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Background: About 50% of ER positive (ERpos) BCs are resistant to hormone treatment. In absence of ligand, ERs are evenly distributed in nuclei in normal tissue. Upon ligand binding, ERs dimerizes and form a discrete focal subnuclear distribution pattern (FDP), which is associated with transcriptional activation of ER and can be visualized with high powered microscopy. We have developed an IHC method to characterize the FDP in archival BC specimens (ASCO 2013 abst#592). We hypothesized that, in BC, the presence/absence of FDP of ER could predict anti-estrogen (anti-E) activity. We could determine two tumor phenotypes for ERpos tumors: a diffuse nuclear ER staining or "D-ER" corresponding to the expression of non-functional ER, which is the pattern observed in vitro or in vivo when no ligand are bound to steroid receptors (SR); D-ER thus is thought to predict lack of treatment effect of anti-E. And an aggregated nuclear pattern which corresponds to a similar pattern observed in vitro or in vivo when ligand is bound to ER; A-ER would suggest that ER is activated and a potential target to anti-Es. Methods: A previously reported study (ASCO 2013 abst #592) was expanded from 254 evaluable cases to 755 with paraffin embedded formalin fixed (PEFF) BC specimens with clinical and pathology data. Specimens were analyzed for standard HES, ER, progesterone receptor (PR) and Ki67. The A-ER and D-ER nuclear patterns were analyzed at 1000x magnification. Results: Mean age; 57 (17 -89). Histology: ductal 85% lobular 13%, other 2%; 82% ERpos and 78% either PRApos or PRBpos, 10% ERpos and 6% PRpos only. 92% of ERpos cases had received anti-Es; Adj. Chemotherapy 36%, Stage: I 47%, II 45%, III 8 %. Grade: I 25%, II 52%, III 23%. Median follow up 42 months. ER status was D-ER in 71% and A-ER in 29% of the specimens. With DFS defined as time to PD or death (5 year cut off), 125/755 events were observed. ERpos was better that ERneg (HR= 0.36, p = 0.00001). Within ERpos tumors group, in univariate analysis, a time-dependent Cox model showed that A-ER pattern was associated with better DFS vs D-ER pattern (HR = 0.03, p=0.02, time interaction = 0.01). A-ER was not correlated with SBR Grade, and was associated with its anisonucleosis (Aniso) index (0.02), with histology (ductal, 0.005) but not age, stage or HER2 status. Ki67 testing is ongoing. In monovariate analysis stage (0.00004), grade (p < 10-6), PR (p < 10-6) were prognostic on DFS, but not histology and HER2. In a time-dependent multivariate Cox model, A-ER remained an independent predictor (p = 0.013), with grade (p =0.001 with Mitotic Index 0.002, Differentiation 0.023, Aniso NS), stage 0.001, time interaction 0.009), PR and HER2 NS. Conclusions: This study supports the hypothesis that anti-Es are mainly active in BC with A-ER pattern, which is targetable by anti-Es. Independent statistical significance was reached after adjusting for well-established prognostic factors. Given the 10-year hormonal treatment adjuvant recommendation guidelines, a better assay than the simple ER status determination would have important implications in BC management. Citation Format: Erard Gilles, Jacques Bosq, Charline Alleaume, Alexander Zukiwski, Emile Hutt, Jacques Bonneterre. Activated form of the estrogen receptor α (ER) in breast cancer (BC) and its correlation with prognosis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-19.

