The Need for Individualized Procedures in ECP, the European Perspective!

Volker Witt, MD St. Anna Kinderspital, Vienna, Austria ECP = extrcorporeal photopheresis

2 3

15.01.2015 2 What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • Preparing a buffy coat • Adding a Photosensitizer • Transferring in a irradiation disposable • Irradiating a “buffy coat” • Retransfusion to the patient

What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • Apheresis • Preparing a buffy coat • Inline systems • Offline systems • Adding a Photosensitizer • To draw peripheral blood • Transferring in a irradiation disposable • Irradiating a “buffy coat” • Retransfusion to the patient

What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • Directly from the Apheresis • Preparing a buffy coat device • Adding a Photosensitizer • Diluted by plasma • Transferring in a irradiation • Diluted by saline solution disposable • Hematocrit • Irradiating a “buffy coat” • Retransfusion to the patient

What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • 8-MOP • Preparing a buffy coat • Final concentration (?) • Adding a Photosensitizer • Transferring in a irradiation disposable • Irradiating a “buffy coat” • Retransfusion to the patient

What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • Chamber • Preparing a buffy coat • Bag • Adding a Photosensitizer • Transferring in a irradiation disposable • Irradiating a “buffy coat” • Retransfusion to the patient

What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • In a pivoting a bag • Preparing a buffy coat • By flowing through a chamber • Adding a Photosensitizer • Constantly • Transferring in a irradiation • Inconstantly disposable • Time • Irradiating a “buffy coat” • Hematocrit • Retransfusion to the patient • Measuring the dose

What is ECP?

STEPS OF THE PROCESS • Harvesting Leukocytes • Immediately • Preparing a buffy coat • After incubating • Adding a Photosensitizer • Transferring in a irradiation disposable • Irradiating a “buffy coat” • Retransfusion to the patient

As often happened in the past, ECP was invoked as a last resort in front of patients with highly unsatisfactory therapeutic proposals. In fact, that was the case of acute and chronic GvHD when ECP was even borrowed from the treatment of cutaneous T cell lymphoma.

Extracorporeal photopheresis for bronchiolitis obliterans syndrome after allogeneic stem cell transplant: An emerging therapeutic approach? Claudia Del Fante∗, Cesare Perotti Transfusion and Apheresis Science 56 (2017) 17–19

A possible explanation of the positive impact on BOS after ECP intensification may be the higher number of cells treated over time and consequently the more intense and long lasting immunomodulation.

Moreover, differently from others, we are convinced that in this setting of patients response to ECP must be considered also stabilization or even slowing of lung function decline over time, considering that nearly all patients meet a rapid and dramatic evolution.

2007 French guidelines (Kanold J. , Transfusion) acute and chronic GvHD, children

2012 Italian guidelines (Pierielli L. , Transfusion) acute and chronic GvHD, adults and children

2014 European Academy of Dermatology and Venerology (Knobler R. , JEADV) Guidelines on the use of ECP (aGVHD, cGVHD included)

2014 COCHRAN Review (Weitz M.) acute and chronic GvHD, children

2015 Britsh Guidelines on the clinical use of apheresis procedures … (Howell C, Transfusion)

2016 US ASFA guidelines (Schwartz J. J Clin Apheresis) acute and chronic GvHD, adults and children

15.01.2015 13

The current methods for delivery of ECP use ‘closed’ or ‘open’ systems. Open ECP necessitates collection of a buffy coat on a cell separator, separation of the buffy coat from the system and subsequent UVA irradiation in a separate device, before reinfusion to the patient. By contrast the closed system incorporates an internal UVA source in the apheresis device with no separation of the buffy coat from the device, before return to the patient. While the open system has the potential advantages of increased cell dose and the ability to manipulate the cells further, there is an increased risk of microbial contamination with this technology. The closed system devices have integrated mononuclear cell collection and 8-methoxypsoralen/UVA irradiation, and regulatory approval for treating patients (Wong, 2012).

