Metastatic melanoma is difficult to treat and it is only recently that therapy has been shown to have an impact on overall survival
[1–3]. DTIC/dacarbazine has been shown in contemporary studies to provide tumor responses in less than 15% of patients, with a median response duration of 3–4 months
[4, 5]. Combination therapies may increase response rates, but without improvement in survival
. High dose interleukin-2 and ipilimumab benefit the minority of patients, albeit with a subset of patients experiencing durable responses
[1, 7, 8]. Although many patients with BRAF-mutated melanoma initially respond to vemurafenib, the only other agent approved by the FDA for this disease, most will ultimately relapse
. Thus, while significant advances in both immune based and molecularly targeted therapies have been made, survival for many patients with metastatic melanoma remains poor. New therapies are still needed for this disease, and the testing of new agents is being driven by an increasing knowledge of melanoma biology.
The vast majority of melanomas have activating mutations in signaling proteins involved in the RAS pathway. Mutations in RAS occur in around 15% of melanomas
[9, 10]. In addition, frequent mutations in downstream RAS effectors have been reported, the most common of which is BRAF which has been reported to be mutated in approximately 50% of cases
[11–13]. Mutated BRAF can be effectively targeted in patients with metastatic melanoma, with impressive response rates in early phase trials
[14–16]. Recent data now demonstrates an improvement in overall survival in patients treated with selective BRAF inhibitors when compared to dacarbazine, although many patients ultimately relapse, further highlighting the importance of understanding the molecular pathogenesis of this disease
[2, 17]. Activation of the PI3 Kinase/Akt pathway has also been implicated in melanoma tumorigenesis, potentially through downregulated expression of the negative regulator PTEN
[18–20]. Interestingly, even in melanoma cells having mutations in downstream effectors, constitutive RAS activation is nonetheless seen, likely through the activity of autocrine or paracrine growth factor secretion
. Transgenic mouse experiments have confirmed the important contribution of activated RAS-based signaling to melanomagenesis in vivo
Targeted inhibition of RAS-based signaling has therefore received significant attention. While kinase inhibitors that interfere with the activity of the downstream molecules PI3 Kinase, RAF, and MEK are in various stages of development, it has been difficult to identify a pharmacologic strategy to inhibit RAS activity directly
. However, the fact that RAS must undergo a lipid post-translational modification for localization to membrane compartments where access to its effectors occurs generated an alternative strategy for inhibiting RAS function. The most important post-translational modification of RAS is farnesylation, which is catalyzed by the enzyme Farnesyltransferase (FT)
. FT inhibitors (FTIs) have been developed as a strategy to block this process, thereby decreasing RAS translocation to membranes and reducing its ability to mediate activation of downstream effectors
. Interestingly, despite the initial motivation of FTI development driven by an interest in inhibiting RAS, FTIs have subsequently been shown to have effects on numerous additional proteins involved in tumor survival and proliferation. These include other GTPases such as Rheb, Ral, RhoC and Rac1, as well as factors involved in regulated protein translation and angiogenesis
[28, 29]. Preclinical data have shown anti-proliferative activity that is independent of Ras mutation status, and mechanistic experiments have implicated alternative farnesylated targets as functionally relevant. Thus, FTIs may in fact target multiple signaling molecules that contribute to malignant transformation and are no longer viewed as pure RAS inhibitors
. Recently, there has also been evidence to suggest that FTIs may enhance the effectiveness of cytotoxic chemotherapy when used in combination, potentially expanding the role of these agents
R115777 is an orally bioavailable methyl-quinolone, which has been shown to be a potent and selective inhibitor of FT in the nanomolar concentration range. Preclinical experiments demonstrated activity against melanoma tumor cell growth both in vitro and in vivo
. Phase I clinical trial testing identified a dose and schedule of R115777 of 300 mg PO BID, given for 21 days of a 28 day cycle which was sufficiently well tolerated for subsequent investigation
[33, 34]. The most common toxicities were myelosuppression, nausea, vomiting, and fatigue. Phase II studies have shown clinical activity as a single agent in patients with hematologic malignancies
[35–39]. Together, these observations coalesced to motivate investigation of the FTI R115777 in patients with advanced melanoma. Inasmuch as there was limited experience in evaluating tumor tissue for effective biochemical target inhibition, an integral part of the current study involved obtaining sizable tumor tissue before and during R115777 administration to measure FT enzymatic activity directly and also to assess effects on specific signaling pathways ex vivo.
Many of the signaling pathways involved in melanomagenesis are also involved in T cell activation, including the RAS pathway
. We recently have shown that cytokine production and proliferation of T cells in response to T cell receptor (TCR) engagement is blocked in vitro by FTIs, suggesting that these compounds could theoretically inhibit T cell function in treated patients
. Given the importance of the immune system to participate in melanoma growth control, the effect of signal transduction inhibitors on lymphocyte function has become a critical parameter to consider in the cancer context
. This may be particularly relevant, given the recent data suggesting that selective inhibition of BRAFV600E may increase T cell recognition of melanoma antigens in vitro
. Therefore, an additional goal of the current study was to assess whether T cell function in treated patients was affected ex vivo.
Patients and methods
This was a multicenter phase II clinical trial of R115777 in patients with metastatic melanoma carried out by the CALGB melanoma working group. The primary objectives were to estimate the clinical response rate and to evaluate the toxicity of this agent in this patient population. The secondary objectives were to measure FT activity and effects on signaling events in tumor tissue, and to assess effects on T cell activation ex vivo from the peripheral blood.
