University of Groningen Long-term adverse effects of cancer treatment Westerink, Nico-Derk Lodewijk

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Long-term adverse effects of cancer treatment

Westerink, Nico-Derk Lodewijk

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Westerink, N-D. L. (2018). Long-term adverse effects of cancer treatment: Susceptibility and intervention strategies. Rijksuniversiteit Groningen.

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101 101

Variation in the HFE gene is

associated with the development of

bleomycin-induced pulmonary

toxicity in testicular cancer patients

G.G.F. van der Schoot1_{, N.L. Westerink}1_{, S. Lubberts}1_{, J. Nuver}1_{, N. Zwart}1_{, A.M.E. Walenkamp}1_{, J.B.} Wempe2_{, C. Meijer}1_{, J.A. Gietema}1

Departments of 1_{Medical Oncology and}2_{Pulmonary Medicine, University of Groningen,} University Medical Center Groningen, Groningen, The Netherlands.

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102

ABSTRACT

Background: Bleomycin and cisplatin are of key importance in testicular cancer treatment.

Known potential serious adverse effects are bleomycin-induced pulmonary toxicity (BIP) and cisplatin-induced renal toxicity. Iron handling may play a role in development of this toxicity. Carriage of allelic variants of the HFE gene induces altered iron metabolism and may contribute to toxicity. We investigated the association between two common allelic variants of the HFE gene, H63D and C282Y, with development of pulmonary and renal toxicity during and after treatment with bleomycin- and cisplatin-containing chemotherapy.

Methods: In 369 testicular cancer patients treated with bleomycin and cisplatin at the University

Medical Center Groningen between 1978 and 2006, H63D and/or C282Y genotypes were determined with an allelic discrimination assay. Data were collected on development of BIP, pulmonary function parameters, renal function and survival.

Results: BIP developed more frequently in patients who were heterozygote (16 in 75, 21%) and

homozygote (2 in 4, 50%) for the H63D variant, compared with those who had the HFE wild-type gene (31 in 278, 11%) (P = 0.012). Overall survival, testicular cancer-related survival and change in renal function were not associated with the H63D variant.

Conclusion: We observed an association between presence of one or both H63D alleles

and development of BIP in testicular cancer patients treated with bleomycin combination chemotherapy. In patients heterozygote and homozygote for the H63D variant, BIP occurred more frequently compared to wild-type patients. When validated and confirmed, HFE H63D genotyping may be used to identify patients with increased risk for pulmonary bleomycin toxicity.

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103

INTRODUCTION

Testicular germ-cell cancer is the most frequently diagnosed cancer type among young men between the ages of 18 and 35.1_{Introduction of bleomycin- and cisplatin-containing chemotherapy} dramatically improved survival of patients with metastatic disease.2_{Unfortunately, a significant} number of testicular cancer patients develop toxicity induced by bleomycin and cisplatin, both during and after completion of treatment.3_{Important adverse events are bleomycin-induced} pulmonary toxicity (BIP) and cisplatin-induced renal toxicity. Signs of BIP occur in roughly 10% of the patients receiving bleomycin and may be fatal in 1 to 3% of these patients.4,5_{Renal toxicity is} both an acute and late adverse effect of cisplatin-containing chemotherapy for testicular cancer6 and is largely prevented by adequate hydration.

The toxic effect of bleomycin on tumor tissue and on healthy tissue can be attributed to its ability to initiate single- and double-strand DNA breaks in the presence of iron (Fe2+_{) and} oxygen (O₂).7,8_{Bleomycin can bind to DNA by forming a complex with Fe}2+_{in presence of O}

2, inducing generation of extremely reactive hydroxyl radicals, which induce DNA damage.8,9_These hydroxyl radicals also lead to apoptosis of healthy epithelial and endothelial cells, although this is restricted due to protective activity of the bleomycin-degrading enzyme bleomycin hydrolase (BLMH).10_{However, in pulmonary tissue, BLMH expression is low. Subsequently, lungs are prone} to develop BIP.7

