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Quantitative real-time reverse transcription-PCR assay for cyclin D1 expression: utility in the diagnosis of mantle cell lymphoma.

Overexpression of the cell cycle regulator cyclin D1 is a hallmark of mantle cell lymphoma (MCL) [4] a tumor of immature B cells that accounts for 5-10% of malignant non-Hodgkin lymphomas in adults (1-3). In most cases, overexpression results from the translocation t(11;14)(q13; q32), which places the gene for cyclin D1 in close proximity to the immunoglobulin heavy chain enhancer (4,5). Detection of the t(11;14) translocation has clinical utility in distinguishing MCL from other small B-cell lymphomas, which can be histologically similar (6). The translocation can be detected by Southern blot, cytogenetics, PCR, or fluorescence in situ hybridization. Of these methods, fluorescence in situ hybridization has the highest reported sensitivity, with the detection of 95-100% of cases reported, and PCR the lowest sensitivity, with the detection of 30-50% of cases (7-12). An alternative approach, quantification of cyclin D1 RNA expression, also permits the detection of MCL with high sensitivity. In a previous study, we described a reverse transcription-PCR (RTPCR) method for detecting cyclin D1 mRNA (13). RT-PCR detected the cyclin D1 message in nearly all MCL specimens tested, but lacked specificity, in that 65% of non-mantle-cell lymphomas also contained detectable cyclin D1 mRNA. By normalizing the cyclin D1 signal to a control generated from the ubiquitously expressed [[beta].sub.2]-microglobulin ([[beta].sub.2]M) mRNA to yield a semiquantitative result, we found that MCLs could be reliably distinguished from other lymphomas by their increased cyclin D1 expression. We now report the development of a quantitative real-time RT-PCR assay for cyclin D1 expression with high sensitivity and specificity for MCL. Advantages of this assay include a significant reduction in labor relative to previous assays (13-15), greater reproducibility, and a reduction in turnaround time from several days to ~4 h.

The assay involves RT-PCR with product detection by the 5'-nuclease (TagMan) 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 ([C.sub.T]) value, is related to the quantity of specific target in the reaction, with larger quantities of starting material leading to lower [C.sub.T] values.

Assuming the same amplification efficiencies, the relative expression of two genes can be estimated from the difference in [C.sub.T] values, the [Delta][C.sub.T]. The OCT value can be normalized to a control by subtracting the [Delta][C.sub.T] value obtained with the control from that obtained with the test specimen to yield a [Delta][Delta][C.sub.T] (18). The assay described in this report involves the determination of a [Delta][Delta][C.sub.T] value. [C.sub.T] values for cyclin D1 and [[beta].sub.2]M 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 [Delta][Delta][C.sub.T] value, the lower the expression of cyclin D1 in the specimen. By choosing an appropriate cutoff value for [Delta][Delta][C.sub.T], the assay achieved a sensitivity and a specificity approaching 100% for MCL.

Materials and Methods


Fifty-one cases were selected for study, including 39 formalin-fixed, paraffin-embedded (FFPE) tissues from 1980 to 1997 and 12 fresh tissue samples. Fourteen of the FFPE tissue cases and 6 of the fresh tissue cases were classified as MCL according to the REAL classification (19). Additional data on the individual mantle cell lymphoma cases, including fixation and cyclin D1 expression as determined by immunochemical stain or Northern blot are available as a supplemental table in the online version of this journal ( Twenty-one cases consisted of non-mantle-cell lymphomas or leukemias (4 follicular lymphomas, 5 B-cell lymphomas, 5 anaplastic large cell lymphomas, 5 T-cell lymphomas, 1 acute promyelocytic leukemia, and 1 acute lymphocytic leukemia). The remaining 10 cases included 2 atypical lymphoid infiltrates and 8 cases of reactive lymphoid hyperplasia, of which 2 were considered to represent florid reactive hyperplasia. HeLa and Raji cell lines were obtained from the American Type Culture Collection. The M02058 MCL cell line was provided by T.C. Meeker (University of Kentucky, Lexington, KY).


Primer and probe sequences for cyclin D1 and [[beta].sub.2]M 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 (3ZM probe with the reporter dye VIC. Probes were purchased from PE Applied Bio-systems 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 [[beta].sub.2]M probes were designed to cross a splice junction to minimize signal generation resulting from amplification of genomic DNA.


To extract RNA from FFPE specimens, six 6-[micro]m sections were placed in a 1.5-mL microcentrifuge tube, and the samples were deparaffinized by the addition of 800 [micro]L of Hemo-DE (Fisher Scientific) and 400 [micro]L 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 [degrees]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 [micro]L 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 [micro]L of diethylpyrocarbonate-treated [H.sub.2]O (Research Genetics), incubated at 55 [degrees]C for 10 min, and stored at -70 [degrees]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 [H.sub.2]O to a concentration of 50 ng/[micro]L.

