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Purpose: This trial aimed to assess the efficacy and safety of olipudase alfa enzyme replacement therapy for non–central nervous system manifestations of acid sphingomyelinase deficiency (ASMD) in adults. Methods: A phase 2/3, 52 week, international, double-blind, placebo-controlled trial (ASCEND; NCT02004691/EudraCT 2015-000371-26) enrolled 36 adults with ASMD randomized 1:1 to receive olipudase alfa or placebo intravenously every 2 weeks with intrapatient dose escalation to 3 mg/kg. Primary efficacy endpoints were percent change from baseline to week 52 in percent predicted diffusing capacity of the lung for carbon monoxide and spleen volume (combined with splenomegaly-related score in the United States). Other outcomes included liver volume/function/sphingomyelin content, pulmonary imaging/function, platelet levels, lipid profiles, and pharmacodynamics. Results: Least square mean percent change from baseline to week 52 favored olipudase alfa over placebo for percent predicted diffusing capacity of the lung for carbon monoxide (22% vs 3.0% increases, P =.0004), spleen volume (39% decrease vs 0.5% increase, P

Copy number for AR-V7 and ARv567es stratified by PSA response AR, androgen receptor; IQ, interquartile; PSA50, 50% reduction from baseline in prostate-specific antigen; SD, standard deviation.

Assay sensitivity in 22RV1 prostate cancer cells spiked-in healthy donor blood run through the GEDI device. AR-FL (blue) and AR-V7 (red) mRNA expression (#copies per sample) was determined by ddPCR in varying amounts of 22RV1 cells in the presence of healthy donor blood run through the GEDI device. The assay, reliably and reproducibly detects both transcripts in single spiked-in cells. The table below the graph shows the raw data (copy number) for each transcript per condition. Healthy donor blood PBMCs alone were used as a control. AR, androgen receptor; ddPCR, droplet digital polymerase chain reaction; FL, full length; GEDI, geometrically enhanced differential immunocapture; PBMC, peripheral blood mononuclear cell.

Copy number for AR-FL, AR-V7 and ARv567es for the evaluable population at baseline AR, androgen receptor; FL, full length.

ddPCR primer and probe sequences AR, androgen receptor; FL, full length.

Visual representation of AR-FL and AR splice variant expression per patient at baseline Data are shown as positive (green for AR-FL; red for AR-V7; blue for ARv567es) or negative (white boxes) for each transcript. AR, androgen receptor; FL, full length

AR-FL and AR-V expression in healthy donor blood run through the GEDI device. The mRNA expression levels of AR-FL (blue), AR-v7 (red) and AR-v567es (green) were analyzed in healthy donor blood from 10 volunteers. As previously described, 1 mL of healthy donor peripheral blood was processed through the GEDI microfluidic device. The table below the graph shows the raw data (copy number) for each transcript per sample. AR, androgen receptor; ddPCR, droplet digital polymerase chain reaction; FL, full length; GEDI, geometrically enhanced differential immunocapture; mRNA, messenger ribonucleic acid.

Venglustat inhibits the enzymatic conversion of ceramide to glucosylceramide, reducing available substrate for the synthesis of more complex glycosphingolipids. It offers a potential new approach to the treatment of patients with Fabry disease (α-Gal A deficiency), in whom progressive accumulation of such glycosphingolipids, including globotriaosylceramide (GL-3), in the lysosomes of a wide range of cell types often leads to vital organ complications in adulthood. An international, open-label, single-arm, Phase 2a uncontrolled 26-week clinical study (NCT02228460) and a 130-week extension study (NCT02489344) were conducted to assess the safety, pharmacodynamics, pharmacokinetics, and exploratory efficacy of 15 mg once daily oral venglustat in treatment-naïve adult male patients with classic Fabry disease. Of 11 patients (18-37 years old) who initially enrolled, nine completed the 26-week study and seven completed the extension study. A total of 169 treatment-emergent adverse events (TEAEs) were reported by nine patients, the majority being mild (73%) and unrelated to the study drug (70%). Nine serious TEAEs (serious adverse events) and 11 severe TEAEs, including a self-harm event, were reported. No deaths or treatment-related life-threatening adverse events were reported. Skin GL-3 scores in superficial skin capillary endothelium (SSCE), estimated by light microscopy, were unchanged from baseline at Week 26 in five patients, decreased in three patients, and increased in one patient. There was no significant change in GL-3 scores or significant shift in grouped GL-3 scores. Five of six patients had reductions from baseline in GL-3 score at the end of the extension study. At Weeks 26 and 156 the mean (standard deviation) changes from baseline in the fraction of the volume of SSCE cytoplasm occupied by GL-3 inclusions, measured by electron microscopy unbiased stereology, were -0.06 (0.03) (p = 0.0010) and -0.12 (0.04) (p = 0.0008), respectively. Venglustat treatment reduced markers in the synthetic and degradative pathway of major glycosphingolipids; proximal markers reduced rapidly and more distal markers (plasma GL-3 and globotriaosylsphingosine) reduced progressively. There were no biochemical or histological indications of progression of Fabry disease over 3 years of follow-up. These findings confirm target engagement and the pharmacodynamic effects of venglustat in adult males with classic Fabry disease. However, further clinical evaluation in larger studies is needed to determine efficacy and safety.

