Supplementary information: Materials and methods
Samples and nucleic acid extraction
Bone marrow (BM) and peripheral blood (PB) samples were obtained from 10
multiple myeloma (MM) patients during standard diagnosis procedure. A
lymphoblastoid cell line CL-Wi-L2-729-HF2 1 was used to validate the quantitative
PCR methodologies.
Samples from healthy PB volunteers were used as negative controls for ASO
PCR. An additional specificity control was performed using different pathological
cellular samples (BM, PB, leukaphereses, lymphnodes) non-related to the patients
for whom an ASO PCR was developed.
PB and BM mononuclear cells (MNC) were separated by density gradient
centrifugation (Lymphoprep, AbCys, Paris, France, density = 1.077).
DNA was isolated using Qiamp mini kit (Qiagen, Courtaboeuf, France) according to
the manufacturer’s recommendations. DNA concentration was established with a
spectrophotometer.
Tumor IgH rearrangement characterization by GeneScanning
A single round IgH consensus PCR was performed with 6 VH-family specific
primers used in combination with JH fluorescent primers under previously described
conditions 2 (Table 1). PCR products were separated via highly sensitive capillary
electrophoresis on an automated sequencer (Applied Biosystems, ABI 310,
Courtaboeuf, France) and analysed by GeneScan software v. 3.1.2 (ABI). This
procedure allowed the detection of MM monoclonal rearrangements.
Table 1: PCR primers and hydrolysis probes
Primers and
probes
location
Albumine
IgH FR1
IgH FR4
Primers
designation
IgH FR4
probes
Primers
lenght
Reference
ALB+
ALB-
CTg AgA Agg AgA gAC AAA TC
TgC TAT CAA gAC TAC AAA CAC
20 bp
21 bp
in-house
design
VH1
VH2
CCT CAg TgA Agg TCT CCT gCA Agg
gAg TCT ggT CCT gCg CTg gTg AAA
24 bp
24 bp
3
VH3
ggT CCC TgA gAC TCT CCT gTg CA
23 bp
3
VH4
TCg gAg ACC CTg TCC CTC ACC TgC
24 bp
3
VH5
gAA AAA gCC Cgg ggA gTC TCT gAA
24 bp
3
VH6
gAA AAA gCC Cgg ggA gTC TCT gAA
24 bp
3
JHD
JHEXT
ACC TgA ggA gAC ggT gAC CAg ggT
g TgA ggA CTC ACC TgA ggA g
20 bp
20 bp
3
JH6
ACC TgA ggA gAC ggT gAC CgT ggT
24 bp
6
JH3i
Agg CAg AAg gAA AgC CAT CTT AC
23 bp
7
CAg AgT TAA AgC Agg AgA gag gTT gT
26 bp
7
JH5i
AgA gAg ggg gTg gTg Agg ACT
21 bp
7
JH6i
gCA gAA AAC AAA GGC CCT AgA gT
24 bp
7
JH1,2,4,5
JH3
CCC Tgg TCA CCg TCT CCT CAg gTg
24 bp
CAA ggg ACA ATg gTC ACC gTC TCT TCA 27 bp
7
JH6
CAC ggT CAC CgT CTC CTC Agg TAA Gaa 27 bp
7
IgH intronic JH4i
JH
Sequence 5'-3'
4
5
7
ASO primer design
Monoclonal purified IgH consensus PCR products were directly sequenced
with the Big Dye terminator reaction kit (Applied Biosystems) and analysed on an ABI
310.
IgH tumor rearrangement sequence was compared to the germline sequence
from Ig specific data bases (VBASE : http : //www.mrc.cpe.cam.ac.uk 8 and IMGT :
http : //www. imgt.cines.fr 9). The ASO primer was derived from the most
hypervariable region CDR3 as shown on figure 1.
Figure 1 : IgH CDR3 junctional sequence.
