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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85 85

Bleomycin-induced pulmonary

changes on restaging computed

tomography scans in two thirds of

testicular cancer patients show no

correlation with ﬁ brosis markers

M.W. den Hollander1_{, N.L. Westerink*}1_{, S. Lubberts*}1_{, A.H.H. Bongaerts}†2_{, R.F.E. Wolf}3_{, R. Altena}1_, J. Nuver1_{, S.F. Oosting}1_{, E.G.E. de Vries}1_{, A.M.E. Walenkamp}1_{, C. Meijer}1_{, J.A. Gietema}1

Departments of 1_{Medical Oncology,}2_{Radiology and}3_{Pulmonology, University of Groningen,} University Medical Center Groningen, Groningen, The Netherlands.

* these authors contributed equally †_deceased

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86

ABSTRACT

Background: In metastatic testicular cancer patients treated with bleomycin, etoposide and

cisplatin (BEP) chemotherapy, bleomycin-induced pneumonitis is a well-known and potentially fatal side effect. We sought to determine the prevalence of lesions as signs of bleomycin-induced pulmonary changes on restaging computed tomography (CT) scans after treatment and to ascertain whether fibrosis markers were predictive of these changes.

Patients and methods: This prospective non-randomized cohort study included metastatic

testicular cancer patients, 18 to 50 years of age, treated with BEP chemotherapy. Restaging CT scans were examined for lesions as signs of bleomycin-induced pulmonary changes by two independent radiologists and graded as minor, moderate or severe. Plasma samples were collected before, during and after treatment and were quantified for transforming growth factor-β1 (TGF-β1), growth differentiation factor-15 (GDF-15) and high-sensitivity C-reactive protein (hs-CRP).

Results: In total, 66 patients were included: forty-five (68%) showed signs of bleomycin-induced

pulmonary changes on the restaging CT scan, 37 of which were classified as minor and 8 as moderate. No differences in TGF-β1, GDF-15 or hs-CRP plasma levels were found between these groups.

Conclusion: Bleomycin-induced pulmonary changes are common on restaging CT scans after

BEP chemotherapy for metastatic testicular cancer. Changes in TGF-β1, GDF-15, and hs-CRP plasma levels do not differ between patients with and without radiological lesions as signs of bleomycin-induced pulmonary changes and are therefore not helpful as predictive biomarkers.

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87

INTRODUCTION

Metastatic testicular cancer has a favorable prognosis; 5-year overall survival is more than 90% when the disease is treated with the current standard of bleomycin, etoposide and cisplatin (BEP) chemotherapy. Bleomycin is considered an essential component of the regimen.1_Although generally well-tolerated, bleomycin-induced pulmonary toxicity is observed in roughly 10% of the patients treated with BEP, with clinical symptoms such as dry cough, dyspnea, crackles during auscultation, abnormalities on chest radiography and fever.2_{Bleomycin-induced pulmonary} toxicity is predominantly a fibrotic lung disease; although its pathogenesis is not clear, the immune system appears to be involved.3,4

Bleomycin-induced pulmonary toxicity occurs during bleomycin treatment but can also develop after a treatment-free interval of weeks to months.5_{In 1 to 3% of the affected patients,} this pulmonary toxicity is fatal.6_{The cumulative bleomycin dose is an important risk factor for} bleomycin-induced pulmonary toxicity, although a safe dose has not been established.7_Other known risk factors are smoking, impaired renal function and older age, but there is currently no test for predicting which patients will develop bleomycin-induced pulmonary toxicity or a therapy to prevent it.8,9_{The standard strategy in case of signs of bleomycin-induced pulmonary} toxicity during treatment is to stop bleomycin administration. The change in diffusion capacity is used in several centers as a decision tool to terminate bleomycin administration, although these changes do not appear to be specifically caused by bleomycin.10

In previous studies, restaging computed tomography (CT) scans after completion of BEP chemotherapy for metastatic testicular cancer were reported to show signs suggesting bleomycin-induced pulmonary changes.11-13_{These radiologic changes may be a good surrogate} endpoint for the development of bleomycin-induced pulmonary toxicity. However, because the effect of bleomycin on CT scans is observed only after completion of treatment, an upfront or early biomarker that identifies patients likely to develop bleomycin-induced pulmonary toxicity would be preferable. These patients could then be treated on a schedule without bleomycin. An early biomarker would support the decision to stop weekly bleomycin administration.

