Maria Karlou

Effects of Abiraterone Acetate on Androgen Signaling in Castrate-Resistant Prostate Cancer in Bone

PURPOSE Persistent androgen signaling is implicated in castrate-resistant prostate cancer (CRPC) progression. This study aimed to evaluate androgen signaling in bone marrow-infiltrating cancer and testosterone in blood and bone marrow and to correlate with clinical observations. PATIENTS AND METHODS This was an open-label, observational study of 57 patients with bone-metastatic CRPC who underwent transiliac bone marrow biopsy between October 2007 and March 2010. Patients received oral abiraterone acetate (1 g) once daily and prednisone (5 mg) twice daily. Androgen receptor (AR) and CYP17 expression were assessed by immunohistochemistry, testosterone concentration by mass spectrometry, AR copy number by polymerase chain reaction, and TMPRSS2-ERG status by fluorescent in situ hybridization in available tissues. RESULTS Median overall survival was 555 days (95% CI, 440 to 965+ days). Maximal prostate-specific antigen decline ≥ 50% occurred in 28 (50%) of 56 patients. Homogeneous, intense nuclear expression of AR, combined with ≥ 10% CYP17 tumor expression, was correlated with longer time to treatment discontinuation (> 4 months) in 25 patients with tumor-infiltrated bone marrow samples. Pretreatment CYP17 tumor expression ≥ 10% was correlated with increased bone marrow aspirate testosterone. Blood and bone marrow aspirate testosterone concentrations declined to less than picograms-per-milliliter levels and remained suppressed at progression. CONCLUSION The observed pretreatment androgen-signaling signature is consistent with persistent androgen signaling in CRPC bone metastases. This is the first evidence that abiraterone acetate achieves sustained suppression of testosterone in both blood and bone marrow aspirate to less than picograms-per-milliliter levels. Potential admixture of blood with bone marrow aspirate limits our ability to determine the origin of measured testosterone.

Molecular Characterization of Enzalutamide-treated Bone Metastatic Castration-resistant Prostate Cancer

BACKGROUND Enzalutamide is a novel antiandrogen with proven efficacy in metastatic castration-resistant prostate cancer (mCRPC). OBJECTIVE To evaluate enzalutamides effects on cancer and on androgens in blood and bone marrow, and associate these with clinical observations. DESIGN, SETTING, AND PARTICIPANTS In this prospective phase 2 study, 60 patients with bone mCRPC received enzalutamide 160mg orally daily and had transilial bone marrow biopsies before treatment and at 8 wk of treatment. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Androgen signaling components (androgen receptor [AR], AR splice variant 7 (ARV7), v-ets avian erythroblastosis virus E26 oncogene homolog [ERG], cytochrome P450, family 17, subfamily A, polypeptide 1 [CYP17]) and molecules implicated in mCRPC progression (phospho-Met, phospho-Src, glucocorticoid receptor, Ki67) were assessed by immunohistochemistry; testosterone, cortisol, and androstenedione concentrations were assessed by liquid chromatography-tandem mass spectrometry; AR copy number was assessed by real-time polymerase chain reaction. Descriptive statistics were applied. RESULTS AND LIMITATIONS Median time to treatment discontinuation was 22 wk (95% confidence interval, 19.9-29.6). Twenty-two (37%) patients exhibited primary resistance to enzalutamide, discontinuing treatment within 4 mo. Maximal prostate-specific antigen (PSA) decline ≥ 50% and ≥ 90% occurred in 27 (45%) and 13 (22%) patients, respectively. Following 8 wk of treatment, bone marrow and circulating testosterone levels increased. Pretreatment tumor nuclear AR overexpression (> 75%) and CYP17 (> 10%) expression were associated with benefit (p = 0.018). AR subcellular localization shift from the nucleus was confirmed in eight paired samples (with PSA decline) of 23 evaluable paired samples. Presence of an ARV7 variant was associated with primary resistance to enzalutamide (p = 0.018). Limited patient numbers warrant further validation. CONCLUSIONS The observed subcellular shift of AR from the nucleus and increased testosterone concentration provide the first evidence in humans that enzalutamide suppresses AR signaling while inducing an adaptive feedback. Persistent androgen signaling in mCRPC was predictive of benefit and ARV7 was associated with primary resistance. PATIENT SUMMARY We report a first bone biopsy study in metastatic prostate cancer in humans that searched for predictors of outcome of enzalutamide therapy. Benefit is linked to a pretreatment androgen-signaling signature. TRIAL REGISTRATION ClinicalTrials.gov identifier NCT01091103.

