KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T3 C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.
Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient’s lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient’s own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib’s effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8+, -CD4+, -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types.
This investigation aimed to develop a biologically relevant murine model of colorectal liver metastases and determine if Kupffer cells (KC) and hepatic natural killer cells (hNKC) regulate tumor growth. The model involves the injection of murine colon adenocarcinoma 26 (MCA 26) tumor cells into the portal vein of female-specific pathogen-free BALB/c mice. Metastases developed in all animals, and the growth was limited entirely to the liver. To determine if KC and hNKC control the development of liver metastases, the in vivo function of these hepatic effector cells was modulated. Tumor growth was quantitated by the uptake of 125I into tumor DNA. Stimulation of the KC and hNKC produced a significant (P less than 0.01) dose-dependent decrease in 125I uptake in the liver in both treatment groups, which was associated with a significant improvement in survival (P less than 0.05). The in vivo cytotoxic function of the liver was inhibited with an intravenous injection of gadolinium chloride (for KC) or asialo GM1 antiserum (for hNKC). Inhibition of KC and hNKC cytotoxic function led to a significant (P less than 0.01) increase in 125I uptake in the liver and a significant decrease in survival (P less than 0.05).
We have previously demonstrated that in vivo activation or inhibition of Kupffer cell (KC) cytotoxic function can reduce or enhance, respectively, the hepatic tumor burden in a syngeneic murine colon adenocarcinoma (MCA26) tumor model. In the current study, we have performed in vitro experiments to define the possible mechanisms of KC cytotoxicity against MCA26 cells. Addition of either anti-tumor necrosis factor (TNF) or anti-interleukin-1 alpha (IL-1 alpha) antisera reduced KC cytotoxicity in coculture against MCA26 targets in a dose-dependent fashion; addition of these sera together resulted in approximately additive inhibition, suggesting the existence of parallel pathways for these effector molecules. Nitric oxide as a mediator of cytotoxicity by KCs in coculture with MCA26 cells was evaluated by two approaches. Activated KCs produced detectable levels of nitric oxide; however, activated KC exerted cytotoxicity against MCA26 targets in the absence of exogenous free L-arginine. Thus, TNF and IL-1 play major roles in producing murine KC cytotoxicity against MCA26 colon cancer cells in vitro, whereas reactive nitric oxides do not.
11/6/20928activity against human colon tumor cell lines. Hopefully, through an understanding of the cytotoxic mechanisms of human KC, strategies to stimulate human KC to prevent or eradicate hepatic metastases can be developed rationally.T h e aim of this investigation is to determine if (1) human KC are cytotoxic against human colon carcinoma in vitro, (2) this cytotoxic activity can be stimulated, and (3) human KC mediate cytotoxicity through the secretion of T N F . MATERIAL AND METHODSIsolation of human KC. At the time of resection for colon cancer in six patients, a small (2 X 2 cm) wedge biopsy specimen of the liver was obtained. The specimen was placed in an enzyme solution (0.05% collagenase and deoxyribonuclease) and incubated in a shaking water bath at 37" C for 35 minutes. The cell suspension in Dulbecco's phosphate-buffered saline solution (DPBS) was placed on a 30% metrizamide gradient and centrifuged for 20 minutes (1400 g at 4" C). The KC were taken from the metrizamide-DPBS interface, washed, and placed in a 96-microwell plate at various effector concentrations (0.5 X lo', 1.0 X lo', and 2.5 X lo5 KC/well). The KC adhered for 2 hours and were washed twice with DPBS. This technique produced a suspension of >95% KC as determined by (1) macrophage morphology in a Wright-Giemsa-stained 400 SURGERY
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