Professional Documents
Culture Documents
Key Words: bcr-abl; Quantitative RT-PCR; real-time RT-PCR; Reverse transcriptase–polymerase chain reaction
DOI: 10.1309/60A9C8WGEGHRNXEE
Our method uses a 5'-exonuclease technique to generate The quantitative real-time PCR was performed in the
fluorescence during the PCR process that is proportional to ABI 7700 with cycling as follows: an initial cycle for 10
the starting quantity of template, which is monitored in real- minutes at 95°C, followed by 40 biphasic cycles of 15
time by using an ABI PRISM 7700 Sequence Detection seconds at 95°C and 1 minute at 60°C. Each 96-well plate
System (Applied Biosystems, Foster City, CA).9,10 Briefly, included standard curves for p210, p190, and GAPD, as well
the principle of this method, called TaqMan (Applied as sensitivity controls, no-template controls, and no-RT
Biosystems), is as follows: A sequence-specific probe is controls. Patient samples were run in triplicate for each target
included in the PCR reaction, which is dual labeled with a (p210, p190, and GAPD), and there was sufficient space for
reporter fluorophore at the 5'-end and a quencher fluo- 4 patient samples on each plate. Each well contained 2.5 U
rophore at the 3'-end. The probe anneals to the region of of TaqGold, 1× PCR buffer, a 5.5-mmol/L concentration of
interest internal to the forward and reverse PCR primers. magnesium chloride, and a 250-µmol/L concentration of
During the extension phase, the 5'→3' exonuclease activity deoxynucleoside triphosphates (all from Applied
of the Taq polymerase will cleave the reporter molecule. Its Biosystems) and 2 µL of cDNA to constitute a final volume
release is accompanied by a fluorescent signal, which the of 25 µL. The GAPD wells included 50 pmol each of GAPD
ABI 7700 instrument measures and records in real time. forward and reverse primers and 10 pmol of GAPD probe.
The assay described herein quantitates the bcr-abl p210 The p210 wells included 10 pmol each of the p210 and abl
and p190 transcripts, as well as a housekeeping gene, GAPD. primers and 10 pmol of abl probe. The p190 wells included
We demonstrated that this assay is highly specific and that its 10 pmol each of the p190 and abl primers and 10 pmol of abl
sensitivity is such that it can reliably detect and quantitate 10 probe. Primers and probes were manufactured by Operon
pg of bcr-abl RNA (0.0001%) in a background of negative Technologies, Alameda, CA.
RNA. The p210 and p190 reaction used the same reverse
primer and probe (for abl), while the forward primer was
specific for the bcr breakpoint region. Each probe included
FAM (6-carboxy-fluorescein; emission, 518 nm) at the 5'-
Materials and Methods
end as the reporter and TAMRA (6-carboxy-tetramethyl-
rhodamine; emission, 582 nm) at the 3'-end as the quencher.
Samples The bcr-abl primer and probe sequences were designed using
We performed quantitative analyses on all specimens Primer Express software (Applied Biosystems). The GAPD
submitted to the Clinical Molecular Diagnosis Laboratory at primer and probe sequences were adapted from the
Stanford University Medical Center, Stanford, CA, from sequences used in Applied Biosystems’ GAPD Control
September 1996 through May 2001 on which our standard Reagents Kit. However, we used FAM as the reporting fluo-
qualitative bcr-abl reverse transcriptase (RT)-PCR was rophore, rather than VIC. Primer and probe sequences are
requested and performed. There were 372 bone marrow, listed in ❚Table 1❚.
blood, and pheresis product specimens from 145 patients for
which sufficient RNA and/or complementary DNA (cDNA) Qualitative RT-PCR
remained in our archives. In addition, we tested peripheral Our qualitative RT-PCR method used for routine clinical
blood samples from 50 patients not known to have any laboratory testing was adapted from Cross.11 It is a multiplex
myeloproliferative disorders. RT-PCR assay using only a single round of amplification.
Quantitative RT-PCR
RNA was isolated from peripheral blood or bone ❚Table 1❚❚
Primer and Probe Sequences for Quantitative bcr-abl
marrow leukocytes by a modified guanidinium-thiocyanate Determination
method according to the manufacturer’s instructions (RNA-
STAT, Tel-Test, Friendswood, TX, or TRIzol, Invitrogen, Primer Sequence
Carlsbad, CA). The reverse transcription reaction contained GAPD forward 5’-GAAGGTGAAGGTCGGAGTC-3’
the following in a 40-µL volume: 300 U of M-MLV enzyme, GAPD reverse 5’-GAAGATGGTGATGGGATTTC-3’
GAPD probe 5’-FAM-CAAGCTTCCCGTTCTCAGCC-TAMRA-3’
a 2.5-mmol/L concentration of dithiothreitol, 1× RT buffer p210 forward 5’-TGCTGACCAACTCGTGTGTG-3’
(all from Invitrogen); 40 U of RNasin (Promega, Madison, p190 forward 5’-AACTCGCAACAGTCCTTCGAC-3’
abl reverse 5’-CCATTCCCCATTGTGATTATAGC-3’
WI); 5-µg random hexamers (Amersham Biosciences, abl probe 5’-FAM-AAGACCCGGAGCTTTTCACCTTTAGTT
Piscataway, NJ); a 1-mmol/L concentration of deoxynucleo- ATGC-TAMRA-3’
side triphosphates (Amersham Biosciences); and 10 µg of
FAM, 6-carboxy-fluorescein; GAPD, glyceraldehyde-3-phosphate dehydrogenase;
total RNA. The reaction was incubated at 37°C for 2 hours. TAMRA, 6-carboxy-tetramethyl-rhodamine.
The multiplex primer set includes primers for the normal bcr bcr-abl–negative RNA with known quantities of targets.
transcript, the p210 fusion transcript, and the p190 fusion Tenfold serial dilutions of plasmid were spiked into 5-µg
transcript. Primer sequences and primers used are given in aliquots of negative RNA (derived from Stanford lymphoma
❚Table 2❚ and ❚Table 3❚. cell line OCI-Ly8) and reverse transcribed as described. The
cDNA then was used as a template for the real-time PCR reac-
Standards tion. The 10-fold plasmid dilutions spanned 7 logs (106 to 100).
All standards described were assayed in duplicate and In addition, we performed the quantitative bcr-abl assay
plotted using the SDS software package for the ABI 7700. on serial 10-fold dilutions of RNA from a p210-positive cell
Standard curves were considered valid if they achieved a line (Meg-01, American Type Culture Collection, Manassas,
correlation coefficient of more than 0.990 with a slope of VA) and a p190-positive cell line (JD1, courtesy of Michael
–3.1 to –3.5. Standard series for each of the 3 targets Cleary, MD, Stanford University) using bcr-abl–negative
(GAPD, bcr-abl p210, and bcr-abl p190) were run on every RNA as the diluent. The undiluted cell line RNA was called
plate in parallel with patient samples. Patient results must 100%, because we assumed that all of the cells in the bcr-abl
fall within the linear range for each target to be quantitated. cell line (ie, 100%) contained the fusion transcript.
For the quantity to be sufficient for testing, the GAPD must Following this model, the 10-fold dilution was called 10%,
yield results of at least 0.5 µg of RNA equivalent. the 100-fold was called 1%, and so on. According to our
Standards for the bcr-abl p210 and p190 assays were procedure, 10 µg of each RNA dilution was used for the
plasmids prepared from the PCR amplicons generated by the reverse-transcription reaction. The serial 10-fold dilutions
described primer sets. Fresh PCR products were cloned into spanned 9 logs, from 100% (containing 10 µg of bcr-
a pCR 2.1 vector using the TOPO-TA cloning kit according abl–positive cell line RNA) to 0.000001% (containing 0.1 pg
to the manufacturer’s instructions (Invitrogen), and the plas- of bcr-abl–positive cell line RNA).
mids were purified using the QIAprep mini-prep kit
(QIAgen, Valencia, CA). The identities of the purified plas- Reproducibility
mids were confirmed by sequencing (ABI BigDye kit and Between-run reproducibility of this method was
ABI310, Applied Biosystems). The plasmids were linearized assessed by performing the assay on 5 different patient
by Hind III digestion (New England Biolabs, Beverly, MA) samples on a single plate, which was repeated on the same
before quantitation and use. Each plate included a series of 6 cDNA samples on 3 different days. Within-run repro-
standards: 10-fold serial dilutions starting at 105 targets per ducibility was evaluated by performing replicate analyses of
well, down to 100 targets per well, for each of the p210 and a single specimen in each well of a 96-well plate.
p190 targets. An example of the amplification plots with the
associated standard curve for the p210 fusion transcript is Data Analysis
shown in ❚Figure 1❚. By using the Sequence Detector System software
The GAPD standards were prepared from commercial package for the ABI 7700, the mean threshold cycles for
human RNA (Stratagene, La Jolla, CA), using the same GAPD, p210, and p190 were obtained for each sample.
reverse transcription protocol as described. The series was Again using the SDS software, the mean threshold cycle was
composed of five 5-fold dilutions of cDNA corresponding to applied to the appropriate standard curve (run in parallel
an initial RNA quantity per RT reaction of 18, 3.6, 0.72, with the sample) to derive the target number (for p210 and
0.144, and 0.03 µg. p190) or micrograms of RNA equivalents (for GAPD). The
values derived from the standard curves then were used to
Sensitivity calculate 2 simple ratios: the number of p210 transcripts per
We assessed the sensitivity of the method in 2 ways. microgram of RNA equivalents and the number of p190
The recombinant bcr-abl plasmids were used to spike transcripts per microgram of RNA equivalents. The term
A B
101 55.00
50.00
45.00
100
Threshold Cycle
40.00
∆ Rn
35.00
30.00
10–1
25.00
A B C D E F 20.00
15.00
10–2 10.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 5.00
Cycle 100 101 102 103 104 105 106
Starting Quantity
❚Figure 1❚ Example of bcr-abl p210 standards. A, Amplification, bcr032801. Standards were as follows: A, 105; B, 104; C, 103; D,
102; E, 101; and F, 100. ∆Rn, change in fluorescence. B, Standard curve, bcr032801. Slope, –3.499; Y-intercept, 33.670;
correlation coefficient, 0.998. Red, unknown; black, standards.
RNA equivalents rather than RNA was chosen because the possibly because the amplicon is larger (284 base pairs [bp]
result represents the amount of signal present in the 2 µL of for the b2a2-p210 or 359 bp for the b3a2-p210 vs 381 bp
cDNA rather than some direct measurement of the RNA itself. for p190), which, according to the manufacturer, can
decrease the amplification efficiency in the TaqMan
system. The 10-pg sensitivity limit is equivalent to a 1 in
106 dilution (0.0001%).
Results
Of the 372 parallel-tested samples, 170 (45.7%) were
negative by the qualitative method. Of the 372 samples, 102
Specificity (27.4%) also were negative by the quantitative method, but
The results of the parallel testing of 372 patient samples 68 (18.3%) showed some p210 positivity by the quantitative
are given in ❚Table 4❚. method, albeit at a low level that cannot be accurately quanti-
In our parallel-tested specimens, the qualitative results tated. In other words, the signal was less than that of our
by breakpoint were as follows: p210-positive, 180 (48.4%); lowest standard, which necessarily defines the linearity limits
p190-positive, 22 (5.9%); and negative, 170 (45.7%). The of the assay. This phenomenon is considered further in the
quantitative results completely correlated by breakpoint. discussion section.
Moreover, 52 (28.9%) of the 180 p210-positive specimens
showed a small amount of p190 positivity as well, as Reproducibility
reported previously to be a common occurrence.12 Between-run reproducibility showed a coefficient of
We performed our quantitative bcr-abl assay on 50 variance of 12.3%, and the within-run reproducibility
samples from people not known to have any myeloproliferative showed a coefficient of variance of 13.8%.
disorders. This entire cohort was negative for both the p210
and p190 fusion transcripts, further demonstrating the speci-
❚Table 4❚❚
ficity of the assay.
Results of 372 Parallel-Tested Clinical Samples*
1,000,000
1,200,000 BMT #2
900,000
p210/µg RNA Equivalents
BMT #1
p210/µg RNA Equivalents
8,000 800,000
DLI
7,000 700,000
6,000 600,000
5,000 500,000
4,000 400,000
3,000 300,000
2,000 200,000
1,000 100,000
0
0
30
0
30
60
d+ 0
0
d+ 0
d+ 0
d+ 0
d+ 0
d+ 0
0
d+ 0
0
21
d+
9
12
15
18
24
27
33
36
30
30
60
90
0
d–
d+
d+
d+
21
d–
12
15
18
24
27
30
d+
d+
d+
d+
d+
d+
d+
d+
d+
d+
d+
d+
❚Figure 2❚ Longitudinal quantitative bcr-abl results. Patient ❚Figure 3❚ Longitudinal quantitative bcr-abl results. Patient with
with no relapse following allogeneic bone marrow relapse at day +100 after allogeneic bone marrow transplan-
transplantation (BMT). tation (BMT), who subsequently received donor lymphocyte
infusion (DLI) but died at day +996 following the second BMT.
