Platelet-derived Growth Factor Receptor Alpha Gene Research Articles

Association between Platelet-Derived Growth Factor Receptor Alpha Gene Polymorphisms and Platelet-Rich Plasma's Efficiency in Treating Lateral Elbow Tendinopathy-A Prospective Cohort Study.

Individual differences in the response to platelet-rich plasma (PRP) therapy can be observed among patients. The genetic background may be the cause of this variability. The current study focused on the impact of genetic variants on the effectiveness of PRP. The aim of the present study was to analyze the impact of single nucleotide polymorphisms (SNP) of the platelet-derived growth factor receptor alpha (PDGFRA) gene on the effectiveness of treating lateral elbow tendinopathy (LET) with PRP. The treatment's efficacy was analyzed over time (2, 4, 8, 12, 24, 52 and 104 weeks after the PRP injection) on 107 patients using patient-reported outcome measures (PROM) and achievement of a minimal clinically important difference (MCID). Four SNPs of the PDGFRA gene (rs7668190, rs6554164, rs869978 and rs1316926) were genotyped using the TaqMan assay method. Patients with the AA genotypes of the rs7668190 and the rs1316926 polymorphisms, as well as carriers of the T allele of rs6554164 showed greater effectiveness of PRP therapy than carriers of other genotypes. Moreover, the studied SNPs influenced the platelets' parameters both in whole blood and in PRP. These results showed that PDGFRA gene polymorphisms affect the effectiveness of PRP treatment. Genotyping the rs6554164 and the rs1316926 SNPs may be considered for use in individualized patient selection for PRP therapy.

Association of PDGFRA polymorphisms with the risk of corneal astigmatism in a Japanese population

Corneal astigmatism is reportedly associated with polymorphisms of the platelet-derived growth factor receptor alpha (PDGFRA) gene region in Asian populations of Chinese, Malay, and Indian ancestry and populations of European ancestry. In this study, we investigated whether these PDGFRA polymorphisms are associated with corneal astigmatism in a Japanese population. We recruited 1,535 cases with corneal astigmatism (mean corneal cylinder power across both eyes: ≤ − 0.75 diopters [D]) and 842 controls (> − 0.75 D) to genotype 13 single-nucleotide polymorphisms (SNPs) in the PDGFRA gene region. We also performed imputation analysis in the region, with 179 imputed SNPs included in the statistical analyses. The PDGFRA SNPs were not significantly associated with the cases with corneal astigmatism ≤ − 0.75 D. However, the odds ratios (ORs) of the minor alleles of SNPs in the upstream region of PDGFRA, including rs7673984, rs4864857, and rs11133315, tended to increase according to the degree of corneal astigmatism, and these SNPs were significantly associated with the cases with corneal astigmatism ≤ − 1.25 D or ≤ − 1.50 D (Pc < 0.05, OR = 1.34–1.39). These results suggest that PDGFRA SNPs play a potential role in the development of greater corneal astigmatism.

Association of MTOR and PDGFRA gene polymorphisms with different degrees of myopia severity

This study aims to investigate the associations of SNPs in the mammalian target of rapamycin (MTOR) and the platelet derived growth factor receptor alpha (PDGFRA) genes with different degrees of myopia severity in Han and Zhuang populations. The SNPs of MTOR (rs1057079, rs1064261, and rs2536) and PDGFRA (rs1800812, rs35597368, rs4358459, rs6554162, and rs7677751) were analyzed among 1347 patients with myopia (849 patients with high myopia and 498 patients with mild to moderate myopia) and 453 controls without myopia in Guangxi, China (collected 2016–2018). Genetic model association analysis was performed on each SNP in different myopia subgroups. The associations of rs1057079 and rs1064261 with mild to moderate myopia were observed under the dominant models (rs1057079: OR = 1.324, 95%CI: 1.005–1.744, P = 0.046; rs1064261: OR = 1.597, 95%CI: 1.099–2.319, P = 0.014). However, the association of SNP rs1057079 could not withstand multiple correction. The number of adverse genotypes in each sample was counted. Results showed that in the high myopia group, the levels of risk of myopia in patients carrying three to four and five to eight adverse genotypes were 1.734 and 2.062 times the level of risk in patients carrying two or lower genotypes, respectively. After the stratified analyses of Han and Zhuang populations, the Zhuang populations consistently had high frequencies of myopia. This study provides evidence suggesting that the MTOR and PDGFRA genes are associated with different degrees of myopia severity and have gene–gene interactions. In addition, this study discovered a new SNP of MTOR (rs1064261) that is associated with myopia. Thus, further longitudinal studies are warranted.

Phosflow assessment of PDGFRA phosphorylation state: A guide for tyrosine kinase inhibitor targeted therapy in hypereosinophilia patients.

