Diagnostic Relevance of Overexpressions of PKC- θand ar. · PDF...

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  • Abstract. Background: We investigated the clinicopatho-logical and immunohistochemical characteristics, geneticaberrations and prognostic factors in 28 patients withextragastrointestinal stromal tumors (EGISTs) from six centersin South Korea. Patients and Methods: Immunohistochemistrywas performed for c-KIT (CD117), PKC- (protein kinase Ctheta), DOG-1 (discovered on GIST-1), CD34, alpha-smoothmuscle actin (-SMA), vimentin, desmin and S-100 protein.Genetic analyses for the KIT gene (exon 9, 11, 13 and 17) andthe platelet- derived growth factor receptor alpha (PDGFRA)gene (exons 12 and 18) were performed by direct sequencing ofPCR products. The relationships of various clinicopathologicalcharacteristics and outcomes were also examined. Results: Ofthe tumor samples, 78.6% (22/28) were located in the intra-abdominal cavity including the omentum and mesentery, and

    10.7% (3/28) were located in the retroperitoneum. All patientswere older than 39 years. The median size of the tumors was10 cm for the maximum diameter. When first detected, 57.1% ofEGISTs were large in size, measuring more than 10 cm.Tumors that were larger than 10 cm were found morefrequently among tumors with more than 10 mitoses per 50high-power fields (HPFs) and this finding was statisticallysignificant (p10 cm, tumor necrosis, obvious nuclear atypia,mitotic counts >10/50 HPFs and epithelioid or mixed cell type(p

  • most common being missense mutations or deletions affectingexon 11 of the KIT gene (n=9) or exon 18 of the PDGFRAgene (n=6). Three cases showed co-existence of both KIT andPDGFRA gene mutations. There were no mutations of exon17 of KIT and exon 12 of PDGFRA genes. Conclusion: The c-KIT, PKC- and DOG-1 antigens are the most sensitive andspecific immunomarkers for confirming EGISTs. PKC- andPDGFRA immunostains are helpful markers for c-KIT-negative EGISTs. Survival analyses indicated that tumor size>10 cm, mitotic rate >10/50 HPFs, tumor necrosis, obviousnuclear atypia, and epithelioid or mixed cell type weresignificant predictors of survival. We found that thecombination of these parameters helped to predict aggressivetumor behavior and may be useful for predicting theprognosis of EGISTs. The majority of gene mutations wereidentified in exon 11 of the KIT gene or exon 18 of thePDGFRA gene. The pattern of KIT and PDGFRA mutationsin EGISTs was essentially similar to the one in GISTs. Fromthe immunohistochemistry and molecular geneticsperspective, EGISTs may be a special subtype of GISTs. Bothimmunohistochemical and molecular evaluation are useful forclassifying tumors as EGISTs.

    Extragastrointestinal stromal tumors (EGISTs) are neoplasmswith histology and immunohistochemistry similar to those ofgastrointestinal stromal tumors (GISTs) but are locatedoutside the gastrointestinal tract, such as in the soft tissue ofthe intra-abdominal cavity, including the omentum,mesentery, retroperitoneum, pancreas, spleen or vulvovaginal/rectovaginal septum. The histogenesis of EGISTs has notbeen elucidated, but the expression of the c-KIT, a tyrosinekinase receptor in EGISTs indicates the presence ofpacemaker cells from outside the gastrointestinal tract (1-9).

    Most EGISTs are thought to be malignant, but due to theirrarity, little is known about their pathogenesis, incidence,prognosis and genetic background. Currently, the malignantpotential of EGISTs is determined by the same parametersused for GISTs, such as tumor size, mitotic rate and presenceof necrosis, but it remains unclear whether this is a rationalapproach. The guidelines for risk assessment of primaryGISTs are different according to the gastrointestinal organ(10), so we anticipate that the guidelines for risk assessmentof primary EGISTs will be different from those for GISTs.As the risk factors of EGISTs remain unknown, comparisonsbetween EGISTs and GISTs are important to determinewhether they have similar clinicopathological behaviors andare caused by the same mutations.

    Most EGISTs express the KIT protein, based on previousstudies demonstrating immunohistochemical staining for c-KIT (CD117), which is thought to a useful marker fordistinguishing EGISTs from other mesenchymal tumors ofsoft tissue, such as desmoid tumors, leiomyosarcomas, andmalignant peripheral nerve sheath tumors (1, 2, 4-7).

    Recently, protein kinase C theta (PKC-), a novel PKCisotype involved in T-cell activation, skeletal muscle signaltransduction, and neuronal differentiation (11, 12), andDOG-1 (discovered on GIST 1) (13), were shown to beoverexpressed in GISTs. PKC- and DOG-1 expression inGISTs has been studied previously, but there have only beena few studies of these two markers in only a small numbersof EGISTs (9).

