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Articles |
Departments of
1
Cellular Pathology and
2
Hematopathology, Armed Forces Institute of Pathology, 6825 16th St. NW, Washington, DC 20306-6000.
3
Department of Pathology, Sir Mortimer B. Davis Jewish
General Hospital and McGill University, Faculty of Medicine, Montreal,
Quebec, H3T 1E2 Canada.
a Author for correspondence. Fax 202-782-7623; e-mail
bijwaard{at}afip.osd.mil.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, ß2-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, M02058.
Results: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes.
Conclusions: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 h. The assay involves RT-PCR with product detection by the 5'-nuclease (TaqMan) technology. Thermocycling and signal detection are performed on the ABI Prism 7700 Sequence Detection System, an integrated system consisting of a thermal cycler, a laser for fluorescence induction, and a charge-coupled device detector for the real-time detection of PCR products. Reactions are carried out in the presence of an oligonucleotide probe labeled with two fluorescent dyes, a reporter at the 5' end and a quencher at the 3' end of the molecule. The structure-specific 5'-nuclease activity (16)(17) of Taq polymerase digests the probe only when annealed to the specific product generated during PCR amplification. Degradation of the probe separates the reporter dye from the quencher, leading to an increase in fluorescence, which is detected quantitatively in real time by the instrument.
The ability of the instrument to measure fluorescence from several dyes simultaneously allows for multiplex amplifications, with simultaneous detection of different targets in the same reaction. The Sequence Detection System software provided with the instrument analyzes the fluorescence data generated during the reaction and calculates the cycle number at which fluorescence crosses a threshold value determined by analysis of data from early cycles in the amplification process. This cycle number, the threshold cycle (CT) value, is related to the quantity of specific target in the reaction, with larger quantities of starting material leading to lower CT values.
Assuming the same amplification efficiencies, the relative expression
of two genes can be estimated from the difference in
CT values, the 
CT. The
CT value can be normalized to a control by
subtracting the CT value obtained with the
control from that obtained with the test specimen to yield a
CT (18). The assay described in
this report involves the determination of a
CT value. CT values
for cyclin D1 and ß2M in the test specimen are
compared with those obtained with the MCL cell line M02058, which
expresses high amounts of cyclin D1. The greater the
CT value, the lower the expression of
cyclin D1 in the specimen. By choosing an appropriate cutoff value for
CT, the assay achieved a sensitivity and a
specificity approaching 100% for MCL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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primer and probe design
Primer and probe sequences for cyclin D1 and ß2M are
presented in Table 1
. Primers and probes were designed using Primer Express software
(PE Applied Biosystems). The cyclin D1 probe was labeled with the
reporter dye 6-carboxy fluorescein and the ß2M probe with
the reporter dye VIC. Probes were purchased from PE Applied Biosystems
or Integrated DNA Technologies, Inc. For both genes, the primers were
placed in different exons to minimize amplification from residual DNA
in the RNA preparations. Both the cyclin D1 and ß2M
probes were designed to cross a splice junction to minimize signal
generation resulting from amplification of genomic DNA.
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rna extraction
To extract RNA from FFPE specimens, six 6-µm sections were
placed in a 1.5-mL microcentrifuge tube, and the samples were
deparaffinized by the addition of 800 µL of Hemo-DE (Fisher
Scientific) and 400 µL absolute ethanol. The tissue fragments were
pelleted by centrifugation, the supernatant was decanted, and the
pellet washed with 1 mL of absolute ethanol. The supernatant was
discarded after centrifugation, and the samples were air-dried. The
tissue pellets were digested overnight at 55 °C in an extraction
buffer containing 20 mmol/L Tris-HCl (pH 7.6), 20 mmol/L EDTA, 10
g/L sodium dodecyl sulfate, and 0.5 g/L Proteinase K
(20). Fresh tissue specimens (20 µL tissue volume) were
digested in the same extraction buffer. RNA was purified from the
digested fresh or paraffin-embedded tissue by TRIzol LS (Gibco/Life
Technologies, Ltd.) according to the manufacturer’s instructions. RNA
was purified from cell lines using the TRIzol reagent. After
isopropanol precipitation, the RNA pellet was hydrated in 30–50 µL
of diethylpyrocarbonate-treated H2O
(Research Genetics), incubated at 55 °C for 10 min, and stored at
-70 °C.