Clonal eosinophilia is a hematologic disorder caused by translocation in growth factor receptor (GFR) genes. Despite the identified molecular mechanisms underlying clonal hypereosinophilia, the distinction between clonal and reactive eosinophilia has remained challenging due to the diversity of partner genes for translocated GFRs. This study aimed to examine the feasibility of phosphoflow cytometry in the diagnosis of clonal hypereosinophilia through evaluating the level of platelet-derived growth factor receptor alpha (PDGFRA) phosphorylation and its correlation with PDGFRA genetic aberration. Blood samples were collected from 45 hypereosinophilia patients and 10 healthy controls. Using phosphoflow cytometry method, the phosphorylation state of PDGFRA was assessed. The specificity of phosflow results was confirmed by western blotting and eventually compared with qRT-PCR expression analysis of 3'-region of PDGFRA. To detect the genetic aberration of PDGFRA, 5'-rapid amplification of cDNA ends (5'-RACE) was performed. Phosflow analysis illustrated that 9 of 45 hypereosinophilic patients had higher level of PDGFRA phosphorylation while sequence analysis of 5'-RACE-PCR fragments confirmed that in seven cases of them, there was a PDGFRA-FIP1L1 fusion. We also verified that two of nine patients with hyperposphorylated PDGFRA hold ETV6-PDGFRA and STRN-PDGFRA rearrangements. Importantly, nine cases also had significantly higher levels of PDGFRA mRNA expression when compared with healthy controls, and cases with no PDGFRA rearrangement. These findings highlight a robust correlation between hyperphosphorylation state of PDGFRA and aberrant PDGFRA gene fusions. This implicates phosflow as an efficient and reliable technique raising an intriguing possibility that it could replace other genomic and cDNA-amplification-based diagnostic approaches with limited effectiveness.

S2922 A Imatinib-Resistant Metastatic GIST Tumor Manifests as Diffuse Pruritus

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the prevalent nonepithelial tumors of the GI tract. GIST can manifest with a plethora of symptoms including early satiety, dysphagia, abdominal pain, bleeding and obstruction from mass effect. The main stay for medical therapy has been Imatinib, a tyrosine kinase inhibitor shutting down the oncogenic signal promoting tumor growth. Most GIST respond to Imatinib, while 10–15% will show disease progression or imatinib resistance. The following case highlights a genetic mutation conferring Imatinib resistance in a metastatic GIST. CASE DESCRIPTION/METHODS: A 64-year-old Nigerian man presented with acute exacerbation and generalized pruritis which was localized and mild for over 10 years. His physical exam only revealed diffuse excoriations on upper extremities, face and back. Labs showed a mixed hepatocellular and cholestatic pattern of transaminases. Hepatitis serology showed previously cleared infection with hepatitis B. Due to concomitant abdominal discomfort he was admitted and underwent colonoscopy and upper endoscopy which were negative. CT of abdomen with intravenous contrast showed numerous and diffusely distributed hypo-attenuated masses with the largest measuring 12.9 × 8 × 8 cm (Figure 1). Interventional radiology guided biopsy of the liver mass was performed with pathology consistent with oval and spindled shaped tumor cells with very rare mitosis. Immunostains of tumor cells were strongly and diffusely positive for GIST-1, weakly positive for KIT(CD117) (Figure 2). Given the liver masses on imaging and convincing pathology findings, he was diagnosed with metastatic GISTs with unknown primary and discharged to outpatient oncology for initiation of Imatinib mesylate. Within a couple weeks of treatment, he was hospitalized due to increased vascularity and hemorrhage within the tumor. Further genetic test was positive for D842V platelet derived growth factor receptor-alpha (PDGFRA) mutation which has been shown to confer resistance to Imatinib. He was then switched to second line agent- Sunitinib considering minimal improvement in lesion size on ultrasound with 3 months treatment with Imatinib. DISCUSSION: Most of the patients who have imatinib-resistance show KIT exon 9 or PDGFRA exon 18 D842V mutations. For patients imatinib-resistant GIST, sunitinib is a second-line drug treatment. This case highlights the need for further precise and targeted research on the various mechanisms of imatinib resistance in those presenting with metastatic GIST.Figure 1.: Core needle Biopsy of liver Panel A: H&E, objective x20; Panel B: H&E, objective x40; Panel C: immunohistochemistry of DOG1, x20 objective. Microscopic description: The entire liver core needle biopsy shows diffuse sheet of tumor cells (Panel A) with oval and spindled cytologic feature (Panel B) with very rare mitosis. Immunohistochemistry shows the tumor cells are strongly and diffusely positive for DOG1(Panel C), weakly positive for CD117 (not shown). It was diagnosed metastatic gastrointestinal stromal tumor to the liver. Molecular study (Quest diagnostics, Chantilly) on core biopsy specimen detected D842V mutant in the PDGFRA gene Exon 18.Figure 2.: Axial cut (left) and coronal cut (right) of a CT of the patient described showing a large liver lesion.

