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Molecular Diagnostics and Genetics |
Departments of
1 Laboratory Medicine and
2 Internal Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.
aAddress correspondence to this author at: Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar St., PO Box 208035, New Haven, CT 06520-8035. Fax 203-688-4111; e-mail brian.smith{at}yale.edu.
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Abstract |
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Methods: We studied 97 fresh specimens (50 blood, 30 bone marrow, 15 lymph node, and 2 other samples) from patients diagnosed with a variety of lymphoproliferative diseases, including 25 cases of MCL. We used real-time quantitative RT-PCR to evaluate cyclin D1 mRNA expression. Because blood and marrow specimens may contain only a minority of potentially malignant cells (as opposed to most lymph nodes) and to increase sensitivity, we normalized the cyclin D1 mRNA concentrations to mRNA of a B-cell-specific marker, CD19, as well as to previously characterized ß2-microglobulin mRNA.
Results: In 16 of 21 cases of MCL with overt disease, the ratio of cyclin D1 mRNA to ß2-microglobulin mRNA was increased, but all 21 cases showed increased ratios of cyclin D1 mRNA to CD19 mRNA. Cyclin D1 mRNA was low or undetectable in various lymphoproliferative diseases, including cases of ambiguous immunophenotype. The mRNA ratios were stable over 3–7 days of sample storage.
Conclusion: Quantitative RT-PCR for cyclin D1 mRNA normalized to CD19 mRNA can be used in the diagnosis of MCL in blood, marrow, and tissue.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3–10% of all NHLs (1). Clinically aggressive, MCLs are refractory to conventional therapies that are effective with other types of NHL. MCL is classified as a specific NHL subtype by the WHO, with distinct immunophenotypic, molecular, cytogenetic, and clinicopathologic properties. Although not 100% discriminating (1), expression of CD5 and lack of CD10 help to distinguish MCL and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) from other B-cell lymphoproliferative diseases, whereas differential CD23 and FMC7 expression help to distinguish MCL from CLL/SLL. Molecular and cytogenetic analyses have revealed that >90% of MCLs are associated with the t(11;14)(q13;q32) translocation (2)(3). This translocation juxtaposes the immunoglobulin heavy chain gene locus at 14q32 with the bcl-1 locus at 11q13, leading to overexpression of cyclin D1 (4). Cyclin D1 overexpression is characteristic of MCL and is rarely found in healthy lymphocytes or other types of B-cell NHL (5) with the exception of hairy cell leukemia. Cyclin D1 regulates the cell cycle G1-S phase checkpoint; therefore, any increase in cyclin D1 expression will drive cells from the G1 phase into the S phase (6). Identification of either the t(11;14)(q13;q32) translocation or overexpression of cyclin D1 determines unequivocally the diagnosis of MCL. Recently, several laboratories have demonstrated the usefulness of quantitative reverse transcription-PCR (RT-PCR) in the diagnosis of MCL (7)(8)(9)(10)(11)(12). The authors of these reports concluded that measurement of cyclin D1 mRNA can distinguish MCL from most other lymphomas in formalin-fixed tissue, especially CLL/SLL, which can be difficult to distinguish from MCL by immunohistochemistry.
The analytical sensitivity of measuring cyclin D1 mRNA is generally not an issue when testing formalin-fixed tissue because the nontumor material can be selectively dissected away from tumor cells. In blood and bone marrow samples, however, the tumor cells may be at significantly low numbers and below the detection limits of current assays. One recent report used a real-time quantitative RT-PCR assay to determine cyclin D1 mRNA expression in whole-blood samples but did not determine the sensitivity of the assay for specimens with low numbers of malignant cells (10).
We have examined specimens from patients with lymphoproliferative diseases (LPDs), including MCL, and normalized results with either ß2-microglobulin or CD19 mRNA. CD19 protein is generally present at similar concentrations in MCL, CLL/SLL, and other B-cell LPDs, whereas other B-lineage-restricted proteins, such as CD20, are present at markedly different concentrations in MCL and CLL/SLL (13)(14). We hypothesized that examining cyclin D1 vs CD19 mRNA expression would permit accurate diagnostic assessment even in samples with low numbers of malignant cells.
During the course of these studies, we also sought to determine how frequently immunophenotyping results suggesting MCL vs CLL/SLL might differ from cyclin D1 results when applied to blood and bone marrow.
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Materials and Methods |
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detection of cyclin d1 transcripts by one-step real-time quantitative rt-pcr
RNA preparation.
