KIT and BRAF heterogeneous mutations in gastrointestinal stromal tumors after secondary imatinib resistance.

BACKGROUND AND AIMS: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the digestive tract and characterized by expression of KIT protein. Imatinib is the frontline therapy for metastatic and unresectable GIST patients showing clinical responses in 80 % of cases. Despite the often long-lasting clinical benefit seen in most patients treated with imatinib, many will eventually suffer disease progression. The most frequent mechanism of imatinib resistance in GIST is the acquisition of secondary mutations in either KIT or PDGFRA. There are also some imatinib-resistant GIST patients lacking an identifiable mechanism of treatment failure. Recently, activating BRAF mutation was detected in a small percentage of GISTs. In this study, we report a case of GIST with acquired resistance to imatinib during therapy. METHODS: Histological, immunohistochemical, Western blot and mutational analyses were performed on GIST tissues before and after imatinib resistance. RESULTS: The imatinib-resistant tumor showed not only heterogeneous mutations of KIT and BRAF besides the primary mutation, but also transdifferentiation into a rhabdomyosarcoma phenotype. According to Western blot analysis, in imatinib-resistant GIST with both KIT V559D and BRAF V600E mutations, the inhibition of KIT V559D by imatinib caused a strong decrease of AKT phosphorylation, while ERK1/2 phosphorylation was not affected. CONCLUSIONS: This finding, in combination with the loss of KIT expression, suggests the possibility of activation of RAS-RAF-MEK-ERK pathways driven by a KIT-independent oncogenic mechanism. Understanding the genetic aberrations beyond KIT and PDGFRA may lead to the identification of additional therapeutic targets for GISTs.

[Establishment and pathologic analysis of imatinib-resistant gastrointestinal stromal tumor xenografts].

OBJECTIVE: To establish and characterize imatinib-resistant gastrointestinal stromal tumor (GIST) xenografts. Further provided an ideal experimental platform through the imatinib-resistant GIST xenografts to investigate the mechanism of resistance to imatinib. METHODS: Imatinib-resistant GIST cells were injected under the skin of athymic nude mice to establish animal models of human imatinib-resistant GIST. The molecular and histopathologic features of GIST xenografts were also analysed and compared with their counterpart of cell lines. RESULTS: The xenograft tumor models had been established by subcutaneously injection of GIST cells into nude mice. Immunohistochemistry results showed CD117 expression was positive in GIST-PR2 xenograft tumor, but negative in GIST-R. In GIST-PR1, tumor areas showing rhabdomyoblastic differentiation were presented next to areas with classic GIST morphology. The rhabdomyoblastic component demonstrated consistently positivity for desmin and myogenin, whereas CD117 was completely negative. The mutation profiles of these xenograft tumors were the same as their counterpart of cell lines. CONCLUSIONS: Human GIST xenografts with mutation in c-kit have been established from imatinib-resistant GIST lines. Those models will enable further studies on mechanisms of resistance, combination therapies and allow testing of novel targeted therapies.

Gene mutations and prognostic factors analysis in extragastrointestinal stromal tumor of a Chinese three-center study.

BACKGROUND: Most 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. METHODS: Paraffin-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. RESULTS: c-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. CONCLUSION: The 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.

Survival and prognostic factors analysis in surgically resected gastrointestinal stromal tumor patients.

