Expression of stem cell factor in gastrointestinal stromal tumors: Implications for proliferation and imatinib resistance.

KIT autophosphorylation caused by mutation of KIT is considered to be a critical mechanism for the oncogenesis of gastrointestinal stromal tumors (GISTs). However, little is known regarding whether stem cell factor (SCF), the KIT ligand, is able to induce the proliferation of GIST cells by activating the wild-type KIT receptor in GISTs. Imatinib, a tyrosine kinase inhibitor, has been demonstrated to be effective as treatment for the majority of GISTs. However, primary resistance to imatinib in GISTs with wild-type KIT and acquired resistance in GISTs with mutant KIT are becoming increasingly significant problems. The aims of this study were to detect the expression and function of SCF in 68 GIST samples, and to explore the relationship between SCF activity and imatinib resistance using immunohistochemical staining and western blot analysis. Results showed abundant expression of SCF in GISTs and demonstrated that SCF is capable of enhancing GIST cell proliferation. Similar to its ineffectiveness in wild-type GISTs, imatinib also failed to inhibit SCF-induced KIT activation in GISTs with mutant KIT. We also found increased SCF expression in GIST cells treated with imatinib. Overall, our results indicated that SCF-induced KIT activation is a novel essential pathway for the proliferation of GISTs. Imatinib was not able to inhibit the activity of SCF, while it promoted the expression of SCF, which may have contributed to acquired imatinib resistance.

A novel PDGFRA mutation in gastrointestinal stromal tumours, L839P, is sensitive to imatinib in vitro.

Evidence suggests that different types of mutation in gastrointestinal stromal tumours (GISTs) correlate with different response rates to imatinib (Glivec, STI571). The purpose of this study was to explore the sensitivity of the PDGFRA(L839P) mutant, a novel gain-of-function mutation isoform related to GISTs, to imatinib in vitro. The eukaryotic expression vectors pcDNA3.1-PDGFRA(Wild), pcDNA3.1-PDGFRA(D842V) and pcDNA3.1-PDGFRA(L839P) were constructed and transfected into Chinese hamster ovary (CHO) cells by liposome methods. The responses of cells with PDGFRA(Wild), PDGFRA(L839P) and PDGFRA(D842V) mutants to imatinib were determined by methyl thiazolyl tetrazolium (MTT) assay, western blotting and apoptosis assays. Reults of the MTT assay revealed that the growth rate of CHO(PDGFRA(L839P)) cells decreased to approximately 60% when exposed to 1 µM imatinib and to approximately 50% with 5 µM imatinib. However, the growth rate of CHO(PDGFRA(D842V)) cells did not significantly change with 5 µM imatinib. Western blot analysis indicated that 1 µM imatinib completely blocked the phosphorylation of PDGFRA(L839P), but did not affect PDGFRA(D842V) phosphorylation. Apoptosis analysis suggested that the percentage of apoptotic CHO(PDGFRA(L839P)) cells increased approximately 4-fold (from 5.90 to 25.2%) with 1 µM imatinib. Although the treatment of CHO(PDGFRA(D842V)) and CHO(PDGFRA(Wild)) cells with 5 µM imatinib resulted in a slight increase in the number of apoptotic cells, the percentage of apoptotic cells remained approximately 10% of the total population. Our findings showed that the PDGFRA gene mutation isoform L839P is sensitive to inhibition by imatinib. Screening for PDGFRA mutations in GISTs is essential to identify the response to treatment with imatinib.

Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth.

AIM: To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth. METHODS: The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry, and the results were correlated with clinicopathological parameters, including the mitotic count, proliferative index (Ki-67 immunohistochemical staining), mitotic index (phospho-histone H3 immunohistochemical staining) and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling). Using primary cultured GIST cells, the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting, methyl thiazolyl tetrazolium (MTT), and apoptosis assays. RESULTS: We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples, respectively, and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis, including larger tumor size (P = 0.0118), higher mitotic count (P = 0.0058), higher proliferative index (P = 0.0012), higher mitotic index (P = 0.0282), lower apoptosis index (P = 0.0484), and increased National Institutes of Health risk level (P = 0.0012). We also found that the introduction of exogenous SCF potently increased KIT kinase activity, stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation, while a KIT immunoblocking antibody suppressed proliferation (P = 0.01) and promoted apoptosis (P < 0.01) in cultured GIST cells. CONCLUSION: SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth. The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

Correlation of fluorodeoxyglucose uptake and tumor-proliferating antigen Ki-67 in lymphomas.