Accumulation of iron occurs in patients with hereditary hemochromatosis, a genetic disorder resulting in altered iron metabolism caused by variants of the HFE gene.11_{In idiopathic pulmonary} fibrosis, carriage of variants in the HFE gene, H63D and C282Y, leads to excessive accumulation of extracellular iron in lungs.12_{Also, iron-dependent generation of reactive oxygen species (ROS)} increased in carriers of HFE gene variants. It is suggested that this altered iron homeostasis leads to microscopic oxidative stress-induced pulmonary injury.12

Iron may also be involved in development of cisplatin-induced renal toxicity. Exposure of cisplatin to renal tubular epithelial cells increases iron levels and the amount of ROS in in vitro and in vivo models.13_{Addition of iron chelators could prevent cisplatin-induced cytotoxicity} and provides protection against cisplatin-induced renal failure.13

The interaction of iron with bleomycin and cisplatin handling suggests that variation in the HFE gene might also contribute to development of pulmonary and renal toxicity. There is an unmet need to identify patients susceptible for serious BIP and cisplatin-induced renal toxicity. Therefore, the aim of this study was to investigate the association between two common allelic variants of the HFE gene, H63D and C282Y, with development of pulmonary and renal toxicity in testicular cancer patients treated with bleomycin- and cisplatin-containing chemotherapy.

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PATIENTS AND METHODS

Study population

Patients with metastatic testicular cancer treated with bleomycin- and cisplatin-containing chemotherapy at the University Medical Center Groningen (UMCG) between 1978 and 2006, aged 16 years or older, from whom genomic DNA was available for genotyping were eligible. After informed consent, genomic DNA was isolated from peripheral blood samples collected into EDTA tubes at the general practitioner or at the outpatient clinic. For deceased patients, genomic DNA was isolated from stored serum samples when available.14,15_{The study protocol} was approved by the local medical ethics committee.

Baseline characteristics

The following baseline characteristics were obtained from patients’ medical records: age at start of chemotherapy, Royal Marsden stage, International Germ Cell Cancer Collaborative Group (IGCCCG) classification, presence of pulmonary metastases, chemotherapeutic regimen, cumulative bleomycin and cisplatin dosages and creatinine clearance (CRCL).

Endpoints

Bleomycin-induced pulmonary toxicity

Occurrence of BIP was examined through patients’ medical records. Severity of BIP was classified as follows: (a) death due to BIP, (b) clinical and/or radiologic signs of BIP, resulting in hospitalization or early discontinuation of bleomycin administration, (c) clinical and/or radiologic signs of BIP after completion of treatment and (d) no signs of BIP.15_{Classification of BIP severity} was performed by an investigator blinded for HFE genotype. Age, cumulative bleomycin dose, pretreatment CRCL, pulmonary metastases and smoking were examined as risk factors for BIP.4 Pulmonary function

Pulmonary function tests were used to investigate bleomycin-induced changes in pulmonary function. Between 1979 and 1993, pulmonary function tests were performed in bleomycin-treated patients prior to chemotherapy and at three-week intervals during chemotherapy. The following parameters were measured: transfer factor of the lungs for carbon monoxide (T_LCO), transfer factor of the lungs for carbon monoxide per unit alveolar volume (K_CO), diffusing capacity of the alveolo-capillary membrane for carbon monoxide (D_M), slow inspiratory vital capacity (VC) and pulmonary capillary blood volume (V_CAP).15

Renal function

Renal function was estimated prior to chemotherapy, six weeks after completion of chemotherapy, one year and ten years after start of chemotherapy by means of serum concentration creatinine (µmol/l) and calculated CRCL (Cockcroft-Gault formula).

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105 Survival

Last follow-up date and vital status were obtained from medical records or via the general practitioner. In case of deceased patients, date and cause of death were collected from medical records or via the general practitioner. Survival time was defined as time between date at the start of chemotherapy and date of death or the date of the last follow-up visit.

