Phase II study of ruxolitinib, a selective JAK1/2 inhibitor, in patients with metastatic triple-negative breast cancer

Abstract

Preclinical data support a role for the IL-6/JAK2/STAT3 signaling pathway in breast cancer. Ruxolitinib is an orally bioavailable receptor tyrosine inhibitor targeting JAK1 and JAK2. We evaluated the safety and efficacy of ruxolitinib in patients with metastatic breast cancer. This was a non-randomized phase II study enrolling patients with refractory, metastatic triple-negative breast cancer. The primary endpoint was objective response by RECIST 1.1. The study was designed to enroll patients whose archival tumor tissue was pSTAT3-positive (T-score >5) by central immunohistochemistry. pSTAT3 staining was available from 171 of 217 consented patients and pSTAT3 T-score was positive in 67/171 (39.2%) tumors, suggesting that JAK-STAT activation is frequent. Twenty-three patients including one patient with inflammatory breast cancer were enrolled. Ruxolitinib was well-tolerated with infrequent grade 3 or higher toxicities with fatigue as the most common toxicity. Among 21 patients who received at least one dose of protocol therapy, no objective responses were observed and the study was closed to further accrual. Pharmacodynamic analyses of baseline vs. cycle 2 biopsies suggest on-target activity, including a significant decrease in the proportion of pSTAT3⁺ cells in three patients with paired biopsies and downregulation of JAK-STAT target genes and signatures via transcriptional analyses of 11 total baseline and four metastatic biopsies. Immuno-FISH analyses demonstrate intratumoral heterogeneity of pSTAT3 and JAK2 amplification. Ruxolitinib, as a single agent, did not meet the primary efficacy endpoint in this refractory patient population despite evidence of on-target activity.

Fig. 1

CONSORT diagram

Fig. 2

Immuno-FISH and CD8+ T-cell infiltrate in primary vs. metastatic samples. a iFISH images of one area of the primary tumor and brain and liver metastases of case 15. Scale bar 100  mm. b Dot plot depicting relative frequencies of the four different cell types in primary and metastatic samples. Error bars, S.E.M. c Immunofluorescence analysis of CD8, GranzymeB (GZMB), and pSTAT3. Images are a montage of nine fields captured from one area of the tissue. Scale bar 100  mm. d, e Graph depicting numbers of CD8⁺ T cells per montage (d) and fraction of GZMB⁺CD8⁺ T cells (e). Significance of the difference between primary and metastatic samples was calculated using the Wilcoxon rank-sum test. f Correlation analysis between the number of infiltrating CD8⁺ T cells and the relative frequencies of specific cell populations in primary and metastatic samples. g Correlation analysis between number of infiltrating CD8⁺ T cells and the Shannon index of diversity in all, primary and metastatic samples. Gray area, 95% confidence interval. Sample sizes were n  =  16 primary tumors and n  =  18 metastases

Fig. 3

Pre-treatment and cycle 2 biopsies reveal on-targets effect of JAK/STAT pathway suppression. a Fraction of cells positive for pSTAT3 in three patients who underwent both pre-treatment and cycle 2 biopsies. b–d Total RNA was isolated from fresh frozen biopsy samples obtained at baseline (n  =  12) and cycle 2 (n  =  4) and RNA sequencing performed. Individual JAK/STAT target genes—including both upregulated (SOCS3, EGFR) and downregulated (LCK)—demonstrate expression changes in cycle 2 concordant with pathway suppression (b). Two independently derived STAT3 pathway gene expression signatures demonstrated suppression at cycle 2 (c). GeneGO analyses of differentially expressed genes (DESeq2 p <  0.001) were identified revealed that Jak–STAT pathways as the top two ontologies (d)

Fig. 4

Immuno-FISH analysis of JAK2 copy number and phospho-STAT3. a Representative images of iFISH for pSTAT3 and JAK2 copy number at baseline and after cycle 2 in case 13. Scale bar 50  mm. b Maps showing topologic differences in the distribution of genetically and phenotypically distinct tumor cells based on their copy number signals for JAK2 and pSTAT3 in three different regions of the tumor. c Summary of cell type frequencies in all patients at baseline and after cycle 2. The graph depicts the mean percentage of each cell type in all areas and the samples are combined. p  < 0.0001 (Chi-square test). d Dot plot depicting the relative frequencies of the four different cell types at baseline and at cycle 2. The significance of the difference between baseline and cycle 2 samples was calculated using Wilcoxon rank-sum test. Error bars, S.E.M. e Shannon index of cellular diversity in matched baseline and cycle 2 samples for n  = 3 pairs