features. In this study, we investigated their clinicopathological and molecular features. Four primary GI mucosal melanomas and 4 atypical GISTs resected from 2017 to 2019 were included. Electronic medical records and histology slides were reviewed and, if needed, immunohistochemical stainings were additionally done. Somatic mutations were analyzed using whole exome sequencing (WES) with tumors and matched normal tissues. By immunohistochemistry, GISTs were positive for CD117 (4 of 4), DOG1 (4 of 4), and CD34 (2 of 4). In contrast, melanomas were positive for CD117 (2 of 4), HMB-45 (4 of 4), Melan-A (4 of 4), and S100 protein (3 of 4). Using WES, the KIT mutation was detected in 2 of 4 GISTs and 1 of 4 melanomas, all of which showed strong CD117 immunoreactivity. PDGFRA mutation was detected in 2 of 4 GISTs but 0 of 4 melanomas. TP53, NF1, RB1, TERT, and KMT2D mutations were detected in 1 of 4 melanomas but 0 of 4 GISTs. A patient with KIT-mutated melanoma was treated with Gleevec and has remained disease-free for 1 year. GISTs and GI melanomas have a wide histopathological spectrum. Appropriate ancillary studies can be helpful for proper diagnosis and optimal treatment.

New C3H KitN824K/WT cancer mouse model develops late-onset malignant mammary tumors with high penetrance

Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

Evolutionary Portrait of Adult Core-Binding Factor Leukemia Patients Treated with a Continuation Therapy with Midostaurin: Preliminary Results

Genetic and Clinical Background The clinical outcome of Core Binding Factor Leukemia (CBFL) has been reported to be influenced by KIT mutations, that was frequent in CBFL, and by co-occurring different spectra of cooperating mutations. In addition, gene expression studies found KIT to be highly expressed in CBFL regardless of its mutational status. In vitro experiments revealed that Midostaurin (Mido) is effective in inhibiting both wild type (WT) and a range of KIT mutants. In vivo studies demonstrate the efficacy in KIT-positive malignancies such as Aggressive Systemic Mastocytosis, Mast Cell Leukemia, and SM with Associated Hematological Neoplasm. Therefore, we designed a Phase II trial to evaluate the safety and efficacy of Mido, in association with Intensive Chemotherapy (IC), in CBFL regardless of KIT mutational status. (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345). 34 adult patients with t(8;21) (n=16) and inv(16) (n=18) were enrolled between December 2018 to July 2022. Method Patients with de-novo CBFL received standard induction therapy with an anthracycline containing regimen ("7+3"-like) + Mido, three cycles of post-remission consolidation chemotherapy with high-dose cytarabine + Mido, and 12 months of Mido as Maintenance. At diagnosis, 32 out of 34 patients were studied by a comprehensive NGS panel targeting 40 DNA genes and 29 RNA fusion driver genes associated with major myeloid disorders (Oncomine Myeloid Research Assay). Aim To study the clonal evolution of CBFLs, NGS and MRD status (performed by qPCR and high-resolution multicolor flow cytometry) were assessed at established check-points during consolidation, maintenance therapy, and at relapse. ResultsKIT was the most frequently mutated gene (28%) and there was an enrichment of FLT3 and NRAS (Figure 1). Additionally, the spectrum of recurrent mutations was different, as codon 816 KIT mutations and FLT3-ITD were common only in RUNX1::RUNX1T1 cases. Overall, the CR rate was 97%. At a median follow-up of 18 months (range 2-43 months), we recorded a RI of 37.5 %, an OS of 85 %, and a DFS of 60%. We performed targeted next generation sequencing for six out of 12 relapsed samples in the cohort t(8;21) (n=5) and inv(16) (n=1), showing loss, retention or gain of somatic mutations (Figure 2). Three out of six relapsed patients showed at diagnosis a CEBPA mutation (Gly104del) co-occurring with KIT in RUNX1::RUNX1T1 cases and with NRAS in CBFB::MYH11. Conclusion In patients with CBFL, we found FLT3 and/or KIT mutations actionable by a regimen consisting of intensive chemotherapy and Mido in 47% of cases. In two out of three relapsed cases the KIT mutation is maintained, although frequency of the mutant allele is lower at relapse, while all FLT3 and NRAS mutated patients lose the mutation upon disease relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Subclonal KIT D816V Mutations Are Prevalent in Chronic Myelomonocytic Leukemia and Correlate with Distinct Phenotypic Features

