WT GIST Research Articles

Taming the Wild-Type Gastrointestinal Stromal Tumor: Improved Tissue Culture.

Wild-type gastrointestinal stromal tumors (WT GIST) are most frequently characterized by succinate dehydrogenase (SDH) deficiency. Reliable ex vivo tumor models have been difficult to develop given the downstream metabolic effects of SDH deficiency. Improved tumor modeling approaches are needed to develop effective systemic treatment options for patients with WT GIST.See related article by Yebra et al., p. 187.

Gene duplication, rather than epigenetic changes, drives FGF4 overexpression in KIT/PDGFRA/SDH/RAS-P WT GIST

Gastrointestinal stromal tumours that are wild type for KIT and PDGFRA are referred to as WT GISTs. Of these tumours, SDH-deficient (characterized by the loss of SDHB) and quadruple WT GIST (KIT/PDGFRA/SDH/RAS-P WT) subgroups were reported to display a marked overexpression of FGF4, identifying a putative common therapeutic target for the first time. In SDH-deficient GISTs, methylation of an FGF insulator region was found to be responsible for the induction of FGF4 expression. In quadruple WT, recurrent focal duplication of FGF3/FGF4 was reported; however, how it induced FGF4 expression was not investigated. To assess whether overexpression of FGF4 in quadruple WT could be driven by similar epigenetic mechanisms as in SDH-deficient GISTs, we performed global and locus-specific (on FGF4 and FGF insulator) methylation analyses. However, no epigenetic alterations were detected. Conversely, we demonstrated that in quadruple WT GISTs, FGF4 expression and the structure of the duplication were intimately connected, with the copy of FGF4 closer to the ANO1 super-enhancer being preferentially expressed. In conclusion, we demonstrated that in quadruple WT GISTs, FGF4 overexpression is not due to an epigenetic mechanism but rather to the specific genomic structure of the duplication. Even if FGF4 overexpression is driven by different molecular mechanisms, these findings support an increasing biologic relevance of the FGFR pathway in WT GISTs, both in SDH-deficient and quadruple WT GISTs, suggesting that it may be a common therapeutic target.

Expression of cell cycle regulators and frequency of TP53 mutations in high risk gastrointestinal stromal tumors prior to adjuvant imatinib treatment.

Despite of multitude investigations no reliable prognostic immunohistochemical biomarkers in GIST have been established so far with added value to predict the recurrence risk of high risk GIST besides mitotic count, primary location and size. In this study, we analyzed the prognostic relevance of eight cell cycle and apoptosis modulators and of TP53 mutations for prognosis in GIST with high risk of recurrence prior to adjuvant treatment with imatinib. In total, 400 patients with high risk for GIST recurrence were randomly assigned for adjuvant imatinib either for one or for three years following laparotomy. 320 primary tumor samples with available tumor tissue were immunohistochemically analyzed prior to treatment for the expression of cell cycle regulators and apoptosis modulators cyclin D1, p21, p16, CDK4, E2F1, MDM2, p53 and p-RB1. TP53 mutational analysis was possible in 245 cases. A high expression of CDK4 was observed in 32.8% of all cases and was associated with a favorable recurrence free survival (RFS), whereas high expression of MDM2 (12.2%) or p53 (35.3%) was associated with a shorter RFS. These results were independent from the primary KIT or PDGFRA mutation. In GISTs with higher mitotic counts was a significantly increased expression of cyclin D1, p53 and E2F1. The expression of p16 and E2F1 significantly correlated to a non-gastric localization. Furthermore, we observed a significant higher expression of p21 and E2F1 in KIT mutant GISTs compared to PDGFRA mutant and wt GISTs. The overall frequency of TP53 mutations was low (n = 8; 3.5%) and could not be predicted by the immunohistochemical expression of p53. In summary, mutation analysis in TP53 plays a minor role in the subgroup of high-risk GIST before adjuvant treatment with imatinib. Strong expression of MDM2 and p53 correlated with a shorter recurrence free survival, whereas a strong expression of CDK4 correlated to a better recurrence free survival.

