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From the Department of Hematologic and Lymphatic
Pathology*
and Division of Molecular
Pathology,
Department of Cellular Pathology,
Armed Forces Institute of Pathology, Washington DC
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper we compared three different methods for detecting markers associated with MCL in formalin-fixed, paraffin-embedded (FFPE) tissue: immunohistochemical profile including immunoreactivity for cyclin D1 and CD5, PCR for bcl-1 rearrangement corresponding to t(11;14), and RT-PCR for cyclin D1 mRNA expression. We found that, although the RT-PCR assay for cyclin D1 was very sensitive for MCL, it was not specific in that cyclin D1 expression was observed in the majority of non-MCLs examined. To further characterize expression of cyclin D1 in MCL from its expression in other lymphomas, a semiquantitative RT-PCR assay was developed. This assay reliably distinguished high levels of cyclin D1 mRNA in MCL from the low levels found in other malignant lymphoproliferative processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cases diagnosed as either MCL, intermediately differentiated lymphocytic lymphoma, or diffuse small cleaved lymphoma from 1980 to 1997 were retrieved from the files of the Armed Forces Institute of Pathology Hematopathology Registry and classified according to the REAL classification. Lymphomas with classic, unequivocal histological features of MCL were used.5,26 These showed either a mantle zone, nodular, or diffuse pattern with small to intermediately sized lymphocytes with slightly cleaved nuclei. No transformed cells or proliferation centers were seen. The blastic variant, which made up a subset of these lymphomas, occasionally showed a starry-sky appearance with diffuse effacement of the lymph node with medium-sized lymphocytes with finely dispersed chromatin. So-called variant forms (large-cell or anaplastic) were also accepted for this study.14,27,28
Non-MCLs were selected, as available, and included examples of follicle
center lymphoma, diffuse large-B-cell lymphoma, peripheral T-cell
lymphoma, or anaplastic large-cell lymphoma. Atypical cases were
selected, as available, and were defined as suspicious for malignant
lymphoma, but they lacked definitive histological features of lymphoma
(see Table 4
). Nonstimulated lymph nodes from node-negative mastectomy
specimens and florid reactive lymph nodes without suspicion of
malignancy were used as controls.
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Five-micron sections from paraffin-embedded tissue blocks were
prepared for immunophenotypic analysis according to the standard
avidin-biotin complex method of Hsu.29
The antibody panel
for each case included CD45RB, CD20, CD45RO, CD3, CD43, bcl-2, CD5,
kappa (Ig light chain), lambda (Ig light chain), CD23 (BU38), and
cyclin D1 (Table 1)
. Of the above
antibodies, kappa, lambda, CD3, and CD23 required predigestion for 20
minutes with 0.4% pepsin (P-7000, Sigma Chemical Co., St. Louis, MO)
in 0.1 mol/L HCl buffer solution at pH 2.0 at 40°C to 42°C.
Microwave antigen retrieval was necessary for CD5.30
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Molecular Diagnostic Studies
PCR for t(11;14) and Immunoglobulin Heavy Chain (IgH)
Molecular diagnostic studies for t(11;14) and IgH were performed
by PCR. DNA was extracted from the FFPE sections and amplified as
previously described.35
A PCR master mix containing PCR
buffer II, pH 8.3 (Perkin-Elmer, Norwalk, CT), MgCl2 (2.5
mmol/L final concentration), 1.25 U of AmpliTaq polymerase
(Perkin-Elmer), and 10 pmol of appropriate primer, listed in Table 2
, was added for a final volume of 50
µl. The reaction mixture was amplified in a 9600 Gene Amp PCR system
(Perkin-Elmer) programmed for a 5-minute denaturation at 94°C,
followed by 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and
72°C for 1 minute, with a final 7-minute extension at 72°C. The
t(11;14) PCR products (18 µl) were separated by electrophoresis
through a 2.5% agarose gel in Tris/borate/EDTA buffer, pH 8.4, and
visualized by ethidium bromide staining. Specific products were
detected by Southern blotting with end-labeled oligonucleotide
probes,36,37
as listed in Table 2
. End-labeling was
performed with T4 polynucleotide kinase (Life Technologies) and
[
-32P]ATP (10 mCi/ml; Amersham, Arlington Hts, IL) or
terminal transferase and digoxigenin-ddUTP (Boehringer Mannheim,
Indianapolis, IN). A positive result for a major translocation cluster
of t(11;14) produced a band of 180 to 280 bp. The positive control for
the assay was the M02058 cell line.38
PCR for IgH was
performed according to previously described methods.39
A
monoclonal IgH rearrangement appeared as a dominant band of 105 to 120
bp.
