University of Groningen

Survivorship care after testicular cancerBoer, Hindrik

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Citation for published version (APA):Boer, H. (2019). Survivorship care after testicular cancer: New insights in late effects of treatment andapproaches to shared-care follow-up. [Groningen]: Rijksuniversiteit Groningen.

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89

Vascular fingerprint and vascular damage markers associated with vascular events

in testicular cancer patients during and after chemotherapy

Chapter 5

5

S. Lubberts1, H. Boer1, R. Altena1, C. Meijer1, A.M. van Roon2, N. Zwart1, S.F. Oosting1, P.W. Kamphuisen2, J. Nuver1, A.J. Smit2, A.B. Mulder3, J.D. Lefrandt2, J.A. Gietema1

Author affi liations: Departments of

1Medical Oncology, 2Vascular Medicine and

3Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

European Journal of Cancer. 2016; 63: 180-188

90

Abstract

BackgroundMetastatic testicular cancer (TC) can be cured with bleomycin, etoposide and cisplatin (BEP) chemotherapy. This comes at the price of an increased cardiovascular disease risk, not only years afterwards, but also during and shortly after chemotherapy. To prevent cardiovascular events, high-risk patients should be identified. Aim of this study was to assess BEP-chemotherapy induced vascular damage and to find risk factors for early vascular events.

Patients and methods A prospective cohort study was performed in (B)EP treated TC patients. Development of venous and arterial vascular events was assessed. Vascular damage markers (vWF, FVIII, IMT) and cardiovascular risk factors were assessed before and until 1 year after chemotherapy. Before start of chemotherapy a vascular fingerprint was estimated. Presence of ≥3 risk factors was defined as high-risk vascular fingerprint: BMI>25 kg/m2, current smoking, blood pressure>140/90 mmHg, total cholesterol>5.1 and/or LDL>2.5 mmol/L or glucose≥7 mmol/L.

ResultsSeventy-three patients were included. Eight (11%) developed vascular events (4 arterial events, 4 pulmonary embolisms). VWF and FVIII increased during chemotherapy, especially in patients with vascular events. Sixteen patients (22%) had a high-risk vascular fingerprint before start of chemotherapy. These patients had arterial events more often (3/16(19%) vs. 1/57(2%);p=0.031) and higher vWF levels and IMT.

ConclusionsEndothelial activation and upregulation of procoagulant activity seem important mechanisms involved in early (B)EP-chemotherapy-induced vascular events. Before chemotherapy, a quarter already had cardiovascular risk factors. A vascular fingerprint could identify patients at risk for arterial events. This vascular fingerprint, when validated, can be used as a tool to select patients who may benefit from preventive strategies.

Chapter 5

91

Introduction

Since the introduction of platinum-based chemotherapy in 1977, the prognosis of metastatic testicular cancer (TC) improved remarkably, with survival rates of 80-90%.1 Treatment consists of orchidectomy followed by bleomycin, etoposide and cisplatin (BEP) combination chemotherapy.2 However, cure rates are compromised by the increased risk of cardiovascular disease (CVD) both during or shortly after treatment (acute onset)3,4 as well as years after treatment (late onset).5,6

The pathophysiology is not fully understood, and probably differs between acute and late CVD onset. One of the contributing factors for both types appears to be occurrence of endothelial dysfunction7, which can occur either as a direct chemotherapy effect or can be mediated by development of cardiovascular risk factors; TC survivorship is generally associated with an unfavorable cardiovascular risk profile.8,9

That early onset CVD is a source of serious treatment-induced morbidity and mortality was recently confirmed by Fung et al10. If high-risk patients could be identified before start of chemotherapy, preventive strategies for this group can be explored to prevent early cardiovascular events.Therefore, the aim of this study was to assess BEP-chemotherapy-induced (subclinical) vascular damage and to find clues to identify patients at risk for early vascular events. To assess vascular damage we prospectively measured different markers for subclinical vascular damage and cardiovascular risk factors. We looked into the relationship of these markers and risk factors with development of both arterial and venous vascular events.

