Myxoid epithelioid gastrointestinal stromal tumor harboring an unreported PDGFRA mutation: report of a case and review of the literature

Activating mutations of platelet-derived growth factor receptor α (PDGFRA) are detected in a significant proportion of gastrointestinal stromal tumors (GISTs), in addition to the more frequent mutation in c-kit. GISTs with PDGFRA mutations have been found to have several characteristic morphological features, sometimes allowing to discriminate them from GISTs with c-kit mutations. Among these, epithelioid morphology in tumor cells and tumor-infiltrating mast cells are powerful predictors of PDGFRA mutations. Although myxoid stroma by itself is not so much a reliable predictor of PDGFRA mutation, myxoid stroma in conjunction with epithelioid morphology in tumor cells is a powerful predictor of mutations in this gene. GISTs showing either weak or negative immunoreactivity for c-kit and epithelioid cells with myxoid stroma are called myxoid epithelioid GISTs, which typically show PDGFRA mutation. Herein, we presented a case of a 59-year-old woman with myxoid epithelioid GIST of the stomach. A unique finding in this case was eosinophil infiltration, probably more numerous than mast cells; mast cell infiltration is known to be usually found in myxoid epithelioid GIST. The existence of a similar mechanism in eosinophil and mast cell recruitment via tumor-producing stem cell factor is speculated. Mutational analyses revealed a PDGFRA exon 18 mutation: D842_H845del, D846N. Combined deletion and substitution mutation has been reported in rare instances, but to the best of our knowledge, D846N has not been documented.     

Gastrointestinal stromal tumors – Summary of mutational status of the primary/secondary KIT/PDGFRA mutations,BRAF mutations and SDH defects

The most important findings revealing pathogenesis, molecular characteristics, genotyping and targeted therapy of gastrointestinal stromal tumors (GISTs) are activated oncogenic mutations in KIT and PDGFRA genes. Imatinib mesylate (IM), which inhibits both KIT and PDGFRA receptors, significantly improved treatment of advanced (metastatic, recurrent, and/or inoperable) GISTs. However, in a significant number of patients the treatment fails due to the primary or secondary resistance to targeted therapy. Most common cause of secondary resistance is a presence of secondary mutations. Approximately 15% of adult patients with GISTs are negative for mutations in KIT or PDGFRA genes. These so-called wild-type GISTs appear to be characterized by other oncogenetic drivers, including mutations in BRAF, RAS, NF1 genes, and subunits of succinate dehydrogenase (SDH) complex.In the present study we investigated 261 tumour specimens from 239 patients with GIST. Primary mutations were detected in 82 % tumor specimens. 66 of them were in KIT, and 16 % in PDGFRA genes. Remaining 18 % were KIT/PDGFRA wild-type. Secondary KIT mutations were detected in 10 from 133 (7 %) patients treated with IM. We examined secondary KIT mutations in exons 13 and 17 and secondary PDGFRA mutation in exon 18 in sixteen progressive tumors and/or metastasis (from overall 22 samples). We identified BRAF V600E point mutation in 4 % of KIT/PDGFRA wild-type GIST patients. Moreover, we analysed SDH complex mutations in 4 younger patients (15, 33, 37, and 45 years old) from 44 patients without KIT, PDGFRA, and BRAF mutations. Two patients (a 37-year old man, and a 33-year old woman) had defects of the SDH complex.Our findings of mutational status of the primary and secondary KIT/PDGFRA mutations in patients with GIST confirm mechanisms of primary and secondary resistance, and also intralesional and interlesional heterogeneity of secondary mutations within and between progressive lesions. Moreover, detection of V600E BRAF mutation and defects of SDH complex in KIT/PDGFRA wild-type GISTs confirm their activation and allow for a selection of targeted therapy.     

