Academiejaar 2018 – 2019

CORNEAL CHANGES IN TRASTUZUMAB-EMTANSINE TREATMENT

Els DEKLERCK

Promotor 1: Prof. dr. Hannelore DENYS

Promotor 2: Dr. Elke KREPS

Masterproef voorgedragen in de master in de specialistische geneeskunde OFTALMOLOGIE

Academiejaar 2018 – 2019

CORNEAL CHANGES IN TRASTUZUMAB-EMTANSINE TREATMENT

Els DEKLERCK

Promotor 1: Prof. dr. Hannelore DENYS

Promotor 2: Dr. Elke KREPS

Masterproef voorgedragen in de master in de specialistische geneeskunde OFTALMOLOGIE

PREFACE I would like to thank all my loved ones for the support during the writing of this work, especially

my husband and twin sister, who showed a lot of patience, understanding and love. I am proud

of the result and I wish the reader a lot of pleasure and enlightenment while going through this

work.

This project has led to a publication titled “Corneal features in trastuzumab-emtansine

treatment: Not a rare occurrence”.

Reference: Deklerck E, Denys H, Kreps EO. Corneal Features in Trastuzumab Emtansine

Treatment: Not a Rare Occurrence. Breast Cancer Res Treat. 2019 Feb 28. doi:

10.1007/s10549-019-05179-y.

5-year Journal Impact Factor: 4.132(Q1-Q2) (Web of Science)

Table of contents I. ABSTRACT .................................................................................................................................. 1

II. INTRODUCTION ........................................................................................................................ 2

A. Clinical Case ................................................................................................................................ 2

B. Literature Research and Context ................................................................................................. 3

1. Breast Cancer and Traditional Treatment ................................................................................ 3

2. Antibody-drug conjugates and T-DMI .................................................................................... 4

3. Trastuzumab-emtansine and adverse events............................................................................ 6

C. Goals and Objectives ................................................................................................................... 9

III. METHODS................................................................................................................................. 9

A. Study Patient Inclusion and Ophthalmological Evaluation ......................................................... 9

B. Statistical Analysis .................................................................................................................... 12

C. Literature Research .................................................................................................................... 12

IV. RESULTS ................................................................................................................................. 14

A. Patient and eye characteristics ................................................................................................... 14

B. Data analysis .............................................................................................................................. 17

1. Treatment duration ................................................................................................................ 17

2. Time between last dose and ophthalmological examination ................................................. 17

3. Cumulative dose .................................................................................................................... 18

4. Subjective symptoms ............................................................................................................. 19

5. Visual function ...................................................................................................................... 19

6. Adverse events ...................................................................................................................... 20

7. Corneal comorbidities ........................................................................................................... 20

V. DISCUSSION ........................................................................................................................... 21

A. General results and remarks ...................................................................................................... 21

B. Study Design and Statistical limitations .................................................................................... 21

C. Pathophysiology ........................................................................................................................ 23

D. Treatment................................................................................................................................... 25

E. Future ADCs .............................................................................................................................. 27

1. Coltuximab Ravtansine (SAR3419) ...................................................................................... 27

2. Trastuzumab Duocarmazine (SYD985) ................................................................................ 28

3. Denintuzumab mafodotin (SGN-CD19A) ............................................................................. 28

4. Indatuximab ravtansine (BT-062) ......................................................................................... 28

5. Mirvetuximab soravtansine (IMGN853) ............................................................................... 28

F. Conclusion ................................................................................................................................. 29

VI. DUTCH RESUME .................................................................................................................. 30

VII. REFERENCES ........................................................................................................................... I

VIII. APPENDIX .............................................................................................................................. VII

LIST OF ABBREVIATIONS

ADC Antibody-Drug Conjugate

AE Adverse events

AS Autologous serum

BCVA Best-corrected visual acuity

EGF Epidermal Growth Factor

HER-2 Human epidermal growth factor receptor-2

LE Left eye

LVEF Left ventricular ejection fraction

MBC Metastatic breast cancer

RE Right eye

SLE Slit-lamp examination

T-DM1 Ado-trastuzumab emtansine

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I. ABSTRACT

OBJECTIVE/PURPOSE:

Trastuzumab emtansine (T-DM1/Kadcyla;Genentech) is an antibody-drug conjugate (ADC)

used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive

metastasized breast cancer. Few studies report a spectrum of corneal changes in patients treated

with this drug. Our goal is to investigate the nature of the corneal changes we see in patients

using this drug, as more ADCs, promising low systemic toxicity, are emerging

METHODS:

In our cross-sectional study, a total of 13 patients, completed a full ophthalmic review. Eleven

patients were currently using Kadcyla, and two patients had recently (<10 weeks) stopped

treatment because of clinical non-response. One patient, our first encountered case of corneal

toxicity following T-DM1, was followed-up while treating the epitheliopathy with autologous

serum eye drops.

Also, we tried to find a correlation with dose and duration of therapy with T-DM1, time between

dosing and examination, subjective symptoms, visual function, other adverse events and

corneal comorbidity. Finally, we compared our results with the contemporary knowledge and

available literature.

RESULTS:

All examined patients, currently on T-DM1-treatment, exhibited coarse cystoid lesions to the

deep corneal epithelial cells, primarily in the midperipheral area, both biomicroscopically and

on confocal microscopy. The two patients who stopped treatment, displayed no corneal

epithelial changes. Only 3 patients reported ocular symptoms. Our one patient who was

followed in time while treating the epitheliopathy with eye drops, did not show significant

corneal changes in time. No significant correlation was found with the dose and duration of

treatment, nor did the extent of toxicity correlate with the ocular symptoms and other adverse

events related to treatment with T-DM1 or corneal comorbidities. During the study period, no

patients developed new ocular symptoms.

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CONCLUSIONS:

In conclusion, this case series shows that asymptomatic, low-grade corneal epithelial changes

are hallmark features in T-DM1- treatment and should not alarm clinicians. These findings seem

to be relatively stationary, reversible and thus do not require ocular treatment or cessation of

systemic treatment. Further long-term research into ocular toxicities of ADCs is warranted in

order to increase our understanding of this emerging field of therapeutics.

II. INTRODUCTION

A. Clinical Case In our ophthalmology department in 2017, we encountered a 40-year old Caucasian woman

referred by the oncology department because of blurred vision in both eyes for several weeks

(1). There was no ocular discomfort or redness, nor did the patient show any ocular

abnormalities in her previous ophthalmic review. She was diagnosed with advanced ductal

breast carcinoma in 2014 and had been treated with several chemotherapeutics, including

trastuzumab and tamoxifen. Because of progressive metastatic disease, she started treatment

with trastuzumab-emtansine 5 weeks before our ophthalmologic examination. Her best-

corrected visual acuity (BCVA) was 1.0 (decimal Snellen) in both eyes with optimal refractive

correction. The subjective blurred vision was for a large amount explainable by an uncorrected

hypermetropia. However, slit lamp examination revealed several corneal intraepithelial

spherical lesions in the midperipheral region, which showed no fluorescein staining (Figure 1).

These lesions were also visible on in vivo confocal microscopy as hyperreflective lesions in the

basal layers of the epithelium. Taking into account the blanc ocular history of this lady (she had

a normal eye examination in a previous ophthalmological consultation elsewhere), the tentative

diagnosis of T-DM1-associated epithelial toxicity was made (1). Because the patient reported

blurred vision and we wanted to prevent an exacerbation of the lesions during continuation of

T-DM1, we initiated empirical treatment with autologous serum 20% eye drops 6 times daily

to both eyes. This treatment showed to have little effect on the extent of the corneal lesions, nor

on the patient’s symptoms, so the treatment was tapered. Up to one year after the first

ophthalmologic examination, the corneal lesions were stable despite continued T-DM1

treatment. This case triggered our interest in assessing the consistency of corneal side effects in

T-DM1 treatment, along with the extent and evolution of corneal changes and symptoms during

treatment and dosing, as well as the necessity of ocular treatment.

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B. Literature Research and Context

1. Breast Cancer and Traditional Treatment Breast cancer is the most common type of cancer worldwide and the third leading cause of

cancer-related death among women in Belgium (2, 3). Five percent of newly diagnosed patients

will present with metastatic disease and 20 to 30% of patients with early-stage breast cancers

will experience relapse with metastatic disease (3). Approximately 15-25% of breast cancers

has overexpression or amplification of human epidermal growth factor receptor 2 (HER2), and

is associated with a more aggressive course and poorer prognosis (3-9). HER2 is a cell

membrane protein of the tyrosine kinase receptor family involved in regulation of cell

proliferation and differentiation (5, 6, 10). Traditional chemotherapeutic drugs (eg taxanes) kill

tumor cells nonspecifically, while monoclonal antibodies as trastuzumab can target tumor-

specific cell membrane proteins (11, 12). Since the introduction of trastuzumab as an adjuvant

in the chemotherapeutic treatment of HER2-positive breast cancer, there is a significant

improvement in the prognosis of these patients (HERA trial(13)).