Background: Activating mutations in KIT are infrequently observed in myeloid neoplasms, but are a pathognomonic finding in systemic mastocytosis (SM) where the hotspot D816V variant is documented in 90-95% of patients. In SM, KIT variants may occur at a low variant allele frequency (VAF) below the limits of detection of most commercial next generation sequencing (NGS) assays necessitating digital droplet PCR (ddPCR) for accurate detection. Aggressive SM often presents concurrently with an associated hematologic neoplasm (SM-AHN). Chronic myelomonocytic leukemia (CMML) is the most frequently co-occurring hematologic malignancy, but KIT mutations are infrequently detected by conventional NGS. The limitations in detecting KIT variants by NGS and increased frequency of CMML in SM-AHN suggest that subclonal KIT mutations may be underrecognized in CMML. Further, identification may have prognostic and therapeutic implications given the emergence of KIT-targeted therapies in SM. Methods: A multicenter, retrospective study was performed to assess the prevalence of subclonal KIT D816V mutations in CMML patients and examine clinical implications. Eligible patients had pathologically confirmed CMML by WHO 2017 criteria, with available bone marrow (n=59) or peripheral blood (n=14) sample obtained prior to therapy. Samples underwent DNA sequencing using a 285-gene NGS panel (QIAseq), with a VAF threshold of 5% utilized for variant calling. KIT D816V mutations were assessed by ddPCR, with limit of detection of 0.03%. Clinical data was collected from baseline and follow up for phenotypic and prognostic comparisons. Fishers exact and Chi-square tests were used to compare categorical variables and the Mann-Whitney test for continuous variables. The Kaplan-Meier method was used to estimate overall survival (OS), with the log rank test used to compare survival between groups. The study was approved by institutional review boards at each institution. Results: A total of 73 patients were included with a median age of 71, and 66% were male. By WHO criteria, 50% were classified as CMML-0, 28% CMML-1, and 22% CMML-2; 59% of patients had proliferative CMML by FAB criteria. KIT D816V mutations were detected in 15 patients (21%) by ddPCR, compared to two (3%) by NGS. The median VAF of mutations detected by ddPCR was 0.06%, whereas the two variants detected by NGS had VAFs of 44.1% and 47.0%. Baseline characteristics of KIT wild-type (WT) and mutated (MUT) patients are detailed in Table 1. KIT MUT patients had lower white blood cell (p=0.007) and absolute monocyte counts (p=0.04), less frequent splenomegaly (p=0.05), more frequent karyotype abnormalities (p=0.14), and lower frequency of proliferative disease by FAB criteria (p=0.01). There was a median of 4 variants (range 0-7) detected per patient by NGS, and the most frequently mutated genes were TET2 (59%), SRSF2 (44%), ASXL1 (33%),RUNX1 (25%), and NRAS (25%). KIT MUT patients had a median of 3 total variants (range 0-6) detected by NGS compared to 4 (range 0-7) in KIT WT (p=0.63). There was a trend toward higher frequency of SF3B1 (23% vs. 5%, p=0.07) mutations in KIT MUT patients. Conversely, no mutations were observed in KRAS or CBL in KIT MUT patients, compared to 14% and 16% in KIT WT, respectively (p=0.34, p=0.20). When evaluating mutations in signaling pathways, there was a trend toward