Methods

PatientsWe performed a prospective cohort study in metastatic TC patients, aged 18-50 years, treated with first-line (B)EP chemotherapy in the University Medical Center of Groningen, the Netherlands. Exclusion criteria were previous chemo-/radiotherapy, previous vascular events, erythropoietin use or glomerular filtration rate <60 mL/min. The ethics committee approved this study. All participants gave written informed consent. Patients received three or four (B)EP courses (etoposide 100 mg/m2, days 1-5, cisplatin 20 mg/m2, days 1-5, with/without bleomycin 30 USP, days 2, 8, 15) every three weeks and were during the first 6 days of every course hydrated with 4L saline per 24h. They received dexamethasone and ondansetron as antiemetics. Vascular events of both arterial and venous origin (WHO ICD-10 I1-I99) were included as possible events. Symptomatic as well as asymptomatic vascular events (discovered on staging CT scans) which developed after start of chemotherapy until two years afterwards were taken into account.The Khorana score, risk prediction model for venous thrombosis18, was assessed for every patient before start of chemotherapy.

Vascular damage markers in testicular cancer patients during and after chemotherapy

92

Plasma biomarkersVon Willebrand Factor (vWF) was measured in citrated plasma as described earlier.4 Coagulation factor VIII (FVIII) was measured in stored citrated plasma (-80 oC) using an automatic hemostasis testing system (ACL TOP 500, IL, the Netherlands), measuring APTT and using FVIII deficient plasma (SynthASil kit, IL, the Netherlands). Plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) were measured in citrated plasma as described previously [4]. Growth differentiation factor 15 (GDF-15; Human GDF-15 Quantikine ELISA kit (R&D Systems, USA)) and high sensitive C-reactive protein (hs-CRP; BNII Nephelometer (Siemens Healthcare Diagnostics BV, the Netherlands)) were measured in stored EDTA plasma (-20 oC).

Intima media thickness (IMT)Posterior wall IMT of the right common carotid artery was measured using an ultrasound device, as described earlier.4 The highest value from three measurements was recorded; maximal IMT best reflects atherosclerotic burden and cardiovascular risk.11,12 High IMT was defined as >97.5th age-specific percentile of healthy males.13

Oxidative stress memory: advanced glycation end products (AGEs)AGEs were determined in triplicate by measuring skin autofluorescence at the right lower arm with the AGE-reader (DiagnOptics Technology BV, the Netherlands).14 Measurements with a variation coefficient >12% or low reflection (R<0.07) were excluded.

Cardiovascular risk factors and the vascular fingerprintWeight, height, waist/hip circumference and blood pressure (sphygmomanometer) were measured. Current smoking behavior was recorded. In fasting blood samples, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, total cholesterol and glucose levels were measured. Metabolic syndrome was defined according to the NCEP.15 Cardiovascular risk was calculated using the European standard model (SCORE).16 However, the SCORE model was not an useful instrument: because of their young age, every patient had the same low 10-year CVD risk. Therefore, we explored which risk factors were needed to modify the SCORE model in our aim to identify high-risk patients, and compiled a vascular fingerprint of five risk factors prevalent in TC patients/survivors9,17: overweight, smoking, hypertension, dyslipidemia and impaired fasting glucose. We used generally accepted cut-off values: BMI >25 kg/m2, current smoking, blood pressure >140/90 mmHg (or using antihypertensive drugs), dyslipidemia (total cholesterol >5.1 mmol/L, LDL >2.5 mmol/L or using lipid lowering drugs), fasting glucose ≥7 mmol/L (or using blood glucose lowering drugs). A high-risk vascular fingerprint was defined as ≥3 risk factors before start of chemotherapy.

Timing of study-related investigationsIMT, AGEs and cardiovascular risk factors were measured before start, one month after completion and one year after start of chemotherapy. Plasma biomarkers were measured at the same time points and additionally 1-2 weeks after start of the third chemotherapy course.

Chapter 5

93

StatisticsData are presented as medians with ranges. Wilcoxon signed-rank tests were used for paired measurements. For comparisons between groups a Mann-Whitney U or Chi- Square test (expected counts <5: Fisher Exact test) was applied, as appropriate. Two- sided P-values <0.05 were considered significant. Statistical analyses were performed in SPSS Statistics 22.0 (IBM SPSS inc, USA). With the 73 patients, 85% power (α=0.05) was reached to detect an IMT difference of 180 μm (SD:160 μm)13 between patients with (n=8) and without events (n=65).