VH
N
V5-51
#01
TGTGCGAGAC
TGTGCGAAAG
AGGATACAGCATTACT
TGTGCGAGACA
TATTTCTCTGCCTTATCGGGGGA
ACGATATTTTGACTGG
TGTGCGAGGGA
V3-74
#06
TATTACTGCGCAAG
TGTGCGAGC
GAGAGAGTGGGACCTAC
ATTACTGTGCGAAA
V3-23
#10
TGTGCGCGAG
TGTGCGAGAG
ACCACCTGCACAATAAC
CTTTGAAGACTGG
JH5
GG
D2-21
CCT
CTTTGACTACTGG
JH4
GGG
D2-08 (inv)
CTGGTAAATGTGATGTG
V1-02
WI
AGATTGTATTAATGGTGTATGCTATAC
CTTACTACTACTACT
JH4
AAGGGGGG
D2-08
ACGGTCCGGGGGTC
ACTACCACTATGG
JH6
GA
D1-26
TGTACTAGAG
V3-30
#09
GGATTCAAGTTTG
TGATATCTGG
JH6
TAGAATCTCTAA
D3-16 or D5-5
GG
V3-49
#08
GCGTTACGATTTGTGGAATGGTTATT
TGACCACTGG
JH3
TCTCCCGGC
D3-03
TAGGGG
V4-04
#07
TAACAAGTCGTCC
TACCTTGACTCCTGG
JH4
CGC
D6-06
CCTGTATGAGG
TTCGGTATGGACGTCTG
JH4
CTGGGGGGGA
D3-9
V3-33
#05
TTTGACTCTTGG
JH6
GCTG
D2-08 (inv)
GGATTGTCCCGGGGG
JH
JH4
A
D3-16
V4-39
#04
GTATAACGATCTTTTGGCGGG
GGAATGATGC
TGTGTCAGAG
V3-23
#03
N
D3-09
CTAA
V3-13
#02
D
CATATTGTGGTTATGACTGCTATT
GATTCGACCCCTGG
JH4
ACTTCTTTGACTACTGG
VH, D and JH genes were determined after alignment with germline sequences from
databanks. All IgH tumor rearrangement sequences were in frame and submitted to
the process of somatic mutations. For the 10 patient rearrangements, a D segment
was identified presenting less than 4 mismatches with the germline D sequence. The
length of the N nucleotides insertion varied from 0 to 23 nucleotides.
The ASO primer (represented in bold and underlined by an arrow in the figure) was
designed with OLIGO 3.4 software (W. Richlik, Molecular Biology Insights, Inc.,
Cascade, CO, USA). The ASO primer choice was guided following definite criteria :
first, it should overlap the V-D or D-J junction; second, the Tm should be equivalent
to that of the consensus primer; third, the 3’end should be located in a N or D gene
segment. Oligonucleotides likely to form secondary structure were avoided. In most
cases, these criteria restricted the ASO primer location possibilities. Primers for IgH
ASO PCR were chosen either with an ASO downstream or upstream oriented primer.
For the CL-Wi-L2-729-HF2 cell line, both primer orientations were assessed. Primers
were purchased by Proligo (Paris, France)
ASO IgH RQ PCR
Two different detection methods were attempted, either with SYBR Green I or
with hydrolysis probes (when applicable). All PCR reactions were run in duplicate
glass capillaries on the Lightcycler (Roche Applied Science, Meylan, France). The
data were analysed by the quantification software (version 3.3, Roche Applied
Science).
 ASO IgH SYBR Green PCR
The PCR reactions were performed in 20 L total volume, with « Quantitect
SYBR Green I » kit (Qiagen, Courtaboeuf, France) including all PCR reagents with a
pre-optimized MgCl2 concentration, 10 pmoles of each primer and 200 ng DNA. The
thermal cycling conditions included 15 minutes at 95°C, followed by 45 cycles of
95°C for 15 seconds, optimized annealing temperature for 30 seconds and 72°C for
30 seconds. The fluorescence signal was measured at the end of each elongation
step. The final PCR cycle was followed by a melting curve analysis to confirm PCR
product identity and to differentiate it from non-specific products.
 ASO primer and consensus hydrolysis probe for IgH PCR
PCR reaction was performed between ASO primer and one of the JH specific
intronic primers with 3 different probes which hybridized to the FR4 region, as
described by Verhagen 7 (Table 1). A maximum of 500 ng DNA was amplified with 2X
“Quantitect reaction mix”, 8 pmoles of each primer and 4 pmoles of probe in a final
volume of 20 µL. The “Quantitect” reaction mix (Qiagen, Courtaboeuf, France)
included the “hot start” polymerase, adequate buffer, dNTP and a pre-optimized
MgCl2 solution. The thermal cycling conditions included 15 minutes at 95°C followed
by 45 cycles of 95°C for 10 seconds and optimized hybridization temperature for 60
seconds. The fluorescence was monitored after the annealing/elongation step.