Increased p210 values correlated with clinical relapse.
quantitation of both the major (p210) and the minor (p190) 30,000
d+150
d–30
d+30
d+90
d+210
d+270
d+330
d+395
d+455
d+515
d+575
d+695
d+760
d+820
d+880
d+940
d+635
Only Kreuzer et al14 included a measurement of the
p190 transcript in their method. We included the p190 tran-
script because a previous report indicated possible prognostic Time From BMT
significance of the p190 transcript in p210-positive patients.22
❚Figure 4❚ Longitudinal quantitative bcr-abl results. Patient
However, to our knowledge, this result has not been reported
with relapse at day +377 after allogeneic bone marrow
by others, and we did not see significant p190 expression in transplantation (BMT), who subsequently received donor
our patients with p210. Therefore, at present, there seems to lymphocyte infusion (DLI) and imatinib mesylate (STI).
be little usefulness in measuring the p190 transcript in Increased p210 values correlated with clinical relapse.
patients for whom p210 is the dominant transcript.
However, a second reason we chose to include the p190
transcript was so that p190-positive patients could be moni- products from multiple clinical sites and patient groups.
tored in our laboratory. Although uncommon in CML, the We established and validated a quantitative bcr-abl
p190 transcript is the dominant transcript in approximately method that is rapid and easy to perform, reproducible,
two thirds of Ph-positive ALL. 23 Hence, a substantial sensitive, and specific and includes the p210 and the p190
number of patients monitored at this institution would be transcripts. The sensitivity, specificity, and reproducibility
excluded from the benefits of quantitative bcr-abl moni- compare favorably with other real-time methods described in
toring were we to eliminate this breakpoint altogether. the literature. There are 2 important differences between our
However, it is not necessary to run the transcripts in tandem method and previously described methods. One is the detec-
for all patients to provide that service. tion of the p190 transcript, which permits us to offer
To our knowledge, this is the first report of a p210 and
p190 TaqMan method that used extensive parallel testing
with the existing method. Saffroy et al17 examined 186 spec- 101
quantitative bcr-abl monitoring to patients with Ph-positive 13. Faderl S, Talpaz M, Kantarjian H, et al. Should polymerase
ALL served by this institution. The second is the extensive chain reaction analysis to detect minimal residual disease in
patients with chronic myelogenous leukemia be used in
parallel testing against an unsorted series of clinical speci- clinical decision making? Blood. 1999;93:2755-2759.
mens. Although not established in the present study, it may 14. Kreuzer KA, Lass U, Nagel S, et al. Applicability of an absolute
be possible in future studies to establish a cutoff value above quantitative procedure to monitor intra-individual bcr/abl
which a CML relapse is imminent, permitting earlier inter- transcript kinetics in clinical samples from chronic myelog-
enous leukemia patients. Int J Cancer. 2000;86:741-746.
vention and, perhaps, better patient outcomes.
15. Mensink E, van de Locht A, Schattenberg A, et al.
Quantitation of minimal residual disease in Philadelphia
From the Department of Pathology, Stanford University School of chromosome positive chronic myeloid leukaemia patients
Medicine, Stanford, CA. using real-time quantitative RT-PCR. Br J Haematol.
1998;102:768-774.
Supported by grant 2PO1CA49605 from the National
Institutes of Health, Bethesda, MD. 16. Olavarria E, Kanfer E, Szydlo R, et al. Early detection of bcr-
abl transcripts by quantitative reverse transcriptase–
Address reprint requests to Dr Zehnder: Dept of Pathology,
polymerase chain reaction predicts outcome after allogeneic
Stanford University, 300 Pasteur Dr, Mail Code 5324, Stanford, stem cell transplantation for chronic myeloid leukemia.
CA 94305. Blood. 2001;97:1560-1565.
Acknowledgment: We are grateful to Daniel Arber, MD, for
17. Saffroy R, Lemoine A, Brezillon P, et al. Real-time
critical reading of the manuscript and helpful suggestions. quantitation of bcr-abl transcripts in haematological
malignancies. Eur J Haematol. 2000;65:258-266.
18. Elmaagacli A, Freist A, Hahn M, et al. Estimating the relapse
stage in chronic myeloid leukaemia patients after allogeneic
References stem cell transplantation by the amount of bcr-abl fusion
transcripts detected using a new real-time polymerase chain
1. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. reaction method. Br J Haematol. 2001;113:1072-1075.
1999;340:1330-1340.
