Search

Lessons Learned The fibrolamellar carcinoma-associated DNAJB1-PRKACA gene fusion transcript RNA codes for the catalytic domain of protein kinase A and, thus, overexpression of Aurora kinase A. ENMD-2076 showed a favorable toxicity profile. The limited results, one patient (3%) with a partial response and 57% of patients with stable disease, do not support further evaluation of ENMD-2076 as single agent. Future studies will depend on the simultaneous targeting approach of DNAJB1-PRKACA and the critical downstream components. Background Fibrolamellar carcinoma (FLC) represents approximately 0.85% of liver cancers. The associated DNAJB1-PRKACA gene fusion transcript RNA codes for the catalytic domain of protein kinase A and overexpression of Aurora kinase A (AURKA). ENMD-2076 is a selective anti-AURKA inhibitor. Methods Patients aged >12 years with pathologically confirmed incurable FLC, with measurable disease, Eastern Cooperative Oncology Group performance status 0–2 or Lansky 70–100, and adequate organ function were eligible. Patients were prescribed ENMD-2076 based on body surface area. The primary endpoint was overall objective response rate by RECIST v1.1, with a null hypothesis of true response rate of 2% versus one-sided alternative of 15%. Secondary endpoints included 6-month progression-free survival (PFS) rate (Fig. 1), median PFS, time to progression (TTP), and overall survival (OS). Safety was evaluated throughout the study. Results Of 35 patients who enrolled and received treatment, 1 (3%) had a partial response (PR) and 20 (57%) had stable disease (SD). Median TTP, PFS, and OS were 5, 3.9, and 19 months, respectively. The most frequently reported drug-related serious adverse event was hypertension in three patients. Three deaths were reported on-study—two due to disease progression and one due to pulmonary embolism not related to ENMD-2076. Conclusion The study provided no rationale for further studying ENMD-2076 as a single agent in FLC.

Objectives With no clinically effective antiviral options available, infections and fatalities associated with dengue virus (DENV) have reached an alarming level worldwide. We have designed this study to evaluate the efficacy of the celecoxib derivative AR-12 against the in vitro replication of all four DENV serotypes. Methods Each 24-well plate of Vero cells infected with all four DENV serotypes, singly, was subjected to treatments with various doses of AR-12. Following 48 h of incubation, inhibitory efficacies of AR-12 against the different DENV serotypes were evaluated by conducting a virus yield reduction assay whereby DENV RNA copy numbers present in the collected supernatant were quantified using qRT-PCR. The underlying mechanism(s) possibly involved in the compound's inhibitory activities were then investigated by performing molecular docking on several potential target human and DENV protein domains. Results The qRT-PCR data demonstrated that DENV-3 was most potently inhibited by AR-12, followed by DENV-1, DENV-2 and DENV-4. Our molecular docking findings suggested that AR-12 possibly exerted its inhibitory effects by interfering with the chaperone activities of heat shock proteins. Conclusions These results serve as vital information for the design of future studies involving in vitro mechanistic studies and animal models, aiming to decipher the potential of AR-12 as a potential therapeutic option for DENV infection.

We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junin, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc.

Introduction Onapristone is a type I progesterone receptor (PR) antagonist, which prevents PR- mediated DNA transcription. Onapristone is active in multiple preclinical models and two prior studies demonstrated promising activity in patients with breast cancer. We conducted a study of extended release (ER) Onapristone to determine a recommended dose and explore the role of transcriptionally-activated PR (APR), detected as an aggregated subnuclear distribution pattern, as a predictive biomarker. Methods An open-label, multicenter, randomized, parallel-group, phase 1 study (target n = 60; NCT02052128) included female patients ≥18 years with PRpos tumors. APR analysis was performed on archival tumor tissue. Patients were randomized to five cohorts of extended release (ER) onapristone tablets 10, 20, 30, 40 or 50 mg BID, or immediate release 100 mg QD until progressive disease or intolerability. Primary endpoint was to identify the recommended phase 2 dose. Secondary endpoints included safety, clinical benefit and pharmacokinetics. Results The phase 1 dose escalation component of the study is complete (n = 52). Tumor diagnosis included: endometrial carcinoma 12; breast cancer 20; ovarian cancer 13; other 7. Median age was 64 (36–84). No dose limiting toxicity was observed with reported liver function test elevation related only to liver metastases. The RP2D was 50 mg ER BID. Median therapy duration was 8 weeks (range 2–44), and 9 patients had clinical benefit ≥24 weeks, including 2 patients with APRpos endometrial carcinoma. Conclusion Clinical benefit with excellent tolerance was seen in heavily pretreated patients with endometrial, ovarian and breast cancer. The data support the development of Onapristone in endometrial endometrioid cancer. Onapristone should also be evaluated in ovarian and breast cancers along with APR immunohistochemistry validation.

Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR-12 has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a potential therapeutic agent for neurodegenerative disorders. In this study, we investigated the role of AR-12 and its derivatives in controlling prion infection. We tested AR-12 in prion infected neuronal and non-neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR-12 and its analogue AR-14 reduced PrPSc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR-12 or AR-14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro and in vivo. Taken together, this study demonstrates that AR-12 and the AR-14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.

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.

Objective Hormonal therapy is generally reserved for patients with endometrial cancers that fail cytotoxic chemotherapy, but there is a lack of sufficiently sensitive diagnostics to identify potential responders. We sought to develop a diagnostic technique to detect activated progesterone receptors (APR) in endometrial cancers using routine immunohistochemistry (IHC) and to correlate the presence of APR with other histopathological features and clinical disease stage. Methods Seventy-two tumor block specimens from patients with endometrial cancer were processed with conventional IHC methods for estrogen receptor-α (ERα), progesterone receptor (PR) and Ki67, a marker of proliferation. Tumor specimens were analyzed for the PR nuclear distribution patterns in individual tumor cells: APR positive (APR pos ) tumors were prospectively defined as any tumor with >5% countable malignant cells with an aggregated nuclear pattern. Tumor APR status was analyzed against other biomarkers including ERα expression, Ki67 and tumor grade. Results Fifty-six of 72 samples were endometrioid. Twenty-six of 49 PR-positive endometrioid tumors (53%; 95% CI 39–67%) were APR pos . Percent of ER pos cells correlated with % PR pos malignant cells (p=0.001, rho=0.44). APR positivity did not correlate with % PR pos cells in a given tumor, nor did it correlate with % Ki67 positivity; APR positivity was independent of disease stage and tumor grade (p=NS). Conclusions In this study, approximately half of endometrioid tumors were APR pos . APR is independent of histopathological and other known risk factors. Refining conventional PR detection has the potential to prospectively identify patients with endometrial cancer who may benefit from anti-progestin therapy.

Onapristone is an antiprogestin with activity in breast cancer and is under investigation for use in endometrial, ovarian and prostate cancers. Megestrol acetate and abiraterone generally show variability in absorption and, depending on the formulation, food effect. This study was conducted to determine the effect of food on 10 mg oral immediate-release (IR) onapristone and to help identify a formulation to minimize variability. This is an open-label, randomized, crossover study to determine the pharmacokinetic profile of onapristone and its main metabolite, N-mono-desmethyl onapristone. Twelve healthy female subjects received 10 mg of oral IR onapristone after an overnight fast, or within 30 min of a high-fat, high-calorie meal with a 2-week washout between dosing periods. Onapristone plasma t1/2 (mean ± SD) was 4.36 ± 0.81 h for the fasted state and 3.76 ± 0.36 h for the fed state. Following food, onapristone tmax was delayed from 1 to 4 h. Food intake was also associated with a small increase in AUC0–∞ of approximately 13 % and a statistically significant decrease in Cmax of approximately 18 %. One subject experienced a 23-day delay in menses after one 10 mg onapristone dose, while another subject experienced transient grade 2 NCI-CTCAE liver enzyme elevation 3 weeks post dose. The results are consistent with previous observations, indicating that there is a small increase in onapristone exposure and a significant decrease in Cmax when taken with food. These changes are within acceptable limits set out by the FDA. Thus, our findings indicate that onapristone could be administered without regard to food.

5071 Background: An urgent need exists for new therapies after progression (PD) onAA and enzalutamide (ENZ). Increased PR expression or progesterone-activating AR mutations have been associated with resistance to AR targeting. We aimed to test ONA, a type I PR antagonist with clinical activity in PRpos cancers, in AA/enz-resistant CRPC. In a prospectively defined exploratory analysis, we aimed to report outcome by plasma AR status ( pAR). Methods: This was a multi-institution, open label phase I/II clinical trial in pts progressing after ENZ/AA. Pts were first treated with single agent (SA) ONA using a randomised dose escalation design. ONA at 2 doses was then combined with AA (1000mg od with pred 5mg bid) in pts progressing on AA. The primary end-points were safety, pharmacokinetics (PK) and anti-tumor activity split by p AR. Archival and metastatic biopsies were collected when possible and tested for PR status. p AR was studied using previous methods (Romanel STM 2015). Results: 21 pts received SA ONA (5 = 10mg/ 5 = 20mg/ 4 = 30mg/ 4 = 40mg /3 = 50mg BID) and 15 pts received ONA-AA combination (5 = 30mg ONA BID, 10 = 50mg ONA BID). There were not DLTs or significant LFT abnormalities and no G3/4 adverse events (AE), no treatment discontinuations due to AEs and no SAEs considered related to ONA. PK in SA ONA observed active plasma concentrations and no interaction with AA. Of 32 evaluated pts 15 had a 2105T > A (p.L702H) or 2632A > G (p.T878A) AR mutation detected in plasma pre-treatment and 1 had AR copy number gain. PSA declines were not observed with SA ONA but in 2 pts with combination (-30%, -7%) who were AR normal. The rPFS on SA ONA was 2.8 months for AR normal and 2.6 for AR aberrant (Hazard ratio (HR) 1.41; 95% CI, 0.62-3.72; P 0.48) and on combination was 4.4 months for AR normal (8/15) and 2.2 for AR aberrant (7/15) (HR 6.08; 95%CI, 6.32-221.9; P < 0.001). Conclusions: ONA is safe in CRPC as SA and in combination with AA. There was no difference in rPFS by p AR status for SA ONA but on the combination with AA, pts who were plasma AR normal had a significantly longer rPFS. Clinical trial information: NCT02049190.

