Epigenetic landscape of gastrointestinal stromal tumors: Mechanistic insights and therapeutic opportunities

49 Background: Gastrointestinal stromal tumours (GISTs) contain oncogenic KIT or PDGFRA tyrosine kinase (TK) mutations leading to disturbance of downstream signaling pathways that contribute to GIST pathogenesis. Additional genetic aberrations were found in GISTs, demonstrating the involvement of other genes important in tumor progression. The aim of the study was to evaluate the prognostic relevance of different TK mutations in GISTs and to analyze the additional genetic aberrations in GISTs according to mutational status. Methods: 180 GIST patients were examined for KIT (9, 11, 13, 17 exons) and PDGFRA mutations (12, 14, 18 exons) by direct sequencing of DNA obtained from microdissected tumor sections. Tumor tissue DNA from 44 GISTs patients was screened for loss of heterozygosity (LOH) at 11 microsatellite loci on 1p, 9p, 14q, 15q and 22q arms. Results: KIT and PDGFRA mutations were identified in 76.1% and 10% of GISTs, respectively. 13.9% GISTs had no mutations (wild type). Most of KIT mutations were located in exon 11 (65%) and exon 9 (9.4%). Prognostic significance of TK mutations was evaluated. There was a trend of better survival for patients with PDGFRA mutation then with KIT mutation or wild type GISTs. The higher overall survival prior to target therapy was shown for patients with duplication or point mutation in KIT exon 11 in comparison to exon 11 deletion. Analysis of LOH revealed allelic deletions in 85% of GISTs, more frequently at 14q arm. There was a different LOH pattern in subgroups of GISTs. GISTs with PDGFRA mutation and with KIT exon 11 point mutation had LOH at 14q, while GISTs with KIT exon 11 deletions revealed high LOH frequency also on 22q, 15q and 1p arms. LOH in wild type GISTs occurred on all chromosomes with low frequency. LOH on 9p was found exclusively in metastatic and recurrent GISTs. Specific gene loci of frequent LOH were identified on 9p, 15q and 22q that may be contributed to GIST progression. Conclusions: Specific mutations are associated with GISTs prognosis. Tumors with point mutations and duplications in KIT exon 11 are associated with a better survival then GISTs with other KIT mutations. Specific pattern of allelic deletions was identified in subgroups of GISTs according to type of mutation and tumor progression. No significant financial relationships to disclose.

30 Background: Immunohistochemical staining for KIT (CD117) is used as part of diagnostic tool for GIST. Approximately 95% of GIST are known to be positive for CD117. Studies have shown that not all CD117-positive GIST carry KIT mutations nor does CD117 negativity rule out KIT or PDGFRA mutations. We performed a systematic review to investigate the positive and negative predictive values of KIT immunostaining for KIT mutations in GIST. Methods: A Medline search of the MeSH terms “KIT mutation” and “GIST” and “prevalence” yielded 54 articles from year 1999 to 2011. English language studies on GIST with data available for KIT immunostaining as well as KIT gene mutations were included. Studies lacking data on either CD117 positivity or KIT mutations were excluded. Immunostaining was done using Dako polyclonal rabbit antibody or PKCh monoclonal mouse antibody in eligible studies. Denaturing high-pressure liquid chromatography (DHPLC) was used to screen mutations in majority of eligible studies, and ABI Prism Genetic Analyzer to sequence DNA. Results: 6 studies including 708 CD117-positive GIST patients were eligible for analysis of positive predictive value of KIT immunostaining for KIT mutations and 5 studies including 47 CD117-negative GIST patients for negative predictive value. Many studies were excluded due to insufficient data on CD117 positivity or KIT mutational analysis. 72.8% of CD117-positive GIST tumors carried KIT mutations (exon 11 62.2%, exon 9 10.2%, exon 13 1.8% and exon 17 1%), and 4.1% harbored PDGFRA mutations. In contrast, 74.5% of CD117-negative GIST tumors did not have KIT mutations. 25.5% of CD117-negative GIST harbored KIT exon 11 mutations (no mutations in exon 9, 13 or 17 were observed). In addition, 30% of CD117-negative GIST were found to have PDGFRA mutations. Conclusions: PDGFRA mutations were found to be 7 times more common in CD117-negative than in CD117-positive GIST (30% vs. 4.1%). Over half (55.5%) of CD117-negative GIST carried KIT exon 11 or PDGFRA mutations. We recommend mutational analysis in all tumors clinically suspected of GIST and that are CD117-negative for two reasons: 1. in order to make a diagnosis, and 2. to help tailor therapy.