For patients with HER2-positive metastatic breast cancer (MBC), current treatment guidelines

recommend early initiation of targeted anti-HER2 therapy, either alone or in combination with

traditional chemotherapy or endocrine therapy, with continuation of antiHER2 therapy

recommended even upon progression on an anti-HER2 agent (2). The combination of

trastuzumab, pertuzumab, and a taxane is considered first-line therapy after disease progression

with previous trastuzumab (6). Of the anti-HER2 targeted agents approved for clinical use in

Figure 1: Slit-lamp findings of our first patient showing presumed trastuzumab emtansine-associated corneal

epithelial toxicity: a midperipheral ring of opaque microspherules (arrow) in the basal epithelium

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HER2-positive breast cancer, trastuzumab is considered the cornerstone of treatment in these

patients, with proven efficacy both as a single agent and in combination with cytotoxic agents

(2, 9). However, treatment resistance is common and the management of toxicity associated

with adjunctive chemotherapy remains a challenge, which emphasizes the need for novel

alternative therapies (2, 3, 8). ADCs, and more specifically, T-DM1 is considered the standard

of care in second line therapy (6).

2. Antibody-drug conjugates and T-DMI ADCs are an emerging class of oncology therapeutics. These products are composed of a highly

specific high-molecular-weight monoclonal antibody, linked to a potent cytotoxic drug (6).

They selectively deliver the conjugated cytotoxic agent (small-molecular-weight) to tumor cells

targeted by the antibody component (6, 12). The functional linker is an essential component of

ADCs and facilitates drug release into targeted cells but has to keep the ADC stable in the

bloodstream (up to weeks) (6, 11, 14). This should reduce systemic toxicity in the treatment of

cancer. On the other hand, emerging data indicate that adverse effects relatively frequently

occur before ADCs have reached their optimal therapeutic dose, which results in a narrow

therapeutic window (15). The toxins incorporated into current ADCs must be very potent to be

efficient: they target either DNA or tubulin and originally failed as stand-alone chemotherapy

drugs because of unacceptable toxicity (11). Three ADCs are currently approved by the US

Food and Drug Administration (FDA)1 (6). T-DM1 was one of the first ADCs to be approved

in February 2013 (3, 7, 16, 17). This is part of the so-called second-generation ADCs, which

have a higher level of cytotoxic drug conjugation and lower levels of naked antibodies with a

more stable linker between the drug and the antibody (18). T-DM1 consists of the monoclonal

antibody trastuzumab linked to an average of 3.5 molecules of microtubule inhibitor

(emtansine) per antibody (6, 8, 11, 17, 19). T-DM1 binds to the extracellular part of the HER2-

receptor on tumor cells and is internalized in the target cell, which is in contrast to regular

therapeutic antibodies, which bind and block the receptor outside the cell (3, 11). Once inside,

the monoclonal antibody component undergoes proteolytic degradation, which releases the

active drug and linker, that disrupts the cell cycle during the G2-M phase and subsequently

causes apoptosis (1, 3, 6).

T-DM1 also maintains the pharmacological activity of trastuzumab, which causes

downregulation of HER2 receptors, inhibition of HER2 dimerization and cleavage, disruption

1 Gemtuzumab ozogamicin was approved by the FDA in 2000 (withdrawn from market in 2010, re-introduced in 2017 for acute myeloid leukemia), and brentuximab vedotin in 2011 for advanced lymphoma (6,12,15-16)

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of intracellular signal transduction, impairment of angiogenesis, and activation of immune

responses against tumor cells (3).

By now, phase III clinical trials comparing the efficacy and safety of T-DM1 with other

available therapies2,3 for MBC, showed a better tolerability profile and improved progression-

free survival in HER2-positive MBC patients previously treated with trastuzumab or lapatinib,

and chemotherapy (e.g. a taxane), and this in different ethnic populations (3, 6, 7, 12, 18, 20-

26). T-DM1 also had a more favorable toxicity profile. For example, in the EMILIA study, the

median progression free survival and median overall survival improved 3, respectively 5

months in favor of T-DM1 (24, 27). The incidence of grade 3 adverse events was higher in the

lapatinib-capecitabine group than in the T-DM1group (57.0% vs 40.8%)3 (24, 27).

The regimen used in these clinical trials is the same as the recommended dosage of T-DM1,

namely 3.6 mg/kg intravenously every 3 weeks (6, 12, 21). Half-life is 3.5 days in this regimen

(21). When toxicity is reported, dosing can be limited to 3.0 or 2.4 mg/kg (17). Weekly dosing

has a tolerability profile generally similar to that of the 3-weekly schedule, despite twofold

higher cumulative exposure to the agent in cycle 1 of the weekly regimen (2, 19).

Further studies will determine its use in the first-line, adjuvant and neoadjuvant setting (6),(3).

One of them, the MARIANNE phase III trial compares the efficacy of T-DM1 with or without

pertuzumab in first-line treatment of HER2-positive metastatic breast cancer with trastuzumab

and a taxane (2, 6, 12, 28). The results were rather disappointing with a non-superiority of T-

DM1 in progression free survival. The duration of response was longer for T-DM1 (21 months

vs 12.5 months for trastuzumab + taxane) and T-DM1 was better tolerated (12, 28).

On the other hand, more recently, the KATHERINE phase III open-label trial, studied patients

with residual invasive disease after receiving neoadjuvant therapy containing a taxane (with or

without anthracycline) and trastuzumab; patients were randomly assigned to receive adjuvant

T-DM1 or trastuzumab (29). At the interim analysis, invasive disease-free survival was

significantly higher in the T-DM1 group than in the trastuzumab group (hazard ratio for

invasive disease or death 0.50). Compared to trastuzumab alone, there were more adverse

events associated with T-DM1 (29).

2 EMILIA trial: Lapatinib-capecitabine or physician’s choice (3,6,12) 3 TH3RESA trial: Physician’s choice (12)

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The consensus now, is that treatment with T-DM1 is indicated for patients with HER2+ MBC,

previously treated with trastuzumab and a taxane, simultaneously or separated and experience

progression or recurrence within 6 months of this previous treatment (3, 17) .

Studies are also ongoing for the use of T-DM1 for other cancers such as HER2-positive gastric

cancer and small cell lung cancer (6). In general, ADCs are accepted to be less efficient for

central nervous tumors because of their large size, that restricts the drug from crossing the

blood-brain barrier (6). It was thought that a similar problem of penetration would be the case

in solid tumors, because of the vascularization of the first few layers (6). But better

developmental techniques provide the ADCs to be effective for solid tumors as well (11).

Meanwhile, development is occurring for ‘third-generation’ ADCs, as there is a booming of

clinical trials for (new) ADCs in the treatment of both solid and hematological tumors (see

‘Discussion’) (18). This stresses the importance of the awareness and knowledge of possible

adverse events, as researchers and clinicians in this way can anticipate possible problems (see

‘Discussion’).

3. Trastuzumab-emtansine and adverse events ADCs in humans are related to a wide range of toxicities, despite their goal to be selective.

Specifically, T-DM1 has similar safety profiles as DM1 alone, which is consistent with the

mechanism of action of DM1 (i.e. microtubule disruption) (5). On the other hand, T-DM1 has

several unique toxicities (due to the addition of emtansine), although generally well-tolerated

in clinical trials and current practice (8). In a preclinical safety profile study of Poon et al., T-

DM1 showed to be tolerable at doses up to 40mg/kg, where in contrast, DM1 alone was only

tolerable to doses of up to 0.2mg/kg, which supports the premise of ADCs to improve the

therapeutic index (5).

Known adverse events are, besides nausea (0.8% ≥ grade3) and fatigue (2.4% ≥ grade 3) ;

thrombocytopenia (12.9% ≥ grade 3) and bleeding, increased liver transaminases and bilirubin

(4.3% ≥ grade 3), and –sometimes- cardiotoxicity (less than 2% ≥ grade 3) (see also

‘Discussion’) ((3), (4, 6, 8, 12, 17, 19). Most AE were graded 1 or 2; the most frequent grade 3

AEs were hypokalemia (8.9%), thrombocytopenia (8.0%) and fatigue (4.5%), according to

Burris et al (4). Thrombocytopenia seems to be the most frequent dose limiting AE in phase I

studies (30). In phase II studies, the percentage of patients reporting or experiencing grade 3

AE varied between 24.2% and 58% (3). The rate of patients discontinuing treatment due to AE

varied between 3.6% and 16% (3). Diéras et al, who combined all available data from single-

agent T-DM1 studies at that time to better define the T-DM1 safety profile, also found that the

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most common grade 3 to 4 AEs were the laboratory abnormalities of thrombocytopenia (11.9%)

and increased AST serum concentration (4.3%) (27). They described 12 AE-related deaths and

17.2% dose reductions due to AEs (27). Overall, grade 3 or greater AEs were infrequent and

typically asymptomatic and manageable (3, 4, 27).

The safety profile seems to be similar across clinically relevant subgroups except for a greater

incidence of grade 3 AEs among patients older than age 65 years and in Asian patients (27, 31).

In the EMILIA trial, comparison of patients aged 65 years versus less than 65 years, showed an

increase in grade 3 AEs in both the T-DM1 (50.0% vs 43.7% respectively) and the lapatinib

plus capecitabine (71.2% vs 57.6% respectively) (32). The increase in grade 3 AEs in Asian

patients was primarily a result of an increased incidence of thrombocytopenia (27).

Given the notable adverse events in platelet production, patients should take regular laboratory

examination (before each dose) of platelet counts (33). Monitoring of left ventricular ejection

fraction (LVEF) every three months and hepatic function before each dose, is advised (6).

However, cardiac AEs did not appear to be as frequent or serious in T-DM1 compared to

trastuzumab by itself (33). In trastuzumab treatment, cardiotoxicity is considered the most

important side effect (2.5%-4.6%) (10). Antibody formation to T-DM1 is seen in 4.5% of

patients but does not seem to have clinical impact (6).

Recommendations for dose reduction or discontinuation due to toxicities are available (3, 8).

