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Development of diffuse telangiectasias (highlighted by blue arrows) occurred on the upper chest, back, and face in a patient with ovarian granulosa cell tumor treated at the 0.75 mg/kg Q2W dose of STM 434.

Purpose:STM 434 is a soluble receptor ligand trap targeting activin A, a protein in the TGFβ family that plays important roles in growth, differentiation, and cancer cachexia. This study evaluated the safety, antitumor activity, and metabolic effects of STM 434 in a first-in-human, multicenter, phase I clinical trial (NCT02262455).Patients and Methods:Patients with advanced solid tumors were enrolled in 8 dose cohorts ranging from 0.25 mg/kg every 4 weeks to 8 mg/kg every 2 weeks via a 3 + 3 dose-escalation design. The primary endpoint was maximum tolerated dose (MTD). Secondary endpoints included safety, pharmacokinetics, and response. As activin A is implicated in metabolism and muscle function, changes in key metabolic parameters, including lean body mass and 6-minute walk test, were serially measured.Results:Thirty-two patients were treated on study. The most common treatment-related adverse events were fatigue (41%) and mucocutaneous bleeding complications including epistaxis (34%) and gingival bleeding (22%), likely related to off-target inhibition of bone morphogenetic protein 9 (BMP9). STM 434 treatment resulted in the expected follicle-stimulating hormone level decreases in most patients and in metabolic parameter changes, including an increase in total lean body mass and 6-minute walk test distance. No responses were observed in the 30 evaluable patients, but the stable disease rate in patients with granulosa cell ovarian cancer was 10 of 12 (80%).Conclusions:Although no direct antitumor efficacy was documented, potentially clinically meaningful dose-related metabolic effects, including treatment of cancer cachexia, were observed that support further exploration of activin A inhibitors that limit BMP9 blockade.See related commentary by Bonilla and Oza, p. 5432

Change and end of treatment values for albumin, hemoglobin, and absolute lymphocytes among the 32 patients are summarized below. Abbreviations: SD, stable disease.

Despite the success of currently authorized vaccines for the reduction of severe COVID-19 disease risk, rapidly emerging viral variants continue to drive pandemic waves of infection, resulting in numerous global public health challenges. Progress will depend on future advances in prophylactic vaccine activity, including advancement of candidates capable of generating more potent induction of cross-reactive T cells and durable cross-reactive antibody responses. Here we evaluated an Amphiphile (AMP) adjuvant, AMP-CpG, admixed with SARS-CoV-2 Spike receptor binding domain (RBD) immunogen, as a lymph node-targeted protein subunit vaccine (ELI-005) in mice and non-human primates (NHPs). AMP-mediated targeting of CpG DNA to draining lymph nodes resulted in comprehensive local immune activation characterized by extensive transcriptional reprogramming, inflammatory proteomic milieu, and activation of innate immune cells as key orchestrators of antigen-directed adaptive immunity. Prime-boost immunization with AMP-CpG in mice induced potent and durable T cell responses in multiple anatomical sites critical for prophylactic efficacy and prevention of severe disease. Long-lived memory responses were rapidly expanded upon re-exposure to antigen. In parallel, RBD-specific antibodies were long-lived, and exhibited cross-reactive recognition of variant RBD. AMP-CpG-adjuvanted prime-boost immunization in NHPs was safe and well tolerated, while promoting multi-cytokine-producing circulating T cell responses cross-reactive across variants of concern (VOC). Expansion of RBD-specific germinal center (GC) B cells in lymph nodes correlated to rapid seroconversion with variant-specific neutralizing antibody responses exceeding those measured in convalescent human plasma. These results demonstrate the promise of lymph-node adjuvant-targeting to coordinate innate immunity and generate robust adaptive responses critical for vaccine efficacy.

While T cell receptor (TCR)-modified T cell therapies have shown promise against solid tumors, overall therapeutic benefits in clinical practice have been modest due in part to suboptimal T cell persistence and activation in vivo, alongside the possibility of tumor antigen escape. In this study, we demonstrate an approach to enhance the in vivo persistence and activation of TCR-T cells through combination with Amphiphile (AMP)-vaccination including cognate TCR-T peptides. AMP-modification improves lymph node targeting of conjugated tumor immunogens and adjuvants, thereby coordinating a robust T cell-priming endogenous immune response. Vaccine combination with TCR-T cell therapy provided simultaneous in vivo invigoration of adoptively transferred TCR-T cells and in situ priming of the endogenous anti-tumor T cell repertoire. The resulting induction of an adoptive and endogenous anti-tumor effect led to durable responses in established murine solid tumors refractory to TCR-T cell monotherapy. Protection against recurrence was associated with antigen spreading to additional tumor-associated antigens not targeted by vaccination. Enhanced anti-tumor efficacy was further correlated with pro-inflammatory lymph node transcriptional reprogramming and increased antigen presenting cell maturation, resulting in TCR-T cell expansion and functional enhancement in lymph nodes and solid tumor parenchyma without lymphodepletion. In vitro evaluation of AMP-peptides with matched human TCR-T cells targeting NY-ESO-1, mutant KRAS, and HPV16 E7 illustrated the clinical potential of AMP-vaccination to enhance human TCR-T cell proliferation, activation, and anti-tumor activity. Taken together, these studies provide rationale and evidence to support clinical evaluation of the combination of AMP-vaccination with TCR-T cell therapies to augment anti-tumor activity.SummaryAMP-vaccination targets the lymph nodes to enhance TCR-T cell therapy resulting in solid tumor eradication and durable protection against recurrence.