Background: Onapristone is a type I PR antagonist, which prevents PR-induced DNA transcription; its anti-cancer activity is well-documented. The reported T1/2 is 2-4h, so an extended-release (ER) tablet was designed to mitigate Cmax spikes, which may be involved in the liver function test (LFT) elevations seen with an immediate-release (IR) onapristone. Materials and methods: The expansion cohort of this multi-center, open-label, randomized, parallel-group, 2-stage Ph1 study (NCT02052128) is ongoing in endometrioid cancers. Female pts ≥18 years with PR-expressing tumors (including endometrial, ovarian, breast) were eligible. The primary endpoint was to recommend a Ph2 dose of ER onapristone (RP2D), with a 57-day DLT observation period; secondary endpoints include: safety, efficacy, and real-time PK. Pts received onapristone ER 10, 20, 30, 40 or 50 mg BID, or IR tablets 100 mg QD until progressive disease or intolerability in Stage 1. Results: 52 pts are enrolled (by 2 December 2014). Validated PK data are available for 35 pts. Onapristone AUC and Cmax are dose-proportional across all dose levels including 100mg IR (Table 1), with coefficients of determination (r2) of 0.76 and 0.79, respectively. The bioavailability of onapristone ER vs IR is high. Steady state is consistently attained at approximately 8 days (200 h), with T1/2 at 8-12 h, longer than previously published, with no evidence of onapristone accumulation through day 57. Table 1.Onapristone PK parameters following first doseOnapristone formERERERERERIRDose (mg)10 bid20 bid30 bid40 bid50 bid100 qdn666566Mean AUC (μg/L*h)51761765015450174403161067980CV%47.7202.722.943.070.555.1Min348029411200038091515033330Median4654860815040207302593062270Max952440000214302667062500125000Mean Cmax (μg/L)226.8676.3767.3641.614594296CV%46.0140.115.693.646.862.8Min922206762914591556Median218403732.547414953726Max4031466988134424927507 Conclusions: This study reveals a longer than anticipated onapristone T1/2, which nevertheless supports using an ER formulation. AUC and Cmax data are dose proportional and allow dosing flexibility. Protracted exposure minimizing Cmax spikes is best achieved with a twice-daily, ER formulation. There were no LFT elevations in the absence of liver metastases in 30 patients exposed to the ER formulation. The RP2D is 50 mg bid based on safety and PK, to be further explored in the endometrioid cohort. Citation Format: Francois Lokiec, Antoine Italiano, Andrea Varga, Jacques Bonneterre, Mario Campone, Alexandra Leary, Keyvan Rezai, Marie-Paule Sablin, Alice Bexon, Stefan Proniuk, Erard Gilles, Joseph Bisaha, Alexander Zukiwski, Paul Cottu. Onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers: PK results from part 1 of a randomized, parallel-dose phase 1 study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2015-4512

Background: Onapristone is a type I PR antagonist, which prevents PR-induced DNA transcription. Onapristone anti-cancer activity is well documented. An extended-release (ER) tablet formulation of onapristone was designed to address the liver function test (LFT) elevations seen with immediate-release (IR) onapristone. A phase 1 study with onapristone in patients with tumors expressing PR is underway. Objectives included determining the PK profile of ER onapristone using a PPK approach. Materials and methods: This is an ongoing multi-center, open-label, randomized, parallel-group, 2-stage ph1 study. Female pts ≥18 yrs with tumors expressing PR are eligible. The Stage 1 primary endpoint is the recommended ph2 dose of ER onapristone; secondary endpoints include: safety, efficacy, and PK. Pts received onapristone ER 10, 20, 30, 40 or 50 mg BID, or onapristone IR tablets 100 mg QD until progressive disease or intolerability. PK blood samples from 8 time points were collected over 12 h post-dose Day 1 for the ER and 9 blood samples over 24 h post-dose for the IR formulation. Onapristone plasma concentrations were measured using validated UPLC with tandem mass spectrometry detection (range 1-250 ng/mL). Monolix V4.1 was used to calculate absorption constant (Ka); apparent clearance (CL/F); inter-compartmental clearance (Q); apparent distribution volume (V1/F), 2nd compartment distribution volume (V2) and bioavailability (F) of ER vs IR. Results: Stage 1 is complete. 