RNA used in Northern blot analysis was isolated from fresh tissue according to the procedure described by Chomczynski and Sacchi (21). Lyophilized RNA was resuspended in diethylpyrocarbonate-treated H2O to a concentration of 50 ng/µL.
M02058 and Raji cells were grown at 37 °C in a 5%
CO2 atmosphere in RPMI-1640 medium (Gibco/Life
Technologies) supplemented with 100 mL/L fetal calf serum, 2 mmol/L
L-glutamine, 0.1 mmol/L sodium pyruvate, 1x MEM
nonessential amino acids, 1x MEM vitamins, 100 kilounits/L penicillin,
and 100 mg/L streptomycin. Cells were pelleted and washed with
1x Dulbecco’s phosphate-buffered saline, counted, and adjusted to
1 x 106 cells/mL. For the experiment shown
in Fig. 2
, serial dilutions of M02058 cells were prepared in Raji
before RNA isolation. The cells were pelleted in sterile 1.5-mL
microcentrifuge tubes, and RNA was extracted using TRIzol as described
above.
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rt-pcr
Assays were performed in MicroAmp optical reaction tubes and caps
(PE Applied Biosystems). Two quantities of each RNA preparation were
tested, 1 and 50 ng for the control M02058, or 1 and 3 µL of
each specimen. Reactions were set up in triplicate at each quantity of
RNA, producing six separate RT-PCR reactions for each specimen. RNA was
reverse-transcribed with random primers in a 10-µL reaction mixture
containing the RNA specimen plus 1x PCR Buffer II (PE Applied
Biosystems), 5.5 mM MgCl2, 100 mM dithiothreitol,
0.125 µg of Random Primers (Gibco/Life Technologies), 30 U of M-MLV
Reverse Transcriptase, 2 units of RNase inhibitor (Gibco/Life
Technologies), and 0.5 mmol/L each dNTP. Reactions were incubated for
60 min at 37 °C, heated for 5 min to 95 °C, and placed at
4 °C.
PCR was performed in a 50-µL reaction containing the 10 µL of the reverse transcription reaction, 1x Universal PCR Master Mix (PE Applied Biosystems), 15 pmol of each primer, and 5 pmol of each probe. The samples were placed in the ABI Prism 7700 Sequence Analyzer, which was set to detect both 6-carboxy-fluorescein and VIC reporter dyes simultaneously. To increase resolution between the two dyes, the spectral compensation feature was used. After initial incubations at 50 °C for 2 min to allow uracil-N-glycosylase digestion and 95 °C for 10 min to activate the AmpliTaq Gold, both of which are provided by the Universal PCR Master Mix, the samples were amplified for 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min.
data analysis
CT values for each reaction were determined
using TaqMan SDS analysis software. For each amount of RNA tested, the
triplicate CT values were averaged. Because
CT values vary linearly with the logarithm of the
amount of RNA, this average represents a geometric mean. The average
CT value for ß2M was subtracted
from the average cyclin D1 CT value to yield the
CT value. The CT
values were then averaged for both amounts of sample. The average
CT value for the positive control was then
subtracted from the average CT value of each
sample to give the CT. Three different
preparations of the positive control RNA yielded
CT values with a range of 0.46 cycles (SD
= 0.23 cycles).