Transforming growth factor-β1 (TGF-β1) is a cytokine involved in many physiological and pathological processes, including immune response, cell proliferation, angiogenesis, fibrosis and oncogenesis.14_{TGF-β1 plays an important role in the development of bleomycin-induced} pulmonary toxicity and fibrosis in animal models.15-17_{In patients treated with radiotherapy, or} patients who have undergone stem cell transplantation, a relationship between TGF-β1 levels and treatment-induced pulmonary toxicity was found.18,19_{Growth differentiation factor-15} (GDF-15) (also known as macrophage inhibitory cytokine-1) is a member of the TGF-β1 superfamily. GDF-15 is a product of activated macrophages and is involved in inflammatory processes.20_The expression of GDF-15 is induced during the process of fibrosis and correlates with pulmonary function impairment in systemic sclerosis patients.21_{GDF-15 levels are also up-regulated in many} cancer types22_{In addition, we evaluated the role of the generally known inflammation marker} high-sensitivity C-reactive protein (hs-CRP).

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In this study, we determined the prevalence of pulmonary changes assumed to be the result of bleomycin treatment on available restaging CT scans in metastatic testicular cancer patients. We also determined whether fibrosis markers TGF-β1 and GDF-15 and the inflammation marker hs-CRP were predictive of these changes.

PATIENTS AND METHODS

Patients

We performed a prospective non-randomized biomarker cohort study with the following eligibility criteria: patients aged 18 to 50 years with metastatic testicular cancer who were treated with BEP chemotherapy at the University Medical Center Groningen. Exclusion criteria were a medical history of cardiovascular disease or a creatinine clearance < 60 ml per minute because primary assessments within the initial biomarker study were related to cardiovascular parameters. The current study on bleomycin-induced pulmonary toxicity was performed within this biomarker study. Inclusion criteria for this analysis were a minimum of at least three bleomycin administrations (dose, 90 U.S. Pharmacopeia [USP]) and availability of a chest CT scan as part of the restaging evaluation after chemotherapy. Patients were treated with a standard regimen of three or four BEP chemotherapy courses, depending on International Germ Cell Consensus Classification prognosis group. During the first six days of each course, patients received daily antiemetic therapy with dexamethasone and ondansetron. The local ethics committee approved the study. All patients gave written informed consent.

CT scans

Post-chemotherapy restaging CT scans were assessed for lesions that are possibly the result of bleomycin treatment by two independent radiologists who were blinded for clinical phenotype. Patients were instructed to inhale during CT of the thorax. CT was started 30 seconds after intravenous injection of contrast medium (55 ml Iomeron 350, Eisai Co., Tokyo, Japan. Scans were obtained in caudal-cranial direction from the deepest costophrenic pleural recesses to above the thorax aperture. For reconstruction, the following parameters were used: 3/1.5 mm on the Sensation-16 and Symbia T16 and 2/1.5 mm on the S-64, Definition and Biograph mCT scanners (kernel B40f) (used scanners: Siemens, Munich, Germany; Philips, Best, The Netherlands; Toshiba, Zoetermeer, The Netherlands; General Electric, Little Chalfont, Buckinghamshire, United Kingdom).

Radiologic criteria for lesions on restaging CT scan that were regarded as bleomycin-induced were that these lesions were newly developed since the start of BEP chemotherapy and could not be readily explained by other factors such as metastases or infection. When available, the next CT scan (follow-up scan), obtained as part of the routine follow-up after completion of treatment, was assessed to judge whether the suspected bleomycin-induced pulmonary changes on the restaging CT scan had diminished or resolved. The type of lesion was described and the lesion

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89 was scored as unifocal or multifocal; the location and number of lobes involved were noted. The extent of the radiologic lesions was graded as minor (involving only the outer third of the lung), moderate (involving both the outer and middle third of the lung, but not extending across the mediastinum) or severe (involving the whole width of the lung from periphery to mediastinum) according to Bellamy et al.11

Measurement of biomarkers

EDTA plasma samples were collected for all biomarkers before start of chemotherapy (day 1), for TGF-β1 on day 1 of every subsequent chemotherapy course, for GDF-15 and hs-CRP on day 8 of the third course and for all measured biomarkers at 4 weeks after the end of chemotherapy (at follow-up). These time points were chosen because these were the dosing days of the bleomycin. EDTA blood samples were centrifuged at 3,000 g at 4°C for 10 minutes and plasma was stored at -20°C. Total TGF-β1 levels were analyzed in these samples after activation with hydrochloric acid using a human TGF-β1 Quantikine ELISA kit (R&D Systems, Abingdon, United Kingdom). GDF-15 levels were measured with the Human GDF-15 Quantikine ELISA kit (R&D Systems). The hs-CRP levels were analyzed by using a nephelometer.