42 pts have validated PK data. A 2-compartment open model adequately described the total onapristone time-concentration curve with linear elimination. Results are in Table 1. Table 1.Estimated PK parameters for onapristone in pts with PR-expressing cancers (n = 42)ParameterValueRelative standard error (%)Ka0.191 h-114CL/F1.51 L/h20Q3.11 L/h25V1/F5.41 L25V241.1 L45F60%20 Conclusions: The PPK modeling described the plasma onapristone time-concentration curves well. A central volume equivalent to the circulating blood volume and a large volume of the deep compartment suggest a large tissue diffusion. PPK/PD modeling to explore safety and efficacy is ongoing, with no overt PK/safety relationship detected. Citation Format: Keyvan Rezai, Paul Cottu, Samuel Huguet, Mario Campone, Antoine Italiano, Andrea Varga, Jacques Bonneterre, Alexandra Leary, Marie-Paule Sablin, Stefan Proniuk, Alice Bexon, Erard Gilles, Joseph Bisaha, Alexander Zukiwski, Francois Lokiec. Population pharmacokinetic (PPK) modeling of onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2015-4523

Background: In absence of ligand, PRA and PRB are evenly distributed in nuclei in cell lines. Upon ligand binding, PRA and PRB dimerizes and form discrete focal subnuclear distribution patterns, which are associated with transcriptional activation of PR. This pattern corresponds to the transcriptionally active form of PR and serves as an APR biomarker. The expression of APR in cell lines was demonstrated to be predictive of onapristone antiproliferative effects (Serin, Abs # 473, NCI-AACR-EORTC 2012). In this study the correlation of APR to the antiproliferative effects of different antiprogestins (Type I & type II, and steroidal/non-steroidal chemical structures) is examined. Method: T47D and CAMA-1, two PR expressing breast cancer cell lines, were grown in either FBS or Steroid Free FBS (SFFBS). In FBS, single agent anti-progestins were studied; in SFFBS, cell lines were stimulated with estradiol (E2) or progesterone (P4) and antiprogestins tested. All experiments were performed in duplicate. APR was analyzed at 6h and 30h using paraffin embedded cellular pellets, and processed with standard IHC techniques. Cellular viability was measured by the MTS assay at 30h, 4d and 7d. Antiprogestin drugs tested included: Aglepristone, Mifepristone, Onapristone, PF02413873, ZK230211, and ZM150271. Results: In the T47D cell line, in FBS between zero and ∼ 40% antiproliferative activity was observed from D1 to D7 for all antiprogestins. In SFFBS, T47D proliferation at D7 was increased >300% by E2 and >200% by P4. All antiprogestins opposed P4 and E2 proliferative effects at D7, with a max of 80% inhibition relative to control. In the CAMA-1 cell line, in FBS weak antiprogestin antiproliferative effect was observed ( Citation Format: Alexander Zukiwski, Erard Gilles, Guillaume Serin, Jacques Bosq, Charline Alleaume. Comparative assessment of in vitro activity and aactivated progesterone receptor (APR) biomarker predictivity for multiple antiprogestins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3471. doi:10.1158/1538-7445.AM2015-3471

Background: Given the poor therapeutic outcomes for triple negative breast cancer (TNBC), diagnostic accuracy is vital. In routine IHC testing, the progesterone receptor (PR) is determined using bispecific antibodies (Ab) that recognize epitopes common to PRA and PRB. PRA and PRB expression can be imbalanced in BC. Relative expression of the PR isotypes appears to be prognostic in BC evaluated with a bispecific Ab (Hopp 2004). Tumors can express PRA or PRB on different cells in the same tumor (Mote 2008), which differs from those expressing ERα (Zukiwski 2013). A true TN phenotype might escape detection with the use of one single Ab to detect both PRA and B epitopes, depending on sensitivity/specificity. This study evaluated the use of two isotype-specific PR Abs to fully characterize the PR status. Methods: 83 dual ERα and HER2 negative archived BC specimens with clinical data were obtained from the Oscar Lambret Cancer Center, Lille, FR. IHC was performed using anti-PRA, anti-PRB, and the bispecific anti-PR Pg636 antibodies. PR tumor positivity was explored using 2 cut-offs, ≥ 1% or ≥ 5% stained tumor cells. PR positive tumors were defined as either PRA or PRB positive. Results: For PR positive tumors with ≥ 1% positive cells, average PRA positivity was 41%, PRB was 38% (PRA vs PRB p = NS), and PRAB 3% (PRA vs PRAB = 0.001, PRB vs PR AB p = 0.0001). Using 1% as positivity cut off PRA and PRB were discordant