This study represents a retrospective subset analysis of available tissues from two prominent and different clinical trials: NSABP B-14, a treatment trial to test the benefit of adjuvant tamoxifen, and NSABP P-1, a prevention trial to test the ability of tamoxifen in preventing breast cancers. Data from the B-14 trial showed that both ER protein and ESR1 RNA levels had a highly significant interaction with tamoxifen and that the maximum benefit from tamoxifen was achieved in the highest tertile of ESR1 mRNA expression levels.
Data from the P-1 trial validated this relationship between ER and tamoxifen. ER protein and ESR1 RNA levels and whole-transcriptome expression analysis of P-1 tumors confirmed the observation that low levels of ER were associated with tamoxifen resistance. ER protein expression was lower in ER-positive tumors that arose in the tamoxifen arm than those in the placebo arm (P < .001). ESR1 expression was underexpressed by 2.5-fold in tumors that arose in the tamoxifen arm compared with tumors that arose in the placebo arm (P = .0005). Expression profiling of the entire human genome (more than 44,000 probes on Agilent arrays) identified ESR1 as the most significantly differentially expressed gene between the placebo and tamoxifen treated tumors. These two different trials with both protein and RNA analyses both indicated that low ER levels were associated with the tamoxifen resistance.
The limitations of subset analysis have been detailed elsewhere and are always a concern in interpretation of data. However, the confirmation of B-14 results by P-1 results supports the conclusion that low levels of ER are at least in part responsible for tamoxifen resistance in breast cancer.
ER is used as a dichotomous variable in clinical decision making. This practice has been appropriate, because the examination of B-14 data never demonstrated a statistically significant interaction between ER protein levels (measured by LBA) and the degree of benefit from tamoxifen,4
and because the current generation of IHC assays is essentially bimodal in distribution as a result of saturation of the assay at relatively low levels of ER expression.14
However, examination of the NSABP B-14 trial with a quantitative assay method using Oncotype
DX demonstrated a clear linear relationship between ESR1
mRNA and the degree of benefit from tamoxifen; patients with lower levels of ESR1
mRNA did not gain significant benefit from adjuvant tamoxifen, even though their tumors were classified as ER positive by the dichotomous clinical definition.9
This finding has important implications, because it suggests that lower expression levels of ESR1
mRNA may be one of the mechanisms responsible for tamoxifen resistance. Cancer events from the P-1 trial provided a unique cohort in which to test this hypothesis developed from B-14, because ER-positive tumors arising in the tamoxifen arm are, by definition, tamoxifen resistant. Microarray gene expression profiling was used to examine differentially expressed genes between the ER-positive tumors that arose in the tamoxifen arm compared with the placebo arm, and an a priori hypothesis stated that ESR1
would be the major differentially expressed gene. The result is in agreement with that hypothesis. The only gene that was significantly differentially expressed between the two cohorts by more than two-fold was ESR1
(2.47-fold). Another gene, GFRA1
, which is associated with the ER-positive phenotype, was also distinctly differentially expressed, although only by 1.79 fold.13
Tamoxifen was more effective in preventing ER-positive tumors with higher levels of ESR1
mRNA but was not effective in preventing those with lower levels. The absence of other significant differentially expressed genes suggests that low ESR1
mRNA levels are associated with tamoxifen resistance in many of these tumors, but other mechanisms are possible.
Because tamoxifen primarily prevented tumors with high ESR1
mRNA, an interpretation could be made that breast cancer chemoprevention with tamoxifen is clinically meaningless, because it only prevents tumors with good prognoses. However, ESR1
itself was not prognostic in untreated patients (Data Supplement), and there was only modest correlation between ESR1
and the 21-gene recurrence score in such a way that even high ESR1
tumors can have a high recurrence scores (Data Supplement).15
Therefore, one could have a tumor high in ESR1
and still have a poor prognosis. Furthermore, prevention of even good-prognosis breast cancer is a desirable and meaningful outcome.
Because tamoxifen is less effective in preventing the occurrence and relapse of ER-positive tumors with low levels of ESR1
expression, it is important to develop strategies to treat and prevent such tumors. A report by the Trans-ATAC (ie, Arimidex, tamoxifen, alone or in combination trial) investigators16
provided an important perspective on this question. In that study, benefit from anastrazole was independent of the quantile levels of ER measured by IHC or ESR1
mRNA. Therefore, it is possible that aromatase inhibitors could be better preventive agents than tamoxifen by inhibiting the occurrence of even low-ER tumors.
has been implicated as an instigator of tamoxifen resistance in some studies.17–19
However, in our two study cohorts, HER2
gene amplification or expression levels did not directly predict tamoxifen resistance; as reported previously,20
there were generally fewer instances of high ESR1
expression among the HER2
-amplified tumors in B-14 (data not shown).
In summary, these data suggest that the low-level expression of ESR1 is associated with tamoxifen resistance in ER-positive breast cancer. Strategies should be developed to identify, treat, and prevent such tumors.