Lenvatinib plus pembrolizumab in patients with advanced endometrial cancer: an interim analysis of a multicentre, open-label, single-arm, phase 2 trial.

BACKGROUND: Lenvatinib is a multikinase inhibitor of VEGFR1, VEGFR2, and VEGFR3, and other receptor tyrosine kinases. Pembrolizumab, an antibody targeting PD-1, has moderate efficacy in biomarker-unselected endometrial cancer. We aimed to assess the combination of lenvatinib plus pembrolizumab in patients with advanced endometrial carcinoma, after establishing the maximum tolerated dose in a phase 1b study. METHODS: In this open-label, single-arm, phase 2 study done at 11 centres in the USA, eligible patients were aged 18 years or older and had metastatic endometrial cancer (unselected for microsatellite instability or PD-L1), had an Eastern Cooperative Oncology Group performance status of 0 or 1, had received no more than two previous systemic therapies, had measurable disease according to the immune-related Response Evaluation Criteria In Solid Tumors (irRECIST), and had a life expectancy of 12 weeks or longer. Patients received 20 mg oral lenvatinib daily plus 200 mg intravenous pembrolizumab every 3 weeks. Treatment continued until disease progression, development of unacceptable toxic effects, or withdrawal of consent. The primary endpoint of this interim analysis was the proportion of patients with an objective response at week 24 as assessed by investigators according to irRECIST in the per-protocol population. This trial is registered with ClinicalTrials.gov, number NCT02501096. FINDINGS: Between Sept 10, 2015, and July 24, 2017, 54 patients were enrolled, 53 of whom were included in the analysis. At the cutoff date for anti-tumour activity data (Dec 15, 2017), median study follow-up was 13·3 months (IQR 6·7-20·1). 21 (39·6% [95% CI 26·5-54·0]) patients had an objective response at week 24. Serious treatment-related adverse events occurred in 16 (30%) patients, and one treatment-related death was reported (intracranial haemorrhage). The most frequently reported any-grade treatment-related adverse events were hypertension (31 [58%]), fatigue (29 [55%]), diarrhoea (27 [51%]), and hypothyroidism (25 [47%]). The most common grade 3 treatment-related adverse events were hypertension (18 [34%]) and diarrhoea (four [8%]). No grade 4 treatment-related adverse events were reported. Five (9%) patients discontinued study treatment because of treatment-related adverse events. INTERPRETATION: Lenvatinib plus pembrolizumab showed anti-tumour activity in patients with advanced recurrent endometrial cancer with a safety profile that was similar to those previously reported for lenvatinib and pembrolizumab monotherapies, apart from an increased frequency of hypothyroidism. Lenvatinib plus pembrolizumab could represent a new potential treatment option for this patient population, and is being investigated in a randomised phase 3 study. FUNDING: Eisai and Merck.

The tumor microenvironment in renal cell cancer.

PURPOSE OF REVIEW: In addition to the provision of nutrients and growth factors that facilitate tumor cell proliferation and metastasis, the tumor microenvironment (MEV) restricts immune surveillance of tumor-associated antigens and limits the efficacy of immune checkpoint inhibitors, tumor vaccines, and other immune therapies. This review will focus on the immunosuppressive mechanisms operative within the tumor MVE of renal cell carcinoma. RECENT FINDINGS: Several of the immunosuppressive mechanisms within the tumor MEV have been identified and are potentially druggable. Clinical trials with agents that target several of these inhibitory pathways are currently underway. SUMMARY: Although renal cell carcinoma is one of several tumor types responsive to immune checkpoint inhibitors, the effectiveness of these agents is likely to be limited by the various tumor-infiltrating bone marrow-derived myeloid cells that comprise the MEV. Several strategies to combat the recruitment of these cells into tumor tissue or to neutralize their immunosuppressive function have shown encouraging results in animal tumor models and clinical trials.

