Toshirou Nishida

Gain-of-function mutations of platelet-derived growth factor receptor α gene in gastrointestinal stromal tumors

BACKGROUND & AIMS Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of c-kit receptor tyrosine kinase (KIT) gene, but some GISTs do not. We investigated the cause of GISTs without KIT mutations. Because GISTs apparently expressed platelet-derived growth factor receptor (PDGFR) alpha, we examined whether GISTs without KIT mutations had a mutation of PDGFR alpha. METHODS Whole coding region of PDGFR alpha complementary DNA (cDNA) was sequenced in GISTs with or without KIT mutations. Mutant PDGFR alpha cDNA was transfected into 293T human embryonic kidney cells, and autophosphorylation of PDGFR alpha was examined. Proliferation of Ba/F3 murine lymphoid cells stably transfected with mutant PDGFR alpha cDNA was estimated by tritium thymidine incorporation. Wild-type KIT cDNA was cotransfected with mutant PDGFR alpha cDNA, and immunoprecipitation by anti-KIT antibody was performed. Inhibitory effect of Imatinib mesylate on activated PDGFR alpha was examined. RESULTS We found 2 types of constitutively activated mutations of PDGFR alpha, Val-561 to Asp or Asp-842 to Val, in 5 of 8 GISTs without KIT mutations but not in 10 GISTs with KIT mutations. Stable transfection of each mutation induced autonomous proliferation of Ba/F3 cells. Constitutively activated mutant PDGFR alpha bound and activated the cotransfected wild-type KIT. The constitutive activation of PDGFR alpha with Val-561 to Asp was inhibited effectively by Imatinib mesylate but that of PDGFR alpha with Asp-842 to Val was inhibited only weakly, even at the concentration of 10 micromol/L. CONCLUSIONS The gain-of-function mutations of PDGFR alpha appear to play an important role in development of GISTs without KIT mutations.

Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial.

BACKGROUND Until now, only imatinib and sunitinib have proven clinical benefit in patients with gastrointestinal stromal tumours (GIST), but almost all metastatic GIST eventually develop resistance to these agents, resulting in fatal disease progression. We aimed to assess efficacy and safety of regorafenib in patients with metastatic or unresectable GIST progressing after failure of at least imatinib and sunitinib. METHODS We did this phase 3 trial at 57 hospitals in 17 countries. Patients with histologically confirmed, metastatic or unresectable GIST, with failure of at least previous imatinib and sunitinib were randomised in a 2:1 ratio (by computer-generated randomisation list and interactive voice response system; preallocated block design (block size 12); stratified by treatment line and geographical region) to receive either oral regorafenib 160 mg daily or placebo, plus best supportive care in both groups, for the first 3 weeks of each 4 week cycle. The study sponsor, participants, and investigators were masked to treatment assignment. The primary endpoint was progression-free survival (PFS). At disease progression, patients assigned placebo could crossover to open-label regorafenib. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01271712. RESULTS From Jan 4, to Aug 18, 2011, 240 patients were screened and 199 were randomised to receive regorafenib (n=133) or matching placebo (n=66). Data cutoff was Jan 26, 2012. Median PFS per independent blinded central review was 4·8 months (IQR 1·4-9·2) for regorafenib and 0·9 months (0·9-1·8) for placebo (hazard ratio [HR] 0·27, 95% CI 0·19-0·39; p<0·0001). After progression, 56 patients (85%) assigned placebo crossed over to regorafenib. Drug-related adverse events were reported in 130 (98%) patients assigned regorafenib and 45 (68%) patients assigned placebo. The most common regorafenib-related adverse events of grade 3 or higher were hypertension (31 of 132, 23%), hand-foot skin reaction (26 of 132, 20%), and diarrhoea (seven of 132, 5%). INTERPRETATION The results of this study show that oral regorafenib can provide a significant improvement in progression-free survival compared with placebo in patients with metastatic GIST after progression on standard treatments. As far as we are aware, this is the first clinical trial to show benefit from a kinase inhibitor in this highly refractory population of patients. FUNDING Bayer HealthCare Pharmaceuticals.