PURPOSE Hematopoietic growth factors have been shown to ameliorate the side effects of chemotherapy. Here we assess the ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to increase the dose-intensity and reduce the side effects of escalated methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) chemotherapy. PATIENTS AND METHODS A prospective randomized trial to compare escalated MVAC versus escalated MVAC with rhGM-CSF was conducted. All patients were treated at The University of Texas M.D. Anderson Cancer Center (UTMDACC) and had a metastatic or unresectable urothelial tumor. Forty-eight patients were randomized (25 to MVAC with rhGM-CSF and 23 to escalated MVAC alone). The clinical characteristics of the study populations were similar (ie, degree of tumor dissemination and performance status). RESULTS The dose-intensity in the two arms of the study did not differ significantly. No difference in the frequency of bacteriologically documented infections occurred between the two study arms. CONCLUSION The use of the hematopoietic growth factor rhGM-CSF did not result in an increased dose-intensity of escalated MVAC. The inability to increase the dose-intensity of MVAC further was a result of nonhematologic side effects of the chemotherapy. Escalation of treatment delivered at its median-tolerated dose is unlikely to result in additional therapeutic benefit for patients with common solid tumors. Future development of therapy may require the development of new agents or concepts, rather than modification of existing therapies.

e14099Background: A type I progesterone receptor (PR) antagonist that prevents PR-induced DNA transcription, ONA is being developed to target PR-expressing tumors. It has shown anti-cancer activity...

Background The progesterone receptor (PR) is expressed by ∼70% of early breast tumours and is implicated in the progression of breast cancer. In cancerous tissues PR may be activated in the absence of a ligand, or when ligand concentrations are very low, resulting in aberrantly activated PR (APR). The presence of APR may indicate that patients with breast cancer are more likely to respond to antiprogestins. The aims of this study were to describe and classify the histological subnuclear morphology of active and inactive PR in archival breast cancer samples. Methods Archived tumour specimens from 801 women with invasive breast cancer were collected. Tissue samples (n=789) were analysed for PR isoforms A and B (PRA and PRB), Ki67 and estrogen receptors (ERα) status, using immunohistochemistry. Medical records were used to determine human epidermal growth factor 2 (HER2) status, tumour stage and grade. Results A total of 79% of tumours stained positive for either PRA or PRB, and of these 25% of PRA-positive and 23% of PRB-positive tumours had PR present in the activated form. APRA was associated with higher tumour grade (p=0.001). APRB was associated with a higher tumour grade (p=0.046) and a trend for a more advanced stage. Patients with PR-positive tumours treated with antiestrogens had better disease-free survival (DFS) than those with PR-negative tumours (p

PURPOSE The purpose of this phase I trial was to determine the toxicity and maximum-tolerated dose (MTD) of murine monoclonal antibody (Mab) 14G2a (anti-GD2) in cancer patients. PATIENTS AND METHODS Following tracer doses of iodine-131-labeled 14G2a to determine tumor uptake, 18 patients with refractory melanoma, neuroblastoma, or osteosarcoma received unlabeled 14G2a at total concentrations of 50, 100, and 200 mg/m2 administered as daily 24-hour infusions for 5 days. RESULTS The overall sensitivity of external immunoscintigraphy was 64 of 74 known metastases (86%). Toxicity from prolonged infusion of 14G2a consisted of severe generalized pain, hyponatremia, fever, rash, paresthesias, weakness, and chronic refractory postural hypotension (two patients). Toxicity was less severe in pediatric patients. The MTD of Mab was 100 mg/m2. Sixteen of 18 patients developed human antimouse antibodies (HAMA) to 14G2a. Terminal-phase half-life (T1/2) of unlabeled Mab was 6.6 +/- 1.8 hours for patients receiving 50 mg/m2 and 39.5 +/- 13.3 hours at the 100-mg/m2 level. Tumor biopsies from six melanoma patients were positive for GD2 antigen, but only two of six had trace amounts of 14G2a present. Three mixed responses (two melanoma, one osteosarcoma) and two partial responses (PRs; neuroblastoma) were observed. CONCLUSION Mab 14G2a has modest antitumor activity at the expense of significant toxicity. Dose-limiting neurologic sequelae may significantly limit phase II studies other than in pediatric patients with neuroblastoma.