Clinical and laboratory features of autoimmune hemolytic anemia associated with immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICPis) are a novel class of immunotherapeutic agents that have revolutionized the treatment of cancer; however, these drugs can also cause a unique spectrum of autoimmune toxicity. Autoimmune hemolytic anemia (AIHA) is a rare, but often severe, complication of ICPis. We identified 14 patients from nine institutions across the United States who developed ICPi-AIHA. The median interval from ICPi initiation to development of AIHA was 55 days (interquartile range [IQR], 22-110 days). Results from the direct antiglobulin test (DAT) were available for 13 of 14 patients: 8 patients (62%) had a positive DAT and 5 (38%) had a negative DAT. The median pretreatment and nadir hemoglobin concentrations were 11.8 g/dL (IQR, 10.2-12.9 g/dL) and 6.3 g/dL (IQR, 6.1-8.0 g/dL), respectively. Four patients (29%) had a preexisting lymphoproliferative disorder, and two (14%) had a positive DAT prior to initiation of ICPi therapy. All patients were treated with glucocorticoids, with three requiring additional immunosuppressive therapy. Complete and partial recoveries of hemoglobin were achieved in 12 (86%) and 2 (14%) patients, respectively. Seven patients (50%) were rechallenged with ICPis, and one (14%) developed recurrent AIHA. Clinical and laboratory features of ICPi-AIHA were similar in DAT positive and negative patients. ICPi-AIHA shares many clinical features with primary AIHA; however, a unique aspect of ICPi-AIHA is a high incidence of DAT negativity. Glucocorticoids are an effective first-line treatment in the majority of patients with ICPi-AIHA, and most patients who are rechallenged with an ICPi do not appear to develop recurrence of AIHA.

Sunitinib activates Axl signaling in renal cell cancer.

Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.

Killer immunoglobulin-like receptor (KIR) and KIR-ligand genotype do not correlate with clinical outcome of renal cell carcinoma patients receiving high-dose IL2.

NK cells play a role in many cancer immunotherapies. NK cell activity is tightly regulated by killer immunoglobulin-like receptor (KIR) and KIR-ligand interactions. Inhibitory KIR-ligands have been identified as HLA molecules, while activating KIR-ligands are largely unknown. Individuals that have not inherited the corresponding KIR-ligand for at least one inhibitory KIR gene are termed the "KIR-ligand missing" genotype, and they are thought to have a subset of NK cells that express inhibitory KIRs for which the corresponding KIR-ligand is missing on autologous tissue, and thus will not be inhibited through KIR-ligand recognition. In some settings where an anticancer immunotherapeutic effect is likely mediated by NK cells, individuals with a KIR-ligand missing genotype have shown improved clinical outcome compared to individuals with an "all KIR-ligands present" genotype. In addition, patients receiving hematopoietic stem cell transplants for leukemia may do better if their donor has more activating KIR genes (i.e., KIR haplotype-B). In a recent multi-institution clinical trial of patients with metastatic renal cell carcinoma receiving high-dose IL2 (HD-IL2), 25 % of patients showed a complete or partial tumor response to this therapy. We genotyped KIR and KIR-ligand genes for these patients (n = 107) and tested whether KIR/KIR-ligand genotypes correlated with patient clinical outcomes. In these analyses, we did not find any significant association of KIR/KIR-ligand genotype (either KIR-ligand missing or the presence of KIR haplotype-B) with patient outcome in response to the HD-IL2 therapy.

A Phase I Trial of Bortezomib and Sorafenib in Advanced Malignant Melanoma.

LESSONS LEARNED: This study is a rare example of effective doses of both targeted agents being both administered and tolerated.This combination should not be used in melanoma. BACKGROUND: Sorafenib and bortezomib affect BCL family member expression. We previously demonstrated that bortezomib augmented sorafenib-mediated cytotoxicity in melanoma cell lines in vitro. We aimed to combine sorafenib 400 mg b.i.d. with increasing doses of weekly bortezomib. METHODS: Patients with metastatic melanoma were enrolled in dose-escalation cohorts to determine the maximum tolerated dose (MTD) of sorafenib (twice daily) in combination with bortezomib (weekly for 3 of 4 weeks). The MTD was defined as the highest dose level at which less than 33% of patients exhibited a dose-limiting toxicity (DLT). Efficacy, as measured by 6-month progression-free survival and response rate per RECIST, was documented. RESULTS: Eleven patients were enrolled at three dose levels. DLTs (fatigue and rash) were seen in two of three patients at the highest dose level. Five patients were enrolled for sorafenib 400 mg b.i.d. and bortezomib 1.0 mg/m(2) weekly for 3 of every 4 weeks; none had DLTs, and this dose level was defined as the MTD. Of 10 evaluable patients, no responses were seen. Two of 11 patients (18%) remained progression free for longer than 6 months. CONCLUSION: The combination of sorafenib and bortezomib is safe but not active in patients with melanoma.