northern blot analysis
Total RNA (5 µg) from eight fresh tissue samples and two cell
lines was electrophoresed through a 1% denaturing formaldehyde agarose
gel and transferred to a Hybond-N nylon membrane (Amersham Pharmacia
Biotech). The RNA was fixed by ultraviolet cross-linking. The
membrane was prehybridized for 2–4 h and hybridized overnight, as
described previously (22). The blot was probed with a 1.4-kb
BCL-1-specific radiolabeled cDNA probe, PL-8 (23) (obtained
from A. Arnold, Massachusetts General Hospital, Boston, MA) in the same
prehybridization solution with the addition of 100 g/L dextran
sulfate. The membranes were rinsed at room temperature in 2x standard
saline citrate (1x = 0.15 mol/L NaCl and 0.015 mol/L sodium
citrate), washed for 1 h at 65 °C in 0.1x standard
saline citrate containing 1 g/L sodium dodecyl sulfate, and rinsed in
0.1x standard saline citrate at room temperature, followed by exposure
to x-ray film at -70 °C for 1–5 days.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CT value obtained from M02058 RNA
varied by less than 0.1 cycle in assays containing total cellular RNA
quantities ranging from 1 to 50 ng. This result indicates nearly equal
amplification efficiencies and validates the
CT approach for quantifying relative
amounts of expression (18). Fig. 1
illustrates typical assay results. Triplicate amplifications at
one level of lysate for the M02058 control and an FFPE MCL produced
nearly identical, overlapping amplification curves, from which
CT values were calculated. In this example, the
M02058 assay yielded average CT values of 21.6
and 21.8 for cyclin D1 and ß2M, respectively, for a
CT of -0.2. The MCL yielded average
CT values of 32.2 and 28.6, for a
CT of 3.6. Therefore, for this specimen, the
CT was 3.6 - (-0.2) = 3.8.
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To evaluate the analytical sensitivity, accuracy, and precision of the
assay, CT values were determined on the
serial dilutions of M02058 into Raji. RNA was purified from
106 cells from each dilution. Aliquots
representing 2 and 6 x 101
cells were
assayed for cyclin D1 expression in the quantitative RT-PCR assay (Fig. 2
). A cyclin D1 signal first became detectable at 1 in 50 000
cells (0.002%). The within-run CV for the
CT values was <3% at high, intermediate,
and low cyclin D1 expression as estimated from sixfold replicate assays
on each of these samples. The SDs of CT
values between runs, estimated by running three dilutions of M02058
into Raji on 5 separate days, were 0.34, 0.34, and 0.56
CT units for samples having
CT values of 0.91, 3.68, and 12.95,
respectively.
The assay was next characterized for its ability to distinguish MCL
from other lymphomas and from reactive conditions. Fifty-one cases,
including 20 MCLs, were tested for cyclin D1 expression (Table 2
; results for individual cases available in the supplemental
table at
http://www.clinchem.org/content/vol47/issue2). The
distribution of CT values obtained (Fig. 3
) indicated that the assay has clinical utility when interpreted
relative to a cutoff value chosen to distinguish specimens that show
cyclin D1 overexpression from those that do not. For this panel of
cases, a cutoff CT value of 4 produced a
sensitivity and a specificity of 100%: All 20 MCL cases were
determined to be positive for cyclin D1 overexpression
(CT <4), whereas none of the other types
of lymphoma or reactive lymph nodes demonstrated
CT values <4. Two non-MCL samples, a
B-cell lymphoma and a splenic peripheral T-cell lymphoma, yielded
CT values between 4 and 5.
Immunohistochemical staining of these specimens for cyclin D1
demonstrated positive staining of epithelial cells but not tumor within
these specimens, an observation consistent with the known pattern of
cyclin D1 expression (24). Because
CT values between 4 and 5 can result from
the presence of epithelial cells in a specimen, values within this
range are reported as indeterminate for cyclin D1 overexpression. The
results of the titration experiment (Fig. 2
) indicated that the
threshold value of 4.0 would be crossed with a minimum representation
of 1.0–2.5% cyclin D1-overexpressing cells (with cyclin D1 expression
equal to or greater than that of M02058) in a background of
nonexpressors.
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To further evaluate the accuracy of the assay, results obtained by
quantitative real-time PCR were compared with Northern blot and
semiquantitative RT-PCR results obtained on a subset of the specimens
(Table 2
). Consistent with previous results, cyclin D1 expression was
observed in all tumors and cell lines tested with the exception of the
Raji cell line, in which no cyclin D1 signal was obtained even after 40
cycles of amplification (13)(25). High-quality
RNA suitable for Northern blot analysis was available for eight cases.