Reference gene normalization
All samples were normalized by a reference gene in order to take into account the
quality and quantity of the DNA input. The albumin gene was chosen for the SYBR
Green strategy and the GAPDH gene for the hydrolysis probe strategy.
Albumin gene PCRs were performed with a previously optimized PCR using the « LC
Hot Start DNA master SYBR Green I » (Roche Applied Science) and 4 mM MgCl2.
The thermal cycling conditions included 10 minutes at 95°C, followed by 45 cycles of
95°C for 10 seconds, 60°C for 10 seconds and 72°C for 5 seconds. At the end, a
melting curve was performed.
The quantification of the GAPDH reference gene was carried out using the
“Quantitect” kit with 8 pmoles primers and 4 pmoles probe from
“GAPDH Endogeneous reference kit” (Eurogentec, Seraing, Belgium). PCR
conditions were as described for the hydrolysis probe ASO IgH RQ PCR with a 60°C
annealing/elongation step.
Specificity and efficiency determination
The specificity of the generated PCR products was determined using several
controls. For each ASO PCR, some amplified products were checked by
electrophoresis on GelStar stained 1.5% Agarose gels. GelStar and Nusieve
agarose were provided by TEBU (Le Perray-en-Yvelines, France). For all ASO
PCRs, the size of ASO LightCycler PCR product, estimated by agarose gel
electrophoresis, matched the expected length according to the IgH rearrangement
sequence (data not shown) thus ensuring the absence of non-specific amplifications
resulting from primer’s binding to other gene locus or from primers dimerization. For
evaluation of co-amplification of normal and tumor rearrangements, 2 other tests
were used : a specificity control was included in each PCR that consisted of a pooled
DNA from 4 healthy PB volunteers. Moreover, a specificity test including cell samples
non-related to the patients studied was performed for each optimized ASO PCR. For
this test, a minimum of 5 samples from different origins (leukaphereses, lymphnode,
BM,…) and from different hematological B-cell pathologies were analysed. Nonspecific amplifications were admitted when the crossing point (Cp) for non-related
samples were at least over 3 cycles from the sensitivity limit 10 and when they were
not frequent (supplemental table 1).
Standard curve was generated in order to determine the PCR efficiency and
the sensitivity limit of each ASO PCR (see supplementary table 2). The DNA from BM
at diagnosis was 8-fold serially diluted in pooled DNA obtained from 4 normal PB
volunteers. A constant total DNA input of 200 ng was used for the dilutions. For
reference gene PCR, initial BM DNA dilutions in sterile water were used for the
standard curve.
Since the accurate proportion of tumor cells is not available in the initial BM sample,
this sample taken during diagnosis procedure was considered as the calibrator
sample for which the tumor load was set to 1 and all samples were referred to it.
Reproducibility of the standard curves for one ASO IgH PCR was tested by running
the calibration curve 3 times over a period of 3 months (see supplemental table 3).
Relative quantification
The relative amount of tumor IgH rearrangement and reference gene were
calculated with the comparative Ct method (when applicable) and the relative
quantification software “RelQuant” v.1.0. (Roche Applied Science).
 The comparative Ct (Cycle threshold) method
The relative concentration of one target compared to another is reflected by
the difference in cycle number necessary to achieve the same level of fluorescence.
In the comparative Ct method which has been described by Applied Biosystems User
Bulletin No.2 (P/N 4303859), the amount of target, normalized to a reference gene
and relative to a calibrator is given by : 2 -Ct (with Ct = Ct calibrator sampleCt unknown sample and Ct = Ct of the target gene – Ct of the reference gene). For this
method to be valid, equivalent efficiencies between target gene and reference gene
PCRs were presumed. Therefore, a validation experiment was conducted by
determining the Ct variation with template dilution (supplemental table 6).