DNA isolation and genotyping

Isolation of genomic DNA was performed according to standard protocols16_{or by use of the} Nucleospin blood XL kit from Marchery-Nagel (Bioké, Leiden, The Netherlands) according to the protocol of the manufacturer. Serum-derived DNA (isolated by using Qiagen Mini kits; Qiagen, Hilden, Germany) was amplified and quality checked (REPLI-g service; Qiagen).

The H63D and C282Y variants in the HFE gene were assessed with an allelic discrimination assay on an Applied Biosystems 7900HT sequence detection system (Applied Biosystems, ThermoFisher Scientific, Breda, The Netherlands). Genotyping of H63D (rs1799945) was performed with a custom Taqman SNP Genotyping Assay (Applied Biosystems) consisting of a forward primer: 5’-AAGCTTTGGGCTACGTGGA and a reverse primer: 5’-ATCTGGCTTGAAATTCTACTGGAA, in combination with a 5’-VIC-Q-MGB wild-type specific probe to identify the C-variant and a 5’-FAM-Q-MGB mutant-specific probe to identify the G-variant of H63D. Genotyping of C282Y (rs1800562) was also performed with a custom Taqman SNP Genotyping Assay consisting of a forward primer: 5’-GGCTGGATAACCTTGGCTGTA and a reverse primer: 5’-CACAATGAGGGGCTGATCC, in combination with a 5’-VIC-Q-MGB wild-type specific probe to identify the G-variant and a 5’-FAM-Q-MGB mutant-specific probe to identify the A-variant of C282Y.

Statistical analysis

Distribution of H63D and C282Y genotypes was tested for Hardy-Weinberg equilibrium. Patients who had the wild-type gene were compared with those who were heterozygote and homozygote for the H63D and/or C282Y variant. Univariate analyses were performed with Chi square tests or Fisher’s exact tests for categorical variables and with Kruskal-Wallis tests for nominal variables. In a combined analysis, individuals with the wild-type gene were compared with those with the H63D variant, the C282Y variant or both variants. Univariate analyses were performed with Mann-Whitney U tests for nominal variables and Chi square tests or Fisher’s exact tests for categorical variables. Kaplan-Meier curves were generated to illustrate survival. Differences in overall and testicular cancer-related survival were analyzed using log-rank tests. Two sided P-values < 0.05 were considered significant. Statistical analysis was performed using SPSS Statistics 22.0 (IBM SPSS Inc., Chicago, IL, U.S.A.).

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RESULTS

Patients and HFE genotyping

Between 1978 and 2006, 676 patients (≥ 16 years) were treated for metastatic testicular cancer. Of these patients, 545 were treated with bleomycin and cisplatin-containing chemotherapy. DNA for genotyping was available in 369 patients with testicular cancer, treated with bleomycin- and cisplatin-based chemotherapy between 1978 and 2006. Determination of the H63D variant was successfully performed in 366 patients. Determination of the C282Y variant was successfully performed in 364 patients (Table 1). Since the variant of C282Y was only present in 28 of 364 patients, this prohibits us from drawing conclusions from the results of this variant. Allelic frequencies of H63D and C282Y were in Hardy-Weinberg equilibrium.

Table 1: HFE genotyping of the H63D variant, the C282Y variant or both variants

H63D, n (%) C282Y, n (%) H63D, C282Y or both, n (%)

Wild-type 285 (77.9%) 336 (92.3%) 257 (71.2%)

Heterozygote 77 (21.0%) 28 (7.7%) 100 (27.7%)

Homozygote 4 (1.1%) 0 (0.0%) 4 (1.1%)

Total 366 364 361

Baseline characteristics

Age, Royal Marsden stage, IGCCCG classification, pulmonary metastases, chemotherapeutic regimen, cumulative bleomycin dose and CRCL did not differ between patients with the wild-type gene and those who were heterozygote or homozygote for the H63D variant. The cumulative cisplatin dose differed between patients with the wild-type gene and those who were heterozygote or homozygote for the H63D variant (P = 0.018) (Table 2). Baseline patient characteristics showed no differences regarding age, Royal Marsden stage, IGCCCG classification, pulmonary metastases, chemotherapeutic regimen, cumulative bleomycin and cisplatin doses and CRCL between patients with the wild-type gene and those who had either the H63D variant, the C282Y variant or both (Table 2, Supplemental Tables A1 and B1).