Purpose: STM 434 is a soluble receptor ligand trap targeting activin A, a protein in the TGFβ family that plays important roles in growth, differentiation, and cancer cachexia. This study evaluated the safety, antitumor activity, and metabolic effects of STM 434 in a first-in-human, multicenter, phase I clinical trial (NCT02262455). Patients and Methods: Patients with advanced solid tumors were enrolled in 8 dose cohorts ranging from 0.25 mg/kg every 4 weeks to 8 mg/kg every 2 weeks via a 3 + 3 dose-escalation design. The primary endpoint was maximum tolerated dose (MTD). Secondary endpoints included safety, pharmacokinetics, and response. As activin A is implicated in metabolism and muscle function, changes in key metabolic parameters, including lean body mass and 6-minute walk test, were serially measured. Results: Thirty-two patients were treated on study. The most common treatment-related adverse events were fatigue (41%) and mucocutaneous bleeding complications including epistaxis (34%) and gingival bleeding (22%), likely related to off-target inhibition of bone morphogenetic protein 9 (BMP9). STM 434 treatment resulted in the expected follicle-stimulating hormone level decreases in most patients and in metabolic parameter changes, including an increase in total lean body mass and 6-minute walk test distance. No responses were observed in the 30 evaluable patients, but the stable disease rate in patients with granulosa cell ovarian cancer was 10 of 12 (80%). Conclusions: Although no direct antitumor efficacy was documented, potentially clinically meaningful dose-related metabolic effects, including treatment of cancer cachexia, were observed that support further exploration of activin A inhibitors that limit BMP9 blockade. See related commentary by Bonilla and Oza, p. 5432

TPS2701 Background: Mutations in KRAS and NRAS occur in one quarter of human solid tumors. The G12D allele is the most commonly occurring variant in pancreatic, colorectal, non-small cell lung, ovarian, biliary and gallbladder cancers. ELI-002 2P is an immunotherapeutic comprised of a lymph-node targeted amphiphile (AMP)-modified G12D and G12R mutant KRAS peptides together with an AMP-modified CpG oligonucleotide adjuvant. In preclinical models, ELI-002 demonstrated increased cytotoxic KRAS-specific T cells compared to non-lymph node targeted controls using the same peptide and adjuvant. Clinical evaluation of adoptively transferred KRAS-specific T cells demonstrated objective antitumor activity (Tran 2016). Circulating tumor (ctDNA) methodology permits identification of patients with minimal residual disease (MRD) following locoregional treatment. In MRD setting, immunotherapy is anticipated to succeed as the ratio of effector T cells to target tumor cells is maximized prior to bulk visible disease. The AMPLIFY-201 study (NCT04853017) is evaluating ELI-002 in patients with KRAS mutated solid tumors with MRD. Methods: AMPLIFY-201 is an open-label, dose-escalation and expansion phase I, first in human, trial evaluating ELI-002 2P in patients with KRAS mutated tumors with MRD following standard of care therapy. The initial phase I cohort enrolls patients with colorectal and pancreatic cancer to receive multiple doses of AMP-peptides 70mcg each (1.4 mg total) admixed with AMP-CpG 0.1 mg, administered once every two weeks. Subsequent phase I cohorts will enroll patients with RAS-mutated pancreatic, colorectal, non-small cell lung, ovarian, bile duct or gallbladder cancer, who will receive a fixed dose of AMP-peptides 70 mcg each, together with escalating doses of AMP-CpG. Safety and efficacy will be summarized with descriptive statistics. The maximum tolerated dose (if any) and the recommended phase II dose (RP2D) will be determined with the dose-response activity of ELI-002 2P in eliciting functional KRAS-specific T cells. Preliminary antitumor activity will be characterized using changes from baseline in ctDNA, serum biomarkers appropriate for tumor type, and progression free survival time. Eligibility includes patients who have received standard of care locoregional treatment according to NCCN guidelines and whom have MRD persistence or relapse (ctDNA positive). Patients with colon and pancreas cancer with Stage IV oligometastatic disease (< 3 lesions in one organ) rendered surgically free of disease and with MRD, are also eligible. The dose escalation portion of the study is currently enrolling. Clinical trial information: NCT04853017.

Background The SARS-CoV-2 pandemic’s public health, economic, and social impacts mandate urgent development of effective vaccines to contain or eradicate infection. To that end, we evaluated a novel amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid-modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain (Spike RBD) protein for immunization (ELI 005) in two mouse models. AMP immunogens are efficiently delivered to lymph nodes, where innate and adaptive immune responses are generated. Methods Female, 6 to 8-week-old C57BL/6J and BALB/c mice and 37-week-old C57BL/6J mice received two or more doses of benchmark (alum or CpG) or AMP-modified vaccines, comprised of Spike RBD protein and AMP-CpG adjuvant, subcutaneously injected into the tail base in two-week intervals. Antigen was dose spared to determine if AMP-CpG would maintain the immune response. Cellular immune responses were determined via ELISpot analysis of IFNγ production by splenocytes, intracellular cytokine staining of peripheral blood and lung-resident T-cells, and flowcytometric bead array analysis of Th1/2/17 cytokines. Humoral immune responses were determined via blood serum ELISAs to determine sera antibody binding titers, and pseudoviral neutralization assays for comparison to human convalescent serum. Results Compared to alum, AMP immunization induced 29-fold higher antigen-specific T cells which produced multiple Th1 cytokines and trafficked into lung parenchyma. Antibody responses favored Th1 isotypes (IgG2bc, IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers >100-fold higher than the natural immune response from convalescent COVID-19 patients; responses were maintained despite 10-fold dose-reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. Conclusions ELI-005 exhibits the qualities of an optimal SARS-CoV-2 vaccine, which should (1) induce robust and durable CD8+ and CD4+ T cell responses, (2) elicit high magnitude neutralizing antibodies, (3) produce Th1 bias in the elicited antibody and T cell responses, (4) potentially expand pre-existing cross-reactive T cells, (5) enable dose-sparing of required immunogens to improve the speed and cost of broad vaccination campaigns, and (6) be efficacious in elderly populations. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.

Lymph node–targeting adjuvant AMP-CpG enhances SARS-CoV-2 RBD vaccine-induced cellular and humoral immune responses., The profound consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mandate urgent development of effective vaccines. Here, we evaluated an Amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid–modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain protein as a candidate vaccine (ELI-005) in mice. AMP modification efficiently delivers CpG to lymph nodes, where innate and adaptive immune responses are generated. Compared to alum, immunization with AMP-CpG induced >25-fold higher antigen-specific T cells that produced multiple T helper 1 (TH1) cytokines and trafficked into lung parenchyma. Antibody responses favored TH1 isotypes (IgG2c and IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers 265-fold higher than natural convalescent patient COVID-19 responses; T cell and antibody responses were maintained despite 10-fold dose reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.

The SARS-CoV-2 pandemic has led to public health, economic, and social consequences that mandate urgent development of effective vaccines to contain or eradicate infection. To that end, we evaluated a novel amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid-modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain protein as a candidate vaccine (ELI-005) in mice. AMP immunogens are efficiently delivered to lymph nodes, where innate and adaptive immune responses are generated. Compared to alum, AMP immunization induced >25-fold higher antigen-specific T cells which produced multiple Th1 cytokines and trafficked into lung parenchyma and respiratory secretions. Antibody responses favored Th1 isotypes (IgG2bc, IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers 265-fold higher than the natural immune response from convalescent COVID-19 patients; responses were maintained despite 10-fold dose-reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.