Mini-COMET (NCT03019406; Sanofi) is a phase 2, open-label, ascending-dose, 3-cohort study, evaluating avalglucosidase alfa safety, pharmacokinetics, and efficacy in individuals with infantile-onset Pompe disease aged18 years who previously received alglucosidase alfa and showed clinical decline (cohorts 1 and 2) or suboptimal response (cohort 3).During a 25-week primary analysis period, cohorts 1 and 2 received avalglucosidase alfa 20 and 40 mg/kg every other week, respectively, for 6 months, whereas cohort 3 individuals were randomized (1:1) to receive avalglucosidase alfa 40 mg/kg every other week or alglucosidase alfa (current stable dose) for 6 months.In total, 22 individuals were enrolled (cohort 1 [n = 6], cohort 2 [n = 5], cohort 3-avalglucosidase alfa [n = 5], and cohort 3-alglucosidase alfa [n = 6]). Median treatment compliance was 100%. None of the individuals discontinued treatment or died. Percentages of individuals with treatment-emergent adverse events were similar across dose and treatment groups. No serious or severe treatment-related treatment-emergent adverse events occurred. Trends for better motor function from baseline to week 25 were observed for 40 mg/kg every other week avalglucosidase alfa compared with either 20 mg/kg every other week avalglucosidase alfa or alglucosidase alfa up to 40 mg/kg weekly.These data support the positive clinical effect of avalglucosidase alfa in patients with infantile-onset Pompe disease previously declining on alglucosidase alfa.

Background and Aims Fabry disease (FD) is a rare, X-linked, genetic disorder caused by mutations in the gene for the lysosomal enzyme alpha-galactosidase A (αGal-A). The progressive accumulation of glycosphingolipids, most notably globotriaosylceramide (GL-3), in lysosomes of a variety of organs and cell types leads, over decades, to renal, cardiovascular, and other clinical manifestations. In a phase 2 study, glucosylceramide synthase inhibition with venglustat led to a progressive reduction in plasma GL-3 to low-normal levels. Scoring by light microscopy showed a reduction in lysosomal GL-3 inclusions in skin capillary endothelial cells (EC) after 3 years of therapy, although not after 6 months. To further characterize the effect of venglustat using more precise methods, we applied unbiased stereological methods to quantitate GL-3 inclusions in skin ECs. Methods Skin biopsies were obtained from classic male Fabry disease patients (N = 11) at baseline and during daily treatment with venglustat in a phase 2 study (NCT02228460, NCT02489344). Images from at least 50 superficial skin capillaries were obtained using transmission electron microscopy at 7,500 X magnification. The fraction of the volume (Vv) of cytoplasm occupied by GL-3 inclusions [Vv(Inc/Endo)] in all ECs in these capillaries was estimated using point counting by a masked reader. Two-sided paired t tests were used to evaluate differences between baseline and post-treatment values at each time point. Results Venglustat therapy led to a significant reduction from baseline in Vv(Inc/Endo) of 21.1% after 6 months and 38.7% after 3 years of treatment (Figure). Conclusions Treatment with venglustat led to a reduction in GL-3 inclusion fractional volume in skin capillary ECs which was detectable after 6 months using precise quantitative methods. This was followed by further reduction over the next 2 1/2 years. We posit that, in the absence of αGal-A activity, inhibition of GL-3 production with venglustat allowed other enzymatic or non-enzymatic mechanisms to progressively reduce EC lysosomal GL-3 content, and that long-term venglustat therapy may therefore prevent or reverse progressive tissue injury in Fabry disease.

