KIT Variants Research Articles

6. Hot-spot D816 KIT has different clinical outcome compared to non-D816 KIT variants in myeloid neoplasms

6. Hot-spot D816 KIT has different clinical outcome compared to non-D816 KIT variants in myeloid neoplasms

Genomic analysis reveals the association of KIT and MITF variants with the white spotting in swamp buffaloes

BackgroundSwamp-type buffaloes with varying degrees of white spotting are found exclusively in Tana Toraja, South Sulawesi, Indonesia, where spotted buffalo bulls are highly valued in accordance with the Torajan customs. The white spotting depigmentation is caused by the absence of melanocytes. However, the genetic variants that cause this phenotype have not been fully characterized. The objective of this study was to identify the genomic regions and variants responsible for this unique coat-color pattern.ResultsGenome-wide association study (GWAS) and selection signature analysis identified MITF as a key gene based on the whole-genome sequencing data of 28 solid and 39 spotted buffaloes, while KIT was also found to be involved in the development of this phenotype by a candidate gene approach. Alternative candidate mutations included, in addition to the previously reported nonsense mutation c.649 C > T (p.Arg217*) and splice donor mutation c.1179 + 2T > A in MITF, a nonsense mutation c.2028T > A (p.Tyr676*) in KIT. All these three mutations were located in the genomic regions that were highly conserved exclusively in Indonesian swamp buffaloes and they accounted largely (95%) for the manifestation of white spotting. Last but not the least, ADAMTS20 and TWIST2 may also contribute to the diversification of this coat-color pattern.ConclusionsThe alternative mutations identified in this study affect, at least partially and independently, the development of melanocytes. The presence and persistence of such mutations may be explained by significant financial and social value of spotted buffaloes used in historical Rambu Solo ceremony in Tana Toraja, Indonesia. Several de novo spontaneous mutations have therefore been favored by traditional breeding for the spotted buffaloes.

Population Analysis Identifies 15 Multi-Variant Dominant White Haplotypes in Horses.

The influence of a horse's appearance on health, sentimental and monetary value has driven the desire to understand the etiology of coat color. White markings on the coat define inclusion for multiple horse breeds, but they may disqualify a horse from registration in other breeds. In domesticated horses (Equus caballus), 35 KIT alleles are associated with or cause depigmentation and white spotting. It is a common misconception among the general public that a horse can possess only two KIT variants. To correct this misconception, we used BEAGLE 5.4-phased NGS data to identify 15 haplotypes possessing two or more KIT variants previously associated with depigmentation phenotypes. We sourced photos for 161 horses comprising 12 compound genotypes with three or more KIT variants and employed a standardized method to grade depigmentation, yielding average white scores for each unique compound genotype. We found that 7 of the 12 multi-variant haplotypes resulted in significantly more depigmentation relative to the single-variant haplotypes (ANOVA). It is clear horses can possess more than two KIT variants, and future work aims to document phenotypic variations for each compound genotype.

Activation of the receptor KIT induces the secretion of exosome‐like small extracellular vesicles

AbstractThe receptor tyrosine kinase (RTK) KIT and its ligand stem cell factor (SCF) are essential for human mast cell (huMC) survival and proliferation. HuMCs expressing oncogenic KIT variants secrete large numbers of extracellular vesicles (EVs). The role KIT plays in regulating EV secretion has not been examined. Here, we investigated the effects of stimulation or inhibition of KIT activity on the secretion of small EVs (sEVs). In huMCs expressing constitutively active KIT, the quantity and quality of secreted sEVs positively correlated with the activity status of KIT. SCF‐mediated stimulation of KIT in huMCs or murine MCs, or of transiently expressed KIT in HeLa cells, enhanced the release of sEVs expressing exosome markers. In contrast, ligand‐mediated stimulation of the RTK EGFR in HeLa cells did not affect sEV secretion. The release of sEVs induced by either constitutively active or ligand‐activated KIT was remarkably decreased when cells were treated with KIT inhibitors, concomitant with reduced exosome markers in sEVs. Similarly, inhibition of oncogenic KIT signalling kinases like PI3K, and MAPK significantly reduced the secretion of sEVs. Thus, activation of KIT and its early signalling cascades stimulate the secretion of exosome‐like sEVs in a regulated fashion, which may have implications for KIT‐driven functions.