Eight of 28 (28%) cancer patients with liver metastases treated by either splenic (four) or hepatic (four) arterial infusion of recombinant interleukin-2 (rIL-2) developed hypersensitivity reactions to iodine-containing radiographic contrast media. These reactions consisted of fever, chills, malaise, nausea and vomiting, skin rash, diarrhea, and occasionally, hypotension. Reactions usually occurred 1 month after the initial arteriographic procedure and rIL-2 infusion, with 1-hour to 4-hour intervals between procedure and reexposure of the patient to the iodine-containing contrast medium (used in conjunction with computerized tomography or repeated arteriography for subsequent courses of rIL-2 infusions) and the onset of symptoms. Prompt administration of corticosteroids during the reaction and premedication of patients who were known to have had a reaction in the past were very effective in stopping reactions or preventing them from reoccurring. The high incidence (28%) of hypersensitivity reactions, the temporal relationship (4 hours) between the arteriographic procedure (utilizing iodine-containing contrast medium) and the initial infusion of rIL-2 (while some of the contrast medium was still present), and the absence of such hypersensitivity reactions among patients receiving systemic (intravenous) rIL-2 (not requiring the use of concomitant iodine-containing contrast medium) provide additional evidence that in the presence of a potentially immunogenic moiety, rIL-2, a potent stimulant of the human immune system, can produce an initial sensitization followed by subsequent anamnestic reaction upon reexposure of the patient to the immunogen (even without the additional rIL-2).

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

5051 Background: An urgent need exists for new therapies after progression (PD) onabiraterone (AA) and enzalutamide (ENZ). PR expression increases with castration resistance [Bonkhoff 2001]. ONA, a...

5593 Background: ONA is a type I PR antagonist, which prevents PR-induced DNA transcription. Immediate release (IR) 100 mg ONA was active in multiple preclinical models and in patients (pts) with b...

e16517 Background: ONA is a type I PR antagonist, which prevents PR-induced DNA transcription. ONA anti-cancer activity is documented in multiple pre-clinical models and clinical studies in pts wit...

TPS5616 Background: ONA is a type I PR antagonist, which prevents PR-induced DNA transcription. Presence of transcriptionally-activated PR (APR), seen as an aggregated subnuclear PR distribution pa...

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.

thought to play an important role in carcinogenesis by protecting cells from apoptosis. Recent publications disclosing FASN inhibitors suggest a pharmacophore common to many chemical series, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that imidazolinone would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug with a spirocyclopentyl-imidazolinone core. This hypothesis led to the discovery of a new series of imidazolinone-based FASN inhibitors. Extensive structure–activity relationship work in our laboratories resulted in numerous molecules with potent enzyme and cell activity, selectivity, and oral bioavailability. One such compound is a potent inhibitor of human FASN enzymatic activity (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells in lipid-reduced medium (IC50 = 13 nM). Addition of palmitate to the medium rescues the cells from FASN inhibition, demonstrating on-target effects. Potent activity is also seen in other cancer cells such as PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM). This lead compound is highly bioavailable (F 61%) in mice and shows good plasma and tumor exposures after oral dosing. In a pharmacodynamics (PD) model in H460 lung xenograft-bearing mice, oral treatment resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This compound and other members of the imidazolinone series potently inhibit the FASN KR domain (IC50 = 54 nM); specific binding to KR was confirmed by co-crystal structures of several examples. In summary, we discovered a new series of FASN inhibitors built upon an imidazolinone scaffold that are potent in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Moreover, rescue of lipid-reduced cellular activity upon addition of palmitate suggests selectivity, and PD studies in tumor-bearing mice confirm target engagement.

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

TPS2643 Background: ONA is a type I PR antagonist, which prevents PR-induced DNA transcription. Presence of transcriptionally activated PR (APR), could indicate potential for ONA anticancer activit...

TPS5097 Background: PR expression in prostate cancer increases with resistance to castration [Bonkhoff 2001]. The transcriptional, activated PR (APR), offers a potential target in advanced CRPC. ON...

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.

Two patients with gastrointestinal leiomyosarcoma metastatic to the liver were treated by hepatic chemoembolization with cisplatin and polyvinyl sponge followed by hepatic arterial infusion of vinblastine. Effective palliation in terms of durable tumor regression was achieved in both patients after two chemoembolization-infusion procedures. These results suggest that regional therapy may offer new hope for the subset of sarcoma patients who have liver metastases resistant to combination systemic chemotherapy.

Twenty-two patients with liver metastases received 45 courses of recombinant tumor necrosis factor (rTNF) by hepatic arterial infusion in doses ranging from 12.5 to 175 micrograms/m2/d for 5 days by continuous infusion. The induction of statistically significant, dose-related, severe, albeit transient, hypophosphatemia (less than 1.0 mg/dl) associated with clinically significant, right-sided myocardial dysfunction and severe lassitude was observed. These side effects were promptly reversed after rTNF was stopped and intravenous phosphate supplementation was started. As no significant or consistent increase in urinary phosphate excretion was detected, the rTNF-induced hypophosphatemia probably resulted from an intracellular shift of phosphate. Since tumor regression was clearly associated with the lowest levels of serum phosphate, hypophosphatemia may be important in the antitumor effects of rTNF.