Howell, Transfusion Medicine, 2015

Atopic (neuro-) dermatitis (atopic ECP III 2C eczema), recalcitrant IA III 2C TPE III 2C Cardiac transplantation ECP Cellular/recurrent rejection II 1B ECP Rejection prophylaxis Desensitization II 2A TPE Antibody mediated rejection II 1C TPE III 2C Cutaneous T-cell lymphoma; mycosis ECP Erythrodermic I 1B fungoides; Sezary syndrome ECP Non-erythrodermic III 2C Dermatomyositis/polymyositis TPE ECP IV 2B IV 2C Graft-versus-host disease ECP Skin (chronic) II IB ECP Non-skin (chronic) II IB ECP Skin (acute) II 1C ECP Non-skin(acute) II 1C Inflammatory bowel disease Adsorptive Ulcerative colitis III/II 1B/2B cytapheresis Adsorptive Crohn’s Disease III 1B cytapheresis ECP Crohn’s Disease III 2C Schwartz, JCA 2016 Lung transplantation ECP Bronchiolitis obliterans syndrome II 1C TPE Antibody mediated rejection III 2C TPE Desensitization III 2C Nephrogenic systemic fibrosis ECP III 2C TPE III 2C Cutaneous T-cell lymphoma; mycosis ECP Erythrodermic I 1B fungoides; Sezary syndrome ECP Non-erythrodermic III 2C Pemphigus vulgaris TPE Severe III 2B ECP Severe III 2C IA Severe III 2C Psoriasis ECP III 2B Ads.cytaph. Disseminated pustular III 2C Lymphocyt III 2C TPE IV 2C Scleroderma (systemic sclerosis) TPE III 2C ECP III 2A

Schwartz, JCA 2016 Why we use ECP in GVHD? main complications of immunosuppressive therapies

 infections  endocrine dysfunction  avascolar necrosis  multiorgan failure  secondary malignancies  quality of life ?

15.01.2015 20 + Corticoid and IS therapy sparing effect needed(!?) Immunological benefits

• Maeda A et al. (2005) Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells. • J Immunol 174: 5968–5976 • Patients receiving ECP respond normally to new immune challenges and do not lose their immunity against infections, gained either from previous exposure to the pathogen or through vaccination. • Suchin KR et al. (1999) • Extracorporeal photochemotherapy does not suppress T- or B-cell responses to novel or recall antigens. • J Am Acad Dermatol 41: 980–986

15.01.2015 22 ECP in children

• Advantages: • Problems: • Selective downregulation of GVHD • Time consuming without increasing infection or • Venous access malignancy relapse • Circulating extracorporeal blood volume • Possibility to reduce general • Priming with blood immunosuppression • Unvalidated procedures • Minimal acute side effects • Few randomized trials • No reported long-term side effects • Experienced staff necessary

15.01.2015 23 Why ECP in paediatric practice merits attention ?

A substantial number of children undergo HSCT for non-malignant disorders and will not benefit from the GvHD.

The toxicities of systemic treatment in a growing child should be considered.

Therefore, addition of an effective pharmacologic-immunosuppression-sparing agent is of crucial importance for long-term outcome of these patients.

Excellent safety profile of ECP

15.01.2015 24 Studies of Photopheresis for Treatment of Acute Graft-vs-Host Disease in Children and Adult

Luca Pierelli RECOMMENDATIONS FOR ECP IN GVHD Transfusion 2013 15.01.2015 25 Messina et,al. BMT 2003 15.01.2015 26 15.01.2015 27 15.01.2015 28 The National Institutes of Health global score predicts the risk of non relapse mortality and disease-free survival in children with chronic graft-versus-host disease Children with severe chronic graft-versus-host disease had significantly higher non relapse mortality and lower disease-free survival Probability of continuing chronic graft-versus-host disease 8 years after onset of severe chronic graft-versus-host disease was 36% Among survivors of more than 5 years, 22% had a performance score below 70% Efforts to lower the risk of severe chronic graft-versus-host disease are necessary in pediatric hematopoietic stem cell transplantation

When we could use it in GVHD? Bone Marrow Transplantation (2010) 45, 1068–1076 15.01.2015 31 These preliminary data may indicate a potential survival advantage with ECP for transplant recipients undergoing standard myeloablative hematopoietic cell transplantation.

Bone Marrow Transplantation (2010) 45, 1068–1076 15.01.2015 32 How to use ECP in GVHD? Indications for ECP in the paediatric setting: guidelines

acute and chronic GvHD 1. not responding to steroid 2. responding with intolerable side effects (ECP combined with 2nd line treatment ) chronic GvHD 3. unsuccessfully treatment with > 3 lines of therapy (resistant, dependent, late-stage disease) 4. limited, regardless of other therapies acute GVHD 5. grade IV with 1st and 2nd line therapies

34 34

Which system?