Antibody-Based and Cell Therapies for Advanced Mastocytosis: Established and Novel Concepts.

Advanced systemic mastocytosis (SM) is a heterogeneous group of myeloid neoplasms characterized by an uncontrolled expansion of mast cells (MC) in one or more internal organs, SM-induced tissue damage, and poor prognosis. Advanced SM can be categorized into aggressive SM (ASM), MC leukemia (MCL), and SM with an associated hematologic neoplasm (SM-AHN). In a vast majority of all patients, neoplastic cells display a KIT mutation, mostly D816V and rarely other KIT variants. Additional mutations in other target genes, such as SRSF2, ASXL1, or RUNX1, may also be identified, especially when an AHN is present. During the past 10 years, improved treatment approaches have led to a better quality of life and survival in patients with advanced SM. However, despite the availability of novel potent inhibitors of KIT D816V, not all patients enter remission and others relapse, often with a multi-mutated and sometimes KIT D816V-negative disease exhibiting multi-drug resistance. For these patients, (poly)chemotherapy, antibody-based therapies, and allogeneic hematopoietic stem cell transplantation may be viable treatment alternatives. In this article, we discuss treatment options for patients with drug-resistant advanced SM, including novel KIT-targeting drugs, antibody-based drugs, and stem cell-eradicating therapies.

Characteristics of genetic variants in 134 patients with Acute myeloid leukemia

To analyze the characteristics of genetic variants in 134 patients diagnosed with Acute myeloid leukemia (AML). Clinical data of the 134 patients with AML (non-acute promyelocytic leukemia) initially diagnosed at the 940th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army from June 2017 to June 2022 were retrospectively analyzed. Potential variants of AML-related genes were detected by next-generation sequencing, and the frequency of variants was analyzed by using SPSS v26.0 software, and likelihood ratio χ2 test and Fisher exact test were used for data analysis. The patients had included 72 males and 62 females, with a gender ratio of 1.7 : 1 and a median age of 51 years (9 ~ 86 years old). One hundred twenty patients (76.1%) had harbored at least one genetic variant, including 26 (19.4%) having a single variant, 27 (20.1%) having two variants, and 49 (36.6%) having >= 3 variants. 32 (23.9%) had no detectable variants. Genetic variants detected in over 10% of the 134 patients had included NPM1 (n = 24, 17.91%), FLT3-ITD (n = 21, 15.67%), DNMT3A (n = 20, 14.93%), CEBPA (single variant; n = 14, 10.45%), TET2 (n = 14, 10.45%), and NRAS (n = 14, 10.45%). The patients were also divided into low risk, intermediate risk and high risk groups based on their chromosomal karyotypes. The mutational rates for genes in different groups have varied, with 19 patients from the low risk group harboring variants of NRAS (n = 4, 21.05%), KRAS (n = 4, 21.05%), and KIT (n = 2, 10.53%); and 96 patients from the intermediate risk group harboring variants of NPM1 (n = 24, 25.00%), FLT3-ITD (n = 20, 20.83%), DNMT3A (n = 18, 18.75%), CEBPA (n = 12, 12.50%), and TET2 genes (n = 12, 12.50%). The mutational frequencies for the 19 patients from the high risk group were ASXL1 (n = 7, 21.05%), NRAS (n = 3, 15.97%), TP53 (n = 3, 15.79%), and EZH2 (n = 2, 10.53%). A significant difference was found in the frequencies of KIT, NPM1, FLT3-ITD, DNMT3A, and ASXL1 gene variants among the low-risk, medium-risk, and high-risk groups. AML patients have a high frequency for genetic variants, with 76.1% harboring at least one variant. The frequency of genetic variants have varied among patients with different chromosomal karyotypes, and there are apparent dominant variants. KIT, NPM1, FLT3-ITD, DNMT3A, and ASXL1 may be used as prognostic factors for evaluating their prognosis.