Significance Sarcopenia, or aging-associated muscle atrophy, increases the risk of falls and fractures and is associated with metabolic disease. Because skeletal muscle is a major contributor to glucose handling after a meal, sarcopenia has significant effects on whole-body glucose metabolism. Despite the high prevalence and potentially devastating consequences of sarcopenia, no effective therapies are available. Here, we show that treatment of mice with an anti-myostatin antibody for just 4 wk increased muscle mass and strength in both young and old mice. In old mice, this increase in muscle mass was accompanied by an improvement in muscle insulin sensitivity. These data provide support for myostatin inhibition as a potential therapeutic strategy for aging-associated sarcopenia and insulin resistance.

Purpose: Abiraterone is an oral inhibitor of CYP17, which is essential for androgen biosynthesis. This multicenter study assessed its efficacy in patients with castration-resistant prostate cancer (CRPC), without prior chemotherapy or CYP17-targeted therapy, and frequency of bone scans discordant with prostate-specific antigen (PSA) and clinical response. Experimental Design: Thirty-three patients received abiraterone acetate 1,000 mg daily with prednisone 5 mg twice daily in continuous 28-day cycles. Patients were evaluated monthly for efficacy and safety. Bone scan flare was defined as the combination, after 3 months of therapy, of an interpreting radiologist's report indicating “disease progression” in context of a 50% or more decline in PSA level, with scan improvement or stability 3 months later. Results: A 50% or more decline in PSA level at week 12 was confirmed in 22 of 33 (67%) patients. Declines in PSA level of 50% or more were seen in 26 of 33 (79%) patients. Undetectable PSA levels (≤0.1 ng/mL) occurred in 2 patients. Median time on therapy and time to PSA progression were 63 weeks and 16.3 months, respectively. Twenty-three patients were evaluable for bone scan flare. Progression was indicated in radiologist's report in 12 of 23 (52%), and 11 of 12 subsequently showed improvement or stability. As prospectively defined, bone scan flare was observed in 11 of 23 (48%) evaluable patients or 11 of 33 (33%) enrolled patients. Adverse events were typically grade 1/2 and consistent with prior published abiraterone reports. Conclusion: Clinical responses to abiraterone plus prednisone were frequent and durable in men with metastatic CRPC. Further investigation is needed to clarify the confounding effect of bone scan flare on patient management and interpretation of results. Clin Cancer Res; 17(14); 4854–61. ©2011 AACR.

Background Nutritional factors are associated with reduced risk of prostate cancer progression, yet mechanisms remain unclear. We examined the effects of lycopene and fish oil supplements versus placebo on the normal prostate microenvironment, among men pursuing active surveillance for low-burden prostate cancer. We hypothesized that lycopene or fish oil supplements would down-regulate insulin-like growth factor-1 (IGF-1) and cyclooxygenase 2 (COX-2) gene expression, respectively, reflecting putative proliferation (IGF-1) and inflammatory (COX-2) pathways relevant to carcinogenesis. Methods We conducted a 3-month randomized, double-blinded, clinical trial comparing prostate tissue gene expression profiles (assessed by qRT–PCR) among men with favorable-risk prostate cancer receiving either 30 mg/day lycopene, 3 g/day fish oil (including 1,098 mg eicosapentaenoic and 549 mg docosahexaenoic fatty acids) or placebo. Results Among 69 men (22 assigned to lycopene, 21 to fish, and 26 to placebo), there was no difference in the change from baseline to the 3 months in IGF-1 expression level between the placebo and lycopene arms (p = 0.93) nor in COX-2 expression between the placebo and fish arms (p = 0.99). Conclusion Compared to placebo, 3-month intervention with lycopene or fish oil did not significantly change IGF-1 and COX-2 gene expression in the normal prostate microenvironment in men with low-burden prostate cancer. Further analysis of global gene expression profiles may shed light on the bioactivity and relevance of these nutrients in prostate cancer.

Background: STM434 is a soluble receptor ligand trap targeting activin A, a protein in the TGF-beta family that plays important roles in growth, differentiation, and cancer cachexia. Activin A overexpression is a common feature in many gynecologic cancers. Granulosa cell tumors also harbor pathognomonic mutations in FOXL2, which specifically promotes dysregulated activin signaling. The role of inhibiting activin A signaling in human cancers has not been previously explored. Methods: Primary endpoint was to establish to maximum tolerated dose of STM434. Dose was initially escalated by accelerated titration and then 3+3 design. Secondary endpoints included safety per CTCAE v4, pharmacokinetics, and response by RECIST v1.1. As Activin A is also implicated in metabolism and muscle function, changes in key metabolic parameters including lean body and fat mass established by DXA scan and 6 minute walk test (6MWT), were serially measured. Results: 32 patients were treated at doses ranging from 0.25 - 8 mg/kg IV every 2-4 weeks. Tumor types included ovarian granulosa cell (n=13), epithelial ovarian (n=7), colon (n=3), pancreatic (n=2), and other solid tumors (n=7). A total of 5 DLTs were observed in 3 pts (all grade 3): hemorrhagic ascites (0.5 mg/kg Q4W) and stomatitis, gastritis, epistaxis, and alkaline phosphatase increase (all 8 mg/kg Q2W). The most common treatment-related AEs were fatigue (n=12), epistaxis (n=11), and gingival bleeding (n=6). Other observed hematologic related-AEs included telangiectasia (n=3), hemorrhagic ascites (n=2), and petechiae (n=2). No treatment-related grade 4 or 5 AEs occurred. STM434 resulted in the expected FSH level decreases in most patients. STM434 also resulted in metabolic parameter changes, including an increase in total lean body mass (1%-20%, 0.5 mg/kg Q2W to 4 mg/kg Q2W cohorts) and 6-minute walk test (25-150 meters in the 0.5 mg/kg Q2W-2 mg/kg Q2W cohort) and decrease in total fat mass (-3% to -18% in the 0.5 mg/kg Q4W- 4 mg/kg Q2W cohorts). One patient with fatty liver on baseline imaging had complete resolution of this finding after 2 cycles of therapy. No responses were observed in the 28 evaluable patients. In granulosa cell patients, the stable disease rate was 9/12 (75%) although the significance of this finding is uncertain. Conclusions: The MTD of STM434 was 4 mg/kg Q2W. STM434 use resulted in mucocutaneous bleeding complications likely related to off-target inhibition of bone morphogenic protein 9 (BMP9). BMP9 bears substantial homology to activin A and inherited inactivation of BMP9 can result in hereditary hemorrhagic telangiectasia which shares a phenotype similar to the AE profile observed here. While no direct antitumor efficacy was documented, potentially clinically meaningful dose-related metabolic effects were observed that support further exploration of Activin A inhibitors that avoid BMP9 blockade in cancer. Citation Format: Jessica J. Tao, Joyce F. Liu, Drew W. Rasco, Willis Navarro, Christopher M. Haqq, David M. Hyman. First in human study of activin-A inhibitor, STM434, in patients with granulosa cell ovarian cancer and other advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT011.