• „However the machine currently approved is designed for patients over 40 kg of body weight. Significant fluid shift and venous access are major concerns when ECP is performed in children. Various modifications of the ECP procedure have been tried to manage patients with los body weight. Expirience with ECP in children is limited but preliminary data also showed favorable response in children with resistant GVHD.“ Chan KW J Clin Apher.2006;21(1):60-4

15.01.2015 36 Extracorporeal volume of apheresis systems

700 650 UVAR 600 480 UVAR XTS 500 CFS Single Needle 400 CFS Double Needle 319 300 236 Fenwal GRANULO/SVCCH 216 200 190 Fenwal SVSCH/SVCCH 165 169 163 COBEspectra 100 137 0 OPTIA AMICUS COM.TEC Extracoporeal Volume

15.01.2015 37 15.01.2015 38 UVAR system

15.01.2015 39 ECP: on-line and off-line methods

On-line (Therakos) Off-line: I° step = MNC collection

Off-line: II° step = 8-MOP and UV-A irradiation

15.01.2015 40

ECP program from 2005 until today

• Patients non responsive to SIT and need for 2nd line treatment • aGVHD • cGVHD • System • > 40 kg UVAR XTS, > 30 kg Cellex (no blood priming)

• < 40 kg AMICUS, Fenwal CS, AMICUS, OPTIA plus COMBI light plus and later on MACOGENIC

• (1x Apherese, 2x Reinfusion)

• in special cases „MINI“ ECP

15.01.2015 42 UVAR Cellex

• Double needle or single needle method applicable • Validation and qualification is done at our department

15.01.2015 43 ECP program „offline method 1 apheresis 2 reinfusions“

day 1 day 2

divided in 2 stored over night +4oC

15.01.2015 44 ECP Methods in Children: Mini-ECP

10 ml/kg 8-MOP bw PB MNC

densitygradient- Centrifugation or simple entrifugation

2,5 J/m² 365 nm

This procedure can only be performed in a GMP laboratory

15.01.2015 45 Slides from Merlin E. © Monday 9h-13h Wednesday 9h-13h Friday 9h-13h

……

Monday 9h-12h • Cryopreservation of mononuclear cells

• Sequential reinfusion of thawed and ECP treated cells

…… Monday 12h- Wednesday 12h- Friday 12h-13h 13h 13h

Reduction of the number of aphereses 46 Cryopreservation for ECP Slides from Merlin E. ©

Sarkar 2003 Feuerstein 2000

Cryopreservation induces lymphocyte apoptosis while retaining the stimulatory function of dendritic cells 47 Cryopreservation for ECP – in vitro assessment Slides from Merlin E. © Methods

• Healthy donors

• Isolation of PBMC by centrifugation on Ficoll-Hypaque gradients (density, 1.077)

• Cryopreservation – DMSO : 3,5 % – Albumin : 10 % – HES : 92,3 % – -80°C without controlled-rate freezing

• Thawing in 40° water until completely melted, then RPMI is added, supplemented with 10% human AB serum. Washed twice to remove DMSO.

• ECP: Vilber-Lourmat irradiator in UVA transparent bag (Macopharma)

48 Cryopreservation for ECP – in vitro assessment Slides from Merlin E. ©

ECP treated cells - Viability (Trypan Blue Exclusion)

Fresh cells 120 Cryopreserved cells

100

40 Viable cells (%) cells Viable

0 Before ECP h1 h24

49 Cryopreservation for ECP – in vitro assessment Slides from Merlin E. © ECP treated cells – Apoptosis (Annexin V – IP)

Fresh cells 35 Cryopreserved cells 30

10 Annexin Annexin (%)cells +

0 1 2 Before ECP H1 after ECP 50 Cryopreservation for ECP – in vitro assessment Slides from Merlin E. © Total apoptosis

1000 FreshFresh cells cells CryopreservedCryoCryopreserved cells cells ECP-treatedECPECP-treated cells cells CryopreservedCryoCryopreserved+ ECP + ECP-treated+ ECP-treated cells cells 10001000

