Gastrointestinal Stromal Tumors Samples Research Articles

Gastrointestinal stromal tumors regulate macrophage M2 polarization through the MIF/CXCR4 axis to immune escape.

The infiltration of immune cells and their roles of the infiltrating-immune cells in gastrointestinal stromal tumor (GIST) is still unclear. We aimed to discover the infiltration cell types and the relationship between the infiltrating-immune cells and the progression of GIST. Single-cell RNA sequencing were performed to discover types of the infiltrating-immune cells and to analyze CellChat between cells. Immunohistochemistry of 80 GIST samples were used to clarify the relation between macrophages and recurrence risk. In vitro, flow cytometry and Real-time PCR were performed to uncover a potential mechanism of tumor cell regulation of macrophages. Tumor cells, macrophages, and T-cells were the predominant cell types. The MIF/CXCR4 axis was the most common ligand-receptor interaction between macrophages and tumor cells. As the risk increased, expression levels of CD68, CD206, MIF, and CXCR4 gradually increased. In vitro, we found that GIST882 was able to secrete MIF and GIST882 cell supernatant upregulated M2 polarization. Real-time PCR showed that expression levels of IL-10 mRNA and Arginase-1 mRNA were also the highest in the GIST882 cell supernatant group. These findings identify that macrophages are the most abundant infiltrating cells in GIST. The MIF/CXCR4 axis is the most common ligand-receptor interaction between macrophages and tumor cells. GIST cells can regulate macrophage M2 polarization through the MIF/CXCR4 axis.

Iodothyronine Deiodinase 3 Gene Expression in Gastrointestinal Stromal Tumors: A Pilot Study to Contribute to Risk Assessment.

In the realm of gastrointestinal stromal tumors (GIST), understanding the molecular landscape and prognostic factors is crucial for effective management. The deiodinase 3 gene (DIO3), known for its role in thyroid hormone regulation and cell proliferation, has emerged as a potential player in GIST pathogenesis. Our study investigated DIO3 expression in GIST samples and its correlation with tumor characteristics, aiming to enhance prognostic stratification and personalized treatment strategies. Using a retrospective design, we analyzed data and formalin-fixed paraffin-embedded (FFPE) samples of patients diagnosed with GIST. The study cohort comprised 33 patients, predominantly female, with a median age of 66 years. The tumor characteristics were meticulously documented, including location, size, mitotic count, risk classification, and immunohistochemical markers. Gene expression analysis of DIO3 was conducted using FFPE samples, with a focus on relative quantification and association with immunohistochemical markers and prognostic risk. DIO3 overexpression was observed in 69.70% of tumors, while underexpression was noted in 30.30% of cases. Association analyses revealed intriguing insights. A notable association was identified between DIO3 expression and the frequency of DOG1, suggesting a potential interplay between these markers in GIST pathobiology. Furthermore, increased DIO3 expression was significantly higher in very low/low-risk prognostic patients, hinting at a possible link between DIO3 levels and tumor progression prognosis. The intricate interplay between DIO3 expression and GIST characteristics uncovered in this study underscores the potential of molecular markers in refining prognostic assessments and therapeutic strategies for GIST patients.

Expression of TGF-β1 in Gastrointestinal Stromal Tumor (GIST) and the Occurrence of Frequent Desmoplasia.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms with variable behavior characterized by differentiation toward the interstitial cells of Cajal occurring anywhere in the gastrointestinal stromal tract. Frequently, GISTs have fibrous stroma within tumor cell proliferation areas, which is unlike other types of malignant tumors. If this desmoplasia is active, there is a possibility that some sort of transmitter exists between GIST cells and cells related to fibrosis in the tumor cell proliferation areas. Transforming growth factor (TGF)-β isoforms, particularly TGF-β1, are critical for fibrosis pathogenesis. TGF-β1 regulation of myofibroblasts and fibroblasts during fibrosis is well described. The induced fibroblast activation resulting in myofibroblast differentiation has been reported as an important source of collagen, glycoproteins, proteoglycans, and matrix metallopeptidases in wound healing and fibrosis. However, there are a few reports on the relationship between TGF-β1 and GISTs. This study aims to clarify TGF-β1 expression in 30 gastric GISTs using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). For comparison, we also enrolled 30 samples of gastric tubular adenocarcinoma (GTAC). We confirmed TGF-β1 expression (H-score ≥50 points) in 57% of GIST and 13% of GTAC samples, a significant difference between the 2 tumor types ( P =0.001). We examined the TGF-β1 mRNA expression of 3 representative GIST samples, each having their respective immunostained areas detected by RT-PCR. Finding TGF-β1 expression may indicate that this cytokine plays a part in the formation of desmoplasia within GIST cell proliferative areas.

