Platelet-derived Growth Factor Receptor-α Research Articles

Expression level of vascular endothelial growth factor receptor-2 in radical nephrectomy specimens as a prognostic predictor in patients with metastatic renal cell carcinoma treated with sunitinib

ObjectivesTo investigate the expression levels of multiple molecular markers in radical nephrectomy specimens from patients with metastatic renal cell carcinoma (RCC) treated with sunitinib in order to identify factors predicting susceptibility to this agent. Materials and methodsThis study included a total of 40 consecutive patients undergoing radical nephrectomy, who were diagnosed as having metastatic RCC and subsequently treated with sunitinib. Expression levels of 10 molecular markers, including Bcl-2, Bcl-xL, Bax, phosphorylated Akt, p44/42 mitogen-activated protein kinase, and signal transducers and activation of transcription 3, vascular endothelial growth factor receptor (VEGFR)-1 and -2, and platelet-derived growth factor receptor-α and -β, in primary RCC specimens were assessed by immunohistochemical staining. ResultsOf several factors examined, tumor grade and the expression level of VEGFR-2 were shown to have significant impacts on response to sunitinib in these 40 patients. Progression-free survival (PFS) was significantly associated with the expression levels of VEGFR-2 in addition to tumor grade, performance status, Memorial Sloan-Kettering Cancer Center risk classification and pretreatment c-reactive protein level on univariate analysis. Of these significant factors, only VEGFR-2 expression appeared to be independently related to PFS on multivariate analysis. In fact, PFS in patients with strong expression of VEGFR-2 was significantly favorable compared with that in those with weak expression of VEGFR-2. ConclusionsCollectively, these findings suggest that it would be useful to consider expression levels of potential molecular markers, particularly VEGFR-2, as well as conventional clinical parameters to select metastatic RCC patients likely to benefit from treatment with sunitinib.

Shared molecular targets in pediatric gliomas and ependymomas

Recent advances in multidisciplinary treatment approaches have improved the overall prognosis of pediatric brain tumors, but some patients remain refractory to treatment and do poorly. Several molecularly targeted therapies are under development for the treatment of brain tumors, and high-grade gliomas in adults are a particular area of study. To better understand if these new therapies can be used in pediatric populations, we examined the expression of the following seven marker genes involved in signaling pathways targeted by new therapies: β-catenin, suppressor of fused (SUFU), erythroblastic leukemia viral oncogene homolog (ERBB) 2, platelet-derived growth factor receptorα (PDGFRα), proliferating cell nuclear antigen (PCNA), secreted protein acid and rich in cysteine (SPARC), and granulocyte colony-stimulating factor receptor (G-CSFR). Samples from 27 patients with the primitive neuroectodermal tumor (PNET)/medulloblastomas (MBs) (n = 8), ependymomas (n = 5), or gliomas (n = 14) were assessed by quantitative real-time PCR. [Correction made here after initial online publication]. We assigned an EXP score to compare across samples and determined the levels of gene expression among tumor cell types. Gene expression varied among the different tumors, but, within a tumor type, clear expression patterns were seen. The expression of SUFU, ERBB2, and PCNA in metastatic MBs were greater than that seen in non-metastatic MBs. Most glioma cases highly expressed PDGFRα and G-CSFR. Additionally, the expression patterns of gliomas and ependymomas were similar (r = 0.77, P = 0.04), but PNET/MBs substantially differed from gliomas (r = -0.37, P = 0.41) or ependymomas (r = 0.23, P = 0.62). The development of new drugs targeting up-regulated pathways may be useful for the treatment of pediatric brain tumors. As new drugs are developed, gliomas and ependymomas may be treated with similar compounds.

