A prospective phase II trial exploring the association between tumor microenvironment biomarkers and clinical activity of ipilimumab in advanced melanoma

Historically, the prognosis for patients with stage IV metastatic melanoma has been very poor, with a median overall survival (OS) of 6-10 months and a 1-year survival rate of ~25% [1, 2]. After decades of disappointments in the search for a melanoma therapy that could extend OS beyond that seen for the standard of care, two phase III randomized controlled trials demonstrated a statistically significant improvement in OS with ipilimumab in combination with dacarbazine in treatment-naïve patients [3] and as monotherapy in previously treated patients [4] with advanced melanoma. Ipilimumab is a fully human, monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4) [5, 6], a molecule that down-regulates pathways of T-cell activation [7]. By blocking CTLA-4, ipilimumab potentiates an antitumor immune response [8‐10].

Unlike conventional cancer therapies, the antitumor responses with ipilimumab may follow an initial period of apparent disease progression [11]. Ipilimumab therapy is associated with mechanism-based, immune-related adverse events (irAEs) that are inflammatory in nature [12], most of which are reversible using treatment guidelines provided in the US prescribing information [13]. However, treatment-related irAEs can be severe and, in rare instances, can be life-threatening [12]. Therefore, the ability to differentiate responders from nonresponders prior to initiation of therapy would help to maximize benefits associated with ipilimumab and minimize potential risks.

Biomarkers, intrinsic patient or tumor characteristics associated with clinical activity and/or toxicity of a given therapy, are increasingly demonstrating value towards the goals of personalized medicine. By allowing the prediction of both beneficial and detrimental responses to a given agent [14], biomarkers such as HER-2 in breast cancer [15] and KRAS in colon cancer [16] are beginning to change treatment paradigms. Promising biomarkers for melanoma include KIT and BRAF gene mutations [17, 18].

Biomarkers of clinical efficacy for immunotherapies to treat melanoma are in early stages of development. A few studies of melanoma patients treated with immunotherapies have demonstrated association between clinical efficacy and gene expression profiles derived from tumor tissue [19]. These gene expression signatures featured many immune-related genes, indoleamine 2,3-dioxygenase (IDO), and FoxP3. The immunotherapies tested in these studies include high- dose interleukin (IL)-2, IL-12, peptide vaccines (including MAGE-A3), and dendritic cells pulsed with peptide vaccines [19]. Due to the fact that only a small number of patients had a response to therapy, there were few data points on which to base conclusions regarding associations between gene expression and efficacy. Previous studies of anti-CTLA-4 therapy in melanoma patients have shown an association between clinical activity and treatment-emergent changes in immune cells, such as increased absolute lymphocyte count and changes in specific T-cell populations (e.g., increased frequency of CD8+ cells) [20‐24]. The primary objective of this exploratory, phase II trial (CA184-004; http://​www.​clinicaltrials.​gov NCT00261365) was the prospective exploration of candidate biomarkers of clinical response to ipilimumab in the tumor microenvironment.

Fourteen sites in seven European, North American, and South American countries enrolled 101 patients; of these, one patient withdrew consent and 18 who no longer met study criteria were not randomized (12 due to CNS metastasis or active CNS disease, one due to stomach hemorrhage, one had no measurable disease, and four did not meet other inclusion/exclusion criteria). The remaining 82 patients were randomized between January 2006 and May 2007 to receive ipilimumab 3 mg/kg (n = 40) or 10 mg/kg (n = 42). Baseline characteristics of randomized patients (Table 1) were generally consistent between dose groups.

Clinical activity and safety results from the present study are similar to those of other clinical trials of ipilimumab in advanced melanoma. In this study, BORRs of 7.5% and 11.9% and DCRs of 32.5% and 19% were observed for the 3 mg/kg and 10 mg/kg groups, respectively. There results are comparable to BORRs of 5.8% to 15.8% and DCRs of 20% to 30% for 10 mg/kg ipilimumab monotherapy in phase II studies [8‐10], and to a BORR of 10.9% and DCR of 28.5% for 3 mg/kg ipilimumab monotherapy in a phase III trial [4]. The 1-year survival rates of 60.9% and 44.2% for 3 and 10 mg/kg ipilimumab, respectively, were also similar to those of previous phase II studies (47.2% to 62.4% for 10 mg/kg ipilimumab) [8‐10] and to the phase III trial (45.6% for 3 mg/kg ipilimumab) [4]. Safety results from CA184-004 are consistent with those from a combined analysis of 325 patients in phase II trials of 10 mg/kg ipilimumab [37]. In the 10 mg/kg arm, drug-related AEs and irAEs occurred in 76.2% and 66.7% of patients, respectively, compared with 84.6% and 72.3% from the pooled analysis. As in previous studies, mechanism-based irAEs affecting the skin and gastrointestinal system were the most common toxicities observed and irAEs were generally reversible using product-specific treatment guidelines [4].

Variations within an individual patient's genome or somatic changes in the tumor DNA can affect disease progression and management. An increased understanding of how genetic variation can influence disease processes may allow physicians to personalize medicine through the design of individualized strategies for monitoring, prevention, and treatment that are the most effective for a given patient. Determination of a patient's genetic profile may contribute to the primary goals of personalized medicine by guiding the selection of treatments that maximize the chance for successful outcomes while minimizing adverse events [38]. Interest in personalized medicine is growing within the oncology community as evidence accumulates showing that differential clinical responses to a number of anticancer drugs can be associated with characteristics of an individual patient.