Pulmonary toxicity

Information on the presence of BIP was available in 357 patients. In 357 patients with H63D determination, 49 patients developed BIP (14%). Of these patients, BIP occurred in 31 in 278 patients with a wild-type gene (11%), in 16 in 75 H63D heterozygote patients (21%) and in 2 in 4 H63D homozygote patients (50%). Classification of the patients in different types of BIP is depicted in Table 3. Development of BIP between these groups differed significantly (P = 0.012) (Table 4). No differences in risk factors for BIP were observed between the groups. In the analysis of C282Y and the combined analysis of the H63D and C282Y variants, BIP equally occurred between patients with a wild-type gene compared with those with the C282Y variant or both the H63D and/or C282Y variant (P = 0.559, P = 0.093, respectively) (Supplemental Tables A2 and B2).

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107 Table 2: Baseline characteristics of patients with and patients without the H63D variant

Wild-type (n = 285) Heterozygote for H63D (n = 77) Homozygote for H63D (n = 4) P-value

Age at start chemotherapy

(yr), median (range) 28 (16-64) 27 (16-54) 33 (24-51) 0.398* Stage (Royal Marsden) 1_{, n (%)}

II 135 (48.2%) 35 (47.9%) 1 (25%)

0.782†

III 30 (10.7%) 6 (8.2%) 0 (0%)

IV 115 (41.1%) 32 (43.8%) 3 (75%)

IGCCCG prognosis group2_{, n (%)}

Good 137 (52.1%) 32 (47.8%) 2 (50%) 0.390† Intermediate 90 (34.2%) 20 (29.9%) 1 (25%) Poor 36 (13.7%) 15 (22.4%) 1 (25%) Metastases Pulmonary metastases3_{, n (%)} _{116 (41.1%)} _{31 (41.3%)} _{3 (75%)} _0.474† Chemotherapy regimen, n (%) BEP 222 (77.9%) 53 (68.8%) 4 (100%) 0.545† PVB/BEP 23 (8.1%) 8 (10.4%) -PVB 31 (10.9%) 14 (18.2%) -Other regimens 9 (3.2%) 2 (2.6%) -Chemotherapy dosage Cumulative dose bleomycine

(USP), median (range) 270 (60-360) 270 (90-360) 270 (210-270) 0.389* Cumulative dose cisplatin

(mg/m2_{), median (range)} 400 (80-1300) 400 (300-950) 350 (300-400) 0.018* Creatinine clearance

CRCL (ml/min), median

(range) 122.0 (65.0-225.2) 123.0 (41.0-174.2) 122.0 (79.1-161.0) 0.944* Two-sided P-values < 0.05 were considered significant. *Kruskal-Wallis test, † Fisher’s exact test.

CRCL: Creatinine clearance calculated with the Cockcroft-Gault formula, IGCCCG= International Germ Cell Consensus Classification Group, BEP: bleomycin - etoposide - cisplatin combination chemotherapy; PVB: cisplatin - vinblastine - bleomycin combination chemotherapy; USP: U.S. Pharmacopeia.

1_{stage (Royal Marsden) missing: wild-type - 5 patients, heterozygote - 4 patients,}2 _{IGCCCG missing: wild-type - 22 patients,}

heterozygote - 10 patients, 3_{pulmonary metastases missing: wild-type - 3 patients, heterozygote - 2 patients.}

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108

Table 3: Classification of BIP in the different H63D groups (total n = 357*) H63D Type of BIP* No signs of BIP (n) Death due to BIP (n)

Early stop of bleomycin administration or hospitalization due to BIP

(n)

BIP after completion of chemotherapy (n) R C R & C R C R & C Wild-type 1 2 3 14 8 - 3 247 Heterozygote 1 1 - 5 7 - 2 59 Homozygote 1 - - - 1 - - 2 Cumulative 3 3 3 19 16 - 5 308

BIP: bleomycin-induced pulmonary toxicity, R: radiologic signs of BIP, C: clinical signs of BIP. *In 357 of 366 patients information of BIP status was available.