Gene Mutations and Prognostic Factors Analysis in Extragastrointestinal Stromal Tumor of a Chinese Three-Center Study

BackgroundMost gastrointestinal stromal tumors (GISTs) have gain-of-function mutation of the c-kit gene, and some have mutation of the platelet-derived growth factor receptor-α (PDGFR-α) gene. Extragastrointestinal stromal tumors (EGISTs) are mesenchymal tumors that occur outside the digestive tract. But the clinicopathologic characteristics of EGISTs are still poorly understood. MethodsParaffin-embedded tissues from 25 cases of EGIST were analyzed for CD117, CD34, Ki-67, S-100, smooth muscle actin, and desmin expression by immunohistochemical method. These cases of EGISTs were also evaluated for the presence of c-kit exons 9, 11, 13, and 17 mutations and PDGFR-α exons 12 and 18 mutations. Survival analysis was used to evaluate the prognostic factors. Resultsc-kit mutations were detected in 44% of EGIST patients and all were exon 11 mutations. PDGFR-α mutations were found in 12% of the 25 cases and all were exon 18 mutations. Survival analysis indicated that mitotic count and Ki-67 labeling index (Ki-67 LI) were significant predictors of survival. ConclusionThe pattern of c-kit and PDGFR-α mutation in EGISTs was essentially similar to that in GISTs. From the molecular genetics aspect, EGISTs may be a special subtype of GISTs. The results also show that the combination of mitotic counts and Ki-67 LI may be useful for predicting the prognosis of EGISTs.

PDGF‐induced proliferation in human arterial and venous smooth muscle cells: Molecular basis for differential effects of PDGF isoforms

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum-starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF-AA, venous SMCs showed no proliferative response to PDGF-AA, but instead demonstrated a significantly greater proliferative response to PDGF-BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5-fold higher (P < 0.05) density of PDGF receptor-α (PDGF-Rα) and a 6.6-fold lower (P < 0.05) density of PDGF-Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF-AA in arterial SMCs was a marked PDGF-Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c-Jun NH2-terminal kinase (JNK), and a significant reduction in expression of the cell-cycle inhibitor p27(kip1). This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF-Rα was detected after venous SMC exposure to PDGF-AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF-BB stimulation of venous SMC resulted in PDGF-Rβ activation as well as transactivation of epidermal growth factor receptor (EGF-R); transactivation of EGF-R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins.

Down-regulation of miR-221 and miR-222 correlates with pronounced Kit expression in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs), are characterized by mutations of the KIT or platelet-derived growth factor receptor-α gene and the constitutive expression of Kit, which is currently being studied as a potential therapeutic target. In this study, we addressed the question of whether the microRNA (miRNA) 221/222 cluster (miR-221/222), which has been shown to be dysregulated in many malignancies, is linked to GIST diagnosis and prognosis, and whether it could provide a basis for possible therapeutic approaches. We analyzed the expression of miR-221 and miR-222 in 54 formalin-fixed and paraffin-embedded GISTs and corresponding peripheral non-tumorous tissue by real-time PCR. The miRNA-expression levels were studied in relation to histomorphological parameters, KIT mutation status and immunohistochemical Kit expression. miR-221 and miR-222, were reduced in most of the GISTs, in contrast to other tumors. No correlation was observed between miR-221/222 expression levels and histomorphological parameters, tumor risk grade, or KIT mutation status. However, we found major differences in miRNA expression among the different groups of immunohistochemical Kit expression, especially between Kit-negative and -positive tumors. The expression levels of miR-221 and miR-222 were significantly repressed in Kit-positive GISTs, compared to normal tissue, whereas Kit-negative GISTs exhibited a completely inverse expression pattern. This study shows for the first time that miR-221 and miR-222 can act as regulators of Kit expression in GISTs and hence reveals a new aspect in the molecular pathogenesis of these tumors. Although miR-221/222 expression does not have an impact on diagnostics, it could be considered as a tool for future therapeutic strategies for GISTs, especially for tumors with secondary resistance to tyrosine kinase inhibitors.

Pediatric and Adult Gliomas: How Different Are They?