The impact of high-resolution magic angle spinning (HR-MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n = 20) or radical prostatectomy surgery (n = 20), snap-frozen on dry ice, and stored at −80°C until use. Twenty-one samples (n = 11 biopsy, n = 10 surgical) underwent HR-MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n = 9 biopsy, n = 10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14-µm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (HE however, one pathologist's scores were significantly lower for biopsy tissues following HR-MAS while the other pathologist's scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR-MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over- or under-expressed genes, including 14 housekeeping genes, between HR-MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR-MAS analysis; however, biopsy tissues are more fragile than surgical tissues. Copyright © 2009 John Wiley & Sons, Ltd.

The histopathological diagnosis of melanoma can be challenging. No currently used molecular markers accurately distinguish between nevus and melanoma. Recent transcriptome analyses have shown the differential expression of several genes in melanoma progression. Here, we describe a multi-marker diagnostic assay using 5 markers (ARPC2, FN1, RGS1, SPP1, and WNT2) overexpressed in melanomas. Immunohistochemical marker expression was analyzed in 693 melanocytic neoplasms comprising a training set (tissue microarray of 534 melanomas and nevi), and 4 independent validation sets: tissue sections of melanoma arising in a nevus; dysplastic nevi; Spitz nevi; and misdiagnosed melanocytic neoplasms. Both intensity and pattern of expression were scored for each marker. Based on the differential expression of these 5 markers between nevi and melanomas in the training set, a diagnostic algorithm was obtained. Using this algorithm, the lesions in the validation sets were diagnosed as nevus or melanoma, and the results were compared with the known histological diagnoses. Both the intensity and pattern of expression of each marker were significantly different in melanomas compared to nevi. The diagnostic algorithm exploiting these differences achieved a specificity of 95% and a sensitivity of 91% in the training set. In the validation sets, the multi-marker assay correctly diagnosed a high percentage of melanomas arising in a nevus, Spitz nevi, dysplastic nevi, and misdiagnosed lesions. The multi-marker assay described here can aid in the diagnosis of melanoma.

RGS1 (regulator of G protein signaling 1) encodes a member of the regulator of G protein family. Recently, RGS1 was found to be overexpressed in gene expression-profiling studies of melanoma. However, no analyses have been reported of its expression at the protein level in melanoma. In this study, the potential impact of RGS1 as a molecular prognostic marker for melanoma was assessed using immunohistochemical analysis of a melanoma tissue microarray containing primary cutaneous melanomas from 301 patients. High RGS1 expression was significantly correlated with increased tumor thickness (P=0.0083), mitotic rate (P=0.04), and presence of vascular involvement (P

Epidemiological and prospective studies indicate that comprehensive lifestyle changes may modify the progression of prostate cancer. However, the molecular mechanisms by which improvements in diet and lifestyle might affect the prostate microenvironment are poorly understood. We conducted a pilot study to examine changes in prostate gene expression in a unique population of men with low-risk prostate cancer who declined immediate surgery, hormonal therapy, or radiation and participated in an intensive nutrition and lifestyle intervention while undergoing careful surveillance for tumor progression. Consistent with previous studies, significant improvements in weight, abdominal obesity, blood pressure, and lipid profile were observed (all P < 0.05), and surveillance of low-risk patients was safe. Gene expression profiles were obtained from 30 participants, pairing RNA samples from control prostate needle biopsy taken before intervention to RNA from the same patient's 3-month postintervention biopsy. Quantitative real-time PCR was used to validate array observations for selected transcripts. Two-class paired analysis of global gene expression using significance analysis of microarrays detected 48 up-regulated and 453 down-regulated transcripts after the intervention. Pathway analysis identified significant modulation of biological processes that have critical roles in tumorigenesis, including protein metabolism and modification, intracellular protein traffic, and protein phosphorylation (all P < 0.05). Intensive nutrition and lifestyle changes may modulate gene expression in the prostate. Understanding the prostate molecular response to comprehensive lifestyle changes may strengthen efforts to develop effective prevention and treatment. Larger clinical trials are warranted to confirm the results of this pilot study.

PurposeWe previously determined that intravenous administration of rituximab results in limited penetration of this agent into the leptomeningeal space. Systemic rituximab does not reduce the risk of CNS relapse or dissemination in patients with large cell lymphoma. We therefore conducted a phase I dose-escalation study of intrathecal rituximab monotherapy in patients with recurrent CNS non-Hodgkin's lymphoma (NHL).Patients and MethodsThe protocol planned nine injections of rituximab (10 mg, 25 mg, or 50 mg dose levels) through an Ommaya reservoir over 5 weeks. The safety profile of intraventricular rituximab was defined in 10 patients.ResultsThe maximum tolerated dose was determined to be 25 mg and rapid craniospinal axis distribution was demonstrated. Cytologic responses were detected in six patients; four patients exhibited complete response. Two patients experienced improvement in intraocular NHL and one exhibited resolution of parenchymal NHL. High RNA levels of Pim-2 and FoxP1 in meningeal lymphoma cells were associated with disease refractory to rituximab monotherapy.ConclusionThese results suggest that intrathecal rituximab (10 to 25 mg) is feasible and effective in NHL involving the CNS.