Table 4: Analysis of the HFE H63D variant: occurence of BIP and risk factors of BIP Wild-type (n=278) Heterozygote for H63D (n=75) Homozygote for H63D (n=4) P-value BIP, n (%)** 31 (11.2%) 16 (21.3%) 2 (50%) 0.012†

Age at start chemotherapy

(yr), median (range) 28 (16-64) 27 (16-54) 33 (24-51) 0.398* IGCCCG prognosis group1_{, n (%)}

Poor 34 (13.3%) 14 (21.5%) 1 (25.0%)

0.165† Intermediate/good 222 (86.7%) 51 (78.5%) 3 (75.0%)

Risk factors Cumulative dose bleomycin

(USP), median (range) 270 (60-360) 270 (90-360) 270 (210-270) 0.389* CRCL (ml/min), median

(range) 122.0 (65.0-225.2) 123.0 (41.0-174.2) 122.0 (79.1-161.0) 0.944* Pulmonary metastases2_,

n (%) 114 (41.1%) 31 (41.3%) 3 (75%) 0.473†

Smokers, current and

previous3_{, n (%)} 153 (61.4%) 39 (59.1%) 1 (33.3%)

0.641† Non-smokers, n (%) 96 (38.6%) 27 ( 40.9%) 2 (66.7%)

Two-sided P-values < 0.05 were considered significant. *Kruskal-Wallis, †Fisher’s exact test, ** In 357 of 366 patients information of BIP status was available.

BIP: bleomycin-induced pulmonary toxicity; CRCL: creatinine clearance calculated with Cockcroft-Gault formula; IGCCCG: International Germ Cell Consensus Classification Group; USP: U.S. Pharmacopeia. 1_{IGCCCG missing: wild-type - 22 patients,}

heterozygote - 10 patients, 2_{pulmonary metastases missing: wild-type - 3 patients, heterozygote - 1 patient,}3

smokers/non-smokers missing: wild-type - 29 patients, heterozygote - 9 patients, homozygote - 1 patient.

We investigated whether the H63D variant influenced pulmonary function during chemotherapy. Data on T_LCO,K_CO,D_M, V_CAP and VC before and after 3 or 4 courses of BEP chemotherapy were available in 49, 46, 45, 57 and 45 patients respectively. Except for V_CAP, change in pulmonary function was similar in patients with a wild-type genotype compared with those with the H63D variant (Table 5). V_CAP decreased in the group with the H63D variant by 23.8 ml and in wild-type patients by 11.6 ml (P = 0.011).

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109 Table 5: Paramet ers of pulmonar y func tion of t

