Pathogenesis of multiple
myeloma
Doc. RNDr. Sabina Ševčíková, Ph.D.
Babak myeloma group
ÚPF LF MU
Monoclonal gammopathies
 Abnormal proteins in serum or urine
 These proteins produced by a clone of plasma or
lymphoid cells
 MGUS
 Multiple myeloma
 Plasma cell leukemia
 Primary amyloidosis
 Solitary plasmocytoma
 Waldenström macroglobulinemia
World Health
Organization
Adam, 2011
Hematopoesis
Plasma cells
 Cells that produce antibodies
 Usually around 5% in the bone marrow
 In MM, they are malignant
 Produce monoclonal immunoglobulin= paraprotein
Antibodies
 To find and destroy foreign objects in the organism
 Eliminate pathogen
 IgG, IgA, IgM, IgE and IgD
 IgG – only one that enters placenta
 IgA – produced in mucous membranes, protects entry to
body
 IgM – produced first after infection – body protection
in the first few days
 IgE – parasites and allergic reactions
 IgD –unclear, co-expressed on surface of mature B cells
Basic structure of immunoglobulin
MGUS
 Precancerosis
 In people over 50 – 3-4%
 Risk of progression into MM > 1% every year
Multiple myeloma
 Second most common hematological malignancy
 10% of hematological malignancies
 Median age at diagnosis 65
 Incidence 5.7/100 000
 More common in men
 Infiltration of bone marrow by malignant PC
 Osteolytic lesions
 Presence of M-Ig in serum and/or urine
Hájek, 2012
Anderson, 2011
PCL
 Most aggressive monoclonal gammopathy
 Incidence 0.04/100 000
 More than 20% of circulating PC and more than 2x109/L
 Median age at diagnosis - 52
 Median survival 1.5 years or shorter
Primary amylodosis
 Amyloid deposits in organs
 Production of abnormal protein filaments
 Rare
 May be present together with MM
 Survival around 40 months
Solitary plasmocytoma
 Solitary lesion of abnormal PC
 Without bone marrow involvement
 Without osteolytic lesions
 Risk of progression into MM – 10% in 3 years
Waldenstrom macroglobulinemia
 Affects lymphocytoplasmoid cells (cells with
characteristics of both PC and lymphoid cells)
 Production of large amount of IgM
 Very rare
 May progress from IgM MGUS
Multiple myeloma
History of MM
Capasso, 2005
Male skull from the bronze age with MM characteristics
History of MM
 1844 – first documented case – Sarah Newbury
(Dr. Solly)
Kyle et Rajkumar, 2008
destruction of sternum fractures of bones destruction of femur
History of MM
 1845 – presence of proteins in urine of patients (Bence
Jones – Bence Jones protein)
 Kahler’s disease – Prague physician Otto Kahler
Kyle et Rajkumar, 2008
Pathogenesis of MM
 Multistep transformation
Špička, 2005
Healthy bone marrow MM bone marrow
www.pathologyatlas.com
Multiple myeloma
the clinician’s viewpoint
(doc. Minařík FNOl)
Causes of MM
 Unknown but
 Decrease of immunity dependent on age
 Hormonal changes
 Chemicals
 Radiation
  changes leading to unstable genome of PC
Manifestation of MM
 1) bone marrow:
  ery  anemia
  leukocytes  decrease of immunity
  thrombocytes  bleeding
Manifestation of MM
 2) osteolytic lesions:
 Bone pain
 Weakening of bone structures
 Spontaneous fractures
 Calcium increase in serum
Manifestation of MM
 3) Defects in immunoglobulins - paraprotein:
 Hyperviscosity
 Accumulation in blood vessels
 Decrease in proper function of immunity
Diagnosis of MM
 Difficult, not specific (pain, weakness, repeated
infections, tiredness – similar to other diseases)
1) Number of MM cells in the BM
2) Presence of abnormal proteins in blood/urine
3) Typical bone changes
Diagnostic methods
 Blood and urine test – electrophoresis of proteins
 Sampling and analysis of bone marrow
 Imaging – X-rays, CT, MRI, PET-CT, bone density
Diagnostic methods - general
 Various indirect parameters:
 Blood count
 Calcium levels
 Kidney function
 Levels of antibodies
 Liver function
 Bone metabolism
Diagnostic methods- special
 Beta-2-macroglobulin
 Cytogenetics
 FACS
 MM cell proliferation
 Antiangiogenic cytokines
 Free light chain ratio
 Albumin
 Staging
Staging and course of disease
 3 stages of disease
 I-III – level of disease
 A/B kidney function
 Course – individual, varies
 From light form of disease to kidney failure, broken
vertebrae, immune deficiency, bleeding….