The insulin-like growth factor (IGF)-1 receptor is currently being targeted in clinical trials in prostate cancer. Despite this targeting, there are conflicting data on the presence of this receptor in human tumor samples, largely because of differences in technique.Immunohistochemistry was used to determine the presence of IGF-1 receptor in frozen normal prostate and prostate cancer specimens. Clinical and pathologic parameters were correlated with IGF-1 receptor intensity and frequency of staining. Only 2-3+ staining on a scale of 0-3 was considered positive in this evaluation.IGF-1 receptor was expressed in normal prostate epithelium in 6 of 6 patients without cancer and in morphologically normal epithelium adjacent to tumor cells in 21 of 22 patients with cancer studied. IGF-1 receptor was present in the prostate tumor epithelium of 28 of 28 primary tumors, 3 of 5 locally recurrent androgen-independent tumors, and in 4 of 5 metastatic lymph nodes. Stromal staining patterns were positive in 2 of 28 specimens near benign epithelium compared to 19 of 30 specimens of stroma surrounding tumor epithelium (P0.0001, Fisher exact test). Stroma adjacent to Gleason gradeor=7 tumors showed higher intensity staining than that adjacent to lower grade tumors (P0.001). Expression of the closely related insulin receptor did not show expression in either normal or cancer epithelium, or in adjacent stroma.This study using frozen tissue shows widespread IGF-1 receptor expression in normal prostate, prostate cancers, and metastases. These data support investigations into IGF-1 receptor as a therapeutic target in prostate cancer.

Introduction The molecular biology involving neoadjuvant chemotherapy (NAC) response is poorly understood. To elucidate the impact of NAC on the breast cancer transcriptome and its association with clinical outcome, we analyzed gene expression data derived from serial tumor samples of patients with breast cancer who received NAC in the I-SPY 1 TRIAL. Methods Expression data were collected before treatment (T1), 24–96 hours after initiation of chemotherapy (T2) and at surgery (TS). Expression levels between T1 and T2 (T1 vs. T2; n = 36) and between T1 and TS (T1 vs. TS; n = 39) were compared. Subtype was assigned using the PAM50 gene signature. Differences in early gene expression changes (T2 − T1) between responders and nonresponders, as defined by residual cancer burden, were evaluated. Cox proportional hazards modeling was used to identify genes in residual tumors associated with recurrence-free survival (RFS). Pathway analysis was performed with Ingenuity software. Results When we compared expression profiles at T1 vs. T2 and at T1 vs. TS, we detected significantly altered expression of 150 and 59 transcripts, respectively. We observed notable downregulation of proliferation and immune-related genes at T2. Lower concordance in subtype assignment was observed between T1 and TS (62 %) than between T1 and T2 (75 %). Analysis of early gene expression changes (T2 − T1) revealed that decreased expression of cell cycle inhibitors was associated with poor response. Increased interferon signaling (TS − T1) and high expression of cell proliferation genes in residual tumors (TS) were associated with reduced RFS. Conclusions Serial gene expression analysis revealed candidate immune and proliferation pathways associated with response and recurrence. Larger studies incorporating the approach described here are warranted to identify predictive and prognostic biomarkers in the NAC setting for specific targeted therapies. Clinical trial registration ClinicalTrials.gov identifier: NCT00033397. Registered 9 Apr 2002. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0582-3) contains supplementary material, which is available to authorized users.

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients’ blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.

Purpose To assess the prognostic significance of nuclear receptor coactivator-3 (NCOA3) overexpression in primary cutaneous melanoma. Patients and Methods NCOA3 expression was assessed using immunohistochemical analysis of a melanoma tissue microarray (TMA) containing primary melanomas from 343 patients with defined histology and follow-up. The impact of the presence or absence of various prognostic factors on relapse-free survival (RFS) and disease-specific survival (DSS) of melanoma patients was assessed using Cox regression and Kaplan-Meier analysis. The impact of presence or absence of various factors on sentinel lymph node (SLN) metastasis was assessed using logistic regression analysis. Results Increasing degree of NCOA3 expression was significantly predictive of SLN metastasis (P = .013) and the mean number of SLN metastases (P = .031). Kaplan-Meier analysis demonstrated a significant association between NCOA3 overexpression and reduced RFS (P = .021) and DSS (P = .030). Logistic regression analysis revealed increasing degree of NCOA3 expression to be an independent predictor of SLN status (P = .017). Multivariate Cox regression analysis showed the independent impact of NCOA3 expression on RFS (P = .0095) and DSS (P = .021). NCOA3 was the most powerful factor predicting DSS, outperforming tumor thickness and ulceration. Conclusion These results identify NCOA3 as a novel, independent marker of melanoma outcome, with a significant impact on SLN metastasis, RFS, and DSS.

Primary CNS lymphoma is an aggressive form of non-Hodgkin lymphoma whose growth is restricted to the central nervous system. We used cDNA microarray analysis to compare the gene expression signature of primary CNS lymphomas with nodal large B-cell lymphomas. Here, we show that while individual cases of primary CNS lymphomas may be classified as germinal center B-cell, activated B-cell, or type 3 large B-cell lymphoma, brain lymphomas are distinguished from nodal large B-cell lymphomas by high expression of regulators of the unfolded protein response (UPR) signaling pathway, by the oncogenes c-Myc and Pim-1, and by distinct regulators of apoptosis. We demonstrate that interleukin-4 (IL-4) is expressed by tumor vasculature as well as by tumor cells in CNS lymphomas. We also identify high expression in CNS lymphomas of several IL-4-induced genes, including X-box binding protein 1 (XBP-1), a regulator of the UPR. In addition, we demonstrate expression of the activated form of STAT6, a mediator of IL-4 signaling, by tumor cells and tumor endothelia in CNS lymphomas. High expression of activated STAT6 in tumors was associated with short survival in an independent set of patients with primary CNS lymphoma who were treated with high-dose intravenous methotrexate therapy.

Metastasis is the spread of tumor cells from a primary site to distant organs. It is the major cause of cancer morbidity and death. In the last few decades, significant advances have been made in surgical techniques, radiation therapy delivery, and chemotherapy including the development of combination regimens and agents inhibiting newly characterized biological targets. Treatment of metastasis, however, remains the most challenging task in cancer therapy because metastatic growth relies on complex interactions between tumor cells and the host and is often resistant to all therapeutic modalities. Management of metastasis in bone is especially challenging given the difficulty of access for therapeutic agents. Contemporary research seeks to explain the striking organ specificity observed in metastasis. In this article, we will examine historic perspectives on site-specific metastasis and review cellular and molecular evidence pertinent to the mechanisms of organ specificity. We will discuss a number of studies that aim to identify gene signatures correlating with organ-selective metastasis using microarray technology. Lastly, we will discuss potential areas of future research including microRNAs, proteomics, and the development of diagnostic and therapeutic interventions.