Familial gastrointestinal stromal tumours with germline mutation of the KIT gene

nature genetics volume 19 august 1998 323 Gastrointestinal stromal tumour (GIST) is the most common mesenchymal tumour of the human gastrointestinal tract. Most GISTs are solitary, and gain-offunction mutations of the KIT protooncogene have been found in these tumours1. We report here a family with multiple GISTs: affected members all have a KIT mutation occurring between the transmembrane and tyrosine kinase domains, which is also the region where mutations have been found in solitary GISTs (ref. 1). The KIT mutation in this family was detected not only in tumours but also in leukocytes, indicating that GISTs constitute a familial cancer syndrome2. Development of multiple GISTs was found in a 60-year-old Japanese woman (Fig. 1a, case 5). Her nephew (case 10) also suffered from multiple benign GISTs. Analysis of the family pedigree revealed many family members suffering from symptoms attributable to development of multiple GISTs (Fig. 1a), including case 9 (a niece of case 5) who underwent surgery for benign and malignant GISTs. The benign GISTs obtained from cases 5, 9 and 10, and the malignant GIST from case 9, all expressed the KIT protein (Fig. 1b–e). DNA was extracted from paraffin-embedded specimens of the tumours3, and the mutation was investigated using single-strand conformation polymorphism analysis4 (SSCP). SSCP of tumours from cases 5 and 10 showed wild-type and mutant bands at exon 11 (Fig. 1f). Direct sequencing of the mutant bands of exon 11 showed deletion of one of two consecutive valine residues (codon 559 and 560, GTTGTT) which are located between the transmembrane and tyrosine kinase domains. Unfortunately, DNA samples suitable for SSCP and direct sequencing were not obtained from tumours of case 9. Next, we obtained DNA from peripheral leukocytes of cases 5 and 10 and their family members. The valine deletion was detected in leukocyte DNA from cases 5, 10 and 15, but not in DNA from other family members. Case 15 is 22 years old and has so far had no abdominal symptoms. We investigated the function of the mutant KIT protein by introducing an analogous mutation into mouse Kit cDNA and transfecting it into the interleukin-3 (IL-3)-dependent Ba/F3 mouse lymphoid cell line1,5–7. Evidence was found for the constitutive phosphorylation and kinase Familial gastrointestinal stromal tumours with germline mutation of the KIT gene

Risk of recurrence of gastrointestinal stromal tumour after surgery: an analysis of pooled population-based cohorts.

BACKGROUND The risk of recurrence of gastrointestinal stromal tumour (GIST) after surgery needs to be estimated when considering adjuvant systemic therapy. We assessed prognostic factors of patients with operable GIST, to compare widely used risk-stratification schemes and to develop a new method for risk estimation. METHODS Population-based cohorts of patients diagnosed with operable GIST, who were not given adjuvant therapy, were identified from the literature. Data from ten series and 2560 patients were pooled. Risk of tumour recurrence was stratified using the National Institute of Health (NIH) consensus criteria, the modified consensus criteria, and the Armed Forces Institute of Pathology (AFIP) criteria. Prognostic factors were examined using proportional hazards and non-linear models. The results were validated in an independent centre-based cohort consisting of 920 patients with GIST. FINDINGS Estimated 15-year recurrence-free survival (RFS) after surgery was 59·9% (95% CI 56·2-63·6); few recurrences occurred after the first 10 years of follow-up. Large tumour size, high mitosis count, non-gastric location, presence of rupture, and male sex were independent adverse prognostic factors. In receiver operating characteristics curve analysis of 10-year RFS, the NIH consensus criteria, modified consensus criteria, and AFIP criteria resulted in an area under the curve (AUC) of 0·79 (95% CI 0·76-0·81), 0·78 (0·75-0·80), and 0·82 (0·80-0·85), respectively. The modified consensus criteria identified a single high-risk group. Since tumour size and mitosis count had a non-linear association with the risk of GIST recurrence, novel prognostic contour maps were generated using non-linear modelling of tumour size and mitosis count, and taking into account tumour site and rupture. The non-linear model accurately predicted the risk of recurrence (AUC 0·88, 0·86-0·90). INTERPRETATION The risk-stratification schemes assessed identify patients who are likely to be cured by surgery alone. Although the modified NIH classification is the best criteria to identify a single high-risk group for consideration of adjuvant therapy, the prognostic contour maps resulting from non-linear modelling are appropriate for estimation of individualised outcomes. FUNDING Academy of Finland, Cancer Society of Finland, Sigrid Juselius Foundation and Helsinki University Research Funds.

Upregulation of miR-196a and HOTAIR Drive Malignant Character in Gastrointestinal Stromal Tumors

Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference-mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs.

Gain-of-function mutation at the extracellular domain of KIT in gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the human gastrointestinal tract. Previous studies of GISTs found gain‐of‐function mutations of the c‐kit gene, which encodes a receptor tyrosine kinase (KIT). All the mutations were confined to exon 11, which encodes the juxtamembrane domain. By further examination of the whole coding region of c‐kit complementary DNA in 35 GISTs, two were found to show the identical mutation at exon 9, which encodes the extracellular domain. The aims of the present study were to examine the frequency of the extracellular domain mutation and to determine whether the mutation is a gain‐of‐function type or not. Genomic DNA was extracted from paraffin‐embedded tissues of 133 GISTs and exon 9 of the c‐kit gene was amplified by polymerase chain reaction. Screening of the mutation was carried out by single‐strand conformation polymorphism analysis and direct sequencing was done. Mutant c‐kit cDNA was transfected into 293T human embryonic kidney cells and the magnitude of autophosphorylation of the mutant KIT was examined with or without the ligand of KIT, stem cell factor (SCF). In total, seven GIST cases (approximately 5%) were found with the identical mutation at exon 9. The mutant KIT exhibited constitutive autophosphorylation without SCF stimulation. The prognosis of the patients with the extracellular domain mutation was comparable to that of the patients with the juxtamembrane domain mutation. Copyright

