Anna C. Ferrari

Sipuleucel-T Immunotherapy for Castration-Resistant Prostate Cancer

BACKGROUND Sipuleucel-T, an autologous active cellular immunotherapy, has shown evidence of efficacy in reducing the risk of death among men with metastatic castration-resistant prostate cancer. METHODS In this double-blind, placebo-controlled, multicenter phase 3 trial, we randomly assigned 512 patients in a 2:1 ratio to receive either sipuleucel-T (341 patients) or placebo (171 patients) administered intravenously every 2 weeks, for a total of three infusions. The primary end point was overall survival, analyzed by means of a stratified Cox regression model adjusted for baseline levels of serum prostate-specific antigen (PSA) and lactate dehydrogenase. RESULTS In the sipuleucel-T group, there was a relative reduction of 22% in the risk of death as compared with the placebo group (hazard ratio, 0.78; 95% confidence interval [CI], 0.61 to 0.98; P=0.03). This reduction represented a 4.1-month improvement in median survival (25.8 months in the sipuleucel-T group vs. 21.7 months in the placebo group). The 36-month survival probability was 31.7% in the sipuleucel-T group versus 23.0% in the placebo group. The treatment effect was also observed with the use of an unadjusted Cox model and a log-rank test (hazard ratio, 0.77; 95% CI, 0.61 to 0.97; P=0.02) and after adjustment for use of docetaxel after the study therapy (hazard ratio, 0.78; 95% CI, 0.62 to 0.98; P=0.03). The time to objective disease progression was similar in the two study groups. Immune responses to the immunizing antigen were observed in patients who received sipuleucel-T. Adverse events that were more frequently reported in the sipuleucel-T group than in the placebo group included chills, fever, and headache. CONCLUSIONS The use of sipuleucel-T prolonged overall survival among men with metastatic castration-resistant prostate cancer. No effect on the time to disease progression was observed. (Funded by Dendreon; ClinicalTrials.gov number, NCT00065442.)

Activation of Rb and decline in androgen receptor protein precede retinoic acid-induced apoptosis in androgen-dependent LNCaP cells and their androgen-independent derivative.

Androgen ablation‐induced prostate cancer regression is transient and ends with the regrowth of androgen‐independent (AI) tumors. To mimic this evolution in culture, we chronically deprived an androgen‐dependent (AD) prostate cancer cell line (LNCaP) of androgen, generating an AI derivative which retained limited hormone proliferative responsiveness and a barely detectable prostate‐specific antigen (PSA) mRNA level. While the cytokeratin 8 (CK8) level was low, the androgen receptor (AR) protein in AI cells was on average tenfold greater than in AD cells. When challenged for susceptibility to undergo apoptosis, the AI cells were more resistant than AD cells to all‐trans retinoic acid (tRA) and two chemotherapeutic agents, Taxol and Adriamycin, requiring higher doses and longer periods of treatment to achieve similar effects. Compared to AD cells, the partially apoptosis‐resistant AI cells expressed four times more Bcl‐2 protein and undetectable levels of p21/WAF1. Induction of apoptosis by tRA in both cell types did not affect their expression but was preceded by the activation of Rb and a pronounced reduction of AR protein level. The kinetics of the Rb activation and AR downmodulation in both cell types matched their tRA sensitivity, suggesting that these events may be required for tRA‐induced apoptosis. The results show that the apoptotic pathway in AI cells, although more difficult to induce, is not irrevocably lost and that targeted reduction of the AR protein level with retinoids in combination with androgen ablation therapy may prolong remissions in advanced prostate cancer patients. J. Cell. Physiol. 179:336–346, 1999.

mTOR Signaling Pathway and mTOR Inhibitors in Cancer Therapy

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase. It is ubiquitously expressed in cells and is a therapeutic target for the cancer treatment arsenal. Despite the great responses obtained in tumors addicted to specific mutations or overactivation of key members of the mTOR pathway (HiF1α in RCC, cyclin D1 in MCL, or TSC in SEGA), mTOR inhibitors as single agents have modest activity. Dual PI3K/mTOR kinase inhibitors have been developed with the idea of overcoming resistance to the mTOR inhibition through preventing the activation of PI3K/Akt as a result of release negative feedback loops.

LEF1 in Androgen-Independent Prostate Cancer: Regulation of Androgen Receptor Expression, Prostate Cancer Growth, and Invasion

A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease.