5'UTR Variant in KIT Associated With White Spotting in Horses.

Mutations in KIT, a gene that influences melanoblast migration and pigmentation, often result in mammalian white spotting. As of February 2023, over 30 KIT variants associated with white spotting were documented in Equus caballus (horse). Here we report an association of increased white spotting on the skin and coat with a variant in the 5'UTR of KIT (rs1149701677: g.79,618,649A>C). Horses possessing at least one alternate allele demonstrate phenotypic characteristics similar to other KIT mutations: clear borders around unpigmented regions on the body, face, and limbs. Using a quantitative measure of depigmentation, we observed an average white score of 10.70 among individuals with rs1149701677, while the average score of the control, homozygous reference sample was 2.23 (P=1.892e-11, n=109, t-test). The rs1149701677 site has a cross-species conservation score of 3.4, one of the highest scores across the KIT 5'UTR, implying regulatory importance for this site. Ensembl also predicted a "moderately impactful" functional effect for the rs1149701677 variant. We propose that this single nucleotide variant likely alters the regulation of KIT, which in turn may disrupt melanoblast migration causing an increase in white spotting on the coat. Alternatively, the rs1149701677 variant may be in linkage with another nearby variant with an as-yet-undiscovered functional impact. We propose to term this new allele "Holiday White" or W35 based on conventional nomenclature.

KIT and PDGFRA Variants and the Survival of Patients with Gastrointestinal Stromal Tumor Treated with Adjuvant Imatinib.

Adjuvant imatinib improves the recurrence-free survival and overall survival (OS) of patients with gastrointestinal stromal tumors (GISTs) who have a high risk of recurrence after surgery and is now considered standard treatment. Yet, OS benefit has been demonstrated in only one randomized study, the Scandinavian Sarcoma Group XVIII/AIO trial, where patients with high-risk GISTs were allocated to either 1 year or 3 years of adjuvant imatinib. SSGXVIII/AIO is also the only randomized trial in which adjuvant imatinib duration exceeding 2 years was evaluated. In this trial, the 3-year treatment led to a 45% reduction in the risk of death during the first 10 years that followed random allocation even though some of the patients did not have GISTs at tumor histology review, had mutations now known to be imatinib-resistant or had non-localized disease at study entry. In the subgroup of patients who had KIT exon 11 deletion/indel mutation, the reduction in the risk of death was 66% in favor of the longer treatment. Proper patient selection is of crucial importance since many patients are cured with surgery. Little evidence for OS benefit is available from randomized trials for patients whose GIST harbors KIT exon 9 mutation, KIT insertion mutation, PDGFRA D842V mutation, or lacks KIT and PDGFRA mutations. Adjuvant imatinib improves OS substantially if high-risk GISTs can be identified, treatment duration is long enough, and GISTs harbor an imatinib-sensitive mutation.

Multi-omic characterization of gastrointestinal stromal tumor (GIST) in a large real-world patient cohort.

