residual disease in multiple myeloma by next generation sequencing Dr Ramon GarciaSanz 19 th March 2015 MRD The Concept 10 12 10 10 10 8 10 6 10 4 10 2 10 0 Relapse ID: 381071
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Slide1
Minimal residual disease in multiple myeloma by next generation sequencing
Dr
Ramon Garcia-Sanz
19
th
March
2015Slide2
MRD: The Concept
10
12
10
10
10
8
10
6
10
4
10
2
10
0
Relapse
CR
MRD
Cure
Maintenance
Induction
TX 2TX ConsolidationSlide3Slide4
Protein analysis
Serum Electrophoresis
(Palmer
1987
,
Bladé
1994)
Immunofixation
(Gay
. 2011; Martínez 2011)
Isoelectrofocusing (
Sádaba 2004)
Serum Free light chain assessment (Rajkumar
, 2011)
Immunophenotyping
Flow cytometry
(San Miguel
2002; Paiva 2008, 2011, 2012, ; Rawstron 2002, 2013)
Immunohistochemistry (
Durie Leukemia. 2006; 20:1467)
PCR amplification of V(
D
)J clonal rearrangements
CLONAL-SIZE BASED METHODs (PAGE,
GeneScanning): López
-Pérez 2002; Martínez-Sánchez
2008; Martínez-López 2013)
ALLELIC SPECIFIC OLIGONUCLEOTIDE PCR (ASO-PCR)Qualitative
(Corradini 2000, Martinelli 2001, Ladetto 2010, Terragna
2010)Quantitative: ASO-RQ-PCR (Sarasquete
2006, Ladetto 2010, Terragna 2010,
Puig 2013)Identification of V(D)J clonal rearrangements by high throughput sequencing
METHODS FOR MRD STUDIES IN MMSlide5
Antigenic
B-
cell
receptor (
Immunoglobulin
)
Membrane
B-
cell
V
H
V
L
C
H1
C
H2
C
H3
C
L
V
H
C
H1
C
H2
C
H3
V
L
C
L
IgH
IgL
h
h
IgH
Heterodímer
Asociated
to
Ig
(CD79)
IgL
InmunoglobulinsSlide6
Light
chain
gene of immunoglobulins
(
IgL
-kappa)
V
k
J
k
C
k
Light
chain
gene of immunoglobulins (
IgL
-lambda
V
l
J
l1
C
l1
J
l2
C
l2
J
l3
C
l3
J
l4
C
l4
Heavy
chain
gene of
immunoglobulin
(IgH)
B-
cell
Receptor genes
B
V
H
D
H
J
H
C
g3
C
d
C
m
C
g1
C
a1
C
g2
C
g4
C
e
C
a2
y
e
Immunoglobulin
heavy
chain
gene
rearrangementSlide7
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide8
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide9
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide10
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide11
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide12
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide13
V
H
D
H
J
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide14
V
H
D
H
J
H
12
23
Immunoglobulin
heavy
chain
gene
rearrangementSlide15
V
H
DJ
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide16
V
H
DJ
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide17
V
H
DJ
H
Immunoglobulin
heavy
chain
gene
rearrangementSlide18
V
H
DJ
H
Cµ
Joining
region
(
coding
extremes)
Excision
circle
(
signaling
extremes)
Immunoglobulin
heavy
chain
gene
rearrangementSlide19
V
H
DJ
H
Cµ
VDJ
H
Joining
region
Immunoglobulin
heavy
chain
gene
rearrangementSlide20
JH primer