Epithelioid variant of gastrointestinal stromal tumor harboring PDGFRA mutation and MLH1 gene alteration: A case report

Gastrointestinal stromal tumors (GISTs) are the most important and common mesenchymal tumors of the gastrointestinal tract, especially in the stomach. GISTs are usually driven by activating mutations in either KIT or PDGFRA genes. It is known that activating gene mutations predicts, to a certain extent, not only the morphology of the tumor cells but also a response to treatment with tyrosine kinase inhibitors. Here, we present a case of an epithelioid variant of GIST harboring PDGFRA and MLH1 gene alterations in the stomach of a 55‐year‐old Japanese woman. The tumor of 98 mm with multiple cysts showed exophytic growth from the gastric fundus. Histopathologically, it consisted of scattered medium‐sized epithelioid tumor cells in a loose myxoid background. Based on c‐kit and DOG‐1 immunoreactivity and a PDGFRA mutation (p.Trp559_Arg560del), the tumor was diagnosed as an epithelioid variant GIST. Interestingly, it had a gene alteration (p.Met524Ile) in the MLH1 gene of unknown pathogenicity. It was assigned to Group 3a (low risk for malignant behavior). After surgery, the patient has been on imatinib therapy and disease‐free for 10 months.     

Presence of PDGFRA and DOG1 mutations in gastrointestinal stromal tumors among Chinese population

Approximately 15% of gastrointestinal stromal tumors (GIST) do not express KIT mutations and of these about 5 to 7% harbor mutations in PDGFRA. DOG1 was specifically expressed in GISTs. These cases require special attention for PDGFRA and DOG1 mutational status. Hundred cases of GIST were diagnosed between August 2007 and October 2012 at the First Affiliated Hospital of Guangxi Medical University. DNA from tumor tissues and normal adjacent tissues was isolated and amplified for the 22 exons of PDGFRA and 26 exons of DOG1. Each PCR product was sequenced. Amino acid sequences were inferred from DNA and aligned to GenBank reference sequences to determine the position and type of mutations. Overall, 16.0% of the samples had a mutation in PDGFRA, and GISTs with mutations in the DOG1 gene were not found. Of the mutations detected, they were in PDGFRA exon 18 (8 cases, 8%), PDGFRA exon 12 (5 cases, 5%), PDGFRA exon 14 (1 cases, 1.0%), PDGFRA exon 11 (1 cases, 1.0%), and PDGFRA exon 8 (1 cases, 1.0%). Of these, Y392S, L521P and T632K mutant occurred in PDGFRA exon 8, exon 11 and exon 14, respectively. The mutation of PDGFRA has been considered as another causative genetic event as PDGFRA mutations were found in most GISTs lacking a KIT mutation. PDGFRA mutations occurred preferentially in exon 18 and exon 12. Mutations occurring in PDGFRA exon 8 (Y392S), exon 11 (L521P) and exon 14 (T632K) also were first identified. The over-expression of DOG1 was not related to DOG1 gene mutation.     

Loss of DOG-1 expression associated with shift from spindled to epithelioid morphology in gastric gastrointestinal stromal tumors with KIT and platelet-derived growth factor receptor α mutations

Most gastric gastrointestinal stromal tumors (GISTs) display spindle cell morphology and coexpress CD117 (KIT), DOG-1, and CD34. Secondary loss of DOG-1 has not been reported. We present two gastric GISTs which showed loss of DOG-1 in the epithelioid component but retained its expression in the minor spindle cell component. Patients were a 67-year-old man and an 80-year-old woman with 4.8-cm and 3.5-cm gastric GISTs harboring mutations in KIT exon 11 (c.1729_1758dup30; p.P577_R586dup) and platelet-derived growth factor receptor α (PDGFRA) exon 18 (c.2527_2538del12; p.I843_D846del), respectively. Both were predominantly epithelioid with a minor microscopic spindle cell component (3-12 mm). The spindle cell component was CD117⁺CD34⁺DOG-1⁺ in both cases. The epithelioid component in case 1 was CD117⁺CD34⁺DOG-1^?. In case 2, the epithelioid component strongly expressed PDGFRA (dot-like) but lost CD117, CD34, and DOG-1. These cases confirm the immunophenotypic heterogeneity as secondary events in GIST. Loss of DOG-1 in KIT-negative PDGFRA mutants should not preclude diagnosis.     

BRAF mutations in KIT/PDGFRA positive gastrointestinal stromal tumours (GISTs): Is their frequency underestimated?