19. Preudhomme C, Revillion F, Merlat A, et al. Detection of
2. Daley GQ, van Etten RA, Baltimore D. Induction of chronic bcr-abl transcripts in chronic myeloid leukemia (CML) using
myeloid leukemia by the p210 bcr/abl gene of the Philadelphia a “real time” quantitative RT-PCR assay. Leukemia.
chromosome. Science. 1990;247:824-830. 1999;13:957-964.
3. Muller AJ, Young JC, Pendergast AM, et al. BCR first exon 20. Elmaagacli AH, Beelen DW, Opalka B, et al. The amount of
sequences specifically activate the bcr/abl tyrosine kinase bcr-abl fusion transcripts detected by the real-time
oncogene of Philadelphia chromosome positive human quantitative polymerase chain reaction method in patients
leukemia. Mol Cell Biol. 1991;11:1785-1792. with Philadelphia chromosome positive chronic myeloid
4. Svarch E, de la Torre E. Myelomonocytic leukaemia with a leukemia correlates with the disease stage. Ann Hematol.
preleukaemic syndrome and Ph1 chromosome in monozygotic 2000;79:424-431.
twins. Arch Dis Child. 1977;52:72-74. 21. Biernaux C, Loos M, Sels A, et al. Detection of major bcr-abl
5. Hoppin EC, Lewis JP. Polycythemia rubra vera progressing in gene expression at a very low level in blood cells of some
Ph1-positive chronic myelogenous leukemia. Ann Intern Med. healthy individuals. Blood. 1995;86:3118-3122.
1975;83:820-823. 22. Serrano J, Roman J, Sanchez J, et al. Molecular analysis of
6. Price CM, Rasool FS, Shivji MK, et al. Rearrangement of the lineage-specific chimerism and minimal residual disease by RT-
breakpoint cluster region and expression of p210 bcr-abl in a PCR of p210 bcr-abl and p190 bcr-abl after allogeneic bone
“masked” Philadelphia chromosome–positive acute myeloid marrow transplantation for chronic myeloid leukemia:
leukemia. Blood. 1988;72:1829-1832. increasing mixed myeloid chimerism and p190 bcr-abl detection
7. Hochhaus A, Weisser A, La Rosee P, et al. Detection and precede cytogenetic relapse. Blood. 2000;95:2659-2665.
quantification of residual disease in chronic myelogenous 23. Butturini A, Arlinghaus RB, Gale RP. bcr/abl acute leukemia.
leukemia. Leukemia. 2000;14:998-1005. Leuk Res. 1996;20:523-529.
8. Lin F, van Rhee F, Goldman JM, et al. Kinetics of increasing 24. Emig M, Saussele S, Wittor H, et al. Accurate and rapid
bcr-abl transcript numbers in chronic myeloid leukemia analysis of residual disease in patients with CML using
patients who relapse after bone marrow transplantation. specific fluorescent hybridization probes for real time
Blood. 1996;87:4473-4478. quantitative RT-PCR. Leukemia. 1999;13:1825-1832.
9. Heid CA, Stevens J, Livak KJ, et al. Real-time quantitative 25. Bolufer P, Sanz GF, Barragan E, et al. Rapid quantitative
PCR. Genome Res. 1996;6:986-994. detection of bcr-abl transcripts in chronic myeloid leukemia
10. Holland PM, Abramson RD, Watson R, et al. Detection of patients by real-time reverse transcriptase polymerase-chain
specific polymerase chain reaction product by utilizing the 5' reaction using fluorescently labeled probes. Haematologica.
to 3' exonuclease activity of Thermus aquaticus DNA 2000;85:1248-1254.
polymerase. Proc Natl Acad Sci U S A. 1991;88:7276-7280. 26. Amabile M, Giannini B, Testoni N, et al. Real-time
11. Cross NCP. Detection of bcr-abl in hematological quantification of different types of bcr-abl transcript in
malignancies by RT-PCR. In: Cotter FE, ed. Molecular chronic myeloid leukemia. Haematologica. 2001;86:252-259.
Diagnosis of Cancer. Totowa, NJ: Humana Press; 1996. 27. Eder M, Battmer K, Kafert S, et al. Monitoring of bcr-abl
12. van Rhee F, Hochhaus A, Lin F, et al. p190 bcr-abl mRNA is expression using real-time RT-PCR in CML after bone
expressed at low levels in p210-positive chronic myeloid and marrow or peripheral blood stem cell transplantation.
acute lymphoblastic leukemias. Blood. 1996;87:5213-5217. Leukemia. 1999;13:1383-1389.