11522 Background: Molecular knowledge of GIST is limited due to its rarity, few genes have been identified as relevant determinants of outcomes, tumor evolution and therapeutic targets. Therefore, we aimed to dissect the GIST molecular landscape in the largest series of real-world patients reported to date. Methods: 946 GIST patient samples (536 localized, 369 metastatic, 41 unknown) underwent next-gen sequencing of DNA (592-gene, N = 495; whole exome, N = 451) and RNA (whole transcriptome, N = 592) at Caris Life Sciences (Phoenix, AZ). Gene expression signatures of proliferation (Cristescu, 2021), cell cycle activation (CINSARC; Chibon, 2010), inflammation (T-cell inflamed; Ayers, 2017) and tumor microenvironments (MCP-counter; Becht, 2016) were examined. Statistical significance tested by χ2, Fisher’s exact, or Mann-Whitney U as appropriate. Results: GIST samples were comprised of 80% (N = 758) KITmut, 8.1% (N = 77) PDGFRAmut, and 11.7% (N = 111) KIT/PDGFRA wild-type (WT), with 14.8% (N = 140) samples harboring a secondary KIT variant suggestive of TKI resistance. Overall median TMB was 2 mutations/MB (range 0-13). WT GIST were identified with mutations in NF1 (33.7%), DNA repair genes (16.7%), SDHX (8.2%), BRAF (6.3%), and PTEN (1.9%), along with NTRK3 fusions (3.1%). Primary KIT variants occurred in exons 11 (83.5%), 9 (13.9%), and 13 (2.6%), and secondary KIT variants (14.6% of total KIT mutations) were distributed across the ATP binding pocket (36.8%) and activation loop (63.2%). Primary PDGFRA mutations were in exons 18 (62.4%), 12 (11.0%), 14 (4.6%) and other (16.5%). KIT/PDGFRAmut GIST infrequently harbored RB1, TP53, SETD2, ARID1A, PIK3CA, PTEN, TSC1, BRCA1, or CHEK2 co-mutations (1-5% each) . Copy number amplification (≥6 copies) was overall uncommon (≤2% for all genes). Proliferation and cell cycle activation signatures were higher in KIT exon 11 indels v. missense mut (1.2-fold, p < 0.05) and KIT resistant v. KIT primary (1.2-fold, p < 0.05), but not in KIT exon 11 557/558 v. others, nor between KIT v. PDGFRA v. WT subgroups. Deletion of tumor progression genes MAX (40.0%), CDKN2A (32.3%), and DEPDC5 (33.9%) was associated with increased proliferative gene expression (1.2-, 1.3-, and 1.2-fold, p < 0.05 each), while DMD deletion (52.5%) was not (1.1-fold, p = 0.37). Compared to KITmut and WT GIST, PDGFRAmut had increased abundance of several immune cell populations (range 1.2-3.7-fold, p < 0.05), along with enhanced inflammation signatures (1.1- and 1-2-fold, p < 0.05). Conclusions: This series provides unprecedented resolution of KIT/PDGFRAmut GIST with features of clinical aggressiveness associated with KIT exon 11 indels and resistance mutations, illustrating a specific cytogenetic genotype with more aggressive growth and malignant behavior. Identification of less common molecular alterations that drive kinase activation and impaired DNA damage repair warrant further investigation.

Comprehensive ctDNA analysis to identify genome-instability and actionable mutation landscape in gynecologic cancers.

e17503 Background: Gynaecology malignancies are the second most prevalent cancers in women worldwide with rapid progression, high relapse, and mortality. Although HRR pathway inhibitors can greatly benefit clinical outcomes, patients without HRR pathway mutations may remain unbenefited while being profiled for tissue DNA or with a small target gene panel. Compelling data suggest that plasma genotyping shows a wide diversity of cancer mutations along genome instability indicators compared to tumor tissue. Here, we analyzed the mutational landscape of ctDNA and tissue DNA (tDNA) obtained from gynecologic cancer patients. We demonstrate the clinical utility of a comprehensive gene panel to identify multiple actionable mutations and genome instability indicators from ctDNA samples. Methods: We retrospectively analyzed the mutational landscape of 22 ovarian and endometrial cancer patients using target-hybridization enrichment of ctDNA and tDNA. Libraries prepared using a custom-designed comprehensive gene panel OncoIndx, targeting 600 cancer-relevant exons including MSI and HRR pathway genes, were sequenced on the Illumina NGS platform in pair-end mode. Variant calling was performed with an in-house developed bioinformatics pipeline. Results: NGS analysis revealed a total of 127 mutations consisting SNVs (45.6 %), truncations (40.94 %), amplifications (4.72 %), splice variants (4.72 %), and frameshift (3.94 %). Genome-wide mutational analysis showed a slightly higher mean TMB (15.7) for endometrial tumors compared to ovarian samples (11.85). The mean HRD score (45 %) was high for 90 % of patients and corroborated well with the HRR pathway gene mutation. 30 % of patients without HRR gene mutations including BRCA 1/2 had a high average HRD score (43 %) (LOH/TAI/LST) and may qualify for PARP inhibitors (PARPi). Alterations in Ras isoforms (H, K, and N) were the most frequent (36 %) followed by TP53 and PI3K (32 %). At the pathway level, DDR gene-variants were most frequently followed by alterations in proliferative signaling genes. ctDNA showed a wider mutational landscape, including clinically significant mutations, compared to tDNA. Additionally, ctDNA analysis revealed the presence of novel variants of SUFU and KIT, which remain unexplored in the context of cancer stem cells and relapse. Conclusions: Comprehensive mutational landscape showed the presence of genome-wide instability and the presence of mutations in the HRR pathway. Our results suggest that patients with high HRD scores and Ras pathway instability may benefit from Ras pathway inhibitors-PARPi combination. Mutational landscapes obtained from ctDNA were significantly wider and revealed multiple actionable targets compared to tDNA suggesting ctDNA as a viable and informative alternative for genotyping of gynecological cancers.