OBJECTIVE: To investigate the correlation between cellular proliferation and the fluorodeoxyglucose (FDG) uptake in positron emission tomography/computed tomography (PET/CT) imaging by comparing 50 cases of different subtypes of lymphoma. MATERIALS AND METHODS: Fifty cases of lymphomas were collected. Each case was labeled with Ki-67 stain, a marker of cellular proliferation, and a PET/CT examination was performed. All lymphoma cases were sorted according to the World Health Organization's classification, and the International Non-Hodgkin's Lymphoma Working Formulation was used to differentiate groups of large and small cell non-Hodgkin's lymphoma. The Ki-67 staining was described as slight, mild, middle, or strong according to the nuclear staining of positive cells. FDG uptake by lesions in PET/CT images was semi-quantitatively analyzed to calculate the average standard uptake value. The statistics software SPSS13.0 was used to calculate the mean and standard deviation of the FDG uptake value of the lymphoma subtypes, the difference between the large and small cell lymphoma group with a Student's t-test, and the correlation between the Ki-67 level and FDG uptake of lesion with a Spearman's analysis. RESULTS: The FDG uptake value of large cell origin lymphoma was significantly higher than that of small cell origin lymphoma (t = 6.19, P < 0.01). The correlation coefficients between the Ki-67 level and FDG uptake value in lymph nodal and extranodal lesions was 0.750 and 0.843, respectively. CONCLUSIONS: Ki-67 staining, a reflection of tumor-proliferation activity, was significantly related to the FDG uptake value in lymphoma lesions.

Value of napsin A and thyroid transcription factor-1 in the identification of primary lung adenocarcinoma.

Napsin A is a newly discovered functional aspartic proteinase that is expressed in normal lung parenchyma in type II pneumocytes and is thought to be associated with primary lung adenocarcinoma. Thyroid transcription factor-1 (TTF-1) is a widely used relatively restricted marker for lung adenocarcinoma. The present study aimed to compare the usefulness of napsin A with TTF-1 for the identification of primary lung adenocarcinoma. Immunohistochemical expression of napsin A and TTF-1 was analyzed in 351 lung cancer tissues, including 27 metastases. Napsin A was expressed in 180 of 212 (84.9%) primary lung adenocarcinomas, while no expression was noted in all 27 metastatic lung cancer specimens, including 19 metastatic adenocarcinomas. In contrast, TTF-1 expression was not only noted in 179 of 212 (84.4%) primary lung adenocarcinomas, but also in 12 of 18 (66.7%) small-cell carcinomas and some of the squamous carcinomas, as well as in one metastatic adenocarcinoma from the thyroid. The sensitivity and specificity of napsin A for primary lung adenocarcinoma (84.9 and 93.8%, respectively) were higher than the sensitivity and specificity of TTF-1 (84.4 and 83.9%, respectively). By combining napsin A and TTF-1, sensitivity increased to 91.0%. Furthermore, the sensitivity and specificity expression was associated with gender, smoking history, performance status, pathological type, primary tumor size and nodal metastasis. Therefore, napsin A is a useful novel marker in the differential diagnosis of primary lung adenocarcinoma.

[Transforming effect of PDGFRA gene mutant on the cell function in gastrointestinal stromal tumor].