Statistical analysis

Differences in biomarker levels were determined between patients with and without pulmonary changes on CT scan and between patients with and without clinical bleomycin-induced pulmonary toxicity. We also analyzed differences between the groups based on the grade of the lesions on CT scan (none, minor, moderate or severe). Biomarker levels were evaluated as absolute values, relative values (normalized to pre-chemotherapy levels) and absolute increases. Mann-Whitney U tests and Kruskal-Wallis tests were used to evaluate differences in biomarker levels between groups. Chi square and Fisher exact tests were used to analyze categorical data. A Spearman correlation test was used to test correlations. A P-value ≤ 0.05 was considered to represent a statistically significant difference. Statistics were calculated with IBM SPSS Statistics 22 (IBM, Armonk, NY, U.S.A.). Graphs were made by using Prism software, version 5.00 (GraphPad, La Jolla, CA, U.S.A.).

RESULTS

Patients

Between May 2006 and June 2012, 78 patients were included in the biomarker study. In total, five patients were excluded from analysis because data were incomplete as a result of missed measurements or withdrawal of consent. Five patients received ≤ 2 BEP courses. Data from 66 patients were analyzed (Fig. 1, Table 1). Five of these 66 patients did not receive all initially planned bleomycin administrations because of clinical signs of bleomycin-induced pulmonary toxicity (n = 3), development of bleomycin skin toxicity (n = 1) and development of a pulmonary embolism (n = 1).

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Fig. 1: Study cohort

Study participation

n=78

Study completed

n=73

Available for analysis

n=66

Study not completed:

- withdrawal of informed consent (n=2) - missed measurements (n=3)

Excluded for this analysis: - EP or bleomycin <90 USP (n=2) - no restaging thoracic CT scan (n=2)

Table 1: Patient characteristics.

Characteristic Median (range) N (%)

Total patients 66

Age (yr) 31 (19-46)

Diagnosis Non-seminoma 54 (82%)

Seminoma 12 (18%)

IGCCC prognosis group

Good 55 (83%)

Intermediate 10 (15%)

Poor 1 (2%)

Cumulative administered bleomycin dose (USP)

150 1 (2%) 180 1 (2%) 240 2 (3%) 270 52 (79%) 330 1 (2%) 360 9 (14%) Pretreatment creatinine clearance (ml/min) 77 (53-107)

Pulmonary metastases Yes 7 (11%)

No 59 (89%) Smoking Yes 29 (44%) No 26 (39%) Ex 10 (15%) Unknown 1 (2%)

IGCCC: International Germ Cell Consensus Classification; USP: U.S. Pharmacopeia.

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Bleomycin-induced pulmonary changes on CT scan

Pretreatment staging CT scans of the chest and abdomen were obtained a median of 22 days (range 1-78 days) before the start of chemotherapy. For evaluation of treatment response, restaging CT scans were obtained shortly after the last course of chemotherapy (median 21 days, range 5-112 days). The CT scan during follow-up after completion of treatment was obtained a median of 175 days (range 70-588) after the restaging CT scan. In 45 of 66 (68%) analyzed patients, radiological lesions of suspected bleomycin-induced pulmonary changes were seen on the restaging scan. Of these 45 patients, 37 were classifi ed as having minor pulmonary changes and eight as having moderate pulmonary changes (Fig. 2). None of the changes were qualifi ed as severe. Most pulmonary changes were multifocal and found in the basal parts of the lungs. Fig. 2: Representative examples of minor-grade (top row) and moderate-grade (bottom row) bleomycin-induced pulmonary changes on pretreatment, restaging, and follow-up computed tomography scans (left to right).