Mast cell leukemia: clinical and molecular features and survival outcomes of patients in the ECNM Registry.

Mast cell leukemia (MCL) is a rare subtype of systemic mastocytosis defined by ≥20% mast cells (MC) on a bone marrow aspirate. We evaluated 92 patients with MCL from the European Competence Network on Mastocytosis registry. Thirty-one (34%) patients had a diagnosis of MCL with an associated hematologic neoplasm (MCL-AHN). Chronic MCL (lack of C-findings) comprised 14% of patients, and only 4.5% had "leukemic MCL" (≥10% circulating MCs). KIT D816V was found in 62/85 (73%) evaluable patients; 9 (11%) individuals exhibited alternative KIT mutations, and no KIT variants were detected in 14 (17%) subjects. Ten evaluable patients (17%) had an abnormal karyotype and the poor-risk SRSF2, ASXL1, and RUNX1 (S/A/R) mutations were identified in 16/36 (44%) patients who underwent next-generation sequencing. Midostaurin was the most common therapy administered to 65% of patients and 45% as first-line therapy. The median overall survival (OS) was 1.6 years. In multivariate analysis (S/A/R mutations excluded owing to low event rates), a diagnosis of MCL-AHN (hazard ratio [HR], 4.7; 95% confidence interval [CI], 1.7-13.0; P= .001) and abnormal karyotype (HR, 5.6; 95% CI, 1.4-13.3; P= .02) were associated with inferior OS; KIT D816V positivity (HR, 0.33; 95% CI, 0.11-0.98; P= .04) and midostaurin treatment (HR, 0.32; 95% CI, 0.08-0.72; P= .008) were associated with superior OS. These data provide the most comprehensive snapshot of the clinicopathologic, molecular, and treatment landscape of MCL to date, and should help further inform subtyping and prognostication of MCL.

Circulating tumor DNA analysis of the phase III VOYAGER trial: KIT mutational landscape and outcomes in patients with advanced gastrointestinal stromal tumor treated with avapritinib or regorafenib