OBJECTIVE: To explore the effect of malignant transformation of the L839P, a new mutation site of the PDGFRA gene, on the pathogenesis of gastrointestinal stromal tumors. METHODS: All recombinant plasmids were stably transfected into CHO cells by liposomes. Western blotting was used to detect the expression of PDGFRA protein. The cell growth curve was plotted by cell counting. Flow cytometry was used to detect the cell cycle and apoptosis of CHO cell, respectively. The stably transformed cells were inoculated subcutaneously into the back of nude mice and the mice were used to observe the tumorigenesis. Transient transfection of the mutant-type plasmids of PDGFRA gene and the wild-type plasmids of kit gene into the CHO cells was performed. Western blot was used to detect the expression of kit protein and its phosphorylated forms. RESULTS: PDGFRA protein expressed in the negative control, experimental group and positive control, except the empty vector. The growth curve showed that it was accelerated in the experimental group and positive control. The ratios of cells in proliferative phase were 28.4% (blank), 24.5% (negative control), 43.8% (experimental group) and 40.9% (positive control). Their apoptotic indexes were 1.8%, 1.9%, 1.5% and 1.6%, respectively. After three weeks, tumors were observed in the nude mice of experimental group and positive control, inoculated with the stably transformed cells. Moreover, the expression of phosphorylated protein of kit was enhanced after cotransfection of the mutant-type plasmids of PDGFRA and the wild-type plasmid of kit. CONCLUSION: The PDGFRA mutant L839P is a gain-of-function mutation and has obviously malignant transforming effect on normal cells, and may activate kit protein accelerating the tumorigenesis. Gastrointestinal stromal tumors;

Microadenocarcinoma of the pancreas.

BACKGROUND: Microadenocarcinoma (MA) of the pancreas is a rare kind of neoplasm, whose status as an independent tumor entity is still a matter of controversy. METHODS AND RESULTS: In this article we investigated two patients with MA from the histological, immunohistochemical and genetic aspects. Morphologically, MA is composed of small, crowded microglandular structures, forming a cribriform pattern, sometimes solid sheets. Cells of MA were morphologically uniform and were less pleomorphic than those of the ductal adenocarcinoma. Immunohistochemistry revealed that MA, though with a certain extent of epithelial differentiation, possesses a different immunological phenotype from those of ductal carcinoma, acinar cell carcinoma, and endocrine tumors. Genetic analysis showed no abnormality of p53, K-ras, and beta-catenin, which were usually mutated in pancreatic ductal adenocarcinoma. CONCLUSION: Therefore, we suggest that MA should be taken as an independent tumor entity rather than a kind of growth pattern, but a final decision should be reached after cautious differential diagnosis of other kinds of pancreatic neoplasms.

[Expression and Clinical Significance of TTF-1 and p63 in NSCLC.].

BACKGROUND: To detect the expressions of thyroid transcription factor-1 (TTF-1) and p63 in non-small cell lung cancer (NSCLC) and to evaluate their clinical significance. METHODS: The expression of TTF-1 and p63 from 404 NSCLC and 28 benign pulmonary disease (BPD) tissue specimens were detected by immunohistochemical EnVision two-step method, together with their clinicopathologic data. RESULTS: The positive rate of TTF-1 and p63 protein in NSCLC tissues was 51.7% (209/404) and 37.9% (153/404), respectively, while negative in the BPD group. There was overexpression of TTF-1 in female gender and non-smoking history (P<0.001) and asymptomatic patients (P=0.015). It was more frequently in adenocarcinoma (AdC) with sensitivity of 84.1% and specificity of 89.8%, especially in well or moderately differentiated AdC (P<0.001). The positive rate of p63 was closely related with male gender and smoking history (P<0.001). Its sensitivity and specificity to squamous cell carcinoma (SCC) was 95.5% and 98.8%, respectively, which was positively correlated with differentiation of SCC (P=0.008), but negatively with tumor stage (P=0.002). Logistic multivariate analysis showed smoking history and histological type were significantly associated with TTF-1 and p63 expression. 93.1% of those represent TTF-1(+)/p63(-) were AdC, while 98.6% of TTF-1(-)/p63(+) were SCC. p63 expression was negatively correlated with TTF-1 (P<0.001). CONCLUSIONS: TTF-1 and p63 might be effective bio-markers for AdC and SCC in NSCLC. They may be a useful marker panel for the identification and differential of lung cancer.

Regional expression of the hypoxia-inducible factor (HIF) system and association with cardiomyocyte cell cycle re-entry after myocardial infarction in rats.