1422TiP - Phase II, singlearm, nonrandomized, and multicenter clinical trial of regorafenib (REG) as a single agent in the firstline setting for patients with metastatic and/or unresectable KIT/PDGFR wild-type GIST. A GEIS and ISG study

Approximately 10-15% of adults with GIST lack KIT or PDGFRA mutations. This KIT/PDGFRA wild-type (wt) GIST is considered a separate pathological entity and is highly clinically heterogeneous. Because most KIT/PDGFRA wt-GISTs do not respond to imatinib, new treatment approaches are needed. REG is an oral multikinase inhibitor that has been shown to improve progression-free survival (PFS) and disease control rate (DCR) vs placebo in patients with advanced GIST progressing after failure of imatinib and sunitinib (Demetri et al. Lancet 2013). This study was designed to assess the effectiveness of REG in patients with metastatic and/or unresectable KIT/PDGFR wt-GIST in the first-line setting. Trial design: Patients ≥18 years of age with histologically confirmed unresectable and/or metastatic KIT/PDGFR wt-GIST (lack of mutations in KIT exons 11,9,13,17 and PDGFR exons 12,18 confirmed by next-generation sequencing) are eligible. Other inclusion criteria include ≥1 measurable lesion (RECIST v1.1), ECOG PS 0-1, adequate bone marrow, liver, and renal function, not fulfilling any exclusion criteria, and written informed consent. FFPE-tumor blocks are to be provided. Collection of blood and fresh tumor samples for a translational substudy is optional. REG 160 mg will be administered once daily in a 3-weeks on, 1-week off schedule until disease progression, unacceptable toxicity, or investigator/patient decision to withdraw. Thirtythree patients (+ 20%) are to be recruited according to Simon Minmax 2stage Phase II design. Main endpoint is DCR relative to the historical control of imatinib in wt-GIST. Hypothesis 0: p0 = 73% (0% CR + 23% PR + 50% SD). Hypothesis 1: achieving a DCR of p1 = 90% with REG. Using error rates of a = 0.05 and b = 0.20, the first-stage sample size is 31 patients, with 26 patients as the upper limit for firststage drug rejection. Secondary outcomes include PFS, overall survival, response (CHOI criteria), correlation with translational research, early metabolic response by PETscan, and safety (CTCAE 4.03). Clinical trial identification: EudraCT Number: 2015-001048-12 Legal entity responsible for the study: Grupo Español de Investigación en Sarcomas (GEIS)

Limits of Dideoxysequencing in the Detection of Somatic Mutations in Gastrointestinal Stromal Tumors

Abstract Detection of mutations in cancer is particularly important in terms of proper treatment and targeted therapy. The aim of this study was the comparison of two methods: allele-specific PCR (AS-PCR) and dideoxysequencing applied for the identification of BRAF gene mutations in wild-type gastrointestinal stromal tumors (WT GISTs). We have optimized the conditions for the detection V600E mutation representing the c.1799 T>A substitution by AS-PCR and have used dideoxysequencing to verify our results. In nine cases, we were able to detect the mutation by AS-PCR approach; however, the mutations have been confirmed by dideoxysequencing in four cases only. AS-PCR is fast and low cost method for the detection of V600E mutation which was validated as a sensitive assay for the identification of the most common BRAF mutation in DNA extracted from paraffin-embedded tissue of WT GISTs.

Gastrointestinal Stromal Tumors (Gist) and Second Malignancies: a Novel ‘Sentinel Tumor"?

ABSTRACT Aim: The association between GIST and second cancers, both synchronous or asynchronous, remains uncertain either in frequency or in molecular pathogenesis and histotypes. The aim of this study was to evaluate the incidence of second malignancies in our series, with particular regard to molecular profiling. Methods: Clinical data were retrospectively collected in 96 patients with GIST treated at our institution between July 2002 and April 2014. Since 2010 molecular analysis of KIT (exon 9, 11 and 13) and PDGFRalfa (exon 12, 14 and 18) genes was performed. Results: Among 96 GIST patients, 33 (34.4%) had a second cancer (19 synchronous [57.6%], 7 methachronous [21.2%] and 7 previous [21.2%]). Most second tumors (19; 57.5%) raised from gastrointestinal (GI) tract (6 gastric, 4 colorectal, 2 esophageal, 2 duodenal, 1 hepatocellular, 1 colangiocarcinoma, 3 pancreatic). Extraintestinal second tumors included lung, breast, endometrial, prostate, urothelial, thyroid, peritoneal mesothelioma. In our series benign neoplasms were also included (1 meningioma; 2 inflammatory fibrous gastrointestinal polips). Molecular analysis was available for 16/33 patients: 3 had a WT GIST, 10 carried a KIT exon 11 mutation, 2 a PDGFR-a exon 14 mutation and 1 a PDGFR-a exon 18 mutation. Interestingly, exon 11 KIT mutations were mainly associated with GI adenocarcinomas (8/10), while WT GISTs were linked with neoplasms other than Gl. The two patients with PDGFR-a exon 14 mutation were both diagnosed to have a germline mutation with inflammatory fibrous gastrointestinal polips. In metachronous cases the mean time interval between GIST and second tumor was 22.8 months. The mean follow up was 48.7 months (range: 6 - 141 months). Conclusions: This is the largest series on this topic. The relation between exon 11 mutations and GI second tumors suggests to investigate a possible common molecular mechanism. Following the short interval between GIST diagnosis and onset of second neoplasms, the authors suggest to perform strict follow-up particularly in the first years after diagnosis. Disclosure: All authors have declared no conflicts of interest.

