c-Met is associated with poor pathologic features and prognosis in RCC. c-Met inhibition demonstrates in vitro activity against clear cell RCC. Further study of ARQ 197 with appropriate biomarker studies in RCC is warranted.

Angiogenesis is required for progression and metastasis of melanoma. Analysis of angiogenic molecules in benign and malignant tissues may allow identification of markers useful for prediction of sensitivity to antiangiogenic agents. We hypothesized that differential expression of VEGF and its receptors VEGF-R1, - R2, and -R3 would be higher in melanomas than nevi and higher in advanced melanoma. Using automated quantitative analysis (AQUA), we quantified VEGF, -R1, -R2 and -R3 expression in melanoma tissue microarrays composed of 540 nevi and 468 melanoma specimens (198 primaries, 270 metastases). VEGF, -R1, -R2 and -R3 expression was significantly higher in melanomas than nevi by unpaired t-tests (p <0.0001). VEGF-R2 expression was higher in metastatic specimens (p <0.0001), but VEGF-R3 expression was higher in primaries (p < 0.0001). VEGF was coexpressed with all three receptors when assessed by Spearman's rank correlation. VEGF, -R1, -R2 and -R3 expression is higher in melanomas than nevi. Higher expression of VEGF-R2 was found in metastases versus primaries, supporting the idea that selection for an angiogenic phenotype in metastatic melanoma is conferred via upregulation of VEGF-R2. However, higher expression of VEGF-R3 was seen on primary lesions, potentially implicating this receptor in initiation of lymphatic tumor spread. Clinical trials using antiangiogenic agents in melanoma should include correlative assays of VEGF, -R1, -R2 and -R3 as biomarkers of response to therapy, preferably using quantitative methods such as AQUA. Such assessments could assist with evaluation of these molecules as therapeutic targets in melanoma, ultimately facilitating improved selection of patients for treatment.

Purpose Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002. Experimental Design Using Automated Quantitative Analysis, we quantified expression of p85 and p110α subunits in 540 nevi and 523 melanomas. We determined the IC50 for LY294002 for 11melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002. Results p85 and p110α tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110α, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser235 and Ser240 were lower in the more resistant cell lines (P = 0.01and P = 0.004, respectively). Conclusions Expression of p85 and p110α subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.

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BackgroundGain of function mutations in B-RAF and N-RAS occur frequently in melanoma, leading to mitogen activating protein kinase (MAPK) pathway activation, and this pathway is the target of drugs in development. Our purpose was to study clinical characteristics of patients with mutations in this pathway and to determine activity of inhibitors of B-RAF and MEK in short term cultures grown from tumors of some of these patients.MethodsClinical and pathologic data were collected retrospectively on melanoma patients tested for B-RAF and N-RAS mutations at the Yale Cancer Center and associations with survival were determined. We studied in vitro activity of the pan-RAF inhibitor, RAF265, and the MEK inhibitor, MEK162, in 22 melanoma short term cultures. We further characterized the effect of MEK inhibition on apoptosis and growth of melanoma cultures.ResultsIn a cohort of 144 metastatic melanoma patients we found that patients with N-RAS mutant melanoma had a worse prognosis. These patients were more likely to have brain metastases at the time of presentation with metastatic disease than their N-RAS-wild-type counterparts. All N-RAS mutant melanoma cultures tested in our study (n = 7) were sensitive to MEK inhibition162. Exposure to MEK162 reduced ERK1/2 phosphorylation, and induced apoptosis. Clonogenic survival was significantly reduced in sensitive melanoma cell cultures.ConclusionsThe prognosis of patients with melanoma expressing constitutively active N-RAS is poor, consistent with studies performed at other institutions. N-RAS mutant melanoma cultures appear to be particularly sensitive to MEK162, supporting ongoing clinical trials with MEK162 in N-RAS mutated melanoma.

Purpose: Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K.Methods: Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110a. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244.Results: Strong coexpression of mTOR and p110a was observed (r ¼ 0.658; P < 0.0001). Less coexpression was seen with p85 (r ¼ 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC 50 values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage.Conclusions: mTOR and p110a are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors. Clin Cancer Res; 16(24); 6029-39.Ó2010 AACR.

CD70 is upregulated in several malignancies, where it induces cytotoxic effects on B and T lymphocytes leading to immune escape. Novel therapeutic agents targeting CD70 have entered clinical trials. We characterized expression of CD70 protein in RCC specimens of various histologic subtypes and assessed their prognostic value and association with clinical/pathological variables. We employed tissue microarrays containing 330 cases using a novel fluorescent immunohistochemistry-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. The mean expression of CD70 was almost twice as high in tumors relative to normal tissue (p<0.0001). When broken into subsets, CD70 was higher in sarcomatoid and clear cell tumors (p<0.0001), and variably elevated in oncocytomas and some papillary tumors. No association was found between CD70 expression and stage or grade. High CD70 expression was associated with decreased survival on univariate analysis in the clear cell subset of RCC, however, it did not retain significance on multi-variable analysis. Given the elevated expression of CD70 in clear cell, sarcomatoid, and some papillary tumors, our findings suggest that CD70 might be a good drug target in RCC. Additional studies are warranted to assess the association between expression of CD70 and response to therapy with CD70 targeting drugs in RCC.

BackgroundPI3K and mTOR are key components of signal transduction pathways critical for cell survival. Numerous PI3K inhibitors have entered clinical trials, while mTOR is the target of approved drugs for metastatic renal cell carcinoma (RCC). We characterized expression of p85 and p110α PI3K subunits and mTOR in RCC specimens and assessed pharmacologic co-targeting of these molecules in vitro.MethodsWe employed tissue microarrays containing 330 nephrectomy cases using a novel immunofluorescence-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. In RCC cell lines we assessed synergism between PI3K and mTOR inhibitors and activity of NVP-BEZ235, which co-targets PI3K and mTOR.Resultsp85 expression was associated with high stage and grade (P < 0.0001 for both). High p85 and high mTOR expression were strongly associated with decreased survival, and high p85 was independently prognostic on multi-variable analysis. Strong co-expression of both PI3K subunits and mTOR was found in the human specimens. The PI3K inhibitor LY294002 and rapamycin were highly synergistic in all six RCC cell lines studied. Similar synergism was seen with all rapamycin concentrations used. NVP-BEZ235 inhibited RCC cell growth in vitro with IC50s in the low ηM range and resultant PARP cleavage.ConclusionsHigh PI3K and mTOR expression in RCC defines populations with decreased survival, suggesting that they are good drug targets in RCC. These targets tend to be co-expressed, and co-targeting these molecules is synergistic. NVP-BEZ235 is active in RCC cells in vitro; suggesting that concurrent PI3K and mTOR targeting in RCC warrants further investigation.