BRAF V600E mutations in GISTs are considered to be one of the mutational events in KIT/PDGFRA negative or positive GISTs, respectively. BRAF mutated GISTs usually do not respond to imatinib treatment, even more GISTs with imatinib sensitive KIT mutation. However, they are almost phenotypically and morphologically identical with KIT/PDGFRA positive GISTs. In general, due to the small number of BRAF mutations in GIST and because of the rarity of concomitant BRAF/KIT or BRAF/PDGFRA mutations, their frequency may be depreciated. The aim of this study was BRAF mutation detection in KIT/PDGFRA positive GISTs and their verification by other molecular methods. We applied the sensitive droplet digital PCR on 35 randomly selected KIT/PDGFRA positive GISTs to detect V600E mutations. We have established two criteria for the evaluation of samples: false positive rate (FPR) based on the negative controls; Limit of Detection (LoD) based on the serial dilution of positive control from RKO cell line harboring heterozygous V600E mutation in constant wild-type DNA background. Results from ddPCR were verified by other molecular methods: allele-specific PCR, dideoxysequencing, competitive allele-specific TaqMan PCR (castPCR). FPR was determined as 5 (∼4.4) positive droplets, and LoD was assessed to 3.4293 copies/μL what is the method sensitivity of 0.0162 %.We identified eight KIT/PDGFRA positive patients with concomitant V600E mutation. The five of them were in coexistence with KIT mutation and three with PDGFRA mutation. We also included the liver metastasis, but data from primary tumour were not available. We achieved the very high sensitivity of the ddPCR method for detecting BRAF mutation in GISTs to have importance from the point of view of therapy.     

Comparative Ultrastructural Analysis and KIT/PDGFRA Genotype in 125 Gastrointestinal Stromal Tumors

GISTs are the most common mesenchymal neoplasms of the digestive tract and are thought to originate from or differentiate toward the interstitial cell of Cajal lineage. Almost all GISTs express KIT protein and the majority show activating mutations in either KIT or PDGFRA proto-oncogenes. Ultrastructurally, these tumors have been shown to have either a smooth muscle, neuronal, dual, or null phenotype. The objective of this study was to investigate the relationship between ultrastructural features and genotype in a large series of 125 histologically confirmed and CD117 positive GISTs. PCR analysis for the presence of KIT exon 9, 11, 13, and 17 and PDGFRA exon 12 and 18 mutations was performed. There were 62 (50%) tumors located in the stomach and 45 (36%) in the small bowel. Overall, KIT mutations were detected in 93 (75%) patients: 86 (69%) in exon 11, and 7 (6%) in exon 9. A PDGFRA mutation was detected in 7 (6%) cases and 25 (19%) cases had no mutation. Ultrastructurally, skeinoid fibers were seen in 55 (44%) cases and were more common in small bowel than stomach GISTs, and occurred in only in 1 of 16 patients with an ITD (KIT) exon 11 or PDGFRA mutation. Focal actin microfilaments were identified in 82 (65%) cases and did not correlate with location or mutation type. Rare neurosecretory-type granules (NS-G) were seen in 34 (27%) of cases, but were seen in most of the cells in only 5 (4%) cases. GISTs showing both NS-G and microtubules were associated with KIT exon 11 genotype and spindle cell morphology. PDGFRA mutated cases were associated with gastric location, predominantly epithelioid morphology and lacked NS-G.     

Gastrointestinal stromal tumors in children and young adults: a clinicopathologic and molecular genetic study of 22 Korean cases