esticular cancer patients with the H63D var

iant Wil d-ty pe H et ero zy got e or hom oz ygot e for H 63 D P-valu e ‡ N Pr e-ch em o-th er ap y, m ed ia n ( IQR ) Po st-ch em o-th er ap y, m ed ia n ( IQR ) M ed ia n d el ta (IQR ) N Pr e-c he m o-th er ap y, m ed ian (IQR ) Po st-ch em oth er ap y, m ed ia n ( IQR ) M ed ia n d el ta (IQR ) TLCO (m m ol/ m in /k Pa ) 38 11 .5 (3 .5 ) 9. 3 ( 3. 6) -2 .6 (3 .0 ) 11 11 .9 (1 .9) 8. 6 ( 3. 2) -2. 6 ( 2. 7) 0. 857 KCO (m m ol/ m in /k Pa /l) 36 1.7 (0 .4) 1.5 (0 .3 ) -0. 3 ( 0. 3) 10 1.7 (0 .3 ) 1.4 (0 .4) -0. 2 ( 0. 5) 0. 76 9 DM (m m ol/ m in /k Pa ) 36 20 .5 (8 .2) 16 .0 (8 .3 6) -3 .9 (9 .0 ) 9 18. 2 ( 8. 2) 14 .0 (6 .4) -2 .9 (6 .7 ) 0. 49 6 VC (l ) 44 5. 2 ( 1.5) 4. 7 ( 1.4 ) -0 .3 3 (0 .8 ) 13 5. 4 ( 0.9 ) 4. 7 ( 1.9 ) -0. 1 ( 0. 9) 0. 662 VCAP (m l) 36 84 .2 (18 .6 ) 77 .7 (2 4. 3) -11 .6 (2 5. 3) 9 97 .1 ( 19. 4) 68 .4 (2 3. 2) -2 3. 8 ( 35 .3 ) 0. 011 Tw o-sid ed P -v alu es < 0. 05 w er e c on sid er ed s ig ni fic an t. ‡ M an n-W hi tn ey U t es t, P -v alu e c on ce rn s m ed ia n d el ta o f w ild -t yp e v s. h et er oz yg ot e o r h om oz yg ot e H 63 D v ar ia nt s. IQ R: i nt er qu ar til e r an ge ; TLCO : t ra ns fe r f ac to r o f t he lu ng s f or c ar bo n m on ox id e; K CO : t ra ns fe r f ac to r o f t he lu ng s f or c ar bo n m on ox id e p er u ni t a lve ol ar v olu m e; D M : d iff us in g c ap ac ity o f t he al ve ol o-ca pi lla ry m em br an e f or c ar bo n m on ox id e; V C: s lo w i nsp ira to ry v ita l c ap ac ity ; VCAP : p ulm on ar y c ap ill ar y b lo od v olu m e.

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110

Survival

Overall, 66 of 369 patients included in this analysis died, with testicular cancer as most common cause (n = 30 in 66; 45%). Other causes of death were cardiovascular disease (n = 10 in 66; 15%), a secondary malignancy (n = 8 in 66; 12%), pulmonary disease (n = 4 in 66; 6%), BIP (n = 3 in 66; 5%), or infectious disease (n = 3 in 66; 5%). In eight patients, the cause of death was unknown. Overall survival and testicular cancer-related survival did not differ between patients with a wild-type gene and those with the H63D variant nor the C282Y variant nor the H63D and/or C282Y variant combined (Figs. 1 and 2, Supplemental Fig. A).

Renal toxicity

Renal function, by means of serum creatinine level and calculated CRCL before chemotherapy, 6 weeks after the last course of chemotherapy, 1 year after start of chemotherapy and 10 years after start of chemotherapy, was similar in patients with or without HFE gene variants (Supplemental Table C).

Fig. 1: Overall survival of testicular cancer patients treated with bleomycin-cisplatin combination chemotherapy according to HFE H63D variant status.

0 10 20 30 40 20 40 60 80 100 Survival (%) Wild-type (n=285)

Heterozygote HFE H63D variant (n=77) Homozygote HFE H63D variant (n=4)

Time after start of chemotherapy (years)

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111 Fig. 2: Overall survival of testicular cancer patients treated with bleomycin-cisplatin combination chemotherapy according to combined HFE H63D and C282Y variant status.

Wild-type (n=257)

HFE H63D or C282Y variant (n=104) 20 40 60 80 100 Survival (%) 0 10 20 30 40

Time after start of chemotherapy (years)

DISCUSSION

This study investigated whether testicular cancer patients with allelic variants of the HFE gene, H63D and C282Y, were more susceptible to develop BIP or cisplatin-induced renal toxicity. We found that patients who were heterozygote and homozygote for the H63D variant developed BIP more frequently than wild-type H63D patients.