in 14% of the cases, PRA and PR AB in 12%. PR B and PR AB were discordant in 2%. Discordance between PR positivity (either A or B) and PRAB positivity was 7% with no PR negative PRAB positive tumors i.e. no positivity was missed using two PR A and B antibodies while all 7% cases were missed by the PRAB antibody. Using 5% as a cut off, the discordance rate was 8% between PRA and PRB, 25% between PRA and PRAB and no PRA negative and PRAB positive case were found, and 26% between PRB and PRAB with no PRB negative PRAB positive cases either. PR positivity (A or B) was missed in 30% of the cases with a PRAB. Patients with tumors identified as PR positive (≥ 5%) using the isotype specific antibodies and PR negative with the PRAB antibody have a better prognosis (DFS). Conclusion: In TNBC, there is a different staining pattern when using isotype-specific vs bispecific anti-PR anti-bodies. The average percent of positive tumors cells is substantially reduced when using a bispecific Ab as compared to isotype-specific AB. This translates into potential false negative PRAB testing which varies from 7% to 30% depending of the cut off criteria. TNBC reclassified by the use of isotype-specific anti-PR antibodies may be appropriate for investigation with anti-progestins. Citation Format: Jacques Bonneterre, Jacques Bosq, Charline Alleaume, Erard Gilles, Philippe Jamme, Alexander Zukiwski. Triple negative breast cancer, the impact of isotype-specific progesterone receptor antibodies on the diagnosis results [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-02-13.

Background: Onapristone is a type I anti-progestin, which prevents the progesterone receptor (PR) monomers from dimerizing, inhibits ligand-induced phosphorylation and prevents association of the PR with its co-activators, thus preventing PR-induced transcription. Onapristone has the potential to treat patients with endometrial cancer, breast cancer, uterine sarcomas, a potential subset of ovarian cancer, prostate cancer and other tumors in which the progesterone receptor plays a role in growth, proliferation and metastasis. [11C]-radiolabeled onapristone and its visualization via PET-scan, coupled with pharmacokinetic (PK) studies, has the potential to determine tissue-specific and blood PK parameters including tumor/tissue and plasma concentrations, whole body distribution and half-life of onapristone. The aim of this study is the development of a rapid Good Manufacturing Product (GMP) synthesis of parentally administered [11C]onapristone. Methods: The production of [11C]-labeled radiopharmaceuticals used a Tracerlab® FX c-Pro (GEMS) synthesis module. Carbon-11 was produced at Institut Curie-Hôpital René Huguenin via the 14N(p,α)11C nuclear reaction using a PETTrace cyclotron (GEMS) equipped with a carbon-11 target. Carbon-11 is delivered from the cyclotron as [11C]CO2 in the synthesis module, and reduced to [11C]CH4. Methane is halogenated to [11C]CH3I and converted to [11C]methyl triflate (CH3OTf). To prepare [11C]onapristone, the module was loaded with 1mg of N-desmethyl-onapristone (Arno Therapeutics) and 500µL of DMSO in the reaction vessel (Sigma®). [11C]CH3OTf was bubbled into the reaction vessel and heated at +50°C for 10 minutes. The reaction mixture was then diluted with 1 ml of mobile phase and purified using a semi-preparative HPLC column: Sunfire C18 5µm 250X10mm (Waters®), mobile phase: acetonitrile (Sigma®)/water (Waters®) 50/50 v/v; flow rate 4 mL/min. The product fraction was collected in 40mL of sterile water and was passed through a C18 SEP PAK (Waters®). This fraction was eluted with isotonic saline (Braun®) and ethanol (Sigma®). The resulting formulation was passed through a 0.22µm sterilizing filter into a sterile dose vial. Results: 10 tests were performed. Time of synthesis was 50 minutes. The amount of carbon-11 delivered was 12-14GBq for the irradiation parameters as follows: 10 min; 10µA. The decay-corrected yield of reducing [11C]CO2 to [11C]CH4 was >99%. The yields of preparing [11C]CH3I were 27-29%. Time retention of [11C]onapristone was 8.5-9.5 minutes and the non-decay-corrected radiolabelling yields were 1-2%. Conclusions: We have successfully developed a fully-automated production of [11C]onapristone ready for use in clinical trials. Citation Format: Olivier Madar, Julien Fouque, Stefan Proniuk, Keyvan Rezai, Samuel Huguet, Alexander Zukiwski, Erard M. Gilles, Alice S. Bexon, François Lokiec. Synthesis of [11C]onapristone for clinical investigation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2014-1646