Recently, I was discussing the treatment of a pediatric patient who had gliomatosis cerebri. A pediatric neuro-oncologist, one of my respectable friends, insisted on chemotherapy with temozolomide. Because the histologic type was anaplastic astrocytoma (as determined by open biopsy), many would consider radiotherapy the primary option; thus, there was an exciting discussion. On another occasion, the same colleague and I were discussing a pediatric patient with a symptomatic diffuse astrocytoma of the posterior thalamus; this time as well, he insisted on a trial of chemotherapy with carboplatin and vincristine. My colleague's philosophy is that because pediatric gliomas are different from adult gliomas, chemotherapy to defer or avoid radiotherapy is reasonable in cases of low-grade and sometimes even high-grade glioma. Maybe my colleague is right regarding the difference between pediatric and adult gliomas. There have been numerous reports showing substantial differences in the molecular features underlying pediatric and adult high-grade gliomas. Pediatric and adult glioblastomas were shown to be clearly distinguished by frequent gain of chromosome 1q and lower frequency of chromosome 7 gain and 10q loss in the pediatric form. Platelet-derived growth factor receptor-α (PDGFR-α) was found to be the predominant gene subject to focal amplification in childhood high-grade gliomas.1,2 Among the pediatric low-grade gliomas, pilocytic astrocytoma is the predominant histologic type and diffuse astrocytomas are quite rare. In this issue of Neuro-Oncology, Stokland et al. from the Children's Brain Tumour Research Centre, University of Nottingham, UK, report on 639 pediatric patients with low-grade gliomas, 63.7% of which were pilocytic astrocytomas and only 5.9% of which were diffuse fibrillary astrocytomas.3 The predominance of the former type is consistent with other evidence. For instance, in the Brain Tumor Registry of Japan, the incidence of pilocytic astrocytomas peaks in patients between 5 and 9 years old, while that of diffuse astrocytomas peaks in patients between 40 and 44 years old. In the Central Brain Tumor Registry of the United States (CBTRUS), 1,834 pediatric patients (those between 0 and 19 years old) with pilocytic astrocytomas are registered, while only 101 cases of diffuse astrocytoma are found in the pediatric population. Therefore, pediatric low-grade gliomas are mostly pilocytic astrocytomas, and adult low-grade gliomas are mostly diffuse astrocytomas. Pilocytic astrocytomas and diffuse astrocytomas are classified as WHO grades I and II, respectively. However, pilocytic astrocytoma is not a precursor of diffuse astrocytoma, as there is a distinct difference between the molecular features of the two. There appears to be no role for either TP53 mutations or aberrant PDGF signaling in the development of pilocytic astrocytomas, in contrast to the role of TP53 mutations and increased expression of PDGF-A and PDGFR-α as common, early events in the formation of diffuse astrocytomas.4 Pilocytic astrocytomas also differ from diffuse astrocytomas in their altered and increased expression of immune response genes. Hierarchical clustering analysis using a set of 1,176 genes distinguished pilocytic astrocytomas from diffuse astrocytomas and oligodendrogliomas.5 Pilocytic astrocytomas are also different from the diffuse astrocytomas clinically, as evidenced by the fact that histology is one prognostic factor in pediatric low-grade gliomas.3 Fear of radiation-induced adverse effects has been used to justify the use of chemotherapy for unresectable pilocytic astrocytomas. A number of chemotherapy regimens have been used to delay the need for radiotherapy, including carboplatin and vincristine. A recent randomized trial, Children's Oncology Group A9952, compared the combination of carboplatin and vincristine with the combination of procarbazine, thioguanine, lomustine, and vincristine and reported 5-year event-free survival rates of 40%-50%. More than 400 low-grade gliomas were registered, and 83% of them were pilocytic astrocytomas.6 However, although a small study is under way, the role of chemotherapy in the management of pediatric diffuse astrocytomas remains unclear. In adult diffuse astrocytomas, a randomized study comparing radiotherapy and temozolomide is now ongoing in Europe and North America. Yes, pediatric and adult gliomas are different. Pediatric and adult high-grade gliomas are biologically different. On the other hand, differences between pediatric and adult low-grade gliomas are mainly due to the difference in their histologies. Whether pediatric diffuse astrocytomas are chemosensitive remains unproven. The UK study reported in this issue also suggests that, because of the small number of diffuse astrocytomas in the pediatric population, international cooperation will be needed to address the issue of the optimal management of this entity.