lower frequency in KIT MUT patients compared to WT (31% vs. 59%, p=0.12). With a median follow up of 48 months, median OS of the cohort was 30 months. KIT MUT patients demonstrated an OS of 27.5 months compared to 30 months in WT patients (HR 1.06, p=0.87). Transformation to AML was observed in 42% of KIT MUT patients compared to 29% of WT patients (p=0.49). A similar proportion of patients were treated with hypomethylating agents (HMAs), with a response rate of 14% in KIT MUT patients compared to 32% in WT (p=0.63), and complete remission rates of 0% vs. 27%, respectively. Conclusions: This study is the first to document the prevalence of subclonal KIT D816V mutations by ddPCR in CMML, revealing a high prevalence in comparison to detection by standard NGS assays. While sample size limited statistical comparisons, KIT mutations were associated with distinct phenotypic features, including less proliferative disease and poor response to HMAs. Given the efficacy of KIT inhibitors in SM-CMML, these findings could represent an avenue for targeted therapy in CMML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of Survival Outcomes and Clinical and Molecular Modulators in Adult Patients with Core-Binding Factor Acute Myeloid Leukemia (CBF-AML) Treated with Hidac Consolidation: An Alliance Legacy Study

Introduction CBF-AML is a specific disease entity defined by the presence of either t(8;21)(q22q22)RUNX1-RUNX1T1, or inv(16)(p13.1q22) or t(16;16)(p13.1;q22) creating the CBFB-MYH11 fusion gene, and is associated with a relatively favorable prognosis. Yet, outcomes are variable and relapse occurs in ~50% of patients (pts). Whereas several clinical and molecular features have been suggested to modulate survival, consensus data are sparse. Moreover, there is persistent controversy about the optimal dose intensity of cytarabine consolidation, which complicates interpretation of correlative studies with respect to outcome associations. Thus, we analyzed outcomes of a large cohort of CBF-AML pts treated with high-dose cytarabine (HiDAC) consolidation with respect to cytogenetic subtypes and co-existing molecular features. We analyzed clinical and molecular characteristics and outcomes of 304 pts with CBF-AML aged 18-59 years [y; inv(16)/t(16;16), n=186; t(8;21), n=118] who were similarly treated and achieved a complete remission upon standard cytarabine/anthracycline induction followed by HiDAC on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols between 1986-2016. All pts had centrally reviewed cytogenetics and samples from 136 pts underwent comprehensive molecular profiling of 80 AML-associated genes for association studies. Kaplan-Meier method was used to estimate probabilities of disease-free survival (DFS) and overall survival (OS), and the log-rank test evaluated differences between survival distributions. Cox proportional hazard regression analyses was used for DFS and OS univariable and multivariable modeling. Results Thirty-eight percent of pts relapsed and 69% were alive 5y after diagnosis (median OS): 12.5y with median follow-up of 8.6y. Outcomes of t(8;21) and inv(16)/t(16;16) AML were similar; relapse rates: 35% vs 40%, 5y OS rates, 64% vs 72%, respectively. Although outcome of pts with inv(16) was not significantly different from outcome of t(16;16) pts, the latter had, by trend, a superior DFS (5y rates, 75% vs 47%, P=0.07); their OS was not significantly different (5y rates, 81% vs 64%, P=0.24). As secondary cytogenetic abnormalities are common and impact on survival has yielded controversial results, we next analyzed the impact on outcomes of our pt cohort. None of the frequently observed secondary abnormalities [i.e., loss of X or Y chromosomes, +8, or del(9q) in t(8;21) pts and +22 or +8 in inv(16)/t(16;16) pts] affected the outcomes of CBF-AML pts we analyzed, which contrasts with some previous reports. The presence of a complex karyotype also did not