The current treatment paradigm of imatinib-resistant metastatic gastrointestinal stromal tumor (GIST) does not incorporate KIT/PDGFRA genotypes in therapeutic drug sequencing, except for PDGFRA exon 18-mutant GIST that is indicated for avapritinib treatment. Here, circulating tumor DNA (ctDNA) sequencing was used to analyze plasma samples prospectively collected in the phase III VOYAGER trial to understand how the KIT/PDGFRA mutational landscape contributes to tyrosine kinase inhibitor (TKI) resistance and to determine its clinical validity and utility. VOYAGER (N = 476) compared avapritinib with regorafenib in patients with KIT/PDGFRA-mutant GIST previously treated with imatinib and one or two additional TKIs (NCT03465722). KIT/PDGFRA ctDNA mutation profiling of plasma samples at baseline and end of treatment was assessed with 74-gene Guardant360® CDx. Molecular subgroups were determined and correlated with outcomes. A total of 386/476 patients with KIT/PDGFRA-mutant tumors underwent baseline (pre-trial treatment) ctDNA analysis; 196 received avapritinib and 190 received regorafenib. KIT and PDGFRA mutations were detected in 75.1% and 5.4%, respectively. KIT resistance mutations were found in the activation loop (A-loop; 80.4%) and ATP-binding pocket (ATP-BP; 40.8%); 23.4% had both. An average of 2.6 KIT mutations were detected per patient; 17.2% showed 4-14 different KIT resistance mutations. Of all pathogenic KIT variants, 28.0% were novel, including alterations in exons/codons previously unreported. PDGFRA mutations showed similar patterns. ctDNA-detected KIT ATP-BP mutations negatively prognosticated avapritinib activity, with a median progression-free survival (mPFS) of 1.9 versus 5.6 months for regorafenib. mPFS for regorafenib did not vary regardless of the presence or absence of ATP-BP/A-loop mutants and was greater than mPFS with avapritinib in this population. Secondary KIT ATP-BP pocket mutation variants, particularly V654A, were enriched upon disease progression with avapritinib. ctDNA sequencing efficiently detects KIT/PDGFRA mutations and prognosticates outcomes in patients with TKI-resistant GIST treated with avapritinib. ctDNA analysis can be used to monitor disease progression and provide more personalized 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.

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

Novel pathogenic variants in KIT gene in three Chinese piebaldism patients.

Piebaldism is a rare autosomal dominant disease, and roughly 75% patients had KIT gene mutations. Up to date, approximately 90 KIT mutations causing piebaldism were reported. To identify KIT gene mutations in three pediatric piebaldism patients from different families and explore the genotype-phenotype correlation, peripheral blood DNA were collected from probands and their parents. Whole-exome sequencing was performed to detect potential disease-causing variants in the three probands. Putative variants were validated by Sanger sequencing. Heterozygous variants of c.2469_2484del (p.Tyr823*), c.1994G > C (p.Pro665Leu), and c.1982_1983insCAT (p.662_663insIle) in KIT gene were detected in three probands. These variants were all novel and classified as pathogenic/likely pathogenic variants according to the interpretation guidelines of American College of Medical Genetics and Genomics and the Association for Molecular Pathology. The probands carrying variants located in tyrosine kinase domain exhibited a more severe phenotype. The piebaldism in three families was caused by novel heterozygous KIT variants. The severity of phenotypes is related with the types and locations of different mutations. Our results further provided evidence for genetic counseling for the three families.

Immunohistochemical Staining to Identify Concomitant Systemic Mastocytosis in Acute Myeloid Leukemia with RUNX1::RUNX1T1.

Systemic mastocytosis with associated hematological neoplasm (SM-AHN) poses diagnostic challenges because of the coexistence of atypical mast cell proliferation and hematological neoplasms. We assessed the presence of SM-AHN in patients with acute myeloid leukemia (AML) with RUNX1::RUNX1T1 from 2014 to 2020. Bone marrow (BM) samples were evaluated for mast cell aggregates using CD117 and CD25 immunohistochemical (IHC) staining. The KIT D816V variant burden at diagnosis and post induction was assessed using droplet digital PCR. Among 23 patients diagnosed as having AML with RUNX1::RUNX1T1, four (17.4%) were also diagnosed as having SM-AHN. No significant differences in clinical characteristics or overall survival (P=0.565) were observed between patients with or without SM-AHN, except for the presence of KIT variants (P=0.040). After induction therapy, IHC staining revealed the presence of mast cell aggregates in the BM, and the KIT D816V variant burden decreased with decreasing blast count and was similar in BM aspirates, smear slides, and sections. Concomitant SM-AHN was not infrequent in AML patients with RUNX1::RUNX1T1. This study showed the importance of CD117 and CD25 IHC staining after induction chemotherapy for SM-AHN screening, especially in patients with KIT variants.

