Characterization of primary cells by cell culture could be validated for relevance to treatment of metastatic disease by functional assessment and comparison of immunohistochemical or (epi)genetic signatures in metastatic tissue samples, including upon autopsy

Characterization of primary cells by cell culture could be validated for relevance to treatment of metastatic disease by functional assessment and comparison of immunohistochemical or (epi)genetic signatures in metastatic tissue samples, including upon autopsy. Integration of functional studies and rational molecular assays should be prospectively studied in the setting of RCC and may accelerate progress toward stratification of patients for molecular targeted drug therapies beneficial to them. for VHL and Src protein expression. Functional evaluation of a patients tumor cells appears feasible in the setting of RCC. strong class=”kwd-title” Keywords: renal cell carcinoma, molecular targeted therapy, tyrosine kinase Introduction The success of imatinib in chronic myelogenous leukemia launched an era of rationally-designed molecular cancer therapies.1 Standard of care in gastrointestinal stromal tumors, malignant melanoma, colorectal cancer, and lung cancer have been significantly altered by the development of therapies that target aberrations in signaling pathways resulting from oncogenic mutations. Since 2005, treatment of metastatic RCC has undergone a remarkable transformation with the availability of multiple effective new agents Hoechst 33258 analog 3 including VEGF-R TKIs, mTOR inhibitors, and anti-VEGF monocolonal antibody in combination with interferon. Despite the availability of these agents and their ability to induce partial responses and to stabilize disease, long-term survival remains poor due to nearly inevitable development of resistance. Some tumors, as in this case, appear to be primary refractory to currently available therapies. Effective therapeutics are particularly needed in this population of patients. Additionally, treatment of metastatic RCC remains relatively empiric, with no predictive markers yet established for choosing a therapy for an individual patient. Recently, leukemia cells from individual patients are being subjected to functional assays of panels of small molecules that are already approved or in development for use in humans, in order to uncover sensitivities and potentially identify new targets for validation.2-4 Such a targeted approach remains in its infancy for epithelial solid tumors. We herein demonstrate the feasibility of identifying agents that could be effective in RCC differentially from VEGF-R inhibitors, through direct effects on tumor-derived cells. Case Report A 61-y-old male presented with gross hematuria. Imaging revealed a left renal mass and he underwent radical nephrectomy one month later, which revealed a 5.8 cm T1b clear cell renal cell carcinoma, Fuhrman grade 4. Distant metastatic workup was negative. Surveillance imaging at 5 mo after presentation revealed peritoneal nodules and mediastinal lymphadenopathy. Biopsy of an omental nodule was consistent with metastatic Hoechst 33258 analog 3 renal cell carcinoma. He began treatment with the VEGF-R TKI pazopanib Hoechst 33258 analog 3 at 6 mo, but received limited exposure due to significant transaminitis requiring dose interruption and reduction. Follow-up imaging at 7 mo revealed tumor progression. He began second line systemic therapy with the mTOR inhibitor everolimus at 8 mo, but follow-up imaging at 10 mo revealed progressive disease. The patient was aware of a publication regarding the potential utility of the Src inhibitor dasatinib in RCC.5 He elected third line treatment with dasatinib which he began at 11 mo. CT scan after 6 weeks showed progressive disease with enlargement of pre-existing tumors and development of liver metastases. Despite subsequent treatment with the VEGF-R TKI sunitinib, the patient developed worsening symptomatic progression of metastatic disease, and died approximately one year and one month after his diagnosis. Results and Discussion Out of 66 agents tested in the initial screens (Fig.?1), Ki-CA tumor cells exhibited hypersensitivity only to dasatinib (hypersensitivity defined based on IC50 for Ki-CA that is 5-fold lower than the median IC50 observed for 150 other primary tumor specimens).4 This was supported by response to PP2 (over 3-fold lower), recognized as a canonical Src inhibitor. Open in a separate window Figure?1. Functional assays of Ki-CA patient tumor derived cells response to molecular targeted small-molecule kinase inhibitors. Two thousand cells in medium without EGF were added to 96-well plates containing each small-molecule inhibitor at four serial dilutions spanning a concentration range that includes the predicted IC50, incubated at 37C for three days and assayed by MTS (Promega). The viability data were adjusted for wells with no cells/inhibitor, and normalized to untreated wells of the cultured cells (OD value for wells with cells without drug treatment = 100% cell viability) and KLF1 to a database of cell lines and tumor samples4 to yield responses and the IC50 in nM for each agent. IC50 values less than 20% of the global median (shown as 100%) are considered responses and are well within clinically achievable concentrations. Dasatinib has been tested in the clinic, and it is approved for resistant chronic myelogenous leukemia (CML). While both dasatinib and its parent drug imatinib inhibit cAbl kinase, imatinib was not effective, suggesting pathways discordant between dasatinib and imatinib are driving growth of the Ki-CA tumor, one of which is the Src kinase, affected by dasatinib but not imatinib. Further dose.