Impact of mutational status and other prognostic factors on survival in patients with advanced GIST treated with standard-dose imatinib (IM): Results from the BFR14 phase III trial of the French Sarcoma Group.

10548 Background: Factors predicting progression free survival (PFS) and overall survival (OS) of patients (pts) with advanced GIST treated with 400 mg daily dose IM were investigated. As mutational status was a secondary objective of the BFR14 study, we wanted to evaluate the added value of these genomic profiles to conventional clinical prognostic factors. Methods: 434 pts were included in this prospective multicenter trial from June 2002 to July 2009. Prognostic factors for survival were investigated in the cohort where the mutational status was available. A multivariate Cox model including baseline characteristics statistically significant in univariate were included in a backward procedure to identify independent prognostic factors for PFS then OS. Results: Mutational analysis were available in 322 pts. Material was insufficient in 55 cases (12.5%), incomplete results were obtained in 39 pts (9%). There were 196 KIT genetic alterations (exon 11: 173 pts, exon 9: 22 pts, exon 17: 1 pt), 6 GIST with a PDGFRA mutations and 26 GIST WT. The analysis of prognostic factors was performed in all mutated and non-mutated GIST (221 pts) except for pts with a KIT exon 17 and PDGFRa mutation. As of January 2013, median follow-up was 73 months (m) (CI95%:63; 86), 147 progressions (67%) were notified and 84 deaths (38%) occurred. The median PFS and OS were 12.3 m (CI95%: 2.1;32.7) and 54.9 m (CI95%:16.5;83.8) for WT GIST, 12.6m (CI95%: 6.1;30.8) and 55m (CI95%:33.5;83.0) for KIT exon 9 and 39.4m (CI95%: 31.4;54.4) and not reached for KIT exon 11, respectively. An initial low tumour volume, gender (female), and CD34 positivity were the three independent prognostic factors of a higher PFS. A higher OS was independently predicted by PS, low neutrophil counts, KIT exon 11 alterations, normal lymphocyte count, lower tumor size and female’s gender. Conclusions: GIST pts harboring a KIT exon 11 mutations have the better outcome but the mutational status is not the only prognostic factor influencing the outcome of pts. The clinical (gender, PS, tumor size) and biological characteristics (lymphocytes and neutrophil counts) remain critical for OS.

SDHA loss of function mutations in a subset of young adult wild-type gastrointestinal stromal tumors

BackgroundA subset of KIT/PDGFRA wild-type gastrointestinal stromal tumors (WT GIST) have been associated with alteration of the succinate dehydrogenase (SDH) complex II function. A recent report identified four non-syndromic, KIT/PDGFRA WT GIST harboring compound heterozygous or homozygous mutations in SDHA encoding the main subunit of the SDH complex II.MethodsNext generation sequencing was applied on five pediatric and one young adult WT GIST, by whole exome capture and SOLiD 3-plus system sequencing. The putative mutations were first confirmed by Sanger sequencing and then screened on a larger panel of 11 pediatric and young adult WT GIST, including 5 in the context of Carney triad.ResultsA germline p.Arg31X nonsense SDHA mutation was identified in one of the six cases tested by SOLiD platform. An additional p.D38V missense mutation in SDHA exon 2 was identified by Sanger sequencing in the extended KIT/PDGFRA WT GIST patients cohort. Western blotting showed loss of SDHA expression in the two cases harboring SDHA mutations, while expression being retained in the other WT GIST tumors. Results were further confirmed by immunohistochemistry for both SDHA and SDHB, which showed a concurrent loss of expression of both proteins in SDHA-mutant lesions, while the remaining WT tumors showed only loss of SDHB expression.ConclusionsGermline and/or somatic aberrations of SDHA occur in a small subset of KIT/PDGFRA WT GISTs, outside the Carney’s triad and are associated with loss of both SDHA and SDHB protein expression. Mutations of the SDH complex II are more particularly associated with KIT/PDGFRA WT GIST occurring in young adults. Although pediatric GIST consistently display alterations of SDHB protein expression, further molecular studies are needed to identify the crucial genes involved in their tumorigenesis.