Background: Given the poor therapeutic outcomes for triple negative BC, diagnostic accuracy is vital. In routine IHC, PR is measured using bispecific antibodies (Ab) that recognize epitopes common to PRA and PRB. PR A and B expression is imbalanced in BC. Relative expression of the PR isotypes appears to be prognostic in tumors evaluated with a bispecific Ab (Hopp 2004). Tumors can express PRA or PRB on different cells in the same tumor (Mote 2008), which differ from those expressing ERα (Zukiwski 2013). A true triple negative phenotype might escape detection with the use of one single Ab to detect both PR A and B epitopes, depending on its sensitivity/specificity. This study evaluated the use of two isotype-specific PR Abs to fully characterize PR status. Methods: 312 archived BC specimens with clinical data came from Oscar Lambret Cancer Center, Lille. HER2 status was previously determined. IHC was performed using anti-ERα, anti-PRA and PRB Abs. ERα and PR tumor positivity was defined as ≥ 1% stained tumor cells. Results: 276 cases had complete data. Table 1 shows ERα, PRA and PRB status. 38/276 cases (14%) were ERα and HER2 negative: 22 were PR A or B negative, 9 were PR A and B positive, 2 were positive only for PRA and 5 only for PRB. Discussion: Bispecific PR Abs rarely report PRA and PRB similarly (Mote 2001). PR Phosphorylation and activation may vary across tumors. Potentially only one PR isotype may drive BC tumor growth. Triple-negative BC status determination could be PR Ab-dependent. These cases are currently being tested with a standard bispecific PR Ab to understand which would be considered triple-negative with routine IHC. Conclusion: Further evaluation of IHC with specific PRA and PRB Abs is warranted to determine if certain patients, currently classified as having triple-negative BC, could respond to antiprogestins, which have shown activity in BC. Table 1% of276 casesPR (PRA or PRB)PRAPRBERαNegativePositiveTotalNegativePositiveTotalNegativePositiveTotalNegative148221742115722Positive77178146579106868Total217910031691002575100 Citation Format: Erard Gilles, Jacques Bosq, Laura Caplier, Alice Bexon, Alexander Zukiwski, Jacques Bonneterre. Impact of progesterone receptor (PR) isotype-specific immunohistochemistry (IHC) on the diagnosis of triple negative breast cancer (BC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5567. doi:10.1158/1538-7445.AM2014-5567

Introduction: Onapristone is an antiprogestin with demonstrated clinical activity in breast cancer, reported to have a t1/2 between 2 and 4 hours. Drugs such as megestrol acetate and abiraterone generally show variability in absorption and, depending on the formulation, food effect (≥10 x Cmax variation; ≥5 x AUC variation). A study was conducted to determine the best formulation of onapristone to minimize this variability. Methods: The aim of this study was to determine the pharmacokinetic profile of onapristone and mono-desmethyl onapristone (M1), with and without food. Using a two-period, two-sequence, random assignment cross-over design, twelve healthy female subjects were given 10 mg of an oral immediate release formulation of onapristone either after an overnight fast, or within 30 minutes after a high-fat high-calorie meal, with a 2 week washout between dosing periods. PK sampling was performed following each oral administration at: 0, 15, 30, 45 and 60 minutes, 2, 4, 6, 8, 12, 16 and 24 hours (h). Parameters were calculated using the linear trapezoidal method. Population PK modeling was conducted using the nonlinear mixed effect model. Results: Onapristone plasma t1/2 (mean ± SD) was 4.36 ±0.81 h for the fasted and 3.76 ±0.36 h for the fed state. The absorption phase appeared linear. Onapristone tmax was delayed from 1 to 4 h after food intake. A small effect on the single dose onapristone pharmacokinetic profile was also observed, with a slightly decreased mean Cmax (18%) and increased AUC0-last (11%). Mean population CL of onapristone was 5.02 ±0.67 L/h and 6.63 ±0.87 L/h for fasted and fed states respectively. There was no food effect on M1 exposure but a decrease in M1 plasma peaks (∼35%) after food intake. The onapristone time-concentration curve was adequately described by a 2-compartment open model with linear elimination using a population PK