BACKGROUND Prostate cancer incidence and mortality rates vary widely among individuals of different ethnic/racial groups. We identified a relationship between a subset of genes and race/ethnicity using gene expression profiling. Estrogen receptor α (ERα) was selected for confirmation due to its plausible biological role in cancer susceptibility. METHODS Quantitative polymerase chain reaction (Q-PCR) was used to verify gene expression results. Protein levels of ERα were determined by quantitative immunohistochemistry in a large-scale tissue microarray study (n = 183). RESULTS ERα was significantly higher in stroma of Hispanic and Asian men than in Caucasian (P

The global physiological effects of glucocorticoids are well established, and the framework of transcriptional regulation by the glucocorticoid receptor (GR) has been described. However, the genes directly under GR control that trigger these physiological effects are largely unknown. To address this issue in a single cell type, we identified glucocorticoid-responsive genes in A549 human lung adenocarcinoma cells by microarray analysis and quantitative real-time PCR. Reduction of GR expression by RNA interference diminished the effects of dexamethasone on all tested target genes, thus confirming the essential role of GR in glucocorticoid-regulated gene expression. To identify primary GR target genes, in which GR is a component of the transciptional regulatory complex, we developed a strategy that uses chromatin immunoprecipitation to scan putative regulatory regions of target genes for sites occupied by specifically bound GR. We screened 11 glucocorticoid-regulated genes, and we identified GR-binding regions for eight of them (five induced and three repressed). Thus, our approach provides a means for rapid identification of primary GR target genes and glucocorticoid-response elements, which will facilitate analyses of transcriptional regulatory mechanisms and determination of hormone-regulated gene networks.

We have constructed a high-resolution genomic microarray of human chromosome 16q, and used it for comparative genomic hybridization analysis of 16 prostate tumors. We demarcated 10 regions of genomic loss between 16q23.1 and 16qter that occurred in five or more samples. Mining expression array data from four independent studies allowed us to identify 11 genes that were frequently underexpressed in prostate cancer and that co-localized with a region of genomic loss. Quantitative expression analyses of these genes in matched tumor and benign tissue from 13 patients showed that six of these 11 (WWOX, WFDC1, MAF, FOXF1, MVD and the predicted novel transcript Q9H0B8 (NM_031476)) had significant and consistent downregulation in the tumors relative to normal prostate tissue expression making them candidate tumor suppressor genes.

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation.

189 Background: Physical activity (PA), in particular longer duration or higher intensity, may reduce the risk of PCa progression and PCa-specific mortality in men diagnosed with clinically localized PCa. However, the molecular mechanism(s) by which PA exerts its protective effect in the prostate remains unknown. We examined the correlation of PA and gene expression patterns in men with low risk prostate cancer who elected to undergo active surveillance. Methods: Morphologically normal prostate tissue was obtained from men who subsequently participated in a clinical trial focused on nutritional supplements (previously published microarray dataset #GSE27140). Of the original sample (n=84), 70 completed a brief PA questionnaire and were dichotomized based usual PA [e.g. any vigorous PA (yes/no), 3+ h/wk vigorous PA (yes/no)]. Differential expression and pathway (gene set) analyses between groups were performed using Significance Analysis of Microarrays. Genes and gene sets with a false discovery rate ≤0.10 and 0.20 were considered significant, respectively. Results: Gene expression analysis detected 184 significant genes that were differentially expressed between men who performed vigorous PA for 3+ h/wk (n=23) and those who did not (n= 47). Up-regulated genes included the known tumor suppressors, BRCA1 and BRCA2. Furthermore, pathway analysis revealed that cell cycle and DNA repair pathways were positively modulated in men who participated in 3+h/wk vigorous PA vs. not. Consistent with the data on vigorous PA and clinical outcomes in men with PCa, the duration of vigorous PA was important; there were no significant genes detected when comparing men who participated in any vigorous PA to men who did none. Conclusions: Prostate gene expression and pathway analyses revealed candidate genes and in vivo pathways that may be modulated by participating in 3+ h/wk of vigorous PA. These data provide mechanistic insight into how 3+ h/wk of vigorous PA may offer PCa-specific benefits. Furthermore, understanding the molecular mechanisms by which such PA affects normal prostate gene expression may aid the development of strategies to prevent or delay PCa progression.

Mullerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.

Haqq, Christopher M., and Patricia K. Donahoe. Regulation of Sexual Dimorphism in Mammals. Physiol. Rev. 78: 1–33, 1998. — Sexual dimorphism in humans has been the subject of wonder for centuries. In 355 BC, Aristotle postulated that sexual dimorphism arose from differences in the heat of semen at the time of copulation. In his scheme, hot semen generated males, whereas cold semen made females (Jacquart, D., and C. Thomasset. Sexuality and Medicine in the Middle Ages, 1988). In medieval times, there was great controversy about the existence of a female pope, who may have in fact had an intersex phenotype (New, M. I., and E. S. Kitzinger. J. Clin. Endocrinol. Metab. 76: 3–13, 1993.). Recent years have seen a resurgence of interest in mechanisms controlling sexual differentiation in mammals. Sex differentiation relies on establishment of chromosomal sex at fertilization, followed by the differentiation of gonads, and ultimately the establishment of phenotypic sex in its final form at puberty. Each event in sex determination depends on the preceding event, and normally, chromosomal, gonadal, and somatic sex all agree. There are, however, instances where chromosomal, gonadal, or somatic sex do not agree, and sexual differentiation is ambiguous, with male and female characteristics combined in a single individual. In humans, well-characterized patients are 46, XY women who have the syndrome of pure gonadal dysgenesis, and a subset of true hermaphrodites are phenotypic men with a 46, XX karyotype. Analysis of such individuals has permitted identification of some of the molecules involved in sex determination, including SRY (sex-determining region Y gene), which is a Y chromosomal gene fulfilling the genetic and conceptual requirements of a testis-determining factor. The purpose of this review is to summarize the molecular basis for syndromes of sexual ambiguity seen in human patients and to identify areas where further research is needed. Understanding how sex-specific gene activity is orchestrated may provide insight into the molecular basis of other cell fate decisions during development which, in turn, may lead to an understanding of aberrant cell fate decisions made in patients with birth defects and during neoplastic change.

Jerome P. Richie*, Boston, MA; Robert B. Montgomery, Seattle, WA; Christopher J. Logothetis, Houston, TX; Glenn J. Bubley, Boston, MA; Bruce L. Dalkin, Seattle, WA; Martin G. Sanda, Massimo F. Loda, Rosina T. Lis, Boston, MA; Lawrence D. True, Seattle, WA; Patricia Troncoso, Houston, TX; Elizabeth M. Genega, Steven P. Balk, Boston, MA; Elahe A. Mostaghel, Seattle, WA; Trevor M. Penning, Philadelphia, PA; Peter S. Nelson, Seattle, WA; Wanling Xie, Boston, MA; Christopher M. Haqq, NamPhuong Tran, Weimin Peng, Los Angeles, CA; Daniel Tamae, Philadelphia, PA; Thian Kheoh, Arturo Molina, Los Angeles, CA; Philip W. Kantoff, Mary-Ellen Taplin, Boston, MA

Background Abiraterone acetate improved overall survival in metastatic castration-resistant prostate cancer at a preplanned interim analysis of the COU-AA-301 double-blind, placebo-controlled phase 3 study. Here, we present the final analysis of the study before crossover from placebo to abiraterone acetate (after 775 of the prespecified 797 death events). Methods Between May 8, 2008, and July 28, 2009, this study enrolled 1195 patients at 147 sites in 13 countries. Patients were eligible if they had metastatic castration-resistant prostate cancer progressing after docetaxel. Patients were stratified according to baseline Eastern Cooperative Oncology Group (ECOG) performance status, worst pain over the past 24 h on the Brief Pain Inventory-Short Form, number of previous chemotherapy regimens, and type of progression. Patients were randomly assigned (ratio 2:1) to receive either abiraterone acetate (1000 mg, once daily and orally) plus prednisone (5 mg, orally twice daily) or placebo plus prednisone with a permuted block method via an interactive web response system. The primary endpoint was overall survival, analysed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00638690. Findings Of the 1195 eligible patients, 797 were randomly assigned to receive abiraterone acetate plus prednisone (abiraterone group) and 398 to receive placebo plus prednisone (placebo group). At median follow-up of 20·2 months (IQR 18·4—22·1), median overall survival for the abiraterone group was longer than in the placebo group (15·8 months [95% CI 14·8—17·0] vs 11·2 months [10·4—13·1]; hazard ratio [HR] 0·74, 95% CI 0·64—0·86; p

Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.

Background Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial. Methods Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients. Results Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001). Conclusions We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients. Trial Registration ClinicalTrials.gov NCT00402285

Purpose Persistence of ligand-mediated androgen receptor signaling has been documented in castration-resistant prostate cancers (CRPCs). Abiraterone acetate (AA) is a potent and selective inhibitor of CYP17, which is required for androgen biosynthesis in the testes, adrenal glands, and prostate tissue. This trial evaluated the efficacy and safety of AA in combination with prednisone to reduce the symptoms of secondary hyperaldosteronism that can occur with AA monotherapy. Patients and Methods Fifty-eight men with progressive metastatic CRPC who experienced treatment failure with docetaxel-based chemotherapy received AA (1,000 mg daily) with prednisone (5 mg twice daily). Twenty-seven (47%) patients had received prior ketoconazole. The primary outcome was ≥ 50% prostate-specific antigen (PSA) decline, with objective response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and changes in Eastern Cooperative Oncology Group (ECOG) performance status (PS) and circulating tumor cell (CTC) numbers. Safety was also evaluated. Results A ≥ 50% decline in PSA was confirmed in 22 (36%) patients, including 14 (45%) of 31 ketoconazole-naïve and seven (26%) of 27 ketoconazole-pretreated patients. Partial responses were seen in four (18%) of 22 patients with RECIST-evaluable target lesions. Improved ECOG PS was seen in 28% of patients. Median time to PSA progression was 169 days (95% CI, 82 to 200 days). CTC conversions with treatment from ≥ 5 to < 5 were noted in 10 (34%) of 29 patients. The majority of AA-related adverse events were grade 1 to 2, and no AA-related grade 4 events were seen. Conclusion AA plus prednisone was well tolerated, with encouraging antitumor activity in heavily pretreated CRPC patients. The incidence of mineralocorticoid-related toxicities (hypertension or hypokalemia) was reduced by adding low-dose prednisone. The combination of AA plus prednisone is recommended for phase III investigations.

Purpose: To determine the prognostic significance of a multimarker assay incorporating expression levels of three molecular markers in primary cutaneous melanoma. Experimental Design: We assessed expression levels of NCOA3, SPP1, and RGS1 using immunohistochemical analysis in a tissue microarray cohort of 395 patients. For each marker, we identified optimal cut-points for expression intensity to predict disease-specific survival (DSS) and, as a secondary endpoint, sentinel lymph node (SLN) status. The cumulative overexpression of all three markers was embodied in a multimarker index, and its prognostic effect on DSS and SLN status was assessed using Cox regression, Kaplan-Meier analysis, and logistic regression. The prognostic effect of this multimarker assay on DSS was assessed in an independent cohort of 141 patients, in which marker expression levels were scored using immunohistochemical analysis of stained tissue sections. Results: Increasing multimarker index scores were significantly predictive of reduced DSS and increased SLN metastasis in the 395-patient cohort. Multivariate logistic regression analysis revealed multimarker expression scores as an independent predictor of SLN status (P = 0.001). Multivariate Cox regression analysis showed the independent effect of the multimarker index on DSS (P < 0.001). The multimarker index was the most significant factor predicting DSS when compared with other clinical and histologic factors, including SLN status (P = 0.002). Multimarker expression scores were also the most significantly predictive of DSS in the independent cohort (P = 0.01). Conclusions: These results describe a multimarker assay with independent prognostic effect on the prediction of survival associated with melanoma in two distinct cohorts. (Clin Cancer Res 2009;15(22):698792)

IkappaBgamma is one member of a family of proteins that can inhibit the nuclear localization of nuclear factor-kappaB. However, the other specific functions of IkappaBgamma are still poorly understood, and its effects on tumor metastasis have not yet been characterized. We examined the consequences of targeting IkappaBgamma in melanoma cells using a hammerhead ribozyme. We developed stable transformant B16-F10 melanoma cell lines that express a ribozyme that targets mouse IkappaBgamma (IkappaBgamma-144-Rz). Tail-vein injection of B16-F10 cells that stably express IkappaBgamma-144-Rz into mice resulted in a significant reduction of the metastatic potential of these cells. IkappaBgamma-144-Rz-expressing B16 cells were shown to have increased transcriptional activity of nuclear factor-kappaB. We then showed that IkappaBgamma-144-Rz-expressing cells demonstrated both reduced invasion and increased apoptosis, suggesting the existence of pathways through which IkappaBgamma promotes melanoma metastasis. Using gene expression profiling, we identified a differentially expressed gene set that is regulated by the stable suppression of IkappaBgamma that may participate in mediating its anti-metastatic effects; we also confirmed the altered expression levels of several of these genes by quantitative real time polymerase chain reaction. Plasmid-mediated expression of IkappaBgamma-144-Rz produced a significant inhibition of the metastatic progression of B16-F10 cells to the lung and resulted in significant anti-invasive and pro-apoptotic effects on murine Lewis lung carcinoma cells. Our results suggest a novel role for IkappaBgamma in promoting the metastatic progression of melanoma.