Identification of single nucleotide variants in gastrointestinal stromal tumor KIT/PDGFRA wild-type (WT GISTs) by massively parallel sequencing.

10046 Background: Recently SDH variations were identified in KIT and PDGFRA wild type GISTs. However the causative genomic alterations of these GISTs still remain unknown. Massively parallel sequencing allows identifying novel putative variants. Methods: Whole transcriptome paired-end RNA sequencing was performed by Illumina GAIIx system using a 75 bases paired-end strategy on tumor samples of two young adults patients (P1 and P2) affected by gastric WT-GIST (age 28 and 30 years). Sequences were aligned with BWA against exon+junction references. SNVmix2 was used for SNP calling, identifying by this 2045 and 1780 coding non-synonymous novel single nucleotide variants (SNVs) in P1 and P2, respectively. After checking misalignments by SAMTools, the variants were filtered to increasing SNPcall confidence: SNVs with quality read score > 30 (error probability of 0.1%), total coverage > 40, and ratio between coverage of alterate base and total coverage >0.3 were labeled high confidence. Single point mutations were translated at the protein level and their likelihood of being disease-associated was computed with SNP&GO and confirmed with Sanger sequencing. Results: In both patients common mutations in SDHA and different private SNV were highlighted. Among the private disease-related SNVs we identified mutations in MYH9 (myosin heavy chain 9) and TPI1 (triosephosphate isomerase 1) in patient P1, and a mutation in OGDHL (oxoglutarate dehydrogenase-like) in P2. MYH9 is involved in cell motility and cytokinesis, while OGDHL is an enzyme of the Krebs cycle, as the SDH complex, and TPI1 is involved in glycolysis. Ongoing validation by Sanger sequencing on DNA from tumor and peripheral blood (PB) already confirmed the presence of heterozygous MYH9 K1775E in P1 and OGDHL V815M in P2, both in tumor and PB, showing that these mutations are germinal. Conclusions: Massively parallel RNA sequencing, followed by data analysis, allows to discover novel single nucleotide variants and to identify new potential target genes in WT-GIST.

A phase II study of dasatinib for patients with imatinib-resistant gastrointestinal stromal tumor (GIST).

10006 Background: Dasatinib is an oral tyrosine kinase inhibitor of KIT, PDGFR, ABL and SRC with a distinct binding affinity for KIT and PDGFR. We performed a phase II trial to assess antitumor activity of dasatinib in patients (pts) with advanced GIST who were refractory to imatinib (IM) and sunitinib malate (SM). Methods: Pts received dasatinib 70mg PO BID. Computer tomographic response rates (ORR) was evaluated every 8 wks by Choi criteria. The primary endpoint was a target progression-free survival at 6 months (PFS-6) of >30% with <10% deemed inactive by a Bayesian approach. Objective response rate (ORR) per Choi criteria was also determined. Results: From 8/2008 to 7/2010, 50 pts were accrued (47 evaluable for response). Median age was 60(range, 20-78), 62% were male, and median ECOG PS=1. The most common site of primary disease was stomach (59%) and metastasis was liver (56%). Primary median tumor size at diagnosis was 8.7 cm(range 3.7-26) and mitotic rate was >5/50 hpf in 74% of patients. Tumor morphology was spindled (63%), epithelioid (18%) or mixed (18%). The median number of prior therapies was 2[range 1-6, IM (100%), SM (80%)]. Genotype was available for 15 pts: 6(40%) KIT exon 11, 6(40%) WT, 2(13%) KIT exon 9, and 1(7%) had PDGFR D842V. The median number of cycles was 2 (range, 1-24). The PR rate was 32% (15/47) by Choi criteria. 21% patients (10/47) were progression-free >6 months. Median PFS and OS were 2.0 months and 19 months. Median PFS for patients with WT GIST was 8.4 months (range 1.2-27). Toxicity was assessed in all pts: 14 grade 3 events (28%; constitutional, GI, respiratory, and myelosuppression) and 1 grade 4 event (2%, pain). There were no deaths related to dasatinib. Conclusions: Dasatinib has significant activity by ORR in IM and SM-refractory GIST but did not meet the predefined 6 month PFS rate of 30%. Mutational and molecular correlates are forthcoming.