No relationship was found between the cumulative dose of bleomycin and the development of suspected bleomycin-induced pulmonary changes on the restaging CT scan. Renal function and smoking frequency did not diff er between groups (Table 2). On the available follow-up scans of patients suspected of having bleomycin-induced pulmonary changes (available in 38 of 45 patients; 84%), these lesions diminished in 14 patients (37%) and resolved in 24 patients (63%). Patient characteristics did not diff er between these two groups (Table 3). In three patients for whom administration of bleomycin was halted because of clinical signs of bleomycin-induced pulmonary toxicity, the restaging CT scan showed moderate (n = 1), minor (n = 1) and no pulmonary changes (n = 1).

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Table 2:

Charac

ter

istics of patients with and without sig

ns of bleom ycin-induced pulmonar y changes on C T scan. Ch ar ac te rist ic Wit ho ut ch an ge s (n = 2 1) M inor ch ange s (n = 3 7) M ode ra te ch ange s (n = 8 ) P-v al ue* M edi an Ra nge N (%) M edi an Ra nge N (%) M edi an Ra nge N (%) Ag e ( yr) 32 19 -4 5 31 20 -45 28 22 -4 6 0. 76 8 W ei gh t p re -c he m oth er ap y ( kg ) 88 70 -13 0 88 62 -13 9 87 65 -1 06 0. 852 Bl eo m yc in c um ul ati ve d os e ( US P) 27 0 18 0-36 0 27 0 15 0-36 0 27 0 24 0-36 0 0. 231 Ser um c re ati ni ne p re -che mo the ra py (m mol /l) 79 63 -1 07 75 52 -1 05 82 64 -89 0. 54 9 Ser um c re ati ni ne 4 w ee ks af ter l as t co ur se o f c he m oth er ap y ( m m ol /l) 82 57 -1 04 78 59 -9 6 82 68 -9 0 0. 63 0 Smok in g 8/ 21 ( 38 % ) 15 /3 6 (4 2%) 3/ 8 ( 38 %) 0. 87 8 *B as ed o n c om pa ris on o f p at ie nt s w ith m od er at e b le om yc in -in du ce d p ulm on ar y c ha ng es o n C T s ca n v s. p at ie nt s w ith n o o r m in or c ha ng es ( M an n-W hi tn ey U t es t). C T: c om pu te d t om og ra ph y; US P: U .S . P ha rm ac op eia .

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Table 3:

Charac

ter

istics of patients with f

ollo

w-up C

T scans and bleom

ycin-induced pulmonar y changes . Ch ar ac te rist ic Pa tie nt s i n whom b le om yc in -ind uc ed ch an ge s d is ap pe ar ed (n =2 4) Pa tie nt s i n whom b le om yc in -ind uc ed ch ange s di m in is he d ( n= 14 ) P-v al ue* T M edi an Ra nge N (%) M edi an Ra nge N (%) Ag e ( yr) 28 20 -43 30 26 -45 0. 215 W ei gh t p re -c he m oth er ap y ( kg ) 88 62 -13 4 92 72 -13 9 0.15 0 Bl eo m yc in c um ul ati ve d os e ( US P) 27 0 15 0-36 0 27 0 27 0-3 60 0. 07 5 Ser um c re ati ni ne p re -c he m oth er ap y (m m ol/ l) 75 62 -1 05 77 52 -93 0. 565 Ser um c re ati ni ne 4 w ee ks af ter l as t c ou rs e of c he m oth er ap y ( m m ol /l) 76 62 -91 76 59 -9 6 0. 693 Smok in g 10 /2 4 (4 2%) 7/ 13 (5 4% ) 0.4 84 M od er at e c ha ng es o n C T s ca n 2/ 24 (8 % ) 4/ 14 (2 9% ) 0.1 03 TG F-β1 ( pg /ml ) 1.1 4, 61 6 55 0-1 6,1 40 5, 447 2,8 61 -1 5,8 42 0. 34 8 2.1 7,8 13 90 9-30 ,130 10 ,3 64 4,15 6-25 ,8 87 0. 256 3.1 9, 818 3, 58 2-3 2, 05 7 9,5 33 3, 32 9-14 ,51 3 0. 68 7 4.1 3, 54 6 1,7 68 -1 8, 911 6, 602 3,6 64 -1 4,1 08 0. 32 7 C 4,9 16 59 5-17, 63 6 5,9 09 3,2 12 -1 4,2 07 0. 315 GD F-15 ( pg /ml ) 1.1 39 6 19 9-1,9 35 403 18 7-69 1 0. 882 3. 8 5, 23 5 2, 47 5-12 ,013 5, 721 4,13 0-10 ,76 1 0. 36 6 C 697 29 9-1 ,4 86 1,0 66 42 1-2 ,6 01 0. 297 hs -CR P ( m g/ l) 1.1 1.7 0. 3-50. 4 1.2 0. 2-36 .1 0. 405 3. 8 0. 5 0. 2-13 2 0. 5 0. 2-2.1 0. 682 C 2. 3 0. 3-29 .1 3. 0 0. 3-2 6. 7 0. 445 *M an n-W hi tn ey U te st . T : t im e po in t re fe rs to co ur se nu m be r, fo llo w ed by da y nu m be r (e .g ., 1.1 re fe rs to co ur se 1, da y 1) ; C : f ou r w ee ks af te r la st co ur se of ch em ot he ra py ; C T: co m pu te d to m og ra ph y; T G F-β1 : t ra ns fo rm in g g ro w th f ac to r-β 1; G D F-15 : g ro w th d iffe re nt ia tio n f ac to r-1 5; h s-CR P: h ig h-se ns iti vi ty C -re ac tive p ro te in ; U SP: U .S . P ha rm ac op eia .