Morphological, immunohistochemical, and genetic analyses of epithelioid gastrointestinal stromal tumors

Epithelioid gastrointestinal stromal tumors (GISTs) are rare and may be confused with other tumors with epithelioid morphology. Therefore, herein, we collected 12 epithelioid GIST samples and summarized their morphological and immunohistochemical characteristics. Through genetic testing, we explored the correlation between morphology and gene mutations. The results showed that eight tumors showed focal or diffuse myxoid stromal changes with less cohesively arranged rhabdoid tumor cells; among these, five showed platelet-derived growth factor receptor alpha gene (PDGFRA) mutations. Signet ring cells with sclerosing stroma and receptor tyrosine kinase type III gene (KIT) mutations were present in two cases, which might be a KIT mutation-associated growth pattern in epithelioid GISTs. Succinate dehydrogenase gene (SDH) mutations were detected in three cases. Simultaneously, PDGFRA mutations were detected in two cases, and the Kirsten rat sarcoma viral oncogene homolog gene (KRAS) mutation was detected in another case. SDH-subunit B (SDHB) expression was partially weak and strongly diffuse in two cases with concurrent PDGFRA and SDHD mutations, respectively. The coexistence of PDGFRA and SDHD mutations may have affected SDHB expression. Altogether, we concluded that PDGFRA mutations may play an important role in co-mutant GIST pathogenesis.

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.

Abstract 3887: ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor

Abstract Introduction: Ripretinib (Rip) is a kinase inhibitor with broad preclinical activity against mutant KIT. Based on the INVICTUS trial, Rip was approved for patients with Gastrointestinal Stromal Tumors (GIST) after treatment with 3 or more kinase inhibitors. Most patients in this ≥4th-line setting progress on Rip within one year. Here, we characterized Rip-progressing GIST samples to identify resistance mechanisms. Methods: Progressing lesions in 25 patients were analyzed by NGS after Rip failure. KIT mutations (muts) were recapitulated by gene editing. Activities of Rip, sunitinib (SU), and novel TKIs were characterized by cell viability assays and immunoblot. Mutagenesis experiments were performed using ENU. SU- and Rip-resistant cell lines were pooled and treated with various regimens, with clonal composition deconvoluted by cDNA-based amplicon sequencing. Results: 19/25 Rip-progressing GISTs displayed muts in the ATP-binding pocket (ATP-BP; e13/14). Four of these had pre-existing muts in the activation loop (AL; e17/18), which were confirmed to be in-cis with the primary and ATP-BP muts (triple in cis; TIC). Mutagenesis screens using a double KIT-mutant GIST line (e11 + AL) as a starting point confirmed that TIC-muts are a predominant escape mechanism when treated with Rip. A GIST subline (T1-triple) with TIC-muts in e11, e18 (A829P), and e13 (V654A) was highly resistant to Rip (GR50 > 2µM). Structural analyses of ATP-BP muts suggest steric interference and loss of van der Waals interactions that impede Rip binding and thereby confer Rip resistance. Another 3/25 Rip-progressing GISTs harbored pathogenic KIT muts in e9 only. A novel GIST cell line with primary KIT e9 mut (T1-e9) was 14-fold less sensitive to Rip than isogenic e11-driven cells (GR50 = 115 vs 8nM). Notably, adding a typical AL mut to T1-e9 (T1-e9-N822K) sensitized the cells to Rip (GR50 = 20nM). Immunoblots showed >95% inhibition of phospho-KIT in AL-mutant cell lines at Rip 100nM, whereas inhibition was weaker in T1-e9 (77%) and T1-V654A (46%), and absent in T1-triple. A compound screen of FDA-approved kinase inhibitors identified Nintedanib (NIN) as the most active compound against T1-triple. Clonal outgrowth assays of mixed cultures revealed SU (93% inhibition), and a weekly switch between Rip and either SU (96%) or NIN (79%) as the most effective inhibitors of pooled cell growth. Conclusions: KIT e9 primary muts and e13/14 (ATP-BP) secondary muts are enriched in post-progression biopsies following Rip treatment. KIT TIC-muts are novel frequent events driving GIST clinical progression and confer a high degree of resistance. Strategies to overcome resistance may include combinations or sequences of approved drugs, and novel drugs that more efficiently inhibit TIC-mutant KIT. Citation Format: Thomas Mühlenberg, Johanna Falkenhorst, Tom Schulz, Benjamin S. Fletcher, Alina Teuber, Dawid Krzeciesa, Isabella Klooster, Jonas Lategahn, Wen-Bin Ou, Meijun Lundberg, Margaret von Mehren, Susanne Grunewald, Alicia I. Tüns, Mehdi Brahmi, Michael C. Heinrich, Cesar Serrano, Hans-Ulrich Schildhaus, Sonja Sievers, Jürgen Treckmann, Lydia Wilson, Chandrajit P. Raut, Adrian Marino-Enriquez, Suzanne George, Daniel Rauh, Jonathan A. Fletcher, Sebastian Bauer. ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3887.

