Abstract

Purpose:

Preclinical data suggest that antiprogestins inhibit the growth of luminal breast carcinomas that express higher levels of progesterone receptor isoform A (PRA) than isoform B (PRB). Thus, we designed a presurgical window of opportunity trial to determine the therapeutic effects of mifepristone in patients with breast cancer, based on their high PRA/PRB isoform ratio (MIPRA; NCT02651844).

Patients and Methods:

Twenty patients with luminal breast carcinomas with PRA/PRB > 1.5 (determined by Western blots), and PR ≥ 50%, naïve from previous treatment, were included for mifepristone treatment (200 mg/day orally; 14 days). Core needle biopsies and surgical samples were formalin fixed for IHC studies, while others were snap-frozen to perform RNA sequencing (RNA-seq), proteomics, and/or Western blot studies. Plasma mifepristone levels were determined using mass spectrometry. The primary endpoint was the comparison of Ki67 expression pretreatment and posttreatment.

Results:

A 49.62% decrease in Ki67 staining was observed in all surgical specimens compared with baseline (P = 0.0003). Using the prespecified response parameter (30% relative reduction), we identified 14 of 20 responders. Mifepristone induced an increase in tumor-infiltrating lymphocytes; a decrease in hormone receptor and pSer118ER expression; and an increase in calregulin, p21, p15, and activated caspase 3 expression. RNA-seq and proteomic studies identified downregulated pathways related to cell proliferation and upregulated pathways related to immune bioprocesses and extracellular matrix remodeling.

Conclusions:

Our results support the use of mifepristone in patients with luminal breast cancer with high PRA/PRB ratios. The combined effects of mifepristone and estrogen receptor modulators warrant clinical evaluation to improve endocrine treatment responsiveness in these patients.

Discussion

MIPRA was the first clinical trial in which patients were categorized according to their PR isoform ratio. In this single-arm study, we demonstrated that mifepristone inhibited the proliferation of breast carcinomas with higher levels of PRA than of PRB. The study met the primary endpoint of a 30% reduction in Ki67 between CNB and surgery. Morphologic evaluation, transcriptomic, and proteomic studies support the therapeutic effect of mifepristone, and suggest that responses may be underestimated with the unique measurement of Ki67. Although transcriptomic studies have been performed to reinforce Ki67 data in different breast cancer WOT studies (28), to the best of our knowledge, this is the first study in which paired samples were characterized by proteomics.

Originally, we estimated that we had to evaluate 100 patients to reach 20 who met the inclusion criteria. However, we recruited 140 patients because, in some cases, the biopsy failed to have sufficient cancer cells, and the sensitivity of WB was not sufficient to obtain a reliable result.

Following the design of a similar study (29), we did not include a placebo group to compare the intrinsic variation in Ki67 expression between CNB and surgical samples. In trials in which control patients have been included, some authors reported a slight increase in Ki67 expression (30–33), while others reported a decrease between the surgical sample and the CNB that never exceeded 20% (12, 34), which is below the decrease obtained in this study (49%). Ki67 evaluation, as a primary endpoint, has become the gold standard in WOT studies (35–37). Our results are similar to those originally reported for tamoxifen treatment in patients with ER⁺ breast cancer (30, 38). In line with other studies, such as the IMPACT (39), POETIC (40), and neoMONARCH (28) trials, MIPRA patients received mifepristone for only 2 weeks to avoid surgery delays.

Although a decrease in tumor size determined by ultrasound was not expected, it was achieved in several cases, even considering that the first ultrasound measurement was registered 49 days (median) before the surgical sample at the time of CNB. Considering that luminal tumors may increase in size by 0.17%–0.21% per day (41), the real decrease in size might be even higher than the one recorded.

Six of the 20 tumors did not meet the prespecified criteria for treatment response. As Ki67 evaluation was the primary endpoint, we named these tumors as unresponsive. Three of these patients had very low Ki67 levels on CNB, suggesting that a possible therapeutic effect might have been masked. Moreover, RNA-seq analysis performed using four of six unresponsive tumors showed the activation of pathways similar to those in responsive tumors. GSVA showed a clear inhibitory effect for patients M090 and M073 and less sharp suppression for M094, whereas M105 had a different response. Several morphologic signs of drug response were also observed in three of six unresponsive cases, and the analysis was performed blinded to the Ki67 data.

RNA-seq and MS studies identified downregulated pathways related to cell proliferation, and although the individual MKI67 mRNA (Ki67 gene) or protein did not appear to be one of the deregulated candidates, proliferating cell nuclear antigen and MCM2–7 proteins, which are also considered surrogate markers of cell proliferation, were downregulated. This highlights the importance of simultaneously evaluating several biomarkers to improve the data accuracy. As Ki67 and RNA-seq/proteomic data originated from different CNB or surgical samples, inclusion of both analyses increased the robustness of our data.

Common enriched pathways found in both proteomic and RNA-seq studies were those related to the innate immune system and those related to cell-matrix organization. This agrees with the increase in TILs observed in most mifepristone-treated tumors and with recent preclinical findings showing that mifepristone may prime PRA-H tumors for a second treatment with an immune checkpoint inhibitor (26), and with those from Werner and colleagues, who suggested the use of antiprogestins to increase immune infiltrates in tumors by targeting PR (42). Proteins related to PD1 signaling were found in the Cyt extracts of mifepristone-treated tumors. Calreticulin/calregulin, a protein related to immunogenic cell death (43), was highly expressed in the cell membranes of several mifepristone-treated samples (IHC assays). Similar immune signatures were observed in trials using CDK4/6 inhibitors (44). However, it may be argued that the CNB procedure elicits an inflammatory effect. In the NeoMONARCH study, CNB and surgical samples were collected after 14 days of single or combined treatment, and an increase in immune-related pathways was only observed in the combined treatment group, ruling out the possible assumption (45).

