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Predictive factors for Carboplatin related ototoxicity in

children treated for retinoblastoma

Crystal N D’Silva, PhD,1, Sameh E. Soliman2,3, Helen Dimaras2, Erik

Dzeneladze1, Mike Jain4, Rob Laister4, Helen Chan5, Brenda L. Gallie1,2,3.

1Department of Medical Biophysics, University of Toronto, Toronto, Canada

2Department of Ophthalmology and visual sciences, Hospital for Sick

Children. Toronto, Canada

3Department of Ophthalmology, Faculty of Medicine, University of Alexandria,

Alexandria, Egypt.

4 ??

5Department of Haematology/Oncology, Hospital for Sick Children. Toronto, Canada

Corresponding author: Dr. Brenda Gallie at the Department of Ophthalmology and

Vision Sciences, the Hospital for Sick Children, 555 University Avenue, Toronto, ON

M5G 1X8, Canada. Email: brenda@gallie.ca. Tel.: (+1) 416-294-9729, Fax 1-866-833-

5157),

A Research Article submitted to Pediatric Blood Cancer.

This work was presented as a paper presentation in ARVO conference in Seattle,

Washington, 5 May 2016.

Word Count: Abstract (242/250), Main text (3220/3500)

Tables and Figures: 4 tables and 2 figures

Supplemental files: 1 file, 4 tables and 2 figures

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Running Title: Carboplatin ototoxicity in retinoblastoma patients

Keywords: Ototoxicity, Carboplatin, Retinoblastoma, Cancer, Genetics and Chemotherapy.

Abbreviation Key:

IIRC International Intraocular

Retinoblastoma classification

AUC area under the curve

TPMT Thiopurine S-

methyltransferase

SIOP International Society of

Paediatric Oncology

COMT Catechol-O-

methyltransferase

CCG Children Cancer Group

ABCC3 ATP-binding cassette

sub-family C member 3

NCI-

CTCAE

National Cancer Institute

Common Terminology

Criteria for Adverse Events

ROC Receiver operating

characteristic

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ABSTRACT: (242/250)

Background. Children with retinoblastoma treated with carboplatin-based chemotherapy are

at risk of moderate to severe, irreversible hearing loss. Based on previous publications, we

hypothesized that clinical parameters and variants in candidate genes in drug metabolism

pathways (methyltransferases TPMT and COMT, and drug transporter ABCC3), would be

predictive of ototoxicity. Purpose. Retrospective review of clinical records of retinoblastoma

patients treated with carboplatin-based chemotherapy recorded age at diagnosis and at

chemotherapy initiation, chemotherapy sessions (number of cycles, drug doses and cumulative

carboplatin dose), and hearing loss defined as ototoxicity ≥ grade 2 by at least one classification

system. Blood samples were genotyped for genetic variants in TPMT (rs12201199,

rs1800460), COMT (rs4646316, rs9332377), and ABCC3 (rs1051640) by real-time PCR

confirmed by allele-specific PCR.

Results: 97 Seventy one patients with retinoblastoma were included (85% had bilateral

disease). Median carboplatin cumulative dose for all patients was 1400 mg/m2 (260-5148

mg/m2). Ototoxicity occurred in 18 patients (25%), significantly associated with age at diagnosis

(p=0.01) and age at chemotherapy initiation (p=0.008). The highest likelihood ratio of hearing

loss was associated with chemotherapy initiation of <4.25 months of age. Ototoxicity was not

associated with any tested genetic variants in TPMT, COMT, and ABCC3.

Conclusions: We report a higher incidence (25%) of ototoxicity in children treated

with carboplatin for retinoblastoma than previously published. Age at treatment initiation

was a risk predictor of carboplatin-induced ototoxicity, with children <4.25 months of age at

highest risk. Carboplatin related ototoxicity was not associated with the studied genetic variants.

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1 | INTRODUCTION

Retinoblastoma is the most common ocular childhood cancer with an incidence

between 1:14,000-1:22,000 live births.1 Tumors originate before or shortly after birth in

one eye (unilateral, two-thirds of cases) or both eyes (bilateral).2 However, 50% of all

children with retinoblastoma carry RB1 germline mutations.3,4 Retinoblastoma treatment

is determined by age at diagnosis, laterality, stage of disease disease at presentation and

overall cancer staging. Treatment regimens encompass different types of

chemotherapy, laser therapy, cryotherapy, radiotherapy and surveillance.5

Carboplatin is a platinum-based chemotherapeutic agent used in most systemic

chemotherapy protocols for retinoblastoma treatment6-9. While carboplatin is less toxic

than cisplatin10,11, there have been reports of ototoxicity in 0.009% to 16.7% of paediatric

patients treated with carboplatin for retinoblastoma.12-14 However, other studies also

report no hearing impairment as a consequence of carboplatin treatment15-19 even on the

long term follow up20 (Table 1). Cumulative carboplatin doses, younger age at treatment

initiation, and radiation therapy have been identified as potential risk factors for

sustained hearing loss post-treatment.13,14,21

Platinum-induced ototoxicity presents as bilateral high-frequency sensorineural

hearing loss, with increasing incidence and severity in response to cumulative

dosage.22,23 Impairment to hearing has undesirable consequences for quality of life,

cognitive development, and learning, particularly in paediatric patients24-26 who will have

a concurrent degree of visual impairment from retinoblastoma or its treatment. Inter-

individual variation in the development of carboplatin-induced hearing loss exists, but

missing are clear predictors of ototoxicity risk prior to treatment initiation.