Total RNA from Ficoll-Paque Plus (Amersham Biosciences)-purified blood was extracted by use of RNeasy reagent (Qiagen) according to the manufacturer’s instructions. The amount of RNA was determined by measuring the absorbance at 260 nm or by cell counts of 5 x 106 peripheral blood mononuclear cells (PBMCs) per 100-µL RNA sample. The RNA was stored in a -70 °C freezer.
Preparation of calibrators.
Total RNA derived from the cyclin D1-overexpressing mantle cell NHL cell line Granta [Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig (17)], was serially diluted into PBMC RNA from a healthy donor to generate a series of calibrators containing different amounts of Granta cell RNA.
One-step real-time quantitative RT-PCR.
All oligonucleotide primers and probes were synthesized by the Oligo Factory (Perkin-Elmer Applied Biosystems Division). Primer and probe sequences are as follows (7)(12): cyclin D1 sense primer, 5'-CCG TCC ATG CGG AAG ATC-3'; cyclin D1 antisense primer, 5'-ATG GCC AGC GGG AAG AC-3'; cyclin D1 probe, 5'-(6-FAM)-CTT CTG TTC CTC GCA GAC CTC CAG CAT-TAMRA-3'; ß2-microglobulin sense primer, 5'-TGA CTT TGT CAC AGC CCA AGA TA-3'; ß2-microglobulin antisense primer, 5'-AAT CCA AAT GCG GCA TCT TC-3'; ß2-microglobulin probe, 5'-VIC-TGA TGC TGC TTA CAT GTC TCG ATC CCA-TAMRA-3'; CD19 sense primer, 5'-ACC TGA CCA TGT CAT TCC ACC T-3'; CD19 antisense primer, 5'-AGA AGA TCA GAT AAG CCA AAG TCA CA-3'; CD19 probe, 5'-(6-FAM)-AGA CCT TCC AGC CAC CAG TCC TCA GC-TAMRA-3', where 6-FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. The cyclin D1, ß2-microglobulin, and CD19 mRNA quantitative RT-PCR assays amplified 0.3 µg of total RNA or 250 000 cell-equivalents prepared from blood. The standard master mixtures for cyclin D1, CD19, and ß2-microglobulin were composed of reagents obtained from Perkin-Elmer Corporation unless otherwise indicated, and included 10% TaqMan 10x buffer; 2.5 mM MgCl2; 0.2 µM each of dATP, dCTP, and dGTP; 0.4 µM dUTP; 0.5 U of AmpErase uracil N-glycosylase; 1.5 U of TaqGold DNA polymerase; 10 units of RNase inhibitor (Roche); 0.6–0.9 µM primers; 0.1 µM probe; 4 U of Moloney murine leukemia virus reverse transcriptase (Roche), and 50 mL/L dimethyl sulfoxide (Sigma Chemical). All reaction mixtures were brought to a 50-µL volume, placed in MicroAmp optical tubes, and covered with MicroAmp optical caps (Perkin-Elmer Corporation). Reverse transcription and PCR were performed in an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Corporation). The reaction was started with 2 min at 50 °C for uracil N-glycosylase activation followed by 45 min of reverse transcription at 48 °C. This was followed by 10 min at 95 °C for TaqGold activation and predenaturation and 45 cycles with each cycle consisting of 15 s at 95 °C and 1 min at 62 °C. Data were normalized to the quencher dye TAMRA and analyzed by the Signal Detection software (Perkin-Elmer Applied Biosystems Division). Critical threshold (Ct) cycle numbers were obtained from amplification of cyclin D1, CD19, and ß2-microglobulin, and 
Ct values were calculated by subtracting the Ct value of either ß2-microglobulin or CD19 from the Ct value of cyclin D1. PCR results were confirmed by agarose gel electrophoresis.
RNA stability experiment.
Granta cells were washed once with 1x Dulbecco phosphate-buffered saline (Invitrogen), added to normal whole blood, and brought up to a concentration of 10% of the white blood cell (WBC) count. At day 0, aliquots of this suspension, representing 5 x 106 cells, were made for each time point and assay condition. RPMI 1640 (Invitrogen) was added to the appropriate tubes at a ratio of 1:1. Aliquots were processed as described with use of the RNeasy reagent set (Qiagen). Briefly, the red blood cells were lysed with Erythrocyte Lysis Buffer, and the remaining WBCs were washed with Dulbecco phosphate-buffered saline. The cells were resuspended in 0.5 mL of RLT buffer and stored at -70 °C for later RNA extraction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ct for both Granta cells and normal PBMCs: Ct = Ct (cyclin D1) - Ct (ß2-microglobulin or CD19). The Ct value was determined by subtracting the Ct value for the sample from the Ct value for normal PBMCs and calculating the RFI of cyclin D1 by the equation: RFI = 2-Ct. The assay was linear over three orders of magnitude, with a R2 value of 0.995 (Fig. 1A
). We hypothesized that the analytical sensitivity of the cyclin D1 assay could be increased if an expressed gene specific for B cells was used instead of ß2-microglobulin as the normalizing transcript. We reasoned that a gene expressed only in B cells and expressed on MCL cells and almost all LPDs would decrease the background variability and thereby increase sensitivity of the cyclin D1 assay. We selected the B-cell-specific transcript CD19 for this purpose. CD19 was selected over several other potential lineage specific proteins because, as opposed to molecules such as CD20, its expression is relatively constant in different LPDs, in particular in MCL and CLL/SLL (13)(14). When we used CD19 mRNA expression to normalize the data, the assay was also linear over three orders of magnitude with a R2 value of 0.995 (Fig. 1B
). The detection limits of both assays was 1 Granta cell in 104 WBCs.
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To use the 2-Ct calculation, the PCR efficiencies of the target and control assays need to be similar (18). Measurement of the relative efficiencies is done by varying the input amount of RNA, measuring the Ct values, and calculating the Ct values for the various assays. Graphing the Ct values relative to input total RNA should give a line with a slope <0.1. This experiment was done, and a slope <0.1 for cyclin D1 relative to both CD19 and ß2-microglobulin for a range of input RNA of 15–250 ng was obtained.
overexpression of cyclin d1 MRNA in mcl assessed by real-time quantitative rt-pcr
Previously we developed an assay for use with formalin-fixed tissue samples (7) and found a significant RFI difference between MCLs and other B-cell diseases. To determine whether fresh peripheral blood, bone marrow, and lymph node samples would also show this difference, we repeated this work with such samples from patients with various LPDs and also compared the approach of testing for cyclin D1 mRNA expression relative to ß2-microglobulin and CD19 mRNA expression. The data are plotted in Fig. 2
, and the means and ranges are detailed in Table 1
. Patient samples were divided into five broad categories: normal/hyperplasia, B-NHL-other (not CLL/SLL or MCL), CLL/SLL, MCL, and MCL in remission. Significant overproduction of cyclin D1 mRNA was observed only in the MCL group, as seen in Fig. 2
, where the RFI was calculated relative to the mean Ct value of PBMC samples from healthy individuals (normal). Cyclin D1 mRNA RFIs were higher in cases of MCL, with mean RFIs of 133 (cyclin D1/CD19) and 2548 (cyclin D1/ß2-microglobulin) compared with CLL/SLL [mean RFIs, 0.75 (cyclin D1/CD19) and 38.6 (cyclin D1/ß2-microglobulin); Table 1
]. One CLL/SLL specimen had an unusually high RFI (cyclin D1/ß2-microglobulin) of 1408, and the mean RFI for cyclin D1/ß2-microglobulin was lowered to 4.4 if that value was deleted from the calculation. In Fig. 2
, lines are drawn to indicate the value that would adequately differentiate CLL/SLL samples from MCL samples. This line is placed at the RFI value of 10 for cyclin D1 mRNA expression normalized to CD19 mRNA expression (Fig. 2B
) and at the RFI value of 100 for cyclin D1 mRNA expression normalized to ß2-microglobulin mRNA expression (Fig. 2A
).
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When we used these discriminants and compared just CLL/SLL vs MCL, the clinical sensitivity and specificity of the cyclin D1/CD19 assay for diagnosis of MCL were both 100% compared with 76% and 98%, respectively, for cyclin D1/ß2-microglobulin. For B-cell LPDs other than CLL/SLL, several subtypes showed a cyclin D1/CD19 result in the low "MCL range" (RFI = 10–50), specifically one of the three hairy cell leukemia samples, one of the five diffuse large B-cell lymphomas, and one of the four follicular lymphomas, and "borderline" (RFI = 5–10) in one of the three hairy cell leukemia samples, three of five diffuse large B-cell lymphomas, one of five acute lymphoblastic leukemia samples, and one of two Burkitt lymphoma samples.
Interestingly, four MCL samples from patients who were in morphologic and clinical remission had RFI values below the cutoff for ß2-microglobulin mRNA expression-normalized cyclin D1 mRNA expression, but three of four were above the cutoff with cyclin D1 mRNA expression normalized to CD19 mRNA expression (Fig. 2
). Clinical follow-up data are not available for these patients to assess whether these findings have any implications for prognosis.
detection limit of quantitative real-time rt-pcr assay
By combining the differentiating cutoff determined in Fig. 2A
and the calibration curve of cyclin D1 mRNA expression normalized to ß2-microglobulin mRNA expression found in Fig. 1A
, we obtained detection limits of 0.5–1% Granta cells. When this analysis was repeated for cyclin D1 mRNA expression normalized to CD19 mRNA expression (Fig. 1B
and Fig. 2B
), the detection limits of the cyclin D1/CD19 assay were 0.04–0.05% Granta cells. This is a >15-fold improvement in the detection limits compared with the cyclin D1/ß2-microglobulin assay.
immunophenotype correlation
Most samples designated as CLL/SLL or MCL by our assay were immunophenotypically "typical" except for a few cases (Tables 2 and 3 in the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol50/issue1/ ). Two cases of MCL were found to be dim positive for CD23. There were roughly equal numbers of MCLs that were positive (n = 11) and negative (n = 7) for FMC7. Of the 41 cases of CLL/SLL, 3 were negative and 2 were weakly positive for CD23. In addition, one case, although phenotypically typical, showed that the amounts of cyclin D1 mRNA were high relative to ß2-microglobulin mRNA and was the only CLL/SLL case above the RFI = 100 cutoff. The amount of cyclin D1 mRNA relative to CD19 was, however, well within the lower range. The CLL case, which was otherwise typical for that disorder, was not examined for a t(11;14) translocation.
relative amounts of cyclin d1 and cd19 MRNA over time in stored samples
In clinical blood samples, the processing time and storage conditions between when the blood is drawn and the RNA is stabilized by guanidine thiocyanate can vary greatly. It is generally suggested that samples be processed within several hours, but this is not always practical because samples are sometimes shipped overnight. A specific concern in the current assay is that because two mRNAs are being tested, it is possible that one mRNA may preferentially degrade more quickly than the other, causing spurious results. We therefore tested mRNA stability by taking normal blood and adding Granta cells at a concentration of 10% at day 0. At several intervals after day 0, cells were removed and the RNA was stabilized. The aliquot of 5 x 106 cells was then tested by the cyclin D1/CD19 assay. We found that the ratios of cyclin D1 mRNA to ß2-microglobulin or CD19 mRNA remained remarkably stable over at least 3 days at room temperature without RPMI medium and for as many as 7 days under other conditions (Fig. 3
).
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reproducibility of cyclin d1/cd19 quantitative real-time rt-pcr assay
We tested the reproducibility of the cyclin D1/CD19 assay by running 35 samples twice (RFI = 0.1–693) and found it to have a within-run CV of 36% and a between-run CV of 41% (results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies determining cyclin D1 mRNA concentrations by real-time quantitative RT-PCR have generally used embedded formalin-fixed tissue as specimens, whereas many initial clinical samples in patients with LPDs are fresh blood or bone marrow. Indeed, in many patients with small-cell LPD, lymph node biopsy may not be part of the diagnostic work-up, depending on patient status. We tested blood and bone marrow samples for cyclin D1 mRNA expression and normalized these concentrations to the concentrations of either CD19 or ß2-microglobulin mRNA and found that the assays were able to partition the normal/hyperplasia and CLL/SLL samples from the MCL patient samples. Normalization with CD19 mRNA expression was more successful than with ß2-microglobulin mRNA expression. One notable sample, immunophenotypically and clinically a CLL/SLL, was clearly in the MCL range when we used ß2-microglobulin (RFI = 1408) as the normalizing mRNA but was not when we used CD19 as the normalizing mRNA (Fig. 2
). This could be explained if there was a simultaneous increase in cyclin D1 mRNA expression and decrease in CD19 mRNA expression or if there was a decrease in ß2-microglobulin gene expression. Increased concentrations of cyclin D1 protein have been found in a small percentage (3–4%) of CLL/SLL patients (23)(24), but it is unknown whether there were changes in CD19 or ß2-microglobulin mRNA expression in these cases. ß2-Microglobulin expression has been shown to fluctuate the least of the mRNAs studied for use as a normalizing transcript (25); we therefore do not have a definitive explanation for the high RFI value in this case. When we used ß2-microglobulin as the normalizing mRNA, the clinical sensitivity of MCL detection decreased compared with normalization to CD19 mRNA. When we normalized with CD19 mRNA, most of the B-NHL-other samples separated away from the MCL samples, except for several patients with hairy cell leukemia, follicular lymphoma, and diffuse large B-cell lymphoma. Previous studies have shown that hairy cell leukemias can have increased concentrations of cyclin D1 (26).
Most samples designated as CLL/SLL or MCL by our assay were immunophenotypically typical, except for a few cases (Tables 2 and 3 in the online Data Supplement). Although immunophenotyping has been a major aid in the differential diagnosis of MCL, several studies have shown that this technique alone does not have 100% reliability for diagnosis. Well-characterized CD5-negative MCLs have been described (27), and although the lack of CD23 and presence of FMC7 are characteristic, several studies have confirmed that CD23 may instead be expressed and/or FMC7 not expressed in 10–20% of cases (28)(29). Similarly, CLL/SLL has been noted to show no or minimal CD23 expression and/or definite FMC7 expression in a similar percentage of cases (30). The data in Tables 2 and 3 of the online Data Supplement further confirm the presence of a sizeable subset of cases of MCL and CLL/SLL in which immunophenotyping is unable to establish a definitive diagnosis.
An important issue when dealing with blood and bone marrow is the problem of heterogeneity, especially when the numbers of tumor cells may be quite low. We hypothesized that we would be able to increase the sensitivity of the assay by use of a marker, CD19, found specifically in B cells. The samples were measured for cyclin D1 mRNA concentrations relative to CD19, and we found that the detection limit for this assay was 15- to 20-fold lower compared with the cyclin D1 assay normalized to ß2-microglobulin mRNA expression. The authors of a previous study using formalin-fixed tissue found that normalizing to another B-cell specific marker, CD20 mRNA, did not increase the sensitivity or segregation of patient samples compared with glyceraldehyde-3-phosphate dehydrogenase (8). It is possible that the marked variability in CD20 mRNA expression in CLL/SLL vs MCL explains that finding or that the use of glyceraldehyde-3-phosphate dehydrogenase as opposed to ß2-microglobulin or formalin-fixed vs fresh tissue is responsible.
The detection limit of our cyclin D1 mRNA expression assay normalized to CD19 mRNA expression was 1 in 10 000 Granta cells diluted into normal PBMCs. This detection limit is similar to the limit reported in a previous study, in which Granta cells were diluted in another cell line (10). When clinical samples were used, a cutoff was established for separating MCL samples from the other LPDs, as seen in Fig. 2
, and when applied to the calibration curves shown in Fig. 1
, the detection limit of the cyclin D1/CD19 assay for clinical samples was 1 in 1000 cells.
We studied four patients who were in remission, and as seen in Fig. 2B
, three still had cyclin D1/CD19 RFI values that were above the cutoff, although three of the samples had no evidence of monoclonal cells based on flow cytometry criteria (Table 2). This suggests that the cyclin D1/CD19 assay could be used for testing for minimal residual disease. Although there are relatively few data available regarding the detection of minimal residual disease after definitive therapy for MCL, it does appear that detection of tumor cell concentrations as low as 10-5 are frequently required to distinguish therapeutic responders from nonresponders. The techniques used were quantitative RT-PCR for detection of bcl-1 and clonal immunoglobulin heavy chain gene rearrangements, and flow cytometry (31)(32)(33). The current cyclin D1/CD19 assay has a detection limit for tumor cells of only 10-3 and therefore might not be sensitive enough to be used for detection of minimal residual disease (Fig. 1
).
In conclusion, real-time quantitative RT-PCR can be an easily applicable diagnostic assay for the delineation of MCL in fresh tissue, blood, and bone marrow. On the basis of recent correlative clinicopathologic data from other groups, demonstration of the t(11;14) translocation and/or overexpression of cyclin D1 mRNA could be an integral part of the diagnosis of MCL.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: MCL, mantle cell lymphoma; NHL, non-Hodgkin lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; RT-PCR, reverse transcription-PCR; LPD, lymphoproliferative disease; PBMC, peripheral blood mononuclear cell; TAMRA, 6-carboxytetramethylrhodamine; Ct, critical threshold; WBC, white blood cell; and RFI, relative fold increase. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ct method. Methods 2001;25:402-408.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) is the baseline cyclin D1 value. 
, hairy cell leukemia; 
, follicular lymphoma; 
, diffuse large B-cell lymphoma; -, Burkitt lymphoma; •, prolymphocytic leukemia B-cell type 1 and lymphoplasmacytic lymphoma; X, acute lymphoblastic leukemia; 
, marginal zone lymphoma; 
, MCL; 
, MCL in remission. The thick dashed horizontal lines are the discriminating lines for MCL vs CLL/SLL as discussed in the text.