A literature review and database of how the primary KIT/PDGFRA variant of a gastrointestinal stromal tumour predicts for sensitivity to imatinib

It is well recognized that the primary KIT or PDGFRA variant of a gastrointestinal stromal tumour (GIST) can predict sensitivity to imatinib. However, these data are currently spread across a wide range of publications and have not been collated as one reference. A broad-ranging literature search was therefore performed to assemble such a database which should help optimize imatinib-based management of GIST patients henceforth. Having excluded wild type GISTs and results for imatinib used as adjuvant therapy, 79 publications (dated August 2001 to March 2022) underwent data extraction. These data on imatinib sensitivity were either derived from in vitro studies, predicted by in silico analysis or based on in vivo clinical patient response. Data interpretation carried some caveats: there was a potential for replication of patient-derived data between older and new publications; only predicted protein sequences were presented; the criteria used to record clinical response were not uniform across all publications; and imatinib dosage could vary between different clinical publications. However, these data showed broad agreement of imatinib sensitivity amongst similar subtypes of KIT or PDGFRA variant. There was also agreement between in vivo versus in vitro/in silico derived sensitivity data for most variants when both data types were available.

A KIT Variant Associated with Increased White Spotting Epistatic to MC1R Genotype in Horses (Equus caballus).

Simple SummaryAlthough over 40 genetic variants are known to influence white spotting and markings on the horse, many still have unknown genetic causes. Furthermore, some seem to be influenced by pigmentation (base coat color) genes. We investigated two horses demonstrating a heritable white spotting pattern of no known genetic cause. Through sequencing of the coding region of candidate genes, we identified a mutation in the KIT proto-oncogene, receptor tyrosine kinase (KIT) gene changing the coded amino acid, predicted to be deleterious to protein function. We further evaluated this variant in a population of 147 horses, characterized using photographs scored by three independent observers using a standardized Average Grade of White score. The KIT mutation is significantly associated with a quantitative increase in white pattern (p = 3.3 × 10−12) and demonstrates an influence of the MC1R Extension locus. We also report a complete link between the previously reported KIT W19 allele and this mutation. We propose to name this mutation W34, following established nomenclature. Given the quantitative effect on white markings and MC1R influence, genetic testing for this allele can be of value for horse owners that desire to select for white patterns.Over 40 identified genetic variants contribute to white spotting in the horse. White markings and spotting are under selection for their impact on the economic value of an equine, yet many phenotypes have an unknown genetic basis. Previous studies also demonstrate an interaction between MC1R and ASIP pigmentation loci and white spotting associated with KIT and MITF. We investigated two stallions presenting with a white spotting phenotype of unknown cause. Exon sequencing of the KIT and MITF candidate genes identified a missense variant in KIT (rs1140732842, NC_009146.3:g.79566881T>C, p.T391A) predicted by SIFT and PROVEAN as not tolerated/deleterious. Three independent observers generated an Average Grade of White (AGW) phenotype score for 147 individuals based on photographs. The KIT variant demonstrates a significant QTL association to AGW (p = 3.3 × 10−12). Association with the MC1R Extension locus demonstrated that, although not in LD, MC1R e/e (chestnut) individuals had higher AGW scores than MC1R E/- individuals (p = 3.09 × 10−17). We also report complete linkage of the previously reported KIT W19 allele to this missense variant. We propose to term this variant W34, following the standardized nomenclature for white spotting variants within the equine KIT gene, and report its epistatic interaction with MC1R.