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A number of studies27-30 have explored the relationship between polymorphisms in the genes

encoding thiopurine S-methyltransferase (TPMT), catechol-O-methyltransferase (COMT), and

ATP-binding cassette sub-family C member 3 (ABCC3) and hearing loss in patients who

received cisplatin treatment, with conflicting results and lack of consensus.27-30 These

associations require further evaluation before being considered for clinical implementation to

stratify patients at risk of ototoxicity when treated with platinum-based chemotherapeutic agents.

The aim of our study was to assess potential clinical and genetic predictive factors for

developing ototoxicity in children with retinoblastoma receiving Carboplatin based

chemotherapy. These factors may be useful to guide therapy.

METHODS

Ethics

This study was scientifically reviewed and approved by the Research Ethics Board of

the Hospital for Sick Children. Consent for research use of leftover DNA was provided

during sample acquisition for retinoblastoma genetic testing. The study is in accordance

with the Declaration of Helsinki.

Sample

Ninety-seven children with retinoblastoma that received carboplatin based

chemotherapy at the Hospital for Sick Children were studied for variants in genes associated

with platinum chemotherapy toxicity, using archived DNA from blood after clinical RB1

mutation detection. Adequate pre- and post-chemotherapy audiograms were available on 71

children who were included in the full analysis.

Clinical data

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Data collected included: age at diagnosis and at start of chemotherapy, sex,

laterality, RB1 mutations (blood, tumor if available), eye stage(s) at diagnosis, chemotherapy

details (number of cycles, drugs used and cumulative carboplatin dose), number of

audiograms and further treatments (radiation or autologous bone marrow

transplantation).

Audiologic assessment

The following toxicity grading systems were used: National Cancer Institute Common

Terminology Criteria for Adverse Events (NCI-CTCAE) version 331, Children's Cancer

Group (CCG), International Society of Paediatric Oncology (SIOP) Boston ototoxicity

scale32, and Brock33 and Chang34 systems to grade ototoxicity (Supplementary Table 1).

Patients with ototoxicity of grade 2 or higher by at least one classification system were

considered to have hearing loss, similar to inclusion criteria employed in previous

studies.32-34

Genetic analysis

Genotyped samples identified as heterozygous or homozygous for one or more of

five minor allele variants [TPMT (rs12201199 and rs1800460), COMT (rs4646316 and

rs9332377), and ABCC3 (rs1051640)] were used as positive controls to optimize real-

time PCR assays for detection of the five variants (Supplementary table 1-3). Detailed

methods and technique of genetic testing is described in supplementary file 1.

Two sets of amplification reactions were designed for each variant using allele-

specific primers and real-time PCR was monitored by SYBR green dye. Differential

amplification efficiency for each allele was determined with 2 different sets of primers

pairs based on reference sequences35 (Supplementary File 1). Allele-specific primers

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were designed based on reference sequences for TPMT (NCBI Reference

sequence: NC_000006.12, Homo sapiens thiopurine S-methyltransferase on

chromosome 6), COMT (NCBI reference sequence: NC_000022, Homo sapiens

catechol-O-methyltransferase on chromosome 22), and ABCC3 (NCBI reference

sequence: NC_000017.1, ATP-binding cassette sub-family C member 3 on

chromosome 17). Primers specific for the variant allele were designed to have a melting

temperature of 5C lower than primers for the wild-type allele in order to enhance allelic

discrimination (Supplementary table 3).

PCR amplification using the SYBR Green assay was performed on the ABI PRISM

7900 HT Sequence Detection System (Applied Biosystems) in duplicate for samples,

and triplicate for genotyped controls. Reaction mixtures without template DNA were

used as negative controls. Each 12.5 μL reaction volume consisted of 5 μL Power SYBR

Green PCR Master Mix, approximately 2 pmol (range: 1-5 pmol) of each primer, and 10

ng of DNA sample. An initial enzyme heat-activation step of 10 min at 95°C, was

followed by 40 cycles of a 3-step amplification profile of 20 sec at 95°C for denaturation,

1 min at 60°C for annealing, and 30 sec at 72°C for extension.