Evaluation of the presence of IGF1R overexpression in wild-type and kinase mutant GI stromal tumors

10506 Background: Most adult GI stromal tumors have gain-of-function mutations in KIT (80%) or PDGFRA (5–7%). These pathogenetic mutations are the target for kinase inhibitor therapy. The pathogenesis of the 10–15% of GISTs lacking kinase mutations (WT GIST) is unknown; this includes most pediatric GISTs. Recently, Tarn et al. (PNAS 2008) identified IGF1R over-expression in all WT GIST in their series of cases, including one pediatric case. IGF1R may represent a novel therapeutic target for WT GISTs. Methods: We developed a quantitative RQ-PCR assay for IGF1R and GAPDH transcripts that is linear over 5 logs (R2 values of 0.99 and 0.98, respectively). We prepared cDNA from RNA isolated from 79 archival, formalin-fixed, paraffin-embedded GIST specimens and quantified IGF1R expression as the % ratio of IGF1R/GAPDH transcripts. We had previously genotyped the tumors to identify any kinase mutations. Results: Adult WT GIST had a 10–15 fold higher expression of IGF1R transcript than kinase mutant GISTs (WT GIST 13.7 ±18, n=35; KIT exon 11 mutant GIST 1.0 ± 1.1, n=26; PDGFRA mutant GIST 0.9 ± 1.0, n=8; KIT 9 or 13 mutation 0.8 ± 0.6, n=5). Interestingly, the WT GIST group could be further divided into tumors with low vs. high IGF1R expression. The low IGF1R expression group (mean 0.6, range 0.1–1.9, n=14), had similar IGF1R expression to kinase mutant GISTs. In contrast, the high expression group had more than 30-fold higher levels of IGF1R transcript (mean 22.5, range 7.3–76.5, n=21). We also examined IGF1R expression in 5 WT pediatric GISTs. The mean IGF1R expression in pediatric GISTs resembled that of the adult high expression group, with 4 of 5 cases showing IGF1R over-expression. Conclusions: WT GISTs are heterogeneous with regard to IGF1R expression and can be divided into two subgroups. One third of the WT GISTs overlap with kinase-mutant GISTs, while the remainder express IGF1R at more than 30-fold higher levels. These results have implications for evolving models of GIST pathogenesis and the clinical testing of IGF1R monoclonal antibodies for treatment of metastatic GIST. [Table: see text]

Association of Platelet-Derived Growth Factor Receptor α Mutations with Gastric Primary Site and Epithelioid or Mixed Cell Morphology in Gastrointestinal Stromal Tumors

Most gastrointestinal stromal tumors (GISTs) carry activating mutations of the KIT gene encoding the receptor tyrosine kinase KIT. In a previous study we were able to show an association between the lack of KIT mutations (wild-type GISTs) and the presence of a significant epithelioid tumor component. A very recent study described the occurrence of PDGFRalpha mutations in KIT wt GISTS. Therefore, we studied a panel of 87 GISTs for mutations in the hot spot regions of the PDGFRalpha gene with single strand conformation polymorphism analysis and sequencing and correlated the PDGFRalpha status with pathomorphological data. We detected 20 cases with exon 18 mutations but none with exon 12 mutations. The mutations were located in the second kinase domain of PDGFRalpha with 16 point mutations, and four larger deletions of 9 to 12 bp. All cases with mutations in the PDGFRalpha gene revealed wild-type KIT in common regions of mutation, ie, exons 9 and 11. Most interestingly, the occurrence of PDGFRalpha mutations was significantly associated with a higher frequency of epithelioid or mixed morphology (18 of 20 cases, P < 0.0001) and gastric location (all cases, P = 0.0008). Our data indicate that GISTs represent distinctive entities, differing in genetic, biological, and morphological features.