Pro-Inflammatory Cytokines Predict Relapse-Free Survival after One Month of Interferon-α but Not Observation in Intermediate Risk Melanoma Patients

<div><p>Background</p><p>E1697 was a phase III trial of adjuvant interferon (IFN)-α2b for one month (Arm B) versus observation (Arm A) in patients with resected melanoma at intermediate risk. We evaluated the levels of candidate serum cytokines, the HLA genotype, polymorphisms of CTLA4 and FOXP3 genes and the development of autoantibodies for their association with relapse free survival (RFS) in Arm A and Arm B among 268 patients with banked biospecimens.</p><p>Methods</p><p>ELISA was used to test 5 autoantibodies. Luminex/One Lambda LABTypeRSSO was used for HLA Genotyping. Selected <i>CTLA4</i> and <i>FOXP3</i> Single nucleotide polymorphisms (SNPs) and microsatellites were tested for by polymerase chain reaction (PCR). Sixteen serum cytokines were tested at baseline and one month by Luminex xMAP multiplex technology. Cox Proportional Hazards model was applied and the Wald test was used to test the marginal association of each individual marker and RFS. We used the Lasso approach to select the markers to be included in a multi-marker Cox Proportional Hazards model. The ability of the resulting models to predict one year RFS was evaluated by the time-dependent ROC curve. The leave-one-out method of cross validation (LOOCV) was used to avoid over-fitting of the data.</p><p>Results</p><p>In the multi-marker modeling analysis conducted in Arm B, one month serum IL2Rα, IL-12p40 and IFNα levels predicted one year RFS with LOOCV AUC = 82%. Among the three markers selected, IL2Rα and IFNα were the most stable (selected in all the cross validation cycles). The risk score (linear combination of the 3 markers) separated the RFS curves of low and high risk groups well (p = 0.05). This model did not hold for Arm A, indicating a differential marker profile in Arm B linked to the intervention (adjuvant therapy).</p><p>Conclusions</p><p>Early on-treatment proinflammatory serum markers (IL2Rα, IL-12p40, IFNα) significantly predict RFS in our cohort of patients treated with adjuvant IFN-α2b and warrant further study.</p></div

The association of clinical factors with relapse free survival (RFS).

<p>The association of clinical factors with relapse free survival (RFS).</p

The Kaplan–Meier (KM) plot of RFS by the dichotomized multi-marker score in Arm B (patients treated with one month of interferon-α).

<p>One month pro-inflammatory cytokines (IL2Rα, IL-12p40 and IFN-α) predict one year RFS.</p

Kaplan-Meier plot of progression free survival (PFS) by the dichotomized (at median) change in the percentage of circulating regulatory T cells (Treg) between baseline and week 6.

<p>Greater increase in circulating Treg (CD4+CD25hi+Foxp3+%) was associated with improved PFS (HR = 0.57, p = 0.034; N = 27 patients). Example of raw data is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087705#pone.0087705.s003" target="_blank">Figure S3</a> where the gating strategy used for Treg is shown.</p

Adverse events (worst grade) that were possibly, probably or definitely related to ipilimumab (N = 35; incidence ≥2)^*.

*<p>One patient with a history of gastroesophageal reflux disease and irritable bowel syndrome developed grade 3 nausea, vomiting, gastroparesis and achalasia after one dose of ipilimumab. One case of grade 4 uric acid elevation was considered possibly related to ipilimumab.</p

Patient Demographics and Baseline Disease Characteristics (N = 35 patients).

*<p>HDI: high dose interferon-α; ECOG: Eastern Cooperative Oncology Group;</p