    Recent advances in the study of the KIT gene and theplatelet- derived growth factor receptor alpha (PDGFRA)gene revealed that GISTs are closely related to gain-of-function mutations in the KIT and PDGFRA genes. KITmaps to chromosome 4q12 and encodes a 109870 Dtransmembrane glycoprotein. KIT gene mutations have beenfound in exon 11, which encodes the juxtamembranedomain; in exon 9, which encodes the extracellular domain;in exon 13, which encodes the tyrosine kinase domain; and inexon 17, which also encodes the tyrosine kinase domain.PDGFRA is located adjacent to KIT and encodes a 122676 Datransmembrane glycoprotein that is highly similar to KIT.The PDGFRA gene mutations are present in exon 12, whichencodes the juxtamembrane domain and in exon 18, whichencodes the tyrosine kinase domain (14, 15).

    The aim of this study was to analyze the immunohisto-chemical markers and the genetic mutations associated withEGISTs and to assess any correlations between clinico-pathological parameters of EGISTs and survival rate.

    Patients and Methods

    Patients. Tumor samples were obtained from 28 patients withEGISTs. We reviewed cases of EGISTs that arose from the intra-abdominal cavity, abdominal wall, prostate and retroperitoneum,collected at Kangbuk Samsung Hospital (Seoul, South Korea), EuljiUniversity Hospital (Daejeon, South Korea), Chungnam NationalUniversity (Daejeon, South Korea), Hanyang University (Seoul,South Korea), Dong-A University Hospital (Pusan, South Korea)and Pusan National University Hospital (Pusan, South Korea) from1997 to 2007. In addition, 28 non-EGIST tumors, includingleiomyomas, leiomyosarcomas, schwannoma, malignant peripheralnerve sheath tumors and desmoid tumors were included as a controlgroup. The follow-up data for these patients were analyzedretrospectively. Clinical information regarding the locations andsizes of tumors, patient status and follow-up period were obtainedby reviewing the medical records. The histopathologic tumor celltypes, mitotic counts per 50 high power fields (HPFs), obviousnuclear atypia and tumor necrosis were reviewed in thehematoxylin-eosin (H-E) stained slides of each case. Overallsurvival (OS) was calculated as the period from surgery until thedate of the death. The study was performed according to theDeclaration of Helsinki and approved by the local Ethics Committeeof the Kangbuk Samsung Hospital.

    Immunohistochemical staining. We produced tissue microarray(TMA) blocks containing 2-mm diameter cores of the EGISTtissues from the enrolled cases. One core was obtained from the

    ANTICANCER RESEARCH 32: 923-938 (2012)

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  • central portion of the mass of each case. The TMA blocks weresectioned at a 4 m thickness and were processed for immuno-histochemistry. Immunohistochemical studies were performed usingDAKOs Envision method. The vendors that supplied the primaryantibodies and dilution factors are listed in Table I. Theimmunostaining was performed using a compact polymer method(from Bond Intense Detection Kit, Leica Biosystems, Newcastle,UK). Diaminobenzidine (DAB) was the chromogen. Clinicalpositive control cases for all markers were included in every stainingbatch. The immunostained slides were scored on the basis of thepercentage of positive tumor cells staining above background, asnegative (0%), weakly stained (50%).

    Scores were entered into a Microsoft Excel spreadsheet. Scoringresults were separated into two categories, either negative (score of0 or 1) or positive (score of 2 or 3). The uninterpretable results wereeliminated from further consideration.

    Polymerase chain reaction (PCR) for the KIT and PDGFRA genes.Genetic analyses of the KIT gene (exons 9, 11, 13 and 17) and thePDGFRA gene (exons 12 and 18) were performed by directsequencing of PCR products. Genomic DNA was extracted fromparaffin-embedded tissues using standard proteinase K digestion andphenol/chloroform extraction. The sequences of each primer aresummarized in Table II.

    Statistical analysis. PASW Statistics 18.0 (SPSS Inc., Chicago, IL,USA) was used for statistical analysis. Survival curves were plottedusing Kaplan-Meier methods, with significance assessed using log-rank tests and based on OS over 10 years. Multivariate analyseswere analyzed using Cox regression model. For correlations, thePearson 2 test was used.

    Results

    Clinicopathological findings. Out of the EGISTs included inthis study, 78.6% (22/28) were located in the intra-abdominalcavity including the omentum and mesentery and 10.7%(3/28) were retroperitoneal. The ages of the patients rangedfrom 39 to 78 years (mean, 58 years). There were no child oradolescent patients. There was a slight male predominancein the study group (M:F=15:13). The sizes of the tumorsranged from 3 to 47 cm (median 10 cm) in maximumdiameter. A total of 5 tumors out of the 28 defined EGISTswere from 2 to 5 cm in size, 7 tumors were 6 to 10 cm and16 tumors were larger than 10 cm. There were no EGISTssmaller than 2 cm. Upon initial detection, 57.1% (16/28) ofEGISTs were large, measuring more than 10 cm. Regarding

    Kim et al: Diagnostic Relevance of Overexpressions of PKC- and DOG-1 and KIT/PDGFRA in EGIST

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