Analysis of microbiome in gastrointestinal stromal tumors: Looking for different players in tumorigenesis and novel therapeutic options.

Preclinical forms of gastrointestinal stromal tumor (GIST), small asymptomatic lesions, called microGIST, are detected in approximately 30% of the general population. Gastrointestinal stromal tumor driver mutation can be already detected in microGISTs, even if they do not progress into malignant cancer; these mutations are necessary, but insufficient events to foster tumor progression. Here we profiled the tissue microbiota of 60 gastrointestinal specimens in three different patient cohorts—micro, low‐risk, and high‐risk or metastatic GIST—exploring the compositional structure, predicted function, and microbial networks, with the aim of providing a complete overview of microbial ecology in GIST and its preclinical form. Comparing microGISTs and GISTs, both weighted and unweighted UniFrac and Bray–Curtis dissimilarities showed significant community‐level separation between them and a pronounced difference in Proteobacteria, Firmicutes, and Bacteroidota was observed. Through the LEfSe tool, potential microbial biomarkers associated with a specific type of lesion were identified. In particular, GIST samples were significantly enriched in the phylum Proteobacteria compared to microGISTs. Several pathways involved in sugar metabolism were also highlighted in GISTs; this was expected as cancer usually displays high aerobic glycolysis in place of oxidative phosphorylation and rise of glucose flux to promote anabolic request. Our results highlight that specific differences do exist in the tissue microbiome community between GIST and benign lesions and that microbiome restructuration can drive the carcinogenesis process.

Risk-Related Genes and Associated Signaling Pathways of Gastrointestinal Stromal Tumors.

PurposeKnowledge on the potential association between differential gene expression and risk of gastrointestinal stromal tumors (GISTs) is currently limited. We used bioinformatics tools to identify differentially expressed genes in GIST samples and the related signaling pathways of these genes.Patients and MethodsThe GSE136755 dataset was obtained from the GEO database and differentially expressed genes (CENPA, CDK1, TPX2, CCNB1, CCNA2, BUB1, AURKA, KIF11, NDC80) were screened using String and Cytoscape bioinformatics tools. Then, three groups of eight patients at high, intermediate and low risk of GIST were selected from patients diagnosed with GIST by immunohistochemistry in our hospital from October 2020 to March 2021. Differential expression of CDK1 and BUB1 was verified by comparing the amount of expressed p21-Activated kinase 4 (PAK4) protein in pathological sections.ResultsSPSS26.0 analysis showed that the expression level of PAK4 in GISTs was significantly higher than in normal tissues and paratumoral tissues and there was significant difference among the three groups of patients (P < 0.01). PAK4 levels in paratumoral and normal tissues were negligible with no significant difference between the tissues.ConclusionCENPA, CDK1, TPX2, CCNB1, CCNA2, BUB1, AURKA, KIF11 and NDC80 gene expression can be used as biomarkers to assess the risk of gastrointestinal stromal tumors whereby expression increases gradually with the increased risk of GIST formation. The genes encode proteins that regulate the division, proliferation and apoptosis of gastrointestinal stromal tumors mainly through PI3K/AKT, MARK, P53, WNT and other signaling pathways.

Cytogenetic and molecular analyses of 291 gastrointestinal stromal tumors: site-specific cytogenetic evolution as evidence of pathogenetic heterogeneity.

Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm with variable behavior. An increased understanding of the tumor pathogenesis may improve clinical decision-making. Our aim was to obtain more data about the overall chromosome aberrations and intratumor cytogenetic heterogeneity in GIST. We analyzed 306 GIST samples from 291 patients using G-banding, direct sequencing, and statistics. Clonal chromosome aberrations were found in 81% of samples, with 34% of 226 primary tumors demonstrating extensive cytogenetic heterogeneity. 135 tumors had simple (≤5 changes) and 91 had complex (>5 changes) karyotypes. The karyotypically complex tumors more often were non-gastric (P < 0.001), larger (P < 0.001), more mitotically active (P = 0.009) and had a higher risk of rupture (P < 0.001) and recurrence (P < 0.001). Significant differences between gastric and non-gastric tumors were found also in the frequency of main chromosome losses: of 14q (79% vs. 63%), 22q (38% vs. 67%), 1p (23% vs. 88%), and 15q (18% vs. 77%). Gastric PDGFRA-mutated tumors, compared with gastric KIT-mutated, had a lower incidence of 22q losses (18% vs. 43%) but a higher rate of 1p losses (42% vs. 22%). The present, largest by far karyotypic study of GISTs provides further evidence for the existence of variable pathogenetic pathways operating in these tumors’ development.

CSPG4 Expression in GIST Is Associated with Better Prognosis and Strong Cytotoxic Immune Response.

Simple SummaryGastrointestinal stromal tumors (GIST) are the most frequent sarcomas of the gastrointestinal tract. Identification of novel prognostic and/or therapeutic targets is a major issue to overcome tyrosine kinase inhibitors resistances. CSPG4, a cell surface proteoglycan, emerged as a potential therapeutic target for immune therapy in different cancers, including sarcomas. CSPG4 expression has never been studied in GIST. In this work we analyzed CSPG4 mRNA expression in a large series of clinical GIST samples given the scarcity of disease (n = 309 patients). We find that high CSPG4 expression is independently associated with disease-free survival, and with an immune landscape favorable to induce strong cytotoxic immune response after NK cell stimulation. Our results suggest the potential value of CSPG4-specific chimeric antigen receptor-redirected cytokine-induced killer lymphocytes treatment in GIST, notably “CSPG4-high” tumors, and calls for preclinical validation, drug testing in vivo, then in clinical trials.The treatment of gastrointestinal stromal tumors (GIST) must be improved through the development of more reliable prognostic factors and of therapies able to overcome imatinib resistance. The immune system represents an attractive tool. CSPG4, a cell surface proteoglycan, emerged as a potential therapeutic target for immune therapy in different cancers, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in sarcomas. CSPG4 expression has never been studied in GIST. We analyzed CSPG4 mRNA expression data of 309 clinical GIST samples profiled using DNA microarrays and searched for correlations with clinicopathological and immune features. CSPG4 expression, higher in tumors than normal digestive tissues, was heterogeneous across tumors. High expression was associated with AFIP low-risk, gastric site, and localized stage, and independently with longer postoperative disease-free survival (DFS) in localized stage. The correlations between CSPG4 expression and immune signatures highlighted a higher anti-tumor immune response in “CSPG4-high” tumors, relying on both the adaptive and innate immune system, in which the boost of NK cells by CSPG4-CAR.CIKs might be instrumental, eventually combined with immune checkpoint inhibitors. In conclusion, high CSPG4 expression in GIST is associated with better DFS and offers an immune environment favorable to a vulnerability to CAR.CIKs.

Imatinib Regulates miR-483-3p and Mitochondrial Respiratory Complexes in Gastrointestinal Stromal Tumors.

Metabolic adaptation to increased oxidative phosphorylation (OXPHOS) has been found in gastrointestinal stromal tumor (GIST) upon imatinib treatment. However, the underlying mechanism of imatinib-induced OXPHOS is unknown. Discovering molecules that mediate imatinib-induced OXPHOS may lead to the development of therapeutic strategies synergizing the efficacy of imatinib. In this study, we explored the role of microRNAs in regulating OXPHOS in GIST upon imatinib treatment. Using a microarray approach, we found that miR-483-3p was one of the most downregulated miRNAs in imatinib-treated tumors compared to untreated tumors. Using an extended series of GIST samples, we further validated the downregulation of miR-483-3p in imatinib-treated GIST samples by RT-qPCR. Using both gain- and loss-of-function experiments, we showed that miR-483-3p could regulate mitochondrial respiratory Complex II expression, suggesting its role in OXPHOS regulation. Functionally, miR-483-3p overexpression could rescue imatinib-induced cell death. These findings provide the molecular link for imatinib-induced OXPHOS expression and the biological role of miR-483-3p in regulating cell viability upon imatinib treatment.

