MEK targeting in N-RAS mutated metastatic melanoma.

BACKGROUND: Gain 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. METHODS: Clinical 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. RESULTS: In 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 inhibition 162. Exposure to MEK162 reduced ERK1/2 phosphorylation, and induced apoptosis. Clonogenic survival was significantly reduced in sensitive melanoma cell cultures. CONCLUSIONS: The 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.

Microvessel area as a predictor of sorafenib response in metastatic renal cell carcinoma.

BACKGROUND: Sorafenib was the first Food and Drug Administration approved anti-angiogenic therapy for renal cell carcinoma (RCC). Currently, there are no validated predictive biomarkers for sorafenib. Our purpose was to determine if sorafenib target expression is predictive of sorafenib sensitivity. METHODS: We used an automated, quantitative immunofluorescence-based method to determine expression levels of sorafenib targets VEGF, VEGF-R1, VEGF-R2, VEGF-R3, c-RAF, B-RAF, c-Kit, and PDGFR-ß in a cohort of 96 patients treated with sorafenib. To measure vasculature in the tumor samples, we measured microvessel area (MVA) by CD-34 staining. RESULTS: Of the markers studied, only high MVA was predictive of response (p = 0.005). High MVA was associated with smaller primary tumors (p = 0.005). None of the biomarkers studied was predictive of overall or progression-free survival. Using the Bonferroni adjustment correcting for 9 variables with an alpha of 0.05, MVA remained significantly associated with sorafenib response. CONCLUSIONS: Our results suggest that high MVA in tumor specimens might be associated with a greater likelihood of response to therapy. Further studies are needed to confirm these results in additional patients and in patients receiving other VEGF-R2 inhibitors, as MVA might be useful to improve patient selection for VEGF-R2 inhibitors.

Vascularity of primary and metastatic renal cell carcinoma specimens.

PURPOSE: Anti-angiogenic therapies are among the most commonly used drugs in renal cell carcinoma. Tumor vascularity, defined by microvessel area, may be associated with response to these drugs. Clinical studies suggest that metastatic sites are more responsive than primary tumors. Our purpose was to characterize microvessel area (MVA) in matched primary and metastatic samples and in samples of different histologies. METHODS: We employed a method of automated, quantitative analysis of in situ tumor components to identify the area of CD-34 staining endothelial cells within renal cell carcinoma tumors. MVA was assessed in corresponding primary and metastatic samples from 34 patients, as well as in 334 primary nephrectomy specimens with variable histologies. RESULTS: MVA measurements from different parts of the same tumor correlated well (R = 0.75), indicating that MVA was fairly uniform within a tumor. While MVA was slightly higher in primary tumors than corresponding metastatic sites, the difference was not statistically significant (P = 0.1). MVA in paired primary and metastatic samples correlated moderately well (R = 0.36). MVA was higher in clear cell than papillary histology and oncocytomas (P < 0.0001 and P = 0.018, respectively). CONCLUSIONS: Lack of significant differences MVA in matched primary and metastatic samples suggests that both types of tumors should respond to anti-angiogenic drugs. This should be confirmed on additional cohorts. Given the small cohort, future predictive biomarker studies entailing MVA measurements should include specimens from both sites. Clear cell carcinomas are more vascular than other histologic subtypes, which may explain the higher response rates to anti-angiogenic therapies in clear cell tumors.

Expression of drug targets in primary and matched metastatic renal cell carcinoma tumors.

BACKGROUND: Targeted therapies in renal cell carcinoma can have different effects on primary and metastatic tumors. To pave the way for predictive biomarker development, we assessed differences in expression of targets of currently approved drugs in matched primary and metastatic specimens from 34 patients. METHODS: Four cores from each site were embedded in tissue microarray blocks. Expression of B-Raf, C-Raf, cKIT, FGF-R1, HIF-2α, mTOR, PDGF-Rß, VEGF-R1, VEGF-R2, VEGF-R3, VEGF, VEGF-B, VEGF-C, VEGF-D, MEK1, and ERK1/2 was studied using a quantitative immunofluorescence method. RESULTS: No significant differences were observed in global expression levels in primary and metastatic renal cell carcinoma tumors, with the exception of MEK, which had higher expression in metastatic than primary specimens. Similarly, more ki67 positive cells were seen in metastatic specimens. Correlations between marker expression in primary and metastatic specimens were variable, with the lowest correlation seen for FGF-R1 and VEGF-D. There were no significant differences in the degree of heterogeneity in primary versus metastatic tumors. CONCLUSIONS: Expression of most of the studied markers was similar in primary and metastatic renal cell carcinoma tumors, suggesting that predictive biomarker testing for these markers can be conducted on either the primary or metastatic tumors for most markers.

Characterization and targeting of phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) in renal cell cancer.

BACKGROUND: PI3K 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. METHODS: We 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. RESULTS: p85 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 IC(50)s in the low ηM range and resultant PARP cleavage. CONCLUSIONS: High 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.

Phosphatidylinositol-3-kinase as a therapeutic target in melanoma.

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 p110alpha subunits in 540 nevi and 523 melanomas. We determined the IC(50) for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002. RESULTS: p85 and p110alpha 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, p110alpha, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser(235) and Ser(240) were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively). CONCLUSIONS: Expression of p85 and p110alpha 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.

CD70 expression patterns in renal cell carcinoma.

CD70 is up-regulated 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 renal cell carcinoma specimens of various histologic subtypes and assessed their prognostic value and association with clinical/pathologic variables. We used tissue microarrays containing 330 cases using a novel fluorescent immunohistochemistry-based method of Automated Quantitative Analysis of in situ protein expression. The mean expression of CD70 was almost twice as high in tumors relative to normal tissue (P < .0001). When broken into subsets, CD70 was higher in sarcomatoid and clear cell tumors (P < .0001) and was 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 renal cell carcinoma; however, it did not retain significance on multivariate 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 renal cell carcinoma. Additional studies are warranted to assess the association between expression of CD70 and response to therapy with CD70-targeting drugs in renal cell carcinoma.

Vertical targeting of the phosphatidylinositol-3 kinase pathway as a strategy for treating 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 p110α. 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 p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 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 p110α 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.

Chemotherapy and biologic therapies for melanoma: do they work?

The incidence of melanoma is increasing, and the therapeutic options for unresectable disease are limited, resulting in an increase in the death rate. Melanoma is usually resistant to standard chemotherapy, and the response rate for any single agent or combination of agents is 15% to 25%. High-dose interleukin-2 results in prolonged responses in a minority of patients, and biochemotherapy (combinations of chemotherapy, interferon, and interleukin-2) is associated with an improved response rate, but no clear effect on overall survival. A number of promising new agents have entered clinical trials in recent years, including monoclonal antibodies and small molecule inhibitors that target either the malignant melanocytes or negative regulators of the immune system. These drugs appear to benefit subsets of patients, and identification of predictors of response is the subject of intense research. This contribution summarizes the risks and benefits of older regimens and discusses the newer, targeted therapies.

C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.

PURPOSE: Raf-kinases include three major isoforms. Although the role of B-Raf in melanoma is well established, little is known about C-Raf. We studied effects of C-Raf knockdown in vitro and assessed expression of C-Raf in a large cohort of melanomas and nevi. EXPERIMENTAL DESIGN: Using specific siRNAs, we knocked down C-Raf expression, and determined the effect on viability, MAP extracellular signal-regulated kinase (ERK)/ERK kinase signaling, and apoptosis in seven melanoma cell lines. We determined the IC(50) of the C-Raf inhibitors sorafenib and GW5074, and studied the effects of GW5074 on cell signaling. Using an automated method to measure in situ protein expression, we quantified C-Raf expression in 263 nevi and 523 melanomas. RESULTS: C-Raf was knocked down in three cell lines with detectable phospho-C-Raf, resulting in decreased viability in two of the three (YULAC and YUROB). This resulted in decreased Bcl-2 expression and phospho-Bad cleavage, without affecting phospho-MEK and phospho-ERK. Sensitivity to sorafenib and GW5074 varied. GW5074 inhibited mitogen-activated protein kinase signaling without Bcl-2 and phospho-Bad down-regulation. C-Raf was highly expressed in melanomas compared with nevi (P < 0.0001), and no nevi had high C-Raf expression. C-Raf expression was higher in metastatic than primary specimens (P = 0.0225). CONCLUSIONS: C-Raf siRNA knock-down results in decreased viability of YULAC (B-Raf(V600K)) and YUROB (B-Raf(WT)) melanoma cells, likely mediated by Bcl-2 inhibition rather than mitogen-activated protein kinase inhibition. Cotargeting C-Raf and parallel pathways might be an effective therapeutic approach for melanoma. C-Raf expression is up-regulated in a subset of melanomas but not in nevi, suggesting that it might be a valuable diagnostic marker and therapeutic target.