Heterogeneous EGFR, CDK4, MDM4, and PDGFRA Gene Expression Profiles in Primary GBM: No Association with Patient Survival.

Background: The prognostic impact of the expression profile of genes recurrently amplified in glioblastoma multiforme (GBM) remains controversial. Methods: We investigated the RNA gene expression profile of epidermal growth factor receptor (EGFR), cyclin-dependent kinase 4 (CDK4), murine doble minute 4 (MDM4), and platelet derived growth factor receptor alpha (PDGFRA) in 83 primary GBM tumors vs. 42 normal brain tissue samples. Interphase FISH (iFISH) analysis for the four genes, together with analysis of intragenic deletions in EGFR and PDGFRA, were evaluated in parallel at the DNA level. As validation cohort, publicly available RNA gene expression data on 293 samples from 10 different GBM patient series were also studied. Results: At the RNA level, CDK4 was the most frequently overexpressed gene (90%) followed by EGFR (58%) and PDGFRA (58%). Chromosome 7 copy number alterations, i.e., trisomy (49%) and polysomy (44%), showed no clear association with EGFR gene expression levels. In turn, intragenic EGFR deletions were found in 39 patients (47%), including EGFRvIII (46%) in association with EGFRvIVa (4%), EGFRvII (2%) or other EGFR deletions (3%) and PDGFRA deletion of exons 8–9 was found in only two tumors (2%). Conclusions: Overall, none of the gene expression profiles and/or intragenic EGFR deletions showed a significant impact on overall survival of GBM supporting the notion that other still unraveled features of the disease might play a more relevant prognostic role in GBM.

Significance of KIT and PDGFRA mutations in gastric gist imatinib - naive surgically treated patients

Background/Aim. KIT (KIT proto-oncogene receptor tyrosine kinase) and PDGFRA (platelet-derived growth factor receptor alpha) gene mutations represent major molecular forces inside the gastrointestinal stromal tumors (GIST). Aim of this study was to evaluate these mutations in the patients who underwent surgical resection of gastric GIST, but without imatinib mesylate treatment. Methods. Retrospective clinical study included patients who were operated on due to gastric GIST from November 2000 till November 2016. A molecular analysis of paraffin embedded tumor tissue was performed, and the patients with the presence of KIT and PDGFRA mutations were further evaluated, with regard to the pathological tumor stage, disease recurrence and overall survival. Results. Out of 45 patients in total, 43 patients had KIT and PDGFRA mutations, and 2 patients were classified as the wild type GIST. After curative resection, 11 patients were classified as a low risk GIST, 8 as an intermediate risk and 26 as a high risk GIST. The KIT mutations were present in 37 patients, most commonly as deletion in exon 11. The PDGFRA mutations were present in 6 patients. The presence of KIT mutation had a strong statistical correlation with the mitotic index (p = 0.021). After the ten-year follow-up, all patients with the PDGFRA mutations were alive, while those with the KIT mutations had a survival rate of 71% (p = 0.31). Conclusion. The presence of KIT exon 11 deletion in the patients with primarily resected gastric GIST is associated with the higher mitotic index and worse overall survival than those present with the PDGFRA mutations. This results suggest prognostic significance towards more aggressive behaviors.

Comprehensive molecular screening by next generation sequencing reveals a distinctive mutational profile of KIT/PDGFRA genes and novel genomic alterations: results from a 20-year cohort of patients with GIST from north-western Greece

IntroductionGastrointestinal stromal tumours (GIST) are mesenchymal neoplasms that usually carry an activating mutation in KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes with predictive and prognostic significance. We investigated the extended mutational status of GIST in a patient population of north-western Greece in order to look at geopraphic/genotypic distinctive traits.Patient and methodsClinicopathological and molecular data of 38 patients diagnosed from 1996 to 2016 with GIST in the region of Epirus in Greece were retrospectively assessed. Formalin-fixed paraffin-embedded tumours were successfully analysed for mutations in 54 genes with oncogenic potential. Next generation sequencing was conducted by using the Ion AmpliSeqCancer Hotspot Panel V.2 for DNA analysis (Thermofisher Scientific).ResultsAmong 38 tumours, 24 (63.16%) and seven (18.42%) of the tumours harboured mutations in the KIT and PDGFRA genes, respectively, while seven (18.42%) tumours were negative for either KIT or PDGFRA mutation. No mutations were detected in five (13.16%) cases. Concomitant mutations of BRAF and fibroblast growth factor receptor 3 (FGFR3) genes were observed in two patients with KIT gene mutation. Two patients with KIT/PDGFRA wild-type GIST had mutations in either KRAS or phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) genes. There was no significant survival difference regarding the exonic site of mutation in either KIT or PDGFRA gene. The presence of a mutation in pathway effectors downstream of KIT or PDGFRA, such as BRAF, KRAS or PIK3CA, was associated with poor prognosis. Adverse prognosticators were also high mitotic index and the advanced disease status at diagnosis.ConclusionsWe report comparable incidence of KIT and PDGFRA mutation in patients with GIST from north-western Greece as compared with cohorts from other regions. Interestingly, we identified rare mutations on RAS, BRAF and PIK3CA genes in patients with poor prognosis.