The liver is a major site of lipoprotein synthesis and metabolism. Liver manifestations of chronic visceral ASMD include hepatomegaly, fibrosis, elevated liver enzymes and a pro-atherogenic lipid profile. Measurements of sphingomyelin (SM) levels in liver biopsies and lyso-SM in plasma were used as pharmacodynamic biomarkers. Five adult patients with chronic visceral ASMD were enrolled in a 26-week phase 1b trial of enzyme replacement therapy (ERT) with olipudase alfa ( NCT01722526 ) followed by an ongoing long-term extension study ( NCT02004704 ). We compare the changes in hepatic SM levels, plasma lyso-SM, and lipoprotein profiles after 42 months of treatment. Progressive clearance of histologic SM storage was observed throughout the trial, along with similar reductions in plasma lyso-SM. Improvements in liver enzymes were observed at 6 months and remained stable at 42 months. Progressive reductions from baseline in pro-atherogenic lipid profiles (total cholesterol, LDL-C, VLDL-C, triglycerides) were observed at month 6 and 42. Conversely, there were progressive increases in anti-atherogenic markers, HDL-C and apolipoprotein A-I, with HDL-C increases up to 200% over baseline levels after 42 months of treatment. These data demonstrate that hepatic clearance of SM during olipudase alfa treatment over 42 months is associated with overall improvements in the lipid profiles of ASMD patients. The clinical relevance of these findings needs to be determined in the future, but we speculate that these improvements may reduce the risk for liver cirrhosis and cardiovascular disease. Trial registration: Clintrials.gov trial registration # NCT01722526

Purpose: Biomarkers aiding treatment optimization in metastatic castration-resistant prostate cancer (mCRPC) are scarce. The presence or absence of androgen receptor (AR) splice variants, AR-V7 and ARv567es, in mCRPC patient circulating tumor cells (CTC) may be associated with taxane treatment outcomes. Experimental Design: A novel digital droplet PCR (ddPCR) assay assessed AR-splice variant expression in CTCs from patients receiving docetaxel or cabazitaxel in TAXYNERGY (NCT01718353). Patient outcomes were examined according to AR-splice variant expression, including prostate-specific antigen (PSA)50 response and progression-free survival (PFS). Results: Of the 54 evaluable patients, 36 (67%) were AR-V7+, 42 (78%) were ARv567es+, 29 (54%) were double positive, and 5 (9%) were double negative. PSA50 response rates at any time were numerically higher for AR-V7− versus AR-V7+ (78% vs. 58%; P = 0.23) and for ARv567es− versus ARv567es+ (92% vs. 57%; P = 0.04) patients. When AR-V mRNA status was correlated with change in nuclear AR from cycle 1 day 1 to day 8 (n = 24), AR-V7+ patients (n = 16) had a 0.4% decrease versus a 12.9% and 26.7% decrease in AR-V7−/ARv567es− (n = 3) and AR-V7−/ARv567es+ (n = 5) patients, respectively, suggesting a dominant role for AR-V7 over ARv567es. Median PFS was 12.02 versus 8.48 months for AR-V7− versus AR-V7+ (HR = 0.38; P = 0.01), and 12.71 versus 7.29 months for ARv567es− versus ARv567es+ (HR = 0.37; P = 0.02). For AR-V7+, AR-V7−/ARv567es+, and AR-V7−/ARv567es− patients, median PFS was 8.48, 11.17, and 16.62 months, respectively (P = 0.0013 for trend). Conclusions: Although detection of both CTC-specific AR-V7 and ARv567es by ddPCR influenced taxane outcomes, AR-V7 primarily mediated the prognostic impact. The absence of both variants was associated with the best response and PFS with taxane treatment. See related commentary by Dehm et al., p. 1696