CALGB developed and coordinated this trial. Institutional review board approval and patient informed consent were required at each participating center. Patient registration and data collection were managed by the CALGB Statistical Center. Data quality was ensured by careful review of data by CALGB Statistical Center staff and by the study chairperson. Statistical analyses were performed by CALGB statisticians.
As part of the quality assurance program of the CALGB, members of the Audit Committee visit all participating institutions at least once every three years to review source documents. The auditors verify compliance with federal regulations and protocol requirements, including those pertaining to eligibility, treatment, adverse events, tumor response, and outcome in a sample of protocols at each institution. Such on-site review of medical records was performed for a subgroup of 6 patients (43%) of the 14 patients under this study.
Patients must have had histologically confirmed melanoma with evidence for metastatic disease, either regional in-transit metastases not amenable to complete surgical resection or distant metastases. Treating physicians were required to discuss available standard therapies including DTIC and IL-2 prior to enrolling patients. Eligibility criteria included: the presence of at least two accessible lesions amenable to excisional biopsy for correlative assays; measurable disease in addition to the lesions planned for biopsy; absence of brain metastases; no allergies to azoles (e.g. ketoconazole); no more than one prior immunotherapy for metastatic disease; no prior chemotherapy for any stage of disease; ECOG performance status of at least 1; at least 18 years of age; non-pregnant and non-nursing; laboratory parameters within the following range: absolute neutrophil count ≥ 1500/μl; platelet count ≥ 100,000/μl; bilirubin ≤ 1.5 mg/dL; creatinine ≤ 2.0 mg/dL.
R115777 was administered orally at a dose of 300 mg twice per day for 21 days of a 28-day cycle. Disease re-staging was performed every 2 cycles. Patients could remain on treatment until unacceptable toxicity or disease progression occurred. Prior to initiation of treatment, and again during week 7, an excisional biopsy was required to be performed for biologic correlates. At the same time points, heparinized blood was obtained for analysis of effects on T cells.
Evaluation of response and toxicities
Disease assessment was performed using RECIST criteria every two cycles. Toxicity evaluation was performed at least once per cycle. Dose reductions were allowed, with dose level −1 at 200 mg BID, dose level −2 at 100 mg BID, and dose level −3 being permanent discontinuation. For neurologic toxicity ≥ grade 2, drug was held until resolution to ≤ grade 1 and continued at a 1 level dose reduction. If the toxicity did not resolve within one week, then drug was permanently discontinued. For hematologic toxicities, if a grade 4 toxicity was observed then drug was held for up to 2 weeks. If resolution occurred to ≤ grade 1, then drug was resumed at a 1 level dose reduction. For other toxicities, if a grade 3 event was attributed to drug, then treatment was held up to 2 weeks. If toxicity resolved to ≤ grade 1, then drug was resumed at a 1 level dose reduction. If toxicity did not resolve within 2 weeks then drug was permanently discontinued. For any grade 4 non-hematologic toxicity attributed to drug, treatment was permanently discontinued.
A 3-stage design was used to allow for early termination if the drug appeared ineffective in this patient population. A maximum of 40 patients was targeted for enrollment, with the null hypothesis that the response rate (complete or partial response) is less than or equal to 0.05 versus the alternative hypothesis that the response rate is greater than or equal to 0.20. If no responses were observed in the first 14 patients then the trial would conclude accepting the null hypothesis. Otherwise, 14 patients would be enrolled in stage 2. If 2 or fewer total responses were observed, then the null hypothesis would be accepted. Else, the third stage would accrue to a total of 40 patients. If 4 or more responses were observed among 40 patients, then the drug would be considered efficacious. This procedure had a power of 0.91 and a significance level of 0.10. The probability of early termination was approximately 0.85 under the null hypothesis. For the correlative assays, descriptive statistics were proposed to describe changes in post-treatment versus pre-treatment specimens.
Tumor biopsies and assays for FT activity and RAS pathway signaling
Excisional biopsies were performed to obtain sufficient tissue for analysis and to minimize sampling error. Tissue was rapidly processed and stored until batched analysis. Proteins were extracted from snap-frozen tumor tissue using a tissue protein extraction reagent from Pierce (200 mg tumor/1 ml reagent). After homogenization at 4°C, the samples were spun at 13,000 x g and the supernatant found between the fatty top layer and the pellet was used for biochemical analysis. The FTase enzymatic assays as well as Western blots for protein level determination were carried out as described previously
[35, 44]. All analyses were performed in the laboratory of Dr. Said Sebti, at Moffitt Cancer Center.
Measurement of FTI action on T cells ex vivo
Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood samples and stored as viable cells in freezing medium until batch analysis. Briefly, cells were thawed, cultured with the superantigen Staphylococcal enterotoxin A (SEA) or with Phorbol Myristate Acetate (PMA)/Ionomycin as a positive control, with or without the addition of R115777 in vitro as a comparison. After overnight culture, supernatants were analyzed for IFN-γ content by ELISA using antibody pairs from Pharmingen. Post-versus-pre-treatment samples were compared using a paired t-test. In parallel, cells were lysed and analyzed by Western blotting for the apparent molecular weight of the farnesylated protein HDJ-2 as described previously