A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors

BACKGROUND & AIMS The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating. METHODS Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice. RESULTS The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice. CONCLUSIONS Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

Clinical practice guidelines for gastrointestinal stromal tumor (GIST) in Japan: English version

Diagnostic and treatment strategies for gastrointestinal stromal tumors (GISTs) have evolved greatly since the introduction of molecularly targeted therapies. Although several clinical practice guidelines are extant, such as those published by the National Comprehensive Cancer Network and the European Society of Medical Oncology, it is not clear as to whether these are appropriate for clinical practice in Japan. Therefore, clinical practice guidelines for the optimal diagnosis and treatment of GIST tailored for the Japanese situation have often been requested. For this reason, the Japanese Clinical Practice Guideline for GIST was proposed by the GIST Guideline Subcommittee, with the official approval of the Clinical Practice Guidelines Committee for Cancer of the Japan Society of Clinical Oncology (JSCO), and was published after assessment by the Guideline Evaluation Committee of JSCO. The GIST Guideline Subcommittee consists of members from JSCO, the Japanese Gastric Cancer Association (JGCA), and the Japanese Study Group on GIST, with the official approval of these organizations. The GIST Guideline Subcommittee is not influenced by any other organizations or third parties. Revision of the guideline may be done periodically, with the approval of the GIST Guideline Subcommittee, either every 3 years or when important new evidence that might alter the optimal diagnosis and treatment of GIST emerges. Here we present the English version of the Japanese Clinical Practice Guideline for GIST prepared by the GIST Guideline Subcommittee.

Absence of c-kit gene mutations in gastrointestinal stromal tumours from neurofibromatosis type 1 patients

Most sporadic gastrointestinal stromal tumours (GISTs) have somatic c‐kit gene mutations that are considered to be causal. Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene and NF1 patients have an increased risk of developing GISTs. Since most neoplasms are considered to develop as a result of the combination of several gene mutations, these findings suggest that GISTs from NF1 patients might have somatic c‐kit gene mutations and that sporadic GISTs from non‐NF1 patients might have somatic NF1 gene mutations. The present study analysed 29 GISTs from seven NF1 patients for c‐kit gene mutations and ten sporadic GISTs from ten non‐NF1 patients for NF1 mutations. Exons 9, 11, 13, and 17 of the c‐kit gene were amplified and directly sequenced after the extraction of genomic DNA from wax‐embedded tissues from 26 GISTs from five NF1 patients. The whole coding region of the c‐kit cDNA and the whole coding region of the NF1 cDNA were amplified and directly sequenced after RNA extraction and cDNA synthesis in three fresh GIST tissues from two NF1 patients and ten fresh GIST tissues from ten non‐NF1 patients. Of the ten sporadic GISTs, eight had heterozygous mutations at exon 11, and one at exon 9, of c‐kit. Heterozygous NF1 gene mutations were detected in GISTs from the two NF1 patients from whom fresh tissues were available. None of the 29 GISTs derived from NF1 patients had detectable c‐kit gene mutations and none of the ten GISTs derived from non‐NF1 patients had detectable NF1 mutations. These results suggest that the pathogenesis of GISTs in NF1 patients is different from that in non‐NF1 patients. Copyright

Surgical strategy for gastric gastrointestinal stromal tumors: laparoscopic vs. open resection

BackgroundBecause wide resections and extensive lymphadenectomies are usually not required, the laparoscopic approach has been considered reasonable for gastrointestinal stromal tumors (GISTs) of gastric origin and has been reported with increased frequency. However, its long-term oncologic outcome has not been fully assessed.MethodsWe reviewed 67 consecutive patients who underwent laparoscopic (LAP) or traditional open (OPEN) resection of gastric GISTs from January 1993 to May 2004.ResultsThere were 39 LAP and 28 OPEN cases. One LAP case was converted to open (2.6%). Patients in both groups had comparable backgrounds. Tumor location, size, and risk classification were similar. There was no difference in operating time and blood loss. Five patients (one in LAP and four in OPEN) showed recurrence and/or metastases after a median followup period of 26 months. Tumor enucleation resulted in a higher recurrence rate in both groups: one after three enucleations in LAP and two after six in OPEN. In cases with tumors that were larger than 5 cm, laparoscopic manipulation became technically challenging, although no recurrence was noted in this subgroup. Overall recurrence rate was comparable in the two groups.ConclusionLaparoscopic surgery is oncologically justified for gastric GISTs, while its indication should be carefully discussed for cases with bulky and high-risk tumors. Tumor enucleations should be avoided whichever approach (open/laparoscopic) is selected.