Clinicopathological value of long non-coding RNA profiles in gastrointestinal stromal tumor.

BackgroundLong non-coding RNAs (lncRNAs) have been implicated in diagnosis and prognosis in various cancers. However, few lncRNA signatures have been established for prediction of gastrointestinal stromal tumors (GIST). We aimed to explore a lncRNA signature profile that associated with clinical relevance by mining data from Gene Expression Ominus (GEO) and Surveillance, Epidemiology, and End Results (SEER) Program.MethodsUsing a lncRNA-mining approach, we performed non-negative matrix factorization (NMF) consensus algorithm in Gastrointestinal stromal tumors (GISTs) cohorts (61 patients from GSE8167 and GSE17743) to cluster LncRNA expression profiles. Comparative markers selection, and Gene Set Enrichment Analysis (GSEA) algorithm were performed between distinct molecular subtypes of GIST. The survival rate of GIST patients from SEER stratified by gender were compared by Kaplan–Meier method and log-rank analysis. lncRNA-mRNA co-expression analysis was performed by Pearson correlation coefficients (PCC) using R package LINC. Somatic copy number alterations of GIST patients (GSE40966) were analyzed via web server GenePattern GISTIC2 algorithm.ResultsA total of four lncRNA molecular subtypes of GIST were identified with distinct biological pathways and clinical characteristics. LncRNA expression profiles well clustered the GIST samples into small size (<5 mm) and large size tumors (>5 mm), which is a fundamental index for GIST malignancy diagnosis. Several lncRNAs with abundant expression (LRRC75A-AS1, HYMAI, NEAT1, XIST and FTX) were closely associated with tumor size, which may suggest to be biomarkers for the GIST malignancy. Particularly, LRRC75A-AS1 was positively associated with tumor diameters and suggested an oncogene in GIST. Co-expression analysis suggested that chromosome region 17p11.2–p12 may contribute to the oncogenic process in malignant GIST. Interestingly, the gender had a strong influence on clustering by lncRNA expression profile. Data from the Surveillance, Epidemiology, and End Results (SEER) Program were further explored and 7983 patients who were diagnosed with GISTs from 1973 to 2014 were enrolled for analysis. The results also showed the favorable prognosis for female patients. The survival rate between male and female with GIST was statistically significant (P < 0.0001). Gene set enrichment analysis (GSEA) indicated distinct pathways between female and male, and malignant GIST was associated with several cancer metabolism and cell cycle associated pathways.ConclusionsThis lncRNAs-based classification for GISTs may provide a molecular classification applicable to individual GIST that has implications to influence lncRNA markers selection and prediction of tumor progression.

Immunohistochemical features of gastrointestinal stromal tumors and the role of expression of p16ink4A, Ki-67, VEGF and MMP-9 in their behavior

Aim: to clarify the prognostic value of cytoplasmic p16ink4A, VEGF, MMP-9 and Ki-67 expressions in gastrointestinal stromal tumors (GISTs) and connection of different levels of these markers expression with aggressive transformation of GISTs. Materials and methods. Our study included 36 samples of primary tumors and 10 relapses of GIST and metastases in liver after primary combined treatment (surgery and chemotherapy with imatinib). The immunohistochemical study was performed with 4 primary antibodies: Ki-67, p16ink4A, VEGF and MMP-9. We used formalin fixed and paraffin embedded (FFPE) tissue samples for immunohistochemical study. Results. In our study we showed significant connection between levels of cytoplasmic expression of p16ink4A in primary GISTs and such markers of tumor aggressive behaviour as Ki-67, MMP-9 and VEGF (Fisher’s exact P-value = 0.000753; 0.000101 and 0.000048 respectively). Between cytoplasmic expression of p16ink4A and VEGF and also between p16ink4A and MMP-9 strong direct correlation was found (γ = 0.829, P &lt; 0.05 and rs = 0.961, P &lt; 0.05 respectively). The correlation between expression of Ki-67 and p16ink4A was also direct and strong (rs = 0.754, P &lt; 0.05), but with some exclusions, that’s why this correlation needs further investigation in larger groups with preciser molecular analysis. Analysis of metastatic GISTs samples showed prominent levels of MMP-9 and VEGF expression. Conclusions. Our study has shown very important role of cytoplasmic expression of p16ink4A in GIST as one of the markers of aggressive behavior, which can be used in complex with other markers for more accurate prognosis of GISTs progression. Prominent levels of MMP-9 and VEGF expression in metastatic GISTs can be a marker of resistance to imatinib. So probably evaluation of MMP-9 and VEGF expression can be used as a tool for correct choice of chemotherapy for patients with GISTs.