 RelQuant software
The RelQuant software was chosen because it takes the efficiency differences
between ASO and reference gene PCRs into account. The efficiencies were
determined once by using dilutions of BM DNA at diagnosis for IgH ASO and
reference gene PCR. The RelQuant software generates a coefficient file in order to
save the standard curve parameters that will be used for the analysis of consecutive
clinical samples. For each residual disease sample, results are expressed as the
target/reference sample ratio divided by the target/reference calibrator ratio and
therefore corrected for PCR variations.
Aliquots of the calibrator samples corresponding to the BM samples at presentation
were frozen in order to guarantee the stability of the calibrator DNA.
Absolute quantification with internal calibration curve
The calculation of data is based on Cp values obtained from LightCycler software 3.5
by the second derivative maximum method. PCR for standard curve and unknown
samples were performed simultaneously in the same run. The LightCycler program
extrapolates sample concentration from the standard curve. All samples were
analysed in duplicate. The mean extrapolated concentration and standard deviation
were calculated automatically. The ratio between ASO PCR concentration and
reference gene concentration was established (supplemental table 5).
Flow cytometry analysis
106 BM MNC were incubated with anti CD45 PerCP and anti CD38 FITC
antibodies (Becton Dickinson, Le Pont de Claix, France) for 30 min at 4°C. After a
red PB cell lysis step, cells were washed twice and 100000 cells were acquired using
a FACS Calibur flow cytometer (Becton Dickinson). Adequate controls were included.
The tumor plasma cells were defined using a combination of high CD38 and low or
intermediate CD45 fluorescence 11.
References
1.Heitzmann JG, Cohn M. The WI-L2-729-HF2 human hybridoma system. Stable
hybrids at high frequency. Mol Biol Med 1983; 1: 235-243.
2.Welterlin V, Debecker A, Tschieb D, Zanetti C, Lange W and Hénon PR.
Improvement of clonality detection rate in multiple myeloma using fluorescent
IgH PCR with different sets of primers. J Hematother Stem Cell Res. 2000; 9:
983-991.
3.Deane M, Norton JD. Immunoglobulin gene “fingerprinting”: an approach to
analysis of B lymphoid clonality in lymphoproliferative disorders. Br J
Haematol. 1991; 77: 274-281. 21
4.Owen RG, Johnson RJ, Rawstron AC, Evans PA, Jack A, Smith GM, child JA,
Morgan GJ. Assessment of IgH PCR strategies in multiple myeloma. J Clin
Pathol. 1996; 49: 672-675. 22
5.Billadeau D, Blackstadt M, Greipp P, Kyle RA, Oken MM, Kay N, Van Ness B.
Analysis of B-lymphoid malignancies using allele-specific polymerase chain
reaction: a technique for sequential quantification of residual disease. Blood.
1991; 78: 3021-3029. 23
6.Aubin J, Davi F, Nguyen-Salomon F, Leboeuf D, Debert C, Taher M, Valensi F,
Canioni D, Brousse N, Varet B. Description of a novel FR1 IgH PCR strategy
and its comparison with three other strategies for the detection of clonality in B
cell malignancies. Leukemia. 1995; 9: 471-479. 24
7.Verhagen OJ, Willemse MJ, Breunis WB, Wijkhuis AJ, Jacobs DC, Joosten SA,
Van Wering ER, Van Dongen JJ, Van der Schoot CE. Application of germline
IGH probes in real-time quantitative PCR for the detection of minimal residual
disease in acute lymphoblastic leukemia. Leukemia. 2000; 14: 1426-1435 9
8.Cook GP, Tomlinson IM. The human immunoglobulin VH repertoire. Immunol
Today. 1995; 16: 237-242.
9.Lefranc MP. IMGT, the international ImMunoGeneTics database. Nucleic Acids
Res. 2001; 29: 207-209.
10. Van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, Van
Dongen JJ. Detection of minimal residual disease in hematologic malignancies
by real- time quantitative PCR: principles, approaches, and laboratory aspects.
Leukemia. 2003; 17: 1013-1034.
11. Witzig TE, Meyers C, Therneau T, Greipp PR. A prospective study of
CD38/CD45 flow cytometry and immunofluoresence microscopy to detect
blood plasma cells in patients with plasma cell proliferative disorders. Leuk
Lymphoma. 2000; 38: 345-350.