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PDF file 90K, Supplementary Figure Legends

PDF file 79K, GAG-HERV-K ch22q11.23 features. Endogenous retrovirus K from chromosome 22 is located on the q11.23 region, nearby the BCR, ZDHHC8P, IGLL1 and RGL4 genes. HERV-K ch22q11.23 has GAG-PRO, POL and ENV genes flanked by LTR sequences. The GAG gene has an ORF encoding a 715aa protein that contains P10, P24 and Zinc finger domains. {delta}: deletions; I: early stop codons; --> primer binding regions; ABS: antibody binding site for monoclonal antibody TI-35 (see Supplementary Fig. S3C)

Supplementary Data from Pharmacodynamic and Clinical Results from a Phase I/II Study of the HSP90 Inhibitor Onalespib in Combination with Abiraterone Acetate in Prostate Cancer

Purpose:Onalespib is a potent, fragment-derived second-generation HSP90 inhibitor with preclinical activity in castration-resistant prostate cancer (CPRC) models. This phase I/II trial evaluated onalespib in combination with abiraterone acetate (AA) and either prednisone or prednisolone (P) in men with CRPC progressing on AA/P.Patients and Methods:Patients with progressing CRPC were randomly assigned to receive 1 of 2 regimens of onalespib combined with AA/P. Onalespib was administered as intravenous infusion starting at 220 mg/m2 once weekly for 3 of 4 weeks (regimen 1); or at 120 mg/m2 on day 1 and day 2 weekly for 3 of 4 weeks (regimen 2). Primary endpoints were response rate and safety. Secondary endpoints included evaluation of androgen receptor (AR) depletion in circulating tumor cells (CTC) and in fresh tumor tissue biopsies.Results:Forty-eight patients were treated with onalespib in combination with AA/P. The most common ≥grade 3 toxicities related to onalespib included diarrhea (21%) and fatigue (13%). Diarrhea was dose limiting at 260 and 160 mg/m2 for regimens 1 and 2, respectively. Transient decreases in CTC counts and AR expression in CTC were observed in both regimens. HSP72 was significantly upregulated following onalespib treatment, but only a modest decrease in AR and GR was shown in paired pre- and posttreatment tumor biopsy samples. No patients showed an objective or PSA response.Conclusions:Onalespib in combination with AA/P showed mild evidence of some biological effect; however, this effect did not translate into clinical activity, hence further exploration of this combination was not justified.

PDF file 204K, Alignment of GAG proteins derived from human endogenous retroviruses and respective epitopes recognized by prostate cancer patient sera. A, Detail alignment from several GAG-HERV-K family members and the TI-35 binding site (red) highlighting the amino-acid differences present in GAG-HERV-K ch22q11.23 (blue). B, Western-blot analysis of immunoprecipitation of GAG-HERV-K from VCaP and LNCaP prostate cancer cell lines. WFP prostate cancer patient serum (WFP) was incubated with VCaP or LNCaP protein extract for 2 hours at 4{degrees}C. Protein-antibody complexes were pulled down using anti-human IgG sepharose beads. The pull-down pellet was submitted to SDS-PAGE and Western-blot analysis. Reactivity to GAG-HERV-K protein was developed by incubating the membrane with TI-35 monoclonal antibody. A pool of healthy donor sera was used as control (Con). C, Summary of ELISA results from epitope mapping using 20mers peptides overlapping by 10aa (71 peptides tested in total from JPT Innovative Peptide Solution, Berlin, Germany) spanning GAG-HERV-K and tested individually for recognition by monoclonal antibody TI-35 and different prostate cancer patient sera seropositive for GAG-HERV-K protein. The square symbol represents a positive reaction to the corresponding peptide (labeled by aminoacid start and end position). A pool of healthy donor sera was used as control (Con pool). Plates were coated with 5 uM peptide and the reaction was performed as previously described in methods

Purpose: Human endogenous retroviruses (HERV) encode 8% of the human genome. While HERVs may play a role in autoimmune and neoplastic disease, no mechanistic association has yet been established. We studied the expression and immunogenicity of a HERV-K GAG protein encoded on chromosome 22q11.23 in relation to the clinical course of prostate cancer.Experimental Design:In vitro expression of GAG-HERV-K was analyzed in panels of normal and malignant tissues, microarrays, and cell lines, and effects of demethylation and androgen stimulation were evaluated. Patient sera were analyzed for seroreactivity to GAG-HERV-K and other self-antigens by ELISA and seromics (protein array profiling).Results: GAG-HERV-K expression was most frequent in prostate tissues and regulated both by demethylation of the promoter region and by androgen stimulation. Serum screening revealed that antibodies to GAG-HERV-K are found in a subset of patients with prostate cancer (33 of 483, 6.8%) but rarely in male healthy donors (1 of 55, 1.8%). Autoantibodies to GAG-HERV-K occurred more frequently in patients with advanced prostate cancer (29 of 191 in stage III–IV, 21.0%) than in early prostate cancer (4 of 292 in stages I–II, 1.4%). Presence of GAG-HERV-K serum antibody was correlated with worse survival of patients with prostate cancer, with a trend for faster biochemical recurrence in patients with antibodies to GAG-HERV-K.Conclusions: Preferential expression of GAG-HERV-K ch22q11.23 in prostate cancer tissue and increased frequency of autoantibodies observed in patients with advanced prostate cancer make this protein one of the first bona fide retroviral cancer antigens in humans, with potential as a biomarker for progression and biochemical recurrence rate of prostate cancer. Clin Cancer Res; 19(22); 6112–25. ©2013 AACR.