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Biomarker plasma levels

Table 4 shows the biomarker plasma levels of all patients at clinically relevant time points. Median plasma levels before start of chemotherapy were 4,788 (range 550-24,369) pg/ml for TGF-β1, 392 (range 187-1,935) pg/ml for GDF-15 and 1.3 (range 0.2-50.4) mg/l for hs-CRP. No correlation was found between TGF-β1, GDF-15, hs-CRP and levels of tumor markers (α-fetoprotein [α-FP], β-human chorionic gonadotropin [β-HCG] and lactate dehydrogenase [LDH]) before start of treatment in patients with elevated tumor markers (data not shown).

No significant differences in absolute or relative levels of TGF-β1, GDF-15 and hs-CRP at the measured time points were found between patients with no, minor or moderate radiological bleomycin-induced pulmonary changes. In addition, no significant differences were found between patients with no or minor bleomycin-induced pulmonary changes and those with moderate pulmonary changes (Table 4). However, more patients who developed pulmonary changes that were observed on CT scan (minor or moderate) had an increased TGF-β1 between day one of the first BEP course (before start of chemotherapy) and day one of the second course (86% vs. 60%, P = 0.047). In addition, GDF-15 levels on day eight of the third course were higher in patients with moderate pulmonary changes on the CT scan than in patients with no or minor changes, although this difference was not statistically significant (7,246 pg/ml vs. 5,222 pg/ml, P = 0.087). All groups showed the same pattern of hs-CRP decrease during chemotherapy and an increase afterward. The biomarker levels of patients with clinical signs of bleomycin-induced pulmonary toxicity did not differ compared with those of other patients (Fig. 3). In patients with pulmonary changes on follow-up scans that had diminished but not disappeared (n = 14) TGF-β1, GDF-15 and hs-CRP levels did not differ compared with the patient group in which pulmonary changes disappeared (n = 24) (Table 3).