(consensus primer)
VH-FR3
VH-FR2
VH-FR1
CDR1
CDR3
CDR2
V
H
VDJ
H
Cµ
AEEFGRRSAISKWYTA
YFGH
YWYYDLV
YYUWNFNN
RVTMLE
SSTTVRWWKLEM
TTERATY
DEFTVIIP
IRACDDTMMSRRTU
ETC…
Immunoglobulin
heavy
chain
gene
rearrangementSlide21
PCR analysis of
IGH
rearrangements
IGH
tube A:
ube B:
ube C:
6 V -FR1 primers + J consensus
7
7
H
V -FR2 primers + J consensus
V -FR3 primers + J consensus
H
H
IGH
IGH
t
t
D
H
J
H
JH primer
(consensus primer)
VH-FR3 primers
VH-FR2 primers
VH-FR1 primers
V
H
V family primers
H
D
H
J
H
D family primers
H
IGH
tube D:
6
D primers + J consensus
D7 primer + J consensus
H
H
IGH
tube E:
D 3
H
D 1
H
D 2
H
D 5
H
D 4
H
D 6
H
D 7
H
JH primer
(consensus primer)
Biomed-2, Van Dongen et al, 2003
HSlide22
Cycles
Copies
0
2
4
6
8
10
14
18
20
24
28
34
40
44
50
10
-3
10
-2
10
-1
10
0
10
1
D
Rn
10
-1
10
-2
10
-3
10
-4
10
-5
CMN
(5-10
Healthy Donors)
UmbralSlide23
N Puig et al,
Leukemia
2013
MRD
by
RQ-
PCR
MRD
by
Flow
cytometry
10
-1
10
-2
10-3
10
-4
10
-5
10
-1
10
-2
10
-3
10
-4
10
-5
R = 0.881
C
orrelation of tumor cells detected by
RQ-PCR and FCM (n=103)Slide24
MRD evaluation by PCR in Multiple Myeloma patients: prognostic
value
N Puig et al,
Leukemia
2013
Progression free survival in patients in VGPR (n=103; 10
-
5
,
Applicability 43
%)
ASO RQ-PCR
rearranged of IgH genes
CMF
84
72
60
48
36
24
12
0
EMR
neg
n=56
MRD pos
n=47
p=0.003
Months
from
starting
therapy
84
72
60
48
36
24
12
0
1,0
0,8
0,6
0,4
0,2
0,0
ASO RQ-PCR
% free of
progression
EMR
neg
n=48
MRDpos
n=55
p=0.001Slide25
Years since diagnosis
10
9
8
7
6
5
4
3
2
1
0
Percentage Alive
1,0
0,8
0,6
0,4
0,2
0,0
CR + MRD <10
-4
n=43
CR + MRD >10
-4
n=19
P
= 0.008
MRD evaluation by PCR in Multiple Myeloma patients: prognostic
value
Overal
Survival
patients in CR
(IFX-, n=63)
ASO RQ-PCR
rearranged of IgH
genes Slide26
Problems ASO-RQ-PCR
Applicability: 50%
Sensitivity: 10
-5 → (10
-4)High specialization
requirementQuality controlLabor-
intensiveCostSlide27
Multiple generations
… for sequencing
1st generation
-
Sanger
sequencing
2nd generation (high-throughput)
c
apture or
amplifcation
step included e.g. 454 (Roche), Hi-/Thru-/MiSeq
(Illumina), IonTorrent PGM (Life)
3rd generation (high-throughput) single
molecule/cell based e.g. Nanopore,
Helicos, Pacific Bio platforms
Slide
kindly
provided
by
A.W. Langerak,
Erasmus MSlide28
Different platforms for NGS - 2nd generation
454 GS Flex, Junior (Roche)
Ion Torrent
, proton
(Life)
HiSeq
,
MiSeq
(
Illumina
)Slide29
High
throughput
sequencing
:
Is
it
applicable to MRD?
Martínez-López et al,
Blood 2014Slide30
V-J Sequence Frequencies. The
LymphoTrack
Bioinformatics Software provides a stacked bar graph depicting the relative frequencies for
the 200
most common V - J rearrangements sequenced and identified in the sample.