Figure 1. Patient accrual. Between May 2006 and June 2012, 78 consecutive patients were included of which 73 were eligible for analysis.

Results

PatientsSeventy-three consecutive patients were available for analysis (Figure 1). Characteristics are described in Table 1. Eight patients (11%) developed vascular events (4 arterial, 4 venous) (Supplementary table 1). Arterial events were a stroke, renal infarction, splenic infarction and one patient had both a stroke and renal infarction. All venous events were pulmonary embolisms, three were asymptomatic and discovered on CT-scans. Patients with vascular events were older (median 38 vs. 30 years (Table 2)). The known risk factors an elevated lactate dehydrogenase (LDH) level as well as the Khorana score18,19 did not differ between patients with and without events (data not shown).

Vascular damage markers in testicular cancer patients during and after chemotherapy

94

Table 1. Patient characteristics (n=73)

Chapter 5

Tables chapter 5

Table 1. Patient characteristics (n=73).

n/median %/range Age at start of chemotherapy, years 30 18 – 46 Histological diagnosis Non-seminoma 59 81 Seminoma 14 19 Disease stage Stage II 57 78 Stage III 7 10 Stage IV 9 12 IGCCCG prognosis group Good 61 84 Intermediate 11 15 Poor 1 1 Chemotherapy regimen 3 cycles BEP 53 73 4 cycles BEP 13 18 4 cycles EP 3 4 4 cycles BEP/EP combination 4 5 RPLND Before (B)EP 1 1 After (B)EP 34 47

Follow-up duration, months 33 5 – 89

Oncological status at last follow-up No evidence of testicular cancer 67 92 Refractory on (B)EP* 1 1 Death due to testicular cancer 1 1 Recurrent testicular cancer * 4 6 IGCCCG: international germ cell cancer collaborative group; RPLND: retroperitoneal lymph node dissection; BEP: bleomycin, etoposide and cisplatin containing chemotherapy; EP: etoposide and cisplatin containing chemotherapy. * These patients received TIP-chemotherapy (paclitaxel, iphosphamide and cisplatin) as second-line treatment.

IGCCCG: international germ cell cancer collaborative group; RPLND: retroperitoneal lymph node dissection; BEP: bleomycin, etoposide and cisplatin containing chemotherapy; EP: etoposide and cisplatin containing chemotherapy.

* These patients received TIP-chemotherapy (paclitaxel, iphosphamide and cisplatin) as second-line treatment.

95

Table 2 . Differences in vascular damage markers and cardiovascular risk factors in patients with versus patients without vascular events

vWF: von Willebrand Factor; FVIII: coagulation factor VIII; IMT: intima glycation end products; BMI: body mass index.

*Mann Whitney U test / Fisher exact test. *** significance disappeared after correction for age. ¶ IMT above the 97.5th percentile of an age-matched male reference group from literature13. ̂ Hypertension is defined as a systolic blood pressure >140 mmHg and/or a diastolic blood pressure >90 mmHg or use of antihypertensive drugs. ** Dyslipidemia is defined as a fasting total cholesterol >5.1 mmol/L, LDL >2.5 mmol/L, HDL < 1.04 mmol/L, ratio total cholesterol/HDL >5 and/or triglycerides >4.5 mmol/L or use of lipid lowering medication.

Vascular damage markers in testicular cancer patients during and after chemotherapy

96

Diabetes mellitus is defined as a fasting glucose ≥7.0 mmol/L or use of blood glucose lowering medication. � Metabolic syndrome is defined according to the NCEP ATP III definition update in 2005 in which three or more of the following factors have to be present: waist circumference ≥102 cm, triglycerides ≥1.7 mmol/L, HDL cholesterol <1.03 mmol/L or use of lipid lowering medication, blood pressure ≥130/85 mmHg or use of antihypertensive drugs, fasting glucose ≥5.6 mmol/L or use of blood glucose lowering medication. Patients with a BMI >30 kg/m2 with an unknown waist circumference were considered to have abdominal obesity and therefore scored as one point for increased waist circumference.