592 Background: About 50% of ER positive (ERpos) BC 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 are associated with transcriptional activation of ER. This ER FDP is observed in BC. We hypothesized that in BC that the presence/absence of ER in the FDP could predict antiestrogen activity. This study describes an immunohistochemistry (IHC) method, by which a biomarker could be developed to investigate this hypothesis. Methods: 303 archived BC biopsies were obtained along with clinical and pathology data. Biopsies were analyzed for standard HES, ER, progesterone receptor (PR) and Ki67. PR positivity was determined with distinct antibodies to the A and B isoforms. The A-ER and D-ER nuclear patterns were analyzed at x1000 magnification. Results: Mean age; 58 (17 -89). Histology: ductal 85% lobular 13%, other 2%; 84% ERpos and 84% either PRApos or PRBpos, 7% ERpos and 7% PRApos or PRBpos only. All but 3 ERpos cases had received AntiEs; Adj. chemotherapy 51%, Stage: I 51%, II 43%, III 6 %. Grade: I 24%, II 51%, III 25%. Median follow up 31 months (mo). Local or distant progression (PD) was 19%. ER status was D-ER in 78% and A-ER in 22% of the biopsies and PD was associated with the D-ER pattern (p = 0.06), e.g. the hypothetically non-functional pattern D-ER was associated with a higher rate of PD (4 vs 35). With DFS defined as time to PD or death (5 year cut off), ERpos was better that ERneg (HR= 0.38, p = 0.016). For ERpos BC, A-ER was numerically better than D-ER (HR =0.3, p=0.24 two-sided). In univariate analyses, A-ER pattern was associated with higher grade (p=0.035), anisonucleosis (p=0.06), mitotic index (p=0.02), and Ki67 (=0.08). No association was found between ER pattern and age, stage or HER2 status. Conclusions: This study supports the hypothesis that AntiEs are mainly active in BC with A-ER pattern, which is targetable by AntiEs. A larger number of events are needed to reach significance in time-dependent analyses and confirmatory studies are warranted.

2608 Background: AR-12 (OSU-03012) is an oral celecoxib analogue lacking COX-2 inhibitory activity that inhibits pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1), AKT and impacts the endoplastic reticulum stress pathway. Preclinical studies indicate antitumor activity of AR-12 in various models and enhanced activity in combination. We completed a first in human clinical trial to determine its safety and tolerability, maximum tolerated dose (MTD) and recommended phase II dose (RD). Secondary objectives included assessment of tumor response, pharmacokinetics (PK) and pharmacodynamics (PD) including food effect. Methods: Patients (pts) with advanced solid tumors, ECOG PS 0-1, and adequate organ function were recruited in a modified 3+3 dose-escalation study. Pts received a run-in dose of AR-12 to analyze PK-PD and food effect, followed by continuous daily (QD) dosing in 28-day cycles. A twice daily (BID) cohort was initiated based on safety data. PD analysis was performed in platelet-rich plasma (PRP) and paired tumor biopsies when feasible. Results: 35 pts received at least one dose of study drug; 30 were evaluable for dose limiting toxicities (DLT) at dose ranges 100-3200mg QD and 800-1600mg BID. No DLT were observed in the QD cohort; DLT in the BID cohort are listed in table 1. Drug-related events (NCI-CTCAE v3) included rash (G2-2pts; G3-1pt), fatigue (G2-2pts; G3-4pts), nausea (G2-7pts; G3-1pt) and bloating (G2-1pt). Cmax after single dose was dose-proportional but high PK variability was observed, likely due to inadequate disintegration and dissolution of the formulation in the stomach. At RD, partial GSK3ß inhibition in PRP after 4 hours suggests AKT-pathway modulation. Best response (RECIST v1.0) was stable disease >6 cycles for 2 pts. Conclusions: The RD based on safety data is 800mg BID. Signs of pathway modulation were observed in concordance with the expected mechanism of action but were short-lasting. Considering limited drug absorption and PK variability, a new formulation of the drug will be developed to overcome these limitations. Clinical trial information: NCT00978523. [Table: see text]