Studies on gastrointestinal stromal tumors (GISTs) in young patients are limited due to their rarity, and none have been conducted in Asian populations. GISTs from patients under the age of 30 were retrospectively reviewed and were analyzed for clinicopathologic features, immunohistochemistry for SDHB (succinate dehydrogenase subunit B), and mutations for exon 9, 11, 13, and 17 of KIT gene and exon 12, 14, and 18 of PDGFRA gene. We found two pediatric (<18 years old) and 20 young adult (18–30 years old) GIST cases. Pediatric GISTs occurred in two girls, both as solitary masses with epithelioid histology in the stomach. Both GISTs were wild type for KIT and PDGFRA genes, were negative for SDHB, and there was no recurrence during follow‐up. Of the 20 GISTs in young adults, 12 (60%) were from extra‐gastric locations (six duodenum, five jejunum, and one esophagus), and 16 (80%) showed a spindle cell morphology. Mutations of KIT or PDGFRA genes were identified in 14 (78%) of the 18 cases. One patient with multiple gastric GISTs with perigastric lymph node metastases at presentation developed multiple distant metastases and died of the disease 7.3 years after diagnosis. Of the 19 R0‐resected young adult patients, one patient with small intestinal GIST harboring KIT exon 11 deletion mutation developed recurrence and showed partial responses for imatinib. In summary, compared with pediatric GIST cases, young adult GISTs are heterogeneous and share the characteristics of both pediatric and adult GISTs. When a mesenchymal tumor is clinically suspected in the small intestine of young adults, a GIST should be included in the differential diagnoses. Further mutation studies and extensive treatments are recommended for these cases.     

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet‐derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10–15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large‐scale genomic alterations, but little is known about the mutation‐negative tumors. Using genome‐wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation‐negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation‐negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation‐negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine‐threonine kinase BRAF. The only other mutation‐negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT‐mutant GISTs identified a minimal overlapping region of deletion of ～0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell‐cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs. © 2009 Wiley‐Liss, Inc.     

High expression of the RNA-binding protein RBPMS2 in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n = 23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p < 0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.     

Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. Despite clinicopathological differences, GISTs share oncogenic KIT or platelet-derived growth factor-alpha ( PDGFRA ) mutations. Imatinib, KIT and PDGFRA inhibitor, has been successfully used in the treatment of metastatic GISTs. There are primary KIT or PDGFRA mutations diagnosed before imatinib treatment, linked to GIST pathogenesis, and secondary mutations detected during treatment, causing drug resistance. KIT exon 11 mutations are the most common. Gastric GISTs with exon 11 deletions are more aggressive than those with substitutions. KIT exon 11 mutants respond well to imatinib. Less common KIT exon 9 Ala502_Tyr503dup mutants occur predominantly in intestinal GISTs and are less sensitive to imatinib. An Asp842Val substitution in exon 18 is the most common PDGFRA mutation. GISTs with such mutation are resistant to imatinib. PDGFRA mutations are associated with gastric GISTs, epithelioid morphology and a less malignant course of disease. GISTs in neurofibromatosis 1, Carney triad and paediatric tumours generally lack KIT and PDGFRA mutations. Secondary KIT mutations affect exons 13–17. GISTs with secondary mutations in exon 13 and 14 are sensitive to sunitinib, another tyrosine kinase inhibitor. KIT and PDGFRA genotyping is important for GIST diagnosis and assessment of sensitivity to tyrosine kinase inhibitors.     

Novel oncogene and tumor suppressor mutations in KIT and PDGFRA wild type gastrointestinal stromal tumors revealed by next generation sequencing

Among gastrointestinal stromal tumors (GISTs) of 10–15% are negative for KIT and PDGFRA, and most of these cases are SDH deficient. Recent studies have provided data on additional molecular alterations such as KRAS in KIT mutant GISTs. We aimed to assess the frequency and spectrum of somatic mutations in common oncogenes as well as copy number variations in GISTs negative for KIT and PDGFRA mutations. GISTs with wild type KIT/PDGFRA were tested via next generation sequencing for somatic mutations in 341 genes. SDHB immunohistochemistry to evaluate for SDH deficiency was also performed. Of 267 GISTs tested for KIT and PDGFRA mutations, 15 were wild type, of which eight cases had material available for further testing. All eight cases had loss of SDHB expression and had various molecular alterations involving ARID1A, TP53, and other genes. One case had a KRAS G12V (c.35G>T) mutation in both the primary gastric tumor and a post‐imatinib recurrence. This tumor had anaplastic features and was resistant to multiple tyrosine kinase inhibitors, ultimately resulting in cancer‐related mortality within 2 years of diagnosis. In conclusion, KRAS mutations occur in rare GISTs with wild type KIT and PDGFRA. These tumors may display immunohistochemical positivity for KIT and primary resistance to tyrosine kinase inhibitors. © 2014 Wiley Periodicals, Inc.     

