Multiple myeloma treatment in the era of novel agents : special reference to minimal residual disease, stem cell mobilization and drug sensitivity testing / Raija Silvennoinen

(1)

DISSERTATIONS | RAIJA SILVENNOINEN | MULTIPLE MYELOMA TREATMENT IN THE ERA OF NOVEL ... | No 376

uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2260-1 ISSN 1798-5706

Dissertations in Health Sciences

THE UNIVERSITY OF EASTERN FINLAND

RAIJA SILVENNOINEN

MULTIPLE MYELOMA TREATMENT IN THE ERA OF NOVEL AGENTS

– Special reference to minimal residual disease, stem cell mobilization and drug sensitivity testing Novel drugs combined with autologous stem

cell transplantation have improved the overall survival in multiple myeloma. The majority of patients will still relapse after successful first-line treatment. We analyzed the impact of minimal residual disease after novel drugs on the patient outcome. The randomized stem cell mobilization study compared two different

mobilization methods. The ex vivo assay study focused on finding new molecules for

treatment of high-risk myeloma.

RAIJA SILVENNOINEN

(2)

Multiple myeloma treatment in the era of novel agents – special reference to minimal residual disease, stem cell mobilization and

drug sensitivity testing

(3)

(4)

RAIJA SILVENNOINEN

Multiple myeloma treatment in the era of novel agents – special reference to minimal residual disease, stem cell mobilization and

drug sensitivity testing

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Canthia 100 Auditorium, Kuopio, on Friday, October 28^th 2016, at 12 noon

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 376

Department of Medicine, Institute of Clinical Medicine, School of Medicine, Faculty of Health Sciences

University of Eastern Finland Kuopio

2016

(5)

Grano Jyväskylä, 2016

Series Editors:

Professor Tomi Laitinen M.D., Ph.D.

Institute of Clinical Medicine, Clinical Radiology and Nuclear Medicine Faculty of Health Sciences

Professor Hannele Turunen, 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. (pharmacy) School of Pharmacy

Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-‐‑70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-‐‑952-‐‑61-‐‑2260-‐‑1 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2261-‐‑8

ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714

ISSN-‐‑L: 1798-‐‑5706

(6)

Author’s address: Department of Medicine/Kuopio University Hospital

PL 100, 70029 KYS

KUOPIO

FINLAND

Supervisors: Professor Kari Remes, M.D., Ph.D.

Department of Hematology/Turku University Hospital University of Turku

TURKU FINLAND

Docent Veli Kairisto, M.D., Ph.D.

Department of Clinical Chemistry/TYKSLAB/Turku University Hospital University of Turku

TURKU FINLAND

Docent Esa Jantunen, M.D., Ph.D.

Department of Medicine/Kuopio University Hospital University of Eastern Finland

KUOPIO FINLAND

Reviewers: Docent Eeva-‐‑Riitta Savolainen M.D., Ph.D.

NordLab, Oulu University Hospital

Department of Clinical Chemistry, University of Oulu OULU

FINLAND

Docent Erkki Elonen M.D., Ph.D.

Helsinki University Central Hospital Comprehensive Cancer Center Helsinki Department of Hematology

HELSINKI FINLAND

Opponent: Assoc.Professor Hareth Nahi, M.D., Ph.D.

Haematology Centre Karolinska, M54 Karolinska University Hospital, Huddinge STOCKHOLM

SWEDEN

(7)

(8)

Silvennoinen, Raija

Multiple myeloma treatment in the era of novel agents – special reference to minimal residual disease, stem cell mobilization and drug sensitivity testing

University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences 376. 2016. 109 p.

ISBN (print): 978-‐‑952-‐‑61-‐‑2260-‐‑1 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2261-‐‑8 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706

ABSTRACT

The introduction of novel agents and autologous stem cell transplantation (ASCT) has improved overall survival (OS) in multiple myeloma (MM). However, the majority of patients will relapse or progress in 2-‐‑3 years and proceed to end-‐‑stage MM in 5-‐‑6 years.

Several novel agents have been launched for the treatment of relapsed/refractory myeloma patients in recent years. The relapse develops from the original clonal minimal residual disease (MRD) even after complete remission (CR) or from a new evolutional clone.

Multiparameter flow cytometry (MFC) and allele-‐‑specific real-‐‑time quantitative polymerase chain reaction (ASO RQ-‐‑PCR) have both been investigated in detecting MRD.

In two prospective studies including a total of 127 newly diagnosed (ND) MM patients responses after 2-‐‑drug (bortezomib + dexamethasone) or 3-‐‑drug (bortezomib + lenalidomide + dexamethasone) induction treatment followed by ASCT, and lenalidomide maintenance in the latter, were studied. Molecular remission was assessed and immunoelectrophoresis (IFE) was compared with the serum free light chain (FLC) ratio, MFC and ASO RQ-‐‑PCR in near CR/CR patients. The role of a conventional mobilization regimen, cyclophosphamide + G-‐‑CSF (arm A) was compared with G-‐‑CSF alone (arm B) in the randomized mobilization substudy after 3-‐‑drug induction. In the translational prospective study applicability of ex-‐‑vivo drug sensitivity and resistance testing (DSRT) was evaluated in 50 bone marrow samples from 43 NDMM and relapsed MM patients. The results were correlated with findings in cytogenetics (fluorescent in situ hybridization) and especially aimed to identify new treatment modalities for high-‐‑risk patients.

In the first trial the molecular remission rate was 28%, whereas MFC remission rate was 38%. These patients had longer progression-‐‑free survival. Neither IFE nor the FLC ratio was predictive for MFC-‐‑ or PCR -‐‑negativity. In the mobilization study there was no statistically significant differences between the study arms. Arm A was superior in terms of one of the secondary end points, the median number of collections needed to reach the yield of 3 x 10⁶/kg. DSRT allowed stratification of patients into four different drug sensitivity groups ranging from group I having a wide sensitivity to targeted therapies to group IV with a resistance to almost all tested drugs. Several signaling pathway inhibitors showed sensitivity for t(4;14) patients but for del(17p) patients BCL2 -‐‑and histone deacetylase inhibitors only showed some ex vivo sensitivity.

PCR was applicable in all patients tested and was more sensitive than the other response assessment methods. With the defined collection target the mobilization arms were comparable after lenalidomide-‐‑based induction. DSRT appears to be a promising new method to find useful drugs for the treatment of relapsed or refractory MM patients.

National Library of Medicine Classification: WH 540, QY 95, QY 102, QU 550.5.P6, QY 265, WH 380, QZ 267, QZ 203, WB 365

Medical Subject Headings: Multiple Myeloma; Neoplasm, Residual; Flow Cytometry; Polymerase Chain Reaction; Immunoelectrophoresis; Hematopoietic Stem Cell Mobilization; Bortezomib; Lenalidomide; Drug Resistance

(9)

(10)

Silvennoinen, Raija

Multippelin myelooman hoito uusien lääkkeiden aikakaudella -‐‑ jäännöstaudin merkitys, omien kantasolujen mobilisaatio ja myelooman lääkeherkkyys

Itä-‐‑Suomen yliopisto, terveystieteiden tiedekunta

Itä-‐‑Suomen yliopiston julkaisuja. Terveystieteiden tiedekunnan väitöskirjat 376. 2016. 109 s.

ISBN (print): 978-‐‑952-‐‑61-‐‑2260-‐‑1 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2261-‐‑8 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706

TIIVISTELMÄ

Myeloomapotilaiden elinaika on pidentynyt uusien lääkkeiden ja omien kantasolujensiirron tuella annettavan korkea-‐‑annossolunsalpaajahoidon ansiosta. Valta-‐‑

osalla potilaista tauti kuitenkin uusiutuu 2-‐‑3 vuoden kuluessa edeten 5-‐‑6 vuodessa myöhäisvaiheeseen. Täydellisenkin remission jälkeen tauti uusiutuu joko alkuperäisen myeloomasolukon lisääntyessä tai uuden muuntuneen myeloomasolukon kautta. Useita uusia lääkkeitä on viime vuonna hyväksytty myeloomapotilaiden hoitoon. Myelooman jäännöstautia tutkivia menetelmiä ovat monivärivirtaussytometria (immunofenotyypitys) ja molekyyligeneettinen tutkimus, alleeli-‐‑spesifinen reaaliaikainen kvantitatiivinen polymeraasi ketjureaktio (ASO-‐‑RQ-‐‑PCR).

Kahdessa prospektiivisessa tutkimuksessa 127 uudella myeloomapotilaalla tutkittiin hoitovasteet kahden (bortetsomibi, deksametasoni) ja kolmen lääkkeen hoitoyhdistelmän (lenalidomidi, bortetsomibi, deksametasoni) ja omien kantasolujensiirron tuella annetun korkea-‐‑annoshoidon jälkeen. Jälkimmäisessä tutkimuksessa potilaat saivat lenalidomidi-‐‑

ylläpitohoitoa. Ensimmäisessä osatyössä analysoitiin hoidon molekylaarinen vaste.

Toisessa osatyössä verrattiin keskenään immunoelektroforeesin tulosta, seerumin vapaita kevytketjuja, monivärivirtaussytometriaa ja ASO-‐‑RQ-‐‑PCR -‐‑tutkimusta. Kolmas osatyö vertasi satunnaistetussa tutkimuksessa omien kantasolujen keräystä edeltäviä mobilisaatiohoitoja: haara A) perinteinen CY + G-‐‑CSF ja haara B) G-‐‑CSF. Neljäs osatyö, translationaalinen prospektiivinen tutkimus, selvitti ex vivo lääkeherkkyystutkimuksen käytettävyyttä 43 myeloomapotilaan luuydinnäytteen soluissa. Tuloksia verrattiin myeloomasolujen kromosomipoikkeavuuksiin ja pyrittiin identifioimaan uusia potentiaalisia lääkkeitä korkean riskin potilaille.

Molekylaarisen remission saavutti 28% potilaista, virtaussytometrialla 38%. Näillä potilailla oli pidempi tautivapaa elinaika kuin huonomman hoitovasteen saavuttaneilla.

Immunoelektroforeesin tai seerumin vapaat kevytketjut -‐‑tutkimuksen tulos ei ollut luotettava ennakoimaan molekylaarista tai immunofenotyyppistä remissiota. Kantasolujen mobilisaatio-‐‑tutkimuksessa tutkimushaarojen välillä ei ollut eroa ensisijaisen päätetapahtuman (keräystulos ≥ 3 x 10⁶/kg) suhteen. Tutkimushaarassa A tulos saavutettiin kuitenkin vähemmillä keräyskerroilla. Lääkeherkkyysnäytteiden tulosten perusteella potilaiden solut pystyttiin jakamaan neljään ryhmään herkistä hyvin vastustuskykyisiin.

Korkean riskin, t(4;14), potilaiden myelomasoluihin näyttivät tehoavan useat signaalireittien estäjät, mutta kromosomi 17 lyhyen haaran puutoksen omaaviin soluihin lähinnä vain BCL2-‐‑ ja histonideasetylaasiestäjät.

Jäännöstautitutkimuksessa PCR-‐‑aluke pystyttiin muodostamaan kaikille analysoitaville potilaille ja PCR oli herkin jäännöstaudin osoittaja. Satunnaistetun tutkimuksen kanta-‐‑

solujenkeräyshaarat olivat keräystavoitteen suhteen verrannollisia. Lääkeherkkyystutki-‐‑

mus on lupaava uusi menetelmä haettaessa uusia lääkkeitä hoitoresistenttiin ja korkean riskin myeloomaan.

Yleinen Suomalainen asiasanasto: myelooma; uusiutuminen; kantasolujen siirto; hoitomenetelmät;

lääkehoito; hoitovaste; lääkeresistenssi

(11)

(12)

Acknowledgements

The work on this thesis was started in 2008 at the Department of Internal Medicine of Tampere University Hospital. The task force for multiple myeloma in Finland, the Finnish Myeloma Group (FMG) started in 2009. Work on this study continued at the Department of Medicine, Kuopio University Hospital 2012 onwards. In 2013 the Finnish Myeloma Group started collaboration with the Institute for Molecular Medicine Finland (FIMM) and the door for translational research in multiple myeloma opened.

I send my sincere gratitude to the former Head of Department of Internal Medicine, Tampere University Hospital, Docent Kari Pietilä, for supporting me when I was starting the FMG-‐‑MM01 study. I express my gratitude to Professor Markku Laakso, Institute of Clinical Medicine, University of Eastern Finland, for his support to my research since 2012.

I thank the former Head of Department of Medicine, Kuopio University Hospital, Docent Seppo Lehto, for supporting me when I started my second investigator initiated study FMG-‐‑MM02. I am grateful to Kirsi Luoto, Maire Anttonen, Arja Halkoaho and Tuomas Selander at the Science Services Center of Kuopio University Hospital. I thank you for always having time for my projects and me.

I sincerely thank my principal supervisor Professor Kari Remes from Turku University Hospital and the University of Turku. You were brave enough to take on the difficult task to educate a clinician to an investigator. You showed me how to think in-‐‑depth and critically about clinical studies with a clear practical view still in mind. You have challenged me with straight questions, but always softened our collaboration with your fascinating humor.

I express my sincere gratitude to my second supervisor, Docent Veli Kairisto, who with the great patience has had time for my never-‐‑ending questions regarding ASO-‐‑PCR. You have pointed out the main questions of molecular genetics to be answered during my studies. I hope that our collaboration will continue at the next generation level.

My third supervisor, Docent Esa Jantunen, will receive my deepest gratitude for his tireless support during my studies. I would like to say that you have saved my mind. While writing my thesis I was suddenly involved with the development of new clinical studies in multiple myeloma and with a much wider network. With your support I have been able to maintain my balance. I admire how you can take a mix of results and data to a clear conclusion. During the last busy weeks your help has been most valuable.

I am deeply grateful for the reviewers of this thesis, Docent Eeva-‐‑Riitta Savolainen and Docent Erkki Elonen, whose valuable comments helped me to improve the work. I wish to thank Docent David Laaksonen for linguistic revision. The multiparameter flow cytometry part of this work would never have been possible without the support and intelligence of Docent Tarja-‐‑Terttu Pelliniemi, who sometimes had to read my thoughts for lack of adequate content to my questions. I am honored to have the President of Nordic Myeloma Study Group, Assoc. Professor Hareth Nahi, as my opponent.

“The sound of science is clear here” encouraged me in the darkest moments. This was a comment of one reviewer after the first review of the second paper. I would like to thank all voluntary anonymous colleagues in scientific communities who offer their free time for reviewing manuscripts.

I have been privileged to be the first chairperson of the Finnish Myeloma Group. In our working group we have had several workshops concentrating on diagnostic guidelines and the development of treatment for myeloma patients in Finland. Meanwhile, in the background, our clinical and laboratory coinvestigators and nurses have treated study patients and these studies have matured. I give my deepest gratitude to all my colleagues and all study nurses and hematological nurses who have been involved in many different

(13)

ways in this research collaboration. The Finnish myeloma patients and their families need an extremely warm thanks for participating in these studies and donating blood and bone marrow for this research, which in all circumstances does not come for their benefit, but hopefully will help the next generation of patients. I am honored by your altruism and commitment.

Professor Kimmo Porkka receives my special thanks for his ultimate support at every turn of my hospital and research work. He has always been on line to help show me the way to overcome difficulties. Without him we would not have any future for hematological research in Finland. I give my warm gratitude to Caroline Heckman and Mamun Majumder, my co-‐‑investigators at FIMM. You have always been available and helped me with this research. In addition, I would like to thank Tuija Lundán for privat lessons on PCR methods and for interpreting the results.

I am very grateful for Ilona Siljander and Jaakko Valtola. Ilona has shown me the way to understand statistics and Jaakko has been my support. I am looking forward with pleasure to our future collaboration. I thank my nearest neighbor Hannu for saving me from dead-‐‑

ends with the files. When work and research are major parts of your life, I have been happy to find friends there. I am forever thankful to Taru, my soulmate, for her inspiring presence and great support in my life.

For my childhood family I would like to give my deepest gratitude. I thank my deceased parents for showing me how the diligence, hard work and not giving up can lead to results.

I am privileged to have spent my childhood in the far countryside with four siblings. I thank all my siblings for sharing our happiness and sorrows during the whole of our lives.

My husband Seppo and I have shared countless adversities, but just as many happy times in our lives. For this part I had to go alone, but when I had time to look around you were always somewhere nearby. You made all of this possible taken responsibility for the daily routines. Sinna, my lovely dog, you earn a warm hug for trying to teach me how to be noiseless and wait with patience. These lessons clearly will go on.

I am most deeply grateful in my life to our sons Jouni and Jonne. All what makes me happy walks in the door with you. During our entire lives together, but in recent times when I have most desperately needed it you have lightened my days with your irresistible, special sense of humor. You fill my life with happiness, gratitude and joy.

I thank Tampere University Hospital for the Research Funding (Grant9M097) and the Research Committee of Kuopio University Hospital Catchment Area for State Research Funding (project 5101424). I thank the Finnish Hematology Association and Research Foundation of Blood Disease for the grants. I also wish to thank Janssen and Celgene for Research Funding.

Kuopio 18^th September 2016 Raija Silvennoinen

(14)

List of the original publications

This dissertation is based on the following original publications:

I Silvennoinen R, Kairisto V, Pelliniemi T-‐‑T, Putkonen M, Anttila P, Säily M, Sikiö A, Opas J, Penttilä K, Kuittinen T, Honkanen T, Lundán T, Juvonen V, Luukkaala T, Remes K. Assessment of molecular remission rate after bortezomib plus dexamethasone induction treatment and autologous stem cell transplantation in newly diagnosed multiple myeloma patients. Br J Haematol 160: 561-‐‑564, 2013.

II Silvennoinen R, Lundán T, Kairisto V, Pelliniemi T-‐‑T, Putkonen M, Anttila P, Huotari V, Mäntymaa P, Siitonen S, Uotila L, Penttilä T-‐‑L, Juvonen V, Selander T, Remes K. Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-‐‑PCR in multiple myeloma. Blood Cancer J 4: e250, 2014.

III Silvennoinen R, Anttila P, Säily M, Lundan T, Heiskanen J, Siitonen T, Kakko S, Putkonen M, Ollikainen H, Terävä V, Kutila A, Launonen K, Räsänen A, Sikiö A, Suominen M, Bazia P, Kananen K, Selander T, Kuittinen T, Remes K, Jantunen E.

A randomized phase II study of stem cell mobilization with cyclophosphamide + G-‐‑CSF or G-‐‑CSF alone after lenalidomide-‐‑based induction in multiple myeloma.

Bone Marrow Transplant 51: 372-‐‑376, 2016.

IV Majumder M.M, Silvennoinen R, Anttila P, Tamborero D, Eldfors S, Yadav B, Karjalainen R, Kuusanmäki H, Lievonen J, Parsons A, Suvela M, Jantunen E, Porkka K, Heckman C.A. Functional screening of multiple myeloma to develop precision treatment strategies and for outcome prediction. Submitted.

The publications were reprinted with the permission of the copyright owners.

(15)

(16)

Abbreviations

AKT Aktin

ASCT Autologous stem cell trans-‐‑

plantation

ASO Allele specific oligonucleotide BCL B-‐‑cell lymphoma

Β2-‐‑miglo Beta-‐‑2 -‐‑microglobulin

BM Bone marrow

BMSC Bone marrow stem cell BRAF Proto-‐‑oncogen B-‐‑Raf

BZM Bortezomib

CD Cluster of differentiation CDK(i) Cyclin-‐‑D-‐‑kinase (inhibitor) CFZ Carfilzomib

CR Complete remission CT Computerized tomography CXCR4 C-‐‑X-‐‑C chemokine receptor

type 4

CY Cyclophosphamide

Dex Dexamethasone

DNA Deoxyribonucleic acid DSRT Drug sensitivity and

resistance testing DSS Drug sensitivity score EBMT European Society for Blood

and Marrow Transplantation EGF Epidermal growth factor ECOG Eastern Cooperative

ERK Extracellular regulated kinase FGFR Fibroblast growth factor

receptor

FIMM Institute for Molecular Medicine Finland FISH Fluoresence in situ

hybridization

FMG Finnish Myeloma Group G-‐‑CSF Granulocyte-‐‑colony

stimulating factor GEP Gene expression profile GVHD Graft-‐‑versus-‐‑host disease FLC Free light chain

HDAC(i) Histonedeacetylase (inhibitor) HDMEL High-‐‑dose melphalan

HR High-‐‑risk

HSP90 Heat-‐‑shock protein 90

IFE Immunofixation

electrophoresis iFISH Interphase FISH

IF Immunofixation

IFN Interferon

IgA Immunoglobulin A IgD, IgE Immunoglobulin D, -‐‑E IGF-‐‑1R Insulin-‐‑like growth factor 1

receptor

IgG Immunoglobulin G

(20)

IGHV IgH variable

IgK Immunoglobulin kappa IgL Immunoglobulin lambda IgM Immunoglobulin M

IL Interleukin

IMiD Immunomodulating drug IRd Ixazomib, lenalidomide,

dexamethasone

ISS International Staging System IMWG International Myeloma

Working Group

κ kappa

Kd Carfilzomib, dexamethasone KRAS Kirsten rat sarcoma viral

oncogene homolog

KRd Carfilzomib, lenalidomide, dexamethasone

λ Lambda

LC Light chain

LDH Lactate dehydrogenase Len Lenalidomide (Revlimid^®) LenDex Lenalidomide,dexamethasone LR Low-‐‑risk

MFC Multiparameter flow cytometry

MGUS Monoclonal gammopathy of undetermined significance MEK Mitogen-‐‑activated protein

kinase

MEL Melphalan

MM Multiple myeloma

MMSET Multiple myeloma SET domain

MNC Mononuclear cell

Mo Months

MoAb Monoclonal antibodies MolR Molecular remission MP Melphalan, prednisolone MPR Melphalan, prednisolone,

lenalidomide

MPT Melphalan, prednisolone, thalidomide

MPV Melphalan, prednisolone, bortetsomib

MR Minimal response MRD Minimal residual disease mRNA Messenger ribonucleic acid MRI Magnetic resonance imaging mTOR Mammalian target of

rapamycin

MYC V-‐‑myc avian myelocytoma-‐‑

tosis viral oncogene homolog nCR Near complete remission ND Newly diagnosed NF-‐‑kB Nuclear factor-‐‑kappa B NGS Next generation sequencing NK Natural killer

NMSG Nordic Myeloma Study Group

NR Not reported

NRAS Neuroblastoma RAS viral oncogene homolog

(21)

ORR Overall response rate OS Overall survival PB Peripheral blood PC Plasma cell

PCD Plasma cell disease PCR Polymerase chain reaction PD Progressive disease PD-‐‑1 Programmed death-‐‑1 PD-‐‑L1 Programmed death ligand-‐‑1 PET Position emission

tomography

PFS Progression-‐‑free survival PI Proteasome inhibitors PI3K Phosphoinositide 3-‐‑kinase

PN Polyneuropathy

PR Partial remission

RAF Raf proto-‐‑oncogene serine/

threonine protein kinase RAS Retrovirus-‐‑associated DNA

sequences

RD Lenalidomide (Revlimid^®) and dexamethasone RB1 Retinoblastoma 1 RNA Ribonucleic acid RR Relapsed/refractory RVD Lenalidomide, bortezomib,

dexamethasone RQ-‐‑PCR Real-‐‑time quantitative

polymerase chain reaction SC Stem cell

sCR Stringent complete remission

SCT Stem cell transplantation SD Stable disease

sDSS Selective drug sensitivity score

S-‐‑FLC Serum free light chain SMM Smoldering multiple

myeloma SR Standard-‐‑risk

T Thalidomide

TD Thalidomide and dexamethasone TP53 Tumor protein 53 TRAF3 TNF receptor-‐‑associated

factor 3

TTNT Time to next treatment TTP Time to progression VAD Vincristine, doxorubicin,

dexamethasone

VCD Bortezomib (Velcade^®), cyclophosphamide, dexamethasone

VelDex Bortezomib (Velcade^®), dexamethasone

VGPR Very good partial response VMP Bortezomib (Velcade^®),

melphalan, prednisone VT Bortezomib (Velcade^®) and

thalidomide

VTD Bortezomib (Velcade^®) thalidomide, dexa-‐‑

methasone

(22)

(23)

1 Introduction

The overall survival (OS) of multiple myeloma (MM) patients has improved significantly with the combination of novel drugs and autologous stem stell transplantation (ASCT) (1, 2, 3). The majority of patients respond to the first-‐‑line treatment usually achieving a median progression-‐‑free survival (PFS) of 2-‐‑3 years. Inevitable subsequential progression will occur, however, resulting in treatment-‐‑resistant end-‐‑stage disease with cytopenias, infections and poor prognosis (4).

The era of novel agents began with the first publication in 1999 on the first immunomodulating agent (IMiD) thalidomide (5, 6), continued with the launching of the first proteasome inhibitor (PI), bortezomib in 2004, followed by the second-‐‑generation IMiD lenalidomide in 2008 and the second-‐‑generation PI carfilzomib in 2012. The third IMiD pomalidomide was approved by European Medicines Agency in 2013 and carfilzomib in 2015. The next important group of new drugs in MM are the monoclonal antibodies (MoAb). The CD38+ MoAb, daratumumab, has showed efficacy even as a monotherapy in resistant MM (7, 8) and another MoAb, elotuzumab, combined with lenalidomide, has confirmed the usefulness of synergy in MM therapy (9).

Numerous new small molecules and cell signaling pathway inhibitors are now in the pipelines and in preclinical and clinical studies, giving hope in the future for improved treatment response with targeted therapy in MM. We have a challenge to combine these new drugs in the most feasible way in different lines of treatment in addition to clarify their role in consolidation and maintenance treatment. This challenge will be best met by better understanding the heterogenous nature of MM between patients and within individual patient (10). Due to clonal heterogeneity the treatment would be better targeted if we knew which clone or clones were responsible for each progression phase of disease.

With the higher quality responses in the era of novel agents it has became more important to explore the differences between novel therapies at the minimal residual disease (MRD) level. An applicable, practical, sensitive and economic method for MRD assessment for clinical studies and further for routine use is needed. Multiparameter flow cytometry (MFC) and allele-‐‑specific oligonucleotide real time quantitative polymerase chain reaction (ASO RQ-‐‑PCR) have been studied in this setting (11, 12). MFC has been shown to be more applicable in practice. MFC still needs standardization, however, and the EuroFlow Consortium is working on that (13, 14). ASO RQ-‐‑PCR is well standardized, but is more time-‐‑consuming and laborious and its applicability is based on the success rate of probe design (11, 12). Nonetheless, when successful it is usually at least one logarithm more sensitive than 4-‐‑colour MFC (11).

ASCT has sustained its role as standard therapy in transplant-‐‑eligible patients.

Mobilization of autologous stem cells needs to be re-‐‑evaluated after induction with novel agents, which lead to a higher proportion of good responses before ASCT. The best mobilization regimen yielding sufficient graft number without major long-‐‑term harmful effects should be evaluated.

We conducted two prospective national clinical studies for newly diagnosed MM patients with novel drugs. The first study included a 2-‐‑drug combination as induction followed by ASCT and the second study a 3-‐‑drug combination followed by ASCT and maintenance. MRD assessment with MFC and ASO RQ-‐‑PCR was systematically included in both studies. In the second study the earlier mobilization standard (low-‐‑dose cyclophosphamide + G-‐‑CSF) was compared with G-‐‑CSF alone in a randomized setting.

The jungle of second-‐‑generation novel agents and small molecules is thick, and we need tools to predict the efficacy of these expensive drugs. We designed the ex-‐‑vivo drug sensitivity and resistance testing (DSRT) study to evaluate its applicability in MM bone

(24)

marrow samples, and combined the results with fluorescence in situ hybridization (FISH) findings. The aim was to identify new possible innovative therapies for high-‐‑risk MM patients who have the poorest prognosis.

(25)

2 Review of the Literature

2.1 MULTIPLE MYELOMA 2.1.1 Epidemiology and risk factors

Multiple myeloma (MM) is the second most common hematological cancer after lymphomas in the Western countries with the annual incidence of 4-‐‑6/100 000 (15). It represents 1% of all cancers and 10% of hematological cancers. MM occurs more commonly in males than in females: in the Finnish Cancer Registry the incidences are 3.4/100 000 and 2.6/100 000, respectively (16). The annual incidence has been 370 new MM cases in years 2009−2013 (including all plasma cell disorders) and 255 deaths due to myeloma have been registered annually between 2009−2013 (16). The age-‐‑adjusted mortality was 3.6/100 000 in Finland (16). In total, 1446 patients were living with the diagnosis at the end of year 2013 according to the Finnish Cancer Registry (16). The median age in MM patients is 65−70 years at diagnosis, but about 10% of patients are less than 55 years of age (16). The median OS has increased during the last two decades mostly in patients less than 65 years of age from 3−4 to 7−8 years due to novel drugs combined to ASCT (17).

The primary cause of myeloma is unknown, but increasing age, male gender, familial background and past history of monoclonal gammapathy of unknown significance (MGUS) have been established as risk factors (18-‐‑19). In addition, environmental exposure to nuclear radiation, petroleum products and pesticides is thought to increase the risk (20-‐‑21). It is overrepresented in farmers, wood and leather manufacturers (20-‐‑21). A family history of other chronic B-‐‑cell malignancies like smoldering multiple myeloma (SMM) and Waldenström disease, has also been noted as a risk factor for myeloma (18, 22). The risk of MGUS or MM is increased 3-‐‑4.25 fold in the first-‐‑degree relatives (18, 22, 23, 24, 25, 26).

Germinal genetic mutations seem to be localized in chromosome 1q and 4q loci (22).

Grass et al. found that hyperphosphorylated form of Paratarg-‐‑7 (P-‐‑7) was a consistent finding in familial and sporadic MGUS and MM, suggesting its role in the pathogenesis of inherited forms (27). Obesity and immune dysfunction have also been proposed to increase MM risk. Several factors associated with obesity have been linked to the risk, like increased oxidative stress, alterations in immunological and metabolic response, and endogenous hormone levels (sex steroids, insulin and insulin-‐‑like growth factor-‐‑1) (18, 28).

2.1.2 Origin of multiple myeloma

The healthy counter-‐‑part of the malignant plasma cell (PC) is postgerminal center antibody producing mature PC representing terminal differentiation of B lymphocytes. These are needed for production a wide range of immunoglobulins, when exposed to different antigens during a lifetime. The contact of an antigen with a virgin B lymphocytes induces them to either proceed to develop into low-‐‑affinity plasma cells or to move into the germinal center. In germinal centers B-‐‑cells undergo affinity maturation of their antibody through somatic hypermutations. This means a rapid proliferation and differentiation of B-‐‑

cells for selected antibodies. During the development of B-‐‑cells into the antibody secreting mature plasma cells they go through the immunoglobulin heavy chain class switch process, which enables the production of different immunoglobulins, mostly immunoglobulins A, G and M. This process requires several new DNA rearrangements and it makes possible for the necessary translocations to appear (29). The functionality of these antibodies is confirmed by class switch recombination (isotype switching), where one switch region is replaced by another allowing the production of different immunoglobulin isotypes (10, 30).

Early genetic mutations occur at the pre-‐‑B stage and later mutations and genetic events at

(26)

There is a suggestion that MM cancer stem cells with self-‐‑renewal capacity both initiate and propagate MM, and are responsible not only for the initial birth of MM, but also for the relapse and progression and ultimately drug resistance. These cells probably do not have one precise phenotype, but might be plastic and functionally bidirectional between non-‐‑

stem and stem-‐‑like compartments (32, 33, 34). Therapy may induce the regeneration of clones, which are able to survive in an inflammatory and hypoxic microenvironment (33, 34, 35). The immunophenotypes of cell populations having such plasticity have been suggested to be clonotypic B cell (CD19⁺CD138^-‐‑), pre-‐‑PC (CD19-‐‑CD38⁺⁺, CD319⁺,CD138^-‐‑) and MM cell (CD38⁺⁺, CD138⁺) (33, 36). Attempts have been made to identify myeloma stem cell (SC) reservoirs to eradicate of minimal residual disease (37).

2.1.3 MGUS transformation to multiple myeloma

Monoclonal gammapathy of undetermined significance (MGUS) phase precedes MM probably in all cases, and it will convert to MM with an annual incidence of 1% (38, 39).

Elevated serum monoclonal protein (M-‐‑component) > 30g/l (a criteria for smoldering myeloma), an abnormal serum free light chain (sFLC) ratio and IgA or IgD subtype are risk factors for progression to MM (40, 41). The percentage of aberrant PCs as assessed with multiparameter flow cytometry (MFC) ≥ 95% of all plasma cells also means increased risk for active myeloma (42, 43). The latest update regarding the risk of smoldering MM (SMM) to proceed to active MM in two next years concluded that an sFLC ratio of involved to uninvolved light chain equal to or greater than 100 or BM plasma cell infiltration of more than 60% or bone-‐‑specific MRI findings to be an indication for treatment (44).

2.1.4 Genetics of multiple myeloma

Myeloma cells have a very instable genome with a heterogeneous mutation profile (Tables 1 and 2a, 2b). MM cells have more mutations than acute leukemias, and are closer to the number of mutations found in solid tumours (45, 46, 47, 48). Standard cytogenetic techniques found already in 1985 that myeloma cells have several karyotypic aberrations like monosomies, trisomies, deletions and translocations (49, 50, 51, 52). Due to failure to stimulate myeloma cells to divide in vitro leads to inappropriate and insufficient material for metaphase analysis with negative findings (53). About half of myeloma patients have a hyperdiploid genome with trisomies in the odd chromosomes 3, 5, 7, 9, 11, 15, 19 and 21.

The rest have nonhyperdiploid genome were the most common translocation is between the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 and one of the following oncogene partners: cyclin D1, t(11;14); cyclin D3, t(6;14); fibroblast growth factor receptor 3, t(4;14); v-‐‑maf avian musculoaponeurotic fibrosarcoma oncogene homolog, t(14;16) and V-‐‑maf musculoaponeurotic fibrosarcoma oncogene homolog B, t(14;20). In these translocations the IgH region with the strong enhancer gene moves beside to these oncogenes leading to overexpression of the targeted proteins, enabling the immortality of the cells (10, 21, 54, 55, 56). These translocations are reported to be already present in the MGUS phase, but in a lower proportion than in more advanced phases (SMM or MM) of the disease. Copy number abnormalities like del(17p), del(13), 1q gain or del 1p increase in SMM and MM patients (10, 21).

Secondary genetic events are activating mutations (Table 2a, 2b). They include pathways involved in proliferation, immortalization and apoptosis resistance of myeloma cells such as MYC, KRAS and NRAS, BRAF, PI3K, and AKT. Deletion (del) of oncosuppressors, such as del(17p) involving the locus of TP53 (tumor protein 53) is also important (10, 21, 54, 55, 56).

Genetic or nongenetic disruption of key regulators of plasma cell differentiation, XBP-‐‑1 (X-‐‑

box binding protein 1), PRDM1 (PR domain containing 1) and IRF-‐‑4 (interferon regulatory factor 4) have proved to be crucial for the pathogenesis of myeloma (57, 58). Generalized gene hypomethylation is associated with the transition between MGUS and MM and hypermethylation of specific target genes has been correlated with progression of MM into

(27)

plasma cell leukemia (10, 47, 21). There is, however, no consensus about the driver mutations in myeloma.

Chapman et al.were the first who performed whole genome/exome sequencing in MM patients and found five genes to be most commonly mutated in MM:, NRAS, TP53, DIS3 and FAM46C (45). The expanded sequencing results from the same group confirmed these results in addition to BRAF (59). Bolli et al. identified new candidate genes like ROBO1, SP140, LTB and EGFR1, and again highlighted the heterogeneity of the myeloma genome (60). KRAS, NRAS, DIS3, FGFR3, IRF4, FAM46C, BRAF, EGR1, TRAF3, LTB, TP53, HIST1H1E, MAX, CYLD and RB1 were the 15 significantly mutated genes found by Walker et al., and his group formulated the international staging system mutation score for the identification of high-‐‑risk patients (61).

Table 1. Primary genetic events in multiple myeloma (10, 54, 55, 56, 62)

Primary genetic events Genes Percentage of tumours Risk

IGH translocations

t(4;14) (p16;q32) FGFR3 and MMSET 11-15 High

t(6;14) (p21;q32) CCND3 <1 Standard

t(11;14)(q13;q32) CCND1 14-16 Standard

t(14;16)(q32;q23) MAF 3-5 High

t(14;20)(q32;q12) MAFB 1.5-2 High

Hyperdiploidy

(chromosomal trisomy)

Chromosome Genes

3, 5, 7, 9, 11, 15, 19 and 21 45-57 Standard

Table 2a. Secondary genetic changes in multiple myeloma

Chromosomes Genes Percentage

of tumors Risk

Secondary translocations

t(8;14) MYC 1 High

Gains

1q CKS1B and ANP32E 40 High

12p LTBR <1

17q Deletions

1p CDKN2C, FAF1 and FAM46C 30 High?

6q 33

8p TRAIL-R1 and TRAIL-R2 25

11q BIRC2 and BIRC3 7

13 RB1 and DIS3 45

14q TRAF3 38

16q CYLD and WWOX 35

17p TP53 8 High

(28)

Table 2b. Secondary genetic changes in multilple myeloma

Chromosomes Genes Percentage of tumors

Epigenetic event

Global hypomethylation Genome-wide methylation arrays

(MGUS to MM) and gene-specific hypermethylation

(MM to PC leukemia)

Molecular hallmarks

G1/S abnormality CDKN2C

RB1 3

CCND1 3

CDKN2A

Proliferation NRAS 21

KRAS 28

BRAF 5

MYC 1

Resistance to apoptosis PI3k, AKT

NF-kB pathway TRAF3 3

CYLD 3

I-kB Abnormal localization/bone disease DKK1

FRZB, DNAH5 8

Abnormal plasma cell differentiation XBP1 3

BLIMP1 (PRDM1) 6

IRF4 5

Abnormal DNA repair TP53 6

MRE11A 1

PARP1

RNA editing DIS3 13

FAM46C 10

LRRK2 5

Epigenetic abnormalities KDM6A 10

MLL 1

MMSET 8

HOXA9, KDM6B

ANP32E, acidic leucine-rich phosphoprotein 32 family, member E; BIRC, babuloviral IAP repeat- containing protein; BLIMP1, B lymphocyte-induced maturation protein 1; BRAF, proto-ongocen B Raf; CCND, cyclin D; CDKN, cyclin-dependent kinase inhibitor; CKS1B, CDC28 protein kinase 1B;

CYLD, cylindromatosis; DIS3, DIS3 homolog; DKK1, dickkoppf1; DNAH, dynein, axonemal, heavy chain; DNMT3A, DNA methyltransferase 3A; FAF1, FAS-associated factor 1; FAM46C, family with sequence similarity 46, member C; FRZB, frizzled-related protein; HOXA9, homeobox A9; IGH, immunoglobulin heavy chain; I-κB, inhibitor of nuclear factor-κB; IRF4, interferon regulatory factor 4; KDM, lysine demethylase; LRRK2, leucine-rich repeat kinase 2; LTBR, lymphotoxin beta receptor;

MAF, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog; MAFB, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B; MLL, mixed-lineage leukemia; MMSET, multiple myeloma SET domain; MRE11A, meiotic recombination 11A; NF-κB, nuclear-factor-κB;

PARP1, poly (ADP-ribose) polymerase 1; PRDM1, PR domain zinc finger protein 1; RB1, retinoblastoma 1; TRAF3, tumour necrosis factor receptor-associated factor 3; TRAIL-R1, tumor necrosis factor receptor superfamily member 10A; WWOX, WW domain-containing oxidoreductase;

XBP1, X box-binding protein 1

Multiple myeloma treatment in the era of novel agents : special reference to minimal residual disease, stem cell mobilization and drug sensitivity testing / Raija Silvennoinen

uef.fi

Dissertations in Health Sciences

RAIJA SILVENNOINEN

MULTIPLE MYELOMA TREATMENT IN THE ERA OF NOVEL AGENTS

RAIJA SILVENNOINEN

Multiple myeloma treatment in the era of novel agents – special reference to minimal residual disease, stem cell mobilization and

drug sensitivity testing

Multiple myeloma treatment in the era of novel agents – special reference to minimal residual disease, stem cell mobilization and

drug sensitivity testing

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the Literature