For patients with severe hepatic or neurological diseases, drugs should be taken under close

surveillance or should not be prescribed (33).

Solitary reports are made of more rare toxicities such as reversible carotenoderma and hand-

foot syndrome (yellow-orange discoloration and desquamation of palms and soles with oral

mucositis) (34); pulmonary arterial hypertension and telangiectasias (35); cutaneous vasculitis

(9). The exact mechanism and thus plausible causative relationship of these rare phenomena is

not proven.

Trials evaluating drug-drug interactions with T-DM1 have, to our knowledge, not been

performed. However, its cytotoxic component is metabolized by CPY3A4 (major) and

CYP3A5 (minor); thus, the potential for interactions with strong CYP3A4 inhibitors exists (3).

In phase I and II studies of T-DM1, ocular adverse effects were reported in 46.4% (19) and

31.3% (4) of patients respectively, in contrast to trastuzumab itself, where ocular side effects

are uncommon (2.5%)(10). These reports did not specify any slit lamp or confocal microscope

images and largely limited themselves to reported symptoms. Reported ocular adverse effects

of T-DM1 were dry eye, increased lacrimation, photophobia, subjective blurring of vision, and

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conjunctivitis (4, 8, 36). These were mostly recognized to be grade 1 and 2, and no severe

corneal complications with visual impairment have been reported (4). In a phase I study, two

patients reported grade 3 events (cataracts, ocular surface disease) (19). Weekly or 3-weekly

dosing did not make any difference in the prevalence of adverse events (19).

Eaton et al provided an overview of known ocular adverse events related to ADCs in 2015.

Ocular adverse events of ADCs were most frequently reported in nonspecific terms such as

blurred vision and ocular dryness, without further details concerning its pathophysiology,

management and need for cessation of ADC treatment (37). As for T-DM1, they stated that

ocular adverse events were reported in one third of the patients, mostly reported as blurred

vision (most common), dry eye, increased lacrimation, and conjunctivitis (37, 38). AEs

commonly involved the ocular surface, but less commonly intraocular tissues (37). Sporadic

encountered AEs include swollen tear duct, optic neuropathy and cortical blindness

(respectively cited in 1 or 2 references) (37).

The clinical methods used to assess affected patients were rarely reported, population numbers

were often small and heterogeneous; and detailed clinical description lacked often, which

makes uniform conclusions difficult (37).

Regarding corneal toxicity, which we will further discuss in the following pages, Tsuda et al.

were the first to describe a case with spectacular corneal toxicity resulting in vision loss (7).

Researchers described irregular, thickened corneal epithelium invading from the limbus in the

superior and inferior corneal areas disturbing the visual axis. A bit later, we (Department of

Ophthalmology and Medical Oncology, Ghent University Hospital) came across a patient with

limited corneal toxicity (see also ‘Goals and Objectives’)(1). The slit lamp image was

substantially different from the irregular epithelium described above by Tsuda et al. It showed

multiple intraepithelial spherical lesions in the corneal midperiphery with little to no staining.

As a side remark, we want to mention that research efforts are being made in drug targeting

using ADC for the treatment of choroidal neovascularization, proliferative vitreoretinopathy

and other ocular diseases (39). Compared to oncology, ADC research in ophthalmology is much

more limited, due to availability of other successful local therapies with limited concern of off-

target drug toxicity (39).

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C. Goals and Objectives Few reports have expanded on the specifics of T-DM1(and ADCs in general)-associated ocular

toxicity and were mostly limited to subjective symptoms , as we can conclude from the findings

above (6, 12, 37).

Tsuda et al. were the first to acknowledge an extensive keratopathy in the treatment with T-

DM1, necessitating discontinuation of treatment (7). On the other hand, we encountered a

patient with rather limited corneal toxicity limited to basal epithelial changes (1). It was not

clear whether these changes were progressive, needed treatment or discontinuation of T-DM1.

This discrepancy, and especially the potentially devastating and vision-threatening findings of

Tsuda et al. triggered our interest in assessing the extent of corneal changes and necessity of

ocular treatment or cessation of T-DM1. As these two publications are the only ones up until

now to describe a case of corneal toxicity linked to the treatment of T-DMI, we can expect this

to be a rather rare phenomenon.

Our goal was thus to investigate the prevalence, consistency and nature of the corneal changes

we see in patients using the drug T-DM1, by examining and describing a study population using

this drug, or recently stopped treatment. We also tried to find a correlation between the duration

and dosing of treatment, as well as a possible link between the extent of the corneal toxicity and

ocular symptoms, corneal comorbidities, and other adverse events related to T-DM1. As more

ADCs will become available, it will become paramount to understand which ocular toxicities

require immediate cessation of treatment and which are harmless. Existing literature, possible

explanations for our findings, consequences for the patient and implications for the daily clinic

and future research will be clarified.

III. METHODS

A. Study Patient Inclusion and Ophthalmological Evaluation

We performed a prospective, cross-sectional study from May 1 to September 30 2017, in a total

of 12 patients (excluding our previously published first case of presumed corneal toxicity

related to T-DMI (1)). Inclusion criteria consisted of current T-DM1 treatment or recent (<2

months) discontinuation of T-DM1 treatment. Patients with relevant ocular comorbidities

preventing adequate assessment of T-DM1 ocular features were excluded. 10 patients were on

treatment with T-DM1, and two patients recently (<10 weeks) stopped treatment. This was

conducted in the Ophthalmology Department of Ghent University Hospital (Belgium), in co-

operation with the Department of Ophthalmology and Medical Oncology. Ocular work-up

included BCVA, slit-lamp examination (SLE) with use of fluorescein, Goldmann applanation

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tonometry (measuring intra-ocular pressure), Schirmer I test (5 min; without topical

anaesthesia), dilated fundoscopy (evaluating the retina and optic nerve), and in vivo confocal

microscopy (Heidelberg Retina Tomograph II, Rostock Corneal Module, Heidelberg

Engineering, Heidelberg, Germany.).

Visual acuity was measured using the Snellen eye chart at 6 meters and noted as decimal

fraction.

Fluorescein-impregnated paper strips are wetted with a small drop of sterile unpreserved saline

and lightly applied to the surface of the eye. Areas with superficial epithelial damage (both

corneal and conjunctival) are typically highlighted by fluorescein when viewed with blue

(cobalt) light filter. Schirmer I testing allows for quantification of tear production. When

applied without topical anesthesia, it measures both the basal and reflex lacrimation. It is

considered abnormal when wetting of the filter paper strip was less than 10 mm after 5 minutes.

Specific grading systems for basal epithelial lesions are lacking. The Oxford grading system is

a commonly used, adequate system for grading ocular surface staining. Therefore, the intensity

of corneal cystoid lesions was graded using a modified Oxford grading system in which the

Oxford graded intensity of staining was applied to the cystoid lesions rather than the staining

pattern (Table 1)(40). We graded the deep corneal, cystoid lesions as mild-moderate (1+) and

severe (2+) for a semi-quantitative assessment of the epithelial lesions.

A confocal microscope is an imaging instrument in which a specimen, such as the cornea, is

illuminated with a focused light spot on a small focal volume. Emitted as well as reflected light

from the illuminated spot is then recollected and detected by a photodetection device,

transforming the light signal into an electrical one that is recorded. The illumination and

observation pathways have a common focal point, and because of that this principle is termed

‘confocal’. The detector aperture obstructs the light that is not coming from the focal point,

resulting in sharper images than those from conventional light microscopy techniques. As such,

a confocal arrangement isolates information from volume elements of only a few micrometers

of depth without the necessity of physical sectioning. Different types of confocal scanning exist:

tandem scanning, slit scanning and laser scanning. The latter is the most performant one, using

the dimmest light intensity source, providing the greatest contrast and scanning the most rapidly

(41). This is the principle our equipment (see above) is based upon.

Confocal microscopy allows a non-invasive in vivo cellular imaging of all layers of the cornea,

enabling the evaluation of the morphological characteristics of corneal abnormalities at the

histological level. It may therefore be helpful in diagnosis, determination of progression and

understanding the pathophysiology of disease (see also ‘Discussion’) (42, 43). It produces

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greyscale images and enables a microstructural insight into the in vivo cornea in both health

and disease (43).

Both eyes of each patient were examined and the cornea was scanned in 5 areas: the central

cornea and the 4 quadrants.

Ethics approval was obtained from the institutional Ethics Committee and written informed

consent was obtained for all included patients (see ‘Appendix’: information and informed

consent form).

Grade 1

Right eye of patient 5 (see Table 2), imaged using sclerotic scatter: scarce opaque lesions can be visualised (as indicated by the arrow). The intensity of opacities is graded in analogy to the intensity of fluorescein staining of the Oxford grading scale. This example constitutes a grade 1.

Grade 2

Left eye of patient 3 (see Table 2), as seen by iris retro-illumination: multiple, spheroid basal epithelial lesions are visualized without fluorescein uptake at increased frequency compared to the picture above. This was categorized as grade 2.

Table 1: Grading Scale, Adapted from Oxford Grading Scale (40)

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B. Statistical Analysis Data processing and statistical analysis were performed with SPSS version 23.0 (SPSS IBM

Inc, Chicago, USA). For all statistical analyses, a significance level of 0.05 (p-value) was used

(taken into account the small study group sample – see also ‘Discussion’). ). The spreadsheet

program Microsoft Office Excel, version 15.0 (2013) (Microsoft Corporation, Washington,

USA) was used for most of the graphics.

General descriptive parameters were listed for demographic and T-DM1-related data; as well

as ophthalmological examination results.