Cycle threshold (Ct) (number of amplification cycles required for the fluorescent signal to

cross a threshold) was used to estimate reaction efficiency, fixed at the exponential phase of

amplification. Positive (specific) amplifications resulted in Ct values below 24 cycles, whereas

negative (non-specific) amplifications yielded Ct values above 24 cycles. Negative controls did

not show any amplification before cycle 35. The difference in Ct values between positive and

negative reactions was always greater than four cycles, indicating that specific amplifications

were at least 16-fold more efficient than non-specific ones. Median threshold cycle (Ct) values

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were recorded from real-time PCR runs with both normal and variant primers. Melt curve

analysis was used to assess the purity of amplicon generated.36

Statistical analysis

Ototoxicity (hearing loss) was treated as a dichotomized variable: grade 0 versus

grade 2 (by at least one classification system). Classification schemes were deemed to

be in agreement if they produced equal grades for one or both ears, as applicable.

Two-tailed Fisher’s exact tests were used to analyse associations between hearing

loss and clinical characteristics including sex, diagnosis with unilateral or bilateral

retinoblastoma, age at diagnosis and age at treatment initiation. Association between

number of audiograms and cumulative carboplatin dose were evaluated using Mann-

Whitney tests. Age at diagnosis and age at treatment initiation were treated as

continuous variables, and the Mann-Whitney test was used to test for differences

between patients who developed ototoxicity and those did that not.

OR

Ototoxicity was treated as a dichotomized variable: grade 0 versus grade 2 hearing loss.

Two-tailed Fisher’s exact tests were used to analyse associations between hearing loss and

clinical characteristics including sex, unilateral or bilateral disease, germline RB1 mutation

status, age at diagnosis and age at treatment initiation. Associations between hearing loss and

number of audiograms and cumulative carboplatin dose were evaluated using Mann-Whitney

tests. Receiver operating characteristic (ROC) curve analysis37 was used to determine age of

treatment initiation which had the highest risk for ototoxicity. An odds ratio was calculated for

risk predictors of carboplatin-induced ototoxicity. The association between TPMT, COMT, and

ABCC3 genotypes and ototoxicity was assessed by Fisher’s exact test for allelic association. All

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statistical analyses were performed with Prism GraphPad and p-values of < 0.05 were deemed

significant.

Receiver operating characteristic (ROC) curve analysis37 was used to determine age of

treatment initiation as a clinical predictor of risk for ototoxicity. The true positive rate

(sensitivity) and false positive rate (100 – specificity) for different cut-off ages at

treatment initiation to distinguish patients with hearing loss (grade 0) versus no hearing

loss (grade 2 by at least one classification system) was used to determine area under

the curve (AUC, a measure of how well age at treatment initiation can distinguish

between children that develop hearing loss and children who retained normal hearing). An

Odds ratio was calculated for risk predictors of carboplatin-induced ototoxicity.

SNP genotypes were coded according to the number of minor alleles as 0, 1, and 2

for wild-type, heterozygous, and homozygous, respectively. Deviation of genotype

frequencies from those expected under Hardy-Weinberg equilibrium was assessed. The

association between TPMT, COMT, and ABCC3 genotypes and ototoxicity was

assessed by Fisher’s exact test for allelic associations. All statistical analyses were

performed with Prism GraphPad and p-values of < 0.05 were deemed significant in all

tests.

RESULTS

The clinical characteristics of the 71 included patients are summarized in table 3.

Incidence of hearing loss

Twenty-four of 71 patients (33.8%) developed slight to complete deafness ( grade 0

at least one classification system) at some time after treatment initiation (median, 40.5

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months; range, 3 months to 13 years). Eighteen patients (25.4%) were considered to

have moderate to extensive hearing loss ( grade 2 at least one classification system)

with a time to detection of ototoxicity ranged between 2 and 75 months (median, 18.5

months) from the start of treatment with carboplatin and were included in the statistical

analysis (table 4). Most patients with hearing loss experienced bilateral (15/18, 83.3%),

grade 1 or grade 2 (12/18, 66.7%) ototoxicity. Three patients (16.7%) also experienced

unilateral grade 1 or 2 ototoxicity. The remainder presented with grade 3 or higher,

bilateral ototoxicity by various classification systems. Eight out of 18 patients (11%) with

hearing loss were less than 6 months of age at treatment initiation.

The median number of audiograms per patient was 7 (range, 1 to 25 evaluations)

including a baseline audiogram performed at the time of treatment initiation (table 3). On

average, patients with hearing loss received more audiograms (median, 11.5 months;

range, 1 to 25 months) than those without hearing loss (median, 5.5 months; range, 1 to

19 months).

Hearing loss was observed to be grade 3 or higher (all classification systems) in 6 of

18 patients (33%).

Two patients had bilateral, low-grade (grade 1) hearing loss by the NCI-CTCAE and

SIOP classification systems, and were assigned grade 0 by the CCG, Brock, and Chang

classification systems. Two patients had grade 1 loss in the left ear by the NCI-CTCAE

classification system only. Similarly, 1 patient had bilateral grade 1 and another patient,

bilateral grade 2 hearing loss by the NCI-CTCAE classification system, they were

assigned grade 0 by all the other classification systems. No information regarding age

at detection of hearing loss was available for 1 patient. These patients were not

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included in the group of 18 patients with hearing loss. Figure 1 demonstrates a Kaplan-

Meyer curve of time of occurrence of hearing loss in ototoxicity patients using the

different grading systems.