IGHV1-18*01- - - - - IGHJ4-01Slide31
IGHV1
-12*01
ATTTCAGGGATTGTAGAATGAATCACATTAACAAATCTGACACAGAACTTCCTCTGAATCAATCTTTGTAAACATCAATTTCCGAATCAATGTTGTAAATAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV1
-14*01 GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGTCAGAGATCTGAGGACATAGATGTGTACTACTGTGCGAGACAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV1-18*02 ACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTAAGATCTGACGACACGGCCGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV1-02*02 GGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV1-03*01 GGGCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV1-45*02 CAGGACAGAGTCACCATTACCAGGGACAGGTCTATGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACAGCCATGTATTACTGTGCAAGATGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV1-67*01 GGCAGAGTCACCATGACCAGGGACACATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCATGTATTACTGTGGGAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV1-69*01 GGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV1-69*09 CCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV2-05*01 GCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACACAGACGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV2-70*03 ATCTCTGAAGACCAGGCTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACGGCCGTGTATTACTGGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG
IGHV3-11*01 AGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV3-13*01 GGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGCAAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV3
-15*01 AGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGIGHV3-19*
01 GGGCCGATTCATCATCTCCAGAGACAATTCCAGGAACTTCCTGTATCAGCAAATGAACAGCCTGAGGCCCGAGGACATGGCTGTGTATTACTGTGTGAGAAAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-21*
01 GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3-23*01
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-25*01 GGACCGATTCAATACCTCCAGAGATAACGCCAAGAACACACTTCATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCCTCTATTAGTGTACCAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV3-30*01 GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV3-30*
13 GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAGGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-32*
01 GGGCAGATTCTCCATCTCCAAAGACAATGCTAAGAACTCTCTGTATCTGCAAATGAACACTCAGAGAGCTGAGGACGTGGCCGTGTATGGCTATACATAAGGTCGGCCAGGGAACCCTGGTCACCGTCTCCTCAGIGHV3-23*01
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-33*
01 GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV3-36*02 GAAGGGTCGATTCACCCTCTCCAGAGATGATGCCAAGAAATCACTGTATCTGCAAATGAACAGCGTCAGAGCCGAGGATAGGTCTGTGTATTACTGTGGTGGGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-37*02 AGACACTGAAGGGTAGATTCACCATCTCTAGAGACAATGGCAAGAACATGCTGTACTTGCAAATGAACAGTCTGAGAGATGAGGACTCGGCTGTGTGAGAGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG
IGHV3-42*01 GAAAGGCAGGTTCACCATCTCAAGAGATGATTCAAAGAACACACTGTATCTGCAAGTGAATACCCTGAAAACCGAGTACACGGCCATCTATTACTGTACTAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV3-43*01 GCCGATTCACCATCTCCAGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAACTGAGGACACCGCCTTGTATTACTGTGCAAAAGATAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3-47*01 ATGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCTTGTATCTTCATATGAACAGCCTGATAGCTGAGGACATGGCTGTGTATTATTGTGCAAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-23*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-48*01 GGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3-49*03 AAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACTAGAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3-50*03 AAGGGCCGATTGACCATCTCCAGAGACAATGCCAAGAACTCCCTCTATCTGCAAGTGAACAGCCTGAGAGCTGAGGACATGACCGTGTATTACTGTGTGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV3-23*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-53*01 AGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV3-54*02 GAGCAGATTCACCATCTCCAAAGAAAATGCCAAGAACTCACTCCGTTTGCAAATGAACAGTCTGAGAGCAGAGGGCACGGCCGTGTATTACTGTATGTGAGGGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-57*01 GGGCACAAATTAACAGTCCCAAGCGACACCTTTTCATGTGCAGTCTACCTTACAATGACCAACCTGAAAGCCAAGGACAAGGCTGTGTATTACTGTGAGGGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG
IGHV3-57*02 TACAAATAAATTAACAGTCCCAAGCGACACCTTTTCATGTGCAGTCTACCTTACAATGACCAACCTGAAAGCCAAGGACAAGGCTGTGTATTACTGTGAGGGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3-23*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-06*01 GGGCCGATTCACCATTTCCAGAGACAATACCAAAAACTCACTGTATCTGCAAATGAACAGACTGAGGGCAGAGGATGCAGCTGCATATGACTCTGTGAGAGAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-64*05 GGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGTTCAAATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGTGAAAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV3-65*02 AGGTTTCACTAAAAACAAAACTAATCGTGGAACAACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGCGATGATTCCAAAAGCATCGCCTATGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3-66*04
AAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-71*
01 AGGCAGATTCACAATCTCAAGAGATGATTCCAAAAGCATCACCTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-73*01 AAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTACTAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV3-23*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3
-74*02 AAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAG
JH primer
(
consensus
primer)
VH-FR3
VH-FR2
IGHV1-18*01- - - - - IGHJ401Slide32
IGHV1
-12*01
ATTTCAGGGATTGTAGAATGAATCACATTAACAAATCTGACACAGAACTTCCTCTGAATCAATCTTTGTAAACATCAATTTCCGAATCAATGTTGTAAATAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV1
-14*01 GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGTCAGAGATCTGAGGACATAGATGTGTACTACTGTGCGAGACA
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGIGHV1-69*01 ACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTAAGATCTGACGACACGGCC
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGIGHV1-02*
02 GGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV1-03*
01 GGGCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV1-45*02 CAGGACAGAGTCACCATTACCAGGGACAGGTCTATGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACAGCCATGTATTACTGTGCAAGATGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV1-67*01 GGCAGAGTCACCATGACCAGGGACACATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCATGTATTACTGTGGGAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV1-69*01 GGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV1-69*09 CCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV2-05*01 GCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACACAGACGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV2
-70*03 ATCTCTGAAGACCAGGCTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACGGCCGTGTATTACTGGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3
-11*01 AGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3
-13*01 GGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGCAAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV3-15*
01 AGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-19*01 GGGCCGATTCATCATCTCCAGAGACAATTCCAGGAACTTCCTGTATCAGCAAATGAACAGCCTGAGGCCCGAGGACATGGCTGTGTATTACTGTGTGAGAAA
GGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-21*
01 GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG
IGHV3-18*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-25*01 GGACCGATTCAATACCTCCAGAGATAACGCCAAGAACACACTTCATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCCTCTATTAGTGTACCAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV3-30*
01 GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3