Gastrointestinal stromal tumor of the digestive organs: a histopathologic study of 31 cases in a single Japanese institute

The author herein reports histopathologic features of 31 surgical cases of gastrointestinal stromal tumor (GIST) of the digestive organs. The 31 cases of GIST were diagnosed in our pathology laboratory. They consisted of 24 cases of gastric GIST, 1 case of hepatic GIST, 1 case of small intestinal GIST, 4 cases of colon GIST, and 1 case of rectal GIST. The age of the patients ranged from 56 year to 84 years with a mean of 71 years. Male to female ratio was 21:10. The presenting symptoms were gastrointestinal bleeding in 13 cases, abdominal pain and discomfort in 13 cases, and asymptomatic in 5 cases. Endoscopy and imaging modalities including US, CT and MRI were useful to detect the tumors in all cases, and biopsies confirmed the GIST diagnosis in 21 cases. The size of GIST ranged from 1 cm to 12 cm with a mean of 4.3 cm. Grossly, 23 cases were submucosal tumors, 6 serosa-side tumors, 1 solid tumor in the liver, and 1 rectal polyp. Histologically, 28 cases were of spindle cell type and 3 of epithelioid type. According to mitotic counts and tumor size, the malignant risk was very low in 4 cases, low in 14 cases, intermediate in 9 cases, and high in 4 cases. Immunohistochemically, all cases were positive for KIT and vimentin, 30 cases for CD34, and 4 cases for α-smooth muscle actin. None were positive for desmin and S100 protein. Ki-67 labeling ranged from 2% to 18%. P53 protein was negative in all cases. PDGFRA was positive in 20 cases among 24 cases examined. Genetic analysis using PCR-direct sequencing method was performed in 5 GISTs; all the 5 GISTs showed point mutations or deletions in KIT gene, but did not in PDGFRA gene. The 5 cases of GIST were positive for PDGFRA protein, suggesting that PDGFRA overexpression is not associated with PDGFRA gene mutations. Four of the 31 cases showed metastases. The chemotherapy was imatinib mesylate in 6 cases, and none in 25 cases. Four cases of high risk died of GIST, and 27 cases are alive now without tumors.     

GISTs with PDGFRA exon 14 mutations represent subset of clinically favorable gastric tumors with epithelioid morphology

Gastrointestinal stromal tumors (GISTs) are common mesenchymal tumors of the gastrointestinal tract. Activating KIT or PDGFRA (platelet-derived growth factor receptor alpha) mutations have been shown to be a major force in GIST pathogenesis. Recently, a previously undescribed N659K PDGFRA exon 14 mutation has been reported in GISTs. The purpose of this study was to evaluate the frequency of GISTs with PDGFRA exon 14 mutations and define the clinicopathologic profile of such tumors. In all, 200 GISTs negative for mutations in KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were evaluated for PDGFRA exon 14 mutations by PCR amplification and direct sequencing. Mutations were found in 11 of 119 (9%) gastric GISTs. None of the 81 GISTs from other than gastric location had such a PDGFRA mutation. A majority of these mutations (eight cases) represented simple 2125C>A or C>G missense mutations, leading to substitution of the lysine for asparagine (N659K). However, in two cases, 2123A>T missense mutations leading to substitution of the tyrosine for asparagine (N659Y) was found instead. Of 11 PDGFRA N659-mutant GISTs, 10 had pure epithelioid morphology. One tumor had mixed, predominantly spindle and focally epithelioid cell morphology. Frequency of PDGFRA N659-mutant GISTs among pure epithelioid GISTs was almost 19%. Immunohistochemically, the majority (64%) of these tumors lacked KIT expression or showed only focal scattered KIT positivity. Tumor size ranged from 2.5 to 16 cm (average 7.1 cm). Low mitotic activity, 5 cm tumors. Based on mitotic activity and tumor size, six tumors were classified as probably benign with very low malignant potential. Low to moderate malignant potential and high malignant potential was suggested in three and two tumors, respectively. In four cases with moderate or high malignant potential GISTs, a long-term follow-up (average 235.5 months) showed favorable course of disease.     