Expression of platelet-derived growth factor-alpha receptor in human osteosarcoma is not a predictor of outcome

The aims of this study were to examine the prognostic relevance of platelet-derived growth factor-α receptor (PDGFRA) expression in human osteosarcomas and to evaluate the mutation status of exon 12 and exon 18 of the PDGFRA gene. PDGFRA expression was examined in 100 human osteosarcomas by immunohistochemistry using paraffin embedded tumour tissues, and capillary sequencing of genomic DNA was performed to search for mutations in exons 12 and 18 of the PDGFRA gene. Ninety-six osteosarcomas showed PDGFRA expression ranging from 4% to 90% (mean 40%, median 37.5%, SD 27.11%). Furthermore, DNA sequence of exon 12 and exon 18 of the PDGFRA gene were not altered in 40 tumours with high PDGFRA expression. Overall and disease-free survival analysis did not reveal any differences between osteosarcoma patients with high PDGFRA expression and patients with low PDGFRA expression. The protein expression is not linked to mutations in exon 12 or exon 18 of PDGFRA gene. Therefore, treatment modalities based on the suppression of PDGFRA tyrosine kinase activity may need further investigation. PDGFRA expression is not a prognostic marker for osteosarcoma patients.

PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas

Gene rearrangement in the form of an intragenic deletion is the primary mechanism of oncogenic mutation of the epidermal growth factor receptor (EGFR) gene in gliomas. However, the incidence of platelet-derived growth factor receptor-α (PDGFRA) gene rearrangement in these tumors is unknown. We investigated the PDGFRA locus in PDGFRA-amplified gliomas and identified two rearrangements, including the first case of a gene fusion between kinase insert domain receptor (KDR) (VEGFRII) and the PDGFRA gene, and six cases of PDGFRA(Δ8, 9), an intragenic deletion rearrangement. The PDGFRA(Δ8, 9) mutant was common, being present in 40% of the glioblastoma multiformes (GBMs) with PDGFRA amplification. Tumors with these two types of PDGFRA rearrangement displayed histologic features of oligodendroglioma, and the gene products of both rearrangements showed constitutively elevated tyrosine kinase activity and transforming potential that was reversed by PDGFR blockade. These results suggest the possibility that these PDGFRA mutants behave as oncogenes in this subset of gliomas, and that the prevalence of such rearrangements may have been considerably underestimated.

Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

A phase II trial of imatinib (IM) in relapsed, nonresectable chondrosarcoma (CS) expressing platelet-derived growth factor receptor-α or -β (PDGFR-α/PDGFR-β): An Italian Sarcoma Group study.

10060 Background: CS is a rare and heterogeneous disease in which, after failure of surgery and radiotherapy, chemotherapy has a marginal role, if any. Different molecular pathways were shown to be activated in CS. Particularly, both PDGFR isoforms may be expressed and phosphorylated (PC Hogendoorn, MS Lagonigro). Since IM was proven effective in other sarcomas sharing the activation of this pathway, we investigated the activity of IM in advanced CS in a phase II trial. Methods: Between January 2007 and June 2009, 26 subjects, median age 61 (23-81), affected by metastatic, not resectable CS were treated with 400 mg of IM b.i.d. until progression or unacceptable toxicity. Eligibility criteria were: ≥1 prior line of chemotherapy, and immunohistochemical expression of either PDGFR-α or PDGFR-β . Primary objective was the percentage of patients free from progression at 4-month (Kaplan and Meier). Secondary objectives were: objective response (RECIST), overall survival, toxicity, clinical benefit (Pain Analgesic Score). Results: In all patients PDGFR was phosphorylated. Planned IM dose was delivered, but 60% (15) needed dose reductions due to manageable and short lasting toxicity [Median administered dose was 83% (74-91%) of full expected-one]. PFS at 4-month was 31% (2.6-7.8 months) (8). Median overall survival was 10.9 months. No objective responses, long lasting freedom from progression or improvement in PAS were observed. Conclusions: Imatinib, as expected, was relatively well tolerated, but it failed to show a meaningful clinical activity in term of both freedom from progression and tumor shrinkage. Advanced CS remains a disease not curable, and effective targeted therapies are still awaited. No significant financial relationships to disclose.