Most of the gastrointestinal stromal tumors (GISTs) have gain-of-function mutations in the KIT gene, which can be used as a prognostic marker for the biological behavior of tumors, predictive marker for the response of tyrosine kinase inhibitors, and diagnostic marker. Researchers have focused on PDGFRA mutations because of both their prognostic and predictive potential and DOG1 positivity for diagnosis on GISTs. The aim of this study is to investigate the effect DOG1, PDGFRA, and KIT mutations on the prediction of the outcome for GIST management. Polymerase chain reaction was performed for KIT gene exons 9, 11, 13, and 17 and PDGFRA gene exons 12 and 18 with the genomic DNA of 46 GIST patients, and amplicons were sequenced in both directions. Immunocytochemical stainings were done by using primary antibodies. Molecular analysis revealed that the KIT mutation was observed in 63% of all cases, while the PDGFRA mutation was observed in 23.9% of cases. Significant relationships were found between age and KIT mutation, tumor location and KIT mutations, and tumor location and PDGFRA mutations (p ≤ 0.05). DOG1 positivity was detected in 65.2% of all GISTs and DOG1-positive cells had a higher KIT mutation ratio than DOG1-negative cells (p ≤ 0.05). KIT gene exon 11 mutations in DOG1-positive cells was higher than DOG1-negative cells (p ≤ 0.05). Conversely, KIT gene exon 13 mutations were higher in DOG1-negative cells than DOG1-positive cells (p ≤ 0.05). In this study, KIT mutation frequency was found similar with the European population; conversely, PDGFRA mutation frequency was similar with an Asian-Chinese-based study. KIT/PDGFRA mutations and tumor location can be used for the prediction of tumor behavior and the management of disease in GISTs. DOG1 positivity might be a candidate marker to support KIT and PDGFRA mutations, due to the higher DOG1 positivity in KIT exon 11 mutant and stomach- and small intestine-localized GISTs.

Aims: To assess the relation between KIT and PDGFRA mutations and the site of origin, histological phenotype, and pathomorphologically determined risk assessment in gastrointestinal stromal tumours (GISTs). Methods: A series...

The diagnosis of gastrointestinal stromal tumor (GIST) is currently based on morphologic features and immunohistochemical demonstration of KIT (CD117). However, some tumors (in our estimation approximately 4%) have clinicopathologic features of GIST but do not express KIT. To determine if these lesions are truly GISTs, we evaluated 25 tumors with clinical and histologic features typical of GIST, but with negative KIT immunohistochemistry, for KIT and PDGFRA mutations using DNA extracted from paraffin-embedded tissue. Most tumors originated in the stomach (N = 14) or omentum/mesentery (N = 5). The neoplasms were composed of epithelioid cells (13 cases), admixed epithelioid and spindle cells (8 cases), or spindle cells (4 cases). Absence of KIT expression was confirmed by immunoblotting in 5 cases. Tumor karyotypes performed in 4 cases were noncomplex with monosomy 14 or 14q deletion, typical of GIST. Mutational analysis revealed PDGFRA and KIT mutations in 18 and 4 tumors, respectively, whereas 3 tumors did not have apparent KIT or PDGFRA mutations. The PDGFRA mutations primarily involved exon 18 (N = 15) and included 11 tumors with missense mutation in codon 842 (PDGFRA D842V or D842Y). In conclusion, a small subset of GISTs with otherwise typical clinicopathologic and cytogenetic features do not express detectable KIT protein. When compared with KIT-positive GISTs, these KIT-negative GISTs are more likely to have epithelioid cell morphology, contain PDGFRA oncogenic mutations, and arise in the omentum/peritoneal surface. Notably, some KIT-negative GISTs contain imatinib-sensitive KIT or PDGFRA mutations; therefore, patients with KIT-negative GISTs should not, a priori, be denied imatinib therapy.

Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract [1]. About 75% of GISTs harbor activating mutations of KIT and About 10% of GISTs harbor PDGFRA activating mutations [2]. PDGFRA is the second most mutated oncogene in GIST and are particularly associated with gastric tumors [3,4]. The introduction of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of GISTs, with specific efficacy seen in tumors with PDGFRA and KIT mutations. However, different PDGFRA mutations can impact the response to these therapies differently [5,6]. For example, the PDGFRA D842V mutation is resistant to imatinib but may respond to newer drugs like avapritinib. Therefore, it is critically important to identify any specific PDGFRA mutations in tumor molecular testing. Here, we report a case with an unusual PDGFRA mutation with a discussion about its NGS data analysis.

10560 Background: KIT or PDGFRA mutations are found in gastrointestinal stromal tumors (GIST) and are correlated with imatinib (IM) effects. Most reports studied only hot-spots of each gene using paraffin sections and extracted DNA. Here we examined full sequence of the genes using frozen section of GI mesenchymal tumors. Methods: Histologic Dx was done with HE and immunohistochemistory (IH). Using RNA obtained from fresh samples of serial 209 pts with GIST (n=174), myogenic (21), neurogenic tumors (11), or GI sarcoma (3), KIT or PDGFRA cDNA was amplified by RT-PCR and fully sequenced. Results: Tumors other than GIST showed no KIT-immunoreactivity, and had no-PCR-products of KIT (32) or wild type (WT) (3). PDGFRA of these tumors is WT. In IH, KIT was positive in 94% of GIST and CD34 in 84%. 7 GIST showed no-PCR-products of KIT, 8 WT of the genes, 142 KIT, or 17 PDGFRA mutations. Mutations are mutually exclusive. No-PCR was associated with IM-responded GIST (6 out of 15 IM-treated pts), in which KIT expression in IH was scarcely seen. WT are found in 3 juvenile GIST or 4 NF-1, and only 1 adult GIST showed WT. KIT mutations are found in exon (Ex) 9 (10 pts), Ex 11 (125), Ex 13 (3), or Ex 17 (4). All Ex 9 mutations are typical insertion mutations, found in small intestine (8) or colon (2), and associated with poor prognosis. Ex 13 and 17 mutations are missense and Ex 11 showed various type of mutations including deletion, insertion and missense mutations. These mutations distributed throughout GI tract. Deletion mutations in Ex 11 are associated with high recurrence rates (38/64 pts) and poor prognosis. 7 pts showed insertion mutation in the distal part of Ex 11. PDGFRA mutations found in Ex 12 (4 pts) or Ex18 (13) are missense mutations and associated with gastric location, epithelioid histology (10/16), low mitotic activity and relatively indolent clinical features. Conclusions: KIT mRNA and protein are sometimes depressed in IM-responded lesions, which causes difficulty in mutation research. Mutations in KIT and PDGFRA are specific for GIST and WT GIST is extremely rare in adult pts except NF-1. No significant financial relationships to disclose.

Mutational analysis guides therapeutic decision-making for patients with advanced-stage gastrointestinal stromal tumors (GISTs). A total of 491 patients with GISTs were retrospectively included, and their genomic profiles were determined by targeted NGS of 73 or 1,021 gene panels. KIT mutations were identified in 84.7% (416/491) of patients with GISTs, and PDGFRA mutations were identified in 4.5% (22/491) of patients. Among the patients with KIT mutant-GISTs, most had KIT mutations in exon 11 (81.3%, 338/416). PDGFRA mutations were located mainly in exon 18 (77.3%, 17/22). In the remaining 11% of patients with GISTs without KIT or PDGFRA mutations (wild-type GISTs), BRAF and NF1 were the most commonly mutated genes. Compared with KIT/PDGFRA-mutant GISTs, wild-type GISTs were associated with younger age, a greater proportion of female patients, and lower levels of copy number variations. Notably, the concomitant alterations of KIT/PDGFRA-mutant or wild-type GISTs were similar when sequencing data from a 73-gene panel and a 1,021-gene panel were analyzed. The genetic landscape of patients with treatment-naïve GISTs at different locations was described by using a 73-gene panel, and compared with patients with gastric GISTs, patients with nongastric GISTs had higher levels of copy number variations but a lower proportion of PDGFRA mutations. Analysis of the genomic profiles before and after imatinib treatment revealed KIT T670I mutation, as well as ATM and JAK2 mutations, as potential underlying mechanisms of resistance. Using a 73-gene panel, we characterized the molecular characteristics of GISTs and revealed a correlation with their clinical features. Moreover, KIT/PDGFRA-dependent and KIT/PDGFRA-independent mechanisms underlying resistance to imatinib were explored. Overall, our 73-gene panel is sufficient for clinical application in cases of GISTs.