MM treatment
 Orange peel and opioids
 Chemotherapy
 Transplants
 Immunomodulatory drugs
 Proteasome inhibitors
Hájek, 2012
Anderson, 2011
MM prognosis
 Untreated patients survive 14 months
 Standard therapy – 3-4 years
 Transplants – 6-7 years
 New drugs – 5-year survival for more than 80% of pts
Hájek, 2012
Chemotherapy and transplants
 Treatment protocol Junior – Senior
 Melphalan
 Alkylating agent
 Prednisone
 Glucocorticoid – apoptosis of heme cells
 Transplants since 1957
 Autologous – up to 65, even tandem
 Allogeneic – in clinical trials
Hájek, 2012
Anderson, 2011
Treatment options in MM
 IMIDs
 Proteasome inhibitors
Treatment options in MM
 IMIDs
 Proteasome inhibitors
Thalidomide
 1953- Chemie Grünenthal
 1957- distribution
 Sedative
 Morning sickness
 Teratogenic properties
 Only tested on rats
 About 10 000 children born – only about 40% survived
 FDA - Dr. Francis Kelsey
Sedlaříková, 2012
White House Archive
Thalidomide
 1964 – Jason Sheskin – leprosy patient
 1993- Judah Folkman – angiogenesis in hematology
 1994 – Bart Barlogie - refractory MM, thalidomide
 Clinical study – 84 pts, 1/3 of pts response
 2006 – FDA – approval
 Unpleasant side effects - neuropathy
Sedlaříková, 2012
 Analogues of thalidomide – lenalidomide a pomalidomide
 Pleiotropic effect on MM:
 T-cell co-stimulation
 Antiangiogenic properties
 Anti-inflammatory properties
 Apoptosis and cell cycle progression
 Inhibition of MM and BM interactions
IMIDs immunomodulatory drugs
Sedlaříková, 2012
Treatment options in MM
 IMIDs
 Proteasome inhibitors
Treatment options in MM
 IMIDs
 Proteasome inhibitors
Proteasome inhibitors
 Very successful treatment of MM
 Tumor cells very sensitive to proteasome inhibitor
 Increased proliferation
 Increased protein synthesis and protein degradation
 Affect:
 Cell cycle
 Induction of apoptosis
 Stress response
 Various signaling pathways
Kubiczková, 2014
Effect of proteasome inhibitors on BM
microenvironment
 Cell signaling –
apoptosis
 Inhibit cell adhesion,
angiogenesis,
interactions
Kubiczková, 2014
New drugs increase life but do not
cure…..yet
Thalidomid (Myrin) Bortezomib (Velcade) Lenalidomid (Revlimid)
Carfilzomib ˃ Bortezomib Carfilzomib+Revlimid˃ Revlimid
 New proteasome inhibitors:
 Carfilzomib (Kyprolis)
 Ixazomib (Ninlaro)
 Monoclonal antibodies:
 Daratumumab (Darzalex)
 Elotuzumab (Empliciti)
 Perspective for newly diagnosed:
 Early diagnostics → early treatment (new criteria 2014)
 New combinations of drugs
New drugs- new hope
What you can buy for 1 year of
treatment:
Myrin
Velcade/
Bortezomib
Revlimid
Imnovid
Differences in treatment
Before
Treatment
After
others MM
Multiple myeloma
the molecular part
MM pathogenesis
 Unknown
 Genetic predisposition – close relatives - 6 times risk
 Pesticides, herbicides, radiation
 Mutations in PC
 Changes in BM microenvironment – PC growth
Hájek, 2012
Anderson, 2011
MM pathogenesis
Cytogenetic
mutations
Dysregulation of
cyclins, oncogenes,
supressors
Loss of
immune
surveillance
Immunosuppression
Cytokines
Cytokines
Hájek, 2014
Flowcytometry
 MM diagnosis - CD19-CD56+CD38+CD138+
 Risk of progression from MGUS to MM
 Detection of MRD – higher PFS and OS in MRD
-
pts
Hájek, 2012
Anderson, 2011
Říhová, 2013
Cytogenetic aberrations in MM
 Unstable genome – deletions, amplifications
translocations
 Changes accumulate in time
 Numerical and structural changes of chromosomes
 Aneuploidy of odd chromosomes and translocation of
IgH locus
Palumbo, 2013
Anderson, 2011
Kuglík, 2012
Němec, 2012
Numerical aberrations in MM
 Non-hyperdiploid (48 < >74) x hyperdiploid (48-74)
 Hyperdiploid:
 Trisomies of 3,5,7,9,11,15,19,21 – better prognosis