Mutation Status and Immunohistochemical Correlation of EGFR Mutations in Gastrointestinal Stromal Tumors.

Being one of the leading causes of cancer deaths worldwide and their resistance to conventional treatment methods, made gastrointestinal stromal tumors (GISTs) one of the hot topics in medical research areas in the past decade. To investigate molecular alterations underlying the tumor is of great importance to be able to develop new, targeted treatment options. In this study, GIST samples obtained from 40 Turkish patients were analyzed for actionable epidermal growth factor receptor (EGFR) mutations that are related to treatment regimes in non small cell lung cancer (NSCLC) to understand whether EGFR expression is altered in GISTs. Established alterations in EGFR can make the use of tyrosine kinase inhibitors possible, which are currently used in cancer therapy, especially in NSCLC. Our results indicated that EGFR mutations are rare in GISTs. Further research is needed to sequence whole coding regions of the gene to investigate new actionable mutations in EGFR in an increased sample size.

RP11-616M22.7 recapitulates imatinib resistance in gastrointestinal stromal tumor

Emerging evidence has shown that long non-coding RNAs (lncRNAs) play crucial roles in human cancers. However, systematic characterization of lncRNAs and their roles in gastrointestinal stromal tumor (GIST) therapy have been lacking. We performed high-throughput RNA sequencing (RNA-seq) of 20 GIST and paired adjacent normal samples. We characterized the transcriptional landscape and dysregulation of lncRNAs in GIST. We identified 866 upregulated and 1,268 downregulated lncRNAs in GIST samples, the majority of which were GIST-specific over other cancer types. Most hallmarks were found to be dysregulated in GIST samples, and lncRNAs were highly associated with cancer-related hallmarks. RP11-616M22.7 was identified to increase in imatinib-resistant samples compared to those in non-resistant samples. Further analysis revealed that RP11-616M22.7 was closely associated with the Hippo signaling pathway. By treating GIST cells with different doses of imatinib, we verified that RP11-616M22.7 knockdown promotes the sensitivity of tumor cells, whereas RP11-616M22.7 overexpression induces resistance to imatinib. We further confirmed reducing of resistance to imatinib by knocking down RP11-616M22.7 in vivo. Additionally, RP11-616M22.7 was observed to interact with RASSF1 protein. Our study revealed that deficiency of RP11-616M22.7 was able to reduce resistance of the GIST cell response to imatinib treatment both in vitro and in vivo.

Fluorescent In Situ Hybridization Must be Preferred to pan-TRK Immunohistochemistry to Diagnose NTRK3-rearranged Gastrointestinal Stromal Tumors (GIST).

Tyrosine kinase inhibitors have revolutionized the treatment of patients with gastrointestinal stromal tumors (GISTs). Nevertheless, some GISTs do not contain any targetable KIT or PDGFRA mutations classically encountered in this field. Novel approved therapies targeting TRK chimeric proteins products of NTRK genes fusions consist in a promising approach to treat some patients with GISTs lacking any identified driver oncogenic mutation in KIT, PDGFRA or BRAF genes. Thus, an adequate testing strategy permitting to diagnose the rare NTRK-rearranged GISTs is required. In this work, we studied about the performances of pan-TRK immunohistochemistry (IHC) and NTRK1/2/3 fluorescent in situ hybridization in a series of 39 GISTs samples. Among 22 patients with GISTs lacking KIT or PDGFRA mutations, BRAFV600E IHC permitted to diagnose 2/22 (9%) BRAFV600E-mutated GISTs and, among the 20 KIT, PDGFRA, and BRAF wild type tumors, 1/20 (5%), NTRK3-rearranged tumor was diagnosed using NTRK3 fluorescent in situ hybridization. Pan-TRK IHC using EPR17341 and A7H6R clones was negative in this NTRK3-rearranged sample. Pan-TRK IHC was frequently positive in NTRK not rearranged tumors without (24 samples analyzed) or with (15 samples analyzed) KIT or PDGFRA mutations with major discrepancies between the 2 IHC clones (intraclass correlation coefficient of 0.3042). Given the new therapeutic opportunity offered by anti-TRK targeted therapies to treat patients with advanced cancers including GISTs, it is worth to extend molecular analysis to NTRK fusions testing in KIT, PDGFRA, and BRAF wild type GISTs. Pan-TRK IHC appears not relevant in this field but performing a simple NTRK3 fluorescent in situ hybridization test consists in a valuable approach to identify the rare NTRK3-rearranged GISTs treatable using anti-TRK therapies.