Recent reports include examples of this from studies of both melanoma and other cancers. Vemurafenib (PLX4032) is a selective inhibitor of the oncogenic V600E mutant BRAF kinase. In a melanoma study of 21 patients who received a sufficiently high dose of vemurafenib, 5 without the V600E BRAF mutation did not respond to the drug; however, of 16 patients with the mutation, 9 responded to vemurafenib and 7 did not [18]. In a phase II study of imatinib for advanced melanoma, a substantial proportion of patients with tumors characterized by mutation or amplification of KIT responded to the drug whereas it had limited activity in a nonselected population of melanoma patients [39]. Results of the CRYSTAL trial indicate that the clinical activity of cetuximab against metastatic colorectal cancer is enhanced in patients with wild-type KRAS compared with individuals having KRAS mutations, none of whom benefited from cetuximab treatment [40]. The identification of predictive biomarkers for immunotherapies such as ipilimumab will likely require interrogation of the immune functional status of the patient in addition to genomic analysis.

The presence of TILs, indicating an antitumor immune response, is a favorable prognostic indicator in a number of cancers [41‐45]; however, the prognostic significance of TILs in melanoma remains unclear [46]. In this study, CA184-004, the clinical activity of ipilimumab was positively associated with an increase in TILs from baseline, suggesting that the infiltrating lymphocytes may have anti-tumor activity, and that changes in the immune microenvironment of the tumor may slow disease progression. There likely are other changes in immune cell demographics, localization, and function that are involved in melanoma progression. Peripheral blood absolute lymphocyte count (ALC) has also demonstrated prognostic value in a number of malignancies [47‐50]. Association between clinical activity of ipilimumab and change from baseline in ALC was studied in the current trial [24] and is being explored further.

In the current study, higher protein levels of IDO and FoxP3 at baseline were significantly positively associated with clinical outcome and therefore may be predictive of ipilimumab clinical activity. Increased expression of IDO has been associated with an immunosuppressive tumor microenvironment or a response to chronic inflammation [51, 52]. In melanoma, baseline expression of IDO by tumors or infiltrates has been associated with faster tumor growth, lower antitumor T-cell responses, and poor prognosis [53]; however, recent data suggest that IDO may induce apoptosis of melanoma tumor cells [54]. Furthermore, the presence of IDO might be a consequence of an active immune microenvironment since IDO expression is induced by pro-inflammatory cytokines such as IL-1, interferon-γ and tumor necrosis factor-α [55‐57]. It is possible that IDO expression is up-regulated in tumors from patients with an ongoing, albeit suboptimal, antitumor immune response and that these individuals may be more likely to respond to ipilimumab. Further investigation of the role of IDO in melanoma and its value as a biomarker for ipilimumab is warranted.

FoxP3 has been identified as a marker of natural and adaptive/induced regulatory T cells (T_regs) [58]. Patients with cancer have higher levels of T_regs in peripheral blood than healthy individuals [59], leading to a suppression of T-cell immunity that has an immunopathologic role in tumor growth [56] and influences prognosis in many cancers [60‐63]. Metabolic degradation of tryptophan by IDO promotes the conversion of naïve T cells to T_regs[64, 65]. Thus, intratumoral expression of IDO may have promoted the accumulation of FoxP3⁺ cells; however, recent findings suggest that FoxP3 may not be specific for T_regs, but can also be expressed by other activated T-cell populations [66]. Other reports suggest that the ratio of effector T cells (T_effs) to T_regs, rather than changes in the absolute number of intratumoral T_effs or T_regs, may be a more specific biomarker of effective antitumor immune responses elicited by CTLA-4 blockade [67, 68].

Results from CA184-004 are similar to those found with tremelimumab, another anti-CTLA-4 antibody. In a pilot study of 15 tumor biopsies from 7 tremelimumab-treated patients, clinical response was associated with increased tumor infiltration by CD8⁺ TILs whereas a more variable association was found with tumor infiltration of CD4⁺ TILs [69]. The presence of FoxP3⁺ regulatory T cells, indicative of an immunosuppressive tumor microenvironment, was also variably associated with positive response. No apparent association between clinical response and intratumoral expression of IDO was seen in that study.

In addition to FoxP3 and IDO, other proteins likely are involved in creating an anti-tumor immune microenvironment. Pharmacodynamic microarray data on mRNA derived from tumor samples showed that ipilimumab led to altered gene expression in the tumor microenvironment, significantly increasing expression of immune-related genes, and significantly decreasing the expression of genes linked to melanoma. This analysis provides a list of candidate genes for further investigation to determine whether changes in gene expression support anti-tumor activity and/or are associated with clinical efficacy. Protein levels do not always correlate with mRNA expression and so candidate genes of interest should be confirmed by protein assays. Analyses of the relationship between expression changes and clinical efficacy are described in a separate manuscript that has been submitted for publication. In a previous study of ipilimumab in melanoma patients, associations between SNPs in the CTLA-4 gene and objective response were suggested [70]; such associations were not observed in the current study. Consistent with a previous study reporting that ipilimumab-treated patients with advanced melanoma have similar survival outcomes regardless of their HLA-A*0201 status [71], the current study found no association between clinical activity and the presence of the *0201 allele or any of the five most common HLA-A alleles.

The associations of clinical activity with increased TILs during treatment and with baseline expression of IDO and FoxP3 suggest that an immune-active tumor microenvironment might be necessary for a favorable response to ipilimumab. The increased expression of immune-related genes after treatment with ipilimumab suggest additional genes to be tested for their involvement in the creation of this immune-active tumor microenvironment.

Baseline expression of immune-related tumor biomarkers and a post-treatment increase in TILs may be positively associated with ipilimumab clinical activity. These preliminary results are hypothesis generating and support the need for larger prospective trials to confirm these findings and further characterize other potential biomarkers for ipilimumab.