e11535 Background: : About 50% of ER positive (ERpos) BC 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 are associated with transcriptional activation of ER. This ER FDP is observed in BC. We hypothesized that in BC that the presence/absence of ER in the FDP could predict antiestrogen activity. This study describes an immunohistochemistry (IHC) method, by which a biomarker could be developed to investigate this hypothesis. Methods: 37 paraffin embedded, formalin fixed archived BC samples were processed using 4 ER-alpha antibodies (Ab) under different IHC conditions to develop and refine this biomarker, the ER nuclear morphology was analyzed with standard microscopy at x1000. Interpretation of the IHC slides was done by an experienced pathologist. Standard ER, progesterone receptor (PR), and Ki67 testing were done. Tumor grade was obtained from the patients’ records. Analysis of the different Ab was with the kappa statistics. Comparison of the IHC technique to immunofluorescence (IHF) was done on 8 samples. Results: Consistent with prior research observations, tumors had two ER nuclear morphologies: 1. Diffuse pattern (D) where the ER was distributed evenly in a fine granular pattern, 2. Aggregate pattern (A) where the ER is distributed in distinct clumps or aggregates. This defined 3 tumor phenotypes: A cells only (A) 1% (4/37), D cells only (D) 65% (24/37), and a heterogenous mix of A + D cells (AD) 18% (7/37), ER Neg 14% (5/37). IHC and IHF analysis for ER nuclear morphology was concordant. The ER activation status (A/AD or D) was not correlated with staining ER intensity, ERpos %, PRpos%, but was correlated with % Ki67 staining (p=0.09) and tumor grade (0.035). Conclusions: ERpos early BC biopsies can be grouped in two categories based on ER nuclear morphology: A group with diffuse and homogenous nuclear staining, and a different group with heterogeneous area of cells having a nuclear pattern consistent with a functional or activated ER. These observations warrant clinical pathological studies to determine if this biomarker is potentially predictive of clinical outcomes.

5602 Background: Endocrine treatments, in general have limited clinical efficacy in endometrial cancer. Upon ligand binding, PRs in normal tissue form discrete focal subnuclear distribution patterns (FDP), which are associated with DNA transcription. FDP are indicative of functionally activated PRs (APRs), and are observed in EC, independently of menopausal status. The feasibility of using an IHC technique to characterize the PR functional status has previously been reported in breast cancer and the APR phenotype in cell lines correlates with anti-progestin activity. The goal of this study is to determine if APR can be identified in EC and to determine if this IHC technique & APR phenotype could be developed as a companion diagnostic to predict anti-progestin efficacy in patients with EC. Methods: 72 archived primary EC specimens were processed with standard IHC for estrogen receptor (ER), PR & proliferation (Ki67). APR status was determined using commercially available antibodies specific to the A and B isoforms of the PR (PRA and PRB) with standard microscopy at 1000x magnification. Results: 56 (78%) tumors were of endometrioid histology. Endometrioid tumors were ER+ (68%) and PR+ (84%). Two PR nuclear distribution patterns were observed: an aggregated pattern (A) which is indicative of APR and a diffuse or finely granular pattern (D) indicative of an inactivated PR. This resulted in three tumor phenotypes: A cells only, D cells only, and a mix of A + D cells. APR was defined as any tumor with more than 5% A cells. An average of 49% of tumor cells were positive with PRA, 35% were positive for PRB. APR was present with PR A in 41% and with PR B in 47% of the PR+ endometrioid tumors. The APR status, for both PR A and PR B, was independent of PR positivity rate, PR staining intensity score and % Ki67 positive. APRpos phenotype was associated with a lower % ERposstaining. When observable, endometrial stromal and normal cells were PR positive (D pattern). Conclusions: APR can be identified using either PRA or PRB, in ~50 % of the EC samples; there was a pattern consistent with the presence of APR positive cells. The IHC technique to identify APR has the potential to be developed as companion diagnostic as a potential predictor of anti-progestin efficacy.

596 Background: The oncology literature indicates that ER and PR are linked and in BC the presence of PR usually indicates functional ER. BCs express both ER and PR to varying degree, but little has been published on expression of ER + PR in individual cancer cells. The goal of this study is to 1. Determine the expression ER and PR at the cellular level, 2. Determine if ER and PR are expressed in the same BC cells. If ER and PR are separate, this may indicate that antiestrogens and antiprogestins may target different cells. Methods: Archived 1° BC specimens were processed using standard IHC techniques for ER/PR testing. The procedure consisted of sequential double staining on the same microtone section, where the PR was visualized through brown staining using HRP/DAB, and the ER was visualized through red staining using AP/Permanent Red. The initial testing on 13 tumor samples utilized a duel anti-PR-A/B antibody (Ab) and an ER Ab. The next 63 tumor samples utilized; 1. anti-ER and anti-PR-A Abs, 2. anti-ER and anti-PR-B Abs. A pathologist experienced with IHC, interpreted and enumerated the cells staining positive for ER only, for PR only and cells staining positive for both ER & PR. The number of cells expected to be stained for both ER & PR by chance can be calculated as the rate of total ER by the rate of total PR and compared with the observed rate (paired rank test). ER/PR positivity is defined as ≥ 5% cells positive. Results: In the first series, 11/13 tumors were ERpos, 13/13 were PRpos, 7/13 tumors had both ER & PR expressed in 5-20% (median 5%) of the same tumor cells. In the 2nd series of 63 tumors; 1. 50/53 were ERpos, 52/53 were PRApos and 44/53 had both ER & PRA expressed in pos, 52/52 were PRBpos and 42/52 tumors had both ER & PRB expressed in