BACKGROUND/AIMS: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the digestive tract and predicting the clinical behavior and prognosis of GISTs has still been problem for both pathologists and clinicians. The aim of this study was to investigate the survival and prognostic factors of gastrointestinal stromal tumors after surgery. METHODOLOGY: Hematoxylin and eosin (H&E) stained histopathological slides of tumors from patients with GISTs were reviewed. Immunohistochemical staining was performed to demonstrate CD117, CD34, platelet -derived growth factor receptor (PDGFR-alpha) and Ki-67 protein expression. Clinicopathologic features (age, sex, tumor location and size, cell type, mitotic count, risk category, necrosis, surgical method, expression of CD117, CD34, PDGFR-alpha and Ki-67 protein) were evaluated by univariate and multivariate analyses in 135 patients with resected primary GISTs to identify independent prognostic factors. RESULTS: The overall disease-specific survival of 135 patients was 94.1% at 1 year, 76.3% at 3 years and 65.9% at 5 years. Multivariate analyses indicated that the tumor size, primary location, mitotic count, risk category, necrosis and Ki-67 index were independent significant predictors of survival (p < 0.05). Ki-67 index was strong poor predictors of survival as tumor size and mitotic count. CONCLUSIONS: Fletcher's biological behavior ranking method was a good approach to predict prognosis of GIST patients and had significant clinical value. It's better to combine other factors such as Ki-67 index and tumor primary location et al to predict prognosis accurately. Accurate prognostic prediction could provide evidence for postoperative adjuvant targeted therapy.

Secondary C-kit mutation is a cause of acquired resistance to imatinib in gastrointestinal stromal tumor.

C-kit gene gain of function mutations are important in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of KIT and achieves a partial response or stable disease in most patients with metastatic GIST, but there is increasing evidence of acquired resistance. We report a case of GIST with acquired resistance to imatinib during therapy and secondary c-kit mutation besides the primary mutation.

Gefitinib-induced hair alterations.

Human epidermal growth factor receptor (EGFR) is an attractive target for anticancer therapy. EGFR tyrosine kinase inhibitors are generally well tolerated and do not have the severe systemic side-effects usually seen with cytotoxic drugs. A specific adverse effect common to this class of agent is a papulopustular rash, usually on the face and upper torso. During prolonged treatment with EGFR inhibitors, changes of the hairs can be noticed. This report describes a rare case of a non-small-cell lung cancer with hair changes after several months of treatment with the EGFR inhibitor gefitinib. The patient's scalp hair grew more slowly and adopted a finer, more brittle and curly aspect. However, the eyelashes, eyebrows and hair of other parts of the face did not display similar changes. Little is known about the aetiology of this kind of hair alteration, and there are no clear evidence-based management recommendations. Histological data indicate that the hair alteration may be caused by EGFR inhibition in skin, although this has not been confirmed. Further studies are needed to investigate the reason for this phenomenon.

[Influence of microencapsulated ovary cells modified with maspin gene on the microvessel density and lung metastasis of breast carcinoma].

OBJECTIVE: To observe the inhibition of maspin on the angiogenesis in tumor and lung metastasis of breast carcinoma and the feasibility of treatment of tumor by microencapsulated transgene cells in vivo. METHODS: Microencapsulated Chinese hamster ovarian epithelial cells (CHO) modified with maspin gene, CHO/pcDNA3.1/maspin cells, were prepared. Twenty BALB/C nude rats underwent subcutaneous injection of breast carcinoma cells of the line Bcap37 to establish tumor-loaded animal models and then randomly divided into 2 groups: maspin group, undergoing subcutaneous injection of CHO/pcDNA3.1/maspin cells next to the transplanted tumor, and control group undergoing subcutaneous injection of microencapsulated CHO/pcDNA3.1 cells. One month later, the rats were killed and the size and microvessel density (MVD) of the transplanted tumor and metastatic tumor in lung were observed. RESULTS: The MVD of the transplanted tumor of the maspin group was 26 +/- 9, significantly lower than that of the control group (60 +/- 16, P < 0.05). The lung metastatic rate of the maspin group was 15%, significantly lower than that of the control group (55%, P < 0.05). CONCLUSION: Maspin may inhibit the MVD in tumor and the occurrence of metastatic tumor in lung. It is feasible to use microencapsulated transgene cells as tumor-killer.

[Effects of microencapsulated CHO cells modified with maspin gene on the motility and adhesiveness of breast carcinoma cells Bcap37].

