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Purpose: Cisplatin-induced hearing loss (CIHL) is common and permanent. As compared with earlier otoprotectants, we hypothesized N-acetylcysteine (NAC) offers potential for stronger otoprotection through stimulation of glutathione (GSH) production. This study tested the optimal dose, safety, and efficacy of NAC to prevent CIHL. Patients and Methods: In this nonrandomized, controlled phase Ia/Ib trial, children and adolescents newly diagnosed with nonmetastatic, cisplatin-treated tumors received NAC intravenously 4 hours post-cisplatin. The trial performed dose-escalation across three dose levels to establish a safe dose that exceeded the targeted peak serum NAC concentration of 1.5 mmol/L (as identified from preclinical models). Patients with metastatic disease or who were otherwise ineligible were enrolled in an observation-only/control arm. To evaluate efficacy, serial age-appropriate audiology assessments were performed. Integrated biology examined genes involved in GSH metabolism and post-NAC GSH concentrations. Results: Of 52 patients enrolled, 24 received NAC and 28 were in the control arm. The maximum tolerated dose was not reached; analysis of peak NAC concentration identified 450 mg/kg as the recommended phase II dose (RP2D). Infusion-related reactions were common. No severe adverse events occurred. Compared with the control arm, NAC decreased likelihood of CIHL at the end of cisplatin therapy [OR, 0.13; 95% confidence interval (CI), 0.021–0.847; P = 0.033] and recommendations for hearing intervention at end of study (OR, 0.082; 95% CI, 0.011–0.60; P = 0.014). NAC increased GSH; GSTP1 influenced risk for CIHL and NAC otoprotection. Conclusions: NAC was safe at the RP2D, with strong evidence for efficacy to prevent CIHL, warranting further development as a next-generation otoprotectant.

Supplementary Fig. 1 Treatment schema & Sampling Timepoints

Supplementary Fig. 4 Evaluation of long-term storage of serum specimens (NAC assay)

Supplementary Fig. 3 Change in serum glutathione concentrations following NAC infusion

Supplementary Fig. 5 Progression-free survival in NAC treated and observation patients

Supplementary Fig. 2 Cumulative incidence of SIOP Grade ≥2 hearing loss

Supplemental Tables and Files

Purpose:Cisplatin-induced hearing loss (CIHL) is common and permanent. As compared with earlier otoprotectants, we hypothesized N-acetylcysteine (NAC) offers potential for stronger otoprotection through stimulation of glutathione (GSH) production. This study tested the optimal dose, safety, and efficacy of NAC to prevent CIHL.Patients and Methods:In this nonrandomized, controlled phase Ia/Ib trial, children and adolescents newly diagnosed with nonmetastatic, cisplatin-treated tumors received NAC intravenously 4 hours post-cisplatin. The trial performed dose-escalation across three dose levels to establish a safe dose that exceeded the targeted peak serum NAC concentration of 1.5 mmol/L (as identified from preclinical models). Patients with metastatic disease or who were otherwise ineligible were enrolled in an observation-only/control arm. To evaluate efficacy, serial age-appropriate audiology assessments were performed. Integrated biology examined genes involved in GSH metabolism and post-NAC GSH concentrations.Results:Of 52 patients enrolled, 24 received NAC and 28 were in the control arm. The maximum tolerated dose was not reached; analysis of peak NAC concentration identified 450 mg/kg as the recommended phase II dose (RP2D). Infusion-related reactions were common. No severe adverse events occurred. Compared with the control arm, NAC decreased likelihood of CIHL at the end of cisplatin therapy [OR, 0.13; 95% confidence interval (CI), 0.021–0.847; P = 0.033] and recommendations for hearing intervention at end of study (OR, 0.082; 95% CI, 0.011–0.60; P = 0.014). NAC increased GSH; GSTP1 influenced risk for CIHL and NAC otoprotection.Conclusions:NAC was safe at the RP2D, with strong evidence for efficacy to prevent CIHL, warranting further development as a next-generation otoprotectant.

Table S1 provides a summary of the genetic association results in the European only cohort

Figure S4 provides examples of audiogram configurations

Figure S2 provides a visualization of the hearing thresholds of patients grouped by ACYP2 and WFS1 genotype

Figure S3 provides a visualization of the interaction between genotype and cisplatin dose

Purpose: Adverse drug reactions such as ototoxicity, which occurs in approximately one-fifth of adult patients who receive cisplatin treatment, can incur large socioeconomic burdens on patients with testicular cancer who develop this cancer during early adulthood. Recent genome-wide association studies have identified genetic variants in ACYP2 and WFS1 that are associated with cisplatin-induced ototoxicity. We sought to explore the role of these genetic susceptibility factors to cisplatin-induced ototoxicity in patients with testicular cancer.Experimental Design: Extensive clinical and demographic data were collected for 229 patients with testicular cancer treated with cisplatin. Patients were genotyped for two variants, ACYP2 rs1872328 and WFS1 rs62283056, that have previously been associated with hearing loss in cisplatin-treated patients. Analyses were performed to investigate the association of these variants with ototoxicity in this cohort of adult patients with testicular cancer.Results: Pharmacogenomic analyses revealed that ACYP2 rs1872328 was significantly associated with cisplatin-induced ototoxicity [P = 2.83 × 10−3, OR (95% CI):14.7 (2.6–84.2)]. WFS1 rs62283056 was not significantly associated with ototoxicity caused by cisplatin (P = 0.39); however, this variant was associated with hearing loss attributable to any cause [P = 5.67 × 10−3, OR (95% CI): 3.2 (1.4–7.7)].Conclusions: This study has provided the first evidence for the role of ACYP2 rs1872328 in cisplatin-induced ototoxicity in patients with testicular cancer. These results support the use of this information to guide the development of strategies to prevent cisplatin-induced ototoxicity across cancers. Further, this study has highlighted the importance of phenotypic differences in replication studies and has provided further evidence for the role of WFS1 rs62283056 in susceptibility to hearing loss, which may be worsened by cisplatin treatment. Clin Cancer Res; 24(8); 1866–71. ©2018 AACR.

Figure S1 provides a visualization of the genetic ancestry of the cohort

Background: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. Methods: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human induced pluripotent stem cell–derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell–derived cardiomyocytes using a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9). Fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of the potential causal variant was done using cytosine base editor. SLC28A3-AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-sequencing after ribosomal RNA depletion. Drug screening was done using the Prestwick Chemical Library (n = 1200), followed by in vivo validation in mice. The effect of desipramine on doxorubicin cytotoxicity was also investigated in 8 cancer cell lines. Results: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore-based fine-mapping and base editing, we identify a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus; its effect is exerted via regulation of an antisense long noncoding RNA ( SLC28A3-AS1 ) that overlaps with SLC28A3. Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. Conclusions: This work demonstrates the power of the human induced pluripotent stem cell model to take a single nucleotide polymorphism from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.