Plasma biomarkersPatterns of biomarker levels are shown in Table 3.Before chemotherapy, 12% had a high vWF level (>150%). Pre-chemotherapy high vWF did not predict for vascular events. Patients with vascular events had higher vWF levels during chemotherapy and one month afterwards compared to patients without vascular events (Figure 2, Table 2).Six patients had an elevated FVIII level (>150%) (n=6/71, 8%) before chemotherapy of whom three (n=3/6, 50%) developed vascular events versus five patients (n=5/65, 8%) with FVIII ≤150% (p=0.017). Patients with vascular events had higher FVIII levels pre-chemotherapy, during chemotherapy and one month afterwards compared to patients without vascular events (Figure 2, Table 2).PAI-1, t-PA, PAI-1/t-PA ratio, GDF-15, and hs-CRP were not associated with the observed vascular events.

IMTIn five out of the 73 patients (7%) no successful IMT measurement was performed before start of chemotherapy due to logistic or technical reasons. Of these 68 evaluable patients, 21 (31%) had a high IMT at start of chemotherapy. Three of the 21 (14%) developed arterial events, compared to none in the remaining 47 patients (p=0.027). Overall, IMT did not change in the first year after chemotherapy (Table 4). Absolute IMT and IMT changes were, after correction for age, not different in patients who developed vascular events (Table 2).

AGEsThe amount of AGEs did not change during the first year after treatment (Table 3). There were no differences in patients with vascular events compared to those without vascular events (Table 2).

Chapter 5

97

Tabl

e 3.

Vas

cula

r mar

kers

bef

ore,

dur

ing

and

afte

r che

mot

hera

py.

vWF:

von

Will

ebra

nd F

acto

r; FV

III: c

oagu

latio

n fa

ctor

VIII

; PAI

-1: p

lasm

inog

en a

ctiv

ator

inhi

bito

r 1; t

-PA:

tiss

ue p

lasm

inog

en a

ctiv

ator

; GD

F-15

: gro

wth

diff

eren

tiatio

n fa

ctor

15;

hs

-CRP

: hig

h-se

nsiti

vity

C-re

activ

e pr

otei

n; IM

T: in

tima

med

ia th

ickn

ess;

AGEs

: adv

ance

d gl

ycat

ion

end

prod

ucts

; BM

I: bod

y mas

s ind

ex.

*: sig

nific

antly

diff

eren

t fro

m m

easu

rem

ent b

efor

e st

art o

f che

mot

hera

py.

Be

fore

star

t of

chem

othe

rapy

Dur

ing

3th

cour

se o

f ch

emot

hera

py

O

ne m

onth

aft

er

chem

othe

rapy

One

yea

r aft

er

chem

othe

rapy

med

ian/

n ra

nge/

%

m

edia

n/n

rang

e/%

med

ian/

n ra

nge/

%

m

edia

n/n

rang

e/%

Pl

asm

a bi

omar

kers

vW

F, %

98

42

-297

160*

51

-379

124*

54

-249

111*

44

-237

FV

III, %

10

0 49

-162

152*

55

-342

118*

73

-192

123*

34

-197

PA

I-1, µ

g/l

28

2-14

3

14*

5-48

27

6-12

6

21*

8-66

t-P

A, µ

g/l

8 4-

19

7*

3-

16

9*

4-

28

9*

4-

20

PA

I-1/t

-PA

ratio

3.

7 1.

0-12

.5

2.

0*

0.8-

7.5

3.

3*

0.6-

12.6

2.6*

0.

7-5.

9

GD

F-15

, pg/

ml

394

187-

1935

5342

* 14

39-1

8959

865*

29

9-47

37

38

2*

247-

981

hs

-CRP

, mg/

L 1.

3 0.

2-50

.4

0.

5*

0.2-

132.

0

2.2

0.3-

29.1

1.5

0.2-

15.1

IM

T, µ

m

586

435-

1097

586

420-

1065

600

400-

1037

AG

Es, A

U 1.

60

1.03

-2.7

0

1.56

0.

99-2

.28

1.