+ + cells (%) 500

500500 Annexine

0 0 0 j0 j0 j0d0 h2 h2 h2h2 j1 j1 j1d1j2 j2 j3d2j2j3 j6d3j6j3 d6j6

Time 51 Cryopreservation for ECP – in vitro assessment Slides from Merlin E. © Functional assessment - MLR

Judgment criterion: proliferation in MLR

Measured with CFSE dye dilution Stimulator cells: A* = mother - 30 Gys Reactive cells : B = daughter – labelled with cfse Tested cells: ECP treated B cells

7 days culture in RPMI-1640 supplemented with human AB serum, Hepes, antibiotics and glutamine

CFSE

A* B B A* B 52 Test Control Cryopreservation for ECP – in vitro assessment Slides from Merlin E. ©

A*+BCFSE 2:1

A*+ BCFSE + BECP 2:1:1

A*+ BCFSE + BECP CRYO 2:1:1

53 Cryopreservation for ECP – in vitro assessment Slides from Merlin E. ©

Proliferating cells cells (%) Proliferating 10

0 Control ECP Cryo + ECP

54 Cryopreserved-cell ECP for GVH in children – Clermont-Fd experience Slides from Merlin E. ©

1 2 3 4 5

Freezing Thawing –DMSO : 3,5 % Washing twice –Albumin : 10 % –HES : 92,3 % –-80°C without controlled-rate freezing

Cryopreserved-cell ECP for GVH in children – Clermont-Fd experience

ECP center Referring hospital

Cell infusion (ECP)

Apheresis

0 10 20 30 40 Days of ECP therapy 56 ECP for GVH in children – Clermont-Fd experience Slides from Merlin E. ©

Immunosuppression Salt-free diet Short term Hypertension Fed up Colitis Venous access Troubles de l’humeur Transfusions Neutropenia Logistical issues

57 ECP for GVH in children – Clermont-Fd experience Slides from Merlin E. ©

Fed up Immunosuppression Venous access Shortterm Salt-free diet Transfusions Hypertension Logistical issues Colitis Troubles de l’humeur Neutropenia

58 MINI ECP → simple method

1. Drawing blood from peripheral vascular access 2. Fluid substitution with cristalloid solutions if needed 3. Centrifugation of the product 4. Squeezing of the buffy coat 5. Reinfusion of the residual erythrocyte fraction 6. Dilution of the buffy coat to hematocrit < 5% 7. Plus 8-Methoxypsoralen 8. Irradiation with UVA 2,0 - 2,5 J/cm² 9. Reinfusion of the treated buffy coat

15.01.2015 59 Time needed

3. + 4. + 6. +7. 8.

1. 2. 5. 9.

180 min

15.01.2015 60 Facility Ressources GMP facility Laboratory

3. + 4. + 6. +7. 8.

1. 2. 5. 9.

Day care center/ ward

15.01.2015 61 MINI ECP in practise

• SCID (Omenn-Syndrom, RAG1 Mutation), 3y alt, multiple premorbidity pre SCT (viral infections, BCG infection, surgery Ileum resection ,...) • SCT MUD (BM) 17.12.2013 • Fulminatn engraftment (d+13) • Severe acut GvHD skin/liver/gut: exanthema Bilirubin increase bloody diarrhea (transfusion dependend)

• Nonresponse to IST with steroid, high dose CSA/FK506 and MMF (MMF ex d +28)  start MINI ECP d +20 (05.01.2014): 2x/week

15.01.2015 62 Mini ECP in very low bodyweight patients(< 10kg BW)

Cave circulatory ! Monitoring: HF, RR

Hickman ZVK single lumen

15.01.2015 63 Bag with ACD-A

Syringe to take blood

pRBC (isovolemic Hemodilution))

HA 5%

Hickman CVCsingle lumen

15.01.2015 64 steps: 1. Drawing 10ml blood from CVC 2. Direct to sample bag 3. Reinfuse pRBC 1:1 with HA 5%

15.01.2015 65 outcome 100 25

90 skin: remission 80 20 liver: remission 70 gut: 60 15 partial response 50 GPT 40 10 Bili

20 5

0 0

Reduction of the liver size Reduction of steroids ECP Decrease of skin rush 15.01.2015 66 15.01.2015 67 15.01.2015 68 Celldose [cellsx10e06/kg bw]