negatively affect the favorable survival of CBF-AML pts. Next, we assessed the impact of recurrent gene mutations on pt outcomes. While KIT mutations have previously been shown to confer inferior survival, our data suggest that presence of a KIT mutation negatively affects DFS of both t(8;21) and inv(16)/t(16;16) AML pts [5y rates, t(8;21) 36% vs 67%, P= 0.03; inv(16)/t(16;16), 21% vs 61%, P<0.001], and also OS in inv(16)/t(16;16) pts (5y rates, 52% vs 74%, P=0.04). Co-existing FLT3-TKD mutations were almost exclusively found in inv(16)/t(16;16) in 16/18 cases. Though a negative prognostic impact of FLT3-TKD has been suggested, this was not found in our cohort (5y rates, DFS, 56% vs 55%, P=0.93; OS, 78% vs 68%, P=0.69). Furthermore, neither the presence of NRAS or KRAS mutations affected survival outcomes. In multivariable analyses for OS, a higher percentage of peripheral blood blasts was the only feature associated with inferior survival [HR, 1.14 (95% CI, 1.04-1.25), P=0.004]. Conclusions Our analysis of a large cohort of younger CBF-AML pts treated on frontline protocols with 7+3-based induction therapy and HiDAC consolidation, showed though these pts are considered favorable risk, they demonstrate varying long-term survival. Clinically, the presence of high blast count at time of diagnosis, was a strong adverse prognosticator for inferior survival. With respect to recurrent co-occurring cytogenetic aberrations or gene mutations, only KIT mutations associated with inferior survival in pts with t(8;21) and in those with inv(16)/t(16;16). Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org. Clinicaltrials.gov: NCT00048958(CALGB 8461), NCT00899223(CALGB 9665), NCT00900224(CALGB 20202) *co-senior authors

Drivers of Deep Molecular Response and Outcomes in Patients with Core Binding Factor Acute Myeloid Leukemia

Introduction: Core-binding factor acute myeloid leukemia (CBF-AML), including t (8;21) and Inv (16), constitutes 15% of all AML cases and generally carries a favorable prognosis. However, relapse rate is ≈ 30% and predictors are not well understood. Although CBF-AML responds to induction chemotherapy, post-remission treatment can be further optimized, particularly with current next generation sequencing (NGS) and measurable residual disease (MRD) detection techniques. Impact of baseline somatic mutations on outcomes remains unclear, as does role of allogeneic stem cell transplant (allo-SCT). In this study, we investigated the potential drivers of treatment response and overall outcomes in CBF-AML. Methods: In this single center retrospective study, clinical, demographic, and molecular data were collected from adult pts with CBF-AML who were treated at Moffitt Cancer Center. We included pts with MRD by polymerase chain reaction (PCR) for CBF abnormality (CBF-MRD) results available from time of induction and consolidation. All statistical analyses were performed in SPSS. Results: Our study included 68 patients (pts), including 26 (38%) t (8;21) AML. The median age at diagnosis was 48 years, and 54% pts were male. Pts with inv (16) AML had significantly more leukocytosis than t (8;21) (10x109/L vs. 23x109/L; p=0.001). Only 5% of pts (n=3) in our cohort had extramedullary disease on presentation. Common mutations identified on baseline NGS (n=60) were KIT (35%), NRAS (27%), KRAS (15%), FLT3-ITD and FLT3-TKD (12% each; 2 pts had both types). Mutation in KIT was more common in t (8;21) AML (50% vs. 25%; p=0.04), whereas inv (16) cases were enriched in NRAS mutations (36% vs. 13%; p=0.04). Overall, 80% of CBF-AML pts had at least one kinase mutation (KIT/RAS/FLT3). Majority of pts were treated with "7+3" (44%) and "7+3+ gemtuzumab ozagamicin (GO)" (40%) as induction regimens (Table 1). Daunorubicin was the predominant anthracycline, used in 80% of pts treated with "7+3" and 48% received dose of 90 mg/m2. Overall, 31 (46%) pts received GO as part of induction