59

1.17

-2.5

1

Card

iova

scul

ar ri

sk fa

ctor

s

Curre

nt sm

oker

20

/73

27

10

/70

14

16

/65

25

O

verw

eigh

t (BM

I >25

kg/

m2 )

41/7

3 56

43/6

9 62

36/6

6 55

Obe

sity

(BM

I >30

kg/

m2 )

14/7

3 19

17/6

9 25

13/6

7 19

Hype

rtens

ion^

14/6

0 23

7/66

* 11

7/65

11

Dys

lipid

emia

**

57/7

0 81

59/7

1 83

54/6

5 83

Dia

bete

s mel

litus

‡ 2/

68

3

3/71

4

2/

61

3

Met

abol

ic sy

ndro

me

□ 14

/62

23

18

/69

26

11

/65

17

Tabl

e 3.

Vas

cula

r mar

kers

bef

ore,

dur

ing

and

afte

r che

mot

hera

py

vWF:

von

Wille

bran

d Fa

ctor

; FVI

II: co

agul

atio

n fa

ctor

VIII

; PAI

-1: p

lasm

inog

en a

ctiva

tor i

nhib

itor 1

; t-P

A: d

iffer

entia

tion

fact

or 1

5; h

s-CR

P: h

igh-

sens

itivi

ty C

-reac

tive

prot

ein;

IMT:

intim

a m

edia

thick

ness

; AGE

s: m

ass i

ndex

.tis

sue p

lasm

inog

en a

ctiva

tor;

GDF-

15: g

row

th a

dvan

ced

glyc

atio

n en

d pr

oduc

ts; B

MI: b

ody

*: sig

nific

antly

diff

eren

t fro

m m

easu

rem

ent b

efor

e sta

rt of

chem

othe

rapy

.^

Hype

rtens

ion

is de

fined

as a

syst

olic

bloo

d pr

essu

re >

140

mm

Hg a

nd/o

r a d

iast

olic

bloo

d pr

essu

re >

90 m

mHg

or u

se o

f ant

ihyp

erte

nsive

dru

gs. *

* Dys

lipid

emia

is d

efine

d as

a

fast

ing

tota

l cho

lest

erol

>5.

1 m

mol

/L, L

DL >

2.5

mm

ol/L

, HD

L < 1

.04

mm

ol/L

, rat

io to

tal c

hole

ster

ol/H

DL >

5 an

d/or

trig

lycer

ides

>4.

5 m

mol

/L o

r use

of l

ipid

low

erin

g m

edica

tion.

‡

Dia

bete

s mel

litus

is d

efine

d as

a fa

stin

g gl

ucos

e ≥7.

0 m

mol

/L o

r use

of b

lood

glu

cose

low

erin

g m

edica

tion.

� M

etab

olic

synd

rom

e is d

efine

d ac

cord

ing

to th

e NCE

P AT

P III

defi

nitio

n up

date

in 2

005

in w

hich

thre

e or m

ore o

f the

follo

win

g fa

ctor

s hav

e to

be p

rese

nt: w

aist

circ

umfe

renc

e ≥10

2 cm

, trig

lycer

ides

≥1.

7 m

mol

/L, H

DL c

hole

ster

ol <

1.03

mm

ol/L

or u

se o

f lip

id lo

wer

ing

med

icatio

n, b

lood

pre

ssur

e ≥13

0/85

mm

Hg o

r use

of a

ntih

yper

tens

ive d

rugs

, fast

ing

gluc

ose ≥

5.6

mm

ol/L

or u

se o

f blo

od g

luco

se lo

wer

ing

med

icatio

n. Pa

tient

s with

a

BMI >

30 kg

/m2

with

an

unkn

own

wai

st ci

rcum

fere

nce w

ere c

onsid

ered

to h

ave a

bdom

inal

obe

sity a

nd th

eref

ore s

core

d as

one

poi

nt fo

r inc

reas

ed w

aist

circ

umfe

renc

e.

98

Cardiovascular risk factors and the vascular fingerprintBefore start of chemotherapy, the most common risk factors were dyslipidemia (81%) and overweight (56%). Twenty-three percent had the metabolic syndrome (Table 3). Separate risk factors and the metabolic syndrome were equally present in patients with and without vascular events (Table 2). Sixteen patients (22%) had a high-risk vascular fingerprint, these patients developed arterial events more often (n=3/16 (19%) vs. 1/57 (2%); p=0.031) (Table 4). Prevalence of risk factors did not change in the first year after chemotherapy, except for a decreased prevalence of hypertension (p=0.021) (Table 3). Detected risk factors at start of chemotherapy were not treated.

