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Table S1. Canadian tissue microarray, histology and relapse rate details. Table S2. Univariate, stratified and multivariate models for prognostic ability of VI, CXCL12 expression. Table S3. Multivariate analyses; alternative models. Figure S1: Kaplan-Meier relapse-free survival curves in combined TE08 and TE22 trial cohorts with (n=190) and without FFPE (n=501). Figure S2: Relapse-free rate by CXCL12 intensity (

Supplementary data from Clinical and Genome-wide Analysis of Cisplatin-induced Tinnitus Implicates Novel Ototoxic Mechanisms

Supplementary Figure 4 shows pharmacokinetic parameters for guadecitabine and decitabine in the dose escalation phase

Purpose:Preclinical data indicate that DNA methyltransferase inhibition will circumvent cisplatin resistance in various cancers.Patient and Methods:SPIRE comprised a dose-escalation phase for incurable metastatic solid cancers, followed by a randomized dose expansion phase for neoadjuvant treatment of T2–4a N0 M0 bladder urothelial carcinoma. The primary objective was a recommended phase II dose (RP2D) for guadecitabine combined with gemcitabine and cisplatin. Treatment comprised 21-day gemcitabine and cisplatin cycles (cisplatin 70 mg/m2, i.v., day 8 and gemcitabine 1,000 mg/m2, i.v., days 8 + 15). Guadecitabine was injected subcutaneously on days 1–5, within escalation phase cohorts, and to half of 20 patients in the expansion phase. Registration ID: ISRCTN 16332228.Results:Within the escalation phase, dose-limiting toxicities related predominantly to myelosuppression requiring G-CSF prophylaxis from cohort 2 (guadecitabine 20 mg/m2, days 1–5). The most common grade ≥3 adverse events in 17 patients in the dose-escalation phase were neutropenia (76.5%), thrombocytopenia (64.7%), leukopenia (29.4%), and anemia (29.4%). Addition of guadecitabine to gemcitabine and cisplatin in the expansion phase resulted in similar rates of severe hematologic adverse events, similar cisplatin dose intensity, but modestly reduced gemcitabine dose intensity. Radical treatment options after chemotherapy were not compromised. Pharmacodynamics evaluations indicated guadecitabine maximal target effect at the point of cisplatin administration. Pharmacokinetics were consistent with prior data. No treatment-related deaths occurred.Conclusions:The guadecitabine RP2D was 20 mg/m2, days 1–5, in combination with gemcitabine and cisplatin and required GCSF prophylaxis. Gene promoter methylation pharmacodynamics are optimal with this schedule. Addition of guadecitabine to gemcitabine and cisplatin was tolerable, despite some additional myelosuppression, and warrants further investigation to assess efficacy.

Purpose:Cisplatin, a commonly used chemotherapeutic, results in tinnitus, the phantom perception of sound. Our purpose was to identify the clinical and genetic determinants of tinnitus among testicular cancer survivors (TCS) following cisplatin-based chemotherapy.Experimental Design:TCS (n = 762) were dichotomized to cases (moderate/severe tinnitus; n = 154) and controls (none; n = 608). Logistic regression was used to evaluate associations with comorbidities and SNP dosages in genome-wide association study (GWAS) following quality control and imputation (covariates: age, noise exposure, cisplatin dose, genetic principal components). Pathway over-representation tests and functional studies in mouse auditory cells were performed.Results:Cisplatin-induced tinnitus (CisIT) significantly associated with age at diagnosis (P = 0.007) and cumulative cisplatin dose (P = 0.007). CisIT prevalence was not significantly greater in 400 mg/m2-treated TCS compared with 300 (P = 0.41), but doses >400 mg/m2 (median 580, range 402–828) increased risk by 2.61-fold (P < 0.0001). CisIT cases had worse hearing at each frequency (0.25–12 kHz, P < 0.0001), and reported more vertigo (OR = 6.47; P < 0.0001) and problems hearing in a crowd (OR = 8.22; P < 0.0001) than controls. Cases reported poorer health (P < 0.0001) and greater psychotropic medication use (OR = 2.4; P = 0.003). GWAS suggested a variant near OTOS (rs7606353, P = 2 × 10−6) and OTOS eQTLs were significantly enriched independently of that SNP (P = 0.018). OTOS overexpression in HEI-OC1, a mouse auditory cell line, resulted in resistance to cisplatin-induced cytotoxicity. Pathway analysis implicated potassium ion transport (q = 0.007).Conclusions:CisIT associated with several neuro-otological symptoms, increased use of psychotropic medication, and poorer health. OTOS, expressed in the cochlear lateral wall, was implicated as protective. Future studies should investigate otoprotective targets in supporting cochlear cells.