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95 Table 4: Biomar ker le vels in r elation t o pr esence of bleom ycin-induced pulmonar y changes on comput ed t omog raph y scan. Bio m ar ke r W ho le gr oup (n =6 6) Wit ho ut ch an ge s ( n= 21 ) M inor ch ange s ( n= 37 ) M ode ra te ch ange s ( n= 8) P-v al ue* T N (%) M edi an Ra nge M edi an Ra nge M edi an Ra nge M edi an Ra nge TG F-β1 ( pg /ml ) 1.1 66 (10 0% ) 4, 78 8 55 0-2 4, 36 9 4, 30 6 1,34 5-24 ,3 69 5, 002 55 0-1 6,1 40 5, 26 8 2, 05 2-13 ,36 1 0. 582 2.1 63 (9 5% ) 9,5 70 90 9-42 ,015 8,9 40 1,38 0-42, 01 5 8, 915 90 9-30 ,130 10 ,8 03 4,15 6-23 ,8 49 0. 397 3.1 59 (8 9% ) 7,8 89 1,4 84 -4 9, 89 7 6, 20 1 2, 37 8-49 ,8 97 8, 52 2 1,4 84 -3 2, 05 7 8, 512 3, 87 8-2 0, 37 2 0. 76 5 4.1 12 (1 8% ) 6, 774 1,7 68 -1 8, 911 6, 774 6, 23 2-7,3 16 3,9 12 1,7 68 -1 4,1 08 13 ,97 7 9, 04 3-18 ,911 0.12 1 C 64 (9 7% ) 5, 22 0 59 5-1 8, 83 9 5, 357 1,6 22 -1 8, 83 9 5, 041 59 5-17, 63 6 5,11 6 3, 223 -1 4, 20 7 0. 527 GD F-15 ( pg /ml ) 1.1 63 (9 5% ) 392 18 7-1,9 35 38 8 19 8-72 1 36 6 19 9-1,9 35 413 18 7-1,8 76 0. 397 3. 8 58 (8 8%) 5,34 2 1,4 39 -1 8,9 59 5, 20 9 1,4 39 -1 4, 40 0 5, 23 5 2, 47 5-12 ,013 7,2 46 4,13 0-18 ,95 9 0. 08 7 C 62 (9 4% ) 880 29 9-2 ,6 01 80 8 35 0-2 ,0 23 840 29 9-1,61 0 1,0 89 44 2-2 ,6 01 0. 07 3 hs -CR P ( m g/ l) 1.1 66 (10 0% ) 1.3 0. 2-50. 4 1.9 0. 2-1 8. 5 1.2 0. 2-50. 4 2.1 0. 3-3 9. 6 0. 813 3. 8 61 (9 2% ) 0. 5 0. 2-13 2. 0 0. 5 0. 2-2 5. 7 0. 5 0. 2-13 2. 0 0.7 0.2 -7 .2 0. 62 6 C 65 (9 8% ) 2. 2 0. 3-29 .1 2.7 0. 4-15 .3 2. 2 0. 3-29 .1 1.9 0.3 -9 .3 0. 54 6 *P -v alu e b as ed o n c om pa ris on o f p at ie nt s w ith m od er at e b le om yc in -in du ce d p ulm on ar y c ha ng es o n C T s ca n v s. p at ie nt s w ith n o o r m in or c ha ng es ( M an n-W hi tn ey U t es ts ). T: t im e po in t r ef er s t o co ur se n um be r, f ol lo w ed b y d ay nu m be r ( e. g. , 1 .1 re fe rs t o c ou rs e 1, d ay 1 ); C : f ou r w ee ks a fte r la st c ou rs e o f c he m ot he ra py ; C T: c om pu te d t om og ra ph y; TG F-β1 : tr an sf or m in g g ro w th f ac to r-β 1; G D F-15 : g ro w th d iffe re nt ia tio n f ac to r-1 5; h s-CR P: h ig h-se ns iti vi ty C -re ac tive p ro te in .

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Fig. 3: Plasma levels of TGF-β1, GDF-15 and hs-CRP respectively in all patients. Results are plotted according to no pulmonary changes on computed tomography (CT) scan (green); minor pulmonary changes on CT scan (orange); moderate pulmonary changes on CT scan (red) and clinical signs of bleomycin-induced pulmonary toxicity (black). Horizontal bars represent median and interquartile ranges. TGF-β1: transforming growth factor-β1; GDF-15: growth differentiation factor-15; hs-CRP: high-sensitivity C-reactive protein.

TGF-β1 (pg/mL) 0 10000 20000 30000 40000 50000 60000

Pre-chemotherapy Course 2, day 1 Month after chemotherapy Course 3, day 1 5000 10000 15000 20000

Pre-chemotherapy Course 3, day 8 Month after chemotherapy

GDF-15 (pg/mL) 0 500 1000 1500 2000 150 0 2 4 6 8 10 50 100

Pre-chemotherapy Course 3, day 8 Month after chemotherapy

Hs-CRP

(mg/L)