 Non-hyperdiploid:
 Monosomies of 8,13,14,16,17,22
Palumbo, 2013
Anderson, 2011
Kuglík, 2012
Němec, 2012
Structural aberrations
 Translocations of locus 14q32 (IgH)
 Primary changes:
 t(11;14) 15-20% cyclin D1
 t(4;14) 10-15% FGFR3/MMSET
 t(14;16) 2-10% c-MAF
 t(6;14) 5% cyclin D3
 Secondary changes:
 Complex karyotypes - MYC
 Deletion or duplication of 1q21
 Deletion or monosomy of chr 13
 Deletion of chr 17 – deletion of TP53 Palumbo, 2013
Anderson, 2011
Kuglík, 2012
Němec, 2012
FISH
 Most MM cells do not cycle – classical cytogenetics
basically useless
 i-FISH – much better results
 Prognosis – t(4;14), t(14;16), t(14;20) and del (17p) –
bad prognosis
Kuglík, 2012
Němec, 2012
Bešše, 2015
Diagnostics
 Bone marrow biopsies
 Painful, unethical to repeat too often
 New marker? Liquid biopsies?
Why liquid biopsies?
Limitations of classic biopsies
 Invasive
 Painful
 One site of tumor – heterogeneity not represented
 In MM – presence of subclones in focal lesions
Liquid biopsies
 Biopsies of PB
 Detection of circulating tumor cells
 Detection of circulating nucleic acids
 Easier sampling
 Entire heterogeneity of the tumor
Liquid biopsies
Crowley et al 2013
healthy tissue
inflammation
tumor
Circulating PC (cPC)
 Prognostic marker
 Loss of dependence on BM microenvironment
 Changes of adhesion molecules, chemokines, aberrations
 Faster progression MGUS to MM
 Higher BM infiltration in MM
 Negative prognostic marker in refractory MM
 5-20% cPC – worse survival regardless of age
 > 5% cPC – prognosis like PCL
Which molecules?
KO suplementum 2017
Non-coding RNAs
 Less than 1.5% of human genome codes for proteins
 More than 90% is transcribed
 Most common non coding RNAs : rRNA, tRNA
(Sana, 2012)
 Basic division:
 Short ncRNA < 200 bp < long ncRNA
Circulating ncRNA
 Body fluids
 Stable and resistant to
RNAses
 Easily accessible
 Cell communications
 Diagnostics
 Relapse monitoring
 MRD monitoring
http://circresearch.com/gallery/tag/circulating-mirna/
New markers for MM
 MicroRNA
 Cell-free DNA
New markers for MM
 MicroRNA
 Cell-free DNA
microRNA
 Short noncoding RNAs
 20-22 nt long
 Post-transcriptional regulation of gene expression
 Physiological processes (proliferation, differentiation)
 Tumorigenesis
Lee, 1993
Calin, 2002
History of miRNA
 Identified in 1993 in C. elegans
 Lin-4 – larval stages of C. elegans
 About 1/3 of human genes regulated by miRNA
 About 2200 of human miRNA
 miR-15a and miR-16 identified in region 13q14 –
potential oncogenes in CLL
Lee, 1993
Calin, 2002
Circulating miRNA
http://circresearch.com/gallery/tag/circulating-mirna/
Exosomes
 50-140 nm vesicles
 Proteins, NA
 Active secretion from
cells
 Change gene expression,
signaling in cells
 Remove chemo from cells
actively
 Support tuorigenesis –
miRNA transport
Our pilot study
 4 miRNA chosen based on MM pathogenesis
 Their presence analyzed in serum of PB
 miR-410 – locus 14q32 – MM translocation
 miR-660 – aberrant expression in MM
 miR-142-5p – aberrant expression in MGUS and MM
 miR-29a – increased expression in PC
Ševčíková, 2012
Results of pilot study
 miR-29a increased in MM
 Specificity 70%, sensitivity 88%, AUC=0.832
Ševčíková, 2012
Circulating miRNA as MG markers
 MM, MGUS and HD
 103 MM at diagnosis
 18 MM at relapse
 57 MGUS
 30 HD
Kubiczková, 2014
Methods
TaqMan Low
Density Arrays
Differential expression analysis
qPCR
Specific TaqMan miRNA assaysData analysis
miRNA isolation
Correlation with
clinically important
parameters
 miR-744, miR-130a, miR-34a, let-7d, let-7e
deregulated in MG vs HD
 Combination of miR-34a and let-7e: MM vs HD vs
MGUS
 No correlation with PC in BM – other pathological
changes in MM?