PDF file 63K, Correlation between antibodies against GAG-HERV-K and group risk or Gleason score in cancer patients. Prostate cancer patients were split (A) in 3 groups according to Gleason score and classified from group risk 1 to 3 and compared to healthy donors (HD), or (B) in 3 groups according to Gleason score as indicated. Antibodies anti-GAG-HERV-K were detected and analyzed as in Fig. 4. Asterisk denotes significantly higher frequency of seropositive patients (titer > 100) in risk group 3 and Gleason {greater than or equal to} 8 (Chi-square test, p

PDF file 341K, qPCR gene expression analysis of chromosome 22q11.23 region in cancer tissues. Total RNA from cancer tissues was submitted to DNAse treatment and reverse transcriptase reaction for cDNA synthesis. The resulting cDNA was used on qPCR to detect the expression of GAG-HERV-K and the neighbor genes BCR, IGGL1, RGL4 and ZDHHC8P. The results are expressed as relative expression to GAG-HERV-K levels in normal prostate. GAG-HERV-K expression is relative to GAPDH. BCR, IGGL1, RGL4 and ZDHHC8P expression is relative to TFRC

In prostate cancer, there is considerable evidence that tumors promote immune tolerance starting early in the disease. By suppressing tumors and activating immune system homeostatic mechanisms, chemotherapy may help overcome this tumor-induced immune tolerance. As such, chemotherapy may therefore support improved results from novel immune-modulating therapies. Prostate cancer is particularly suited for active immunotherapy because prostate tumor cells express a number of distinctive surface antigens. Sipuleucel-T, which has recently been approved in the United States, is an active immunotherapy that triggers T-cell responses against prostate cancer. An exploratory analysis of phase III trial participants found a substantial survival benefit to receiving docetaxel some months after sipuleucel-T. However, VITAL-2, a phase III trial investigating a prostate cancer therapeutic vaccine plus concurrent docetaxel versus standard docetaxel therapy in advanced prostate cancer, observed lower overall survival with the vaccine regimen. This trial highlights major unresolved questions concerning the optimum choice, dosing, and timing of chemotherapy relative to active immunotherapy. Patient characteristics, prostate cancer disease stage, and treatment history also may influence the response to combined therapy. Advances in biomarker validation and trial design are needed to efficiently investigate these issues.

The purpose is to determine a safe weekly dose of i.v. estramustine phosphate (EMP) to combine with weekly paclitaxel and monthly carboplatin in patients with advanced prostate cancer.Patients with advanced prostate cancer (castrate and noncastrate) were administered escalating doses of weekly 1-h infusion of i.v. EMP (500-1000-1500 mg/m(2)) in combination with weekly paclitaxel (100 mg/m(2) over 1 h) and i.v. carboplatin (area under the curve 6 mg/ml-min every 4 weeks). Four weeks of therapy were considered one cycle. In the first three cohorts, EMP was given i.v. 3 h before paclitaxel. Cohorts 4 and 5 reversed the administration order: EMP (doses 1000-1500 mg/m(2)) was given immediately after the end of paclitaxel infusion. Plasma levels of EMP and its metabolites, estramustine and estromustine, were monitored at time 0, at 120 min, and approximately at 20, 21, and 168 h from the start of EMP infusion. Paclitaxel concentrations were determined at basal (0), 30, 60, 90, and 120 min and 18 h after the start of paclitaxel infusion, and a concentration-time curve was estimated. Pharmacokinetic evaluation was performed in cycles 1 and 2 during the first week of therapy.Nineteen patients were entered on the initial three dose levels (cohorts 1-3). Dose-limiting transient hepatic toxicity was encountered in cohort 3 (EMP = 1500 mg/m(2)). An additional 13 patients were treated with paclitaxel (100 mg/m(2)) first, followed by i.v. EMP at 1000 mg/m(2) (cohort 4), and 1500 mg/m(2) (cohort 5). No dose-limiting toxicities were seen, and cohort 5 was determined safe for Phase II studies. Thromboembolic events were observed in 9% of patients (no prophylactic coumadin was used). Plasma concentrations of EMP and metabolites increased proportionally with dose. In all cohorts, there was a slight decrease in EMP and estramustine plasma concentrations between cycles 1 and 2. Although not significant, higher levels of estromustine at cycle 2 were observed in comparison to cycle 1. Decreased clearance of paclitaxel leading to higher than expected paclitaxel plasma concentrations was observed during the first cycle of therapy. Paclitaxel plasma concentrations were lower during cycle 2. In 17 patients with androgen-independent disease, 59% had/=50% posttherapy decline in PSA and 22% showed measurable disease regression.The regimen of weekly i.v. EMP in combination with paclitaxel and carboplatin can be safely administered with hepatic toxicity being transient and reversible. Pharmacokinetic results suggest that EMP competitively inhibits the biotransformation of paclitaxel after the first administration. This effect is counterbalanced, after repeated administrations, by a possible induction of the metabolic system caused by EMP. Phase II testing is ongoing to evaluate the efficacy of this combination.

SLOVIN-ELA, SUSAN, and KOHEN-RAZ, R. Developmental Differences in Primary Reaching Responses of Young Infants from Varying Social Backgrounds. CHILD DEVELOPMENT, 1978, 49, 132-140. Infants 4 and 7 months old, from disadvantaged and middle-class homes and from institutions, were tested for various reaching and manipulation responses to a static and moving red ring. Results showed that at each age level infants from middle-class homes had more developed reaching and manipulation responses than infants in unstimulating disadvantaged homes and institutions, demonstrating that the developmental lag of the disadvantaged infants can be detected as early as 4 months. By 7 months of age all disadvantaged and institutionalized infants had developed the reaching behaviors present in the middle-class infants at 4 months, thus indicating that sociocultural factors may change the rate of development but not its course.