approach. A significant food effect (p Conclusion: Clinically meaningful Cmax can be reached with a 10 mg onapristone dose. The results are consistent with prior observations, indicating that food delayed absorption and increased AUC when taken concomitantly. Food effect is minimal compared to other drugs in class, but onapristone should preferably be administered 2 hours before or 1 hour after meals. This food effect is unlikely to have clinical consequences. As absorption is linear, a sustained release formulation would probably reduce Cmax by ∼25% while not substantially modifying AUC. Given the estimated t1/2 of 2-4 hours, adequate concentrations should be maintained with twice a day dosing. Citation Format: Keyvan Rezai, Stefan Proniuk, Alex Zukiwski, Erard Gilles, Didier Chassard, Caroline Denot, Haydee L. Ramos, Alice S. Bexon, François Lokiec. Pharmacokinetic (PK) food effect study of immediate-release onapristone and its primary metabolite (M1) in healthy female subjects: implications for design of a new formulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4636. doi:10.1158/1538-7445.AM2014-4636

Background: Hormonal therapy is considered one of the most effective treatment options for patients with estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancers (BC); however only a 50% objective response rate is observed as 1st line therapy of ER positive (ERpos) tumors (Bonneterre, 2001). An IHC diagnostic test to identify the activated form of the ER and PR has previous been described (ASCO 2013 abst # 592, 593 & 5602). The nuclear morphological of distribution of the ER and PR can be categorized in two patterns, a presumably not-activated D (diffuse pattern) or activated A (aggregated pattern). This classification is correlated with antiestrogen treatment outcome (ASCO 2013, abst # 592), and correlated with antiprogestin activity in vitro (AACR 2013, abst # 1317). Clinical activity of antiprogestins has been previously described (Robertson 1999, Jonat 2002). It is generally accepted that expression of PR is under the transcriptional control of ER, thus targeting ER can potentially suppresses PR expression. However, in breast cancer tissues although both ER and PR are present in the tumor specimen, there is low level of co-expression of ER/PR at the cellular level (ASCO 2013, abstract # 596). The goal of the study is to elucidate whether or not the activated form of ER and PR were coexpressed. Method: 293 ERpos or PRpos BC were obtained from the Oscar-Lambret Cancer Center, Lille, France. Each sample was analyzed with specific antibodies for ERα, PR A or PR B; a subset (53 for PR A and 49 for PR B cases) was analyzed by immunochemical duel staining on the same paraffin tumor section for ERα and PR A or PR B. Tumors were deemed receptor positive if >10% of the tumors cells (tc) were stained. Samples processed for duel staining came from the same dataset and were selected if they had both ERα and PR A and/or PR B present in more than 10% and less than 80% of stained tc. The distribution of ERα, PR A, PR B tc was described. Determination of the activated form of ERα (AERpos) and PR A (APRpos A), PR B (APRpos B) was performed using the previously described IHC technique at 100X magnification. Results: Standard receptor positivity (% of tumors tested) was as follows; ERα 85%, PR A 61% and PR B 65%, either PR A or B 68%. Duel staining receptor positivity (% of tumors) showed for the PR A subset: 72% ER pos, 74% PR A pos, and 9% ER/PR (% of tumors co-expressing); for the PR B subset: ER 60%, PR B 100%, and 13% ER/PR (% of co-expressing); Subnuclear Morphology: 216 cases were ER and PR positive, and of these, 23% were AERpos, 23% were APRpos A, 24%, APRpos B% and 31% APRpos for either A or B. In the 216 case dataset, 18% of the tumors had both AER and APR A or APR B; However, using the duel staining method no cases had more than 10% of the tc co-expressing i.e. AERpos and APRpos, indicating that the co-expression was principally at the tumor level and not at the cellular level. Conclusion: As known, BC commonly express both ER and PR; however the co-expression of ER and PR in malignant cells is uncommon, and this co-expression of ER and PR could be explained by chance. Although AER and APR are expressed within the same tumor, the activated forms of the ER and PR are not co-expressed at the cellular level. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-13.