Single-cell transcriptome analysis revealed the heterogeneity and microenvironment of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. In this study, we performed single‐cell RNA sequencing (RNA‐seq) on intra‐ and peri‐tumor tissues from GIST patients with the aim of discovering the heterogeneity of tumor cells in GIST and their interactions with other cells. We found four predominating cell types in GIST tumor tissue, including T cells, macrophages, tumor cells, and NK cells. Tumor cells could be clustered into two groups: one was highly proliferating and associated with high risk of metastasis, the other seemed “resting” and associated with low risk. Their clinical relevance and prognostic values were confirmed by RNA‐seq of 65 GIST samples. T cells were the largest cell type in our single‐cell data. Two groups of CD8+ effector memory (EM) cells were in the highest clonal expansion and performed the highest cytotoxicity but were also the most exhausted among all T cells. A group of macrophages were found polarized to possess both M1 and M2 signatures, and increased along with tumor progression. Cell‐to‐cell interaction analysis revealed that adipose endothelial cells had high interactions with tumor cells to facilitate their progression. Macrophages were at the center of the tumor microenvironment, recruiting immune cells to the tumor site and having most interactions with both tumor and nontumor cells. In conclusion, we obtained an overview of the GIST microenvironment and revealed the heterogeneity of each cell type and their relevance to risk classifications, which provided a novel theoretical basis for learning and curing GISTs.

HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) arises from interstitial cells of Cajal (ICC) or their precursors, which are present throughout the gastrointestinal tract. Although gastric GIST is commonly indolent and small intestine GIST more aggressive, a molecular understanding of disease behavior would inform therapy decisions in GIST. Although a core transcription factor (TF) network is conserved across GIST, accessory TFs HAND1 and BARX1 are expressed in a disease state-specific pattern. Here, we characterize two divergent transcriptional programs maintained by HAND1 and BARX1, and evaluate their association with clinical outcomes. We evaluated RNA sequencing and TF chromatin immunoprecipitation with sequencing in GIST samples and cultured cells for transcriptional programs associated with HAND1 and BARX1. Multiplexed tissue-based cyclic immunofluorescence and IHC evaluated tissue- and cell-level expression of TFs and their association with clinical factors. We show that HAND1 is expressed in aggressive GIST, modulating KIT and core TF expression and supporting proliferative cellular programs. In contrast, BARX1 is expressed in indolent and micro-GISTs. HAND1 and BARX1 expression were superior predictors of relapse-free survival, as compared with standard risk stratification, and they predict progression-free survival on imatinib. Reflecting the developmental origins of accessory TF programs, HAND1 was expressed solely in small intestine ICCs, whereas BARX1 expression was restricted to gastric ICCs. Our results define anatomic and transcriptional determinants of GIST and molecular origins of clinical phenotypes. Assessment of HAND1 and BARX1 expression in GIST may provide prognostic information and improve clinical decisions on the administration of adjuvant therapy.