A genome-wide association study of corneal astigmatism: The CREAM Consortium

To identify genes and genetic markers associated with corneal astigmatism. A meta-analysis of genome-wide association studies (GWASs) of corneal astigmatism undertaken for 14 European ancestry (n=22,250) and 8 Asian ancestry (n=9,120) cohorts was performed by the Consortium for Refractive Error and Myopia. Cases were defined as having >0.75 diopters of corneal astigmatism. Subsequent gene-based and gene-set analyses of the meta-analyzed results of European ancestry cohorts were performed using VEGAS2 and MAGMA software. Additionally, estimates of single nucleotide polymorphism (SNP)-based heritability for corneal and refractive astigmatism and the spherical equivalent were calculated for Europeans using LD score regression. The meta-analysis of all cohorts identified a genome-wide significant locus near the platelet-derived growth factor receptor alpha (PDGFRA) gene: top SNP: rs7673984, odds ratio=1.12 (95% CI:1.08-1.16), p=5.55×10-9. No other genome-wide significant loci were identified in the combined analysis or European/Asian ancestry-specific analyses. Gene-based analysis identified three novel candidate genes for corneal astigmatism in Europeans-claudin-7 (CLDN7), acid phosphatase 2, lysosomal (ACP2), and TNF alpha-induced protein 8 like 3 (TNFAIP8L3). In addition to replicating a previously identified genome-wide significant locus for corneal astigmatism near the PDGFRA gene, gene-based analysis identified three novel candidate genes, CLDN7, ACP2, and TNFAIP8L3, that warrant further investigation to understand their role in the pathogenesis of corneal astigmatism. The much lower number of genetic variants and genes demonstrating an association with corneal astigmatism compared to published spherical equivalent GWAS analyses suggest a greater influence of rare genetic variants, non-additive genetic effects, or environmental factors in the development of astigmatism.

Association of PDGFRA gene polymorphisms and early-onset myopia in South Sumatera, Indonesia

Purpose: Myopia is a refraction error that may caused by corneal curvature (CC) anomaly. Platelet-derived growth factor receptor alpha (PDGFRA) gene was determined to have an effect on CC. The purpose of this study was to find a correlation between single nucleotide polymorphisms (SNP) in PDGFRA gene and early-onset myopia in South Sumatera tribes, a part of Indonesian ethnic group.&#x0D; Design: Using a random sampling method, population-based, case control study included 100 subjects aged 18-40 years from Palembang, South Sumatera, Indonesia.&#x0D; Methods: Visual acuity was measured by Snellen chart and CC was measured by manual keratometer. DNA sample from buccal swab was investigated with Amplification Refractory Mutation System (ARMS) PCR and visualized in agarose gel.&#x0D; Results: Median of CC for the right eye was 7.73 (7.07-8.63) mm and the left eye was 7.73 (7.04-8.69) mm. There was no difference between CC in myopic and normal subjects. Distribution of mutant allele in rs17084051, rs7677751, rs7682912, and rs2114039 were higher in myopic subject compare to those of normal control. Significant association between PDGFRA gene polymorphism and early-onset myopia was found only in rs17084051 (p = 0.009) and rs7677751 (p = 0.001).&#x0D; Conclusions: Mutant type allele A of rs17084051 and mutant type allele T of rs7677751 of PDGFRA gene polymorphism are associated with early-onset myopia in South Sumatera tribes in this study.