Abstract 1468: Antagonism of platelet-derived growth factor receptor-α as a potential therapeutic for prostate cancer bone metastases

Abstract Prostate adenocarcinoma is the second leading cause of cancer death among men, due primarily to the fact that the majority of prostate cancers will eventually spread to the skeleton. Metastatic dissemination requires a complex series of coordinated events that result in cells that escape from the primary tumor into the circulation and eventually colonize distant organs. The ability of disseminated cancer cells to evolve into metastases of clinically significant size depends strongly on their compatibility with, and ability to utilize, this new micro-environment. We previously showed that bone-metastatic prostate cancer cells exposed to human bone marrow respond by activation of cell survival pathways, such as PI3K/Akt., and that these events are mediated by the alpha-receptor for platelet-derived growth factor (PDGFRα). We also have shown that prophylactic antagonism of this receptor using IMC-3G3, a monoclonal, humanized antibody currently in phase II trials, was able to significantly reduce tumor burden in an in vivo model of skeletal metastasis. Here we show that IMC-3G3 can be used in a curative manner to reduce the growth of previously established skeletal lesions. In addition, we have successfully combined this antibody with zoledronic acid, a bisphosphonate commonly used to treat skeletal metastases, with encouraging results. Interestingly, treatment with a monoclonal antibody targeting only host PDGFRα failed to show any significant difference over saline treated mice. Lastly, we sought to dissect the mechanism by which PDGFRα may exert its role within the bone microenvironment. We provide evidence that soluble components of human bone marrow can activate PDGFRα through a mechanism that does not require the canonical binding of PDGF ligand(s) to the receptor. To establish the relevance of this phenomenon in vivo, we employed a PDGFRα mutant lacking the extracellular ligand-binding domain. Our studies show that this truncated PDGFRα was able to restore bone-metastatic potential of prostate cancer cells as effectively as the full-length form of the receptor. In addition, we have found that expression of PDGFRα, and its related signaling events, results in the regulation of a specific set of genes that we believe may aid in the survival and growth of metastatic prostate cancer cells within the bone. Studies to understand the contribution of several of these putative bone metastatic genes are currently underway in our laboratory. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1468.

β‐Catenin loss promotes DEN‐induced liver cancer via oxidative stress &amp; PDGFRα/PI3K signaling

β‐Catenin, the central effector of canonical Wnt pathway is a recognized ‘oncogene?for its role in tumors, including hepatocellular carcinoma (HCC). We investigated whether β‐catenin liver conditional knockout mice β‐cat KO) were protected from chemically induced HCC. Intriguingly, β‐cat KO mice were more susceptible to N‐nitrosodiethylamine (DEN)‐induced hepatocarcinogenesis than controls Wildtype animals. This susceptibility correlated to unrestricted oxidative stress, higher apoptosis and compensatory hepatocyte proliferation, secondary to increased platelet‐derived growth factor receptor‐α; (PDGFRα) and Phosphoinositide 3‐kinase (PIK3CA)/Akt activation. By in vivo and in vitro study, we found activation of nuclear factor κB (NF‐κB) in absence of β‐catenin to be accounting for the upregulation of PDGFRα. Short‐term treatment with PDGFRα inhibitor or long‐term feeding of N‐acetyl‐L cysteine (NAC)‐ an antioxidant and NF‐κB inhibitor, blocked PDGFRα/PIK3CA/Akt pathway, significantly impacting hepatocarcinogenesis. These results demonstrate an important role of β‐catenin as a regulator of oxidative stress in liver and highlights novel compensatory role of PDGFRα signaling in promoting hepatocarcinogenesis in absence of β‐catenin.

