Antitumor Activity of Eribulin After Fulvestrant Plus CDK4/6 Inhibitor in Breast Cancer Patient-derived Xenograft Models

Antitumor activity of CDK4/6 inhibitor, endocrine therapy and chemotherapy in 4 luminal-type breast cancer patient-derived xenograft (PDX) models. PDX tumor-bearing mice were randomized into each treatment group (n=2 to 4 per group), and treated with 5 mg/mouse fulvestrant (Flv; purple) on a Q7D×3; 100 mg/kg palbociclib (Palb; green) on a Q1D×21; a combination of Flv plus Palb (orange); 0.5-0.57 mg/kg of eribulin (Eri-L; pink), and 2.0-2.26 mg/kg of eribulin (Eri-H; red) on a Q4D×3; or 250 mg/kg capecitabine (Cape; blue) on a Q1D×14. Untreated control is shown in black. The same control group data are shown in the upper (CDK4/6 inhibitor and endocrine therapy) and lower (chemotherapy) panels. (A) Relative tumor volumes are shown for days after treatment initiation. Minimum relative tumor volumes (RTVs) for individual mice, which were lower than 0.01 are shown as 0.01. (B) Survival of mice. Kaplan–Meier estimates of mouse survival combined across the 4 PDX models are shown (n=8 to 12 per treatment group). Mouse survival was defined as the number of days t until the tumor volume reached four times the initial tumor volume. Differences in survival were examined by using the log-rank (Mantel–Cox) test. *p<0.05 vs. Control by log-rank test. ^#p<0.05 vs. Cape by log-rank test. (C) Waterfall plots. The best overall response as percentages from baseline across the 4 PDX models are shown. The best overall response was determined by comparing the tumor volume change at day t with the baseline tumor volume at day 0 for t≥21 (after end of treatment period). Maximum values of best percentage change from baseline are shown as 200%. Best percentage changes from baseline that exceeded 200% are represented as 200%. The same control data are used in the left (CDK4/6 inhibitor and endocrine therapy) and right (chemotherapy) panels.

Molecular profiles of the 4 luminal-type breast-cancer patient-derived xenograft (PDX) models. RNA was extracted and purified from resected tumor tissues from untreated mice (n=1 per model). After synthesis of cDNA, quantitative RT-PCR was performed, and mRNA expression levels of genes relative to ones in the OD-BRE-0438 model were calculated. Ct values for each sample were measured in duplicate and mean Ct values were used for analysis. nd: Not detected.

Antitumor activity of endocrine therapy plus CDK4/6 inhibitor combination treatment in the OD-BRE-0438 model. (A) Scheme of treatment with fulvestrant (Flv), palbociclib (Palb), or Flv plus Palb in the 1^st and 2^nd experiments. (B) Antitumor activity of Flv, Palb, or Flv plus Palb combined for an initial 2 weeks, days 0 and 14 (1^st experiment). OD-BRE-0438 tumor-bearing mice were treated with Flv (purple, 5 mg/mouse, Q7D×2), palbociclib (green, 100 mg/kg, Q1D×14), or their combination (orange). Untreated control is shown in black. n=6 per group. Data shown are means+SEM. *p<0.05 vs. control by repeated measures Dunnett’s multiple comparisons test using log-transformed values. (C) Antitumor activity of Flv plus Palb for 2 weeks, days 14 and 28 (2^nd experiment). Mice treated with Flv plus Palb for 2 weeks were randomized into two groups (n=6 per group). One group continued treatment with Flv plus Palb (Flv+Palb→Flv+Palb, orange) and the other group received no further treatment (Flv+Palb→no treat, black). Data shown are means+SEM. (D–G) Tumor samples were collected on day 14 (untreated (control) or Flv+Palb) or on day 28 (Flv+Palb→Flv+Palb or Flv+Palb→no treat). Representative immunohistochemical images of estrogen receptor (ER) (D) and E-cadherin (F) expression, and quantification of ER in tumor cells (E) and E-cadherin on cell membranes of tumor cells (G). ER expression in tumor cells (E) or E-cadherin expression on membranes of tumor cells (G) was classified into levels that were high (3+), moderate (2+), low (1+) or negative (0+) and H-scores were calculated. n=6 per group. Data shown are means±SEM. *p<0.05 vs. Control by Dunnett’s multiple comparisons test. ^#p<0.05 vs. Flv+Palb by Dunnett’s multiple comparisons test.