Persistent, Biologically Meaningful Prostate Cancer After 1 Year of Androgen Ablation and Docetaxel Treatment

PURPOSE Clinicians are increasingly willing to treat prostate cancer within the primary site in the presence of regional lymph node or even limited distant metastases. However, no formal study on the merits of this approach has been reported. We used a preoperative clinical discovery platform to prioritize pathways for assessment as therapeutic targets and to test the hypothesis that the primary site harbors potentially lethal tumors after aggressive treatment. PATIENTS AND METHODS Patients with locally advanced or lymph node-metastatic prostate cancer underwent 1 year of androgen ablation and three cycles of docetaxel therapy, followed by prostatectomy. All specimens were characterized for stage by accepted criteria. Expression of select molecular markers implicated in disease progression and therapy resistance was determined immunohistochemically and compared with that in 30 archived specimens from untreated patients with high-grade prostate cancer. Marker expression was divided into three groups: intracellular signaling pathways, stromal-epithelial interaction pathways, and angiogenesis. RESULTS Forty patients were enrolled, 30 (75%) of whom underwent prostatectomy and two (5%) who underwent cystoprostatectomy. Twenty-nine specimens contained sufficient residual tumor for inclusion in a tissue microarray. Immunohistochemical analysis showed increased epithelial and stromal expression of CYP17, SRD5A1, and Hedgehog pathway components, and modulations of the insulin-like growth factor I pathway. CONCLUSION A network of molecular pathways reportedly linked to prostate cancer progression is activated after 1 year of therapy; biomarker expression suggests that potentially lethal cancers persist in the primary tumor and may contribute to progression.

Hedgehog signaling inhibition by the small molecule Smoothened inhibitor GDC-0449 in the bone forming prostate cancer xenograft MDA PCa 118b

Hedgehog signaling is a stromal‐mesenchymal pathway central to the development and homeostasis of both the prostate and the bone. Aberrant Hedgehog signaling activation has been associated with prostate cancer aggressiveness. We hypothesize that Hedgehog pathway is a candidate therapeutic target in advanced prostate cancer. We confirm increased Hedgehog signaling in advanced and bone metastatic castrate resistant prostate cancer and examine the pharmacodynamic effect of Smoothened inhibition by the novel reagent GDC‐0449 in an experimental prostate cancer model.

Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling.

Tzelepi V, Karlou M, Wen S, Hoang A, Logothetis C, Troncoso P & Efstathiou E
(2011) Histopathology 58, 1037–1047
Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling

Integrated Hedgehog signaling is induced following castration in human and murine prostate cancers

The interplay between androgen and Hedgehog (Hh) signaling pathways may be associated with prostate cancer progression and resistance to therapy.

Abstract 2696: Modulation of intracrine androgen signaling in bone metastatic castrate resistant prostate cancer (bmCRPC) by MDV3100

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background Androgen signaling is a central driver in CRPC. We performed a study to determine the effect of the antiandrogen MDV 3100 on bone marrow infiltrating CRPC. Methods From February 2010 to June 2011, we performed an open-label, observational study of 60 bmCRPC patients (pts), who underwent transilial bone marrow biopsy (BMB) at baseline and 8 weeks of treatment. Pts received MDV3100 160mg orally once daily. The primary objective was to evaluate androgen signaling in bone marrow infiltrating cancer and testosterone in blood (BT) and bone marrow (BMT) and correlate findings with clinical observations. Androgen receptor (AR), CYP17 and phospho Src (pSrc) expression were assessed by immunohistochemistry, and BT and BMT by liquid chromatography mass spectrometry. Findings Maximal PSA decline of Δ50% occurred in 29 (48%) of 60 pts, and α90% in 13 (22%). Tumor involvement in BMB was detected in 28 (47%) pretreatment BMBs. Paired tumor infiltrated BMBs were found in 23 (38%) pts. Pretreatment androgen signaling and pSrc tumor expression is shown in Table 1. Pretreatment intense nuclear AR expression combined with α10% CYP17 tumor expression or increased BMT correlate with Δ50% PSA decline (p value 0.02). AR subcellular localization shift from dominant nuclear to cytoplasmic correlate with Δ50% PSA decline (p value 0.05). Increased pretreatment p-Src expression is associated with lack of PSA decline (p value 0.002). Increase in BMT and BT is observed after 8 weeks of MDV3100. (Mean Pretreatment BMT 0.03 ng/ml, Wk8 BMT 0.04 in 33pts, p value 0.0009) (Pretreatment BT 0.05, wk8 BT 0.066 in 37 pts, p value 0.0001) Interpretation Pretreatment expression profile is consistent with persistent androgen signaling in bmCRPC. MDV3100 induces ‘pharmacodynamic’ changes in Androgen Signaling. AR changes likely account for the reported therapeutic effect of MDV3100. These data prompt exploratory combinations of MDV3100 with Androgen Biosynthesis Inhibitors and src inhibitors. ![Figure][1] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2696. doi:1538-7445.AM2012-2696 [1]: pending:yes

Abstract A2: A Study of the effects of MDV3100 in the tumor microenvironment of bone metastatic castrate resistant prostate cancer (bmCRPC)