Succinate dehydrogenase deficient gastrointestinal stromal tumor: a clinicopathologic analysis of eight cases

To study the clinicopathologic features, diagnosis and differential diagnosis of succinate dehydrogenase (SDH) deficient gastrointestinal stromal tumors (GISTs) as a unique tumor subtype. SDHB and SDHA immunohistochemistry was performed in 120 gastric GISTs, in addition to CD117, DOG-1, CD34, smooth muscle actin (SMA), desmin, S-100 protein, cytokeratin (CK) and Ki-67. Subset of the cases was further evaluated for the presence of mutations in CKIT exons 9, 11, 13 and 17 mutations and platelet derived growth factor receptor alpha(PDGFRA) exons 12 and 18. Eight of 120 (6.6%) GIST cases were found SDH-deficient including 3 male and 5 female patients (median age of 36.2 years; ranging 16 to 65 years of age). The tumors involved antrum (6 cases), lesser curvature (1 case) and fundus (1 case). Macroscopically, the dominant tumor masses varied from 3 to 10 cm in diameter with a multinodular or plexiform pattern involving the gastric wall. Microscopically,tumor cells had predominantly epithelioid morphology, with occasional mixed spindle cell nodules. Lymphovascular invasion was identified in 5 cases. Immunohistochemistry for SDHB was negative in all 8 cases, and SDHA was negative in 5 cases. All 8 SDHB negative cases also expressed CD117, DOG-1 and CD34, but were negative for SMA, desmin, S-100 and CK. All 8 cases were found to have wild-type CKIT and PDGFRA genes. Available clinical follow-up were obtained in 7 cases, ranging from 2 to 60 months (median follow-up 23.3 months), and all patient were alive. Three cases were found to have liver metastases at their first diagnosis, and one developed omental and mesenteric metastases in 17 months. SDH-deficient GIST is a distinct subtype of GIST, with a predilection to occur in young and female patients. Characteristic pathological findings include multinodular gastric wall involvement, epithelioid cell morphology, frequently lymphovascular invasion with occasional lymph node and liver metastases, but an overall indolent clinical behavior. Immunohistochemistry for SDHB is required for the diagnosis.

An Unusual Case of Sigmoid Colon Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumors (GIST), uncommon mesenchymal tumors, are driven by KIT or plateletderived growth factor receptor alpha (PDGFRA) mutations. They are most commonly diagnosed in older adults (median 62-63 years) presenting with GI bleeding or peptic ulcer-like symptoms, and most are found in the stomach (approximately 60%). GISTs are much more rarely diagnosed in the colon ( < 1%), with the sigmoid colon most commonly affected. We review the case of a 90-year-old white man who presented with one day of painless bright red blood per rectum. He denied abdominal pain, nausea, and vomiting. Past medical history included atrial fibrillation on warfarin, stroke, prostate cancer with retropubic radical prostatectomy, and hypertension. The patient was hemodynamically stable with a benign abdominal examination. Rectal examination revealed maroon blood but no hemorrhoids, fissures, or masses. Laboratory evaluation revealed hemoglobin 7.3 g/dL, WBC 7.6x10(9), INR 2.73, and creatinine 0.6 mg/dL. Electrolytes were unremarkable. A CT abdomen/pelvis revealed extensive bowel wall thickening focally, at the junction of the sigmoid and descending colon, with adjacent stranding (Figure 1). The patient underwent his index colonoscopy revealing a nearly-obstructing, necrotic, ulcerated mass with overlying clot 45 cm from the anal verge. (Figure 2). Several cold forceps biopsies were taken diagnosing a gastrointestinal stromal tumor, spindle cell subtype. Within the biopsy specimen there was no mucosal ulceration or mitoses and the ki-67 proliferative index was < 1%. By immunohistochemistry, the tumor cells expressed CD117 (c-kit), CD34, and SMA, and were negative for S100. Neither the c-KIT nor PDGFRA gene mutation was detected. Given the extent of disease and the patient's comorbidities, surgical resection was not pursued and he was initiated on imatinib 400 mg daily. At six months the patient was tolerating therapy well aside from lower extremity edema, a common side effect of imatinib.Figure 1Figure 2An estimated 12-15% of adult GIST lack KIT and PDGFRA mutations, which are mutually exclusive, and are classified as wild type. This case highlights the importance of using several histopathology markers to aid in the diagnosis of GIST. Over 95% of GIST express CD117 and 70-90% also express CD34, as was seen in this case. Those with the mutations are more likely to respond to imatinib therapy, a tyrosine kinase inhibitor., which allows therapy to be individualized.

Platelet-derived growth factor alpha mediates the proliferation of peripheral T-cell lymphoma cells via an autocrine regulatory pathway

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

Diagnostic significance of DOG-1 and PKC-θ expression and c-Kit/PDGFRA mutations in gastrointestinal stromal tumours