Time to detection of hearing loss was higher in the 6 patients with slight to moderate

ototoxicity (grades 1 and 2) based on the SIOP and NCI-CTCAE, or the NCI-CTCAE

classification system only, and none (grade 0) based on the other classification

systems; 1 year 6 months, 3, 4, 9, and 13 years after treatment initiation. With regard to

current hearing status, 3 patients wore hearing aids, while the remaining 21 patients

(including those identified with grade 1 hearing loss only by the NCI-CTCAE, and by the

NCI-CTCAE and SIOP classification systems) were recorded as having no hearing

devices.

Agreement among classification systems

The greatest agreement was observed between the Brock and Chang classification

systems for ototoxicity (68 of 71 patients, 95.8%). Among the 18 patients with hearing

loss, Brock and Chang grades were identical in 15 patients. In the remaining 3 patients,

the Chang system had higher grades for 2 and the Brock system a higher grade for 1

patient.

The SIOP and Chang systems were second highest in agreement (67 out of 71

patients, 94.4%). Among the patients with hearing loss, SIOP and Chang grades were

identical in 16 patients. For the remainder of the patients with hearing loss, the SIOP

system had a higher grade for one patient and the Chang system, a higher grade for the

other.

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The SIOP/CCG and Chang/CCG systems of classification were both in agreement

for 66 out of 71 patients (93%), but the CCG system was in agreement with SIOP for 15

out of 18 patients with hearing loss, and in agreement with Chang for 13 out of 18

patients with hearing loss. In the former case, for the remaining 3 patients with hearing

loss, SIOP had a higher grade for 2 patients and CCG for one. For the latter, CCG had

a higher grade for 2 patients and the Chang system for 3 patients with hearing loss.

The Brock system of classification for ototoxicity was in agreement with CCG for 65

out of 71 patients (91.6%), and SIOP for 64 patients (90.14%). For patients with hearing

loss, the Brock system was in agreement with SIOP for 13 out of 18 patients and in

agreement with CCG for 12 patients. For the remaining 5 patients with hearing loss, in

the former case, the SIOP system had a higher grade for 3 patients and the Brock

system for two. For the remaining 6 patients with hearing loss in the latter case, the

Brock system had a higher grade for 3 patients and the CCG system for three.

Overall, the NCI-CTCAE system was least often in agreement with the other

classification systems. The NCI-CTCAE system was highest in agreement with the

Brock system (57 out of 71 patients, 80.3%). For patients with hearing loss, the NCI-

CTCAE system was in agreement with the Brock system for 10 patients. For the

remaining 8 patients with hearing loss, the NCI-CTCAE system had a higher grade in all

cases. The NCI-CTCAE system was next highest in agreement with the SIOP and

Chang systems (55 out of 71 patients, 77.6%). For patients with hearing loss, the NCI-

CTCAE system was in agreement with SIOP system for only 6 out of 18 patients. In the

remaining 12, the NCI-CTCAE system had a higher grade for 8 patients and the SIOP

system for four. Similarly, for patients with hearing loss, the NCI-CTCAE system was in

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agreement with the Chang system for 8 out of 18 patients. For the remaining 10, the

NCI-CTCAE had a higher grade for 7 patients and the Chang system for three. The

least agreement was observed between the NCI-CTCAE and CCG systems (54 out of

71 patients, 76%). Out of 18 patients with hearing loss, there was agreement between

these two systems in 7 patients. The NCI-CTCAE system had a higher grade for 8 out

of the remaining 11 patients with hearing loss, and the CCG system had a higher grade

for three.

Clinical and genetic risk factors for hearing loss

Potential clinical and genetic predictors of ototoxicity were assessed. In univariate

analyses, the only significant risk factors were age at diagnosis (p = 0.01) and age at

treatment initiation (p = 0.008) (Table 3).

ROC analysis identified age at treatment initiation of less than 4.25 months to have

the highest likelihood ratio for the development of ototoxicity post-treatment with

carboplatin in retinoblastoma patients. The AUC was calculated to be 0.7059 (p =

0.008). The sensitivity, specificity, and likelihood ratios for different cut-off ages at

treatment initiation to distinguish patients with hearing loss (grade 0) versus no hearing

loss (grade 2 by at least one classification system) are listed in Supplementary table

4. This finding was further assessed by constructing three different Kaplan-Meier curves

for the development of ototoxicity after treatment with carboplatin in children younger

than 4.25 months (Figure 2a), 6 months as reported by Qaddoumi et al.,1 (Figure 2b),

and the median age at treatment initiation, 10 months (Figure 2c). The log-rank test was

used to assess the difference between the two groups based on the cut-off age at

treatment initiation as specified. A statistically significant difference was noted when the

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cohort of patients that developed hearing loss was separated into groups of younger

than 6 months (p = 0.0045) and 4.25 months (p = 0.0027), but not younger than 10

months (p = 0.2103). Age at treatment initiation younger than 4.5 months and 6 months

conferred odds ratios of 4.99 (95% CI, 1.3960 to 17.8002; p = 0.01) and 3.36 (95% CI,

1.0559 to 10.6922; p = 0.04), respectively.