Gastrointestinal Stromal Tumors: Disease and Treatment Update

In the present study, we investigated the association of PDGFRA and KIT mutations as well as PDGFRA immunohistochemical expression with clinicopathologic features and prognosis in a series of gastrointestinal stromal tumors (GISTs). Tumor DNA from 40 GISTs was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17, and in PDGFRA exons 12 and 18. Tissue sections were stained with polyclonal anti-PDGFRA antibody. KIT mutations occurred in 26 cases. There were 13 deletions, 6 substitutions, 3 deletion-substitutions, 3 duplications and 1 insertion. Tumors with KIT deletions/insertion were large with a high mitotic index (MI), and were associated with a high rate of symptoms at diagnosis, invasion into adjacent organs, distant metastasis, relapse and a short disease-free survival (DFS). PDGFRA mutations occurred in 6 gastric GISTs. There were 4 deletions and 2 substitutions. Tumors with PDGFRA mutations were small, with a low MI and Ki67 score, and were associated with a very low rate of symptoms at diagnosis, invasion into adjacent organs and distant metastasis. PDGFRA immunopositivity was found in 23 cases: a peculiar 'dotlike' staining was found in 5 out of 6 PDGFRA mutated cases. Patients with positive PDGFRA immunostaining had a longer DFS than those with negative staining. Our data confirm that the type of KIT mutation is associated with various clinicopathologic features of GISTs, and indicate that PDGFRA mutations are associated with rather indolent tumors. PDGFRA immunopositivity reflects PDGFRA mutational status and is associated with a favorable outcome.

9010 Background: IM is effective in the majority of pts with GIST, but limited data exist on KIT- GIST. S0033 aimed to evaluate differences between two initial dose levels of IM (400 vs. 800 mg/day) in pts with GIST expressing CD117 (KIT+), a subset of pts with KIT- GIST were treated. This abstract describes the outcomes of these pts and compares them with the KIT+ pts treated. Methods: Central pathology review was performed by a single pathologist on tumor specimens retrospectively, and data from S0033 (746 pts randomized) was analyzed. Pre-treatment tumor samples from 344 pts were also examined for mutations of KIT or PDGFRA. Results: Pathology has been reviewed on 414 pts: 377 (91%) had KIT+ GIST, 14 (3%) KIT- GIST, and 16 (4%) leiomyosarcoma/other subtype. Eight of the KIT- GISTs were genotyped and mutations in KIT or PDGFRA were noted in 4 and 3, respectively. (mutation frequency 87.5% for KIT- GISTs). Twelve non-GIST sarcomas had no mutations. The response rate and PFS in the pts with KIT- GISTs was not significantly different than that seen in KIT+ GISTs (43% vs. 49%, estimated PFS at 2 yrs). Pts with leiomyosarcoma or other histology have a significantly worse outcome than either KIT+ or KIT- GIST pts, with PFS 13% at 2 yrs and a median survival of about eight months compared to GIST pts for whom median survival has still not been reached. A significant difference in overall survival was noted in favor of the KIT+ GIST vs. KIT- GIST (77% vs. 57%, estimated OS at 2 yrs, p<0.01), although this was an unplanned subset analysis. Toxicities of all grades were similar across in the various histological groups. Conclusion: Our results indicate that IM therapy can provide clinical benefit for pts with KIT- GIST. The high incidence of kinase mutations in KIT- GIST support a therapeutic trial of IM for all GIST pts regardless of CD117 expression. There is no evidence of significant activity with IM for the non-GIST malignancies treated on this study. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis

Gastrointestinal stromal tumours (GISTs) typically harbour KIT or PDGFRA mutations; 15% of adult GISTs and >90% in children lack such mutations ('wild-type' GISTs). Paediatric and occasional adult GISTs show similar, distinctive features: multinodular architecture and epithelioid morphology, indolent behaviour with metastases, and imatinib resistance. Recent studies have suggested that these tumours can be identified by loss of succinate dehydrogenase subunit B (SDHB) expression. The aim of this study was to validate the predictive value of SDHB immunohistochemistry in a large genotyped cohort. SDHB expression was examined in GISTs with known genotypes: 179 with KIT mutations, 32 with PDGFRA mutations, and 53 wild type. Histological features were recorded without knowledge of genotype or SDHB status. SDHB was deficient in 22 (42%) wild-type GISTs. All other tumours showed intact SDHB expression. All SDHB-deficient GISTs with known primary sites arose in the stomach, and had multinodular architecture and epithelioid or mixed morphology. None of the wild-type GISTs with intact SDHB showed multinodular architecture, and only four (13%) had epithelioid morphology. SDHB-deficient GISTs are wild-type gastric tumours with distinctive histology. Immunohistochemistry for SDHB can be used to confirm the diagnosis of this tumour class. SDHB expression is retained in all GISTs with KIT and PDGFRA mutations.

GI stromal tumors (GISTs) are mesenchymal neoplasms with variable natural histories, originating in the GI tract, most commonly in the stomach. They are frequently characterized by KIT or platelet-derived growth factor receptor alpha (PDGFRA) oncogenic mutations. Surgical resection remains the cornerstone treatment for localized GISTs. Clinical trials have demonstrated the benefits of adjuvant imatinib in patients selected on the basis of recurrence risk and gene mutations, although the optimal duration of therapy is yet to be established. Some data suggest that longer durations of adjuvant imatinib (>3 years) may provide additional benefit, which is being investigated in ongoing trials. Management of imatinib-related adverse effects is essential during treatment, and longitudinal abdominal imaging is mandatory both during and after adjuvant therapy. Once GISTs are more advanced and unresectable, KIT- and PDGFRA-directed tyrosine kinase inhibitors (TKIs) become the key treatment in most patients with KIT mutation. Several TKIs have regulatory approval for advanced GISTs, but in most patients, resistance to TKIs eventually emerges, mainly from secondary resistance mutations in KIT. Each TKI has different coverage of oncogenic KIT mutations, suggested by preclinical and clinical findings, which has given rationale to an ongoing clinical trial that includes molecular selection as eligibility criteria. Furthermore, novel treatment approaches, from TKI combinations to an antibody-drug conjugate, are being investigated. Despite the significant advance in managing GISTs with KIT mutations, those without KIT or PDGFRA mutation, which consists of 10%-15% of patients with GIST, can be a clinical challenge in the advanced setting. These non-KIT/PDGFRA GISTs could be driven by genomic or epigenomic alterations in SDHx, NF1 mutations, and other genomic alterations. Non-KIT/PDGFRA GISTs are less responsive to currently available TKIs than GISTs driven by KIT/PDGFRA mutations, and each subset of non-KIT/PDGFRA GIST has distinctive biology and clinical features. Therefore, individualized, multidisciplinary, biology-based management and consideration for clinical trial enrollment are critical for non-KIT/PDGRFA GISTs.

Gastrointestinal stromal tumours (GIST) have an incidence of ~1.2 per 105 individuals per year in most countries. Around 80% of GIST have varying molecular changes, predominantly mutually exclusive activating KIT or PDGFRA mutations, but other, rare subtypes also exist. Localized GIST are curable, and surgery is their standard treatment. Risk factors for relapse are tumour size, mitotic index, non-gastric site and tumour rupture. Patients with GIST with KIT or PDGFRA mutations sensitive to the tyrosine kinase inhibitor (TKI) imatinib that are at high risk of relapse have improved survival with adjuvant imatinib treatment. In advanced disease, median overall survival has improved from 18 months to >70 months since the introduction of TKIs. The role of surgery in the advanced setting remains unclear. Resistance to TKIs arise mainly from subclonal selection of cells with resistance mutations in KIT or PDGFRA when they are the primary drivers. Advanced resistant GIST respond to second-line sunitinib and third-line regorafenib, as well as to the new broad-spectrum TKI ripretinib. Rare molecular forms of GIST with alterations involving NF1, SDH genes, BRAF or NTRK genes generally show primary resistance to standard TKIs, but some respond to specific inhibitors of the activated genes. Despite major advances, many questions in both advanced and localized disease remain unanswered.