Kubiczková, 2014
Circulating miRNA as MG markers
Low levels of miR-744 and let-7e shorter
OS
Kubiczková, 2014
New markers for MM
 MicroRNA
 Cell-free DNA
Cell-free DNA
 Short fragments of DNA (180 bp) in PB
 First described in 1949 (Mandel et Métais)
 1977 – described in tumor patients (Leon et al)
 Higher levels in pts than controls
 Higher levels in metastases
 Lower levels after radiotherapy
 1994 – cfDNA carrying RAS mutation in MDS
 Used in prenatal diagnostics
cfDNA levels
 Physiologically low (10-100 ng/ml)
 Change of quantity and quality in pathology
 Higher levels (1000 ng/ml) in tumors, inflammations – but
not diagnostic
http://biomarkerinsights.qiagen.com/2016/08/17
How are cfDNA released from cells?
 Apoptosis
 Necrosis (tumor cells)
 Active release (cell signaling)
Schwarzenbach et al 2014
Our cfDNA project
 Detect a patient specific VDJ rearrangement of the IgH
locus in BM
 Check the rearrangement in cfDNA
 Follow the dynamics of the molecules after treatment
 Analyzed 85 pts, follow up up to 2 years after start of
treatment
Summary - cfDNA
 cfDNA carry various reaarangments
 Possible to follow MRD in MM?
Summary
 MM disease of older people
 New drugs dramatically improved survival
 Need more specific easily accessible markers
 Several possibilities – miRNA, cfDNA, lncRNA…
 Improvement in diagnostics and follow-up of patients
Liquid biopsies in the future?
 PB biopsies – all
heterogeneity of the
tumor
 Less painful
 Several molecules
 Great potential
Acknowledgments
Babákova myelomová
skupina,
Ústav patologické fyziologie,
Lékařská fakulta, MU
Sabina Ševčíková
Veronika Kubaczková
Lenka Sedlaříková
Božena Bollová
Martina Valachová
Jana Gregorová
Veronika Kelbichová
Kamila Novosadová
Oddělení klinické
hematologie,
Fakultní nemocnice Brno
Miroslav Penka
Martina Almáši
Renata Bezděková
Božena Hanáková
Lucie Říhová
Barbora Sáblíková
Renata Suská
Pavla Všianská
Interní hematologická a
onkologická klinika
Fakultní nemocnice Brno
Zdeněk Adam
Marta Krejčí
Luděk Pour
Viera Sandecká
Martin Štork
Institut biostatistiky a
analýz
LF MU
Jiří Jarkovský
Lucie Brožová
Oddělení klinické hematologie
Fakultní nemocnice Ostrava
Roman Hájek
Tomáš Jelínek
Fedor Kryukov
Elena Kryukova
Zuzana Kufová
Laboratoř molekulární
cytogenetiky
Ústav experimentální
biologie, PřF MU
Petr Kuglík
Aneta Mikulášová
Jan Smetana
Markéta Wayhelová
Ústav patologické fyziologie
Lékařská fakulta, MU
Anna Vašků
Thanks for your attention