Concerning inferential statistics, the Spearman correlation test was used to find a possible link

between the corneal image and duration and dosing of treatment, as the data were organized as

an interval scale and non-parametric (Shapiro-Wilk test was used to test normality distribution

of parameters). Mann-Whitney U testing supports the findings of the Spearman correlation

tests, as they are used when the difference between two groups (patients with Oxford grade 1,

respectively Oxford grade 2 keratopathy) on some continuous non-parametric variable is tested.

Furthermore, Fisher’s Exact test was used to compare the categorical data of ocular symptoms,

general T-DM1-related adverse events and pre-existing ocular comorbidities with the extent of

the corneal lesions.

For clarity, we opted to conduct the data analysis without the patient that was the first to be

recognized as a possible case of T-DM1-related toxicity in our department, as we started

treatment in this case and tracked the evolution of the lesions in time (1). This patient was seen

before the start of this prospective study, and therefore was not included.

C. Literature Research

We performed a systematic literature search through the search engine ‘Pubmed’ using the

following search terms:

- Antibody drug conjugate AND eye

- Kadcyla AND side effects

- Cornea AND antibody drug conjugate

- Antibody drug conjugate AND corneal toxicity

In the following flowchart (Figure 2 – based upon the modified PRISMA flowchart (44)) the

selection process of the relevant articles is clarified. The additional 21 records that were added

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for the screening process were identified by checking the reference lists of the collected articles

through the search query.

After removal of duplicates, two articles were excluded from screening because of non-

availability. Of the screened articles, 86 articles were excluded based on the content of the title

and/or abstract. Another 12 articles were excluded after examining the full text (mostly because

of inadequate study design or irrelevant focus). The last search results were gathered in May

2018.

Figure 2: Flowchart of article selection process for systematic review (modified PRISMA flowchart – (44))

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IV. RESULTS

A. Patient and eye characteristics

We identified and examined, in collaboration with the Medical Oncology Service of Ghent

University Hospital, 10 patients who were currently on treatment with T-DMI. Dose

specifications and results of ocular work-up of the 10 patients on T-DM1 treatment are listed

in Table 2. Their mean age was 55.6 ±10.9 years (range 35-71). Prior treatment consisted of

taxanes and trastuzumab (Herceptin), next to a variety of other cytotoxic and monoclonal

agents. By the time of their ocular exam, they had received a median cumulative dose of 4566,8

mg of T-DM1 (range 1324,8 – 14385,6) during a median of 19 cycles (range 5-74). Our

assessment took place a varying amount of days after the last treatment cycle (range 1-21 days).

Seven patients received normal dosing of the therapeutic agent, 3 had 25-33% dose reduction

because of non-ocular side effects (elevation of liver parameters and thrombocytopenia in two

patients, one because of severe thrombocytopenia only).

We performed a thorough ocular exam including an inspection of the eyelids and –lashes,

conjunctiva and sclera, cornea, anterior chamber, iris and lens, retina and optic nerve. In all

patients, there were no signs of eczema, conjunctivitis, anterior chamber inflammation, optic

nerve anomalies or fundus abnormalities. Intra-ocular pressure was within normal limits in all

patients. Schirmer I was abnormal in 12 of 20 eyes (60%).

Three patients reported symptoms. One patient complained of dry eyes and had a BCVA of 0.9

in the right eye (RE) and 0.8 in the left eye (LE) (Decimal Snellen), which could be explained

by keratitis sicca (Schirmer score 7mm (RE), 9mm (LE)). There was fluorescein staining not

related to the deeper corneal lesions.

Another patient reported blurred vision and had a BCVA of 0.8 and 0.6, which was compatible

with vision loss due to cataracts. The third symptomatic patient also complained of blurred

vision, but showed a vision of 1.0 (RE) and 0.9 (LE) (most probably due to lens sclerosis). Only

2 patients requested lubrication treatment .

Each of the 20 eyes revealed a varying degree of corneal epithelial lesions on slit-lamp

examination. Lesions were predominantly observed in the midperipheral cornea. They

consisted of multiple small spheroid intra-epithelial inclusions lacking fluorescein staining

(Figure 3). According to the modified Oxford Grading Scale (see ‘Materials and Methods’), 11

of 20 eyes showed a grade2 keratopathy (55%). In all patients but one (patient number 6), the

intensity of epithelial opacities was very similar in both eyes.

15

Confocal microscopy confirmed these hyperreflective microcysts to be located in the basal

epithelium, with sparing of the superficial epithelium and the deeper corneal layers (Figure 4).

The foci did not extend to the subbasal plexus, Bowman’s layer or anerior stroma. The central

cornea showed little to no hyperreflective intra- or extracellular deposits, whereas the

midperiphery in all quadrants exhibited lesions. At the level of the subbasal nerve plexus, an

increased number of dendritic cells was remarked, mainly adjacent to higher intensity of

opacities.

Figure 3: Anterior segment photographs of the T-DM1 associated corneal microcystoid epitheliopathy. Panel on

the left shows the midperipheral lesions with iris retroillumination (see arrow). Cystoid lesions are

highlighted by fundus retroillumination (middle panel). A lack of fluorescein staining is characteristic (right panel).

Figure 4: In vivo confocal microscopy (images are 400 x 400 m). Panel A and B respectively show normal superficial corneal epitheium (depth 0 m)

and normal epithelial wing cells (depth 20 m). Panel C to E show increasing density of hyperreflective lesions at the level of the deeper wing

cells (depth 30 m). Panel F (depth 40 m) demonstrates the increased presence of dendritic cells at the level of the subbasal nerve plexus.

16

The previously published case (1) was followed-up as well, and showed no increase of corneal

changes upon tapering of her autologous serum eye drops despite continuation of T-DM1

treatment. During the study period, no patients developed new ocular symptoms that required

further ophthalmological follow-up.

Additionally, we examined two patients who stopped T-DMI treatment (>1 month at time of

exam: respectively 8 and 9 weeks following cessation) due to progression of disease. One

patient received a total of 12 cycles (cumulative dose 7261.2 mg) of T-DM1, the other 34

(cumulative dose 2710.08). These two patients did not show any sign of keratopathy.

Table 2: Patient characteristics of 10 patients on T-DM1 treatment (45). Best-corrected visual acuity was measured using the Snellen eye chart at 6 meters and noted as decimal fraction. A grading scale was based on a modified Oxford grading scale

(see ‘Materials and Methods’ (40)).

17

B. Data analysis To try to find an insight in the pathophysiology of the lesions, we analyzed the acquired data to

uncover a possible correlation with several parameters. In what follows, the different test results

are published, taking into account the small study sample and thus limited statistical value. The

figures below can be useful to indicate a direction of correlation visually.

1. Treatment duration First, we tried to find a correlation between the treatment duration and the extent of corneal side

effects. The Spearman correlation coefficient rs was -0.28 (p= 0.43). This was supported by a

non-significant Mann-Whitney U test (U=8 , p=0.39), testing the difference between the Oxford

grade 1 and Oxford grade 2 group.

2. Time between last dose and ophthalmological examination Second, we conducted a test to find a possible link between the time that has passed since the

last dosing of T-DM1 and the first ophthalmological examination; and the extent of the lesions.

The Spearman correlation coefficient rs was 0.29 (p=0.42). Mann-Whitney U testing was also

non-significant (U=8 , P=0.39).

Figure 5: Relationship between treatment duration and grade of keratopathy

18

3. Cumulative dose Third, a relationship between the cumulative dose of T-DM1 and the grade of keratopathy was

tested. The Spearman correlation coefficient rs was -0.07 (p=0.85). Mann-Whitney U test

resulted in a U value of 11 (p-value 0.83). Both non-significant as depicted in the figure below.

Figure 6: Relationship time between last dosing and eye examination; and grade of keratopathy

Figure 7: Relationship between cumulative dose and grade of keratopathy

19

4. Subjective symptoms Fourth, a relationship between the reported symptoms and the grade of keratopathy was tested.

These parameters are tested with the Fisher’s Exact-test. P-value was 0.57. An insignificant

result, published for completeness (see ‘Discussion’). The columns below show symptomatic

keratopathy is as much frequent as asymptomatic keratopathy in the patients with grade 2

keratopathy. Keratopathy without symptoms is more frequently encountered in patients with

grade 1 keratopathy compared to symptomatic lesions.

5. Visual function Fifth, a relationship between the visual acuity and grade of keratopathy was tested. The Fisher’s

Exact-test p-value was 0.08, again non-significant. The figure below shows that a BCVA of

less than 1.0 is more frequent in the group of patients with grade 2 keratopathy, compared to a

perfect vision in all those with grade 1 keratopathy.

0

1

2

3

4

Symptoms No symptoms

Symptoms and Grade of Keratopathy

Oxford grade 1 Oxford grade 2

0

1

2

3

4

5

BCVA less than 1,0 Normal BCVA

Vision and Grade of Keratopathy

Oxford grade 1 Oxford grade 2

Figure 8: Relationship between subjective symptoms and grade of keratopathy

Figure 9: Relationship between visual function and grade of keratopathy

20

6. Adverse events Sixth, a relationship between the frequency of other (non ocular) AEs and grade of keratopathy

was tested. Adverse events of all kinds were included, most frequently thrombocytopenia,

elevated liver transaminases and nausea. The Fisher’s Exact-test p-value was 0.53. Again non-

significant.

7. Corneal comorbidities Seventh, a relationship between the frequency of corneal comorbidities and grade of

keratopathy was tested. Comorbidities included ocular surface disease such as meibomianitis,

sicca keratopathy, guttae or a history of ocular infectious disease. The Fisher’s exact-test p-

value was 1; a useless result and non-significant, as shown in the figure below.