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DISCUSSION

In this study, we determined the prevalence of pulmonary changes - assumed to be the result of bleomycin treatment - on available restaging CT scans after BEP chemotherapy in patients with metastatic testicular cancer. We also determined whether fibrosis markers TGF-β1, GDF-15 and inflammation marker hs-CRP were predictive for these changes. In 68% of these patients, radiological lesions on the restaging CT scan - interpreted as signs of bleomycin-induced pulmonary changes - were observed. However, bleomycin administration was halted prematurely in only 5% of the patients because of clinical presentation that was suspected as being the result of bleomycin-induced pulmonary toxicity during treatment. This discrepancy between clinical presentation of bleomycin-induced pulmonary toxicity and radiological signs of bleomycin-induced toxicity on CT scans is notable. Previous studies reported a much higher incidence of abnormalities on restaging CT scans as well, compared with the 10% of the patients who classically develop clinical signs of bleomycin-induced pulmonary toxicity.11-13_However, these radiological abnormalities were not well documented and have not been reported in more recent clinical trials in which bleomycin is a component of the combination regimen for metastatic testicular cancer.23

The clinical relevance of radiological signs of bleomycin-induced pulmonary abnormalities on restaging CT scans remains unclear. Most of these abnormalities on CT scans were without accompanying symptoms and resolved spontaneously based on subsequent CT scans obtained several months later during follow-up. Our research question was whether post-treatment radiological findings are accompanied by biochemical signs of active fibrosis which could potentially be used as early markers of the toxic effect of bleomycin on the lung.

In the current study, we did not find a difference in TGF-β1 levels between patients with and without radiological signs of bleomycin-induced pulmonary changes. However, patients who developed bleomycin-induced pulmonary changes on CT scan showed increased TGF-β1 levels from pre-chemotherapy to day one of the second course. This may indicate involvement of the TGF-β1 pathway in development of pulmonary abnormalities due to bleomycin administration. The TGF-β1 plasma levels found in our patients at baseline had a broad range, as did these values in previous studies among healthy volunteers.24,25_{Measured TGF-β1 levels in our patient} group may also be tumor-derived rather than selectively the result of bleomycin-induced pulmonary changes.26_{However, no correlation between tumor markers in testicular cancer} (α-FP, β-HCG and LDH) and TGF-β1 levels before treatment was found. An interesting approach to solve this problem would be to assess TGF-β1 levels during bronchoalveolar lavage to assess only lung TGF-β1 levels.27

Although high levels of GDF-15 were present on day eight of the third course, we found no differences in GDF-15 levels in patients with bleomycin-induced pulmonary changes on their CT scan compared with those without pulmonary changes. The increase in GDF-15 levels were larger in patients with moderate pulmonary changes on CT scans, but this difference was not significant. The number of patients with moderate changes (n = 8) might have been too small to detect significant differences.

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Although, hs-CRP could be an easily accessible biomarker for subclinical inflammation, levels did not differ between patients with and without bleomycin-induced pulmonary changes. Moreover, because of the concurrent administration of dexamethasone (antiemetic drug), hs-CRP is not a usable biomarker for bleomycin-induced pulmonary toxicity during chemotherapy.

Our study has several limitations. First, no high-resolution CT scans were obtained in any patient, which precludes observations of interstitial pneumonitis. Second, we did not collect platelet-poor plasma, although TFG-β1 might have been released from platelets during collection and analyses of the samples.28_{TGF-β1 levels might have been falsely raised in our} observation. However, this does not preclude comparison of TGF-β1 levels within our patient group. In addition, GDF-15 is also known as an apoptosis marker20_{, which might limit its distinctive} value during cancer therapy. Nevertheless, it could be worth examining GDF-15 levels in a larger patient group with moderate pulmonary changes on CT scans after BEP chemotherapy.

CONCLUSION

Radiological pulmonary changes suspect for bleomycin-induced pulmonary toxicity are very common on restaging CT scans after treatment with BEP chemotherapy for metastatic testicular cancer, occurring in two thirds (68%) of the patients. Most of these radiological abnormalities appear to resolve on follow-up CT scans. TGF-β1, GDF-15 and hs-CRP do not seem usable as early biomarkers for pulmonary bleomycin-induced toxicity or the corresponding radiological pulmonary changes.

Acknowledgements

We thank Nynke Zwart and Gerrie Steursma for their technical and administrative support. This work was supported by the Dutch Cancer Society (DCS 2009-4365). The funding source had no role in the study design, collection, analysis, and interpretation of data, in the writing of the manuscript or in the decision to submit the manuscript for publication. This study was presented in part as a poster at the 2015 American Society of Clinical Oncology annual meeting in Chicago, IL, U.S.A.

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