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. 2018 Sep 18;6(1):90.
doi: 10.1186/s40425-018-0408-9.

A whole-blood RNA transcript-based gene signature is associated with the development of CTLA-4 blockade-related diarrhea in patients with advanced melanoma treated with the checkpoint inhibitor tremelimumab

Philip Friedlander  1 , Kevin Wood  2 , Karl Wassmann  3 , Alan M Christenfeld  3 , Nina Bhardwaj  4   5 , William K Oh  4
Affiliations

A whole-blood RNA transcript-based gene signature is associated with the development of CTLA-4 blockade-related diarrhea in patients with advanced melanoma treated with the checkpoint inhibitor tremelimumab

Philip Friedlander et al. J Immunother Cancer. .

Abstract

Background: Anti-CTLA-4 immune checkpoint blockade is associated with immune-related adverse events (irAEs). Grade 3-4 diarrhea/colitis is the most frequent irAE requiring treatment discontinuation. Predicting high-risk diarrhea/colitis patients may facilitate early intervention, limit irAE severity, and extend treatment duration. No biomarkers currently predict for anti-CTLA-4 immunotherapy related severe diarrhea.

Methods: Whole-blood was collected pre-treatment and 30 days post-treatment initiation from patients with stage III or IV unresectable melanoma who received 15 mg/kg tremelimumab at 90 day intervals in two clinical trials. The discovery dataset was a phase II study that enrolled 150 patients between December 2005 and November 2006. The validation dataset was a phase III study that enrolled 210 patients between March 2006 and July 2007. RT-PCR was performed for 169 genes associated with inflammation, immunity, CTLA-4 pathway and melanoma. Gene expression was correlated with grade 0-1 versus grade 2-4 diarrhea/colitis development.

Results: Pre-treatment blood obtained from the discovery dataset (N = 150) revealed no gene predictive of diarrhea/colitis development (p < 0.05). A 16-gene signature (CARD12, CCL3, CCR3, CXCL1, F5, FAM210B, GADD45A, IL18bp, IL2RA, IL5, IL8, MMP9, PTGS2, SOCS3, TLR9 and UBE2C) was identified from 30 days post-tremelimumab initiation blood that discriminated patients developing grade 0-1 from grade 2-4 diarrhea/colitis. The 16-gene signature demonstrated an AUC of 0.814 (95% CI 0.743 to 0.873, p < 0.0001), sensitivity 42.9%, specificity 99.2%, positive predictive value (PPV) 90.0%, and negative predictive value (NPV) 91.4%. In the validation dataset (N = 210), the 16-gene signature discriminated patients developing grade 0-1 from grade 2-4 diarrhea/colitis with an AUC 0.785 (95% CI 0.723 to 0.838, p < 0.0001), sensitivity 57.1%, specificity 84.4%, PPV 57.1% and NPV 84.4%.

Conclusion: This study identifies a whole-blood mRNA signature predictive of a clinically relevant irAE in patients treated with immune checkpoint blockade. We hypothesize that immune system modulation induced by immune checkpoint blockade results in peripheral blood gene expression changes that are detectable prior to clinical onset of severe diarrhea. Assessment of peripheral blood gene expression changes in patients receiving anti-PD-1/PD-L1 immunotherapy, or combination anti-CTLA4 and anti-PD-1/PD-L1 immunotherapy, is warranted to provide early on-treatment mechanistic insights and identify clinically relevant predictive biomarkers.

Trial registration: Clinicaltrials.gov , NCT00257205 , registered 22 November 2005.

Keywords: Biomarker; CTLA-4; Diarrhea; Immunotherapy; Melanoma; Predictive; Tremelimumab; irAE.

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Conflict of interest statement

Ethics approval and consent to participate

Separate ethical approval is not needed for the current submission. This manuscript presents data analyzing RNA obtained from blood of patients enrolled in two different Pfizer sponsored clinical trials. RNA analysis of the Pfizer study data was contemplated in the original protocol consents and therefore this study does not need ethical approval as the subjects had consented to participate in the two studies and agreed to have their blood drawn and gene expression analyzed. Furthermore the investigators remain totally blinded as to the patient’s identifiers with HIPPA protected data.

Consent for publication

not applicable.

Competing interests

PF: Advisory Board Member for Seattle Genetics, Regeneron, Aspyrian Therapeutics, Array Biopharma, EMD Serono and Castle Biosciences. Stock equity in MACK, AGN, INCY, ADVM, and CLVS.

KW M.D.: no conflicts.

KW: President and CEO of CPS Companion Diagnostics. Equity interest in CPS Companion Diagnostics which holds patent related to content of the manuscript.

AMC: Chairman of CPS Companion Diagnostics. Equity interest in CPS Companion Diagnostics which holds patent related to content of the manuscript.

NB: Consultant for Neon. Advisory Board Member for CPS Companion Diagnostics and Curevac. Equity interest in CPS Companion Diagnostics which holds patent related to content of the manuscript.

WKO: Chief Scientific Officer and Chairman of the Science Advisory Board CPS Companion Diagnostics. Equity interest in CPS Companion Diagnostics which holds patent related to content of the manuscript.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Main clinical trial enrollment and study population exclusions
Fig. 2
Fig. 2
Graphic presentation of the difference in expression of each of the eight genes with statistical significance (defined by p value < 0.05) in discriminating patients who develop grades 0–1 versus 2–4 diarrhea. T-test analysis performed on blood samples obtained 30 days post initiation of tremelimumab treatment
Fig. 3
Fig. 3
The 16-gene signature discriminates in the 30-days post-treatment discovery dataset the 129 patients who experienced grade 0–1 from the 21 patients who experienced grade 2–4 diarrhea with an AUC of 0.814 (95% CI 0.743, 0.873; p < 0.0001). a Accuracy of patient classification of as a function of diarrhea grade using the 16 gene signature. b Dot diagram depicting the 16-gene signature results in discovery dataset patients (N = 150) experiencing grade 0–1 diarrhea (designated as 0) versus grade 2–4 diarrhea patients (designated as 1) with a 0.15 cut-off. c Receiver operating characteristic curve plotting sensitivity of the 16-gene signature on the y axis and 100-specificity on the x axis in discovery dataset patients
Fig. 4
Fig. 4
The 16-gene signature discriminates in the validation dataset the 154 patients who experienced grade 0–1 from the 56 patients who experienced grade 2–4 diarrhea with an AUC 0.785 (95% CI 0.723, 0.838; p < 0.0001). a Accuracy of patient classification of as a function of diarrhea grade using the 16 gene signature. b Dot diagram depicting the 16-gene signature results in discovery dataset patients (N = 150) experiencing grade 0–1 diarrhea (designated as 0) versus grade 2–4 diarrhea patients (designated as 1) with a 0.15 cut-off. c Receiver operating characteristic curve plotting sensitivity of the 16-gene signature on the y axis and 100-specificity on the x axis in validation dataset patients
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