Biological effects of fulvestrant on estrogen receptor positive human breast cancer: short, medium and long-term effects based on sequential biopsies

Abstract

We report the first study of the biological effect of fulvestrant on ER positive clinical breast cancer using sequential biopsies through to progression. Thirty-two locally/systemically advanced breast cancers treated with first-line fulvestrant (250 mg/month) were biopsied at therapy initiation, 6 weeks, 6 months and progression and immunohistochemically-analyzed for Ki67, ER, EGFR and HER2 expression/signaling activity. This series showed good fulvestrant responses (duration of response [DoR] = 25.8 months; clinical benefit = 81%). Ki67 fell (p < 0.001) in 79% of tumours by 6 months and lower Ki67 at all preprogression time-points predicted for longer DoR. ER and PR significantly decreased in all tumours by 6 months (p < 0.001), with some declines in ER (serine 118) phosphorylation and Bcl-2 (p = 0.007). There were modest HER2 increases (p = 0.034, 29% tumours) and loss of any detectable EGFR phosphorylation (p = 0.024, 50% tumours) and MAP kinase (ERK1/2) phosphorylation (p = 0.019, 65% tumours) by 6 months. While ER remained low, there was some recovery of Ki67, Bcl-2 and (weakly) EGFR/MAPK activity in 45-67% patients at progression. Fulvestrant's anti-proliferative impact is related to DoR, but while commonly downregulating ER and indicators of its signaling and depleting EGFR/MAPK signaling in some patients, additional elements must determine response duration. Residual ER at fulvestrant relapse explains reported sensitivity to further endocrine therapies. Occasional modest treatment-induced HER2 and weakly detectable EGFR/HER2/MAPK signaling at relapse suggests targeting of such activity might have value alongside fulvestrant in some patients. However, unknown pathways must drive relapse in most. Ki67 has biomarker potential to predict fulvestrant outcome and as a quantitative measure of response.

Keywords: biology; breast cancer; endocrine resistance; fulvestrant.

Figure 1

Box and whisker plots displaying marker profile (unmatched data) across T1 (pre‐treatment) and T2, T3 and T4 (6 week, 6 month and at progression on fulvestrant, respectively) for the All‐patient cohort. (a) ER H Score; (b) PR H Score; (c) % Bcl‐2; (d) pER (serine 118 phosphorylation) H Score; (e) HercepTest™ score; (f) HER2m (membrane) and HER2c (cytoplasmic) H Scores; (g) pHER2m (membrane) and pHER2c (cytoplasmic) H Scores; (h) EGFRm (membrane) and EGFRc (cytoplasmic) H Scores; (i) pEGFRm (membrane) and pEGFRc (cytoplasmic) H Scores; (j) pMAPK H Score and (k) % Ki67 staining (o = outliers and *= extreme outliers with values more than 3× the box height).

Figure 2

Line plots of individual changes in markers (matched data) across T1 (pretreatment) and T2, T3 and T4 (6 week, 6 month and at progression on fulvestrant respectively) for each patient. Wilcoxon Paired Signed‐Rank test compared staining between time‐points using paired sample data from each patient. Significance between T1‐T2, T1‐T3, T2‐T3, T1‐T4 or T3‐T4 is indicated by , , , and , respectively. To aid visualisation of profile over the multiple time‐points in these plots, marker data have been connected in the same patient if any sample was unavailable. (a) ER H Score (T1‐T2, T1‐T3, T2‐T3 falls p < 0.001; T1‐T4 fall p = 0.001); (b) PR H Score (T1‐T3 p < 0.001, T2‐T3 falls p = 0.006; T1‐T4 fall p = 0.012); (c) Bcl‐2% positivity (T1‐T3 fall p = 0.007; T3‐T4 increase p = 0.066); (d) pER (serine 118 phosphorylation) H Score; (e) HER2m (membrane) and HER2c (cytoplasmic) H Scores; (f) pHER2m (membrane) and pHER2c (cytoplasmic) H Scores (NB. no measurable staining was detected in any sample from 20 patients for HER2c and 16 patients for pHER2c); (g) EGFRm (membrane) and EGFRc (cytoplasmic) H Scores; (h) pEGFRm (membrane; T1‐T3 fall p = 0.024; T3‐T4 increase p = 0.012) and pEGFRc H Scores (cytoplasmic; T3‐T4 increase p = 0.041); (i) pMAPK H Score (T1‐T3 fall p = 0.019); (j) Ki67% positivity (T1‐T2 p = 0.001, T1‐T3 p = 0.012, T2‐T3 falls p = 0.048; T1‐T4 fall p = 0.028); (k) Tumour cellularity % (T1‐T2 fall p = 0.019; T1‐T3 fall p = 0.003; T3‐T4 increase p = 0.065).

Figure 3

Kaplan–Meier association (using the median staining level from Supporting Information Table S2 at each time point as a cut‐point) between biomarkers and duration of response (DoR) to fulvestrant (*p < 0.05 using Log Rank test). Responses to any other treatments following fulvestrant progression were not a component of these response data. (a) PR H Score at T2; p = 0.008 for higher PR [mean DoR= 48.2 months (95% CI=36.9–59.5; n = 14)] vs. lower PR [mean DoR= 23.4 months (95% CI= 11.6–35.1; n = 14)]; (b) Bcl‐2% at T2; p = 0.01 for higher Bcl‐2% [mean DoR= 48.4 months (95% CI=38.5–58.4; n = 13)] vs. lower Bcl‐2% [mean DoR= 27.5 months (95% CI= 15.3–39.6; n = 14)]; (c) pHER2c (cytoplasmic) H Score at T1; p = 0.018 for any detectable pHER2c [mean DoR= 16.8 months (95% CI= 10.1–23.5); n = 9] versus no pHER2c [mean DoR= 39.1 months (95% CI= 28.5–49.7; n = 22)]; (d) pEGFRm (membrane) H Score at T2; p = 0.007 for any detectable pEGFRm [mean DoR= 21.1 months (95% CI= 12.5–29.7; n = 11)] vs. no pEGFRm [mean DoR= 48.1 months (95% CI= 36.0–60.3; n = 16)]; (e) Ki67% at T1; p = 0.01 for higher Ki67 [mean DoR= 22.2 months (95% CI= 13.2–31.3; n = 15)] vs. lower Ki67 [mean DoR= 43.1 months (95% CI= 30.6–55.5; n = 16)]; (f) Ki67% at T2; p = 0.027 for higher Ki67 [mean DoR= 24.7 months (95% CI= 11.6–37.9; n = 12)] vs. lower Ki67 [mean DoR= 47.1 months (95% CI= 36.2–58.0; n = 15)].