Mitotic recombination as evidence of alternative pathogenesis of gastrointestinal stromal tumours in neurofibromatosis type 1

Background

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder resulting in the growth of a variety of tumours, and is inherited in an autosomal dominant pattern. Gastrointestinal stromal tumours (GISTs) are mesenchymal tumours that commonly harbour oncogenic mutations in KIT or PDGFRA and are thought to arise from the interstitial cells of Cajal (ICC; the pacemaker cells of the gut).

Aim

To characterise two patients with NF1 and GISTs.

Methods

Two patients were genotyped for germline mutations in NF1. GISTs from both patients were genotyped for somatic mutations in KIT and PDGFRA. Loss of heterozygosity (LOH) of NF1 in one GIST was assessed by genotyping seven microsatellite markers spanning 2.39 Mb of the NF1 locus in the tumour and in genomic DNA. The known germline mutation in NF1 was confirmed in GIST DNA by sequencing. The copy number of the mutated NF1 allele was determined by multiplex ligand‐dependent probe amplification.

Results

GISTs from both patients were of wild type for mutations in KIT and PDGFRA. In the GIST with adequate DNA, all seven markers were informative and showed LOH at the NF1 locus; sequencing of NF1 from that GIST showed no wild‐type sequence, suggesting that it was lost in the tumour. Multiplex ligand‐dependent probe amplification analysis showed that two copies of all NF1 exons were present.

Conclusions

This is the first evidence of mitotic recombination resulting in a reduction to homozygosity of a germline NF1 mutation in an NF1‐associated GIST. We hypothesise that the LOH of NF1 and lack of KIT and PDGFRA mutations are evidence of an alternative pathogenesis in NF1‐associated GISTs.     

KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs)

Mutually exclusive KIT and PDGFRA mutations are central events in GIST pathogenesis, and their understanding is becoming increasingly important, because specific treatment targeting oncogenic KIT and PDGFRA activation (especially imatinib mesylate) has become available. KIT mutations in GIST are clustered in four exons. Most common are exon 11 (juxtamembrane domain) mutations that include deletions, point mutations (affecting a few codons), and duplications (mostly in the 3' region). The latter mutations most often occur in gastric GISTs. Among gastric GISTs, tumors with deletions are more aggressive than those with point mutations; this does not seem to hold true in small intestinal GISTs. Exon 9 mutations (5-10%) usually are 2-codon 502-503 duplications, and these occur predominantly in intestinal versus gastric GISTs. Lesser imatinib sensitivity of these tumors has been noted. Kinase domain mutations are very rare; GISTs with such mutations are variably sensitive to imatinib. PDGFRA mutations usually occur in gastric GISTs, especially in the epithelioid variants; their overall frequency is approximately 30% to 40% of KIT mutation negative GISTs. Most common is exon 18 mutation leading Asp842Val at the protein level. This mutation causes imatinib resistance. Exon 12 and 14 mutations are rare. Most mutations are somatic (in tumor tissue only), but patients with familial GIST syndrome have consitutitonal KIT/PDGFRA mutations; >10 families have been reported worldwide with mutations generally similar to those in sporadic GISTs. GISTs in neurofibromatosis 1 patients, children, and Carney triad seem to lack GIST-specific KIT and PDGFRA mutations and may have a different disease mechanism. Secondary mutations usually occur in KIT kinase domains in patients after imatinib treatment resulting in resistance to this drug. Mutation genotyping is a tool in GIST diagnosis and in assessment of sensitivity to kinase inhibitors. This is a US government work. There are no restrictions on its use.     

Strong PDGFRA positivity is seen in GISTs but not in other intra-abdominal mesenchymal tumors: Immunohistochemical and mutational analyses.

