Increased Soluble PD-1 Predicts Response to Nivolumab plus Ipilimumab in Melanoma

SIMPLE SUMMARY: Checkpoint inhibitors have emerged as an effective therapy for patients with metastatic melanoma significantly improving survival for these patients. Despite this, many patients do not respond to the therapy and no current biomarkers can identify responders from non-responders. Using machine learning, we analysed cytokine levels in serially collected liquid biopsy to identify cytokine changes associated with response to checkpoint inhibitors in advanced-stage melanoma patients. The results presented here highlight that serial measurements of cytokine levels are a strong predictor of treatment response. Particularly, we demonstrate that high increases of soluble PD-1 measured from baseline to on-treatment is significantly associated with superior PFS in patients treated with nivolumab plus ipililumab. These results suggest that monitoring cytokine levels using serial samples is informative of treatment response and can improve guidance of treatment modality and the outcome of cancer patients. ABSTRACT: Background: Checkpoint inhibitors have revolutionized the treatment of metastatic melanoma, yielding long-term survival in a considerable proportion of the patients. Yet, 40–60% of patients do not achieve a long-term benefit from such therapy, emphasizing the urgent need to identify biomarkers that can predict response to immunotherapy and guide patients for the best possible treatment. Here, we exploited an unsupervised machine learning approach to identify potential inflammatory cytokine signatures from liquid biopsies, which could predict response to immunotherapy in melanoma. Methods: We studied a cohort of 77 patients diagnosed with unresectable advanced-stage melanoma undergoing treatment with first-line nivolumab plus ipilimumab or pembrolizumab. Baseline and on-treatment plasma samples were tested for levels of PD-1, PD-L1, IFNγ, IFNβ, CCL20, CXCL5, CXCL10, IL6, IL8, IL10, MCP1, and TNFα and analyzed by Uniform Manifold Approximation and Projection (UMAP) dimension reduction method and k-means clustering analysis. Results: Interestingly, using UMAP analysis, we found that treatment-induced cytokine changes measured as a ratio between baseline and on-treatment samples correlated significantly to progression-free survival (PFS). For patients treated with nivolumab plus ipilimumab we identified a group of patients with superior PFS that were characterized by significantly higher baseline-to-on-treatment increments of PD-1, PD-L1, IFNγ, IL10, CXCL10, and TNFα compared to patients with worse PFS. Particularly, a high PD-1 increment was a strong individual predictor for superior PFS (HR = 0.13; 95% CI 0.034–0.49; p = 0.0026). In contrast, decreasing levels of IFNγ and IL6 and increasing levels of CXCL5 were associated with superior PFS in the pembrolizumab group, although none of the cytokines were individually predictors for PFS. Conclusions: In short, our study demonstrates that a high increment of PD-1 is associated with superior PFS in advanced-stage melanoma patients treated with nivolumab plus ipilimumab. In contrast, decreasing levels of IFNγ and IL6, and increasing levels of CXCL5 are associated with response to pembrolizumab. These results suggest that using serial samples to monitor changes in cytokine levels early during treatment is informative for treatment response

Regulatory dissection of the CBX5 and hnRNPA1 bi-directional promoter in human breast cancer cells reveals novel transcript variants differentially associated with HP1α down-regulation in metastatic cells

BACKGROUND: The three members of the human heterochromatin protein 1 (HP1) family of proteins, HP1α, HP1β, and HPγ, are involved in chromatin packing and epigenetic gene regulation. HP1α is encoded from the CBX5 gene and is a suppressor of metastasis. CBX5 is down-regulated at the transcriptional and protein level in metastatic compared to non-metastatic breast cancer. CBX5 shares a bi-directional promoter structure with the hnRNPA1 gene. But whereas CBX5 expression is down-regulated in metastatic cells, hnRNAP1 expression is constant. Here, we address the regulation of CBX5 in human breast cancer. METHODS: Transient transfection and transposon mediated integration of dual-reporter mini-genes containing the bi-directional hnRNPA1 and CBX5 promoter was performed to investigate transcriptional regulation in breast cancer cell lines. Bioinformatics and functional analysis were performed to characterize transcriptional events specifically regulating CBX5 expression. TSA treatment and Chromatin Immunoprecipitation (ChIP) were performed to investigate the chromatin structure along CBX5 in breast cancer cells. Finally, expression of hnRNPA1 and CBX5 mRNA isoforms were measured by quantitative reverse transcriptase PCR (qRT-PCR) in breast cancer tissue samples. RESULTS: We demonstrate that an hnRNPA1 and CBX5 bi-directional core promoter fragment does not comprise intrinsic capacity for specific CBX5 down-regulation in metastatic cells. Characterization of transcriptional events in the 20 kb CBX5 intron 1 revealed existence of several novel CBX5 transcripts. Two of these encode consensus HP1α protein but used autonomous promoters in intron 1 by which HP1α expression could be de-coupled from the bi-directional promoter. In addition, another CBX5 transcriptional isoform, STET, was discovered. This transcript includes CBX5 exon 1 and part of intron 1 sequences but lacks inclusion of HP1α encoding exons. Inverse correlation between STET and HP1α coding CBX5 mRNA expression was observed in breast cancer cell lines and tissue samples from breast cancer patients. CONCLUSION: We find that HP1α is down-regulated in a mechanism involving CBX5 promoter downstream sequences and that regulation through alternative polyadenylation and splicing generates a transcript, STET, with potential importance in carcinogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-016-2059-x) contains supplementary material, which is available to authorized users

Additional file 3: Figure S1. of Regulatory dissection of the CBX5 and hnRNPA1 bi-directional promoter in human breast cancer cells reveals novel transcript variants differentially associated with HP1α down-regulation in metastatic cells

CBX1 and CBX3 and correlation of expression analyses. A) Schematized view of CBX3 and hnRNPA2B1 (not drawn to scale). Arrows indicate direction of transcription. The coding region is indicated by black colouring. pA indicates the localization of poly-A signals. A2UCOE represents localization of the characterized insulator element. B) Correlation analysis of CBX3 and hnRNPA2B1 expression in the NCI-60 breast cancer cell panel. The analysis presented as heat map was performed using the CellMiner database, http://discover.nci.nih.gov/cellminer/ , with red symbolizing positive and blue negative correlation. C) Correlation analysis of CBX1, CBX3, CBX5, hnRNPA1 and hnRNPA2B1 expression in the NCI-60 breast cancer cell panel. The analysis presented as correlation coefficients estimated using the CellMiner database with red numbering symbolizing significant expression correlation. D) Schematized view of CBX1 and neighboring SNX11 (not drawn to scale). Arrows indicate direction of transcription. The coding region is indicated by black coloring. The position of a promoter overlapping CpG island is shown. (PDF 380 kb

Additional file 1: Table S1. of Regulatory dissection of the CBX5 and hnRNPA1 bi-directional promoter in human breast cancer cells reveals novel transcript variants differentially associated with HP1Îą down-regulation in metastatic cells

Sequences for primers used in RT-qPCR. (DOCX 17 kb

Additional file 2: Table S2. of Regulatory dissection of the CBX5 and hnRNPA1 bi-directional promoter in human breast cancer cells reveals novel transcript variants differentially associated with HP1Îą down-regulation in metastatic cells

Expression data extracted from Affymetrix microarray experiments. (DOCX 17 kb