-30*13 GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAGGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV3-32*01 GGGCAGATTCTCCATCTCCAAAGACAATGCTAAGAACTCTCTGTATCTGCAAATGAACACTCAGAGAGCTGAGGACGTGGCCGTGTATGGCTATACATAAGGTCGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-18*01
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-33*01 GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV3-36*02 GAAGGGTCGATTCACCCTCTCCAGAGATGATGCCAAGAAATCACTGTATCTGCAAATGAACAGCGTCAGAGCCGAGGATAGGTCTGTGTATTACTGTGGTGG
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGIGHV3-37*02 AGACACTGAAGGGTAGATTCACCATCTCTAGAGACAATGGCAAGAACATGCTGTACTTGCAAATGAACAGTCTGAGAGATGAGGACTCGGCTGTGTGAGAGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3-42*01 GAAAGGCAGGTTCACCATCTCAAGAGATGATTCAAAGAACACACTGTATCTGCAAGTGAATACCCTGAAAACCGAGTACACGGCCATCTATTACTGTACTAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3-43*01 GCCGATTCACCATCTCCAGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAACTGAGGACACCGCCTTGTATTACTGTGCAAAAGATA
GGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3-47*01 ATGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCTTGTATCTTCATATGAACAGCCTGATAGCTGAGGACATGGCTGTGTATTATTGTGCAAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGIGHV3-18*01
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-48*01 GGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3
-49*03 AAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACTAGAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3-50*03 AAGGGCCGATTGACCATCTCCAGAGACAATGCCAAGAACTCCCTCTATCTGCAAGTGAACAGCCTGAGAGCTGAGGACATGACCGTGTATTACTGTGTGAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV3-18*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-53*01
AGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAG
IGHV3-54*02 GAGCAGATTCACCATCTCCAAAGAAAATGCCAAGAACTCACTCCGTTTGCAAATGAACAGTCTGAGAGCAGAGGGCACGGCCGTGTATTACTGTATGTGAGGGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV3-57*01 GGGCACAAATTAACAGTCCCAAGCGACACCTTTTCATGTGCAGTCTACCTTACAATGACCAACCTGAAAGCCAAGGACAAGGCTGTGTATTACTGTGAGGGA
GGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGIGHV3-57*
02 TACAAATAAATTAACAGTCCCAAGCGACACCTTTTCATGTGCAGTCTACCTTACAATGACCAACCTGAAAGCCAAGGACAAGGCTGTGTATTACTGTGAGGGAGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3-23*01
GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGIGHV3-06*
01 GGGCCGATTCACCATTTCCAGAGACAATACCAAAAACTCACTGTATCTGCAAATGAACAGACTGAGGGCAGAGGATGCAGCTGCATATGACTCTGTGAGAGAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-64*05 GGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGTTCAAATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGTGAAAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG
IGHV3-65*02 AGGTTTCACTAAAAACAAAACTAATCGTGGAACAACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGCGATGATTCCAAAAGCATCGCCTATGGGCCAAGGGACAATGGTCACCGTCTCTTCAGIGHV3
-66*04 AAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG
IGHV3-71*01 AGGCAGATTCACAATCTCAAGAGATGATTCCAAAAGCATCACCTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGIGHV3
-73*01 AAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTACTAGAGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGIGHV3
-18*01 GGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAG
IGHV3-74*02 AAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAG
JH primer
(
consensus
primer)
VH-FR3
VH-FR2
IGHV3-18*01- - - - - IGHJ4-01Slide33
Martinez-Lopez
et al
,
Blood 2014
Deep sequencing of rearranged BCR genes (
Lymphosight
™)
N 133
cases in CR or VGPR
(
sensitivity ≥10
-5), GEM00,05,10; Applicability 91%
62/101 (
63%)
samples were positive with both techniques
22/101
(22%) samples were negative with two techniques17/101 (17%)
were discordant:FCM+SEQ-: 5
FCM-SEQ+: 12
HTS vs. FCM
MRD evaluation by HTS in Multiple Myeloma patients: clinical valueSlide34
Martínez-López
et
al,