Predictive biomarkers in renal cell cancer: insights in drug resistance mechanisms.

INTRODUCTION: VEGF-targeted therapy is currently the first line treatment for patients with metastatic clear cell renal cell carcinoma (ccRCC), but most patients either display primary (intrinsic) resistance or acquire drug resistance. In recent years multiple mechanisms of resistance to VEGF-targeted therapy emerged from preclinical research, but it is currently unknown to what extent these drug resistance modalities play a role in the clinic. Here we reviewed the current literature on biomarkers that predict treatment outcome in patients with ccRCC to gain insight in clinical drug resistance mechanisms. METHODS: A search syntax was compiled by combining different synonyms of "biomarker" AND "renal" AND "cancer". MEDLINE was accessed through PubMed, where this syntax was entered and used to search titles and abstracts of publications. Articles were selected based on three criteria: (1) description of patients with clear cell RCC, (2) treatment with VEGF targeted therapy and (3) discussion of biomarkers that were studied for potential association with treatment response. RESULTS: The literature search was performed on March 4th 2014 and yielded 1882 articles. After carefully reading the titles and abstracts based on the three previously mentioned criteria, 103 publications were evaluated. Backward citation screening was performed on all eligible studies and revealed another 24 articles. This search revealed that (1) High glucose uptake and low contrast enhancement on PET- and CT-imaging before start of treatment may correlate with poor response to therapy, (2) Low dose intensity due to treatment intolerance is related to shorter progression free survival. (3) Acquired resistance appears to be associated with rebound vascularization based on both longitudinal monitoring of contrast enhancement by CT and blood vessel counts in tumor tissue, and (4) Based on plasma cytokine and single nucleotide polymorphism (SNP) studies, interleukin-8, VEGFR-3, FGFR2 and HGF/MET emerged as potential clinical markers for chemoresistance. CONCLUSION: Low dose intensity, specific tumor-imaging techniques and potential biological biomarkers may be predictive for response to VEGF-targeted therapy in ccRCC. Some of these plausible biomarkers may also provide more insight into the underlying mechanisms of resistance such as altered glucose metabolism and rapid rebound vascularization.

PI3K inhibition potentiates Bcl-2-dependent apoptosis in renal carcinoma cells.

Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3-K/Akt inhibitors is therefore of great interest. As PI3-K inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl-2 family inhibitor ABT-737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl-1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl-1 and Bim levels. Our results suggest that the combination of PI3-K inhibitors with BH3-mimetics may be a viable therapeutic strategy in RCC.

Effects of HDM2 antagonism on sunitinib resistance, p53 activation, SDF-1 induction, and tumor infiltration by CD11b+/Gr-1+ myeloid derived suppressor cells.

BACKGROUND: The studies reported herein were undertaken to determine if the angiostatic function of p53 could be exploited as an adjunct to VEGF-targeted therapy in the treatment of renal cell carcinoma (RCC). METHODS: Nude/beige mice bearing human RCC xenografts were treated with various combinations of sunitinib and the HDM2 antagonist MI-319. Tumors were excised at various time points before and during treatment and analyzed by western blot and IHC for evidence of p53 activation and function. RESULTS: Sunitinib treatment increased p53 levels in RCC xenografts and transiently induced the expression of p21(waf1), Noxa, and HDM2, the levels of which subsequently declined to baseline (or undetectable) with the emergence of sunitinib resistance. The development of resistance and the suppression of p53-dependent gene expression temporally correlated with the induction of the p53 antagonist HDMX. The concurrent administration of MI-319 markedly increased the antitumor and anti-angiogenic activities of sunitinib and led to sustained p53-dependent gene expression. It also suppressed the expression of the chemokine SDF-1 (CXCL12) and the influx of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) otherwise induced by sunitinib. Although p53 knockdown markedly reduced the production of the angiostatic peptide endostatin, the production of endostatin was not augmented by MI-319 treatment. CONCLUSIONS: The evasion of p53 function (possibly through the expression of HDMX) is an essential element in the development of resistance to VEGF-targeted therapy in RCC. The maintenance of p53 function through the concurrent administration of an HDM2 antagonist is an effective means of delaying or preventing the development of resistance.