Abstract 5161: Exome-scale longitudinal tracking of emerging therapeutic resistance in GIST via analysis of circulating tumor DNA

Abstract Gastrointestinal stromal tumors (GIST) are lethal tumors characterized by constitutively activating mutations to KIT or PDGFRA. Transient disease control in the first-line setting is achieved via inhibition of tyrosine kinase signaling using the KIT inhibitor imatinib. As patients progress through subsequent lines of therapy a molecularly heterogeneous disease evolves, characterized by distinct subtypes and shifting repertoires of exon-specific KIT variants which directly impact treatment outcomes. Here, we use tumor-informed exome-scale liquid biopsy to identify and track the evolution of multiple resistance mechanisms in patients receiving tyrosine kinase inhibitors (TKIs) to address the unmet need of comprehensive understanding of GIST evolution in response to therapy. Matched tumor, normal and serial plasma samples were obtained from 15 heavily pretreated metastatic GIST patients. Following baseline sample collection, all patients received systemic TKI therapy, and were monitored until disease progression. Exome-scale detection of somatic variants in cfDNA from longitudinal matched plasma samples was achieved using the NeXT Liquid BiopsyTM platform. The ImmunoID NeXT PlatformⓇ, an augmented exome/transcriptome platform and analysis pipeline which generates comprehensive tumor and immune data was used to profile paired tumor and normal samples. Longitudinal whole exome sequencing of plasma identified dynamic shifts in existing clones harboring exon-specific KIT mutations, and evolution of new KIT mutations arising prior to identification of tumor progression using standard imaging techniques. We detected a correlation between the number of damaging mutations detected in baseline ctDNA and tumor exon 11 KIT mutation status, suggesting that plasma mutation profiles may be KIT-variant dependent. ctDNA from patients with shorter overall survival (OS) was enriched for variants in the PI3K-AKT and MAPK pathway, potentially contributing to immune evasion observed in those patients. Additional associations were observed between gene copy-number changes and OS (P = .0097). Previous studies have demonstrated that immune infiltration and activity may be KIT variant specific, here we broaden those findings, identifying a significant correlation between TCRɑ clonality and variants detected only in plasma (P = .04), as well as a significant association between TCRβ diversity and OS (HR = 2.55, log rank P = .04). Comprehensive profiling of paired tumor tissue (WES and RNA-Seq) and WES of serially collected ctDNA sensitively and repeatedly identified evolving KIT mutations and other molecular alterations prior to radiologically confirmed disease progression. These findings suggest plasma-based monitoring of late-stage GIST malignancies may be useful for non-invasive disease tracking, providing treatment guidance prior to traditional approaches. Citation Format: Charles W. Abbott, Niamh Coleman, Jing Wang, Josette Northcott, Jason Pugh, Dan Norton, Fábio C. Navarro, Lee D. McDaniel, Eric Levy, Rachel Marty Pyke, John Lyle, Jason Harris, Gabor Bartha, Filip Janku, John West, Richard O. Chen, Sean Boyle. Exome-scale longitudinal tracking of emerging therapeutic resistance in GIST via analysis of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5161.

Positive Selection and Adaptive Introgression of Haplotypes from Bos indicus Improve the Modern Bos taurus Cattle

Complex evolutionary processes, such as positive selection and introgression can be characterized by in-depth assessment of sequence variation on a whole-genome scale. Here, we demonstrate the combined effects of positive selection and adaptive introgression on genomes, resulting in observed hotspots of runs of homozygosity (ROH) haplotypes on the modern bovine (Bos taurus) genome. We first confirm that these observed ROH hotspot haplotypes are results of positive selection. The haplotypes under selection, including genes of biological interest, such as PLAG1, KIT, CYP19A1 and TSHB, were known to be associated with productive traits in modern Bos taurus cattle breeds. Among the haplotypes under selection, we demonstrate that the CYP19A1 haplotype under selection was associated with milk yield, a trait under strong recent selection, demonstrating a likely cause of the selective sweep. We further deduce that selection on haplotypes containing KIT variants affecting coat color occurred approximately 250 generations ago. The study on the genealogies and phylogenies of these haplotypes identifies that the introgression events of the RERE and REG3G haplotypes happened from Bos indicus to Bos taurus. With the aid of sequencing data and evolutionary analyses, we here report introgression events in the formation of the current bovine genome.