treatment, with equal distribution of "day 1 only" and "day 1,4,7" schedules at 3mg/m2. Median number of consolidation cycles was 3, and GO was incorporated in consolidation (commonly with high dose cytarabine) for 29 (43%) pts. After induction, 16% pts experienced CBF-MRD negativity which increased to 73% following consolidation, with no significant difference between t (8;21) and inv (16) AML. Use of GO during induction or consolidation was associated with significantly higher CBF-MRD negativity rate at completion of induction and consolidation (83% vs. 50%; p= 0.021). However, we did not find any significant correlation between groups based on presence of baseline kinase mutation/s and CBF-MRD negativity at end of induction or consolidation. In FLT3-positive cases, 100% of pts (5/5 evaluated;4 ITD and 1 TKD) achieved FLT3-MRD negativity by PCR (sensitivity of 5X10-5) following consolidation. Overall, 35% of pts (n=24) underwent allo-SCT in our cohort, including 15 in first remission (CR1). Kinase mutations were present at baseline in majority of these pts (75%; 87% in CR1 group). Notably, 9 pts (7 in CR1 group) in the allo-SCT cohort had positive CBF-MRD at the end of consolidation. Seven pre-transplant CBF-MRD positive cases turned MRD negative following allo-SCT. Relapse rate was low (n=10; 15%) in our CBF-AML pt population. Among pts with relapse, 6 were CBF-MRD positive post-induction and 3 had MRD even after consolidation. Use of GO in induction was associated with significantly lower relapse rate (0% vs. 28%; p= 0.001). Presence of kinase (KIT/RAS/FLT3) mutation at diagnosis was associated with shorter relapse free survival (RFS) in these pts (median RFS- 7.7 vs. 12.9 months (mo); p= 0.014; Figure 1). After a median follow up of 33.4 mo, median OS was 94.1 mo in our pt cohort. There was no significant OS difference between subgroups based on CBF-AML subtype, baseline mutations, or receipt of GO during treatment. Conclusions: CBF-AML continues to be a favorable risk disease, with a median OS >7 years in our study population. Post-treatment CBF-MRD negativity rate was 73% and was predicted by the incorporation of GO in induction or consolidation. Rate of relapse rate was low at 15%, and all occurred in pts who did not receive GO during induction or consolidation. Baseline kinase mutations (KIT/RAS/FLT3) on NGS was associated with a shorter RFS in our cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RAS Pathway Alterations in Pediatric AML Patients

RAS pathway alterations are highly prevalent in hematologic malignancies1,2, often involving gain of function KRAS, NRAS or HRAS mutations and have been linked to tumorigenesis and associated with poorer prognosis in different cancer entities3-5. In adult myeloid leukemia, RAS mutations are one of the most common mutations, yet show no clear prognostic value6. In a study of the Japanese pediatric AML cohort, NRAS mutations were associated with a favorable outcome, the effect of KRAS mutations could not be analyzed due to the limited patient size7. Here we report the outcome of 372 pediatric AML patients of the German AML-BFM study between 2016 and 2021 that were characterized by morphology, multicolor flow cytometry, classical cytogenetics and mutation analysis via panel sequencing. In total, 27% (n=102) of the patients showed a RAS pathway alteration, involving n=32 patients (9%) with a KRAS mutation and n=75 (20%) with a NRAS mutation. Notably, no HRAS mutation was detected in our cohort. Age distribution did not differ between the groups (Fig. A). Interestingly, the KRAS cohort consisted mostly of male patients (72%, p=0,0087), while the overall cohort was balanced (48,7% male patients). NRAS and KRAS mutations were strong predictors of therapy response and early death. Only 1/75 (1,3%) of all patients harboring a NRAS mutations showed poor response to induction therapy compared to patients without a NRAS mutation (10,10%, p=0,01). KRAS mutations on the opposite were strongly associated with non-response (21,9%; p<0,01) and subsequent early deaths (n=6/32; p=0,001) compared to patients without KRAS mutations (3,2%). Of note, only 1/75 (1,3%) of patients with NRAS mutation and 17/372 (4,6%) of all AML patients suffered early deaths. Risk stratification taking into account re-stratification after poor therapy response showed a significant higher proportion of high-risk patients in the KRAS-mutated cohort (43,8 %) compared to 20% in the patients with NRAS mutations (p=0,0022). KRAS mutations correlated with KMT2A-MLLT1 (15,6%; p=0,01) translocations, which were significantly rarer in patients without KRAS mutations (3,5%). NRAS mutations occurred frequently with CBFA2T3-RUNX1 (14,7%; p=0,0002) and CBFB-MYH11 translocations (12%; p=0,0068%) as well as KIT mutations (12%; p=0,026). Existence of RAS mutations showed overall no significant impact on event-free survival (EFS=69,6% ±4,8%) and overall survival (OS=82,8% ±4,2%) compared to RAS wildtype (EFS=66,4% ±3,1% ; OS=76,5% ±2,9%) (Fig. B). Multivariate analysis comparing outcome for several subgroups also showed no significant differences. In summary, we show worse therapy response and increased early death rates in patients with KRAS mutations, while NRAS mutations are associated with better therapy response. These findings warrant further analyses and longer follow-up on the cohort to determine long-term significances of RAS pathway alterations in pediatric AML patients. 1.de Luca, A., Maiello, M. R., D'Alessio, A., Pergameno, M. & Normanno, N. The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets16 Suppl 2, (2012). 2.Hobbs, G. A., Der, C. J. & Rossman, K. L. RAS isoforms and mutations in cancer at a glance. Journal of Cell Science129, 1287-1292 (2016). 3.Hsu, H. C. et al. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients. Oncotarget7, 22257 (2016). 4.Vauthey, J., Zimmitti, G., Kopetz, S., ... J. S.-A. of & 2013, undefined. RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases. ncbi.nlm.nih.gov. 5.Dinu, D. et al. Prognostic significance of KRAS gene mutations in colorectal cancer - preliminary study. Journal of Medicine and Life7, 581 (2014). 6.Neubauer, A. et al. Patients With Acute Myeloid Leukemia and RAS Mutations Benefit Most From Postremission High-Dose Cytarabine: A Cancer and Leukemia Group B Study. Journal of Clinical Oncology26, 4603 (2008). 7.Kaburagi, T. et al. Clinical significance of RAS pathway alterations in pediatric acute myeloid leukemia. Haematologica107, 583-592 (2022). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FLT3-ITD Degrades CEBPA Via the Ubiquitin-Proteosome Pathway

FLT3 internal-tandem-duplication (FLT3-ITD) mutations are highly recurrent in acute myeloid leukemias (AML). Although secondary messenger impacts of these receptor tyrosine kinase mutations are well characterized, downstream mechanisms by which myeloid precursors are transformed remain unclear. One clue is that FLT3-ITD and CEBPA bi-allelic mutations are mutually exclusive, suggesting that FLT3-ITD might impact CEBPA, a master transcription factor mandatory for granulo-monocytic lineage-differentiation. In fact, FLT3-signaling has been shown to phosphorylate CEBPA at serine 21 (S21-P), and inhibition of this post-translational modification resumed granulocytic-differentiation of FLT3-ITD AML cells (Radomska et al, J Exp Med 2006). How S21-P post-translational modification of CEBPA blocks granulocyte lineage-differentiation, however, was unknown. To examine FLT3-ITD impacts on CEBPA function in primary AML cells, we examined expression of CEBPA target genes (24 genes identified by CEBPA ChIP-seq, Jakobsen et al, Genome Res 2013): CEBPA target genes positively correlated with CEBPA mRNA expression in normal granulopoiesis and in primary FLT3/CEBPA-wildtype (WT) AML cells (n=112) (Pearson correlation coefficient [ρ] 