OBJECTIVE: To investigate the effects of microencapsulated Chinese hamster ovary (CHO) cells modified with maspin gene on the motility and adhesiveness of breast carcinoma cells Bcap37 and to explore the possibility and feasibility of its clinical application in treatment of malignant tumors. METHODS: After the Bcap37 cells were co-cultured with the microencapsulated CHO cells modified with maspin gene, their motility and adhesion to vascular endothelial cells (ECV304), changes in CD44v6 and E-cadherin expression were examined. RESULTS: After the treatment, the motility of Bcap37 cells, their adhesion to vascular endothelial cells ECV304 and the CD44v6 expression were significantly reduced. The adhesiveness of Bcap37 cells and their E-cadherin expression were significantly enhanced. CONCLUSION: The microencapsulated CHO cells modified with maspin gene decrease motility and adhesiveness of breast carcinoma cells Bcap37, which help explain the anti-metastatic effects of maspin.

[Subcutaneous transplantation of microencapsulated Chinese hamster ovary (CHO) cells/pcDNA 3.1/mIL-12 and subcutaneous transplantation of microencapsulated CHO/pcDNA3.1/mIL-12 combined with 5-fluoro-uracil in treatment of tumor-burdened mice].

OBJECTIVE: To investigate the effect of subcutaneous transplantation of microencapsulated Chinese hamster ovary cells (CHO)/pcDNA3.1/mIL-12 and subcutaneous transplantation of microencapsulated CHO/pcDNA3.1/mIL-12 combined with 5-fluoro-uracilum (5-FU) in treatment of tumor-burdened mice. METHODS: CHO/pcDNA3.1/mIL-12 and CHO/pcDNA3.1 were suspended in solution of sodium alginate and made into microcapsules. Sixty mice were divided into 6 groups of 10 mice: (1) IL-12 group, microencapsulated CHO/pcDNA3.1/mIL-12 was injected subcutaneously, (2) combined treatment group: CHO/pcDN with 5-FU, (3) 5-FU group: 5-FU was injected intraperitoneally, (4) blank vector group: CHO/pcDNA3.1 was injected subcutaneously, (5) tumor-burdened group: without any treatment, and (6) blank control group: normal mice without any treatment. Except the mice of the blank control group, all mice were injected subcutaneously into the inner side of right thigh with mice colonic adenoma cells. The volume of tumor was measured every third day. 20 days after the treatment, 5 mice in each group were killed to examine the serum Th1 type cytokines: interferon (IFN)-gamma, IL-12, and Th2 type cytokins: IL-4, IL-10 by double antibody sandwich ELISA. The spleens were made into suspension of lymphocytes to examine the activity of natural killer cell (NK) and cytolytic T lymphocyte (CTL). The survival period of the remaining 5 mice in each group was observed till the 60th day. RESULTS: The activity of NK and CTL were significantly much more in the IL-12 group than in other groups. The activity of NK was significantly much more in combined treatment group than in the tumor-burdened group. The levels of Th1 type cytokines were the lowest in the tumor-burdened group. There was no difference in the levels of Th1 type cytokine between the tumor-burdened group and 5-FU group. However, the levels of Th1 type cytokine were significantly higher in the IL-12 group than in other groups. The levels of Th2 type cytokines were rather high in the tumor-burdened group. There was no difference in the levels of Th2 type cytokine between the tumor-burdened group and 5-FU group. However, the levels of Th2 type cytokine were the lowest in the IL-12 group than in other groups. There was no significant difference in volume of tumor among the tumor-burdened group, blank vector group, and 5-FU group. The mean diameters of tumor in the IL-12 group and combined treatment group were significantly smaller than in the 5-FU, tumor-burdened, and blank vector groups (P < 0.05), however, without a difference between the IL-12 group and combined treatment group. The survival periods of the IL-12 group and combined treatment group were significantly longer than those in the blank vector, tumor-burdened and 5-FU groups (P < 0.05). CONCLUSION: Microencapsulated CHO/pcDNA3.1/mIL-12 transplanted subcutaneously significantly improves the immune function of tumor-burdened mice and partially overcomes immune suppression caused by chemotherapy, and is effective in slowing the growth of tumor and lengthening the survival period of tumor-burdened ice.

Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy.

AIM: To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene. METHODS: Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated. RESULTS: Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-gamma, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice. CONCLUSION: The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy.