ANU PARTANEN IMPACT OF MOBILIZATION METHOD AND PREVIOUS LENALIDOMIDE EXPOSURE IN AUTOLOGOUS STEM CELL TRANSPLANTATION

Transkriptio

1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences ANU PARTANEN IMPACT OF MOBILIZATION METHOD AND PREVIOUS LENALIDOMIDE EXPOSURE IN AUTOLOGOUS STEM CELL TRANSPLANTATION

2

3 IMPACT OF MOBILIZATION METHOD AND PREVIOUS LENALIDOMIDE EXPOSURE IN AUTOLOGOUS STEM CELL TRANSPLANTATION

4

5 Anu Partanen IMPACT OF MOBILIZATION METHOD AND PREVIOUS LENALIDOMIDE EXPOSURE IN AUTOLOGOUS STEM CELL TRANSPLANTATION To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Auditorium 2, Kuopio University Hospital, Kuopio, on Friday, May 31st 2019, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences No 508 Department of Medicine, Kuopio University Hospital and Institute of Clinical Medicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland Kuopio 2019

6 Series Editors Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences Associate professor (Tenure Track) Tarja Kvist, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI Kuopio, Finland Grano, 2019 ISBN (print): ISBN (PDF): ISSNL (print): ISSN (print): ISSN (PDF):

7 Author s address: Doctoral programme: Supervisors: Department of Medicine Kuopio University Hospital KUOPIO FINLAND Clinical research Professor Esa Jantunen M.D., Ph.D. Siunsote -North Karelia Hospital District, Joensuu Institute of Clinical Medicine, Internal Medicine University of Eastern Finland KUOPIO FINLAND Ville Varmavuo M.D., Ph.D. Department of Medicine Kymenlaakso Central Hospital KOTKA FINLAND Reviewers: Docent Marjatta Sinisalo M.D., Ph.D. Department of Internal Medicine Tampere University Hospital TAMPERE FINLAND Docent Urpu Salmenniemi M.D., Ph.D. Department of Clinical Hematology and Stem Cell Transplantation Unit Turku University Hospital TURKU FINLAND Opponent: Professor Maija Itälä-Remes M.D., Ph.D. Department of Clinical Hematology and Stem Cell Transplantation Unit Turku University Hospital University of Turku TURKU FINLAND

8

9 Partanen, Anu Impact of mobilization method and previous lenalidomide exposure in autologous stem cell transplantation Kuopio: University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland Dissertations in Health Sciences , 127 p. ISBN (print): ISSNL (print): ISSN (print): ISBN (PDF): ISSN (PDF): ABSTRACT High-dose therapy supported by autologous stem cell transplantation (auto-sct) is an increasingly common treatment approach in multiple myeloma (MM) and in non- Hodgkin lymphoma (NHL) patients. Granulocyte colony -stimulating factors (G- CSFs) alone or combined with chemotherapy are mainly used mobilization methods to achieve adequate blood grafts for auto-sct. However, the best and most costeffective mobilization method is not known, albeit new drugs for mobilization, including plerixafor (PLER), have been launched. The present series of studies are based on the GOA study (Graft and Outcome in Autologous stem cell transplantation), which aims to investigate the impact of various mobilization methods on the blood graft cellular composition and post-transplant outcome of the patients after auto-sct. The possible negative effect of previous lenalidomide (LEN) use on CD34 + mobilization has raised a concern. Mobilization with low-dose cyclophosphamide combined with G-CSF resulted in an adequate number of blood CD34 + cells collected in all MM patients after limited LEN exposure. However, previous LEN use resulted in higher number of apheresis sessions needed to achieve target collection yields. LEN use did not hamper the tempo of engraftment. In contrast, it improved the progression-free survival (PFS) but not overall survival (OS) in the patients after auto-sct. In the majority of the hard-to-mobilize NHL patients pre-emptive PLER added to chemotherapy plus pegfilgrastim (PEG) as a G-CSF could overcome mobilization problems, and 94 % of the patients proceeded to auto-sct. The analysis of graft cellular composition revealed higher numbers of both T lymphocytes and NK cells after PLER use in the mobilization. Lower blood CD34 + cell counts on the first apheresis day were reflected in significantly lower total apheresis yields in PLER mobilized patients. Early haematological recovery was slower in PLER mobilized 7

10 patients whereas the outcome after auto-sct was comparable with PLER-naïve patients. Lipegfilgrastim (LIPEG) combined with chemotherapy to mobilize blood grafts for transplant purposes resulted in higher blood CD34 + cell counts and superior CD34 + yields on the first apheresis day compared to filgrastim (FIL) or PEG. LIPEG use achieved collection targets with less apheresis and cost. The early hematological recovery was faster in LIPEG mobilized patients. Neither PFS nor OS of the patients was affected by the choice of G-CSF for the mobilization. To conclude, limited lenalidomide exposure does not prevent effective mobilization of CD34 + cells for auto-sct in MM patients. Plerixafor added to chemotherapy plus pegfilgrastim to mobilize blood grafts in NHL patients was safe and effective without an impact on long-term outcomes. Finally, the non-randomized comparison of various G-CSFs added to chemotherapy mobilization in NHL patients showed lipegfilgrastim to be superior to filgrastim or pegfilgrastim clearly suggesting a need for randomized mobilization studies with this G-CSF. National Library of Medicine Classification: QU 325, WH 540, WH 525, WH 140, WO 660 Medical Subject Headings: Stem Cell Transplantation; Transplantation, Autologous; Multiple Myeloma; Lymphoma, Non-Hodgkin; Hematopoietic Stem Cell Mobilization; Granulocyte Colony-Stimulating Factor; Lenalidomide; Heterocyclic Compounds; Filgrastim; Antigens; CD34; Treatment Outcome; Prognosis; Graft Survival; Survival Rate, Humans 8

11 Partanen, Anu Mobilisaatiomenetelmän ja aiemman lenalidomidihoidon vaikutus autologisessa kantasolujensiirrossa Kuopio: Itä-Suomen yliopisto, terveystieteiden tiedekunta Publications of the University of Eastern Finland Dissertations in Health Sciences , 127 s. ISBN (print): ISSNL (print): ISSN (print): ISBN (PDF): ISSN (PDF): TIIVISTELMÄ Potilaalta aiemmin kerättyjen kantasolujen palautus (autologinen kantasolujensiirto) korkea-annoksisen solunsalpaajahoidon (intensiivihoito) jälkeen on yhä suositumpi myeloomaa (MM) tai non-hodgkin lymfoomaa (NHL) sairastavien potilaiden hoitomenetelmä. Valkosolukasvutekijöitä käytetään mobilisaatiohoidossa yksin tai yhdessä solunsalpaajahoidon kanssa riittävän siirteen saamiseksi. Parasta ja kustannustehokkainta mobilisaatiomenetelmää ei tiedetä, vaikka on kehitetty uusia lääkeaineita, kuten pleriksafori, jotka pystyvät tehostamaan kantasolujen mobilisaatiota. Tämä tutkimussarja perustuu prospektiiviseen GOA-tutkimukseen (Graft and Outcome in Autologous stem cell transplantation), jonka tarkoituksena on selvittää eri mobilisaatiomenetelmien vaikutusta kantasolukeräyksessä saatavan siirteen solukoostumukseen sekä potilaiden selviytymiseen autologisen kantasolujensiirron jälkeen. Lenalidomidihoidon vaikutus CD34 + -solujen mobilisaatioon on herättänyt huolta. Kantasolujen mobilisaatio matala-annoksisella syklofosfamidilla yhdessä valkosolukasvutekijän kanssa kestoltaan lyhyehkön lenalidomidin käytön jälkeen mahdollisti riittävän CD34 + -solujen keräyssaaliin kaikilla MM potilailla. Keräystavoitteen saavuttamiseksi tarvittu keräyspäivien määrä oli kuitenkin suurempi lenalidomidia käyttäneillä. Aiempi lenalidomidihoito ei hidastanut verisolujen toipumista kantasolujensiirron jälkeen, mutta se pidensi tautivapaata aikaa vaikuttamatta kokonaiseloonjäämiseen. Huonosti mobilisoivilla NHL-potilailla pleriksaforin liittäminen solunsalpaajahoidon ja pegfilgrastiimi-kasvutekijän yhdistelmään vähensi mobilisaatio-ongelmia ja 94 % potilaista eteni kantasolujensiirtoon. Kantasolusiirteiden solunanalyysissä todettiin mobilisaatiossa käytetyn pleriksaforin lisänneen sekä T-lymfosyyttien että luonnollisten tappajasolujen määrää siirteessä. Ensimmäisenä kantasolukeräyspäivänä mitatut matalammat 9

12 veren CD34 + -solumäärät heijastuivat merkittävästi pienempään kokonaiskeräyssaaliiseen pleriksaforia saaneilla. Varhainen hematologinen toipuminen kantasolujensiirron jälkeen oli hitaampaa, mutta tautivapaa- sekä eloonjäämisaika oli siirron jälkeen verrokkiryhmää vastaava pleriksaforia saaneilla. Veren kantasolusiirteen mobilisaatiossa solunsalpaajahoitoon yhdistetyllä lipegfilgrastiimi-valkosolukasvutekijällä saavutettiin filgrastiimiin tai pegfilgrastiimiin verrattuna korkeampi veren CD34 + -solujen määrä sekä suurempi CD34 + -solujen saalis ensimmäisenä keräyspäivänä. Lipegfilgrastiimia käyttäneillä ennalta määritelty CD34 + -solujen keräyssaalis saavutettiin vähemmillä keräyskerroilla sekä pienemmillä kustannuksilla. Varhainen hematologinen toipuminen oli lipegfilgrastiimia saaneilla nopeampaa. Mobilisaatiohoidon kasvutekijävalinta ei vaikuttanut potilaiden ennusteeseen kantasolujensiirron jälkeen. Kestoltaan lyhyehkö lenalidomidin käyttö ei estänyt veren CD34 + -solujen tehokasta mobilisaatiota myeloomapotilailla. Pleriksaforin lisäys kemoterapian ja pegfilgrastiimin yhdistelmään kantasolujen mobilisoinnissa osoittautui turvalliseksi ja tehokkaaksi, mutta sillä ei ollut merkittävää vaikutusta pitkäaikaiseen selviytymiseen. Tämä ei-satunnaistettu tutkimus eri kasvutekijöiden yhdistämisestä kemoterapiaan kantasolujen mobilisaatiossa non-hodgkin lymfoomapotilailla osoitti lipegfilgrastiimin tehokkaammaksi filgrastiimiin tai pegfilgrastiimiin verrattuna. Satunnaistettuja mobilisaatiotutkimuksia lipegfilgrastiimilla tarvitaan. Luokitus: QU 325, WH 540, WH 525, WH 140, WO 660 Yleinen suomalainen asiasanasto: kantasolujen siirto; kantasolut; myelooma; non- Hodgkin- lymfoomat; mobilisaatiohoito; sytostaattihoito; lääkehoito; pleriksafori; kasvutekijät; valkosolut; toipuminen; henkiinjääminen 10

13 ACKNOWLEDGEMENTS This thesis was carried out during principally at the Department of Medicine, Kuopio University Hospital and at the Department of Clinical Microbiology, University of Eastern Finland. These studies are based on the Graft and Outcome in Autologous stem cell transplantation (GOA) study, which included patients from Kuopio, Oulu, Tampere and Turku University Hospital districts. I owe my sincere thanks for the patients, colleagues, research nurses and other personnel who participated in the GOA study. I wish to express my deepest gratitude to my principal supervisors, the architects of the GOA study Professor Esa Jantunen, M.D., Ph.D., and Ville Varmavuo, M.D., Ph.D., for their continuous support, intelligent advice and the patience in navigation of a clinician to the fascinating and immersive world of scientific research. I have felt privileged for having such remarkable experienced and talented scientists with clinical orientation besides me, guiding by discreet suggestions, counselling critical scientific thinking and writing as well as encouragement to overcome challenges. I cannot thank you enough for the time you have devoted to this project. I owe my sincere gratitude to Docent Tapio Nousiainen, M.D., Ph.D., the former Chief in Hematology at the Department of Hematology in Kuopio University Hospital and Docent Taru Kuittinen, M.D., Ph.D., the present Chief in Hematology at the Department of Medicine in Kuopio University Hospital for their encouraging attitudes towards my study and for providing the facilities to perform it. I especially wish to thank Tuomas Selander, MSc, for statistical guidance and Jaakko Valtola, M.D., Ph.D., for practical and valuable advice. I also wish to warmly thank all the other colleagues at the Department of Hematology, Kuopio University Hospital, the Hematological family for the delightful working atmosphere, support and inspiring discussions. I wish to warmly thank all my co-authors Leena Keskinen, M.D., Professor Outi Kuittinen, M.D., Ph.D., Hanne Kuitunen, M.D., Ph.D., Docent Pentti Mäntymaa, M.D., Ph.D., Docent Tapio Nousiainen, M.D., Ph.D., Professor Jukka Pelkonen, M.D., Ph.D., Karri Penttilä, M.D., Ph.D., Mervi Putkonen, M.D., Ph.D., Marja Pyörälä, M.D., Ph.D., Antti Ropponen, B.S., Marja Sankelo, M.D., Ph.D., Tuomas Selanader, MsC, Docent Timo Siitonen, M.D., Ph.D., Raija Silvennoinen, M.D., Ph.D., Jaakko Valtola, M.D., Ph.D., Kaija Vasala, M.D., Ph.D. and Lasse Ågren, M.D. I owe my deepest gratitude to the official reviewers of this thesis, Docent Marjatta Sinisalo, M.D., Ph.D., and Docent Urpu Salmenniemi, M.D., Ph.D. for their constructive criticism and wise suggestions that helped me to improve this work. I wish to warmly thank Docent David Laaksonen for the excellent linguistic revision. I m honored to have Professor Maija Itälä-Remes, M.D., Ph.D., as my opponent. I express my heartfelt thanks to my wonderful friends, dear relatives and family for the invaluable support and interest in my work. I am very grateful to my cousins Vesa and Matti for technical advice and my friend Leena for revision of Finnish 11

14 language. My warmest thanks belong to my sister Taina and her family for support and eventful moments we have spent together. Finally, I express my deepest gratitude to the persons of utmost importance to me -my closest family. My beloved sons Lauri and Juho have delighted my life with their boyish energy and joy, and they have also kept my feet on the ground during this project. Most of all I am grateful to my loving husband Ismo for your patience, support and care. I am fortunate to have you beside me. This thesis was financially supported by grants from Cancer Foundation of North Savo, the Finnish Society of Hematology, the Research Foundation of Hematological Diseases, the Kuopio University Hospital Research Foundation and the Finnish Cultural Foundation. Kuopio, April 2019 Anu Partanen 12

15 LIST OF ORIGINAL PUBLICATIONS This dissertation is based on the following original publications, which are later referred by their Roman numerals: I Partanen A, Valtola J, Silvennoinen R, Ropponen A, Siitonen T, Putkonen M, Sankelo M, Pelkonen J, Mäntymaa P, Varmavuo V, Jantunen E. Impact of lenalidomide-based induction therapy on the mobilization of CD34(+) cells, blood graft cellular composition, and post-transplant recovery in myeloma patients: a prospective multicenter study. Transfusion 57(10): , II Partanen A, Valtola J, Ropponen A, Vasala K, Penttilä K, Ågren L, Pyörälä M, Nousiainen T, Selander T, Mäntymaa P, Pelkonen J, Varmavuo V, Jantunen E. Preemptive plerixafor injection added to pegfilgrastim after chemotherapy in non-hodgkin lymphoma patients mobilizing poorly. Ann Hematol 96(11): , III Partanen A, Valtola J, Ropponen A, Kuitunen H, Kuittinen O, Vasala K, Ågren L, Penttilä K, Keskinen L, Pyörälä M, Nousiainen T, Selander T, Mäntymaa P, Pelkonen J, Varmavuo V, Jantunen E. Comparison of filgrastim, pegfilgrastim and lipegfilgrastim added to chemotherapy for mobilization of CD34 + cells in non-hodgkin lymphoma patients. Transfusion 59(1): , The publications were adapted with the permission of the copyright owners. 13

16 14

17 CONTENTS ABSTRACT... 7 TIIVISTELMÄ... 9 ACKNOWLEDGEMENTS INTRODUCTION REVIEW OF THE LITERATURE Autologous stem cell transplantation (auto-sct) Indications in patients with multiple myeloma Indications in patients with non-hodgkin lymphoma Lenalidomide Pharmacology Use in transplant-eligible myeloma patients Impact of lenalidomide on mobilization of CD34 + cells Mobilization of CD34+ cells in autologous setting Mechanism of CD34+ cell mobilization Mobilization methods Chemotherapy plus G-CSF G-CSF alone Plerixafor added to G-CSF with or without chemotherapy Type of G-CSF used in the mobilization of CD34+ cells Filgrastim and biosimilar filgrastim Pegfilgrastim Pharmacology Pharmacokinetics and pharmacodynamics Mobilization studies Lipegfilgrastim Pharmacology Pharmacokinetics and pharmacodynamics Clinical studies Plerixafor in the mobilization of CD34 + cells Pharmacology Mobilization studies Algorithms for plerixafor use Collection, analysis and processing blood grafts in autologous setting Enumeration of blood CD34+ cells

18 2.6.2 Clinical aspects of leukapheresis Enumeration of CD34 + cells in the apheresis product Processing of blood grafts Blood graft cellular composition in autologous transplantation CD34 + cells CD34 + cell subclasses Lymphocytes B lymphocytes T lymphocytes NK cells Factors affecting graft cellular composition Post-transplant recovery after auto-sct Engraftment Hematological and immune recovery AIMS OF THE STUDY SUBJECTS AND METHODS Study outlines Patients Characteristics of myeloma patients (I) Characteristics of non-hodgkin lymphoma patients (II-III) Clinical methods Mobilization of blood grafts G-CSF use in the mobilization Plerixafor use in the mobilization Collection of blood grafts High-dose therapy Assessment of post-transplant recovery Laboratory methods Blood and leukapheresis CD34 + cell assessment Processing of the blood grafts Flow cytometric analysis of thawed grafts Cost evaluation of mobilizing G-CSF and apheresis Quality control Data collection Statistical methods Approvals RESULTS Impact of lenalidomide exposure on blood CD34 + cell mobilization, graft cellular composition and post-transplant outcome in myeloma patients (I) 65 16

19 5.2 Plerixafor added to pegfilgrastim after chemotherapy in NHL patients mobilizing poorly CD34 + cells (II) Comparison of filgrastim, pegfilgrastim and lipegfilgrastim added to chemotherapy for mobilization of CD34 + cells in NHL patients (III) DISCUSSION The main findings Patients and study design Methods Mobilization of blood grafts High-dose therapy CD34 + measurements and graft analysis Impact of previous lenalidomide exposure on the mobilization of CD34 + cells and cellular composition of blood grafts in MM patients (I) Mobilization with plerixafor added to pegfilgrastim plus chemotherapy in hard-to-mobilize NHL patients (II) Effect of G-CSF type after chemotherapy for the mobilization of CD34 + cells and graft cellular composition in NHL patients (III) Future perspectives CONCLUSIONS REFERENCES

20 18

21 ABBREVIATIONS CEOP cyclophosphamide, epirubicin, vincristine, 7-AAD 7-aminoactinomycin D prednisone ALC-15 absolute lymphocyte count on day 15 after transplantation CHOEP cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone Ara-C cytarabine CHOP cyclophosphamide, auto-sct autologous stem cell transplantation doxorubicin, vincristine, prednisone B blood CIBMTR Center for International B-CD34 + number of CD34 + cells in peripheral blood Blood and Marrow Transplant Research BEAC carmustine (BCNU), CK1α kasein kinase 1-alfa etoposide, cytarabine, CNS central nervous system cyclophosphamide CR complete remission BEAM carmustine (BCNU), CRBN cereblon etoposide, cytarabine, CY cyclophosphamide melphalan CXCL C-X-C chemokine ligand BM bone marrow CXCR C-X-C chemokine receptor CAR-T chimeric antigen receptor- d day derived T cells DHAP dexamethasone, high-dose- CD cluster of differentiation cytarabine, cisplatin 19

22 DMSO dimethylsulfoxide ICE ifosfamide, DLBCL diffuse large B-cell lymphoma IFNγ carboplatin, etoposide interferon gamma EBMT European Society for Blood IK2F1 Ikaros and Marrow IK2F3 Aiolos Transplantation IMWG International Myeloma EMA European Medicine Agency Working Group EMN European Myeloma IRF4 interferon regulatory Network factor 4 ESMO FIL European Society for Medical Oncology filgrastim ISHAGE International Society of Hematotherapy and Graft Engineering FIMEA Finnish Medicine Agency IVAC ifosfamide, etoposide, high- FL follicular lymphoma dose cytarabine FMG G-CSF Finnish Myeloma Group granulocyte colony- LA-CD34 + number of CD34 + cells in leukapheresis yield stimulating factor LD-CY low-dose GOA Graft and Outcome in cyclophosphamide Autologous stem cell transplantation LEN LIPEG lenalidomide lipegfilgrastim HD HDT high-dose high-dose therapy 20

23 MATRIX methotrexate, cytarabine, thiotepa, rituximab PFS PLER progression-free survival plerixafor MCL mantle cell lymphoma PR partial response MEL melphalan PTCL peripheral T cell lymphoma MINE 2-mercaptoethane sulfonate, R rituximab ifosfamide, mitoxantrone, RD lenalidomide, etoposide dexamethasone MM multiple myeloma RVD lenalidomide, bortezomib, MM-02 Multiple Myeloma 02 study dexamethasone (by the FMG) SC subcutaneous MRD minimal residual disease SDF-1 stromal cell-derived factor 1 NCCN National Comprehensive Treg regulatory T cell Cancer Network UEF University of Eastern NHL non-hodgkin lymphoma Finland NK natural killer VCAM vascular cell adhesion OS overall survival molecule PCNSL primary central nervous VGPR very good partial response system lymphoma VLA4 very late antigen 4 PCR polymerase chain reaction WBC white blood cell PD-1 programmed cell death-1 PEG pegfilgrastim 21

24 22

25 1 INTRODUCTION Autologous stem cell transplantation (auto-sct) is an effective and potentially curative procedure with increasing utilization observed both by European Society for Blood and Marrow Transplantation (EBMT) registry [Passweg et al. 2019] and by the Center for International Blood and Marrow Transplant Research (CIBMTR) registry [D Souza et al. 2017] especially in patients with hematological malignancies. Multiple myeloma (MM) is the most common indication for auto-sct followed by non-hodgkin lymphomas (NHL) [Passweg et al. 2019, D Souza et al. 2017]. Inadequate grafts are, however, collected in % of the patients intended to proceed to auto-sct [Pusic et al. 2008, Wuchter et al. 2010, Jantunen et al. 2012]. The well-known factors that impact on the mobilization of CD34 + cells for auto- SCT include previous chemotherapy, platelet counts just before the mobilization procedure, age and BM (bone marrow) involvement of lymphoma [Kuittinen et al. 2004, Bensinger et al. 2009, Wuchter et al. 2010]. The possible hampering effect of the lenalidomide (LEN) exposure in MM patients has raised a concern [Kumar et al. 2007, Popat et al. 2009]. To some extent contradictory views of effects of LEN utilization on the number of CD34 + cells collected have also been observed in some studies [Roussel et al. 2014, Silvennoinen et al. 2016]. Chemotherapy plus G-CSF or G-CSF alone have been the most commonly used mobilization methods to collect grafts for auto-sct. Almost for three decades recombinant filgrastim (FIL) has been utilized as a G-CSF. The more feasible and convenient long-acting G-CSF pegfilgrastim (PEG) has also been found to be a reliable product in mobilization studies [Nosari et al. 2006, Russell et al. 2008, Putkonen et al. 2009, Simona et al. 2010, Herbert et al. 2013], although it is not officially approved for mobilization purposes. Published studies of lipegfilgrastim (LIPEG) use in the mobilization of CD34 + cells for auto-sct are lacking. Plerixafor (PLER), a quite novel member in the mobilization family can mobilize CD34 + cells safely and efficiently in combination with G-CSF [DiPersio et al. 2009a, DiPersio et al. 2009b] or chemotherapy and G-CSF [Dugan et al. 2010, d Addio et al. 2011, Jantunen 2011, Lanza et al. 2014]. Several algorithms for pre-emptive use of costly PLER have been developed for hard-to-mobilize patients [Sancho et al. 2012, Abhyankar et al. 2012, Jantunen et al. 2012]. Very limited data exist on the addition of PLER in patients mobilizing poorly with chemotherapy and PEG as practically all studies evaluating the efficacy of PLER have been carried out with daily FIL. The type of mobilization procedures used has an impact not only on CD34 + cells but also on other cells in the grafts collected, e.g. CD34 + subtypes and lymphocyte subsets [Jantunen and Fruehauf 2011, Saraceni et al. 2015]. The only surrogate marker for the blood graft quality has been the number of CD34 + cells collected [Jantunen and Fruehauf 2011]. Grafts including higher CD34 + cell numbers have been correlated with faster early engraftment after high-dose therapy [Weaver et al. 1995, Ketterer et al. 1998, Siena et al. 2000, Allan et al. 2002, 23

26 Stiff et al. 2011], prolonged progression-free survival (PFS) and even overall survival (OS) benefit in some studies [Pavone et al. 2006, Bolwell et al. 2007], but contradictory results of the impact of the higher CD34 + counts on OS have also been presented [Stockerl-Goldstein et al. 2000]. In addition, the number of more primitive CD34 + CD38 - cells have been associated with early engraftment after high-dose therapy (HDT) [Zubair et al. 2006]. These series of studies are based on the prospective and non-randomized GOA study (Graft and Outcome in Autologous stem cell transplantation), which aimed to evaluate the impact of various mobilization methods on the graft cellular composition and on the outcome in MM or NHL patients after auto-sct. The impact of premobilization LEN exposure on the mobilization efficiency and graft composition in MM patients was investigated in the first study. The second study evaluated the feasibility and efficiency of PLER use in patients who mobilize poorly with chemotherapy plus PEG. Finally, the efficacy of three types of G-CSFs (FIL, PEG and LIPEG) used for mobilization purposes and their effects on post-transplant hematological recovery was evaluated in NHL patients intended for auto-sct. 24

27 2 REVIEW OF THE LITERATURE 2.1 AUTOLOGOUS STEM CELL TRANSPLANTATION (AUTOSCT) Indications in patients with multiple myeloma For the last two decades auto-sct has been a standard of care for transplant-eligible patients with MM, a malignancy of clonal B-cell derived plasma cells. Although MM accounts only 1% of all malignances, it is the most popular indication for auto-sct, with over transplantations in 2017 according to the EBMT registry [Passweg et al. 2019]. The acknowledged status of auto-sct is based on its superiority in previous randomized studies regarding PFS and OS compared to conventional therapy before the approval of new MM drugs [Attal et al. 1996, Child et al. 2003]. Novel drugs launched for the treatment of MM combined with auto-sct have substantially improved the survival of transplant-eligible younger patients [Kristinsson et al. 2014]. The most recent meta-analysis, which included three randomized phase III studies, concluded that up-front auto-sct in the era of novel drug induction regimens produced higher proportion of complete responses (CR) and improved PFS [Dhakal et al. 2018]. However, a crucial prognostic marker regarding PFS and OS in newly diagnosed MM patients was observed not only to be achievement of CR, but also minimal residual disease (MRD) negativity [Lahuerta et al. 2017, Usmani et al. 2018]. At present, auto-sct is preferred in newly-diagnosed MM patients below years of age as a consolidation after induction regimens. However, in a retrospective study auto-sct improved OS also in older patients [Wildes et al. 2015], which encourages reconsideration of the age limits regarding transplantation. Upfront transplantation was superior to later transplantation in regard to PFS [Attal et al. 2017, Aggarwal et al. 2018]. In the context of relapsed MM, rescue auto-sct is considered beneficial [Gay et al. 2018]. A consensus of the international expert group suggested to consider salvage auto-sct in relapsed MM if the patient had a prolonged (> 18 months) response to the first transplant or is transplant-naïve [Giralt et al. 2015]. Of note, data from the ongoing studies of novel regimens in MM treatment may impact on the role of auto-sct in the future. A widely used practice is to collect CD34+ cells for two transplants in MM patients [Giralt et al. 2015], but some centers collect for three transplants in fit patients, intending to carry out upfront tandem transplant and rescue in the future. However, the Cochrane Database meta-analyses concluded that OS of MM patients was not impacted by tandem transplant [Naumann-Winter et al. 2012] in concordance with the most recent phase III trial [Mai et al. 2016] and the update of the randomized STAMINA study [Stadtmauer et al. 2019]. 25

28 2.1.2 Indications in patients with non-hodgkin lymphoma Auto-SCT has been a widely accepted treatment approach for patients with relapsed or refractory aggressive NHL after the impressive results of the classical PARMA study [Philip et al. 1995]. NHL has been the second most common as well as an increasing indication for auto-sct in Europe with over 6500 transplantations in 2017 [Passweg et al. 2019]. With novel mobilization methods the majority of patients achieve adequate grafts to proceed to auto-sct. Diffuse large B-cell lymphoma (DLBCL) accounts one third of all lymphomas and presents also the biggest lymphoma subtype in patients proceeding to auto-sct. A previous meta-analysis including 3079 randomized NHL patients also concluded that auto-sct produced more remissions without an impact on survival in the upfront setting [Greb et al. 2008]. The revolution in the use of biologic prognostic factors may reveal some subgroups of newly diagnosed DLBCL patients who might benefit from auto-sct in the era of the improved first-line therapies [Jantunen and Sureda 2012]. In patients achieving partial response (PR) after immunochemotherapy, second-line chemotherapy followed by auto-sct may be considered. The randomized PARMA study showed that the patients with relapsed DLBCL who proceeded to auto-sct had significantly longer PFS and better OS compared to patients treated with chemotherapy alone [Philip et al. 1995]. The more recent CORAL study suggested comparable results with ICE- (ifosfamide-carboplatinetoposide) or DHAP- (dexamethasone-hd-cytarabine (HD-Arac)-cisplatin) induction therapies combined with rituximab plus auto-sct in relapsed or refractory DLBCL patients [Gisselbrecht et al. 2010]. The EBMT registry study highlighted the benefit of auto-sct on the outcome in patients with their first lymphoma relapse [Mounier et al. 2012]. In addition, the most recent data suggested that auto-sct is also a feasible and effective treatment option for elderly patients with relapsed lymphoma [Chihara et al. 2014]. Follicular lymphoma (FL) accounts for one fourth of all NHLs and is the most common indolent B-cell lymphoma [Teras et al. 2016]. In the randomized European CUP study, auto-sct produced in relapsed or refractory FL patients longer PFS and better OS than conventional treatment [Schouten et al. 2003]. The FL2000 study concluded that FL patients with auto-sct as a consolidation therapy after the first relapse had a better OS [Le Gouill et al. 2011], in concordance with the most recent study, which highlighted the benefits of auto-sct at first or at least second relapse [Oh et al. 2016]. In addition, Casulo and co-workers (2018) showed improved 5-year OS in FL patients who proceeded to auto-sct within 12 months after early relapse compared to transplant-naïve patients. Also, patients with an aggressive transformed FL gain an outcome benefit when treated with auto-sct according to a study by a Canadian group [Villa et al. 2013]. A meta-analysis of randomized studies of patients with newly diagnosed FL showed that also after rituximab exposure autosct after conventional induction therapy enhanced PFS but not OS [Schaaf et al. 2012]. The results of the GOELAMS study with extended follow-up time showed a 26

29 plateau of the PFS curve suggesting a possible cure in some FL patients with upfront auto-sct [Gyan et al. 2009]. The ESMO guidelines [Dreyling et al. 2017] suggest that auto-sct is a treatment option in FL patients with early relapse with prior rituximab exposure, whereas it should be avoided in newly diagnosed chemosensitive patients. Also, the National Comprehensive Cancer Network (NCCN) recommended auto- SCT in the context of relapsed or refractory FL patients with previous rituximab exposure [Evens et al. 2013]. Peripheral T cell lymphomas (PTCL) have a poor outcome except for the ALKpositive anaplastic large cell lymphoma. These rare lymphomas do not have a widely accepted standard treatment. To date, the randomized studies of PTCLs concerning upfront auto-sct are still lacking. In prospective studies upfront auto-sct improved the 3-year PFS and overall survival in PTCLs [Reimer et al. 2009] in concordance with the previous observations with extended 12-year follow-up of the Italian group [Corradini et al. 2006]. A large prospective phase II study by Nordic Lymphoma Group showed the efficacy of upfront auto-sct in PTCL, manifested as a 5-year OS in half of the patients [d Amore et al. 2012]. In line with that the most recent prospective COMPLETE study suggested significantly prolonged PFS and OS in the patients with angioimmunoblastic subtype of PTCL who underwent auto-sct in the first CR [Park et al. 2019]. The retrospective observations by the CIBMTR showed 53 % 3-year OS curves in relapsed PTCL patients [Smith et al. 2013] in line with the findings of the EBMT registry study in angioimmunoblastic T -cell lymphoma patients [Kyriakou et al. 2008]. The promising results of these studies lead at least to consider this option for some relapsed PTCL patients as a supplement to the common use of auto-sct in the upfront setting if this treatment has not been used as a consolidation after first-line therapy [Kharfan-Dabaja et al. 2017]. Mantle cell lymphoma (MCL) is a rare B-cell lymphoma entity (< 5 % of NHLs) with relatively poor prognosis. A previous randomized study comprising of MCL patients in first CR concluded that auto-sct consolidation produced a longer PFS than maintenance with alpha-interferon [Dreyling et al. 2005]. The registry data study of EBMT and Autologous Blood and Marrow Transplant Registry showed that half of the MCL patients survived 5 years after auto-sct [Vandenberghe et al. 2003]. In the era of rituximab, the Nordic MCL-2 study showed with extended follow-up time (6 years) extremely high OS curves of up to 70 % after upfront auto-sct [Geisler et al. 2012] in concordance with the observations of the CIBMTR data which highlighted the efficacy of upfront auto-sct in chemosensitive transplant-eligible patients [Fenske et al. 2014]. In addition, the most recent phase III randomized study by French group suggested the efficacy of rituximab maintenance after auto-sct to improve both PFS and OS [Le Gouill et al. 2017]. Primary central nervous lymphoma (PCNSL), an aggressive extranodal B cell malignancy, accounts for only 1% of all NHL patients. The optimal treatment schema of PCNSL is unknown. The randomized phase II IELSG32 study concluded MATRIX (methotrexate-cytarabine-thiotepa-rituximab) to be the preferable treatment in patients < 70 years [Ferreri et al. 2016]. Auto-SCT has been suggested in several phase 27

30 II studies to prolong PFS and 2-year OS after high-dose methotrexate-based induction treatment in PCNSL patients [Han and Batchelor 2017]. In addition, a previous study showed in an extended 10-year follow-up that upfront auto-sct consolidation resulted in 35 % OS rates [Kiefer et al. 2012]. 2.2 LENALIDOMIDE Pharmacology LEN targets to the interferon regulatory factor INF4 CRBN receptor E3 ubiquitin ligase complex of cereblon (CRBN) resulting in the downregulation of CRBN substrates [Zhu et al. 2011]. The prerequisite transcription factors for B-cell differentiation and survival of MM cells, Ikaros (IKZF1) and Aiolos (IKZF3) and casein kinase 1 alpha (CK1α) are ubiquitinated and degraded by LEN [Krönke et al. 2014]. Downregulation of the target of CRBN, interferon regulatory factor 4 (IRF4), both inhibits the growth and kills MM cells [Krönke et al. 2014]. The direct mechanism of action of LEN against MM cells include apoptotic signalling via suppression of the activity of nuclear factor-kappab and activation of caspase 8 [Mitsiades et al. 2002]. LEN also hampers the interaction of MM cells and the microenvironment in BM by inhibiting the adhesion molecules covering the MM cells and stromal cells [Zhu et al. 2013]. The inhibition of transcription factor PU.1 by LEN results in reduced osteoclastogenesis [Anderson et al. 2006, Pal et al. 2010]. LEN has also antiangiogenetic properties caused by the reduced expression of vascular endothelial growth factor and fibroblast growth factor and possibly by suppressed Argonaute 2 [Gupta et al. 2001, Xu et al. 2016]. The expression of immune checkpoint inhibitor Programmed cell death-1 (PD-1) is downregulated in NK (natural killer) cells, CD4 + T cells and CD8 + T cells as well as in MM cells by LEN use [Görgün et al. 2015]. LEN induces CD4 + and CD8 + T cell proliferation and cytotoxicity and increases interferon (IFN)-γ and interleukin-2 production by cytotoxic CD8 + cells in MM [Luptakova et al. 2013]. Regulatory T -cells (Tregs) and proinflammatory cytokines e.g. tumor necrosis factor-α and interleukin- 6 released from blood mononuclear cells are suppressed by LEN [Muller et al. 1996, Galustian et al. 2009]. LEN exposure impairs myeloid maturation by downregulating the critical transcription factor PU.1, which leads to excess development of immature myeloid precursor cells especially promyelocytes and as a concequence neutropenia [Anderson et al. 2006, Pal et al. 2010]. The risk of venous thromboembolism during LEN treatment is increased and caused by the excess of promyelocytes that secrete high levels of serine protease cathepsin G, a platelet aggregation agonist [Pal et al. 2010]. However, the self-regulated system of intrinsic G-CSF tries to overcome the arrest of granulocyte differentiation after LEN use [Pal et al. 2010]. 28

31 2.2.2 Use in transplant-eligible myeloma patients LEN was initially approved for clinical use based on randomized phase III studies in patients with relapsed or refractory MM [Weber et al. 2007, Dimopoulos et al. 2007]. LEN was approved by the FDA in 2006 and the EMA in Subsequently also first-line studies in MM patients were started with LEN both in transplant eligible (SWOG-study) [Durie et al. 2017] and non-eligible patients (FIRST-study) [Facon et al. 2018]. In transplant-eligible patients a phase I/II study suggested that LEN combined with bortezomib and dexamethasone was an effective combination with manageable toxicity [Richardson et al. 2010]. LEN-based regimen before auto-sct lead to improved OS compared to LEN-naive patients in a retrospective study (Chakraborty et al. 2017). A triple combination of LEN, bortezomib and dexamethasone (RVD) combined with auto-sct resulted in CR in up to half of the patients before LEN maintenance and 3-year PFS was 77 % [Roussel et al. 2014]. The retrospective data by Mayo Clinic showed extremely high overall response rates at 3 months after transplantation with a similar RVD combination followed by auto-sct, and CR was obtained in 62 % of the patients during the followup [Sidiqi et al. 2018] in congruence with the findings of the previous IFM 2009 study [Attal et al. 2017]. The synergism of LEN and proteasome inhibitors in the treatment of MM is possibly a consequence of different pharmacokinetics as well as timetables of the administration of the drugs [Stewart et al. 2015, Fink and Ebert 2015]. Auto-SCT prolonged PFS compared to the extended melphalan-prednisonelenalidomide (MPR) -consolidation therapy after four cycles of LEN-based regimen as did also LEN maintenance regardless the former regimens used [Palumbo et al. 2014]. In a randomized multicenter study auto-sct consolidation significantly enhanced PFS in newly diagnosed MM patients compared to cyclophosphamide plus LEN consolidation after four LEN-based cycles combined with LEN maintenance ± prednisolone [Gay et al. 2015]. The most recent phase III study concluded that autosct improved PFS and OS compared to LEN-based consolidation [Mina et al. 2018]. LEN maintenance improved PFS compared to no maintenance [Mina et al. 2018] in concordance with a previous randomized phase III study [Attal et al. 2012]. Another randomized phase III study in a similar setting showed prolonged PFS and also better OS with LEN maintenance after auto-sct [McCarthy et al. 2012]. More recent analysis of the CALGB study showed that time to progressive disease was significantly longer (57.3 months vs months) with LEN maintenance after autosct [Holstein et al. 2017]. Further, a recent meta-analysis on prolonged LEN maintenance after auto-sct showed a significant benefit in both PFS and OS compared to placebo [McCarthy et al. 2017]. LEN maintenance and VGPR (very good partial) response after auto-sct have correlated with superior survival [Aggarwal et al. 2018]. In addition, recent data suggested that post-transplant therapy had greater impact on the outcome of MM patients than the regimens used before auto-sct [Cornell et al. 2017]. Currently LEN maintenance has been suggested as a standard of care in MM patients after auto-sct both by the European Society for Medical Oncology (ESMO) and European Myeloma Network (EMN). 29

32 2.2.3 Impact of lenalidomide on mobilization of CD34 + cells Incongruous results have been published in retrospective studies concerning the effect of the previous LEN use in the mobilization of CD34 + cells. Mazumder et al. (2018) concluded in a retrospective study that the number of premobilization LEN courses did not correlate with the CD34 + cell yields and also adequate CD34 + cell yields for tandem transplantation for the most of patients were collected after limited number of LEN cycles in a large prospective study [Cavallo et al. 2011]. LEN use before mobilization with G-CSF alone resulted in higher numbers of apheresis sessions needed, a lower amount of CD34 + cells collected with the first apheresis and lower total CD34 + yields [Kumar et al. 2007]. In retrospective series LEN use compromised the apheresis yields after mobilization with G-CSF alone, denoted by high failure rates [Paripati et al. 2008, Popat et al. 2009]. The Finnish Myeloma Group (FMG) showed in a prospective study that mobilization with G-CSF alone resulted in adequate grafts for a single transplant in all patients although cyclophosphamide (CY)-based mobilization was a bit more effective after LEN use [Silvennoinen et al. 2016]. In addition, a retrospective study by the Mayo Clinic found that pretransplant therapy including LEN did not hamper engraftment kinetics in MM patients [Kumar et al. 2007], in line with later studies (Paripati et al. 2008, Roussel et al. 2014). Polymerase chain reaction (PCR) and Western blot analysis showed that LEN administration increased the C-X-C chemokine receptor 4 (CXCR4) protein translocation to the surface of CD34 + cells by N-glycosylation of CXCR4 [Pal et al. 2010, Li et al. 2013]. LEN supported also the SDF-1α-stimulated migration of CD34 + cells, which possibly hampers the stem cell mobilization from bone marrow niche [Li et al. 2013]. Pre-emptive PLER combined with G-CSF can mobilize adequate grafts for auto-sct after LEN-based induction therapy in MM patients [Costa et al. 2012a] as PLER overcomes the LEN-induced binding of CXCR4 to SDF-1α by blocking the CXCR4 [Li et al. 2013]. The optimal timing of mobilization after LEN exposure is unclear. M.D. Anderson Cancer Center recommended mobilization after three courses of LEN [Popat et al. 2009] and Kumar et al (2007) within six months after the beginning of the MM treatment. Consensus guidelines by the American Society for Blood and Marrow Transplantation suggested early-phase mobilization and collection of CD34 + cells before the fourth LEN course [Duong et al. 2014] in line with IMWG (The International Myeloma Working Group) consensus view [Kumar et al. 2009], which also suggested to avoid pauses in LEN-based therapy before mobilization procedures. 30

33 2.3 MOBILIZATION OF CD34 + CELLS IN AUTOLOGOUS SETTING Mechanism of CD34 + cell mobilization Pluripotent hematopoietic stem cells are precursor cells that encompass the properties of self-renewal, differentiation into all lineages of blood cells and quiescence [Yu and Scarren 2016]. The majority of stem cells are located near the perivascular sinusoidal endothelium of endosteal sites in BM niche and during steady-state only a few quiescent stem cells are in peripheral blood [Anthony and Link 2014]. Stem cells express CXCR4 receptors, whereas SDF-1 is excreted by stromal cells, osteoclasts and endothelial cells [Alvarez et al. 2013, Mosaad 2014]. Leptin-receptor stromal cells, nestin-gfp + stromal cells and especially CXCL12- abundant reticular mesenchymal progenitor cells express CXCL12, which is one of the potent impactors of the actions of stem cells [Alvarez et al. 2013, Anthony and Link 2014, Morrison and Scarren 2014]. Holding of stem cells in the BM niche depends critically on chemotactic axis, which consist of the interference between SDF-1/ CXCL12 and CXCR4 [Lapidot et al. 2005]. The mobilization of stem cells to the circulation is based on the disturbance of the orchestra of stem cells and the microenvironment, which depends on the interaction between adhesion molecules and proteases, chemokines and chemokine receptors as well as intracellular signaling [Alvarez et al. 2013]. The homing of stem cells is influenced by chemotherapy, which disturbs endothelial function enhancing the secretion of chemokines and their receptors, cytokines, selectins (P-selectin, E-selectin) activated integrins and adhesion molecules e.g. vascular cell adhesion molecule 1 (VCAM-1), which regulate the traffic of infused stem cells to BM [Alvarez et al. 2013, De Grandis et al. 2016]. In steady state only 0.06 % of the hematopoietic cells are in the circulation [Körbling and Anderlini 2001]. Different mobilization procedures block the binding of stem cells to BM microenvironment resulting in release of stem cells to peripheral blood [Fruehauf and Tricot 2010], which has been the source of hematopoietic stem cells in the majority auto-scts (over 98 %) according to most recent EBMT registry analysis [Passweg et al. 2019] Mobilization methods Chemotherapy plus G-CSF Historically CY (up to dose 7 g/m 2 ) has been used to mobilize blood grafts for transplant purposes. More recently disease-specific chemotherapy has been used especially in lymphoma patients. The advantages of disease-specific chemotherapy include the potential to both decrease the tumor load and to stimulate mobilization. The drawbacks of chemomobilization include the time-consuming procedure, hospitalization during chemotherapy, poorer predictability of the apheresis timing 31

34 and toxicity of mobilization regimen possibly requiring supportive care. Infectious complications have also been observed after chemomobilization [Meldgaard Knudsen et al. 2000, Toor et al. 2001, Damon et al. 2008]. In a prospective mobilization study in NHL patients, low-dose (LD)-CY 1.5 g/m 2 was recommended due to less toxicity [Ahn et al. 2005]. The combination of CY plus G-CSF in the mobilization of CD34 + cells is more efficient than mobilization with G- CSF alone [Milone et al. 2003, Bensinger et al. 2009]. However, NHL patients may benefit from intensive mobilization chemotherapy regardless of the toxicity of treatment [Damon et al. 2008]. Although CY is mostly used in MM patients, HD- AraC, platinum-derived regimens such as DHAP or ICE are employed in NHL patients for CD34 + cell mobilization. In patients with MM low-dose cyclophosphamide (LD-CY) plus G-CSF has resulted in higher CD34 + yields compared to mobilization with G-CSF alone, although both methods have produced adequate grafts [Silvennoinen et al. 2016] G-CSF alone The action of G-CSF is based on the stimulation of BM resulting in enhanced protease function of cathepsin G and neutrophil elastase, which disrupts the adhesion interference of Very Late Antigen 4 (VLA4)/VCAM-1 and CXCR4/SDF-1 [Levesque et al. 2003, Bensinger et al. 2009], regulates the level of SDF-1 [Lapidot et al. 2005] and leads to the upregulation of CXCR4 in BM [Petit et al. 2002]. Mobilization with cytokine alone has less toxicity, accomplishes as outpatient treatment and appears to allow more predictable timing of apheresis [Watanabe et al. 2006]. Apheresis takes place on d 5 from the start of mobilization with G-CSF and short-acting G-CSF injections continue daily until the predetermined apheresis target is reached. The most widely used G-CSF in the mobilization setting is officially approved recombinant FIL [Duong et al. 2014], and since 2008 also biosimilar formulations of FIL have received the same indication [Lefrere et al. 2011, Schmitt et al. 2014, Becker et al. 2016, Schmitt et al. 2016, Bhamidipati et al. 2017]. In the randomized study in lymphoma patients mobilized with FIL alone produced adequate grafts albeit the apheresis yields were lower than in chemomobilized patients [Narayanasami et al. 2001]. The observations of more recent study have confirmed these findings [Watanabe et al. 2006]. A recent meta-analysis concluded that biosimilar FIL was comparable to the original product in the mobilization of CD34 + cells [Schmitt et al. 2016]. The off-label use of a long-acting pegylated FIL named PEG as a single injection was suggested to be comparable to FIL in mobilization procedures regarding collection yields and engraftment in recent meta-analyses [Kim et al. 2015, Kuan et al. 2017]. Published data on another long-sustained pegylated G-CSF called LIPEG in the mobilization setting are lacking. 32