Objective. To investigate discovered on gastrointestinal stromal tumor (GIST)-1 (DOG-1) and protein kinase C-θ (PKC-θ) expression in a series of GISTs and determine the sensitivity, specificity, and diagnostic value of these two antigens. Methods.Immnunohistochemistry (IHC) was used to detect CD117, DOG-1, PKC-θ, CD34, Ki-67, α-smooth muscle actin (SMA), S100, and Desmin expression in 147 GISTs and 51 non-GISTs. c-Kit gene (exons 9, 11, 13, and 17) and platelet-derived growth factor receptor-alpha (PDGFRA) gene (exons 12 and 18) mutations were also detected. Results.About 94.5% GISTs were CD117 positive, 96% were DOG-1 positive, and 90.5% were PKC-θ positive. DOG-1 had a specificity of 100%, while CD117 and PKC-θ had a specificity of 90% and 80%, respectively. There was no significant difference between DOG-1 and PKC-θ expressions when compared to CD117 expression. In 30 out of 42 (71.5%) GISTs, a c-Kit gene mutation was found, and in 3 out of 42 cases (7%), PDGFRA was mutated. Wild-type c-Kit/PDGFRA genes accounted for 21.5% (9/42). Most c-Kit gene mutations were found to be located at exon 11, mainly as in-frame deletions. Mutations in exon 9 were all missense mutations. Most PDGFRA gene mutations were found in exon 18, codon 842. c-Kit gene mutations in exons 13 and 17, and the PDGFRA gene mutation in exon 12 were not detected. Conclusions.Compared to CD117, DOG-1 is a biomarker with higher sensitivity and specificity. The combination of CD117 and DOG-1 can be used to improve the diagnosis of GIST. Although PKC-θ has a lower specificity than DOG-1, it can be a useful biomarker, especially in CD117− and/or DOG-1− cases.

A subset of gastrointestinal stromal tumors previously regarded as wild-type tumors carries somatic activating mutations in KIT exon 8 (p.D419del)

About 10–15% of gastrointestinal stromal tumors (GISTs) carry wild-type sequences in all hot spots of KIT and platelet-derived growth factor receptor alpha (PDGFRA) (wt-GISTs). These tumors are currently defined by having no mutations in exons 9, 11, 13, and 17 of the KIT gene and exons 12, 14, and 18 of the PDGFRA gene. Until now, the analysis of further exons is not recommended. However, we have previously published a report on a KIT exon 8 germline mutation, which was associated with familial GIST and mastocytosis. We therefore investigated whether KIT exon 8 mutations might also occur in sporadic GIST. We screened a cohort of 145 wt-GISTs from a total of 1351 cases from our registry for somatic mutations in KIT exon 8. Two primary GISTs with an identical exon 8 mutation (p.D419del) were detected, representing 1.4% of all the cases analyzed. Based on all GISTs from our registry, the overall frequency of KIT exon 8 mutations was 0.15%. The first tumor originating in the small bowel of a 53-year-old male patient had mostly a biphasic spindled-epithelioid pattern with a high proliferative activity (14 mitoses/50 HPF) combined with a second low proliferative spindle cell pattern (4/50 HPF). The patient developed multiple peritoneal metastases 29 months later. The second case represented a jejunal GIST in a 67-year old woman who is relapse-free under adjuvant imatinib treatment. We conclude that about 1–2% of GISTs being classified as ‘wild type’ so far might, in fact, carry KIT mutations in exon 8. Moreover, this mutational subtype was shown to be activating and imatinib sensitive in vitro. We therefore propose that screening for KIT exon 8 mutations should become a routine in the diagnostic work-up of GIST and that patients with an exon 8 mutation and a significant risk for tumor progression should be treated with imatinib.

Tyrosine-kinase mutations in c-KIT and PDGFR-alpha genes of imatinib naïve adult patients with gastrointestinal stromal tumours (GISTs) of the stomach and small intestine: relation to tumour-biological risk-profile and long-term outcome

The identification of activating mutations in either c-KIT cell surface growth factor receptor or platelet-derived growth factor receptor alpha (PDGFRA) has lead the way for the development of novel agents that selectively inhibit key molecular events in gastrointestinal stromal tumour (GIST) pathogenesis. The aim of this study was to investigate the role of c-KIT and PDGFRA gene mutations in primary resectable, imatinib naïve GISTs located in the stomach and small intestine. All adult patients with GIST located in either stomach or small intestine who underwent surgical resection without prior imatinib (Glivec) treatment were included. DNA extraction and mutational analysis were performed. Mutational analyses were performed for c-KIT (exons 9, 11, 13, and 17) and the PDGFRA genes (exons 12, 14 and 18). Clinical and pathological parameters were analyzed in relation to the mutations in c-KIT and PDGFRA. A total of 38 patients who underwent surgery for GIST located in either the stomach (n=24) or in the small intestines (n=14) were included. Mutations were found in 31 of 38 (81.6%) patients, with 24 (63.2%) located in c-KIT and 7 (18.4%) in the PDGRFA exons, respectively. Seven patients (18.4%) were wildtype (WT). The most common mutation was in c-KIT exon 11. Incidentally found GISTs were significantly smaller (size >5cm in 15% for incidental vs. 71% for symptomatic; OR of 13.4, 95% CI 2.3-76.5; P=0.001) and had lower mitotic rate (0% for incidental vs. 44% of the symptomatic; OR 0.52, 95% CI 0.36-0.75; P=0.005). Accordingly, the Fletcher grade was significantly better for incidental cases, with most having very low or low risk (85%) in contrast to 19 of 25 (76%) symptomatic cases showing moderate to high-risk features (OR 17.4, 95% CI 2.98-101.7; P<0.001). However, the distribution of c-KIT, PDGFRA and WT was not differently distributed between incidental and symptomatic GISTs. Long-term survival up to 25years (median: 8years) was best determined by Fletcher risk-score in the multivariate model (HR 14.1, 95% CI 1.7-114.5; p=0.013). Long-term survival in resected GISTs of the stomach and small intestine is best determined by Fletcher risk-score. Mitotic activity appears related to tumour size and young age at onset. Mutational status did not influence the clinical or tumour-specific features in this cohort.