Androgen receptor level controlled by a suppressor complex lost in an androgen-independent prostate cancer cell line

Androgen receptor (AR) overexpression is one of the characteristics of prostate cancer (PC) that progresses to hormone independence. An androgen-independent (AI) derivative, with much higher AR-mRNA and protein levels than the parental LNCaP cell line, whose proliferation was androgen dependent (AD), was used to explore the mechanism of AR overexpression. We found that a suppressor element (ARS), previously identified in mouse AR and located in the 5′-untranslated region of human AR gene, malfunctions in AI cells. Transfection of constructs that included ARS element into AD cells reduced the transactivating activities of both AR promoter and a heterologous SV40 promoter. The deletion of ARS resulted in an eightfold increase in AR-promoter activity in AD cells, but had no effect in AI cells. Moreover, the nuclear extracts of AD cells contained proteins that produced a specific, ARS-binding complex, while this complex appeared to have been lost from AI cells. Most importantly, treatment of AI cells with a demethylating agent or histone deacetylase inhibitors restored the lost ARS-binding complex. The restoration of the complex coincided with a reduced expression of AR-mRNA and protein and a reduced rate of AR-gene transcription, determined by nuclear run-on experiment. Thus, epigenetic transcriptional silencing of the suppressor protein(s) may be responsible for AR overexpression in AI cells, and its reversal in hormone-independent PC may normalize AR levels and restore their hormone dependence.

A phase II randomized study of cixutumumab (IMC-A12: CIX) or ramucirumab (IMC-1121B: RAM) plus mitoxantrone (M) and prednisone (P) in patients (pts) with metastatic castrate-resistant prostate cancer (mCRPC) following disease progression (PD) on docetaxel (DCT) therapy.

97 Background: Vascular endothelial growth factor (VEGF)-mediated angiogenesis and insulin-like growth factor (IGF-IR)-mediated signaling contribute to mCRPC growth. CIX and RAM are fully human IgG1 human monoclonal antibodies targeting IGF-IR and VEGF receptor-2 (VEGFR-2) respectively. We investigated the safety and efficacy of CIX or RAM in combination with M + P in mCRPC pts with PD on DCT. METHODS Eligible pts had mCRPC and PD during/within 120 days of DCT, ECOG PS 0-2, PSA ≥ 2 ng/mL, and adequate organ function. All pts received M 12 mg/m2 IV every 3 weeks (w) + P 5 mg PO BID for up to 12 cycles and were randomized to either CIX 6 mg/kg or RAM 6 mg/kg IV q w. Tumor assessments were after the first 3 cycles and then q6w. Primary endpoint was composite progression-free survival (cPFS: either RECIST PD, bone scan PD or new skeletal events). Other endpoints included safety, response and overall survival (OS). Sample size was based on a targeted 50% increase in median (mdn) cPFS from 2.6 months (m) to 3.9 m. RESULTS 132 pts were treated; 66 each to CIX or RAM. Mdn age and baseline PSA was 65 yr and 129 ng/mL for pts treated with CIX and 68 yr and 111 ng/mL for RAM. Involvement of sites other than bone was CIX: 79% and RAM: 70%. The most frequent Grade ≥3 related adverse events for CIX/M/P: fatigue 17%, leukopenia 12%, and neutropenia 8%, and for RAM/M/P: leukopenia 8%, neutropenia 8% and hypertension 8%. Left ventricular dysfunction/CHF: 12% for CIX (0% G3) and 23% for RAM (8% G3). Mdn number of Rx cycles were 5 for CIX and 6 for RAM. Mdn follow-up was 22.7 m for CIX and 21.8 m for RAM. PSA response was 18.4% (8.8-32% 95% CI) on CIX and 22.0% (11.5-36% 95% CI) on RAM. Mdn cPFS and OS were 4.1 m (3.0-5.6 m 95% CI) and 10.8 m (6.5-13.0 m 95% CI) for CIX and 6.7 m (4.5-8.3 m 95% CI) and 13.0 m (9.5-16.0 m 95% CI) for RAM. CONCLUSIONS CIX/M/P and RAM/M/P were reasonably tolerated and achieved the primary endpoint. Preliminary cPFS and OS of RAM/M/P appear encouraging; sustained disease control was observed in pts on both rx arms. Correlation of serum markers of IGF and VEGF activity with clinical endpoints is planned.

KLF6 Loss of Function in Human Prostate Cancer Progression Is Implicated in Resistance to Androgen Deprivation

Inactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy.

Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Purα‐mediated decrease in androgen receptor levels

Increased androgen receptor (AR) levels support resistance to apoptosis and hormone therapy in advanced prostate cancer (PC). We recently linked the overexpression of AR in androgen‐independent LNCaP cells (AI‐cells) and tissues from castration‐resistant patients to decreased nuclear levels of Pur‐alpha (Purα) and loss from a protein complex bound to repressor sequences (ARS) in the 5′‐UTR of AR. Strategies to regain control of increased AR transcription may overcome resistance of AI‐cells and improve treatment outcomes.

A randomised non-comparative phase II trial of cixutumumab (IMC-A12) or ramucirumab (IMC-1121B) plus mitoxantrone and prednisone in men with metastatic docetaxel-pretreated castration-resistant prostate cancer

BACKGROUND Cixutumumab, a human monoclonal antibody (HuMAb), targets the insulin-like growth factor receptor. Ramucirumab is a recombinant HuMAb that binds to vascular endothelial growth factor receptor-2. A non-comparative randomised phase II study evaluated cixutumumab or ramucirumab plus mitoxantrone and prednisone (MP) in metastatic castration-resistant prostate cancer (mCRPC). PATIENTS AND METHODS Men with progressive mCRPC during or after docetaxel therapy received mitoxantrone 12 mg/m(2) on day 1 and prednisone 5mg twice daily and were randomised 1:1 to receive either cixutumumab or ramucirumab 6 mg/kg intravenously weekly in a 21-day cycle. Primary end-point was composite progression-free survival (cPFS). Secondary end-points included safety, response, radiographic progression-free survival (PFS) and overall survival (OS). Sample size was based on a 50% increase in median cPFS from 2.6 (MP) to 3.9 months (either combination). RESULTS 132 men were treated (66 per arm). Median cPFS was 4.1 months (95% confidence interval (CI), 2.2-5.6) for cixutumumab and 6.7 months (95% CI, 4.5-8.3) for ramucirumab. Median time to radiographic progression was 7.5 months for cixutumumab and 10.2 months for ramucirumab, with a median OS of 10.8 and 13.0 months, respectively. Fatigue was the most frequent adverse event (AE). Incidence of most non-haematologic grade 3-4 AEs was <10% on both arms. Grade 3 cardiac dysfunction occurred in 7.6% of patients on ramucirumab. CONCLUSION Combinations of cixutumumab or ramucirumab plus MP were feasible and associated with moderate toxicities in docetaxel-pretreated men with mCRPC. Of the two regimens, the ramucirumab regimen is worthy of further testing based on the observed cPFS relative to the historical control.

13-cis Retinoic Acid and Complete Androgen Blockade in Advanced Hormone-Naive Prostate Cancer Patients: Report of a Phase II Randomized Study

PURPOSE 13 cis Retinoic acid (isotretinoin) is a retinoid with preclinical evidence of anti-prostate cancer activity. This phase II, cross-over, randomized study of advanced, predominantly androgen-dependent prostate cancer patients was designed to assess primarily the effect on prostate-specific antigen (PSA) decline and toxicity of adding isotretinoin to hormonal therapy and, secondarily, the potential antitumor activity of the combination. PATIENTS AND METHODS Thirty-seven D0 to D2 patients were randomized soon after initiating luteinizing hormone-releasing hormone agonist with antiandrogen treatment to add (arm 1) or not (arm 2) isotretinoin from weeks 1 to 12. After cross-over on week 13, patients in arm 1 discontinued while patients in arm 2 added isotretinoin from weeks 14 to 25. Observation on hormonal therapy alone continued until week 49. RESULTS Baseline and randomization median PSA for 30 assessable patients were, respectively, 34 and 18.2 ng/mL for arm 1 and 31 and 13.4 ng/mL for arm 2. Median PSA at week 13 was 0.5 ng/mL (range, < 0.05 to 136 ng/mL) for arm 1 and 0.7 ng/mL (range, < 0.05 to 4.4 ng/mL) for arm 2; at week 25, 0.1 ng/mL (range, < 0.05 to 121 ng/mL) and 0.4 ng/mL (range, < 0.05 to 3.1 ng/mL), respectively. At week 49, arm 1 had median PSA of 0.1 ng/mL (range, < 0.05 to 345 ng/mL) and arm 2, 0.3 ng/mL (range, < 0.05 to 8.8 ng/mL); seven of 15 and three of 15 patients, respectively, had undetectable PSA levels (P =.12). Frequent isotretinoin-related toxicity included grade 1 cheilitis (76%), skin dryness (43%), and elevated triglycerides (50%). CONCLUSION Isotretinoin does not impair PSA decline or add significant toxicity to hormonal therapy. An adequately powered, randomized study would be required to determine whether the combination is superior to standard hormonal treatment.