M02058 and Raji cells were grown at 37 [degrees]C in a 5% [CO.sub.2] 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, 1 x MEM nonessential amino acids, 1 x MEM vitamins, 100 kilounits/L penicillin, and 100 mg/L streptomycin. Cells were pelleted and washed with 1 x Dulbecco's phosphate-buffered saline, counted, and adjusted to 1 x [10.sup.6] 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.


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 [micro]L 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-[micro]L reaction mixture containing the RNA specimen plus 1 x PCR Buffer II (PE Applied Biosystems), 5.5 mM Mg[Cl.sub.2], 100 mM dithiothreitol, 0.125 [micro]g 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 [degrees]C, heated for 5 min to 95 [degrees]C, and placed at 4 [degrees]C.

PCR was performed in a 50-[micro]L reaction containing the 10 [micro]L of the reverse transcription reaction, 1 x 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 [degrees]C for 2 min to allow uracil-N-glycosylase digestion and 95 [degrees]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 [degrees]C for 15 s, followed by 60 [degrees]C for 1 min.


[C.sub.T] values for each reaction were determined using TaqMan SDS analysis software. For each amount of RNA tested, the triplicate [C.sub.T] values were averaged. Because [C.sub.T] values vary linearly with the logarithm of the amount of RNA, this average represents a geometric mean. The average [C.sub.T] value for [[beta].sub.2]M was subtracted from the average cyclin D1 [C.sub.T] value to yield the [Delta][C.sub.T] value. The [Delta][C.sub.T] values were then averaged for both amounts of sample. The average [Delta][C.sub.T] value for the positive control was then subtracted from the average [Delta][C.sub.T] value of each sample to give the [Delta][Delta][C.sub.T]. Three different preparations of the positive control RNA yielded [Delta][C.sub.T] values with a range of 0.46 cycles (SD = 0.23 cycles).


Total RNA (5 [micro]g) 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 2 x standard saline citrate (1 x = 0.15 mol/L NaCI and 0.015 mol/L sodium citrate), washed for 1 h at 65 [degrees]C in 0.1 x standard saline citrate containing 1 g/L sodium dodecyl sulfate, and rinsed in 0.1 x standard saline citrate at room temperature, followed by exposure to x-ray film at -70 [degrees]C for 1-5 days.


A quantitative real-time RT-PCR assay was designed for the ABI Model 7700 Sequence detection system using the 5'-nuclease (TagMan) technology for signal detection. Primers and probes were designed to avoid amplification and signal generation from genomic DNA. When tested with either 90 ng of HeLa cell or 50 ng of human tonsil genomic DNA, no signal was detected for either [[beta].sub.2]M or cyclin D1, indicating that neither the [[beta].sub.2]M nor the cyclin D1 genes or possible pseudogenes that might be present interfered with this assay. The [Delta][C.sub.T] 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 [Delta][Delta][C.sub.T] 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 [C.sub.T] values were calculated. In this example, the M02058 assay yielded average [C.sub.T] values of 21.6 and 21.8 for cyclin D1 and (3ZM, respectively, for a [Delta][C.sub.T] of -0.2. The MCL yielded average [C.sub.T] values of 32.2 and 28.6, for a [Delta][C.sub.T] of 3.6. Therefore, for this specimen, the [Delta][Delta][C.sub.T] was 3.6 - (-0.2) = 3.8.

To evaluate the analytical sensitivity, accuracy, and precision of the assay, [Delta][Delta][C.sub.T] values were determined on the serial dilutions of M02058 into Raji. RNA was purified from [10.sup.6] cells from each dilution. Aliquots representing 2 and 6 x [10.sup.4] 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 [Delta][Delta][C.sub.T] 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 [Delta][Delta][C.sub.T] values between runs, estimated by running three dilutions of M02058 into Raji on 5 separate days, were 0.34, 0.34, and 0.56 OOCT units for samples having [Delta][Delta][C.sub.T] 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 The distribution of [Delta][Delta][C.sub.T] 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 [Delta][Delta][C.sub.T] value of 4 produced a sensitivity and a specificity of 100%: All 20 MCL cases were determined to be positive for cyclin D1 overexpression ([Delta][Delta][C.sub.T] <4), whereas none of the other types of lymphoma or reactive lymph nodes demonstrated [Delta][Delta][C.sub.T] values <4. Two non-MCL samples, a B-cell lymphoma and a splenic peripheral T-cell lymphoma, yielded [Delta][Delta][C.sub.T] 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 [Delta][Delta][C.sub.T] 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 Y102058) in a background of nonexpressors.