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

Based on preclinical studies which reveal enhanced antitumor activity of tumor necrosis factor (TNF) when combined with actinomycin D in human prostate cancer cell lines, we performed a phase I clinical study combining TNF and actinomycin D. All patients had metastatic prostatic carcinoma exhibiting androgen-independent growth. Patients were treated with a combination of a short infusion of actinomycin D followed by a TNF infusion daily for five consecutive days. Soluble TNF receptor p60 was not modulated by treatment but p80 receptor increased significantly following treatment with a combination of TNF and actinomycin D (baseline median 3.4 ng/ml) range 2.5-6.6 ng/ml follow up (9.3 ng/ml) range 6-24 ng/ml. We concluded that the maximum tolerated dose of continuous infusion TNF and short infusion actinomycin D is 400 micrograms/m2 of actinomycin D and 400 micrograms/m2 of TNF. The increased soluble receptor isoform (p80) may account for the lack of clinical activity seen in this trial. Should these results be confirmed, a strategy focused on overcoming the upregulation of the TNF soluble receptor will be required before further study of TNF should be considered.

We conducted a phase II trial to confirm the activity of fixed, low dose gamma-interferon in metastatic renal cell carcinoma. A total of 35 patients with metastatic renal cell carcinoma, who had not received prior immunotherapy and who had a Zubrod performance status of 2 or less, was enrolled in this study. Primary tumors were controlled by nephrectomy or embolization before treatment began. gamma-Interferon was administered weekly as a subcutaneous injection at a fixed dose of 100 micrograms. Toxic effects were limited to low grade fever, chills and myalgias within 24 hours of injection. There were no incidences of grade 3 or 4 toxicity. Responses could be evaluated in 34 patients. There were 1 complete and 4 partial responses, for an objective response rate of 15% (95% confidence interval 5 to 32%). Durations of response to date are 21+, 17+, 13+, 9 and 2 months. We conclude that gamma-interferon is an active agent for metastatic renal cell carcinoma when administered according to this dose and schedule. The response rate compares favorably with those of alpha-interferon and interleukin-2, and toxicity is minimal. gamma-Interferon has excellent potential for use in combination with other biological or chemotherapeutic agents and in the adjuvant setting.

PURPOSE A phase II clinical trial was performed to assess the antitumor activity and toxicity of ketoconazole in combination with doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) in patients with androgen-independent prostate cancer (AI PCa). PATIENTS AND METHODS Thirty-nine consecutive patients whose disease progressed following castration were treated with oral ketoconazole (1,200 mg) daily and Adriamycin (20 mg/m2 in a 24-hour infusion) once weekly. Antitumor activity was assessed by the level of prostatic-specific antigen (PSA) decline. RESULTS PSA levels decreased > or = 50% from baseline in 21 (55%; 95% confidence interval, 38% to 71%) of 38 assessable patients. We observed partial responses (PRs) in seven (58%) of 12 patients with measurable soft tissue disease (in the lung, lymph nodes, and liver). Two patients with history of atherosclerotic heart disease had a sudden cardiac death. Serious toxic reactions included grade III to V stomatitis and grade III to IV acral erythema in 11 patients (29%), and grade III to IV anal and urethral mucositis in five patients (13%). Grade III to IV neutropenia occurred in 11 patients (29%). Seventeen patients (45%) required hospitalization for complications. Fifteen patients (39%) developed hypokalemia, and 24 patients (63%) developed clinical adrenal insufficiency. CONCLUSION The combination of ketoconazole and Adriamycin has a 55% PSA response rate in patients with AI PCa and is worthy of additional study. This treatment results in frequent adrenal insufficiency. Therefore, future studies should incorporate routine corticosteroid replacement. The cardiac complications caused by this combination should be studied further before it is widely used.

Interferon-alpha (IFN-alpha) and interleukin-2 (IL-2) each has produced a 15%-20% response in metastatic renal cell cancer. Combining IFN-alpha with either IL-2 or 5-fluorouracil (5-FU) enhanced IFN-alpha activity. We have therefore conducted a Phase I Study combining IL-2, IFN-alpha, and 5-FU. The patients were continuously infused with IL-2 (1-3 x 10(6) u/m2) and 5-FU (600-750 mg/m2) for a 5-day period every 28 days, and IFN-alpha (4-5 x 10(6) u/m2) was injected subcutaneously daily. Lymphokine-activated killer (LAK) and natural killer (NK) cell activity was measured on days 0 and 8. Twenty-one patients received 76 courses. All primary tumors were controlled by surgery (81%) or angioinfarction. Hematologic toxicity was mild; median nadir of platelets was 117 K/microL and of granulocytes was 1.2 K/microL. Dose-limiting toxicity included mucositis, liver damage, and hypotension. No treatment-related death occurred, and only one patient required intensive-care-unit support. Two patients had an objective response, one of which was a complete response. Increased LAK cell and NK cell activity occurred at all IL-2 dose levels. Simultaneous delivery of IL-2, IFN-alpha, and 5-FU is safe and shows antitumor and biologic activity. 5-FU did not appear to suppress IL-2-induced LAK and NK cell activation. Maximum tolerated dose of the three-drug combination is IL-2, 2 x 10(6) u/m2, 5-FU 600 mg/m2, and IFN-alpha, 4 x 10(6) u/m2.