There was no association between genetic variants in TPMT, COMT, ABCC3 and

ototoxicity (Supplementary Figure 1-2).

Discussion

Platinum-induced ototoxicity presents as bilateral high-frequency sensorineural

hearing loss, with increasing incidence and severity in response to cumulative

dosage.22,23 Impairment to hearing has undesirable consequences for quality of life,

cognitive development, and learning, particularly in paediatric patients24-26 who will have

a concurrent degree of visual impairment from retinoblastoma or its treatment. Inter-

individual variation in the development of carboplatin-induced hearing loss exists, but

missing are clear predictors of ototoxicity risk prior to treatment initiation.

In the current study, age at diagnosis and age at treatment initiation were identified

as the only statistically significant variables associated with hearing loss. Age at

treatment initiation was also found to be a risk predictor of carboplatin-induced

ototoxicity, where younger patients were more likely to develop ototoxicity than older

patients. This is in accordance with previously published reports of clinical predictors of

carboplatin-induced hearing loss in paediatric patients.13,38

Previous studies have reported predominantly bilateral (90%), grade 3 or higher

(90%) ototoxicity in retinoblastoma13, albeit with a higher cumulative carboplatin dose

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(median, 3,576 mg/m2) received during chemotherapy than in our study (median, 1,400

mg/m2). The incidence of ototoxicity following carboplatin-based treatment in

retinoblastoma patients is higher in our study (25%) than formerly reported12-19 (0% –

16.7%, Table 1). Differences in methodology, definition of ototoxicity, and heterogeneity

between the cohorts have made it difficult to draw comparisons. Furthermore, chronic

cyclosporine A usage has also been associated with the development of hearing loss,39

and might explain the higher incidence of ototoxicity in our cohort as all patients

received Cyclosporine A to reduce the evolution of multidrug resistance tumour cells.9

Our study is the third13,40 to employ multiple grading systems for ototoxicity, including

the most recently introduced Chang system of classification34. We demonstrate high

agreement (90.2 – 95.8%) among the SIOP, CCG, Brock, and Chang classification

systems. The NCI-CTCAE classification system has been reported to underestimate the

frequency of platinum therapy-induced ototoxicity.40 However, our review of the

audiologic results of 71 patients treated with carboplatin revealed that in patients with

mild to severe hearing loss (grade 1 or higher) by the five classification systems used in

this study, use of the NCI-CTCAE classification grading alone would result in an

overrepresentation of ototoxicity in our cohort.

The NCI-CTCAE grade was lower than at least two other classification systems in 4

out of 18 patients (22.2%), the same as at least two other classification systems in 6 out

of 19 patients (33.3%), and higher than at least two other classification systems in 8 out

of 18 (44.4%) patients. Furthermore, patients that were assigned grade 0 (no hearing

loss) by all other classification systems (except 2 patients with grade 1 ototoxicity by the

SIOP classification system) were assigned grade 1 or grade 2 ototoxicity based on the

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NCI-CTCAE classification system. The inclusion of these 6 patients in the group of

patients with hearing loss would result in an 8.8% increase (25% to 33.8%) in the

incidence of ototoxicity in this cohort (Figure 1.12). Given that ototoxicity was detected

in 4 of these patients (out of 5 with available data) over 5 years after chemotherapy, it is

possible that the slight to moderate ototoxicity (grade 1, 83.3%; grade 2, 16.7%) noted

might be on account of reasons unrelated to carboplatin treatment. Baseline

audiograms at the start of treatment ensured that the ototoxicity identified in patients in

our study was only after treatment with carboplatin and not pre-existing hearing loss

unrelated to chemotherapy.

Genetic variants in TPMT and COMT were initially discovered as predictors of

cisplatin-induced ototoxicity in a candidate gene study of 166 paediatric patients with

different types of cancers27. Subsequent validation studies of the same variants in TPMT

and COMT revealed inconsistent results. Pussegoda et al.28 replicated the association

between TPMT variants and cisplatin-induced ototoxicity in a cohort of 155 patients.

However, in another study, no association between TPMT and COMT variants and

ototoxicity was reported in 213 paediatric patients with medulloblastoma.23 Ratain et

al.,41 expressed concerns regarding the two published reports that demonstrated an

association between TPMT, COMT, and ABCC3 variation and cisplatin-induced hearing

loss27,42.They fail to support former provisional patent applications, evince discrepancies

in the data, and may have overestimated the significance of associations due to

inadequate correction for population variation in the polymorphisms that were

examined.

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Finally, a meta-analysis of previously published studies27-29, in addition to two

independent cohorts of 100 Dutch and 38 Spanish patients with osteosarcoma, yielded

no statistically significant association between genetic variants in TPMT and COMT and

cisplatin treatment-related ototoxicity30. Our results do not support the hypothesis that

genetic variation in TPMT, COMT, or ABCC3 is associated with carboplatin-induced

hearing loss in retinoblastoma.