Mutation of the platelet-derived growth factor receptor-alpha (PDGFRA) gene has been well documented as an alternative oncogenic mechanism in a subset of gastrointestinal stromal tumors (GISTs) lacking c-kit mutations. However, the role of PDGFRA immunohistochemistry in the diagnosis of GISTs has not been well studied. We investigated PDGFRA immunoreactivity in GISTs and in other intra-abdominal mesenchymal tumors, and correlated PDGFRA expression with CD117 positivity and with the mutational status of PDGFRA and c-kit genes. In addition, expression of phosphorylated AKT, an activated downstream molecule in the PDGFRA and c-kit signaling pathways, was correlated with PDGFRA and CD117 status. A total of 39 GISTs and 20 other mesenchymal tumors in the abdomen were included in this study. Thirty-five of 39 GIST cases (89.7%) were positive for PDGFRA and 19 of these 35 positive cases were strongly positive. Five of 20 non-GIST lesions (25%) were positive for PDGFRA, but none of these cases were strongly positive. With one exception, PDGFRA-positive cases were also positive for CD117. Phosphorylated AKT positivity was not associated with the immunoreactivity or mutation of PDGFRA and c-kit, suggesting that the activation of AKT is probably independent of the activation of PDGFRA and c-kit in GISTs. Of 14 GISTs assayed, 4 had mutations in c-kit at exons 11 or 17, and 4 had mutations in PDGFRA at exons 12 or 18. Three of 4 GIST cases with PDGFRA mutations show epithelioid morphology and strong PDGFRA immunoreactivity with prominent perinuclear dotlike accentuation (so-called Golgi pattern). In conclusion, strong PDGFRA positivity with Golgi pattern is a useful adjunct in the diagnosis of GISTs with PDGFRA mutation.     

Molecular analysis of c-Kit and PDGFRA in GISTs diagnosed by EUS

Gastrointestinal stromal tumors (GISTs) are characterized by overexpression and mutations of c-Kit. Approximately 80% of c-Kit mutations occur in exon 11, being a response factor to imatinib (Gleevec) therapy. Mutations of platelet-derived growth factor receptor-alpha (PDGFRA) are observed in a subset of GISTs lacking c-Kit mutations.We aimed to assess whether c-Kit and PDGFRA mutation analysis of GISTs obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) could be routinely performed. Mutation analysis of c-Kit hotspot exons (9, 11, 13 and 17) and PDGFRA hotspot exons (12 and 18) was performed in aspirates of 33 GISTs and 18 non-GIST mesenchymal tumors.Of the GIST cases, 19 (58%) of 33 contained a mutation in exon 11, 1 (3%) in exon 9, and none in exons 13 and 17. No activating c-Kit mutations were identified in non-GIST cases. No PDGFRA mutation was detected.Mutation analysis is possible in these FNA cell blocks and can assist in the diagnosis and therapeutic decisions in GIST cases/     

Multiple sporadic gastrointestinal stromal tumours arising at different gastrointestinal sites: pattern of involvement of the muscularis propria as a clue to independent primary GISTs

Multifocal sporadic gastrointestinal stromal tumours (GISTs) may be misinterpreted as recurrent or metastatic disease, leading to inappropriate treatment. As molecular analysis is generally not available in routine practise, histological criteria that would facilitate diagnosis of multiple primary GISTs in routine slides are needed. We studied 14 GISTs (mean size, 2.7 cm) from six men and one woman (mean age, 70 years) applying morphological features and direct sequencing of KIT, PDGFRA, BRAF, and KRAS. Diagnosis was synchronous in five and metachronous in two patients. Paired tumours originated in stomach/small bowel (n = 5), duodenum/jejunum (n = 1), and stomach/oesophagus (n = 1) and revealed spindle (n = 10) and mixed spindle and epithelioid (n = 4) phenotype. Tumours were well circumscribed and have involved the muscularis propria in a pattern typical of primary GISTs. Different somatic KIT mutations were found in tumours from four patients. One patient had a KIT-mutated and a BRAF-mutated (V600E) tumour. Two patients had wild-type tumours. No PDGFRA or KRAS mutations were detected. Our results underscore the molecular heterogeneity of sporadic multifocal GISTs. The characteristic involvement of the muscularis propria and the site-typical morphology and immunophenotype facilitated the diagnosis of primary GISTs in all cases and correlated with molecular findings, emphasising the value of conventional histology in recognising independent primary GISTs.