Low ETV1 mRNA expression is associated with recurrence in gastrointestinal stromal tumors

Although the majority of gastrointestinal stromal tumors (GISTs) possess KIT mutations that induce constitutive signal transduction, the clinical outcomes are variable. The ETS translocation variant 1 (ETV1) gene encodes a transcription factor that is reported to cooperate with KIT in GISTs. However, the clinical role of ETV1 is largely unknown. The aim of this study was to examine ETV1 expression and its associations with clinical features in GISTs. We conducted a cohort study involving 64 patients with GISTs who underwent surgical resection between October 2008 and February 2015. ETV1 mRNA expression was compared with that in non-GISTs and was analyzed among risk classifications or clinical outcomes. The GIST samples exhibited significantly higher ETV1 mRNA expression than the non-GIST samples (P < 0.0001). Sixty-four GISTs were stratified into high or low ETV1 mRNA expression groups based on the median relative abundance of ETV1 mRNA. The multivariate analysis showed that low ETV1 expression, as well as tumor size and mitotic index, was an independent factor of recurrence (hazard ratio: 8.1). Patients with high ETV1 expression achieved significantly longer recurrence-free survival (RFS) times than those with low ETV1 expression (P = 0.025). Our study revealed that low ETV1 expression is an independent factor of recurrence after surgery in patients with GISTs, and thus, low ETV1 expression might be a marker of more aggressive malignant GISTs.

Abstract 5181: Therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumor (GIST), with DS-6157a, an antibody-drug conjugate (ADC)

Abstract More than 85% of GISTs are driven by activating mutations in KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Currently, the only approved treatments for GIST are KIT directed tyrosine kinase inhibitors (TKIs). However, treatment with approved TKIs eventually results in disease progression most often due to the development of secondary resistance mutations in KIT. In addition, these agents have limited activity in PDGFRA mutant GIST and KIT/PDGFRA wild type (WT) GIST as primary therapy. Therefore, it is essential to develop novel therapeutic strategies with different modes of action in advanced GIST. G protein-coupled receptor 20 (GPR20) is an orphan GPCR selectively and abundantly expressed in GIST. GPR20 expression is regulated by FOXF1 and ETV1, transcription factors that play critical roles in KIT-driven GIST initiation, proliferation, and survival. We hypothesize that GPR20 is a potential therapeutic target for ADC development for the treatment of GIST. In this study, 1) GPR20 and KIT protein expression was assessed by IHC staining on GIST samples from DFCI (n=144) and NCCHE (n=100) as well as on normal and malignant tissue microarrays obtained commercially, and 2) an anti-GPR20 ADC (DS-6157a) was generated to evaluate antitumor activity in GIST models and to assess safety. GPR20 was expressed in more than 88% of the GIST samples analyzed, with higher expression levels in samples that: (I) received multiple treatment lines compared to naïve/early treated samples, (II) expressed higher KIT levels, (III) were small intestinal GIST, and/or (IV) had no KIT mutation, including succinate dehydrogenase (SDH) deficient GIST and neurofibromatosis type 1 (NF1)-associated GIST. The interstitial cells of Cajal were the only normal cells positive for GPR20. Normal mast cells expressed KIT but not GPR20. DS-6157a is an ADC composed of a humanized anti-GPR20 antibody, a Gly-Gly-Phe-Gly tetra-peptide-based linker, and a DNA topoisomerase I (TOP1) inhibitor Dxd. DS-6157a exhibited GPR20 expression-dependent cell growth-inhibitory activity and induced tumor regression with dosing at 3 to 10 mg/kg in multiple GIST xenograft models. In addition, DS-6157a showed antitumor activity in a GIST patient-derived xenograft model that was resistant to imatinib, sunitinib, and regorafenib. In vitro, DS-6157a induced TOP1 inhibitor-associated markers of DNA damage (phosphorylation of Chk1) and apoptosis (cleaved PARP) in GPR20 expressing cells. In preclinical toxicology studies using rats and cynomolgus monkeys, the pharmacokinetics and safety profile of DS-6157a were favorable at up to 200 mg/kg and 30 mg/kg, respectively. These data support the clinical development of DS-6157a as a potential novel GIST therapy with activity in patients that are resistant, refractory, or intolerant to approved TKIs. Citation Format: Kenji Iida, Amr H. Abdelhamid, Akiko Kawano Nagatsuma, Tomoko Shibutani, Satoru Yasuda, Michiko Kitamura, Chiharu Hattori, Manabu Abe, Jun Hasegawa, Takuma Iguchi, Tsuyoshi Karibe, Takashi Nakada, Koichiro Inaki, Reiko Kamei, Yuki Abe, Jessica L. Andersen, Sandro Santagata, Matthew L. Hemming, Suzanne George, Toshihiko Doi, Atsushi Ochiai, George D. Demetri, Toshinori Agatsuma. Therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumor (GIST), with DS-6157a, an antibody-drug conjugate (ADC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5181.