Genotype-phenotype associations, particularly between genetic variants and drug

toxicities, are often confounded by myriad non-genetic factors. Increased cisplatin

dosage,23 younger age,43-45 cranial irradiation,44,46 and use of aminoglycosides45,47,48 and

vincristine45,49,50 have been reported to affect platinum-induced ototoxicity.

The genetic basis of carboplatin-induced ototoxicity has yet to be determined. One

study identified a missense mutation in eIF3, which encodes the largest subunit of

eukaryotic translation initiation factor 3 (EIF3) and plays a role in DNA repair, as a

potential biomarker for cisplatin- and carboplatin-related nephrotoxicity and ototoxicity in

lung cancer patients.51 Several genetic pathways regulate the uptake, transport, and

clearance of platinum.52 As a result, genetic risk factors for platinum-related deafness

might well include more than one gene. In a comprehensive review of platinum-induced

ototoxicity in paediatric patients, Brock et al.,30 suggest the use of novel methodologies

for a “polygenic approach” to identify genetic determinants of hearing loss.32 Large-scale

genome approaches such as next generation sequencing (NGS) might elucidate

genetic risk factors for treatment-related ototoxicity. The use of NGS for the

identification of variants associated with ototoxicity after treatment with cisplatin or

carboplatin is limited. A recent genome-wide association study of 238 paediatric

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patients with brain tumours discovered that variant rs1872328 in the enzyme-coding

ACYP2 gene was overrepresented in children who developed ototoxicity after treatment

with cisplatin.53 The authors replicated their findings in a cohort of 68 children treated

with cisplatin using targeted gene resequencing.

In summary, we show that treatment with carboplatin is accompanied by a significant

risk of bilateral, irreversible hearing loss, particularly in younger children and additional

pharmacogenetic studies are required to ascertain genetic determinants of treatment-

related ototoxicity in retinoblastoma.

Conflict of interests

No financial conflicting relationship exists for any author. BLG is an unpaid medical

director for Impact Genetics Inc.

Acknowledgements

?

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24. Brabyn JA, Schneck ME, Haegerstrom-Portnoy G, et al: Dual sensory loss:

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25. Simon HJ, Levitt H: Effect of dual sensory loss on auditory localization:

implications for intervention. Trends Amplif 11:259-72, 2007

26. Pierson SK, Caudle SE, Krull KR, et al: Cognition in children with sensorineural

hearing loss: etiologic considerations. Laryngoscope 117:1661-5, 2007

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are associated with hearing loss in children receiving cisplatin chemotherapy. Nat Genet

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genetic variants highly associated with cisplatin-induced hearing loss in children. Clin Pharmacol

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29. Yang JJ, Lim JY, Huang J, et al: The role of inherited TPMT and COMT genetic

variation in cisplatin-induced ototoxicity in children with cancer. Clin Pharmacol Ther 94:252-9,

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30. Hagleitner MM, Coenen MJ, Patino-Garcia A, et al: Influence of genetic variants

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38. Musial-Bright L, Fengler R, Henze G, et al: Carboplatin and ototoxicity: hearing

loss rates among survivors of childhood medulloblastoma. Childs Nerv Syst 27:407-13, 2011

39. Marioni G, Perin N, Tregnaghi A, et al: Progressive bilateral sensorineural

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Coordinating Complex-Induced Ototoxicity., 2008

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47. Veenstra DL, Harris J, Gibson RL, et al: Pharmacogenomic testing to prevent

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patient, and economic outcomes. Genet Med 9:695-704, 2007

48. Waguespack JR, Ricci AJ: Aminoglycoside ototoxicity: permeant drugs cause

permanent hair cell loss. J Physiol 567:359-60, 2005

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50. Aydogdu I, Ozturan O, Kuku I, et al: Bilateral transient hearing loss associated

with vincristine therapy: case report. J Chemother 12:530-2, 2000

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severe toxicity caused by platinum-based chemotherapy in non-small cell lung cancer patients.

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with cancer. Eur J Cancer 33:1823-8, 1997

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susceptibility to cisplatin-induced hearing loss. Nat Genet 47:263-6, 2015

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Figure Legends

Figure 1: Kaplan-Meier plot of time to development of ototoxicity (months) in

retinoblastoma patients treated with carboplatin. The majority of hearing loss (grade

0 or higher) was detected between 3 and 30 months from treatment initiation with

carboplatin-based chemotherapy (n = 23). Each curve represents ototoxicity definition

by different classification systems: NCI CTCAE ≥ 0 (orange), Brock ≥ 0 (red), SIOP ≥ 0

(green), CCG ≥ 0 (violet), and Chang ≥ 0 (blue).

Figure 2: Kaplan-Meier (KM) plot of time to development of ototoxicity

(months) in retinoblastoma patients treated with carboplatin separated by age.