Alteration in interactions between tumor-infiltrating lymphocytes (TILs) and tumor cells after chemotherapy or immunotherapy was studied in metastatic melanoma patients. Tumors were harvested from surgical specimens 17 days after the end of chemotherapy with cisplatin, vinblastine, and dacarbazine (CVD). Tumors of nonlymph-node metastases from two responders yielded neither TILs nor tumor cells, whereas those from all four nonresponders had both TILs [(1.1-13.8) x 10(6) cells/g tumor] and tumor cells [(2.8-30.8) x 10(6) cells/g tumor). Tumors of lymph node metastases from nine patients yielded substantial numbers both of TILs and tumor cells, regardless of different clinical responses, except with one complete responder, whose tumor did not contain tumor cells. The mean increase of TILs from these tumors (n = 14) 3-4 weeks after incubation with 200 U/ml recombinant interleukin-2 (rIL-2) was 2.5-fold, whereas there was a 56-fold increase in TILs from untreated tumors (n = 3). CD3+ T cells predominated in TILs before and after expansion with IL-2. IL-2-activated TILs from five of six tumors tested displayed higher cytotoxicity against autologous tumor cells than against cells from any of three allogeneic tumors. Mean tumor cell numbers (10(6) cells/trial) obtained by serial needle biopsies for the same tumor in five patients decreased from 1.2 before therapy to 0.25 at day 4 of therapy (interferon alpha alone), and to 0.02 at day 8 (interferon alpha and IL-2). This decrease did not correlate with clinical responses. Yields (x 10(6) cells/g tumor) of TILs and tumor cells in subcutaneous melanomas obtained by excisional biopsies in one nonresponder under IL-2 therapy were respectively 0.2 and 1.1 before therapy (day 0), 0.1 and less than 0.01 during (day 7), 0.2 and less than 0.01 at the end of therapy (day 21), and 0.5 and 0.5 at the time of tumor progression (day 66). Yields of TILs and tumor cells in the other nonresponder were respectively 3 and 26 before (day 0), 16 and 3 during (day 7), and 0.4 and less than 0.01 at the end of IL-2 therapy (day 17), and 2.5 and 6 at the time of progression (day 62). TILs in these two patients before therapy proliferated well in culture with IL-2 (570- and 720-fold, respectively), and showed higher cytotoxicity against autologous tumor cells, whereas none of those from the five tumors biopsied during or at the end of IL-2 therapy proliferated. TILs at the time of progression showed modest proliferation (54- and 76-fold, respectively) and showed major-histocompatibility-complex-nonrestricted cytotoxicity. In summary, a decrease in the number of live tumor cells did not always correlate with clinical response in either therapy. CVD chemotherapy may simply impair IL-2-induced proliferation of TILs. IL-2 therapy may induce transient unresponsiveness of TILs to IL-2.

In an attempt to improve the therapeutic index of recombinant interleukin-2 (rIL-2) by generating or activating lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes (TIL) regionally and/or in situ, we randomly assigned 28 patients with liver metastases to receive rIL-2 by continuous infusion for 5 days via either the splenic artery or the hepatic artery. Clinically significant and lasting tumor regression was observed only in two of 28 patients (7%), one in each of the two treatment arms. The maximum-tolerated daily dosage of rIL-2 was 3 x 10(6) U/m2; beyond this dosage, toxicity was excessive. Peripheral LAK cell activity measured in vitro and clinical tumor regression did not correlate. This observation, coupled with the equal distribution of regressions between the two treatment arms, raises the possibility that tumor regression, rare though it may be in response to rIL-2 administration, is largely mediated by TIL activation and not by LAK cell generation.

The clinical data that have been accumulated so far suggests a significant influence of IL-2 dose and schedule on the immunobiological effects and clinical toxicities observed with this cytokine. Consequently, the series of Phase I and Phase II clinical trials conducted at the University of Texas M. D. Anderson Cancer Center in patients with advanced malignant melanoma investigating the use of IL-2 in combination with other cytokines, monoclonal antibodies, or ex vivo activated effector cells have used a common dose and schedule of IL-2 administration for which abundant immunobiological information already exists. This approach allows cross-trial comparison of experience with toxicities, immunobiological observations and clinical activity by a group of investigatiors within a single institution, and more rapid and valid evolution towards combination biological therapy, which preclinical data suggest will have greater activity than single agent therapy.