Apoptosis has not been identified as a hierarchical pathway involved in the success of endocrine therapies, showing only mild increases after tamoxifen or fulvestrant treatment (46). However, in preclinical studies in which mifepristone induced almost complete tumor regression involving differentiation and/or tissue remodeling, a significant increase in apoptosis was observed after 24 or 48 hours of mifepristone administration (47). Thus, we expected to find an increase in apoptotic cells in mifepristone-treated tumors. In the MIPRA trial, a modest increase in apoptosis was observed morphologically, as confirmed by IHC and proteomic studies, in which an increase in several proapoptotic proteins was observed.

In our study, we also included patients with advanced stages that were naïve to any other treatment for this cancer, which makes this study unique compared with other trials using mifepristone, in which patients who failed to respond to other treatments were included. Romieu and colleagues (8) and Klijn and colleagues (6) enrolled patients with tamoxifen resistance. Perrault and colleagues identified PR⁺ patients who received no other treatment for recurrence but were previously treated for their primary tumors (7). In preclinical models, most endocrine-resistant tumors are PRB-H (3), which may partially explain the poor responses observed in the aforementioned clinical trials.

Different reasons may explain why some patients in our cohort were unresponsive to mifepristone, and they should be considered in future studies. Two of the unresponsive tumors, M026 and M105, and the responsive M140 tumor were classified as PRA-H based on the WB of the Nuc extract; however, in the three tumors, the Cyt fraction was PRB-H. It may be suggested that tumors in which both the Nuc and Cyt fractions are PRA-H may represent the best candidates for mifepristone treatment, as they truly represent the total protein ratio. Tumor M140 showed good responsiveness in the Ki67 assay, a decrease in tumor size, an increase in the immune bioprocess pathways and apoptosis, but unexpectedly, an increase in proliferative pathways. This may be because the frozen CNB sample also contained adjacent nontumor mammary cells, which may have masked the mifepristone-induced effects.

Among Ki67 responsive patients, a HER2⁺ patient (CNB evaluation) was cataloged as negative in the surgical sample by the Hospital Pathology Department. We also found a decrease in membrane staining for HER2 (IHC), and HER2/ERBB2 was one of the downregulated proteins reported in the Cyt extracts (proteomics). Similar observations were made by Lee and colleagues, who used TLP to show a decrease in HER2-related genes in TLP-treated tumors. Moreover, they proposed that HER2⁺ luminal tumors might respond best to antiprogestin therapy (12).

IHC for p53 indirectly detects p53 mutations (48). The only tumor that was positive for p53 was sensitive to mifepristone, as was the case with T47D xenografts that have a pathogenic p53 mutation (49) and respond to mifepristone treatment (50). RNA-seq analysis allowed us to investigate possible mutations in the eight tumors studied. It is worth mentioning the missense mutation in ESR1, which, as reported previously, is associated with endocrine resistance (27). As this patient was responsive to mifepristone, the effect of mifepristone treatment in patients with activated ESR1 mutations deserves further investigation.

The decrease in ER observed by IHC was consistent with the possible displacement of ER from the nuclear compartment to the cytoplasm, as observed in proteomic studies. ER is usually phosphorylated at Ser118 in response to MAPK activation or estradiol binding, whereas Ser167 is phosphorylated by Akt, RSK, and casein kinase II in addition to MAPK (51, 52). As we only observed a decrease in pSer118ER expression following mifepristone treatment, it can be speculated that mifepristone treatment compromises MAPK-mediated ER signaling.

As mentioned previously, mifepristone, in addition to its antiprogestin action, exerts antiglucocorticoid effects (53). Taking advantage of the immunomodulatory effects of mifepristone, an ongoing clinical trial intends to exploit this property therapeutically using high mifepristone doses in patients with breast cancer (NCT03225547). Using experimental PRA-H tumor models, we showed that mifepristone inhibits tumor growth even when tumors are transplanted into immunosuppressed mice (26). Moreover, in vitro, mifepristone exerts antiproliferative effects only in PRA-H+ cells (16), suggesting that the direct antiprogestin effect of mifepristone is the prevailing effect, without ruling out a contribution from its antiglucocorticoid or its antiandrogenic effects (reviewed in ref. 15).

In summary, our clinical study showed that preclinical findings are valid in patients with breast cancer, suggesting that mifepristone can be used for the treatment of luminal PRA-H breast cancer. Further studies are needed to evaluate the effects of combined treatment with antiprogestins and tamoxifen in PRA-H patients. Preclinical studies have suggested that a stronger response can be obtained with this combination (54). Aromatase inhibitors or ER degraders may not work together with mifepristone, because they inhibit PR expression. Because we have already shown that lymph node metastases maintain the same PR isoform ratio as the primary tumor (15), it may be speculated that this treatment might prove suitable in an adjuvant setting, or alternatively, in a neoadjuvant setting, if further immunotherapy is suggested. Ongoing studies are currently evaluating the combination of mifepristone and CDK4/6 inhibitors in preclinical PRA-H models. Finally, because WB does not seem to be an ideal method for discriminating PRA-H patients in hospital facilities, companion diagnostic tools to improve screening should be developed.

Strengths

Originality, proof of concept after years of preclinical research, primary endpoint met, combination of approaches: standard Ki67 supported by exhaustive morphologic evaluation, IHC validation, WB, transcriptomics, and proteomics. Most secondary outcomes were met, including studies based on frozen samples and studies using different formalin-fixed samples. The evaluation of mifepristone in plasma guarantees treatment compliance.

Limitations

This study had a small number of patients, no placebo group, limited material from CNB for further analysis, and a short treatment period.