KM curves were generated for carboplatin-treated retinoblastoma patients and

separated by age at treatment initiation. (A) Younger than 4.25 months (blue) and older

than 4.25 months (red). (B) Younger than 6 months (blue) and older than 6 months

(red). (C) Younger than 10 months (blue) and older than 10 months (red). P-values are

indicated in boldface below each figure legend.

Suppl. Figure 1 (a-e): SYBR-based real-time PCR allele discrimination for a)

TPMT rs12201199, b) TPMT rs1800460, c) COMT rs4646316, d) COMT rs9332377, e)

ABCC3 rs1051640. Normal primers, red square; variant primers, green triangle; Ct = 24,

threshold between primer-specific amplification (Ct < 24) and non-specific amplification

(Ct of > 24); samples that amplified below threshold with normal primers (red square)

and above threshold with variant primers (green triangle) were scored homozygous

normal; samples that amplified below threshold with both normal (red square) and

variant (green triangle) primers were scored heterozygous; samples that amplified

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below threshold with variant primers (green triangle) and above threshold with normal

primers (red square) were scored homozygous variant.

Suppl. Figure 2: The distribution of carboplatin-induced ototoxicity by TPMT,

COMT, and ABCC3 genotype. The percentage of patients with ototoxicity is plotted for

each genotype: (a) TPMT rs12201199, (b) TPMT rs1800460, (c) COMT rs4646316, (d)

COMT rs9332377, and (e) ABCC3 rs1051640. P-values determined by Fisher’s exact

test for allelic association.

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Table 1: Published reports of carboplatin-induced ototoxicity in retinoblastoma

 

Frie

dman et

al

200015

Sm

its et

al

200616

La

mbert

et al

200817

Jeh

anne et

al

200914

Bha

gat et

al

201019

Liber

man et al

201118

Qadd

oumi et

al 201213

Ba

tra et

al

201512

Geurts

en et al

201620

Current

study

No. of patients 47 25 116 175 10 15 6011

622 71

Bilateral RB 38 19 71 136 6 14 48 29 17 61

Ototoxicity                    

No. of patients

(%)

0

(0%)

0

(0%)

0

(0%)

8

(4.6%)

0

(0%)

1

(6.7%)

10

(16.7%)

1

(1%)

1

(4.5%)18 (25%)

Age at

treatment initiation

Median

(months)

Bi,

5; Uni,

7.5

7 10 8 7.6 19.2 8.6 36 11 12

Time to

ototoxicity, Median

na na na 44. na Rx 9 48* 55.8* 25.5

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(months) 4 end **

Mean

cumulative

carboplatin dose

(mg/m2)

na2,2

40

3,3

48

2,8

80

1,6

602,802 3,920

3,3

602,023 1,400

Grading system na na naBro

ckna Brock

Brock

, CCG,

NCI-

CTCAE

Br

ock

Brock,

SIOP

Brock,

CCG, NCI-

CTCAE,

SIOP, Chang

Abbreviations: Bi, bilateral; Rx, treatment; CCG, Children's Cancer Group; SIOP, International Society of Paediatric Oncology;

na, not applicable; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse Events, version 3; RB,

retinoblastoma; Uni, unilateral.*Only one patient was identified with hearing loss post-treatment with carboplatin24. **Time to

detection of ototoxicity following treatment with carboplatin not specified18. Adapted from20 “Long term audiologic follow-up of

carboplatin-treated children with retinoblastoma” by Geurtsen et al Ophthalmic Genet. 2016 Apr 6:1-5. [Epub ahead of print]

Table 2: Definition of ototoxicity according to classification system.

G

radeSIOP (HL) Brock (HL) NCI-CTCAE (ototoxicity) CCG (HL)

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0≤ 20 dB HL at all

frequencies

< 40 dB HL at all

frequenciesNot defined No HL

1≥ 20 dB HL at 6 kHz

and/or 8 kHz≥ 40 dB HL at 8 kHz only

Threshold shift or loss of 15-25 dB

relative to baseline

≥ 40 dB HL at 6 kHz and/or 8

kHz

2> 20 dB HL SNHL at

4,000 Hz and above

≥ 40 dB HL at 4 kHz and

aboveThreshold shift or loss of > 25-90 dB

> 25 dB HL at 3 kHz and/or 4

kHz

3> 20 dB HL SNHL at 2

kHz and above

≥ 40 dB HL at 2 kHz and

above

Hearing loss sufficient to indicate

therapeutic intervention> 25 dB HL at 2 kHz

440 dB HL at 2 kHz and

above

≥ 40 dB HL at 1 kHz and

above

Audiologic indication for cochlear

implant and requiring additional speech-

language related services

≥ 40 dB HL at 2 kHz

G

rade Chang (Sensorineural Hearing Thresholds)

 Abbreviations: HL, hearing loss; SIOP, International Society of Paediatric Oncology; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse Events, version 3; CCG, Children's Cancer Group. Adapted from13 “Carboplatin-associated Ototoxicity in Children with Retinoblastoma” by Qaddoumi et al, Journal of Clinical Oncology 2012; 30(10), p1037.