Hypoxia-inducible factor (HIF)-1alpha and-2alpha have diverse actions on the myocardium, but the importance of direct effects on cardiac myocytes is unclear. To define their regional accumulation and association with cardiomyocyte cell cycle change after myocardial infarction (MI), a rat MI model was established by occluding the coronary arteries. To further prove a causative relationship between HIF and cell cycle regulation, cultured cardiomyocytes were transfected with adenoviral vectors carrying HIF-1alpha and HIF-2alpha. Two weeks after MI, both HIF-1alpha and HIF-2alpha mRNA were moderately increased in the infarcted left ventricle and noninfarcted left ventricle; HIF-2alpha amplification was also detected in areas of the interventricular septum and the right ventricle. In concordance with the changes in mRNA levels, immunohistochemistry signals of HIF-1alpha and HIF-2alpha were characterized by different regional distributions. In the myocardium adjacent to the infarcted tissue, a significant correlation between HIF-1alpha or HIF-2alpha and Ki-67 labeling index was observed (P < 0.001). Immunohistochemical double staining showed that HIF positive cardiomyocytes underwent DNA synthesis. Cardiomyocytes treated with HIF-1alpha or -2alpha expressed Ki-67, phosphohistone H3, and bromodeoxyuridine effectively in vitro. In conclusion, HIF-1alpha and HIF-2alpha had a distinct spatial expression pattern in a rat model of ischemic heart disease. Both HIF subunits might be potent stimuli for cardiomyocytes to re-enter the cell cycle and initiate DNA synthesis.

[Expression of Oct2 and its significance in lymphoma diagnosis].

OBJECTIVE: To investigate the specificity and sensitivity of Oct2 protein expression in lymphoma cells and its significance in diagnosis and classification of lymphoma. METHODS: Formalin-fixed and paraffin-embedded materials from 129 cases of lymphoma and 10 cases of reactive lymphoid hyperplasia (RLH) were studied by EnVision immunohistochemistry for Oct2 protein. RESULTS: Oct2 was mainly expressed in germinal center cells of RLH. It was diffusely expressed in B-cell lymphoma cells. 97.7% cases (85/87) of B-cell lymphoma and 3.8% cases (1/26) of T-cell lymphoma were positive for Oct2 protein. In comparison, the expression rates for CD20 and CD79alpha in B-cell lymphomas were 90.8% (79/87) and 84.7% (61/72) respectively. The difference in expression rates between Oct2 protein and CD20 was not statistically significant (P > 0.05) There was, however, significant difference in expression rates between Oct2 protein and CD79alpha (P < 0.05). The expression rates of Oct2 protein in nodular lymphocyte-predominant Hodgkin lymphoma and classic Hodgkin lymphoma were 3/3 and 46.2% (6/13) respectively. The difference in expression rates of Oct2 protein in these two groups showed no statistical significance (P > 0.05). CONCLUSION: As a relatively sensitive and specific marker for B cells, Oct2 can serve as a useful antibody for the diagnosis and differential diagnosis of lymphoma.

Prognostic value of KIT mutation in gastrointestinal stromal tumors.

AIM: To examine the prevalence and prognostic significance of C-kit gene mutation and analysis the correlation of C-kit gene mutation and the clinicalpathologic parameters of GISTs. METHODS: Eighty-two GISTs were studied for the mutation of C-kit gene by PCR-SSCP, DNA sequence. Statistical comparison were used to analysis the correlation of C-kit gene mutation and clinicalpathology, clinical behavior, recurrence. RESULTS: (1) Mutation-positive and mutation-negative GISTs were 34 and 48,respectively; (2) Among these patients with C-kit mutation remained a significantly poor prognosis associated with 59% 3-year survival compared to those whose tumors did not; (3) Tumor size, PCNA index, mitotic cell number, presence of necrosis, microscopic invasion to adjacent tissues, recurrence and distant metastasis among mutation-positive and mutation-negative GISTs were significantly different. CONCLUSION: C-kit mutation is a undoubtedly pivotal event in GIST and may be associated with poor prognosis. Evaluation of C-kit gene mutation may have both prognosis and therapeutic significances.