Supplementary methods
Genomic long range PCR
Expand Long Template PCR reagents (Roche) were used. A 20 ng aliquot of genomic
DNA was amplified in a reaction mix containing 1x PCR buffer, 1 U DNA
polymerase mix, 500 μM dNTPs, and 400 nM forward and reverse primers. The
thermal cycling conditions were: 95 C for 2 min; followed by 10 cycles of 94 C for 20
s, 66 C for 30s, 68 C for 10 min; followed by 25 cycles of 94 C for 20s, 66 C for 40s,
68 C for 10 min +10 s increment each cycle; then 68 C for 9 min. PCR products were
resolved by electrophoresis in 0.6% agarose gel stained with ethidium bromide.
Quantification of the GUSB control gene
DNA was extracted from fresh normal peripheral blood leukocytes following red cell
lysis, and used to prepare a GUSB standard. The amount of DNA in the standard was
determined by spectrophotometry, and this value was used to assign a pg amount of
amplifiable DNA to the standard. Real-time Q-PCR was performed using 2.5 μL of
sample (or standard) in a total reaction volume of 25 μL on an ABI Prism 7500
thermal cycler. The reaction mix contained 1x Universal Master Mix (Applied
Biosystems), 200 nM forward and reverse primers, and 100 nM hydrolysis
(TaqMan®) probe. The thermal cycling conditions were: 50 C for 2 min; followed by
95 C for 10 min; followed by 40 cycles of 95 C for 15s and 60 C for 1 min.
Fluorescence was measured during the 60 C extension phase of each PCR cycle. The
oligonucleotide sequences were: forward primer 5’GAAAAAATGAGGACGGGTACGT-3’, reverse primer 5’-
ATTTTGCCGATTTCATGACTGA-3’, and hydrolysis probe FAMATCCCATGAGCCAAACTGCCACTTACAC-TAMRA. The normal DNA was
serially diluted in buffered salmon sperm diluent and assayed to construct a standard
curve. Assays were performed in duplicate (within a single Q-PCR) and rejected if
there was greater than 2-fold variation between duplicates.
Patient-specific BCR-ABL1 DNA PCR
Genomic DNA was extracted from peripheral blood. If the blood sample was not
available, or its amplifiable DNA concentration was low, the corresponding bone
marrow sample was used. Three bone marrow samples were used (Patient #14 at 4
months; Patient #7 at 12 months and 18 months).
BCR-ABL1 DNA was amplified from 500 ng patient DNA in a total reaction volume
of 50 μL. Each reaction contained 1x AmpliTaq buffer, 1.25 U TaqGold DNA
polymerase (Applied Biosystems), 200 μM dNTPs, and 200 nM forward and reverse
primers. The thermal cycling conditions were: 95 C for 10 min; followed by 30 cycles
of 94 C for 30 s, 59 C for 30 s, 72 C for 30 s; then 72 C for 10 min.
A 2.5 μL aliquot of each first round PCR product was used in real-time PCR (total
reaction volume 25 μL) as described above, except that the hydrolysis probe
concentration was 300 nM. Patient-specific BCR-ABL1 DNA standard curves were
constructed using the serially diluted DNA from the diagnostic sample in which the
breakpoint was detected. The BCR-ABL1 standard DNA was amplified as described
for the follow-up samples. All positive results gave a threshold cycle number (Ct) <3
cycles after the Ct predicted to result from a single copy of BCR-ABL1, extrapolated
from the standard curve.
Semi-quantitative results were calculated using the Ct values of the positive patient
samples and the proportion of positive samples. The Ct for each positive replicate was
calculated as a pg amount of BCR-ABL1 DNA (using the standard curve for that
individual assay). The pg amounts of BCR-ABL1 DNA for every replicate were then
expressed as a number of cells and added together. The proportion of BCR-ABL1
DNA was then expressed as the total amount of BCR-ABL1 DNA divided by the total
amount of amplifiable DNA used.
Calculated sensitivity
The lower limit of detection of each individual assay is dependent on the amount of
nucleic acid used (dependent on the number of replicates performed), the integrity of
the nucleic acid, the presence of PCR inhibitors in the sample, and the efficiency of
the assay. Due to fundamental differences between mRNA and genomic DNA the
lower detection limit was calculated differently for RQ-PCR and for DNA PCR. The
calculated sensitivity values for all RNA and DNA samples prior to relapse are shown
in Table S2.
Definition of calculated sensitivity for DNA PCR
The number of amplifiable genomes (cells) in the sample was estimated using Q-PCR
for genomic GUSB. Using the GUSB control gene value we were able to calculate the
equivalent number of cells tested in each assay. The BCR-ABL1 and GUSB genes are
present in a constant ratio of 1:2. Therefore, we could accurately assign the lower
detection limit of each sample. Up to 20 replicates of 500 ng each were tested, giving
a maximum of 10 μg amplifiable DNA per sample. As a single cell contains 5.7 pg
amplifiable DNA this is approximately equivalent to 1.75 x 106 cells. Hence, the
lower detection limit was 6.24-log (log10 1.75 x 106). The number of cells tested is the
base 10 antilogarithm of the calculated sensitivity.
Definition of calculated sensitivity for RQ-PCR
Unlike a DNA-based assay, in RQ-PCR the number of copies of the mRNA control
gene cannot be used to determine the number of patient cells tested. The number of
BCR transcripts per cell is not known, and might vary between cell types (e.g.
myelocytes vs neutrophils). Methodological factors such as RNA extraction efficiency
and the efficiency of cDNA synthesis could also contribute to the indirect relationship
between cell number and BCR transcripts. This problem was approached by the EAC
by expressing the sensitivity relative to the baseline BCR-ABL1 ratio.1
Calculated sensitivity was defined by the formula log10 (control gene copy number x
standardized baseline BCR-ABL1 ratio/minimum number of copies detectable).1 The
laboratory-specific baseline BCR-ABL1 ratio defines the number of copies of BCRABL1 mRNA per copy of the control gene. Our laboratory uses the BCR control gene,
and the standardized baseline ratio is 0.8. The minimum number of copies of BCRABL1 mRNA that can reliably be detected in our assay is 10 copies (the lowest
number of copies of BCR-ABL1 in the standard curve). This threshold of 10 copies
helps to overcome: 1) the occurrence of weak positive signals in negative control
samples, and 2) sampling error.
Hence, if a sample contains 400,000 copies of the BCR control gene the (average)
maximum possible number of copies of BCR-ABL1 when using the standardized
baseline ratio is 0.8 x 400,000 (i.e. 320,000). With a detection limit of 10 copies a
positive result would be expected if the result were >1/32,000 relative to the pretreatment level of BCR-ABL1. In this case the estimated lower limit of detection
(calculated sensitivity) is 4.5-log (log10 32,000) below the standardized baseline.
Reference
1.
Gabert J, Beillard E, van der Velden VH, et al. Standardization and quality
control studies of 'real-time' quantitative reverse transcriptase polymerase chain
reaction of fusion gene transcripts for residual disease detection in leukemia - a
Europe Against Cancer program. Leukemia. 2003;17:2318-2357.