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All RT-PCR studies were performed as previously described37 and according to the guidelines of the College of American Pathologists checklist for molecular pathology with appropriate positive and negative controls.
RNA was extracted from the formalin-fixed, paraffin-embedded tissue for
RT-PCR as previously described.37
RT-PCR for the
ubiquitously expressed ß2-microglobulin
(ß2M) gene was used as a control for the presence of
amplifiable RNA. Reverse transcription was performed with a specific
antisense primer, listed in Table 2
. The RT step was performed on 1-
and 5-µl templates at 37°C for 60 minutes in 20-µl mixtures
containing First Stand Buffer (Life Technologies, Gaithersburg, MD), 10
mmol/L dithiothreitol, 60 U of Moloney murine leukemia virus reverse
transcriptase (Life Technologies), 0.2 mmol/L each deoxynucleotide
triphosphate, and 15 pmol of antisense primer. A PCR master mix
containing PCR buffer II, pH 8.3 (Perkin-Elmer), MgCl2 (2.5
mmol/L final concentration), 1.25 U of AmpliTaq polymerase
(Perkin-Elmer), and 15 pmol of sense primer (Table 2)
was added for a
final volume of 50 µl. The reaction mixture was amplified in a 9600
Gene Amp PCR System (Perkin-Elmer) programmed for a 5-minute
denaturation at 94°C, followed by 40 cycles of 94°C for 1 minute,
55°C for 1 minute, and 72°C for 1 minute, with a final 7-minute
extension at 72°C. Cyclin D1 RT-PCR products (18 µl) were separated
by electrophoresis through a 2.5% agarose gel in Tris/borate/EDTA
buffer, pH 8.4, and visualized by ethidium bromide staining. Specific
products were detected by Southern blotting with an end-labeled
oligonucleotide probe as listed in Table 2
. A positive result for
ß2M produced a band of 158 bp. A positive result for
cyclin D1 produced a band of 134 bp. The positive control for the
cyclin D1 assay was RNA extracted from the t(11;14)-bearing cell line
M02058.38
For semiquantitative RT-PCR, 5 µl of lysate was reverse transcribed as described above for cyclin D1 and ß2M. As absolute RNA amounts in FFPE lysates cannot be easily quantitated, a semiquantitative assay was designed in which the relative expression of cyclin D1 was compared with the expression of ß2M. To determine the linear range of amplification, control lysates were amplified by RT-PCR for ß2M with 20, 25, 30, and 40 cycles. It was determined that 30 cycles of PCR was still in the linear range and was optimal for this assay. For quantification, the amplified samples were subjected to gel electrophoresis and Southern blotting as above. Southern blots were visualized and bands were quantitated using a Storm 860 PhosphorImager equipped with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). For cyclin D1 quantitation, the ratio of cyclin D1/ß2M was calculated for each case. The resultant ratios were normalized to the cyclin D1/ß2M ratio of the positive control, cell line M02058, arbitrarily set as 1.0.
Statistics
The Statistics Program for Social Sciences (STSS 7.5) for windows (STSS, Chicago, IL) was used for determining statistical significance between the cyclin D1/ß2M ratios of MCLs and non-MCLs with a two-tailed t-test for equality of means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other cases examined included 17 non-mantle-cell lymphomas (9 follicle
center lymphomas, 3 peripheral T-cell lymphomas, 3 diffuse large-B-cell
lymphomas, and 2 anaplastic large-cell lymphomas) and 7 atypical
lymphoid proliferations (Table 4)
. Twenty
axillary lymph nodes from node-negative breast carcinoma cases and nine
floridly reactive lymph nodes were also examined for cyclin D1
expression and t(11;14).
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The immunohistochemistry is summarized in Table 3
. All MCLs were
B-cell immunophenotype with CD20 immunoreactivity and CD3 and/or CD45RO
negativity. Cyclin D1 was immunoreactive in 16 of 23 cases (70%).
Results of other immunohistochemical markers included the following: 20
of 22 showed CD43 coexpression (91%); 21 of 21 MCLs were bcl-2
immunoreactive (100%); and 15 of 19 MCL were CD5 positive (79%). All
cases tested were CD23 negative. All cases of non-MCLs studied were
negative with cyclin D1.
Molecular Diagnostic Studies
All cases of MCL showed a monoclonal IgH rearrangement by PCR (24
of 24, 100%). PCR for t(11;14) translocation was performed on all MCLs
and showed a distinct band in 9 of 24 cases (38%); no band was seen in
15 of 24 cases. Cyclin D1 mRNA as detected by RT-PCR was expressed in
23 of 24 cases (96%) of MCL (Table 3)
. All nine t(11;14)-positive MCL
cases were cyclin D1 positive. Of the 15 MCL cases negative for
t(11;14) by PCR, 14 (93%) were cyclin D1 positive. One case with a
negative result for t(11;14), cyclin D1 mRNA and protein, had results
showing CD20+, CD43+, CD5+,
CD23-, and bcl-2 immunoreactivity and was considered MCL
by morphology and immunohistochemistry. Results for two MCL cases are
shown in Figure 1
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Representative cases are also shown in Figure 2
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, the MCL cases showed
significantly higher expression of cyclin D1 than did other non-MCLs.
There was no statistically significant difference between the cyclin
D1/ß2M ratio of MCLs that were t(11;14) positive and
those that were t(11;14) negative. Mean relative cyclin
D1/ß2M ratio of MCL ± SEM was 1.44 ± 0.25.
Mean relative cyclin D1/ß2M ratio for non-MCLs was
0.17 ± 0.05 (P < 0.001, two-tailed
t-test for equality of means).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With the advent of the REAL classification, there has been an augmentation of knowledge and treatment of lymphomas. With this expanding knowledge, there is increasing necessity to subclassify small-B-cell lymphomas. In the case of MCL, this is especially justified because the natural history of MCL, which has low-grade histological features, has proven to be worse than its more indolent simulators, and its clinical behavior is more similar to intermediate histological grade.1-4 The recent development of immunohistochemical markers, such as CD5,44 cyclin D1,5,13,14 and CD23,33 has made the classification of small-B-cell lymphomas considerably easier, although there are still cases that are difficult to classify unequivocally by immunohistochemistry alone.
MCL has been associated with the cytogenetic abnormality t(11;14)(q13;q32). This translocation, which involves the bcl-1 breakpoint and cyclin D1, appears to be restricted to MCLs with occasional exceptions.13,17,20-22,45 This translocation leads to deregulation of the bcl-1 gene and plays a role in the overexpression of cyclin D1 mRNA and subsequent overexpression of the cyclin D1 protein.1,8-10,12,13 Cyclin D1 mRNA overexpression in MCL has been demonstrated by in situ hybridization, by Northern blot,11,12,23,24 and more recently by competitive RT-PCR, with cyclin D2 and D3, in fresh tissue.25 The cyclin D1 protein overexpression has been demonstrated with both Western blot and immunohistochemical studies in fresh and fixed tissue.13,14,17,34
The bcl-1 locus is rearranged in 50% to 80% of MCLs when using Southern blot or 75% to 80% using cytogenetics,13,18 but using a direct visualization method with DNA fiber fluorescence in situ hybridization, this translocation can be seen in as many as 95% of MCLs.19 As a result of the characteristic t(11;14), the bcl-1 locus, on chromosome 11, is juxtaposed to the immunoglobulin heavy chain (IgH) gene locus located on chromosome 14. The breakpoints are widely scattered on chromosome 11q13, but it has been shown that 70% to 80% of the breakpoints are localized to a 1-kb DNA segment known as the major translocation cluster (MTC).18,24 Within the MTC, the breakpoints occur in a relatively small region of approximately 80 bp on chromosome 11 and the 5' area of one of the IgH joining (JH) regions on chromosome 14.46 Because of the localization of these breakpoints, the translocation is amenable to PCR techniques for detection, although some of the breakpoints falling outside the MTC cannot be identified with PCR. This technique has been demonstrated to be sensitive when the MTC is present,46 although because of the low percentage of MCLs exhibiting the MTC, the technique has a low sensitivity for MCL overall. It is estimated that only in 33% to 50% of patients with MCL can the breakpoints be detected by PCR using primers in the region of the MTC.18,47,48 Our findings in this study fall within this expected range of detection.
Although there is low sensitivity for the t(11;14) molecular counterpart of the bcl-1 rearrangement by PCR,47 the expression of the cyclin D1 protein in MCL is similar to that of the presence of t(11;14) itself, approximately 50% to 90%.13,16,17 This can be demonstrated by immunohistochemical studies.13,49 In the current study, cyclin D1 protein expression was observed in 70% of MCLs, which may reflect the variability of fixation and processing of cases submitted to the Armed Forces Institute of Pathology. Cyclin D1 is associated with progression of the cell cycle through G1 but is generally not found in normal lymphoid tissue or B-cell lines without t(11;14).50 As with the translocation, the presence of this protein is highly characteristic of MCL, although it has been demonstrated occasionally in other hematopoietic malignancies, including hairy cell leukemia, splenic marginal zone lymphoma, multiple myeloma, and plasmacytoma.20-22,45 Nonhematopoietic neoplasms, such as parathyroid adenoma,51 breast carcinoma, and squamous cell carcinomas52 have also demonstrated cyclin D1. Although the weak nuclear expression of this protein, detected by the anti-cyclin-D1 antibody, and high background in some cases can hinder interpretation, we feel that in conjunction with other immunohistochemical stains and diagnostic modalities, it is extremely helpful in definitive classification of MCL.
Because cyclin D1 protein appears helpful in diagnosis of MCL, we speculated that cyclin D1 mRNA would also be useful, even at low levels. We investigated the use of RT-PCR in FFPE tissue to demonstrate the presence of cyclin D1 mRNA and create a specific and sensitive test for MCL. The use of RT-PCR is often helpful in demonstrating the presence of low-level target mRNA in tissue, often lower than can be detected with Northern blot or in situ analysis. This was recently demonstrated in a study by Uchimaru et al in which competitive RT-PCR was used to detect cyclin D1 in MCL and lymphoid cell lines.25 The finding of cyclin D1 in non-MCL cell lines was also demonstrated in the study by Uchimaru et al but was seen rarely and in conjunction with the presence of t(11;14).25 Our study shows that cyclin D1 can be demonstrated in MCL in a high percentage of cases by RT-PCR from paraffin-embedded tissue. The presence of cyclin D1 mRNA by RT-PCR was highly sensitive, but the extensive presence of cyclin D1 in non-MCL was an unexpected finding. In contrast to our study of cyclin D1 expression by RT-PCR, other studies, in which cyclin D1 expression was examined, used Northern blot and demonstrated only rare non-MCLs to be positive: 3/122 (2.5%),10 0/10 (0%),53 and 1/50 (2%).11 Only one study by De Boer et al also found low levels of expression in 36/56 (64%), a high percentage of non-MCLs.9 In our current study and that of de Boer and colleagues, cyclin D1 expression (without quantitation) did appear to be a sensitive marker for lymphoid malignancy but not specific for MCL as has been previously reported.25,11 Quantitation of the cyclin D1 expression was essential in differentiating MCL from non-MCL. Although Uchimaru used comparative quantitation,25 to our knowledge, the present study is the first to use a semiquantitative, comparative method in paraffin-embedded tissue.
Cyclin D1 mRNA has also rarely been demonstrated in non-neoplastic lymphoid tissues by Northern blot.11,17,22,50,53 Only a single study by de Boer et al9 demonstrated low levels of cyclin D1 expression in widespread non-neoplastic lymphoid tissue, including normal lymph node, spleen, and tonsil. This finding is unique to the study of de Boer et al and has not been demonstrated by other authors, nor was it seen in our study. In our study, all of the non-neoplastic lymph nodes we examined, both nonstimulated and floridly reactive, were negative for cyclin D1 mRNA. The differences in sensitivity between the Northern blot assays developed by these different groups and our RT-PCR assay are difficult to assess.
Our study showed that the majority of non-MCLs expressed cyclin D1 mRNA, albeit at low levels. The presence of cyclin D1 expression in our study appears to be a sensitive adjunctive marker for malignancy in lymphoproliferative processes, but it is not specific for MCL. Because cyclin D1 expression is not seen in normal hematopoietic cells, the demonstrated expression of cyclin D1 in lymphomas other than MCL may indicate that cyclin D1 is a ubiquitous oncogene, however, at significantly lower levels than is seen in MCL. As the vast majority of these lymphomas do not possess the t(11;14) responsible for cyclin D1 overexpression in MCL, the mechanism for cyclin D1 expression/overexpression in these cases likely is different from that of MCL. However, using the semiquantitative assay described herein, cyclin D1 overexpression in MCL can be readily distinguished in paraffin-embedded tissues from its low expression/overexpression in non-MCLs. This finding may be helpful in diagnosis of morphologically and immunophenotypically ambiguous cases of suspected MCL. This method of examining differential expression of mRNA, although somewhat arduous, may also be helpful in other studies using paraffin-embedded fixed tissue where there is a necessity to quantitate mRNA expression.
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Acknowledgements |
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Footnotes |
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Supported by the intramural funds of the Armed Forces Institute of Pathology.
Accepted for publication September 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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