Background: Antihormonal agents (AH) are some of the most clinically useful anticancer drugs. AH resistance and the availability of alternative treatments have made patient selection increasingly important for AH drug development. We hypothesized that the progesterone receptor (PR) may be constitutively active because of the aberrant biological pathways and that selecting patients with baseline tumor PR activation would predict for antiprogestin activity. Activated PR (APR) can be visualized in nuclei as fluorescent aggregates via fluorescent green protein engineered PR cell lines in vitro and by immunofluorescence (IF) in tissues. The detection of PR foci has been associated with transcription and gene activation in vitro. PR phosphorylation (phos) is induced by a variety of pathways. We have developed a IHC method that allows APR determination in tumor biopsies on a routine basis (SABCS 2012). The predictive nature of the observation of APR by IHC in vitro has been tested with onapristone (ONA) for cell survival and proliferation. Methods: 8 human breast cancer (BT-474, CAMA-1, EVSA-T, HCC-1954, MCF-7, MDA-MB-231, T-47D, ZR-75-1) and 2 human endometrial cancer (Ishikawa, HEC-1-A) cell lines were studied. Cytoblocks for APR analysis were made for each time point and experimental condition, baseline ER/PR expression was determined by IHC and PR phos was analyzed with specific antibodies (pSER162, 190, 294, 400, 554) by Western Blotting and IHC. Culture conditions: normal and stripped FBS, +/- ONA and growth factor/hormone exposure (EGF, FGF2, E2 and P4) in triplicate. Analysis was performed at baseline (BL), 6h, 96h and 7 days. Cell viability was assessed by MTS assay, proliferation was studied by Ki67 analysis. APR foci were determined by IHC and read by a pathologist blinded to the experimental conditions. Results: At BL only two cell lines were PR pos/APR pos (T47D, CAMA-1) and all other cell lines where PR pos/APR neg (BT-474, EVSA-T, HCC-1954, MCF-7) or PR neg/APR neg (Ishikawa, HEC-1-A, MDA-MB-231, ZR-75-1). ONA exerted inhibitory effect only in APR pos cell lines. In stripped FBS at 6h, T47D APR pos status was converted to APR neg by ONA except with E2 stimulation which required longer treatment. The CAMA-1 data are not yet available. No APR neg cell lines were converted into APR pos except in one experiment after protracted exposure to EGF. ONA generally decreased phos; phos decrease measured by WB did not correlate with growth inhibition. Phos status determined by IHC and Ki67 proliferation assessment are currently in process and will be presented with the confirmatory data. Conclusions: Baseline APR positivity is predictive of ONA activity in vitro. ONA converts APR pos cells to APR neg within 6h in most cases, a time frame consistent with PR biology. The growth inhibitory effects of ONA were not correlated to PR phos in the cell lines and conditions tested. The APR IHC methodology may be applicable in the clinical setting. Citation Format: Erard Gilles, Laura Caplier, Alexander Valent, Guillaume Serin, Anne Gompel, Jacques Bosq, Alexander Zukiwski. Antiprogestin drug development: in vitro validation of a potential clinical biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2013-1317