0

1

2

3

4

5

no AE AE

AE and Grade of Keratopathy

Oxford grade 1 Oxford grade 2

0 0,5 1 1,5 2 2,5 3 3,5

No corneal comorbidities

Corneal comorbidities

Corneal Comorbidities and Keratopathy

Oxford grade 2 Oxford grade 1

Figure 10: Relationship between adverse events of T-DM1 and grade of keratopathy

Figure 11: Relationship between ocular comorbidities and grade of keratopathy

21

V. DISCUSSION

A. General results and remarks

In short, all ten of our studied patients on T-DM1 showed typical corneal lesions, closely

described above. The keratopathy is clearly asymptomatic, as most of the subjective symptoms

such as dry eyes and blurred vision are explainable by other ocular factors (see ‘Results’ and

‘Pathophysiology’). Remarkably, patients who stopped treatment, did not show any signs of

keratopathy < 10 weeks after the cessation of T-DM1. This phenomenon could be due to two

reasons. Either the epitheliopathy is reversible and heals after cessation of treatment; or, on the

other hand, the corneal toxicity never developed in these patients. They both had therapy for

over one year, which gave them enough time to possibly develop corneal toxicity, and which

suggests initial therapy response. Since these patients stopped treatment because of disease

progression, it is plausible that the effectiveness of T-DM1 is related to its toxicity (see also

‘Pathophysiology’). Maybe the absence of corneal lesions pleads for a variant in the

HER2/EGFR pathway or expression, which makes the cornea less susceptible to the effects of

T-DM1, and makes it a less effective treatment for these patients as well. Our limited statistics

could not find a link between corneal toxicity and other T-DM1-related adverse events, which

are not always linked to normal tissue expression of targeted receptors (see ‘Pathophysiology’).

We assume that the lesions are stationary, because of the universal image in our patients,

irrespective of treatment duration, cumulative dosing and timing of ophthalmological review in

the treatment cycle (see also ‘Data analysis’). This remains an assumption, as our study is not

conducted in a longitudinal manner. This is partly an ethical decision, since these patients

already have to go through a lot of time- and energy-consuming hospital-related examinations.

In our protocol, we agreed to see patients back when referred from the Oncology service when

there was subjective ocular detoriation. This was not the case during the course of the study,

despite continuation of their T-DM1 treatment.

B. Study Design and Statistical limitations

We acknowledge a number of limitations to our study. As it was a monocentric study limited

in time, the number of patients is rather low (10 patients). We acknowledge that statistical

analysis on these numbers is of limited to no value. No scientifically or clinically reliable and

significant conclusions can be made concerning a relationship with parameters such as

cumulative dose of T-DM1, treatment duration, time between last dose and ophthalmological

evaluation, other adverse events, subjective symptoms, visual function and corneal

comorbidities. This does not mean that a possible correlation doesn’t exist, as shown in some

22

figures (see ‘Results’). It only suggests the need for further research to define the influencing

factors and thus eventually the pathophysiology in T-DM1-related keratopathy. However,

despite the low number of patients, the universal detection of similar lesions in all 20 eyes of

our examined patients supports a strong association between the basal epithelial findings and

T-DM1 treatment.

Secondly, no data were available concerning the patients’ ocular state before the T-DM1

treatment. As our patients are of a certain age and have general health issues, systemic and

ocular comorbidities are likely to be frequent, and could possibly affect the phenotype of the

toxicity, although our limited statistics could not find an association (37). The expression of

EGFR (and possibly related receptors) in the conjunctival epithelium is significantly greater in

eyes with keratoconjunctivitis sicca than in normal eyes (see also ‘Pathophysiology’) (46).

Also, pre-existing dry-eye syndrome (frequently caused by previous chemotherapy due to

damage to tear-film producing ocular structures), could exacerbate the toxicity by the use of T-

DM1 (46). Patients were pretreated with a variety of cytotoxic and monoclonal agents; mostly

including tamoxifen, which might have been a confounding factor causing the corneal toxicity.

Most of the by now well-studied pre-T-DM1 treatment products are not known to be associated

with a similar microcystic epitheliopathy except for tamoxifen, which is associated with corneal

depositions in 72% of patients, even at low doses (42, 47). However, the typically reversible

and asymptomatic brownish, linear to whorl-like subepithelial deposits in the inferior cornea

differ substantially from the corneal image in our patients and are merely similar to amiodarone-

and chloroquine-associated toxicities (47). As the lesions persist even after more than 1 year

after cessation of tamoxifen, a T-DM1-related toxicity is more likely. Cytarabine (an

antimetabolite) also demonstrates similar reversible corneal cystoid changes as seen in our

patients. However, this toxicity is associated with severe ocular discomfort due to the central

corneal location of these lesions, including the superficial epithelial layers (1, 38, 48, 49).

Confocal microscopy in this setting was useful to confirm, objectify, quantify and identify the

T-DM1- associated lesions. It allows us to evaluate the microstructure of the deposits and thus

can differentiate between certain differential diagnosis (38, 50). For example, in contrast to the

corneal toxicity we encountered, no pathologic alterations on in vivo confocal microscopy are

generally found with tamoxifen, except for one patient with crystalline deposits in the deeper

stromal layers (38, 42, 51). Confocal microscopy can also reveal subclinical corneal changes

undetectable on slit-lamp evaluation (38, 51).

As a third remark, densitometry (as available on the Pentacam HR Scheimpflug topography)

would be a valuable device to further objectify and quantify the level of microcystic changes to

23

the basal epithelium. It allows for detailed assessment of the level of backward scattering of

light induced by various corneal layers, as measured in different concentric circles.

Unfortunately, this module was unavailable to our department at the time of the study.

C. Pathophysiology

Normal tissue expression of the target molecule does not always drive ADC toxicity (15, 52).

This is illustrated by the fact that T-DMI does not have the same adverse events as reported

with the unconjugated antibody trastuzumab, or at least not to the same extent. A fearful side

effect of trastuzumab is the HER2-expression-related cardiotoxicity. By contrast, the dose

limiting toxicity of T-DM1 is thrombocytopenia, thought to be an off-target toxicity (delivery

of an unconjugated cytotoxin)(37). Toxicity of the heart seems to be present, but less frequently

encountered (15). However, many ADCs targeting molecules such as EGF receptors, are

associated with some degree of corneal toxicity, which seems to be an on-target effect

(antibody-mediated delivery) (37). They’re mainly related to epithelial lesions ranging from

punctate keratopathy to corneal ulcers, melting and perforation (1, 7, 53). Examples include

erlotinib and gefitinib (EGFR tyrosine kinase inhibitors; the latter only at high doses not used

in clinical practice); and panitumumab and cetuximab (EGFR monoclonal antibodies) (36, 46,

53-57).

Lack of detailed clinical description may underrepresent distinguishing features of certain

toxicities (37). The typical pattern of the epitheliopathy encountered (midperipheral and basal

epithelium) suggests a unique functional role regarding the receptor (in this case HER2/ErbB2)

the ADC (in this case T-DM1) targets.

Normal corneal epithelium consists of 5 to 6 cell layers of epithelial cells and measures 50 mm

centrally. New epithelial cells develop from the limbal stem cells at the corneal periphery,

located at the limbus, the junction of the clear cornea and it surrounding conjunctiva and sclera.

These stem cells undergo centripetal migration within the basal epithelial layers (ie, the

transient amplifying cells), and subsequently differentiate to intermediate (so-called wing cells)

and superficial epithelial cells (1, 43). Rapid cycling cells, such as the transient amplifying cells

located within the basal cell layer of the corneal epithelium, are most sensitive to the actions of

cytotoxins (49). Most cytotoxicity of ADCs or traditional chemotherapeutics targeting EGF

receptors is likely to be related to intracellular accumulation of the active metabolite in normal

corneal epithelial cells (38).

EGF is the primary endogenous growth factor responsible for corneal epithelial homeostasis

(36, 53-55, 58). There are four receptors identified to be related to the EGFR (receptor tyrosine

24

kinase family): EGFR, ErbB2 (HER2), ErbB3,and ErbB4 (7, 10, 58). Of these, EGFR is mainly

expressed by the basal cells of corneal epithelium that have the greatest proliferative potential

(1, 46, 58). In contrast, HER2/ErbB2 (the targeted molecule by T-DM1) is preferentially

expressed by the superficial differentiated ocular surface epithelia (58). HER2 seems to be

expressed at low levels in all healthy adult epithelial tissues (58). The ocular surface epithelium

(cornea and conjunctiva) shows stronger expression compared to other epithelial tissues and it

is proven to be crucial to the migration of corneal epithelial cells (10, 59). EGFR and related

receptors are thus most likely to be involved in the corneal toxicity found in our T-DM1 treated

patients (1, 10, 58, 59). Namely, in all of our studied patients, there was a preponderance for

the corneal midperiphery whereas the central cornea was clear. The transient amplifying cells

in the corneal periphery display some of the characteristics of stem cells, who tend to have long

cell cycles and thus are less vulnerable to the effects of cytotoxins (49). A functional difference

in behaviour between this peripheral zone of epithelium and the central zone is suggested

clinically by findings in some of the corneal dystrophies (eg Granular, Lattice, Avelino and

Honeycomb dystrophies); the peripheral epithelium is clear in these disorders (49). The cysts

could develop as a result of toxicity to the limbal stem cells (transient amplifying cells), which

in turn results in altered centripetal differentiation (37). However, ocular toxicities have

been observed in ADCs against different targets (52). These findings reveal a different

mechanism for nonreceptor-mediated toxicities of ADCs by macropinocytosis, and thus

internalization of the ADC in the epithelial cell causing damage by the cytotoxic agent (52).