Abstract Background: Androgen signaling remains a central driver in castrate-resistant prostate cancer. We performed a study to determine the effect of the antiandrogen MDV 3100 on bone marrow infiltrating CRPC. Methods: From February 2010 to June 2011, we performed an open-label, observational study of 60 bmCRPC patients (pts), who underwent transilial bone marrow biopsy (BMB) at baseline, 8 weeks and end of study. Pts received MDV3100 160mg orally once daily. The primary objective was to evaluate androgen signaling in bone marrow infiltrating cancer and testosterone in blood (BT) and bone marrow (BMT) and correlate findings with clinical observations. Androgen receptor (AR) c-terminus n-terminus, AR Variant 7 (ARV7), ERG, CYP17, phospho Src (pSrc), expression and mitotic index (Ki67 ) were assessed by immunohistochemistry, AR copy number by qPCR and BT and BMT by liquid chromatography mass spectrometry. Initial Findings: Maximal PSA decline of ≥50% occurred in 29 (48%) of 60 pts, and ≥90% in 13 (22%). Tumor involvement in BMB was detected in 28 (47%) pretreatment BMBs. Paired tumor infiltrated BMBs were found in 23 (38%) pts. Increased androgen receptor expression (n-terminal antibody used for IHC) was observed at pretreatment with heterogeneous CYP17 and pSrc extent of expression. Pretreatment intense nuclear AR expression combined with ≥10% CYP17 tumor expression or increased BMT correlate with ≥50% PSA decline (p value 0.02). AR subcellular localization shift from dominant nuclear to cytoplasmic and regression of bone marrow tumor infiltration correlate with ≥50% PSA decline (p value 0.05). Increased pretreatment p-Src expression is associated with lack of PSA decline (p value 0.002). Increase in BMT and BT is observed after 8 weeks of MDV3100 (mean pretreatment BMT 0.026 ng/ml, Wk8 BMT 0.04 in 33pts, p value 0.0001) (pretreatment BT 0.041, wk8 BT 0.066 in 37 pts, p value Interpretation: Pretreatment expression profile is consistent with persistent androgen signaling in bmCRPC. MDV3100 induces pharmacodynamic changes in androgen signaling. AR changes likely account for the reported therapeutic effect of MDV3100. These data prompt exploratory combinations of MDV3100 with androgen biosynthesis inhibitors and src inhibitors. Citation Format: Eleni Efstathiou, Patricia Troncoso, James Mohler, Christopher J. Logothetis, Mark Titus, Dimitra Tsavachidou, Sijin Wen, Anh Hoang, Robynne Ashe, Maria Karlou, Craig Berman. A study of the effects of MDV3100 in the tumor microenvironment of bone metastatic castrate-resistant prostate cancer (bmCRPC) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A2.

Abstract C253: Effective inhibition of Hedgehog signaling pathway with small molecule Smoothened inhibitor GDC‐0449 in a bone producing prostate cancer xenograft

Background: The tumor microenvironment is involved in prostate cancer progression. Several stromal‐epithelial‐interacting pathways associated with prostate cancer (PCa) progression in the bone, are also considered candidates of resistance to current therapeutic strategies applied. Hedgehog (Hh) signaling has been associated with PCa aggressiveness. Therefore Hh pathway could be a novel diagnostic and therapeutic target, also contributing to therapy against prostate carcinoma metastasis in bone. Experimental work on specific chemical inhibitors of Hh signaling suggests that they may represent an entirely new class of therapeutic agents. In this study we tested the efficacy of Hh pathway inhibition with Smoothened inhibitor GDC‐0449 in 118b prostate cancer xenograft model. Experimental Design: We employed the xenograft MDA PCa 118b, derived from the bone metastasis of a patient with castrate‐resistant prostatic adenocarcinoma. This is characterized by unique bone producing feature when injected subcutaneously in SCID mice. Bone is the most common site for metastasis of prostate carcinoma, therefore a model with the new bone formation distinctive characteristic is really informative. Following MDA PCa 118b sc injection, animals were treated with Smoothened inhibitor GDC‐0449 (oral administration, for 21 days). Hh pathway components expression was measured both in the tumor cells and stromal compartment using species specific primers for real‐time PCR, western blot analysis and immunohistochemistry. Results: Paracrine Hedgehog signaling was confirmed in the tumor xenograft microenvironment. Baseline Shh expression was present in human tumor cells and significantly higher compared to stromal compartment, while nuclear Gli1 (a hallmark of pathway activation) and Ptch1 expression in the stromal compartment were present and significantly higher compared to human tumor cells. Smoothened inhibition resulted in attenuation of stromal expression of Gli1 and Ptch1, in the treated group compared to the control thus confirming the pharmacodynamic effect of the agent. A trend for slower tumor growth at the treated group was observed, while a decline in proliferation rate was noted when Hh pathway was suppressed in the stromal compartment. Conclusion: Hh signaling pathway demonstrates paracrine activation in the 118b prostate cancer bone producing xenograft model. GDC‐0449 is effective in blocking Hh signaling in PCa. Future directions: A combination of Hedgehog signaling inhibition and cytotoxic therapies, such as androgen ablation would likely increase efficacy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C253.