0.36, p<0.0001), but not in primary FLT3-ITD AML cells (n=35)(ρ =-0.15, p=0.4). Moreover, CEBPA target genes were almost 2-fold less expressed in the FLT3-ITD AML cells (p<0.0009), despite CEBPA mRNA expression comparable to the WT AML cells (RNA-sequencing, TCGA). To investigate the disconnect between CEBPA mRNA expression and CEBPA target gene activation, we analyzed CEBPA protein, and found very low amounts in FLT3-ITD AML cells MOLM13 and MV411 compared to large CEBPA protein amounts detected in WT THP1 AML cells, both by Western blot and immuno-fluorescence, again despite similar CEBPA mRNA expression. This observation complemented published data from others that FLT3-ITD introduction into murine 32D cells suppressed total Cebpa protein (Mizuki et al, Blood 2003; Zheng et al, Blood 2004). The FLT3-inhibitors sunitinib and gilteritinib decreased CEBPA S21-P, increased CEBPA total protein measured by Western blot and immunofluorescence, and induced terminal granulocytic differentiation in the FLT3-ITD MOLM13 and MV4-11 AML cells, but not FLT3-WT THP1 or OCI-AML3 cells. Moreover, the proteosome inhibitors bortezomib or MG132 increased S21-P-, ubiquitinated-, and total-CEBPA protein in the FLT3-ITD but not the FLT3-WT AML cells. We therefore further investigated interactions of endogenous CEBPA with the ubiquitin-proteosome system (UPS): unbiased analyses by immunoprecipitation (IP)-LCMS/MS demonstrated 3-4 fold enrichment of the deubiquitinase USP7 and E3 ubiquitin ligase UHRF1 in the CEBPA interactome of FLT3-ITD versus FLT3-WT AML cells. Reverse analyses of the endogenous USP7 protein interactome by IP-LCMS/MS and IP-Western blot confirmed USP7 interactions with CEBPA and UHRF1 and demonstrated USP7 phosphorylation at serine-18 (S18-P), a candidate for regulation by FLT3. The small molecule P22077 alters and inhibits USP7 catalytic site conformation: P22077 shifted USP7 deubiquitinase interactions from UHRF1 to CEBPA, decreased UHRF1 and increased total CEBPA (Figure). FLT3-ITD AML cells were >2-fold more sensitive to P22077 than FLT3-wt AML cells (IC50 5.8 and 7.1 µM for MV411 and MOLM13 versus 13.65 and 21.7 µM for THP1 and K562 cells) (Figure). Another mystery in FLT3-ITD AMLs is relative chemo-resistance even though p53 is wild-type - USP7 regulates p53, and thus FLT3-ITD regulation of USP7 may contribute to chemo-resistance, a link we are currently consolidating. In sum, FLT3-ITD signals for CEBPA protein degradation via USP7 and UHRF1, to thereby decouple proliferation from onward lineage-maturation in committed granulo-monocytic progenitors. Overall, this pathway explains FLT3-ITD: (i) origin in committed granulo-monocytic progenitors; (ii) link to suppressed CEBPA-target genes; (iii) mutual exclusivity with bi-allelic CEBPA mutations; (iv) differentiation-syndrome upon treatment with FLT3-inhibitors; (v) sensitivity to UPS inhibitors; (vi) mutual exclusivity with TP53-mutations yet chemo-resistance, and can inform understanding of how other receptor tyrosine kinase mutations, e.g., KIT mutations, transform committed lineage-progenitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Response and Prolonged Treatment-Free Remission after a Short-Course of Modified Intensive Chemotherapy and Venetoclax in Elderly AML: An Updated Analysis of the Caveat Trial

Background Venetoclax (VEN) plus azacitidine has an established role in the management of older patients (pts) with AML (DiNardo, NEJM 2020). To explore the feasibility of intensive chemotherapy combined with VEN in older pts with newly diagnosed AML, we conducted the CAVEAT trial (Chua et al, JCO 2020). We now present an updated analysis of this trial (with >3 years median follow-up), reporting on the optimal dose of VEN with chemotherapy, with and without posaconazole (POSA), the deliverability of consolidation therapy, characteristics of treatment failure and observation of substantial treatment-free