This study showed that 49 in 357 patients (14%) in whom the genotyping of H63D was performed developed BIP. BIP was fatal in 1% of the cases: one wild-type patient (1 in 285), one patient with the heterozygote variant (1 in 77) and one patient with the homozygote variant of H63D (1 in 4). Other reported studies have comparable figures of BIP prevalence and BIP as cause of death.4,5,7_{BIP developed in 11% of the patients with the wild-type gene. Of patients} who were heterozygote for the H63D variant, 21% developed BIP and 50% of patients who were homozygote for H63D developed BIP. The number of patients with homozygote H63D variant is low, limiting the value of this remarkable finding. However, there seems to be a ‘dose-effect’ considering the increasing percentage of BIP development inherent to the number of mutations in H63D alleles. No differences in age at start of chemotherapy, cumulative bleomycin dose, pre-treatment CRCL, pulmonary metastases and smoking were found between the groups, which were considered as risk factors for development of BIP.17

There may be a potential selection bias of the studied group, due to the retrospective design of this study. Especially, testicular patients with short survival interval after start of chemotherapy were potentially underrepresented in the study population. Considering the possibility that some of these patients died due to BIP, our analysis might represent an underestimation of the true number of patients who developed BIP. This can also be said for other causes of early death.

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The association of variation in the HFE gene and development of BIP could be explained by involvement of the HFE gene in iron metabolism. Excessive body iron leads to toxicity and cell death, due to increased free radical formation and lipid peroxidation.18_{The HFE gene encodes} a protein which plays a role in the regulation of iron transport across the cell.19_{The HFE protein} provides protection against iron overload by binding to transferrin receptors and reducing its affinity for iron-bound transferrin.11_{Variation of the HFE gene, like H63D and C282Y, alter the HFE} protein structure, which disrupts binding to transferrin receptors.20_{Subsequently, iron-bound} transferrin can bind more easily with transferrin receptors, leading to elevated levels of iron in blood serum. Elevated levels of iron might increase risk of ROS-induced DNA damage. Bleomycin might enhance this process through induction of extremely reactive oxygen radicals by complex formation with iron (Fe2+_).

Pulmonary function before and after chemotherapy was similar regarding T_LCO, K_CO, D_M and VC between patients with the wild-type gene and those with the H63D variant. However, we found a more pronounced decrease in pulmonary capillary blood volume (V_CAP) in patients who were heterozygote or homozygote for the H63D variant. This is in line with the study of Sleijfer et al.21_{, which describes that decreases in VC and V}

CAP are most specific for bleomycin-induced pulmonary alterations. The decrease in V_CAP might be explained by bleomycin-induced endothelial damage of the lung vasculature.22,23

Data on iron metabolism, such as iron, transferrin saturation and serum ferritin, were not regularly determined between 1978 and 2006 in testicular cancer patients in the UMCG and therefore, iron status was not available. As a consequence, it was not possible to link occurrence of BIP with iron metabolism. In future studies, it may be interesting to examine whether the presence of the H63D polymorphism is associated with alterations of a patient’s iron status and metabolism.

Patients with an H63D variant developed BIP more frequently. If the same results are found in the validation study, it is plausible that testicular cancer patients carrying H63D variants are predisposed to develop BIP and thereby, have a higher risk of treatment-induced morbidity/ mortality. If H63D heterozygosity or homozygosity is known before start of chemotherapy, altered regimes could be considered. Alternative options are four courses of etoposide and cisplatin (EP) in case of good prognosis disease or four courses of etoposide, ifosfamide and cisplatin (VIP) in case of intermediate or poor prognosis disease instead three or four courses of bleomycin, etoposide and cisplatin. This way development of BIP might be reduced or prevented, leading to a decrease in BIP as defined in this study, among the in general young testicular cancer patients. In conclusion, we observed an association between the H63D variant of the HFE gene and development of BIP in testicular cancer patients treated with bleomycin-cisplatin combination chemotherapy. In patients who were heterozygote and homozygote for the H63D variant, BIP occurred more frequently compared with wild-type patients. When validated and confirmed, HFE H63D genotyping may be used to identify patients with an increased risk for pulmonary bleomycin toxicity.

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Role of the funding source

Supported by grant CVZ 01-211 from the Health Care Insurance Board/Dutch Association of University Medical Centers, The Netherlands and by grant RUG2000-2177 from the Dutch Cancer Society, The Netherlands.

Conflict of interest statement

None declared.

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