The 16 patients with a high-risk vascular fingerprint had a higher IMT, higher vWF levels, FVIII levels more often >150% and higher AGEs before chemotherapy (Figure 2, Table 4). Adding pre-chemotherapy FVIII >150% as risk factor to the vascular fingerprint, defining presence of ≥3 out of 6 risk factors as high-risk, led to detection of all arterial events: 18 patients (n=18/71, 25%) had a high-risk vascular fingerprint with FVIII added, of whom 4 (n=4/18, 22%) developed arterial events, compared to no arterial events in the 53 patients without high-risk vascular fingerprint (p=0.003).

Persistently high vWF and FVIIIAfter one year, 29% still had high vWF and/or FVIII (>150%). These patients already had higher vWF and FVIII levels pre-chemotherapy (vWF: median 136% (range:84-219) vs. 82% (range:42-297); p<.001 and FVIII: median 123% (range:56-162) vs. 93% (range:49-152); p=0.001) and during chemotherapy and were older (Supplementary table 2).

Chapter 5

99

Figu

re2.

vWF

and

FVIII

leve

ls in

pat

ient

s with

vers

us w

ithou

t hig

h-ris

k va

scul

ar fi

nger

prin

t. Th

e re

d lin

es re

pres

ent p

atie

nts w

ith a

rter

ial v

ascu

lar e

vent

s, th

e bl

ue lin

es re

pres

ent p

atie

nts w

ith ve

nous

vasc

ular

even

ts. N

umbe

rs co

rresp

ond

with

the n

umbe

rs in

the l

eft c

olum

n of

Sup

plem

enta

ry Ta

ble 1

. The

gre

y are

a re

pres

ents

refe

renc

es va

lues

. Erro

r bar

s rep

rese

nt m

edia

ns w

ith in

terq

uart

ile ra

nges

. vW

F, vo

n W

illeb

rand

fact

or; F

VIII,

coag

ulat

ion

fact

or V

III; B

EP, b

leom

ycin

, et

opos

ide

and

cisp

latin

100

Supplementary table 2. Patients with persistently high vWF and/or FVIII after one year versus patients with normalized vWF and FVIII after one year (n=65 patients with measured plasma biomarkers after one year).

vWF and/or FVIII >150%

after one year n=19

Normalized vWF and VIII

after one year n=46

median/n range/% median/n range/% p*

Age at start of chemotherapy, years 36 25-45 30 18-46 .011

Cisplatin dose, mg/m2 300 300-700 300 300-400 .895 Biomarkers during 3th course vWF, % 203 144-379 142 51-276 <.001 FVIII, % 173 79-249 143 55-277 .004 PAI-1, µg/l 16 7-37 13 6-48 .416 t-PA, µg/l 9 5-16 7 3-16 .066 PAI-1/t-PA ratio 2.0 0.8-6.9 2.1 0.8-7.5 .493 GDF-15, pg/ml 5500 2475-14400 5235 3060-18959 .880 Hs-CRP, mg/L 0.7 0.2-132.0 0.4 0.2-9.6 .110 IMT change between start and one year after chemotherapy, µm 83 -31-300 3 -300-317 .181 Pre-chemotherapy high-risk vascular fingerprint 5 26 9 20 .418

vWF: von Willebrand Factor; FVIII: coagulation factor VIII; CT: chemotherapy; PAI-1: plasminogen activator inhibitor 1; t-PA: tissue plasminogen activator; GDF-15: growth differentiation factor 15; hs-CRP: high-sensitivity C-reactive protein; IMT: intima media thickness. *Mann Whitney U test / Fisher exact test.

Table 4 . Patients with versus patients without high-risk vascular fingerprint before start of chemotherapy

vWF: von Willebrand Factor; FVIII: coagulation advanced glycation end products.

*Mann Whitney U test / Fisher exact test.