The qualitative amounts cyclin D1 expressed, as determined by Northern
blot, were concordant in each case with the results obtained with the
quantitative RT-PCR assay. Thirteen of the MCLs, 9 of the
non-mantle-cell lymphomas and reactive lymph nodes, and 2 cell lines
were assayed by the semiquantitative expression assay reported
previously (13). With the single exception of HeLa, which
yielded a CT value <4.0 but was considered
to express an intermediate amount of cyclin D1 by the other methods,
there was complete concordance of results among the three assays.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many of the chromosome 11q13 breakpoints in MCL map within a 1-kb region designated the major translocation cluster. The clustering of breakpoints at both 11q13 and 14q32 make detection of the translocation amenable to PCR-based assays (4)(26). However, the 11q13 breakpoints lie within the major translocation cluster in only 30–50% of cases. Consequently, most PCR-based assays designed to amplify the translocation breakpoint will yield false-negative results for >50% of MCLs because of breakpoints lying outside of the major translocation cluster. However, when detectable by PCR, the t(11;14) breakpoint provides a molecular marker unique to tumor cells, thereby allowing the detection of minimal residual disease. Whereas the quantitative RT-PCR assay detected cyclin D1 overexpression with 1–2.5% high-expressing cells in a background of nonexpressing cells, direct detection of the t(11;14) breakpoint has the potential to detect one tumor cell per 101 –105 nucleated cells in a specimen.
Another alternative approach for assessing cyclin D1 expression involves direct detection of the protein, by either Western blot or immunohistochemistry. Both of these methods have substantial limitations. In our previous study, only 70% of MCLs demonstrated cyclin D1 protein expression by immunohistochemistry, indicating a lack of sensitivity (13). The qualitative nature of these methods often does not permit distinction between low and high cyclin D1 expression. Cyclin D1 protein expression has been demonstrated by immunohistochemistry or Western blot in a variety of lymphoproliferative disorders other than MCL, including hairy cell leukemia, plasmacytoma, multiple myeloma, B-cell chronic lymphocytic leukemia, B-prolymphocytic leukemia, plasma cell leukemia, and splenic marginal zone lymphoma (4)(27)(28)(29)(30)(31)(32). Thus, nonquantitative assays for cyclin D1 protein generally lack specificity for MCL. The results presented in this report demonstrate that this lack of specificity can be overcome by quantitative real-time PCR technology.
In the application of this and other quantitative cyclin D1 assays, it
is important to separate lymphoid from epithelial components of the
specimen that would typically express cyclin D1 (24). This
potential problem is illustrated by the transbronchial biopsy specimen
included in this study, which yielded a CT
near the cutoff value of 4.0 because of epithelial cell expression of
cyclin D1. A similar phenomenon was observed with an adenoid submitted
to our laboratory, in which a CT <4.0 was
obtained because of expression in the squamous epithelium overlying the
lymphoid tissue. It is also important to recognize that, in addition to
MCL, cyclin D1 overexpression can occur in other tumors. Overexpression
occurs with high frequency in parathyroid adenomas because of an
inv11(q13;p13) translocation, which juxtaposes the cyclin D1 gene with
the parathyroid hormone gene on 11p13 (23). Overexpression
of cyclin D1 has also been demonstrated in some subsets of a variety of
solid tumors, including carcinomas of the breast, lung, head and neck,
esophagus, and colon
(24)(27)(29)(31)(33)(34)(35)(36)(37).
In conclusion, we have described a quantitative real-time RT-PCR assay for cyclin D1 expression. The assay was shown to have a sensitivity and a specificity of nearly 100% on lymphoid tissue specimens. The assay is rapid and easy to perform relative to alternative technologies and has clinical utility in distinguishing MCL from other lymphomas and reactive lymphoid proliferations.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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