remission in subsets of pts using this time-limited, short-course regimen. Methods The CAVEAT study enrolled pts aged ≥65 years with de novo or secondary/therapy-related AML (sAML) considered suitable for intensive chemotherapy. Five VEN dose escalation cohorts (50-100-200-400-600 mg) were explored initially. Two additional cohorts examined the safety of adjusted dosing of VEN (50 or 100 mg) during anti-fungal prophylaxis with the strong CYP3A4 inhibitor POSA. For induction, VEN was administered over 14 days, commencing with a 7-day pre-chemo dose ramp-up, followed by a 7-day overlap with 5+2 chemotherapy (cytarabine 100mg/m2/d IVI d1-5 and idarubicin 12mg/m2 IV d2-3). Consolidation (CONS) with 2+1 chemotherapy (below), followed by up to 7 cycles of VEN monotherapy maintenance was planned. Results Data cut-off: 31MAR2022. A total of 69 pts are included in this analysis (first pt 17JUL2016, last pt 15MAY2020). Median age was 71 years (range 63-80). Poor risk features included sAML (42%), adverse karyotype (28%) and prior HMA (23%). From the dose escalation phase, the VEN 600 mg cohort was well tolerated (30-day mortality 6%). Induction with VEN-POSA (n=18) was also well tolerated (30-day mortality 0%) with 3 DLTs observed: 2 hematologic (1 in each cohort) and 1 due to pulmonary infiltrates occurring on d2 induction (suspected to be allopurinol hypersensitivity). No clinical TLS was observed. Although hematologic recovery was satisfactory after VEN-POSA induction, recovery was considerably longer in the VEN (100mg)-POSA cohort after CONS1 compared to VEN (50mg)-POSA for both neutrophils (median 64 vs 44d to 0.5 x109/L) and platelets (median not reached vs 26d to 50 x109/L). Therefore, if POSA is used concurrently, VEN 50 mg is recommended. Alternatively, if VEN 600 mg is used, POSA should commence only after completion of VEN. In the VEN 600 mg cohort, significant delays in hematological recovery were also observed after multiple rounds of attenuated CONS (VEN d1-14, bolus doses of cytarabine 100 mg/m2 d1-2, idarubicin 12 mg/m2 on d1), limiting deliverability (median CONS cycles 1; range 1-3). By intention-to-treat, the overall response (CR/CRi) rate was 73%. CR/CRi was 90% in de novo AML (vs 51% in sAML), 84% in intermediate karyotype (vs 69% in adverse), 76% and 83% in NPM1 and IDH2 mutant AML, respectively. With a median follow-up of 37 months (m), the median overall survival (OS) was 15.4m. Median OS was 31.3m in de novo AML (40% alive at 36m) and 30.3m in intermediate karyotype AML (38% alive at 36m). In the VEN 600 mg cohort, median OS was 24.1m (36% alive at 36m). As CAVEAT delivered just one intensive cycle of therapy, followed by limited post remission therapy (median time on therapy 7.7m), we next assessed the duration of treatment-free remission (TFR) among responders. Among pts achieving CR/CRi, 20/51 (39%) experienced TFR (Fig 1). The median TFR was 22.1m (range 4.6-48.3m). At data cut-off, 12 (24%) have ongoing TFR. Notably, 15/20 (75%) with TFR had an NPM1 or IDH2 mutation at diagnosis. 11 out of 14 evaluable pts with NPM1 mutation had mutation clearance by RT-qPCR, including 3 with NPM1 negative disease at time of failure (1 refractory, 2 relapse). Two pts with persistently mutated NPM1 at relapse had a concomitant FLT3-ITD or KIT mutation. The mutation landscape at relapse is shown (Fig 1) Conclusion VEN combined with modified intensive chemotherapy achieved high response rates in older pts with newly diagnosed AML. With long-term follow-up, the time-limited CAVEAT schedule enabled pts, especially with mutated NPM1 or IDH2, to experience a durable treatment-free remission. Cumulative hematological toxicity is limiting, prompting us to now explore non-anthracycline CONS (low dose cytarabine 20mg/m2 SC d1-10 + VEN 400mg d1-7, for up to 4 cycles), followed by VEN monotherapy (400mg D1-14 q28d cycle x7). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal