Abstract P5-21-10: Phase 2 study and correlative analyses of ruxolitinib, a selective JAK1/2 inhibitor, in patients with metastatic, triple-negative breast cancer
Authors: Stover DG , Gil Del Alcazar CR , Tolaney SM , Bardia A , Guo H , Balko JM , Overmoyer BA , Gelman RS , Lloyd M , Wang V , Brock JE , Winer EP , Polyak K , Lin NU 
Published: February 2018

Abstract
Background: Preclinical data supports a role for the IL-6/JAK2/STAT3 signaling pathway in breast cancer (BC). Ruxolitinib is an orally bioavailable receptor tyrosine inhibitor targeting JAK1 and JAK2. We evaluated the safety and efficacy of ruxolitinib in patients with metastatic BC and performed correlative analyses.
Methods: This was a non-randomized, phase 2 study of patients with refractory, metastatic, triple-negative BC (TNBC). Patients with inflammatory BC (IBC) of any subtype were also enrolled. The primary endpoint was objective response by RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. The study was designed to enroll only patients whose archival tumor tissue was pSTAT3 moderately to strongly positive in the tumor epithelial cells by central immunohistochemistry (IHC). 16 patients underwent pre-treatment biopsy, of whom 4 also had a second biopsy prior to cycle 2. Biopsy samples and paired primary tumor samples (when available) were subjected to multi-color immunofluorescence and/or immune-FISH for leukocyte markers, pSTAT3, and JAK2. RNA sequencing was performed on available on-study frozen biopsy specimens. 17 patients had plasma collected with cell-free DNA (cfDNA) extracted and subjected to low coverage whole-genome sequencing.
Results: Of 217 patients who consented to archival tumor testing, T-score for pSTAT3 was 'high' (&gt;5) in 69 patients (31.8%), demonstrating frequent activation of the JAK/STAT pathway in metastatic TNBC or IBC. 23 pSTAT3 high patients were enrolled. Ruxolitinib was generally well-tolerated. The most commonly observed adverse events (any grade) were anemia, neutropenia, thrombocytopenia, constipation, nausea, and increased AST/ALT. Grade 3 or higher toxicities were uncommon. No objective responses were seen among 21 evaluable patients, therefore the study was closed to accrual based on study design. Intensive correlative analyses revealed important insights regarding ruxolitinib effects. Pharmacodynamic analyses of baseline versus cycle 2 biopsies demonstrate downregulation of JAK2 target genes, STAT3 signatures, and JAK/STAT gene ontology gene sets, suggesting on-target activity. There was evidence of immune microenvironment modulation: gene set enrichment analysis implicated reduced macrophage/myeloid phenotypes after treatment and CIBERSORT analysis of inferred immune cell subsets demonstrated reduced monocyte/macrophage proportion after treatment (t-test p=0.013). Multi-color immunofluorescence analyses of immune microenvironment are ongoing and will be reported. 17 patients underwent cfDNA analysis with 8 patients (47%) demonstrating gain or amplification of JAK2.
Conclusions: Ruxolitinib, as a single agent, did not meet the primary efficacy endpoint in this refractory patient population. Correlative studies demonstrate evidence of on-target activity and immune microenvironment modulation. Frequent JAK/STAT pathway activation and JAK2 locus chromosomal gains in this cohort suggest that the JAK/STAT pathway remains a potential therapeutic target in BC.
Citation Format: Stover DG, Gil Del Alcazar CR, Tolaney SM, Bardia A, Guo H, Balko JM, Overmoyer BA, Gelman RS, Lloyd M, Wang V, Brock JE, Winer EP, Polyak K, Lin NU. Phase 2 study and correlative analyses of ruxolitinib, a selective JAK1/2 inhibitor, in patients with metastatic, triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-10.