Supplementary Materials & Methods
ZAP70 expression
Expression was assessed using two methods: ‘Caltag’ (Caltag-Medsystems, Buckingham, UK ) and
‘Upstate’ (Upstate Biotechnology, Milton Keynes, UK). Using the Caltag method, cells were
incubated with anti-CD2-PE (DakoCytomation, Denmark) prior to use of the ‘‘Fix and Perm’’ kit
(Caltag-Medsystems) according to the manufacturer’s instructions. Anti-ZAP-70 Alexa Fluor 488
antibody (test) or mouse IgG1-Alexa Fluor 488 antibody (control) was added with reagent B. Using
the Upstate method, lymphocytes were fixed and permeabilized using paraformaldehyde and ethanol,
and then incubated with the primary antibody (ZAP-70, clone 2F3.2, Upstate Biotechnology) or
isotype control (Mouse IgG2a, Dako, Glostrup, Denmark). Subsequent staining was carried out with
the secondary antibody (Sheep anti-mouse FITC, BD Biosciences, San Diego, USA) and then antiCD2 PE. For each analysis method, 10,000 cells were acquired on a Facscaliber flow cytometer (BD
Biosciences). Samples with greater than 10% positive cells were classed as ZAP70 positive.(1, 2)
CD38 expression
Cells were incubated for 15 minutes with FITC labeled anti-CD5 (clone DK23, Dako), phycoerythrin
(PE)–labeled anti- CD38 (clone HB7, Becton Dickinson, Oxford, UK), and R-phycoerythrin-cyanine 5
(RPE-Cy5) labeled anti-CD19 (clone HD37, Dako). Red cells were lysed and at least 10 000 cells
were acquired on a FACSCalibur flow cytometer (Becton Dickinson). For each sample an
appropriate isotype control was used to define the negatively stained cells. Samples with greater
than 30% positive cells were classed CD38 positive.
IgVH mutation analysis
Prior to 2004 cDNA was PCR amplified using a mixture of oligonucleotide 5¢ primers specific for
each leader sequence of the VH1 to VH6 families or a consensus 5¢ FW1 region primer, together
with either a consensus 3¢ primer complementary to the germ line JH regions or a 3¢ primer
complimentary to the constant region.(3) From 2004 onwards gDNA was amplified using 6VH
framework 1 primers combined with one JH consensus primer.{Hamblin, 2008 #63; van Dongen,
2003 #47} Genomic DNA or cDNA samples were directly sequenced and nucleotide sequences
were aligned to EMBL/GenBank, V-BASE sequence directory IMGT/VQUEST, using MacVector 4.0
sequence analysis software (International Biotechnologies, New Haven, USA) and Lasergene
(DNASTAR, Madison, USA). Percentage homology was calculated by counting the number of
mutations between the 5¢ end of FR1 and the 3¢ end of FR3. A cut-off of ≥98% germ-line homology
was taken to define the unmutated sub-set.(3)
Affymetrix SNP6.0
500ng of genomic tumour DNA was split into two aliquots and digested with either Nsp I or Sty I
restriction enzymes (New England BioLabs (NEB), Ipswich, MA, USA). Following restriction enzyme
inactivation, the sticky ends of the digested DNA were ligated to Nsp I or Sty I specific
adaptor/primers (Affymetrix, Santa Clara, USA) using T4 DNA ligase (NEB). Four and three aliquots
from the Nsp I and Sty I ligated products, respectively, were amplified using the TITANIUM DNA
amplification kit (Clontech, Mountain View, CA, USA). The products of all seven PCR reactions
were combined and purified using magnetic beads (Beckman Coulter, Takeley, UK). The amplified
DNA yield was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific,
Wilmington, USA), and the size distribution was assessed by gel electrophoresis. Subsequently, DNA
was fragmented to less than 200 bp (Affymetrix) and the size distribution of the fragmented
amplicons was assessed by agarose gel electrophoresis. Samples were end-labeled with a biotinylated
nucleotide using Terminal Deoxynucleotidyl Transferase (Affymetrix). The end-labeled products
were added to a hybridization solution, denatured, and applied to a Genome-Wide Human SNP
Array 6.0. Each array was placed in a GeneChip® Hybridization Oven 640, and incubated at 50°C
for 16hrs. Following hybridization, the DNA sample was removed from each microarray and
replaced with Array Holding Buffer. Arrays were then washed and stained on the Fluidics Station
450 (Affymetrix) using the GenomeWideSNP6_450 protocol
Agilent 8x60K aCGH
250ng of normal genomic reference (Promega, Southampton, UK) and tumor DNA was heat
fragmented and labeled with either Cy3 or Cy5 dyes, using the ULS labeling kit (Agilent
Technologies, Stockport, UK). Labeled DNA was filter purified using kreapure columns (Agilent).
Yield and degree of labeling or specific activity was measured using a NanoDrop ND-1000 (Thermo
Scientific). The labeled DNA was added to a hybridization solution, denatured and applied to an
8x60K Agilent array, according to manufacturers’ instructions, using a gasket slide and microarray
hybridization chamber (Agilent). Arrays were hybridized for 24hrs at 65°Cprior to post
hybridization washing with wash buffers 1 and 2 (Agilent) and acetonitrile (Sigma, St Louis, MO,
USA). Arrays were dried using stabilization and drying solution (Agilent) and scanned (Agilent Cscanner and Agilent Scanner Control software v8.3). Arrays were analyzed using Genomic
Workbench Standard edition 5.0 (Agilent). All achieved the manufacturers’ quality cut-off scores
(Derivative log ratio spread (DLRS) <0.3). Preliminary analysis identified statistically significant groups
of probes, based on z-scoring and a 2Mb genomic window size. The aberration detection method 1
(ADM1, Agilent) algorithm was used for a robust estimation of the position and extent of the
aberration, defined as a minimum of five consecutive probes that deviated by ±0.25 from a
normalized ratio of zero.
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