Purpose: Up to 50% of patients diagnosed with stage I nonseminomatous germ cell tumors (NSGCTs) harbor occult metastases. Patients are managed by surveillance with chemotherapy at relapse or adjuvant treatment up front. Late toxicities from chemotherapy are increasingly recognized. Based on a potential biologic role in germ cells/tumors and pilot data, our aim was to evaluate tumor expression of the chemokine CXCL12 alongside previously proposed markers as clinically useful biomarkers of relapse.Experimental Design: Immunohistochemistry for tumor expression of CXCL12 was assessed as a biomarker of relapse alongside vascular invasion, histology (percentage embryonal carcinoma), and MIB1 staining for proliferation in formalin-fixed paraffin-embedded orchidectomy samples from patients enrolled in the Medical Research Council's TE08/22 prospective trials of surveillance in stage I NSGCTs.Results: TE08/TE22 trial patients had a 76.4% 2-year relapse-free rate, and both CXCL12 expression and percentage embryonal carcinoma provided prognostic value independently of vascular invasion (stratified log rank test P = 0.006 for both). There was no additional prognostic value for MIB1 staining. A model using CXCL12, percentage embryonal carcinoma, and VI defines three prognostic groups that were independently validated.Conclusions: CXCL12 and percentage embryonal carcinoma both stratify patients' relapse risk over and above vascular invasion alone. This is anticipated to improve the stratification of patients and identify high-risk cases to be considered for adjuvant therapy. Clin Cancer Res; 22(5); 1265–73. ©2015 AACR.

Supplementary Tables and Methods

Supplementary Figure 1 shows a schematic of the treatment cycle

Supplementary Figure 2 shows CONSORT diagrams for both phases of the trial

Supplementary Figure 3 shows promotor methylation status for the indicated genes in cfDNA

We have previously identified amplification at 4q12 in testicular germ cell tumors of adolescents and adults centered around the KIT gene encoding a tyrosine kinase transmembrane receptor. Analysis of primary testicular germ cell tumors totaling 190 cases revealed 21% of the seminoma subtype with an increased copy number of KIT whereas this change was rarely found in the nonseminomas. In most cases, gain of KIT did not include the immediately flanking noncoding DNA or the flanking genes KDR and PDGFRA. Increased copy number of KIT was not found in the putative precursor lesion, carcinoma in situ (CIS), adjacent to tumor with this change. KIT overexpression was found independent of gain and KIT immunostaining was stronger in selected cases with gain of KIT compared to those without. Taken together with activating mutations of KIT in exon 17 identified in 13% of seminomas, this suggests that the KIT gene product plays a role in the progression of CIS towards seminoma, the further understanding of which may lead to novel less toxic therapeutic approaches.

Testicular germ cell tumors (TGCTs) are the most common cancer in young men. Although cure rates for TGCT are generally high, due to the sensitivity of malignant testicular germ cells to platinum-based chemotherapies, this comes at the cost of an increased risk of metabolic syndrome, infertility and secondary cancer. Furthermore, there are limited options for patients who are platinum resistant, a group for whom the long-term survival rate is less than 15%. Improved targeted therapies for TGCT are predicated on a greater understanding of mutational profile of these tumours, including identification of driver mutations and pathways disrupted. We recently published the first large-scale analysis of whole exome sequencing of TGCTs (comprising 42 tumors), which revealed a uniformly low mutation rate; some ∼ten times lower than average rates observed in other adult solid tumors, consistent with the embryological origin of TGCT. Aside from mutations in the previously known driver gene KIT, no additional novel genes with mutation frequency greater than 15% were associated with tumorigenesis. In parallel The Cancer Genome Atlas consortium is sequencing an additional series of TGCTs. Given the apparent absence of high frequency driver genes aside from KIT, several lower frequency oncogenic pathways may exist, requiring identification through larger sample size studies and/or pooled analysis. Here we present meta-analysis across multiple datasets with variant calling re-processed through a consistent bioinformatic pipeline. We systemically searched for novel recurrent mutational events at three levels:- (i) driver genes (combined sample size was empowered to detect recurrent gene mutations of frequency >5% with 89% power), ii) pathway based associations and iii) focal copy number alterations. In addition we assessed for clinicopathological-molecular associations, with particular focus on mutational events distinguishing treatment responsive and resistant tumors. Comprehensive cataloguing of the mutational spectra, through analyses of our data in combination with other initiatives such as TCGA, is leading to increasing insights into this biology of this cancer. Citation Format: Kevin Litchfield, Richard S. Houlston, Robert Huddart, Brenda Summersgill, Janet Shipley, Clare Turnbull. Meta-analysis of whole exome sequencing data reveals the mutational spectrum of testicular germ cell tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2986. doi:10.1158/1538-7445.AM2015-2986