SUPPLEMENTARY DATA
METHODS
Tissue Banking
Samples submitted to the MMRC tissue bank are all processed in a uniform
fashion and following good laboratory practices. The complete process is
standardized including the collection kits, shipment conditions, purification and
extraction methodology. Rigorous quality control criteria are applied to these
samples at multiple steps, and those not meeting minimum standard are not
accepted for future use. Likewise application of rigorous standards for derived
products to be used in research projects (e.g. RNA) are applied to material
distributed in support of scientific projects. In brief, samples are shipped in a
temperature controlled package that is kept at 4oC using a cooling insert. On
arrival the samples have red cells removed via ACK lysis, cytospins are made on
unsorted cells for future FISH and frozen for subsequent RNA and DNA
extraction in Trizol. Remaining cells are immediately incubated with anti CD138
magnetic beads. An automated robotic separator is used to segregate the
positive and negative fraction. The quality of the positive fraction is determined
using
an
immunofluorescence
based
assay
to
asses
purity
and
clonality. Additional metrics for the RNA derived from these samples are applied
including a qualitative analysis in gel, NanoDrop™ concentrations and
parameters, and Agilent Bioanalyzer™ quality determinations.
Flow Cytometry
For flow cytometry, 180 ul of buffer (PBS, 1% FBS, 0.1%NaN3) was added to 20
ul of unsorted bone marrow and split. 1 ul rabbit anti human FGFR3 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA: clone H100, cat.# sc9007) was added to
one tube and 1 ul normal rabbit IgG (Santa Cruz, cat.# sc2027) to the other.
Following incubation at room temperature for 15 min, cells were washed once
with buffer and goat anti rabbit Ig-PE and anti-CD138 FITC added to each tube.
Following a second room temperature incubation of 15 minutes in the dark, cells
were washed again with buffer. Following buffer removal 0.5 - 1ml ACK red cell
lysis buffer was added and samples were run on a FACScan using Cellquest
Software for analysis. We gated on CD138+ cells with appropriate side and
forward scatter characteristics and examined that population for FGFR3
expression. In later experiments, we have employed an anti-FGFR3-PE
conjugated monoclonal mouse IgG1 clone 136334 (R and D systems,
Minneapolis, MN, USA) at 1:50 dilution in combination with anti CD138-PC5
conjugated mouse IgG1 clone BB4 (Immunotech, Marseille, France) at
1:10
dilution on ACK lysed bone marrow mononuclear cells pre-incubated with 2%
human serum to block non specific Fc receptor binding. Events were acquired on
a CyanADP instrument using Summit software and analyzed on FloJo.
Polymerase Chain Reaction
Mononuclear cells from bone marrow or peripheral blood were isolated following
ACK lysis.
RNA is extracted using Trizol reagent (Invitrogen, Carlsbad, CA,
USA) and sample concentrations were adjusted to 100 ng/ul. RNA integrity was
assessed by the Agilent Bioanlyzer (Agilent Technologies,
Santa Clara, CA, USA).
RT-PCR was performed on 100ng/ul of template using C.therm. Polymerase
One-Step RT-PCR System (Roche Applied Science, Indianapolis, IN, USA) using
the following primers: 5’ACCACGGTCACCGTCTCCTCA-3’ (sense primer) from
JH
and
5’CCTCAATTTCCCTGAAATTGGTT-3’ (antisense primer) from
MMSET exon 6. The RT-PCR reaction was carried out in the following manner:
60oC for 30 minutes (transcription), 95oC for 2 minutes (initial denaturation); 10
cycles of 95oC for 30 seconds, 60 oC for 30 seconds and 72 oC for 60 seconds;
followed by 25 cycles 95oC for 30 seconds, 60 oC for 30 seconds and 72 oC
increasing 5 seconds each additional cycle; 72oC for 7 minutes (final extension),
and finally a 4 oC hold. Rearrangements fall into three breakpoint clusters, MB4-1
(1007 bp) MB4-2 (381 bp), and MB4-3 (218 bp), depending on the position of
the breakpoint on the MMSET gene. Notably, this strategy will not allow the
detection of the rare t(4:14) translocation breakpoints occurring downstream of
the MMSET exon 6. To assess the sensitivity of the assay, the same RT-PCR
reaction was conducted on100ng/ul from the myeloma cell lines JMW (JHMMSET positive) and
MM-1 (JH-MMSET negative) mixed in the following
concentrations, 100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.1%, 0.01%, 0.001%,
0.0001%, and 0%. A faint band was visible at 0.1% dilution which translates into
1 t(4;14) positive cell in 1 million t(4;14) negative.
Immunohistochemistry
Cytospin slides with myeloma cells were fixed in acetone for 10 minutes, treated
with 1% hydrogen peroxide for 10 min, and incubated with anti-FGFR3 antibody
(SC-13121; Santa Cruz Biotechnology; Santa-Cruz, CA) at 1:200 dilution
overnight at 4C. This step was followed by a 30 minutes incubation with a
biotinylated linking reagent (ID Labs, London, On, Canada) and a subsequent
30-min incubation with horseradish peroxidase-conjugated Ultra Streptavidin
labeling reagent (ID Labs). The immunoreaction was visualized with freshly
prepared NovaRed Solution (Vector Laboratories, Burlington, ON, Canada); the
slides were counterstained with Mayer hematoxylin and evaluated with an
Olympus BX50 microscope (Olympus, Melville, NY, USA). The t(4;14) positive
myeloma cell line –KMS11 was used as a positive control for aberrant expression
of FGFR3. As a negative control, non immune mouse serum was substituted for
primary antibody. We considered cells FGFR3 positive if the membrane/or
cytoplasm of at least 20% of the plasma cells stained (ref. Chang et al, Blood,
106:353-355, 2005)