The way the drug reaches the corneal epithelium is not fully understood. Possible routes are

tear secretion, the anterior chamber or the aqueous (48).

The epitheliopathy described in our study has a very specific phenotype: rather than targeting

the most superficial layer of the corneal epithelium, it is mainly the basal epithelial layer of

cells that show low-grade toxic changes. The fact that the superficial epithelial layer and

visually important central cornea is spared, may explain why these patients experience few

visual disturbance or other ocular symptoms. Blurred vision or other complaints could be

related to other non-T-DM1 related factors (see ‘Results’). This in contrast to some other

chemotherapeutic-induced corneal toxicity (eg cetuximab), where a lack of symptoms is

attributed to a reduced corneal sensibility, which was not tested in our patients (56). We do not

believe these opacities to represent active metabolite accumulation as described in the current

literature for other cytotoxic agents (eg cytarabine), as no deposits were seen on confocal

microscopy (37, 38). The hyperreflective lesions are likely to represent necrotic cell matter (37).

Trastuzumab binding and subsequent emtansine-induced cellular toxicity to the transient

25

amplifying cells likely caused the intraepithelial lesions observed (1). There is also an increased

presence of dendritic cells surrounding the opacities in te subbasal plexus, further supporting

the hypothesis that this corneal image is T-DM1-associated, causing a low-grade local

subclinical inflammatory response (38). It should be kept in mind that relatively older patient

populations (such as our study group) have systemic or local comorbidity such as diabetes and

cardiovascular diseases which complicate the microenvironment in the eye, making them more

prone to inflammation (33). Why some patients have a more pronounced toxicity (eg the case

of Tsuda et al.) than others, is not clear. Since we cannot find a relationship with dosing or

duration of T-DM1 treatment (see ‘Statistical Limitations’), we have no arguments to lower

dosing in favor of the cornea.

The 2 patients examined shortly following cessation of T-DM1 treatment showed no residual

corneal changes, both in the basal and superficial epithelium. This suggests a self-limiting

nature of the epithelial findings after cessation of treatment, but conflicts with the existent

literature concerning chemotherapeutic-related corneal toxicity and its reversibility. For

example, cetuximab (an EGFR monoclonal antibody) can cause typical corneal epithelial

microcysts (possible deposits of metabolites of the drug) that progress from the basal to the

apical layers to eventually desquamate (37, 38). Cytarabine-induced corneal epitheliopathy

shows a similar reversible course with necrotic foci progressing from basal to apical epithelial

layers in 1-2 weeks (37, 48). In the phenotype we encountered, the apical layers were never

involved, so the process described above is probably not the case.

It should be noted that T-DM1 is more likely to exhibit ocular adverse effects compared to

trastuzumab alone. Conjunctivitis was reported as an adverse effect in only 2.5% of patients in

a clinical trial for trastuzumab (compared to the 31.3% of patients in T-DM1, see ‘Introduction’)

(7). This can partially be explained by the selective delivery of cytotoxic agents in T-DM1,

besides only blocking the HER2-signaling pathway as trastuzumab does (7).

D. Treatment We had opted to treat our first case of T-DM1-related corneal epitheliopathy with autologous

serum drops in order to prevent an increase of lesions in the assumption that these lesions were

a rare occasion of toxicity (1). During follow-up of this patient, we gradually tapered the

autologous serum treatment and detected no changes to the opacities. The patient remained

asymptomatic and continued her T-DM1 treatment. We therefore did not initiate any topical

treatment in the 10 actively successfully treated patients included in this study as they were

asymptomatic during the course of the study and the lesions did not threaten the visual acuity

26

or ocular integrity. This in contrast to cytarabine-induced toxicity (see ‘Study Design and

Statistical Limitations’), in which routine prophylaxis with topical steroids is now an

established part of the treatment protocol (49). Possibly, topical corticosteroid make the corneal

epithelial cells less susceptible to the effects of cytotoxic drugs by reducing DNA replication

(48, 49). The rationale behind the use of autologous serum (AS) drops is illustrated by Orlandi

et al in a case report during the treatment of trastuzumab. Persistent bilateral corneal marginal

infiltrates may occasionally arise as a side effect of trastuzumab treatment (10). In this

particular patient, lesions disappeared without scarring after 7 days of treatment with AS three

drops twice a day (10). Foerster et al. published a similar case in a patient treated with

cetuximab, an EGFR receptor antibody in the treatment of colorectal cancer, with persistent

corneal erosions. This was successfully treated by topical EGF (55). The rationale behind

treatment is that AS could antagonize the inhibitory effect of the HER2 receptor antibody on

the corneal epithelial cells by competitively providing ligands of HER heterodimers. As such,

completion of the HER2 signaling complex could restore normal proliferation and migration of

the corneal epithelial cells (10, 53-55). Alternatively, in ADCs such as T-DM1, the mechanism

of corneal damage is also mediated by antibody-dependent cell cytotoxicity. AS could prevent

antigen recognition by the HER2 antibody by receptor internalization following ligand binding

(10). This principle cannot be applied to low-molecular weight inhibitors such as erlotinib (an

EGFR inhibitor for lung cancer) (53, 54). These act intracellularly at the tyrosine kinase portion

of the EGFR (54). Also, caution needs to be taken in the timing of acquiring the blood sample

for the AS preparation, to avoid a significant concentration of the toxic drug in it. Orlandi et al

proposed to postpone the chemotherapeutic treatment temporarily according to the half-life of

the drug (which is 3-4 days in T-DM1) (10, 21). C-Cbl, which targets the EGFR for lysosomal

degradation by ubiquitylation, is a new therapeutic target in research to enhance EGFR-

mediated corneal epithelial homeostasis. By antagonizing c-Cbl, EGFR-mediated corneal

epithelial homeostasis could be enhanced in a totally different way than the previous treatment

options (60) .

The prophylactic use of nonpreserved artificial tears would be an appropriate first measure to

protect corneas in patients on T-DM1 treatment, to prevent keratoconjunctivitis sicca as an

predisposing factor for developing corneal problems (see ‘Pathophysiology’: overexpression of

EGFR)(46). A prophylactic use of topical steroids is not generally accepted due to the risk of

herpetic or fungal keratitis or other ocular complications in immunocompromised patients (48).

In cases with development of worse and symptomatic lesions (such as the case in Tsuda et al.

27

or chemotherapeutics targeting EGFR –see above), the step-up therapeutic options should

contain steroids, or autologous serum if treatment can be delayed for some time (eg 1-2 weeks).

Bandage lenses for persistent corneal erosions should be incorporated in the treatment protocol

as well. In extreme situations, as was the case described by Tsuda et al., dose restriction or

therapy switch should be considered. In this apparently exceptional case of visually disturbing

limbal lesions, 0.3% hyaluronic acid solution and 0.1 fluorometholone suspension was

administered four times daily, as well as chemotherapy switch from T-DM1 monotherapy to a

combination of classic chemotherapeutics including trastuzumab (see ‘Introduction’) (7). The

lesions regressed, but never fully healed, which could be partially explained by the continued

use of trastuzumab, a related molecule (7).

Because of the benign character of the more common ocular toxicity encountered in our

patients, dose restriction or cessation of the possible life-saving treatment with T-DM1 is

neither necessary.

E. Future ADCs Several reports have been made concerning potential adverse effects of ADCs, which mutes the

initial enthusiasm associated with their selective approach. The report of Eaton et al. and

Raizman et al. are useful as an overview for ocular and other AEs related to ADCs known up

until the time of their publication (see also ‘Introduction’) (37, 38). As more and more ADCs

are being developed and going into phase II and III studies , there will be an increasing need

for both ophthalmologists and oncologists to understand the various phenotypic expressions of

corneal epithelial toxicity and to develop treatment strategies in order to enable continuation of

these drugs without risking prolonged vision loss or ocular damage. Some of these ADCs, such

as Trastuzumab Duocarmazine (SYD 985; Synthon), Sacituzumab Govitecan ((IMMU-132;

Immunomedics), an anti-Trop 2 antibody (61), and Tisotumab Vedotin (targeting tissue factor

in the treatment of cervical cancer (62)), already incorporate preventive measures (lubricant,

steroids and/or eye cooling pads) in their treatment protocol (63). In what follows, we discuss

a handful investigational ADCs with reported ocular and corneal toxicity.

1. Coltuximab Ravtansine (SAR3419) It is a monoclonal antibody against CD19 associated with a maytansine derivative. CD19 is

expressed on the surface of B-cell precursors, which is why coltuximab ravtansine has a role in

the treatment of diffuse large B cell lymphoma. It has documented ocular side effects such as

reversible late blurred vision with corneal midperipheral deposits/microcysts in two reported

28

cases, and one case of optic neuropathy, reversing within 2-6 weeks (6, 37, 38). For the

microcystic lesions, Eaton and colleagues theorize that “mechanisms could include focal

separation between the epithelium and basement membrane or toxicity to the limbal cells,

resulting in altered centripetal differentiation”, similar to the presumed pathophysiology in our

patients (37). Optimization of the dosing schedule (lowering and staggering dosing) could

reduce the incidence of corneal toxicities (37, 38).