Blood
2014
Clinical
value
of MRD
evalauted
by
sequencig in MM
patients with MM
Deep
sequencing
by
Ig
rearrangement
(
LymphoSight™)
N 62 cases in sCR
(sensitivity ≥10
-5)
N 110 cases in CR or VGPR (
sensitivity ≥10-5)Applicabiliity 91%
Overall
survival
(all patients)
Time
to
tumor progression (Only CR)
Percentage
alive
Alive
without
progrression
Months
MonthsSlide35
Comparison
of
different
methodologies
ASO PCR
of BCR genes
HTS of BCR genes
Flow Cytometry
Sensitivity
10
-
5
-10
-6
10
-5
-10
-6
1
0
-4
-10
-5
Applicability
Low
High
Very High
Time-consuming
Very High
Very High
High
Specific reagents
Yes
No
No
Cost
300 €
900 €
150 €
Clonal evolution detectable
No
Yes*
No**
Expertise
Very High
Very High
Very High
Availability
Low
Low
High
*No clonal evolution has been shown in MM at the BCR gene rearrangement;
**FCM can detect aberrant PC subsets with an Ag profile different to diagnosisSlide36
Future
Technology
Problem
Solution
FCM
Sensitivity
Specificity
Expertise
requirement
Increase the input cells (107 cells)
Go up to 8 colorsNew softwareASO-PCRSensitivity
ApplicabilityExpertise requirement
Time consuming, costIncrease input DNA (6 mcg, 106 cells)
Discard SC, use DCT methodStandardizationOptimization
HTSSensitivityApplicability
Expertise requirementTime consuming, costAvailability
Increase input DNA (6-60mcg, 106-107
cells)Increase targets (mutations)New softwareOptimization
Competition; Lymphosight™, Lymphotrack™,
Euroclonality/EuroNGS
Clinical validation in Prospective StudiesSlide37
Conclusions
MRD
detection
by
molecular
technology
is applicable
to MM patients rendering
similar results or
even superior to FCM
MRD detection by
NGS is an useful
method for stratifying
patients and can be used to
assess molecular response in MM
With this methodology,
today it
is posible to
go close
to personalized
therapeutic strategies:
MRD (‒): treatment
reductionMRD (+):
treatment increase or
continuous therapySlide38
Myeloma
Team
Ramón García Sanz
Norma
C. Gutiérrez
Enrique M. Ocio
M. Victoria Mateos
Noemí Puig
Marcos GonzálezRamón García-SanzLuis MarínRocío Corral
Molecular Laboratory Team
Carmen ChillónAna BalanzateguiM. Eugenia SarasqueteMiguel Alcoceba
Elena SebastiánCristina JiménezIsabel LópezMaría GarcíaSlide39
Grupo Español de Mieloma (GEM)
Hospitales
Clínico de Barcelona
12 Octubre (Madrid)
Clínico de Salamanca
Clínico de San Carlos (Madrid)Hospital de Badalona
Clínico de AsturiasFr. Peset (Valencia)Universitario de Canarias
Rio Ortega (Valladolid)Cínico de ZaragozaHospital General de Jerez
Ramón y Cajal (Madrid)Morales Meseguer (Murcia)La Fe (Valencia)C.U. de Navarra
Galdakao (Vizcaya)Clínico de ValladolidSant Pau (Barcelona)
Arnau Vilanova (Lérida)Universitario de SantiagoGeneral Universitario de ValenciaUniversitario de Getafe (Madrid)
Insular de las PalmasH. de La Princesa (Madrid)Severo Ochoa (Madrid)Juan XIII (Tarragona)
ToledoGandía (Valencia)Vall D´Hebrón (Barcelona)San Jorge (Huesca)Verge de la Cinta (Tortosa)
Alarcos (Ciudad Real)Mataró (Madrid)Juán Canalejo (Coruña)Ferrol
HospitalesGeneral de Segovia
Cruces (Bilbao) St. Coloma de Gramanet (Barcelona)Gregorio Marañon (Madrid)Carlos Haya (Málaga)
H. Tauli (Gerona)HuescaPalenciaAlcira (Valencia)
H. Del Mar (Barcelona)Mahón (Baleares)Clínico de Málaga
Xeral Cies (Vigo)PlasenciaCáceres
AlgecirasÁvilaJaén
S. Pau i Sta Tecla (Tarragona)General de GuadalajaraSagunto (Valencia)
Son Dureta (Mallorca)CuencaAlicante SUS
M. Valdecilla (Santander)AlbaceteH. Del BierzoFundación Jiménez Díaz (Madrid)
Elda (Alicante)V. Del Rosel (Cartagena)Castellón
Mutua TarrasaConsorcio TarrasaC. Corachán (Barcelona)
Salamanca
Group
:
Puig
,
Vidriales
,
Sarasquete
,
Gutierrez
,
Mateos,
Jinénez
,
Orfão
, González.
(Paiva
,
San Miguel)
Madrid (12O)
Group
:
Lahuerta
, Martínez-López, Montalbán
, MA
Montalban
, Martin,
Fernandez