Lymphocyte Monocyte Granulocyte MINI 5,37 2,44 3,99 (n=65) OFFLINE (n=136) 46,7 20,3 14,9 INLINE*) (n=134) 25,4 6,2 21,8

*)INLINE = UVAR XTS

15.01.2015 69 ECP FOR ACUTE GVHD WITH DIFFERENT METHODS (MINI; OFFLINE; INLINE) IN PEDIATRIC PATIENTS Volker Witt, Herbert Pichler, Christina Peters, Susanne Matthes, Anita Lawitschka, Wolfgang Holter

St. Anna Kinderspital,UKKJ Medical University Vienna, Austria, Kinderspitalgasse 6, 1090 Vienna, Austria, [email protected]

Poster EBMT 2014, Milano 15.01.2015 71 aGHVD ECP method

12 13

n= 6 6 4 4 2

0 MINI offline inline

15.01.2015 72 n ECP / patient

15.01.2015 73 aGVHD method of ECP

Chi-Quadrat Degree of freedom P

0,047293083 2 0,976630847 15.01.2015 74 aGVHD response at 3 months to ECP

15.01.2015 75 Conclusion aGVHD

• ECP is in our hands an effective second line therapy in acute GVHD. Neither the method used nor the individualized schedule applied seems to influence the outcome. Due to the small patient number, this report could be only a step forward to prospective randomized trials, bringing hopefully an answer to these questions. Interestingly we could show in our collective, that the response to ECP treatment for aGVHD is a predictor of overall survival.

15.01.2015 76 Conclusion MINI ECP

• Interesting method • No international aggreement or acceptance whether blood, tissue or ATMP regulations should be applied • Low technical efforts, low economic expenses => ? Low budget ECP? • Data registry needed

15.01.2015 77 Outcome

Acute GVHD Chronic GVHD • 14/14 improved after in median • 17 improved after 11 treatments • 17 skin • Organ involvement • 3 gut • 14 skin (14 improved) • 3 liver • 6 liver (5 improved) • 6 mucosa • 4 GI (4 improved) • 7 did not improved • 4 died due to infection and MOF • 4 skin (but in 2 skin improved) not due to the apheresis procedure (Mini ECP) and improvement could • 3 mucosa not be evaluated. • 1 liver • 1 gut

16.01.2015 78 c duration of immunosuppressiv treatment in cGVHD

3000

2500

2000

1500 Tage 1. nach ECP 1000

500 verstorben verstorben

verstorben

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Patient

KS CSA Tacrolimus MMF Rituximab Imatinib

16.01.2015 79 Outcome from all patients with acute, overlap and chronic GVHD from 1999 until today

1 0.9 0.8 improved 0.7 0.6 0.5 0.4 0.3 0.2 not improved probability overall survival overallprobability 0.1 0 0 2 4 6 8 10 12

Interval from date of Tx [years]

acute GVHD chronic GVHD 1 1 0.9 0.9 0.8

0.8 0.7 0.7 improved 0.6 0.6 0.5

0.5 not improved probability probabilitiy 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 0.5 1 1.5 2 2.5 3 3.5 0 2 4 6 8 10 12

patients with acute graft versus host disease patients with chronic graft versus host disease interval first ECP => outcome 16.01.2015 Intervall first ECP => outcome 80 Survival and Improvement in patients with cGVHD

p = 0,818744365

1,2

1 timepoint:

0,8 Ausfälle 1.ECP + 1 month 0,6 n y

0,4 Wahrscheinlichkeit 0,2 p = 0,001948581 0 0 1000 2000 3000 4000 5000 1,2 d_survival_1.ECP 1

0,8 Ausfälle 0,6 n y

End point +3 0,4 Wahrscheinlichkeit months 0,2

0 0 1000 2000 3000 4000 5000 d_survival_1.ECP 16.01.2015 81 Conclusions ECP in children:

• ECP is an approach to GvHD therapy that appears to offer control of GvHD without generalized immunosuppression and its associated complication • Outcome is associated with response to ECP • Despite different harvest and photochemotherapy methods, response to treatment seems to be independent • High response rates in cutaneous and extracutaneous GVHD manifestations • Steroid-sparing effect • Improved quality of life and OS • No negative effect on GvL • Well tolerated, minimal side-efects during therapy and no reported long term side effects

16.01.2015 82 Thank you for your attention