Purpose The TAXYNERGY trial ( ClinicalTrials.gov identifier: NCT01718353) evaluated clinical benefit from early taxane switch and circulating tumor cell (CTC) biomarkers to interrogate mechanisms of sensitivity or resistance to taxanes in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer. Patients and Methods Patients were randomly assigned 2:1 to docetaxel or cabazitaxel. Men who did not achieve ≥ 30% prostate-specific antigen (PSA) decline by cycle 4 (C4) switched taxane. The primary clinical endpoint was confirmed ≥ 50% PSA decline versus historical control (TAX327). The primary biomarker endpoint was analysis of post-treatment CTCs to confirm the hypothesis that clinical response was associated with taxane drug-target engagement, evidenced by decreased percent androgen receptor nuclear localization (%ARNL) and increased microtubule bundling. Results Sixty-three patients were randomly assigned to docetaxel (n = 41) or cabazitaxel (n = 22); 44.4% received prior potent androgen receptor–targeted therapy. Overall, 35 patients (55.6%) had confirmed ≥ 50% PSA responses, exceeding the historical control rate of 45.4% (TAX327). Of 61 treated patients, 33 (54.1%) had ≥ 30% PSA declines by C4 and did not switch taxane, 15 patients (24.6%) who did not achieve ≥ 30% PSA declines by C4 switched taxane, and 13 patients (21.3%) discontinued therapy before or at C4. Of patients switching taxane, 46.7% subsequently achieved ≥ 50% PSA decrease. In 26 CTC-evaluable patients, taxane-induced decrease in %ARNL (cycle 1 day 1 v cycle 1 day 8) was associated with a higher rate of ≥ 50% PSA decrease at C4 ( P = .009). Median composite progression-free survival was 9.1 months (95% CI, 4.9 to 11.7 months); median overall survival was not reached at 14 months. Common grade 3 or 4 adverse events included fatigue (13.1%) and febrile neutropenia (11.5%). Conclusion The early taxane switch strategy was associated with improved PSA response rates versus TAX327. Taxane-induced shifts in %ARNL may serve as an early biomarker of clinical benefit in patients treated with taxanes.

134 Background: A better understanding of taxane sensitivity/resistance in mCRPC is needed to optimize treatment. Preclinically, taxane efficacy has been linked to the ability of microtubules (MT) to inhibit AR nuclear trafficking. In this prospective biomarker study, we used circulating tumor cells (CTCs) from patients (pts) in TAXYNERGY to perform real-time analysis of AR nuclear localization (ARNL) and MT stabilization (bundling; MTB) in order to predict taxane sensitivity. Methods: TAXYNERGY (NCT01718353) is a phase 2 trial randomizing chemo-naïve mCRPC pts 2:1 to docetaxel or cabazitaxel, with a switch to the alternative taxane in the absence of a ≥ 30% PSA drop by C4. Here we present the co-primary biomarker endpoints. CTCs at baseline (C1D1) were compared to CTCs after 1 week of taxane treatment (C1D8), and were analyzed by multiplex confocal microscopy for %ARNL (integrated AR intensity in the cell and nuclear areas) and MTB (assessed for increase compared to C1D1 on a scale from 0–3 from no to most MTB increase). Associations between %ARNL and MTB with clinical outcomes were sought. Results: Of 63 randomized pts, 26 had evaluable CTCs both at C1D1 and C1D8. At C1D8, mean %ARNL was significantly lower in pts achieving a ≥ 50% PSA drop by C4 vs those without (44% vs 64%; p = 0.004). A taxane-induced decrease in mean %ARNL (C1D8 vs C1D1) was associated with a higher rate of ≥ 50% PSA response (73% vs 13%; p = 0.009); mean %ARNL decreased by 18% in responders and increased by 2% in non-responders (p = 0.02). Finally, a taxane-induced increase in mean MTB trended higher in pts achieving a ≥ 30% PSA drop by C4 vs those without (0.69 vs 0.09; p = 0.09); increase in mean MTB score was indicative of response and observed in pts who did not require a taxane switch after C4 (0.75 vs 0.09; p = 0.06). Conclusions: We provide the first prospective data suggesting that taxane-induced shifts in ARNL and MTB (measured in CTCs) may serve as an early biomarker of taxane sensitivity. Consistent with preclinical data, AR nuclear exclusion caused by microtubule bundling may be a clinically-actionable marker of taxane efficacy. Funding: Sanofi Genzyme. Clinical trial information: NCT01718353.

5007Background: TAXYNERGY (NCT01718353), a randomized phase II trial, evaluated early taxane switch with CTC biomarkers to study mechanisms of sensitivity/resistance in men with chemo-naive mCRPC. ...

5031 Background: Microtubule-targeted therapy with taxanes is the only chemo with survival benefit in advanced PC. Emerging molecular evidence suggests sensitivity/resistance to taxanes may relate ...