2. Trastuzumab Duocarmazine (SYD985)

This drug is a HER2-targeting ADC that is based on trastuzumab attached to the duocarmycin

prodrug seco-DUBA with a cleavable linker. By now, it has demonstrated antitumor activity in

breast and gastric cancer models (63). A similar image to T-DMI, with microcyst-related

corneal toxicity in the midperipheral region is encountered in patients using this drug (in clinical

practice).

There is a big similarity between SYD985 and T-DMI, with both of them containing

trastuzumab and a linked cytotoxic drug. The difference lies within the linker: which is a

reducible disulfide bond in SYD985, and a non-reducible thioether in T-DMI. Reducible linkers

tend to also have a bystander cytotoxicity, whereas non-reducible linkers exhibit no bystander

effect (18). One can thus deduce that the possible corneal toxicity related to SYD985 might be

more pronounced compared to T-DMI, if the pathophysiology is linked to the cytotoxic effect.

3. Denintuzumab mafodotin (SGN-CD19A) Denintuzumab mafodotin is being investigated as a component of combination therapy in

diffuse large B-cell lymphoma. It produces a superficial and symptomatic microcystic

keratopathy in 84% of patients, manageable with topical steroids and dose modification (38).

4. Indatuximab ravtansine (BT-062) Indatuximab ravtansine (BT-062) treatment results in epithelial corneal damage with crystal

inclusions and symptoms as blurred vision and dry eye. It is used for multiple myeloma

treatment.

5. Mirvetuximab soravtansine (IMGN853) Mirvetuximab soravtansine (IMGN853) causes corneal presentations of punctate keratitis or

more rarely epithelial microcysts (1 patient). It is used for refractory ovarian cancer.

29

F. Conclusion In peer-reviewed literature, 2 case reports have emerged on the specific topic of T-DM1

associated corneal toxicity (1, 7). As publications on this topic are scarce, one can assume that

T-DM1-related corneal toxicity is a rare phenomenon, as noted in the Introduction. On top of

that, the level of toxicity differed substantially in both case reports: one patient had extensive

superficial corneal toxicity necessitating treatment discontinuation (7) whereas the other

reported case only featured asymptomatic basal epithelial spheroid opacities (1).

The findings of this study indicate that the basal epithelial spheroid opacities reported in the

latter are highly common in patients on T-DM1 treatment. Interestingly, we have not observed

the much more severe phenotype as reported by Tsuda et al (7). Contrary to our findings of

basal epithelial changes, this type of toxicity does appear to be a rare occurrence.

In conclusion, our findings support that low-grade corneal epithelial changes are hallmark

features in T-DM1 treatment and should not alarm clinicians. These findings are relatively

stationary, reversible, asymptomatic and thus do not require ocular treatment. This association

is important both to ophthalmologists and medical oncologists to be aware of, so as not to

erroneously discontinue life-saving treatment. As such, drug development and medical

professionals alike can be aware of these clinical features, which facilitates early recognition

and intervention in both preclinical and clinical investigations, if necessary. Further research is

needed to expose the exact pathophysiology, since this previously unpublished corneal finding

could be the first of many other uncovered systemic or local toxicities.

30

VI. DUTCH RESUME

T-DM1 is een ADC die wordt gebruikt voor de behandeling van HER2-positieve

gemetastaseerde borstcarcinomen. Hoewel ADCs worden beschouwd als zeer selectieve

behandelingsopties, lijkt er steeds meer evidentie te komen voor belangrijke systemische en

lokale bijwerkingen. T-DM1 wordt beschouwd als een pionier in de ADC-ontwikkeling en -

toepassing gezien het één van de eerste FDA-goedgekeurde producten op de markt was in 2013.

Op heden is er inconsistente literatuur beschikbaar omtrent de corneale bijwerkingen bij T-

DM1 (en andere ADCs). Eaton et al. waren de eerste die geassocieerde corneale toxiciteit voor

het eerst beschreven op een weinig gedocumenteerde en eerder symptoomgebaseerde manier.

De hierin beschreven (sicca-)keratopathie leek goedaardig en te verwaarlozen. Tsuda et al.

beschreef in 2016 echter een patiënt met een dramatisch corneaal beeld met ernstig visusverlies

en noodzaak tot stop van T-DM1-behandeling. In 2018 werd door Kreps et al. dan weer een

asymptomatisch microcystisch corneaal beeld gezien, en voor het eerst ook gedocumenteerd en

verder geanalyseerd met confocale microscopie. Het doel van dit werk was dan ook om binnen

dit beschreven spectrum van corneale veranderingen een consistente conclusie te kunnen

vormen omtrent het fenotype en de prevalentie van corneale veranderingen tijdens T-DM1

behandeling, en zo een advies voor research en klinische praktijk te kunnen formuleren. In onze

cross-sectionele studie van tien actueel behandelde patiënten met T-DM1 werd met behulp van

biomicroscopie en confocale microscopie bij allen een typische corneale epitheliopathie

weerhouden met midperifere fluonegatieve microcysten. Pathofysiologisch is een link met een

verstoorde epitheliale migratie plausibel op basis van de kliniek en de kennis omtrent het belang

van EGFR in corneale homeostase. Slechts drie patiënten rapporteerden symptomen die konden

gelinkt worden met oculaire comorbiditeiten. Twee patiënten die recent (<10weken) waren

gestopt met T-DM1 toonden geen corneale veranderingen, wat pleit voor een reversibel

karakter. Eén patiënt, reeds beschreven door Kreps et al., werd gevolgd tijdens topische

behandeling met autoloog serum, en toonde geen significante corneale evolutie. Er kon geen

statistisch significante correlatie aangetoond worden tussen de graad van keratopathie enerzijds,

en de dosis of duur van behandeling, oculaire symptomen, en systemische bijwerkingen

anderzijds. Tijdens de studieperiode van zes maanden ontwikkelden zich geen nieuwe oculaire

symptomen bij onze patiënten. Dit toont dus aan dat asymptomatische, stationaire en reversibele

laaggradige corneale epitheliale veranderingen eigen zijn aan de behandeling met T-DM1 en

geen therapeutische consequenties vereisen. Gezien meer en meer ADCs worden ontwikkeld,

is bewustzijn hiervan in (pre-)klinische trials nodig voor verdere inzichten omtrent de

prevalentie, pathofysiologie en corneale evolutie bij langdurig of hooggedoseerd gebruik.

I

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VII

VIII. APPENDIX

Informatiebrief voor de deelnemers aan experimenten

1. Titel van de studie: EVALUATIE VAN CORNEALE BIJWERKINGEN GEÏNDUCEERD DOOR TRASTUZUMAB-EMTANSINE

2. Doel van de studie: Men heeft u gevraagd om deel te nemen aan een studie. Deze studie heeft als doel het in kaart brengen van mogelijke bijwerkingen aan de ogen bij mensen die behandeld worden met trastuzumab-emtansine. Voordat u akkoord gaat om aan deze studie deel te nemen, vragen wij u om kennis te nemen van wat deze studie zal inhouden op het gebied van organisatie, zodat u een welbewuste beslissing kunt nemen. Dit wordt een “geïnformeerde toestemming” genoemd.

Wij vragen u de volgende pagina’s met informatie aandachtig te lezen. Hebt u vragen, dan kan u terecht bij de arts-onderzoeker of zijn of haar vertegenwoordiger.

3. Beschrijving van de studie: Trastuzumab-emtansine is een medicijn gebruikt bij mensen met vormen van uitgezaaid borstkanker waarbij de kwaadaardige cellen specifieke receptoren dragen die de cellen sneller doen vermenigvuldigen. Deze medicatie is heel specifiek gericht op een dergelijke receptor, zodat de kwaadaardige cellen kunnen onderscheiden worden van goedaardige cellen. Deze receptor is ook aanwezig in de cellen van het hoornvlies (= cornea). Het is momenteel nog niet in kaart gebracht in hoeverre deze medicatie een verstoring geeft van het hoornvlies. Nieuwe kennis hierover kan bij oogklachten een meer gerichte behandeling opleveren. Wij wensen deze onderzoeken uit te voeren om de kennis over mogelijke hoornvliesveranderingen bij deze behandeling te verruimen. Tijdens een bezoek aan de dienst oogheelkunde combineren wij een standaard oogheelkundig nazicht met een in vivo confocale microscopie. Met dit toestel maken we beelden van de verschillende lagen van uw hoornvlies. Dit is een onderzoek waarbij laserlicht, niet schadelijk, zonder contraststoffen, gebruikt wordt om uw hoornvlies te fotograferen. Om ook gedetailleerde foto's van het hoornvlies te nemen (kleurenfoto's met normaal licht), gebruiken we oogdruppels (Tropicol® en Phenylephrine®, 1 druppel van elk in beide ogen) die uw pupillen verwijden gedurende een drietal uur, waardoor U tijdelijk niet met de wagen mag rijden. We vragen u tevens om gegevens uit uw medisch dossier te mogen verzamelen zodat we ze kunnen combineren met de gegevens van andere patiënten met dezelfde behandeling en kunnen linken aan de toestand van uw hoornvlies. Hiervoor hoeft u niets te doen, deze gegevens betreffen enkel uw huidige en vroegere oncologische behandeling.

VIII

De verwachte totale duur van de studie is circa één uur waarvan u ongeveer 40 min onderzocht wordt en 20 min om de druppels te laten inwerken. Het duurt vervolgens 2 uur om de druppels te laten uitwerken. Deze laatste 2 uur hoeft u niet meer op onze dienst te blijven. Er zullen in totaal ongeveer 50 personen gevraagd worden om aan deze studie deel te nemen.