Differential modulatory effects of GSK-3ß and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis) in melanoma.

BACKGROUND: GSK-3ß phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3ß can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3ß and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3ß on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. RESULTS: MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3ß-dependent in that they were blocked with a GSK-3ß shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3ß-S9A). These modulatory effects of GSK-3ß on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3ß activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis. CONCLUSIONS: Our data demonstrate a complex partnership between GSK-3ß and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3ß and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.

High dose intermittent sorafenib shows improved efficacy over conventional continuous dose in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) responds to agents that inhibit vascular endothelial growth factor (VEGF) pathway. Sorafenib, a multikinase inhibitor of VEGF receptor, is effective at producing tumor responses and delaying median progression free survival in patients with cytokine refractory RCC. However, resistance to therapy develops at a median of 5 months. In an effort to increase efficacy, we studied the effects of increased sorafenib dose and intermittent scheduling in a murine RCC xenograft model. METHODS: Mice bearing xenografts derived from the 786-O RCC cell line were treated with sorafenib according to multiple doses and schedules: 1) Conventional dose (CD) continuous therapy; 2) high dose (HD) intermittent therapy, 3) CD intermittent therapy and 4) HD continuous therapy. Tumor diameter was measured daily. Microvessel density was assessed after 3 days to determine the early effects of therapy, and tumor perfusion was assessed serially by arterial spin labeled (ASL) MRI at day 0, 3, 7 and 10. RESULTS: Tumors that were treated with HD sorafenib exhibited slowed tumor growth as compared to CD using either schedule. HD intermittent therapy was superior to CD continous therapy, even though the total dose of sorafenib was essentially equivalent, and not significantly different than HD continuous therapy. The tumors exposed to HD sorafenib had lower microvessel density than the untreated or the CD groups. ASL MRI showed that tumor perfusion was reduced to a greater extent with the HD sorafenib at day 3 and at all time points thereafter relative to CD therapy. Further the intermittent schedule appeared to maintain RCC sensitivity to sorafenib as determined by changes in tumor perfusion. CONCLUSIONS: A modification of the sorafenib dosing schedule involving higher dose intermittent treatment appeared to improve its efficacy in this xenograft model relative to conventional dosing. MRI perfusion imaging and histologic analysis suggest that this benefit is related to enhanced and protracted antiangiogenic activity. Thus, better understanding of dosing and schedule issues may lead to improved therapeutic effectiveness of VEGF directed therapy in RCC and possibly other tumors.

Addition of gemcitabine at the time of sunitinib resistance in metastatic renal cell cancer.

OBJECTIVE: •To investigate the role of adding gemcitabine at the time of sunitinib failure in patients with metastatic renal cell cancer (mRCC). PATIENTS AND METHODS: •A retrospective chart review of 13 patients with mRCC who experienced disease progression on sunitinib and received gemcitabine 750 mg/m(2) i.v. on days 1 and 8 and sunitinib 37.5 mg/day on days 2-15 every 21 days from September 2006-2009 was carried out. •Responses were assessed using the Response Evaluation Criteria in Solid Tumors. •The median age was 59, male to female ratio 10:3, Eastern Cooperative Oncology Group performance status 1-3; the number of prior regimens ranged from one to five, and duration on sunitinib alone ranged from 3 months to >1 year. RESULTS: •Twelve out of 13 patients received more than two cycles with one documented partial response, five stable disease and seven progressive disease. •The median time to progression was 21 weeks. •We documented grade 4 pulmonary emboli (n= 1), grade 3 neutropenia (n= 2), anaemia (n= 2) and thrombocytopenia (n= 1). CONCLUSION: •The combination of sunitinib and gemcitabine in patients with mRCC may delay disease progression in some patients exhibiting resistance to sunitinib. This regimen merits further prospective investigation in this patient population.

CXCR4 inhibition modulates the tumor microenvironment and retards the growth of B16-OVA melanoma and Renca tumors.

To determine whether blockade of the chemokine receptor CXCR4 might alter the tumor microenvironment and inhibit tumor growth, we tested the efficacy of the CXCR4 antagonist X4-136 as a single agent and in combination with various immune checkpoint inhibitors in the syngeneic murine melanoma model B16-OVA. We also tested its activity alone and in combination with axitinib in the renal cancer model Renca. We found that X4-136 exhibited potent single agent antitumor activity in the B16-OVA model that was additive to that of an anti-PDL1 antibody. The antitumor activities were associated with a reduction in the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells and an increase in the number of tumor-specific CD8/perforin cells in the tumor-microenvironment. Apart from these immune effects, X4-136 alone and in combination with checkpoint inhibitors inhibited the Akt/FOXO-3a cell survival pathway in vitro and in vivo, suggesting that it might have antitumor activity independent of its effects on immune cell trafficking. Similar effects on tumor growth and cytotoxic T lymphocytes infiltration were observed in the Renca model. These studies show that the effects of CXCR4 blockade on immune cell trafficking might serve as a useful adjunct to immune checkpoint inhibitors and other therapies in the treatment of cancer.

Rapid evolution of acute kidney injury after initial infusion of pembrolizumab in a melanoma patient concurrently treated with RAF/MEK inhibitors.

The use of either immune checkpoint blockade or RAF/MEK inhibitors represents standard of care treatment options for metastatic melanoma. Each class of these drugs has distinct response kinetics, adverse effects, and unique clinical challenges. Combination of immune checkpoint blockade and RAF/MEK inhibitors may result in rapid and durable responses, however, the potential adverse effects of such combinations are poorly characterized. Here, we describe the case of a patient with BRAF-mutant melanoma who received an initial infusion of anti-PD-1 therapy while taking RAF/MEK inhibitors and experienced severe acute kidney injury, an otherwise infrequent side effect of any of these drugs alone. Treatment with corticosteroids rapidly reversed this process, indicating an underlying immune-mediated complication. A deeper understanding of potential adverse effects of combination therapies and their potential mechanisms should be carefully considered in the treatment landscape for melanoma and other cancers.

Lenvatinib Plus Pembrolizumab in Patients With Advanced Endometrial Cancer.

PURPOSE: Patients with advanced endometrial carcinoma have limited treatment options. We report final primary efficacy analysis results for a patient cohort with advanced endometrial carcinoma receiving lenvatinib plus pembrolizumab in an ongoing phase Ib/II study of selected solid tumors. METHODS: Patients took lenvatinib 20 mg once daily orally plus pembrolizumab 200 mg intravenously once every 3 weeks, in 3-week cycles. The primary end point was objective response rate (ORR) at 24 weeks (ORRWk24); secondary efficacy end points included duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Tumor assessments were evaluated by investigators per immune-related RECIST. RESULTS: At data cutoff, 108 patients with previously treated endometrial carcinoma were enrolled, with a median follow-up of 18.7 months. The ORRWk24 was 38.0% (95% CI, 28.8% to 47.8%). Among subgroups, the ORRWk24 (95% CI) was 63.6% (30.8% to 89.1%) in patients with microsatellite instability (MSI)-high tumors (n = 11) and 36.2% (26.5% to 46.7%) in patients with microsatellite-stable tumors (n = 94). For previously treated patients, regardless of tumor MSI status, the median DOR was 21.2 months (95% CI, 7.6 months to not estimable), median PFS was 7.4 months (95% CI, 5.3 to 8.7 months), and median OS was 16.7 months (15.0 months to not estimable). Grade 3 or 4 treatment-related adverse events occurred in 83/124 (66.9%) patients. CONCLUSION: Lenvatinib plus pembrolizumab showed promising antitumor activity in patients with advanced endometrial carcinoma who have experienced disease progression after prior systemic therapy, regardless of tumor MSI status. The combination therapy had a manageable toxicity profile.

Effect of melanoma on immune function in the regional lymph node basin.

PURPOSE: To determine if melanoma within the tumor microenvironment will result in immunosuppression within the draining lymph node as measured by down-regulation of T-cell receptor zeta (TCR zeta) expression. EXPERIMENTAL DESIGN: Patients with clinical stage I to III melanoma undergoing wide local excision and sentinel lymph node biopsy or therapeutic lymph node dissection were consented to have a portion of their lymph node sampled. Lymph nodes were classified as macroscopically involved (TI), microscopically involved (MI), noninvolved with positive wide excision (NI+), or noninvolved with negative wide excision (NI-). Lymphocytes were stained using antibodies to TCR zeta and other immune cells and analyzed via flow cytometer. Reverse transcription-PCR was used to assess for mediators of immunosuppression. RESULTS: Fifty patient lymph node samples (15 TI, 7 MI, 9 NI+, and 19 NI-) were evaluated. Increasing involvement of tumor in the lymph node was associated with decreasing TCR zeta chain expression (TI 56%, MI 76%, and NI- 89%). Differences between TI and MI (P = 0.005), TI and NI- (P = 0.0001), and MI and NI- (P = 0.019) were statistically significant. There was also a significant difference between TCR zeta chain expression of NI+ and NI- (73% versus 89%; P = 0.0016). A trend toward increased arginase expression in tumor-involved lymph nodes was detected by reverse transcription-PCR. CONCLUSIONS: Melanoma involvement of regional nodes is associated with loss of TCR zeta expression that is inversely related to tumor burden. Residual melanoma within the wide local excision specimen is associated with TCR zeta loss in noninvolved sentinel lymph nodes, suggesting that immune modulation precedes tumor spread.

The role of mammalian target of rapamycin inhibitors in the treatment of advanced renal cancer.

Inhibitors of the mammalian target of rapamycin (mTOR) have shown promising efficacy in early-stage trials in patients with advanced renal cell carcinoma (RCC). Most RCCs have been shown to possess biallelic alterations in the von Hippel-Lindau (VHL) gene, resulting in accumulation of hypoxia-inducible factors 1alpha and 2alpha, as well as their downstream targets including vascular endothelial growth factor (VEGF). The observed clinical efficacy of mTOR inhibitors in patients with RCC may be mediated in part by the dependence of efficient hypoxia-inducible factor translation on the mTOR pathway. mTOR inhibitors have entered more advanced phase clinical trials either as single agents or in combination with other targeted agents or IFN, which might ultimately result in regulatory approval of one or more agents. Given the likely nonoverlapping mechanism of action of mTOR inhibitors and VEGF pathway-targeted agents, mTOR inhibitors may prove useful if administered in combination or after resistance to VEGF inhibitors. With an increasing number of active agents for treatment of patients with RCC, efforts must continue to develop patient selection models based on predictive biomarkers to direct therapy to appropriate patients.

The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells.

Mitogen-activated protein kinase (MAPK) is activated in the majority of melanomas, and its activity is essential for cell survival. In this report, we examined the effects of a novel raf inhibitor BAY 43-9006 on melanoma cell viability and intracellular signaling and found that it induces apoptosis through a caspase-independent mechanism. At concentrations that suppress extracellular signal-regulated kinase (ERK) phosphorylation, BAY 43-9006 dephosphorylates Bad on Ser(75) and Ser(99), activates Bak and Bax, and reduces the mitochondrial transmembrane potential. BAY 43-9006 (sorafenib) down-modulates the levels of Bcl-2 and Bcl-X(L) in a MAPK-independent manner in A2058 and SKMEL5 melanoma cells but not in the more resistant A375 cells. Of the three lines tested, only A375 cells were rescued from BAY 43-9006-induced apoptosis by knocking down Bad. BAY 43-9006 induced poly(ADP-ribose) polymerase cleavage and the mitochondrial release of cytochrome c and SMAC. However, the pan-caspase inhibitor Z-VAD-fmk had only a modest protective effect against the drug, suggesting that BAY 43-9006-induced apoptosis is largely caspase independent. BAY 43-9006 but not the MAP/ERK kinase inhibitors PD98059 or U0126 induced the nuclear translocation of apoptosis-inducing factor (AIF) in A2058 and SKMEL5 cells, and the introduction of a small interfering RNA (siRNA) for AIF partially protected these cells from BAY 43-9006-induced apoptosis. The AIF siRNA had little effect in A375 cells, in which drug-induced AIF release was negligible. These data indicate that in sensitive cell lines, BAY 43-9006-induced apoptosis is independent of Bad dephosphorylation and caspase activation and largely mediated through the nuclear translocation of AIF.