Neuroendocrine carcinoma of the esophagus: a case report with immunohistochemical and molecular genetic analyses of KIT and PDGFRA

Neuroendocrine carcinoma of the esophagus including small-cell carcinoma and large-cell carcinoma is a very rare entity. The author herein reports a neuroendocrine carcinoma of the esophagus with an emphasis on KIT and PDGFRA. An 81-year-old man presented as dysphagia. An endoscopic examination revealed an ulcerated large tumor in the middle esophagus. A biopsy of the esophageal tumor identified an undifferentiated carcinoma consisting of malignant cells with hyperchromatic nuclei without differentiations. An immunohistochemical study revealed that the tumor cells were positive for cytokeratins, neuron-specific enolase, synaptophysin, p53 protein, KIT, and platelet-derived growth factor receptor-α (PDGFRA). The Ki-67 labeling was 100%. A pathologic diagnosis of neuroendocrine carcinoma with expression of KIT and PDGFRA was made. A genetic analysis using PCR direct sequencing method identified no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes. The patient underwent radiation and cisplatin-based chemotherapy. However, he developed systemic metastases and died of respiratory failure 8 months after the first manifestation. The present case is the first one of esophageal neuroendocrine carcinoma with an examination of protein expressions and gene mutations of KIT and PDGFRA.

Poly(ADP-ribose) polymerase-1 (PARP-1); Platelet derived growth factor receptor-α (PDGFRA); Platelet derived growth factor-α (PDGFA; PDGF1)

Poly(ADP-ribose) polymerase-1 (PARP-1); Platelet derived growth factor receptor-α (PDGFRA); Platelet derived growth factor-α (PDGFA; PDGF1)

Pleomorphic phenotypes of gastrointestinal stromal tumors at metastatic sites with or without imatinib treatment

Secondary resistance of gastrointestinal stromal tumors (GISTs) to tyrosine kinase inhibitors occurs after several years' administration. However, the mechanism of resistance has not been fully clarified. In this study, we analyzed the genotypes and the histologic and immunohistochemical phenotypes of metastatic GISTs with and without imatinib treatment, and clarified the pleomorphic nature of metastatic GISTs. We examined 31 autopsy cases in which the patients died of multiple metastases of GISTs, and two surgically resected specimens with and without imatinib treatment. A total of 152 primary and metastatic lesions in 33 cases of GISTs were examined for histologic and immunohistochemical expression of KIT and CD34. We analyzed the expression of other receptor tyrosine kinases (RTKs) in KIT-negative lesions, including human EGFR-related 2 (HER2), epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (MET), platelet-derived growth factor receptor-alpha (PDGFRA), and platelet-derived growth factor receptor-beta (PDGFRB). Fifteen lesions in seven cases (9.9%) lacked KIT expression, and 74 (49%) in 22 cases lacked CD34 expression. Eight KIT-negative lesions in five cases expressed PDGFRB, one of which also expressed EGFR, and three lesions in one case expressed MET. Results for the other RTKs were negative. Missense point mutations at PDGFRB gene exon 12 were detected in one PDGFRB-positive case. Our results indicate that histomorphology, immunohistochemical phenotypes, and genotypes of metastatic GISTs vary among lesions, even in cases without imatinib treatment. A KIT-independent mechanism, such as activation of other RTKs, might participate in the proliferation of late-stage GISTs and might be a cause of secondary imatinib resistance.

Induced NG2 expressing microglia in the facial motor nucleus after facial nerve axotomy

Chondroitin sulfate proteoglycan (NG2) expressing cells, ubiquitously distributed in the CNS respond to injured or diseased neurons; however, their behaviors toward injured neurons have remained to be fully explored. In the present study, along with astrocytic and microglial responses, NG2 expressing cells reacted swiftly and robustly in the facial motor nucleus (FMN) subjected to axotomy. With time, hypertrophic NG2 expressing cells gradually adhered to and enwrapped the axotomized motoneurons. Tight encapsulations around axotomized motoneurons were eventually formed at 7, 14, and 28 days after axotomy. NG2 positive processes appeared to interpose between synapsin-1 immunoreactive nerve terminals and surfaces of axotomized motoneurons. Double labeling results showed that NG2 expressing cells encapsulating axotomized facial motoneurons were mainly microglia marked by OX42 and lectin; only a few of them were positive to platelet-derived growth factor-α receptor and none of them positive to ED-1. No Rhodamine particle was detected in the FMN ipsilateral to axotomy after venous injection of the particles. The results suggest that activated microglia in lesioned FMN were induced to express NG2 molecules. It is concluded that axotomized FMN showed two types of NG2 expressing cells namely constitutive NG2 cells and induced-NG2 expressing microglia.

Primary pulmonary small leiomyosarcoma arising from bronchial smooth muscles

A 74-year-old woman was admitted in an hospital because of abnormal lung shadow in chest X–P. Imaging modalities including CT, MRI, and PET demonstrated a small (12 mm in diameter) mass in the periphery of the right upper lobe. Although biopsy and cytology were negative, wedge resection was performed. Grossly, the lesion was a round white tumor measuring 10 × 10 × 9 mm located beneath the pleura. Microscopically, the tumor consisted of proliferation of atypical spindle cells with hyperchromatic nuclei. The tumor cells were continuous with bronchial smooth muscle. Mitotic figures were recognized in 20 per 10 high power fields. There were foci of necrosis and deposition of dusts and hemosiderin. Residual bronchi were present within the tumor. Immunohistochemically, the tumor cells were positive for vimentin, α-smooth muscle actin, caldesmon, and Ki-67 (labeling = 50%), but negative for cytokeratins (AE1/3, CAM5.2, KL-1, and polyclonal wide), cytokeratin 5/6, cytokeratin 7, cytokeratin 20, epithelial membrane antigen, melanosome, TTF-1, desmin, CD34, calreninin, D2-40 (podoplanin), S100 protein, p53 protein, myoglobin, KIT, and platelet-derived growth factor receptor-α (PDGFRA). The author diagnosed the lesion as leiomyosarcoma. After the pathological diagnosis, CT, MRI and PET of the whole body were performed, but did not reveal any tumors including the uterus. The patient was followed up, and three years later a recurrent lesion (20 mm in diameter) was identified in the right upper lobe near the previous tumor. Biopsy showed leiomyosarcoma with histology and immunohistochemical findings similar to the previous tumor. Further operation was impossible because of her medical conditions. She was treated by chemotherapy and followed up. She was now alive with the small pulmonary tumor, which shows no rapid growth.

Expression of platelet-derived growth factor-alpha receptor and c-kit in giant cell tumours of bone

Platelet-derived growth factor-alpha receptor (PDGFRA) and c-kit are tyrosine kinase receptors expressed in many neoplasms. We investigated protein expression in 34 giant cell tumours of bone and two specimens of lung metastases. The expression of PDGFRA and c-kit was analysed by immunohistochemistry. Additionally, capillary sequencing of genomic DNA was performed to search for mutations in therapeutically relevant exons 12 and 18 of the PDGFRA gene and exons 9 and 11 of the c-kit gene. PDGFRA expression was detected in all specimens and all cellular components such as reactive osteoclast-like giant cells and neoplastic stromal cells. C-kit expression was found in six cases of giant cell tumour and one specimen of lung metastasis in all cellular components ranging from 5 to 50% (mean 4.8%, SD 12.4). A further 13 cases showed labelling of osteoclast-like giant cells. DNA sequence of exons 12 and 18 of the PDGFRA gene and exons 9 and 11 of the c-kit gene were not altered in these tumours. PDGFRA and c-kit play a functional role in the growth of giant cell tumours of bone but genetic changes of therapeutically relevant exons of the PDGFRA gene and the c-kit gene are not seen in these tumours.

Primary small cell carcinoma of the pleura: a case report with immunohistochemical and molecular genetic analyses of KIT and PDGFRA genes

An extremely rare case of primary small cell carcinoma of the pleura with an emphasis on KIT and platelet-derived growth factor receptor-α (PDGFRA) genes is reported here. A 67-year-old man underwent left testicular orchiectomy because of a testicular tumor. The tumor was immunohistochemically shown to be diffuse large B-cell lymphoma. The patient was treated with chemotherapy and radiation and followed up in our hospital. Eight years after the orchiectomy, the patient (75years old) developed left pleural tumor and pleural effusion, and a biopsy was performed. The biopsy revealed a medullary malignant tumor consisting of small round and spindle cells. The following three possibilities were considered: recurrent lymphoma, mesothelioma, and small cell carcinoma. Immunohistochemically, the tumor cells were positive for cytokeratin, synaptophysin, CD56, KIT, and PDGFRA, but negative for CEA, cytokeratin 5/6, neuron-specific enolase, chromogranin, CD45, CD3, CD20, CD45RO, CD15, CD30, calretinin, WT-1, B72.3, D2-40, and TTF-1. Therefore, a diagnosis of pleural small cell carcinoma was made. A molecular genetic analysis using PCR-direct sequencing identified no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes. The patient was treated with chemotherapy (cisplatin) and radiation (50 Gray). The present case is the first reported case of primary small cell carcinoma of the pleura with an examination of KIT and PDGFRA expressions and KIT and PDGFRA gene mutations.

Injury-induced Platelet-derived Growth Factor Receptor-α Expression Mediated by Interleukin-1β (IL-1β) Release and Cooperative Transactivation by NF-κB and ATF-4: IL-1β FACILITATES HDAC-1/2 DISSOCIATION FROM PROMOTER

Platelet-derived growth factors are a family of potent mitogens and chemoattractants for fibroblasts and other cells of mesenchymal origin. Platelet-derived growth factor (PDGF) dimeric ligands (composed of A-, B-, C-, and D-chains) exert their biological activity through high affinity interactions with cell surface receptor subunits (alpha and beta). PDGF-receptor-alpha is widely implicated in the pathogenesis of hyperplastic fibrotic disease, yet the molecular mechanisms controlling its expression in response to injury are poorly understood. Here we show that PDGF-R alpha expression is induced in fibroblasts by mechanical injury and interleukin (IL)-1beta, which was abolished by neutralizing IL-1beta antibodies in the culture supernatant or inhibitors of NF-kappaB. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the existence of a new NF-kappaB binding site at -531/-521 bp in the PDGF-R alpha promoter. We have recently shown that ATF-4 is also induced by injury (Malabanan, K. P., Kanellakis, P., Bobik, A., and Khachigian, L. M. (2008) Circ. Res. 103, 378-387), and we demonstrate here that ATF-4 binds a novel element -259/-254 and stimulates PDGF-R alpha transcription. ATF-4 and NF-kappaB interact, occupy the PDGF-R alpha promoter, and induce PDGF-R alpha transcription in a cooperative manner. IL-1beta facilitates the dissociation of histone deacetylase (HDAC)-1/2 from the PDGF-R alpha promoter, whereas the HDAC inhibitors suberoylanilide hydroxamic acid and trichostatin A potentiate IL-1beta induction of PDGF-R alpha transcription. These findings, taken together, demonstrate that injury stimulates IL-1beta secretion by fibroblasts, which activates NF-kappaB and ATF-4 and stimulates interaction with the PDGF-R alpha promoter, triggering PDGF-R alpha transcription. Physical and functional interactions between NF-kappaB and ATF-4 have not been reported in any gene. This is also the first report of HDAC regulation of PDGF-R alpha transcription.