Discussion

This study shows that (B)EP chemotherapy for metastatic TC induces endothelial activation and procoagulant activity. Both mechanisms were related to development of early vascular events. It also turned out that about a quarter of the TC patients already carries cardiovascular risk factors at start of chemotherapy. The vascular fingerprint combines these risk factors. Metastatic TC patients with a high-risk vascular fingerprint before start of (B)EP chemotherapy developed arterial vascular events more often (19% vs. 2%).

During chemotherapy a clear upregulation of endothelial activation (vWF) and a procoagulant effect (FVIII, PAI-1/t-PA ratio) was seen. The PAI-1/t-PA ratio decrease indicates a tumor-related pro-thrombotic state before start and fibrinolysis during chemotherapy. GDF-15 levels increased 13-fold during treatment, which is partly explained by GDF-15 being an apoptosis marker20, but the pattern does not suggest a relationship with cancer activity. GDF-15 is also known to participate in vascular pathological processes.21 In a preclinical model for chemotherapy-induced endothelial damage its genes were highly upregulated22. GDF-15 thus seems to reflect endothelial activation during chemotherapy. However, since all patients showed an increase, its distinguishing value is limited. Hs-CRP levels fell during treatment, correlating with dexamethasone administration.

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VWF and FVIII levels were higher in patients with vascular events, strongly suggesting that endothelial activation and procoagulant activity are part of underlying mechanisms behind early onset vascular disease. The only pre-chemotherapy biomarker related to vascular events was FVIII>150%. During and one month after chemotherapy, both vWF and FVIII were higher in patients with vascular events. The course of VWF and FVIII therefore can also be helpful in detecting high-risk patients. VWF is a known predictor for arterial events in the general population23 and FVIII has earlier been shown to be related to venous events in cancer patients.24 Both vWF and FVIII were not associated with venous events only in this TC cohort. Venous events probably have a different pathophysiology. Known risk factors are venous access devices, retroperitoneal tumor mass size, high lactate dehydrogenase (LDH) levels, and a high Khorana score.18, 19, 25 In our study, none of the patients with vascular events had venous access devices. Retroperitoneal masses, if any, were of limited size . LDH levels were not different in patients with or without vascular events. The Khorana score was not different between patients with or without vascular events.

IMT did not change in the first year after chemotherapy. In the male general population an increase of 5.2 μm/year is estimated.13 As inter-measurement variability is 5-10% [26], one year seems too short to observe IMT changes. A remarkable finding was that 31% had at start of chemotherapy a high IMT compared to age-matched peers from the healthy male group of a large Dutch cohort (n=1993).13 These patients developed more often arterial vascular events. Furthermore, a large part of the patients had cardiovascular risk factors before start of chemotherapy. Not only TC survivorship is thus associated with an unfavorable cardiovascular risk profile: at start of chemotherapy about a quarter of the patients seem already prone to development of CVD. Presence of cardiovascular risk factors did not change within the first year after chemotherapy, except for blood pressure. The higher blood pressure before start of chemotherapy is consistent with earlier findings4 and is probably related to stress of the recent cancer diagnosis and oncology ward admission.

The recent study of Fung et al. shows excess CVD deaths in the first year after chemotherapy for TC10. This underlines the importance of identifying high-risk patients before start of treatment, as standard application of prevention strategies also carries risk. We provide the vascular fingerprint as an easy tool to identify high-risk patients. The vascular fingerprint consists of traditional cardiovascular risk factors: overweight, smoking, hypertension, dyslipidemia and impaired fasting glucose. Patients with ≥3 risk factors were considered to have a high-risk vascular fingerprint (n=16, 22%). Adding pre-chemotherapy FVIII>150% as risk factor to the vascular fingerprint led to detection of all arterial events. Main differences with the metabolic syndrome –which was not associated with early onset vascular events– are the addition of smoking behavior and a more rigorous cut-off for overweight. The TC population is relatively young, it is assumable that a BMI of 25-30 kg/m2 is an additional risk factor.

The vascular fingerprint appears to pinpoint TC patients at increased risk for early onset arterial vascular events. The metabolic syndrome is more likely involved in late onset atherosclerotic

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CVD, because its definition is based on insulin resistance eventually leading to atherosclerosis.27 Although results are statistically significant and the vascular fingerprint is supported by known vascular damage markers (vWF, FVIII, IMT and AGEs), the current data especially are hypothesis-generating. Therefore a validation study is needed to test the performance of this selection tool in an independent larger cohort with newly diagnosed disseminated TC patients requiring cisplatin combination chemotherapy (validation study: Clinicaltrials.gov NCT02573584). When validated, intervention trials should be initiated in which TC patients with a high-risk vascular fingerprint before start of chemotherapy are randomized for a prophylactic intervention, for example with LMWHs.

One year after chemotherapy, 29% had a high vWF and/or FVIII level. These patients also had higher vWF and FVIII levels before start and during chemotherapy. This persistent vascular activation status possibly contributes to late onset CVD, in which the accelerated atherosclerotic phenotype plays a role. However, longer follow-up is needed to determine this effect. When this is done and the data available, possible intervention strategies could be explored but for now, these patients should be monitored closely during follow-up.

In conclusion, endothelial activation and increased procoagulant activity are important underlying mechanisms involved in the development of vascular events during and shortly after chemotherapy. Before start of chemotherapy, about a quarter of the TC patients already has an increased CVD risk, as demonstrated by presence of cardiovascular risk factors and a high IMT. Traditional cardiovascular risk factors were clustered in a pre-chemotherapy vascular fingerprint. This vascular fingerprint, when validated, can be used as a tool to select patients who may benefit from preventive strategies.

FundingThis work was supported by the Dutch Cancer Society (grant 2009-4365). The funding source had no role in study design, collection, analysis and interpretation of data and also no role in writing this manuscript.

DisclosuresAJS is founder and shareholder of Diagnoptics Technologies, The Netherlands, the company which developed the AGE reader. JG received institutional grants from Roche, AbbVie and Siemens. The other authors have no disclosures to report.

AcknowledgementsWe like to acknowledge Anne van Gessel, Annet Possel-Nicolai, Margreet Teune-Weesjes, Marianne Bruin and Wietze Kuipers for performing the IMT and AGE measurements. We thank Gerrie Steursma for her administrative support.

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Supplementary tables and figures

Supp

lem

enta

ry ta

ble

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106

Supplementary table 2. Patients with persistently high vWF and/or FVIII after one year versus patients with normalized vWF and FVIII after one year (n=65 patients with measured plasma biomarkers after one year)

vWF: von Willebrand Factor; FVIII: coagulation factor VIII; CT: chemotherapy; PAI-1: plasminogen activator inhibitor 1; t-PA: tissue plasminogen activator; GDF-15: growth differentiation factor 15; hs-CRP: high-sensitivity C-reactive protein; IMT: intima media thickness.

*Mann Whitney U test / Fisher exact test.

Supplementary table 2. Patients with persistently high vWF and/or FVIII after one year versus patients with normalized vWF and FVIII after one year (n=65 patients with measured plasma biomarkers after one year).

vWF and/or FVIII >150%

after one year n=19

Normalized vWF and VIII

after one year n=46

median/n range/% median/n range/% p*

Age at start of chemotherapy, years 36 25-45 30 18-46 .011

Cisplatin dose, mg/m2 300 300-700 300 300-400 .895 Biomarkers during 3th course vWF, % 203 144-379 142 51-276 <.001 FVIII, % 173 79-249 143 55-277 .004 PAI-1, µg/l 16 7-37 13 6-48 .416 t-PA, µg/l 9 5-16 7 3-16 .066 PAI-1/t-PA ratio 2.0 0.8-6.9 2.1 0.8-7.5 .493 GDF-15, pg/ml 5500 2475-14400 5235 3060-18959 .880 Hs-CRP, mg/L 0.7 0.2-132.0 0.4 0.2-9.6 .110 IMT change between start and one year after chemotherapy, µm 83 -31-300 3 -300-317 .181 Pre-chemotherapy high-risk vascular fingerprint 5 26 9 20 .418

vWF: von Willebrand Factor; FVIII: coagulation factor VIII; CT: chemotherapy; PAI-1: plasminogen activator inhibitor 1; t-PA: tissue plasminogen activator; GDF-15: growth differentiation factor 15; hs-CRP: high-sensitivity C-reactive protein; IMT: intima media thickness. *Mann Whitney U test / Fisher exact test.

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