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 [Delta][Delta][C.sub.T] 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.



The quantitative real-time RT-PCR assay described in this report represents a major improvement over other assays designed to quantify cyclin D1 expression in tissue specimens. The assay is rapid, technically less demanding than previously available methods, and demonstrates a sensitivity and a specificity approaching 100% on lymphoid tissue. The assay therefore has clinical utility in distinguishing MCL from other lymphomas and reactive lymphoid proliferations. An additional advantage provided by this assay is its applicability to FFPE tissue. Neither genomic Southern blotting nor Northern blotting of tumor RNA can be performed successfully with the degraded nucleic acids obtained from fixed and embedded tissue specimens.

Many of the chromosome 11g13 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 [10.sup.4]-[10.sup.5] 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 (4,27-32). 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 [Delta][Delta][C.sub.T] 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 [Delta][Delta][C.sub.T] <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(g13;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, 33-37).

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.

This work was supported by intramural funds of the Armed Forces Institute of Pathology. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. This is a US Government work; there are no restrictions on its use.

Received August S, 2000; accepted December 5, 2000.


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[4] Nonstandard abbreviations: MCL, mantle cell lymphoma; RT-PCR, reverse transcripfion-PCR; [[beta].sub.2]M, [[beta].sub.2]-microglobulin; [C.sub.T], threshold cycle; and FFPE, formalin-fixed, paraffin-embedded.


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

* Author for correspondence. Fax 202-782-7623; e-mail
Table 1. Primers and probes.

Primer/Probe Sequence

Cyclin D1 (86-bp product)
 Cycl-304F 5'-CCG TCC ATG CGG AAG ATC-3'
 Cycl-389R 5'-ATG GCC AGC GGG AAG AC-3'
 Cycl-334TR probe 5'-[6-FAM] a CTT CTG TTC CTC
[[beta].sub.2]M (85-bp product)
 [[beta].sub.2]M-246F 5'-TGA CTT TGT CAC AGC CCA AGA TA-3'
 [[beta].sub.2]M-330R 5'-AAT CCA AAT GCG GCA TCT TC-3'
 [[beta].sub.2]M-275R probe 5'-[VIC] TGA TGC TGC TTA CAT GTC

(a) 6-FAM, 6-carboxy-fluorescein; TAMRA, 6-carboxy-tetramethylrhodamine.

Table 2. Cases and results

 No. of

 MCL 20
 Follicular lymphoma 4
 Anaplastic large cell lymphoma 5
 Acute lymphocytic leukemia 1
 Acute promyelocytic leukemia 1
 T-cell lymphoma 5
 B-cell lymphoma 5
 Atypical lymphoid infiltrate 2
 Reactive hyperplasia 6
 Floridly reactive 2
Cell lines
 Raji (Burkitt lymphoma)
 HepG2 (Hepatocellular carcinoma)
 HeLa (adenocarcinoma)

 Average [Delta][Delta][C.sub.T]
 for cyclin D1 (range)

 MCL 1.92 (-0.57 to 3.69)
 Follicular lymphoma 6.39 (5.24-8.68)
 Anaplastic large cell lymphoma 7.43 (6.8-7.88)
 Acute lymphocytic leukemia 6.67
 Acute promyelocytic leukemia 15.94
 T-cell lymphoma 6.69 (4.17-8.87)
 B-cell lymphoma 7.31 (4.48-13.37)
 Atypical lymphoid infiltrate 11.97 (11.16-12.78)
 Reactive hyperplasia 8.16 (6.65-8.87)
 Floridly reactive 5.88 (5.02-6.73)
Cell lines
 Raji (Burkitt lymphoma) >0.2
 HepG2 (Hepatocellular carcinoma) -0.23
 HeLa (adenocarcinoma) 2.49


 MCL Positive (4/4)
 Follicular lymphoma
 Anaplastic large cell lymphoma
 Acute lymphocytic leukemia
 Acute promyelocytic leukemia Negative
 T-cell lymphoma
 B-cell lymphoma Negative (1)
 Atypical lymphoid infiltrate Negative (2/2)
 Reactive hyperplasia
 Floridly reactive
Cell lines
 Raji (Burkitt lymphoma) Negative
 HepG2 (Hepatocellular carcinoma) Strongly positive
 HeLa (adenocarcinoma) Positive
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Title Annotation:Molecular Diagnostics and Genetics
Author:Bijwaard, Karen E.; Aguilera, Nadine S.I.; Monczak, Yury; Trudel, Michel; Taubenberger, Jeffery K.;
Publication:Clinical Chemistry
Date:Feb 1, 2001
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