Abstract 3586: CP-868,596, a highly potent PDGFR inhibitor, inhibits phosphorylation of the imatinib-resistant PDGFRA D842V activating mutation associated with advanced GIST

Abstract Background: 8-10% of gastrointestinal stromal tumors (GIST) have activating mutations of the platelet-derived growth factor receptor alpha (PDGFRA) kinase. The most common PDGFRA mutation is the D842V substitution that is encoded by exon 18. This gain-of-function mutation results in auto-phosphorylation and constitutive activation of PDGFRA kinase activity. Type II receptor tyrosine kinase (RTK) inhibitors, such as imatinib and sunitinib, which only bind to the inactive conformation of the RTK, have little or no in vitro activity against this mutation. Clinically, these drugs are not effective for the treatment of GIST patients with D842V mutation. In addition, the D842V mutation can develop as a secondary imatinib resistant mutation during treatment of GISTs with primary imatinib-sensitive PDGFRA mutations (e.g. primary exon 12 mutations), or treatment of patients with hypereosinophillic syndrome with translocations involving PDGFRA. CP-868,596 is an orally bioavailable, highly potent and selective PDGFR TKI. CP-868,596 is a benzimidazole compound that has IC50s of 0.9 nM and 1.8 nM for PDGFRA and PDGFRB, respectively. Phase I trials of CP-868,596 have shown good oral bioavailability, a favorable toxicity profile, and achievable serum concentrations as high as 2000 nM. Methods: Mutant PDGFRA isoforms were expressed by transient transfection of Chinese Hamster ovary cells. The transfected cells were treated with various concentrations of CP-868,596 before preparation of protein lystates. PDGFRA protein was assayed for activation status (phosphorylation) by immunoprecipitation using an anti-PDGFRA antibody, followed by sequential immunoblotting for phospho PDGFRA (using antiphosphotyrosine antibody) or total PDGFRA (anti-PDGFRA monoclonal antibody). IC50 was measured by densitometry of the phospho PDGFRA bands and normalization using total PDGFRA expression (to correct for variations in loading of PDGFRA protein in the various lanes). Results: CP-868,596 inhibited the phosphorylation of wild type PDGFRA at an IC50 of 10 nM and PDGFRA D842V with an IC50 between 10 to 30 nM. Imatinib was ineffective in blocking PDGFRA D842V phosphorylation in these experiments (IC50 &amp;gt; 1000 nM). Profiling of CP-868,596 against other GIST-relevant primary and secondary PDGFRA mutations is ongoing and will be reported. Discussion: GISTs due to D842V activating mutations of PDGFRA gene are clinically resistant to imatinib, sunitinib, and other commercially available tyrosine kinase inhibitors. CP-868,596 blocks phosphorylation of the PDGFRA D842V mutant at clinically achievable concentrations, providing a potentially new therapeutic modality for GIST patients. A clinical trial in GIST patients with primary or secondary PDGFRA D842V mutations is being initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2011-3586

Unusual metastases of gastrointestinal stromal tumor and genotypic correlates: Case report and review of the literature.