BACKGROUND. Osteopontin has been suggested as a marker of disease progression in patients with melanoma because of its overexpression in recent microarray analyses. However, its prognostic role in melanoma has not been fully defined. METHODS. Osteopontin expression status was examined using immunohistochemical analysis of a tissue microarray that contained primary cutaneous melanomas from 345 patients. The correlation between osteopontin expression and several histologic markers for melanoma was assessed by using the Chi-square test and the Le directional test. The impact of osteopontin expression on recurrence-free survival (RFS) and disease-specific survival (DSS) of patients with melanoma was examined using Cox regression and Kaplan-Meier analyses. The impact of increasing osteopontin expression on sentinel lymph node (SLN) metastasis was assessed using logistic regression analysis. RESULTS. High osteopontin expression was associated with increased tumor thickness (P = .037), Clark level (P = .035), and mitotic index (P = .046). Kaplan-Meier analysis demonstrated an association between osteopontin expression and reduced RFS (P < .03) and DSS (P = .05). Multivariate Cox regression analysis demonstrated that high osteopontin immunostaining had an independent impact on the DSS of this melanoma cohort (P = .049). In addition, osteopontin expression was significantly predictive of SLN metastasis (P = .009) and SLN burden, as assessed by the mean number of SLN metastases (P = .0025). Multivariate logistic regression analysis demonstrated an independent role for osteopontin expression in predicting SLN status (P = .0062). CONCLUSIONS. The current results validated the role of osteopontin as an independent prognostic marker for melanoma and provided new evidence for its predictive role in melanoma lymph node metastasis. Cancer 2008. © 2007 American Cancer Society.

The androgen receptor (AR) mediates the physiologic and pathophysiologic effects of androgens including sexual differentiation, prostate development, and cancer progression by binding to genomic androgen response elements (AREs), which influence transcription of AR target genes. The composition and context of AREs differ between genes, thus enabling AR to confer multiple regulatory functions within a single nucleus. We used expression profiling of an immortalized human prostate epithelial cell line to identify 205 androgen-responsive genes (ARGs), most of them novel. In addition, we performed chromatin immunoprecipitation to identify 524 AR binding regions and validated in reporter assays the ARE activities of several such regions. Interestingly, 67% of our AREs resided within ∼50 kb of the transcription start sites of 84% of our ARGs. Indeed, most ARGs were associated with two or more AREs, and ARGs were sometimes themselves linked in gene clusters containing up to 13 AREs and 12 ARGs. AREs appeared typically to be composite elements, containing AR binding sequences adjacent to binding motifs for other transcriptional regulators. Functionally, ARGs were commonly involved in prostate cell proliferation, communication, differentiation, and possibly cancer progression. Our results provide new insights into cell- and gene-specific mechanisms of transcriptional regulation of androgen-responsive gene networks.

Recent studies have demonstrated a role for telomerase in driving tumor progression, but its mechanism of action remains unclear. Here we show that stable, ribozyme-mediated suppression of mouse telomerase RNA reduced telomerase RNA expression, telomerase activity, and telomere length, which significantly reduced tumor invasion and metastatic potential. Our studies reveal that previously unidentified effects of telomerase may mediate its tumor-promoting effects. First, reducing telomerase activity induced a more dendritic morphology, accompanied by increased melanin content and increased expression of tyrosinase, a key enzyme in melanin biosynthesis. Second, gene expression profiling revealed that telomerase targeting down-regulated expression of several glycolytic pathway genes, with a corresponding decrease in glucose consumption and lactate production. Thus, telomerase activity controls the glycolytic pathway, potentially altering the energy state of tumor cells and thereby modulating tyrosinase activity and melanin production. These studies have important implications for understanding the mechanisms by which telomerase promotes tumor invasion and metastasis.

Little is known about the mechanism of bladder smooth muscle differentiation. We hypothesize that epithelial-mesenchymal signaling induces the expression of smooth muscle proteins in bladder mesenchyme resulting in smooth muscle differentiation. We confirmed that smooth muscle differentiation in the mouse urinary bladder occurs first at gestational day 14 (E14) based upon immunohistochemical localization of smooth muscle alpha-actin (SMAA). To investigate murine bladder smooth muscle differentiation and epithlelial-mesenchymal signaling in the developing bladder, we analyzed gene expression profiles of intact embryonic murine bladders and separated epithelial and mesenchymal components at embryonic days E13, E14, E15, E16, and postnatal day 1 (P1). Using cDNA microarray, we identified regulators of vascular smooth muscle differentiation in bladder mesenchyme, including serum response factor (SRF) and its cofactors, ELK1 and SRF accessory protein (SAP)1, as well as two SRF-associated pathways, angiotension receptor II and transforming growth factor- beta2. Immunohistochemistry showed diffuse expression of SRF in the bladder at E12 with localization of expression to the peripheral mesenchyme at E13 and E14. Our results suggest that bladder smooth muscle differentiation may share a similar gene expression program as occurs during vascular smooth muscle differentiation. The unique structure of the urinary bladder makes it an ideal model for studies of smooth muscle differentiation and epithelial-mesenchymal signaling.

Because of the paucity of available tissue, little information has previously been available regarding the gene expression profiles of primary melanomas. To understand the molecular basis of melanoma progression, we compared the gene expression profiles of a series of nevi, primary melanomas, and melanoma metastases. We found that metastatic melanomas exhibit two dichotomous patterns of gene expression, which unexpectedly reflect gene expression differences already apparent in comparing laser-capture microdissected radial and vertical phases of a large primary melanoma. Unsupervised hierarchical clustering accurately separated nevi and primary melanomas. Multiclass significance analysis of microarrays comparing normal skin, nevi, primary melanomas, and the two types of metastatic melanoma identified 2,602 transcripts that significantly correlated with sample class. These results suggest that melanoma pathogenesis can be understood as a series of distinct molecular events. The gene expression signatures identified here provide the basis for developing new diagnostics and targeting therapies for patients with malignant melanoma.