0 ≤ 20 dB HL at 1,2, and 4 kHz

1a ≥ 40 dB HL at any frequency between 6-12 kHz

1b >20 and <40 dB HL at 4kHz

2a ≥ 40 dB HL at 4 kHz and above

2b >20 and <40 dB HL at any frequency below 4kHz

3 ≥ 40 dB HL at 2 or 3 kHz and above

4 ≥ 40 dB HL at 1 kHz and above

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Table 3: Summary of clinical characteristics and univariate analysis.

   

Total

(n=71)

Hearing loss

(n=18)

No hearing

loss (n=53)     

 

 

Characteri

stic

N

o.%   No. %   No. %  

p-

value 

 

  Sex                        

   Male

3

1

4

4  10 56   21

4

0 

0.3a

 

  

   Female

4

0

5

6  8 44   32

6

0   

 

 

RB

Laterality                     

 

   

Unilate

ral8

1

1  1 6   7

1

3 

0.4

a 

 

  

Bilater

al

6

0

8

5  17 94   43

8

1 

0.2

a 

 

  

Trilater

al2 3   0 0   2 4  

0.4

a 

 

 

Age at

diagnosis

(months)

                     

 

   <7

2

2

3

1  8 44   14

2

6 

0.2

a 

 

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   7 to 36

4

7

6

6  10 56   37

7

0 

0.4

a 

 

    >36 2 3   0 0   2 4   1 a    

 

 Media

n (range)

11

(0.2-

104)

 8

(0.4-16) 

12

(0.2-104) 

0.0

1 b 

 

 

Age at treatment

initiation (months)                   

 

   <7

1

9

2

7  8 44   11

2

1 

0.0

6 a 

 

   7 to 36

4

9

6

9  10 56   39

7

3 

0.1

a 

 

   >36 3 4   0 0   3 6  

0.6

a 

 

 

 Media

n (Range)

12

(0.54-

106)

 8

(0.5-30) 

13.5

(0.9-106) 

0.0

08 b 

 

 

Number of

Audiograms                     

 

   

Media

n (Range)

7

(1-25) 

11.5

(1-25) 

5.5

(1-19) 

0.0

003 b 

 

  Cumulative

carboplatin dose

                     

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(mg/m2 )

   

Media

n (Range)

1,400

(260-

5,148)

 1,269

(260-5,148) 

1,504

(330-

4,861)

 0.3

b 

 

  Radiation                        

   Yes

1

3

1

8  5 28   8

1

5  0.3

a

 

  

   No

5

8

8

2  13 72   45

8

5   

 

 

Bone

marrow

transplant

                     

 

    Yes 4 6   1 6   3 6  1 a

 

   

   No

6

7

9

4  17 94   50

9

4   

 

  TPMT                        

rs1220

1199

AA

AT

TT

5

6

1

2

3

7

9

1

7

4

15

3

0

83

17

0

41

9

3

7

7

1

7

6

0.7

c

rs1800

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460

AA

6

5

9

217 94 48

9

1

AG

GG

5 7 1 5 4 8  1 c

1 1 0 0 1 2

   

COMT

rs4646

316

       

 

CC

CT

TT

4

2

5

912 67 30

5

70.6

c2

4

3

44 22 20

3

8

5 7 2 11 3 6

  

rs9332

377       

 

CC

CT

TT

6

0

8

516 89 44

8

3

1

0

1

42 11 8

1

5

0.7

c

1 1 0 0 1 2

 

 

ABCC3

rs1051

640

5

2

7

3

 

13

5

72

28

 

39

13

7

4

   

 

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AA

AG

GG

2

5

1

0 0 1

2

5

2

1

81 c

1

                             

aFisher’s exact test; bMann-Whitney test; cFisher’s exact test for allelic association.

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Table 4: Clinical characteristics of patients with moderate to severe hearing loss (n = 18).

Patie

nt

Number

Age at

treatment

initiation

(months)

Age at

detection of

hearing loss

(months)

Cumulative

carboplatin

dose (mg/m2 )

Chemothe

rapy

Number

of

chemotherap

y cycles

1 0.53 3 436C/V/VM-

266

2 2 4 900C/V/VM-

266

3 3 15 1,325C/V/VM-

269

4 3.5 45 850 CEV 5

5 4 6 1,516 CEV 7

6 4 7 633 CEV 3

7 4 7.5 260C/V/VM-

262

8 5.5 36 730C/V/VM-

263

9 8 47 2,195C/V/VM-

268

10 8 83 1,400C/V/VM-

268

11 9 41 5,148 C/V/VM- 12

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26

12 9 36 2,151 CEV 6

13 10 84 1,060C/V/VM-

265

14 12 40 1,213 CEV 4

15 13.5 16 2,784C/V/VM-

268

16 15 40 3,540C/V/VM-

2610

17 18 58 715C/V/VM-

262

18 30 36 1,650 CEV 5

Abbreviations: CEV, carboplatin + etoposide + vincristine; C/V/VM-26, carboplatin +

vincristine + teniposide.