Gastrointestinal stromal tumors (GISTs) are defined as mesenchymal tumors of the gastrointestinal tract and are characterized by positive CD117 staining, and in most cases positive CD34 staining, with compatible gross features and microscopic findings of a highly cellular mesenchymal tumor of the gastrointestinal tract composed of spindle cells, epithelioid cells or a combination of both (1). They are usually derived from a mutation of the KIT (CD117) or PDGFRA (platelet derived growth factor receptor alpha) gene. Distinguishing GIST from other mesenchymal derived tumors was historically a challenge, since both can arise from the interstitial cells of Cajal, or GI pacemaker cells that form the interface between the autonomic innervation and smooth muscle of the bowel wall (2). The distinction of GISTs based on molecular etiology was described by Hirota et al in 1998, with discovery of a mutation in c-KIT encoding a pro-oncogenic receptor tyrosine kinase (KIT) (3). It is estimated that 4500 to 6000 new cases of GIST are diagnosed in the United States annually and most occur in the stomach (50%-70%) or small intestine (20%-30%) (4). GISTs are often asymptomatic and discovered incidentally during surgery, endoscopic procedures, or imaging studies. However, the clinical presentation of some GISTs may include overt GI bleeding, abdominal mass, abdominal pain, or bowel obstruction and acute abdomen (2). The most common metastatic sites of gastrointestinal stromal tumors are the liver (65%) and peritoneum (21%); GISTs rarely metastasize to lymph nodes (6%), bone (6%), lung (2%) (2),(5), and soft tissue (less than 1%) (6),(7). We report the case of a female diagnosed with GIST with subsequent metastases to the liver, peritoneum, lung, bone, and soft tissue.

Mutation-Free Expression of c-Kit and PDGFRA in Phyllodes Tumors of the Breast

Purpose: Phyllodes tumors (PTs) of the breast have been classified as benign, borderline, or malignant based on their histopathologic features. However, predicting clinical behavior based on these features has proven to be difficult given that local recurrence occurs in both benign and malignant PTs. Recurrence has been shown to mirror the histologic pattern of the primary tumor or to show dedifferentiation. The aim of this study was to assess the value of the histopathologic parameters, expression or mutation of c-Kit and platelet derived growth factor receptor alpha (PDGFRA) in predicting tumor recurrence. Methods: Representative areas from 39 benign, 16 borderline, and 12 malignant PTs were selected for construction of tissue microarrays. Immunohistochemical analyses for p53, Ki-67, c-Kit, and PDGFRA were performed and SSCP-PCR analysis was carried out to identify mutations in exons 9, 11, 13, and 17 of the c-Kit gene and exons 12 and 18 of the PDGFRA gene. Clinicopathologic features, including tumor recurrence and margin status, were also evaluated. Results: Of the 67 PTs, 11 cases (16.4%) recurred from 3 to 92 months following initial diagnosis (4 benign, 2 borderline, and 5 malignant). One benign PT case recurred as a borderline tumor and two borderline PT cases recurred as malignancies. Three patients died of malignant PT. No mutations of the c-Kit or PDGFRA genes were found and there was no statistically significant association of either p53 or p16 immunostaining with recurrent disease (p>0.05). However, histologic grade (p=0.033), margin status (p< 0.001), Ki-67 (p=0.012), c-Kit (p=0.002), and PDGFRA (p= 0.007) stromal immunopositivity were significantly correlated with recurrence. Conclusion: Even though positive or close margins were significantly associated with tumor recurrence, stromal c-Kit, PDGFRA positivity, and the Ki-67 index were useful for predicting recurrent PTs. Despite this, no c-Kit or PDGFRA mutations were found.

Sarcomatoid carcinoma of the urinary bladder: a case report with immunohistochemical and molecular genetic analysis

The author reports a rare case of sarcomatoid carcinoma with an emphasis on immunohistochemical features. A 79-year-old man was admitted to our hospital because of hematuria. An endoscopy revealed a large polypoid tumor in the bladder, and urine cytology demonstrated malignant cells. A cystectomy was performed. The patient is now alive without metastasis 4 months after the operation. Grossly, a large polypoid tumor (5 x 6 x 5 cm) was present in the bladder. Microscopically, the tumor consisted of high-grade transitional cell carcinoma element (10% in area) and sarcomatoid element (90% in area). There was a gradual transition between the two. The tumor cells were invaded into peribladder tissue (pT3b). Immunohistochemically, the sarcomatoid element was positive for four types of pancytokeratins, high-molecular weight cytokeratin (CK), CK5/6, CK7, CK18, CK19, epithelial membrane antigen (EMA), vimentin, p53 protein, p63, Ki-67 (labeling = 92%), neuron-specific enolase (NSE), and platelet-derived growth factor receptor-alpha (PDGFRA). It was negative for CK14, CK20, melanosome, carcinoembryonic antigen (CEA), desmin, S100 protein, myoglobin, alpha-smooth muscle antigen (ASMA), CD34, chromogranin, synaptophysin, CD56, CD68, and KIT. The transitional cell carcinoma element showed similar immunoreactivity except for negative CK5/6, positive CK20, and negative vimentin. A molecular genetic analysis of KIT gene (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) gene with the use of PCR-direct sequencing showed no mutations. The present case is the first report of sarcomatoid carcinoma of the urinary bladder demonstrating extensive immunohistochemistry and mutational status of KIT and PDGFRA genes. The sarcomatoid carcinoma in the present case may be derived from sarcomatous differentiation of high-grade transitional cell carcinoma.