4. Wat wordt verwacht van de deelnemer? Voor het welslagen van de studie, is het belangrijk dat u meewerkt met de onderzoeker en dat u zijn/haar instructies nauwlettend opvolgt. Er zijn overigens geen verdere vereisten of restricties.

5. Deelname en beëindiging: De deelname aan deze studie vindt plaats op vrijwillige basis. Deelname aan deze studie brengt voor u geen onmiddellijk therapeutisch voordeel. Uw deelname in de studie kan helpen om in de toekomst patiënten beter te kunnen helpen. U kan weigeren om deel te nemen aan de studie, en u kunt zich op elk ogenblik terugtrekken uit de studie zonder dat u hiervoor een reden moet opgeven en zonder dat dit op enigerlei wijze een invloed zal hebben op uw verdere relatie en/of behandeling met de onderzoeker of de behandelende arts. Uw deelname aan deze studie zal worden beëindigd als de onderzoeker meent dat dit in uw belang is. U kunt ook voortijdig uit de studie worden teruggetrokken als u de in deze informatiebrief beschreven procedures niet goed opvolgt of u de beschreven items niet respecteert. Als u deelneemt, wordt u gevraagd het toestemmingsformulier te tekenen.

6. Procedures:

6.1. Procedures: Vooreerst voeren we een basis oogheelkundig nazicht uit: bepalen van de gezichtsscherpte, spleetlamponderzoek en het meten van de oogdruk. Nadat de pupillen verwijd zijn, nemen we kleurenfoto's van uw hoornvlies (beide ogen). Tevens gebruiken we een ander soort licht om gedetailleerde beelden van uw hoornvlies te nemen. U dient hiervoor gedurende enkele minuten te kijken naar een lichtje in het toestel.

IX

6.2. Studieverloop: De studie houdt een een- of tweemalige visite in op de dienst oogheelkunde waarbij alle onderzoeken op dezelfde dag doorgaan.

7. Risico’s en voordelen: De druppels die gebruikt worden om de pupil te verwijden, werken gedurende een drietal uur. Deze druppels hebben een theoretisch risico op het afsluiten van de voorkamerhoek met een oogdrukstijging als gevolg. Uw ogen worden voor het indruppelen gecontroleerd. Indien het risico op afsluiten van de ooghoek plausibel wordt geacht, zullen geen oogdruppels worden toegediend. Tijdens de periode na het indruppelen van deze pupilverwijdende druppels kunt u een waziger zicht hebben en hinder ondervinden van fel licht. U mag zelf geen voertuig besturen tijdens deze periode. Deze studie houdt eveneens een basis oogheelkundig nazicht in. Verdere rechtstreekse voordelen zijn er niet aan de studie gebonden. We hopen in de toekomst anderen te kunnen helpen door op basis van de ogen een beter inzicht in de oogproblemen te krijgen. U hebt het recht op elk ogenblik vragen te stellen over de mogelijke en/of gekende risico’s, nadelen van deze studie. Als er in het verloop van de studie gegevens aan het licht komen die een invloed zouden kunnen hebben op uw bereidheid om te blijven deelnemen aan deze studie, zult u daarvan op de hoogte worden gebracht. Mocht u door uw deelname toch enig nadeel ondervinden, zal u een gepaste behandeling krijgen. Deze studie werd goedgekeurd door een onafhankelijke Commissie voor Medische Ethiek verbonden aan het UZ Gent en wordt uitgevoerd volgens de richtlijnen voor de goede klinische praktijk (ICH/GCP) en de verklaring van Helsinki opgesteld ter bescherming van mensen deelnemend aan klinische studies. In geen geval dient u de goedkeuring door de Commissie voor Medische Ethiek te beschouwen als een aanzet tot deelname aan deze studie.

8. Kosten: Uw deelname aan deze studie brengt geen extra kosten mee voor U.

9. Vergoeding: Er is geen specifieke vergoeding verbonden aan deze studie. Een oogheelkundig nazicht is inbegrepen.

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10. Vertrouwelijkheid: In overeenstemming met de Belgische wet van 8 december 1992 en de Belgische wet van 22 augustus 2002, zal uw persoonlijke levenssfeer worden gerespecteerd en zal u toegang krijgen tot de verzamelde gegevens. Elk onjuist gegeven kan op uw verzoek verbeterd worden. Vertegenwoordigers van de opdrachtgever, auditoren, de Commissie voor Medische Ethiek en de bevoegde overheden hebben rechtstreeks toegang tot Uw medische dossiers om de procedures van de studie en/of de gegevens te controleren, zonder de vertrouwelijkheid te schenden. Dit kan enkel binnen de grenzen die door de betreffende wetten zijn toegestaan. Door het toestemmingsformulier, na voorafgaande uitleg, te ondertekenen stemt U in met deze toegang. Als u akkoord gaat om aan deze studie deel te nemen, zullen uw persoonlijke en klinische gegevens tijdens deze studie worden verzameld en geanonimiseerd (hierbij kan men uw gegevens niet terug koppelen naar uw persoonlijk dossier). Verslagen waarin U wordt geïdentificeerd, zullen niet openlijk beschikbaar zijn. Als de resultaten van de studie worden gepubliceerd, zal uw identiteit vertrouwelijke informatie blijven.

11. Letsels ten gevolge van deelname aan de studie: De onderzoeker voorziet in een vergoeding en/of medische behandeling in het geval van schade en/of letsel tengevolge van deelname aan de studie. Voor dit doeleinde is een verzekering afgesloten met foutloze aansprakelijkheid conform de wet inzake experimenten op de menselijke persoon van 7 mei 2004. Op dat ogenblik kunnen uw gegevens doorgegeven worden aan de verzekeraar.

12. Contactpersoon: Als er letsel optreedt tengevolge van de studie, of als U aanvullende informatie wenst over de studie of over uw rechten en plichten, kunt U in de loop van de studie op elk ogenblik contact opnemen met: Prof. Hannelore DENYS Kliniekhoofd Medische Oncologie UZ Gent Tel 09 332 5184

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Toestemmingsformulier Ik, _________________________________________ heb het document “Informatiebrief voor de deelnemers aan experimenten” pagina 1 tot en met 4 gelezen en er een kopij van gekregen. Ik stem in met de inhoud van het document en stem ook in deel te nemen aan de studie. Ik heb een kopij gekregen van dit ondertekende en gedateerde formulier voor “Toestemmingsformulier”. Ik heb uitleg gekregen over de aard, het doel, de duur, en de te voorziene effecten van de studie en over wat men van mij verwacht. Ik heb uitleg gekregen over de mogelijke risico’s en voordelen van de studie. Men heeft me de gelegenheid en voldoende tijd gegeven om vragen te stellen over de studie, en ik heb op al mijn vragen een bevredigend antwoord gekregen. Ik stem ermee in om volledig samen te werken met de toeziende arts. Ik zal hem/haar op de hoogte brengen als ik onverwachte of ongebruikelijke symptomen ervaar. Men heeft mij ingelicht over het bestaan van een verzekeringspolis in geval er letsel zou ontstaan dat aan de studieprocedures is toe te schrijven. Ik ben me ervan bewust dat deze studie werd goedgekeurd door een onafhankelijke Commissie voor Medische Ethiek verbonden aan het UZ Gent en dat deze studie zal uitgevoerd worden volgens de richtlijnen voor de goede klinische praktijk (ICH/GCP) en de verklaring van Helsinki, opgesteld ter bescherming van mensen deelnemend aan experimenten. Deze goedkeuring was in geen geval de aanzet om te beslissen om deel te nemen aan deze studie. Ik mag me op elk ogenblik uit de studie terugtrekken zonder een reden voor deze beslissing op te geven en zonder dat dit op enigerlei wijze een invloed zal hebben op mijn verdere relatie met de arts. Men heeft mij ingelicht dat zowel persoonlijke gegevens als gegevens aangaande mijn gezondheid worden verwerkt en bewaard gedurende minstens 20 jaar. Ik stem hiermee in en ben op de hoogte dat ik recht heb op toegang en verbetering van deze gegevens. Aangezien deze gegevens verwerkt worden in het kader van medisch-wetenschappelijke doeleinden, begrijp ik dat de toegang tot mijn gegevens kan uitgesteld worden tot na beëindiging van het onderzoek. Indien ik toegang wil tot mijn gegevens, zal ik mij richten tot de toeziende arts, die verantwoordelijk is voor de verwerking. Ik begrijp dat auditors, vertegenwoordigers van de opdrachtgever, de Commissie voor Medische Ethiek of bevoegde overheden, mijn gegevens mogelijk willen inspecteren om de verzamelde informatie te controleren. Door dit document te ondertekenen, geef ik toestemming voor deze controle. Bovendien ben ik op de hoogte dat bepaalde gegevens doorgegeven worden aan de opdrachtgever. Ik geef hiervoor mijn toestemming, zelfs indien dit betekent dat mijn gegevens doorgegeven worden aan een land buiten de Europese Unie. Ten alle tijden zal mijn privacy gerespecteerd worden.

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Ik ben bereid op vrijwillige basis deel te nemen aan deze studie. Naam van de vrijwilliger: _________________________________________ Datum: _________________________________________ Handtekening: Ik bevestig dat ik de aard, het doel, en de te voorziene effecten van de studie heb uitgelegd aan de bovenvermelde vrijwilliger. De vrijwilliger stemde toe om deel te nemen door zijn/haar persoonlijk gedateerde handtekening te plaatsen. Naam van de persoon die voorafgaande uitleg heeft gegeven: _________________________________________ Datum: _________________________________________ Handtekening: