Quantitative Measurement of Cyclin D1 mRNA, a Potent Diagnostic Tool to Separate Mantle Cell Lymphoma From Other B-cell Lymphoproliferative Disorders : Diagnostic Molecular Pathology

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00019606-200803000-00007ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008 p 39-50Quantitative Measurement of Cyclin D1 mRNA, a Potent Diagnostic Tool to Separate Mantle Cell Lymphoma From Other B-cell Lymphoproliferative DisordersOriginal ArticlesBrizova, Helena MSc; Kalinova, Marketa MSc; Krskova, Lenka PhD; Mrhalova, Marcela PhD; Kodet, Roman MD, PhDDepartment of Pathology and Molecular Medicine, 2nd Medical School, Charles University in Prague, and Faculty Hospital in Motol, V Uvalu 84, Prague, Czech RepublicSupported by the Internal grant of faculty hospital in Motol No. 9756, Grant GAUK 200055,46/2006/C/2.LF, and the Research project of the Ministry of Health No. 00064203/6704.Reprints: Helena Brizova, MSc, Department of Pathology and Molecular Medicine, 2nd Medical School, Charles University in Prague, and Faculty Hospital in Motol, V Úvalu 84, 150 06, Prague 5, Czech Republic (e-mail: [email protected]).AbstractCyclin D1 overexpression as a result of t(11;14) is a specific marker for diagnosis of mantle cell lymphoma (MCL) and plays an important role in MCL pathogenesis. To set a highly reliable cutoff value that discriminates MCL from other B-cell lymphoproliferative disorders, we performed a retrospective study of cyclin D1 expression. We established cyclin D1 expression level in 116 frozen and formalin-fixed, paraffin-embedded primary tumors from patients diagnosed with a variety of B-cell lymphoproliferative disorders. We used real time quantitative reverse-transcription polymerase chain reaction to quantify levels of cyclin D1 mRNA. The range of cyclin D1 expression in MCLs exceeded the range found in other lymphomas and reactive lymph nodes by a considerable margin. Cyclin D1 overexpression was found in 60/61 MCLs and in none of the other lymphomas, except for 12/19 mucosa-associated lymphoid tissue lymphomas from the lungs and stomach, which also revealed cyclin D1 overexpression. As epithelial tissues are known to express cyclin D1, an admixture of non-neoplastic epithelial cells present in the extranodal specimens probably influenced the quantitative reverse-transcription polymerase chain reaction result. Quantitative cyclin D1 monitoring provides a diagnostic test and an approach for studying MCL pathogenesis and may be of clinical importance.Mantle cell lymphoma (MCL) is a distinct entity with characteristic clinicopathologic, morphologic, immunophenotypical, and molecular genetic features. MCL accounts for 3% to 10% of B-cell non-Hodgkin lymphomas (B-NHLs) in adults.1 MCL has an unfavorable prognosis due to a more rapid disease progression than is seen in other small cell B-NHLs, and it is refractory to conventional therapies used to treat patients with other types of B-NHL.2 Consequently, its recognition from other morphologically similar small B-cell lymphomas is of clinical relevance.The diagnosis of MCL relies largely on histopathologic recognition and immunophenotyping using either immunohistochemistry (IHC) or flow cytometry. Although a morphologic investigation, together with immunophenotyping, is often sufficient, additional studies (cytogenetical and/or molecular) are sometimes necessary to make a definitive diagnosis.MCL is associated with a range of genetic alternations. The most consistent and diagnostically useful finding is a reciprocal translocation t(11;14)(q13;q32).3–5 Detection of t(11;14) has clinical utility in distinguishing MCL from other small B-cell lymphomas, which are histologically similar. The translocation t(11;14)(q13;q32) probably reflects an error in the V(D)J recombination during a precursor B-cell development.6,7 As a result, the proto-oncogene CCND1 becomes juxtaposed under the control of the immunoglobulin heavy chain (IgH) gene enhancer, resulting in a transcriptional up-regulation by the IgH enhancer and leading to an overexpression of the oncogene and its protein product, cyclin D1.8–11D-type cyclins are expressed in a tissue-specific manner.12 It is reported that the cyclin D1 expression is absent in normal lymphoid tissues, lymphocytes, myeloid cells, and many hematopoietic cell lines, which lack t(11;14).13 Therefore, any significant up-regulation of cyclin D1 mRNA or protein in lymphoid cells is abnormal and may virtually be equated with a malignancy, irrespective of the molecular mechanism that led to the cyclin D1 overexpression. Cyclin D1 overexpression plays an important role, especially in the pathogenesis of MCL.2 In MCL cells, cyclin D1 is expressed constitutively and independently of external factors owing to the translocation t(11;14)(q13;q32); cyclin D1 replaces cyclin D2 and D314,15 and is detected in 50% to 90% of MCLs depending on the methodology. Thus, the overexpression of cyclin D1 is a hallmark of MCL, and the detection of cyclin D1 mRNA is of diagnostic value.The CCND1 expression was detected at the protein level by immunocytochemical analysis,16 by immunohistochemical methods in approximately 85% of MCLs,17,18 and by Northern blot analysis19 at the RNA level. However, by the use of highly sensitive method [qualitative reverse-transcription polymerase chain reaction (RT-PCR)], several authors have demonstrated positive results in reactive lymph nodes and B-NHLs other than MCL,19–22 which has also been tested and confirmed in our laboratory. Thus, nonquantitative assays for cyclin D1 generally lack specificity for discriminating MCL. Epithelial cells, especially in proliferative zones, inherently express high levels of CCND1.13 As such cells may be found in many extranodal sites examined for B-NHL, it is not surprising that the nonquantitative method does not adequately distinguish between signals rendered by stromal, epithelial, or lymphoid cells and does not discriminate MCLs from other B-cell lymphoproliferative disorders. A semiquantitative PCR assay detects cyclin D1 mRNA in most MCLs22 and MCL may be separated from other B-NHLs using this approach. Competitive RT-PCR demonstrated cyclin D1 overexpression in all MCLs regardless of the capability to demonstrate t(11;14).21,23As cyclin D1 protein levels correlate well with mRNA levels,24,25 quantitative real time RT-PCR (qRT-PCR) of cyclin D1 mRNA is a useful tool for examination of the cyclin D1 expression level. We examined a series of formalin-fixed, paraffin-embedded, and frozen samples of B-NHLs and reactive lymphoid tissues by qRT-PCR. Advantages of the qRT-PCR assay include a significant reduction in labor, a greater reproducibility and sensitivity, and a reduction in turnaround time, in comparison with other techniques analyzing the cyclin D1 expression, such as blotting techniques, IHC, semiquantitative, or competitive RT-PCR. The qRT-PCR method requires small quantities of a starting material and it is not hampered by translocation breakpoints and does not produce falsely positive results if only low expression levels of the target sequence are present. Therefore, disadvantages encountered with Northern blot analysis, PCR from DNA, or qualitative RT-PCR are avoided. In addition, using the normalizing transcript enables a simultaneous quantitative analysis of samples with highly different starting concentrations. Thereby, qRT-PCR combines the speed and ease of the PCR-based system with an accurate and reproducible quantification methodology. Therefore, it has the potential to become a routine diagnostic tool.MATERIALS AND METHODSTissue SamplesA total of 116 primary tumor specimens diagnosed at the Department of Pathology and Molecular Medicine from 2000 to 2006 (70 lymph nodes, 2 spleens, 8 tonsils, and 36 other tissues) from 102 patients with a variety of B-NHLs were tested in this study. There were 63 specimens of MCL and 53 cases of other B-NHLs in the study. Of the 53 B-NHLs, 29 samples were diagnosed as mucosa-associated lymphoid tissue (MALT) lymphoma; 12 patients had follicular lymphoma; 3 had diffuse large B-cell lymphoma; 3 had nodal marginal zone B-cell lymphoma; 1 had small lymphocytic lymphoma; 1 had B-cell lymphoblastic lymphoma; and 4 had B-NHL not otherwise specified. The B-NHL specimens that were not otherwise specified were excluded to be MCL by a morphologic, immunophenotypical, and fluorescence in situ hybridization (FISH) examination. Therefore, they were incorporated in the group of B-NHLs other than MCL. Forty specimens of MCL and 17 specimens of MALT lymphoma had only formalin-fixed, paraffin-embedded tissue available, and for all remaining cases the nucleic acids were isolated from frozen tissues. Lymph node tissue of 5 patients with a reactive lymphadenopathy was used as a control. The diagnosis was based on a combined morphologic and immunophenotypical examination using IHC and/or flow cytometry and FISH on the interphase nuclei to demonstrate the t(11;14). Three MCL specimens had no molecular or cyclin D1 immunohistochemical support for the diagnosis. In specimen no. 17, the histologic material was not available for the molecular or cyclin D1 IHC examination. However, all the morphologic and immunophenotypical features supported MCL diagnosis. Moreover, another biopsy specimen from the same tumor from the patient revealed PCR, FISH, and IHC support for MCL diagnosis. Specimen no. 38 was poorly preserved for the FISH and IHC analysis. The specimen no. 59 failed to reveal malignant cell population. Therefore, the diagnoses were established from consecutive biopsy specimens from the same tumor, which were FISH and IHC positive. All patients, included in the study have, in addition to a careful examination, molecular or immunohistochemical support for MCL diagnosis. Thus, a well-characterized group of patients was used for the study.Detection of t(11;14), t(11;18) and IHCThe translocation t(11;14)(q13;q32) was examined by FISH using locus specific dual color, dual fusion translocation probes (IgH SpectrumGreen/CCND1 Spectrum Orange, Vysis Inc, IL) hybridizing to the locus 14q32 (IgH) and to the locus 11q13 (CCND1). We also investigated the translocation t(11;14)(q13;q32) by PCR using primers covering the major translocation cluster (MTC) at 11q13 and the JH region of the IgH gene at 14q32. The cyclin D1 overexpression was tested in 95 cases by immunohistochemical methods (primary antibody clone DCS-6, Dako, Glostrup, Denmark). The FISH, PCR, and IHC methodology were performed as described previously.26The translocation t(11;18) occurring in some MALT lymphomas was determined by FISH using locus specific dual color, dual fusion translocation probes [API2/MALT1 t(11;18)(q21;q21), Vysis Inc], or by PCR using specific primers.27RNA Extraction and cDNA SynthesisIn case of frozen tissues, RNA was extracted from 10 tissue sections (5-μm thick) using Tri-Reagent (Invitrogen Ltd, Carlsbad, CA) according to the manufacturer's instructions.In case of formalin-fixed, paraffin-embedded tissues, RNA was extracted from 10 tissue sections (5-μm thick) using “High Pure RNA Paraffin Kit” (Roche Applied Science, Pfenzberg, Germany) according to the manufacturer's instructions.Ten microliters of isolated total RNA was incubated for 5 minutes at 70°C to release the possible secondary structure. Reverse transcription was carried out in a reaction mixture containing 4 μL of 5× First Strand Buffer, 2 μL of dithiothreitol (0.1 M), 1 μL of random hexamers (250 pM), 0.2 μL of dNTP each (100 mM), 1 μL of MMLV reverse transcriptase (200 U/μL) (Invitrogen), and 10 μL of total denaturated RNA in a final volume of 20 μL. Synthesis of cDNA was performed at 37°C for 60 minutes.As a control of the reverse transcription process and quality of cDNA, we used amplification of the gene c-abl by PCR. The PCR mixture contained 1.5 μL of MgCl2 (25 mM) (Top Bio, Prague, Czech Republic), 2.5 μL of 10× concentrated buffer (Top Bio), 2 μL of each dNTP (2 mM) (Invitrogen), 0.25 μL of both primers (A2: 5′ TTC AGC GGC CAG TAG CAT CTG ACT T and A3: 5′ TGT GAT TAT AGC CTA AGA CCC GGA GCT TTT) (100 μM) (Invitrogen), 0.3 μL of Taq polymerase (0.1 U/μl) (Top Bio), 2 μL of cDNA in a final volume of 25 μL. The amplification program consisted of denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 60 seconds, annealing at 57°C for 60 seconds, extension at 71°C for 60 seconds, and final extension at 72°C for 10 minutes. The PCR products were separated by electrophoresis in a 1.5% agarose (Amresco, Solon, OH) gel containing ethidium bromide (Fluka Biochemika, Buchs, Switzerland).Quantitative Real Time RT-PCRThe assay used in this study involves RT-PCR with the product detection by a TaqMan technology. Using the TaqMan template specific probe not only increases the PCR stringency but also eliminates nonspecific products. Probes and primers were previously reported by Bijwaard et al.28 Both the cyclin D1 and β2-microglobulin probes were designed to cross a splice junction to avoid a signal generation, resulting from amplification of genomic DNA in the RNA preparations. Target sequences were kept small (85 and 84 bp) to ensure the detection of fragmented and partially degraded RNA. We adapted the quantitative method of Bijwaard et al28 to measure the expression of cyclin D1 in the Light Cycler 2.0 instrument (Roche Applied Science). The reactions were performed in 20 μL light cycler capillaries (Roche Applied Science). The reaction mixture contained 6 μL of 2× concentrated TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA), 1 μL of both primers (15 μM) (Invitrogen), 0.5 μL of probe (5 μM) (PE Applied Biosystems), 0.5 μL of bovine serum albumin (10 mg/mL), and 1 μL of cDNA in a final volume of 20 μL. Samples were amplified in duplicates.To assess the reproducibility of the assay, we repeated qRT-PCR 1 week, 1 month, and 6 months after the original measurements and compared the crossing point values individually and also the normalized crossing point values for each transcript analyzed.Data AnalysisQuantification of mRNA was accomplished by analysis of fluorescence curves and determination of a crossing point for each sample. The cycle at which the fluorescence of a sample rises above the background fluorescence is called the “crossing point” (Cp) of the sample. The Cp of a sample depends on the initial concentration of DNA/cDNA in the tested specimen. Samples with a higher preamplification target concentration show an earlier crossing point. The higher the Cp value, the lower the expression of cyclin D1 in the specimens. Cp values for each reaction were determined using the LightCycler 4.0 software. For each amount of cDNA tested, the duplicate Cp values were averaged.As we wanted to know whether the expression level of cyclin D1 differs between MCLs and other B-NHLs, we used relative quantification. As it is reported that the level of β2-microglobulin mRNA expression is identical in various tissues and diseases and does not even change owing to the treatment, we can use it as an external standard without determining the exact number of cells in each specimen.29 Thus, the cyclin D1 mRNA expression was normalized to that of β2-microglobulin mRNA. Normalization is necessary to avoid the impact of variability in the RNA quality and quantity and variability in reverse transcription efficiency among samples. The relative expression of cyclin D1 was determined by subtracting the average Cp value for β2-microglobulin from the average cyclin D1 Cp value yielding the ΔCp value. The ΔCp value can be used to compare the relative amounts of cyclin D1 between various samples. The advantage of using a reference gene is that this method circumvents the need for accurate quantification and does not deplete the starting material, which is convenient if the starting material is limited.In this study, we required that β2-microglobulin Cp should be less than 35 to be considered as an interpretable specimen because a higher Cp for β2-microglobulin indicates an insufficient template quality, quantity, and/or presence of inhibitors.Statistical AnalysisWe used the JMP IN 5.1 software (SAS Institute, Cary, NC) for statistical analysis. To establish whether cyclin D1 mRNA differences were statistically significant, we used a nonparametric one-way analysis of variance, the Kruskal-Wallis test, and the Mann-Whitney test. For the correlation analysis, nonparametric Spearman rank correlation coefficient was calculated.RESULTSThe patients' age, biopsy sites, definitive diagnoses, results of FISH and/or PCR analysis of translocation t(11;14), results of immunohistochemical detection of cyclin D1, and the relative cyclin D1 levels as determined by qRT-PCR are summarized in Tables 1 to 3. For 11 patients, 2 (specimens nos. 5,6; 21,22; 24,25; 26,27; 38,39; 59,60; 100,101; and 107,108) or 3 (specimens nos. 12,13,14; 17,18,19; and 102,103,104) different biopsy samples from the same patient were analyzed. The MCL group of patients included 35 men and 18 women.JOURNAL/dimp/04.03/00019606-200803000-00007/table1-7/v/2021-02-17T195944Z/r/image-tiff Clinical Data of Patients With MCL, Biopsy Site, Result of PCR and/or FISH Analysis of t(11;14), IHC Detection of the Cyclin D1 Protein, and Relative Cyclin D1 mRNAJOURNAL/dimp/04.03/00019606-200803000-00007/table2-7/v/2021-02-17T195944Z/r/image-tiff Clinical Data of “non-MCL” B-NHL Patients, Biopsy Site, Result of PCR and/or FISH Analysis of t(11;14), IHC Detection of the Cyclin D1 Protein, and Relative Cyclin D1 mRNAJOURNAL/dimp/04.03/00019606-200803000-00007/table3-7/v/2021-02-17T195944Z/r/image-tiff Clinical Data of Patients With MALT Lymphomas, Biopsy Site, Result of PCR and/or FISH Analysis of t(11;18), Results of FISH Analysis of t(11;14), IHC Detection of the Cyclin D1 Protein and Relative Cyclin D1 mRNACyclin D1 Expression Level in MCLs, Reactive Lymph Nodes, and B-NHLs Other Than MALT LymphomasThe individual relative cyclin D1 mRNA values were very low and tightly clustered in reactive lymph nodes and B-NHLs other than MALT lymphomas if β2-microglobulin was used as a normalizer. The distribution of the ΔCp values measured for groups mentioned in the preceding section is shown in Figure 1. A lower ΔCp value in an assay corresponds to a greater amount of cyclin D1 mRNA in the specimen. In MCL, the normalized values were much higher and showed a significantly different distribution from other B-NHLs and reactive lymph nodes. We were able to measure the cyclin D1 mRNA level in all 63 specimens from patients with MCL as defined by IHC and FISH studies, with no false positivities. Two specimens from the 63 patients with MCL did not contain the tumor cells as was properly recognized by the low cyclin D1 expression. Thus, MCL specimens with a tumor population were present in 61 cases. The cyclin D1 overexpression was observed in 60/61 MCLs. Only 1/61 MCLs expressed an unusually low amount of cyclin D1 mRNA, a result similar to values observed in B-NHLs other than MCL, and by criteria used in this study the case did not display the cyclin D1 overexpression. The resultant ΔCps showed a sharp distribution into 2 categories. In all, 98.36% MCLs showed the overexpression of cyclin D1 with ΔCp values below 4 (range, −8.8 to 3.71), whereas all other B-NHLs, except MALT lymphomas, exceeded 4 (range, 4.28 to 14.18). As the data points were not normally distributed, the medians were used to characterize the diagnostic groups. The median cyclin D1 ΔCp value of B-NHLs other than MCL was 6.695; the value found in reactive lymph nodes was 6.215; and the median cyclin D1 ΔCp in MCLs was 1.040. These data indicate that the median value of ΔCp in the MCL group was 6.2 fold lower than the median values in B-NHLs other than MCL and in reactive lymph nodes investigated in this study. A statistical analysis using the Kruskal-Wallis test for a non-normally distributed data demonstrated that there was a significant difference (P<0.0001) between MCLs, reactive lymph nodes, and B-NHLs other than MCL.JOURNAL/dimp/04.03/00019606-200803000-00007/figure1-7/v/2021-02-17T195944Z/r/image-jpeg Distribution of ΔCP values in “non-MCL” B-NHLs (B-NHL), MCLs, reactive lymph nodes (RLN), and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 6.695 for non-MCL B-NHLs, 1.040 for MCLs, and 6.215 for RLNs. The result of the Kruskal-Wallis test was P<0.0001. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances.Cyclin D1 Expression Level in MCLs and MALT LymphomasFigure 2 shows a comparison of the ΔCp values between MCLs and MALT lymphomas. We found low expression in MALT lymphomas rising from sites other than the lungs or stomach (salivary glands, thyroid gland, conjunctiva, colon, and mediastinum) and in 7/19 MALT lymphomas from the lungs and stomach (Table 3). Low expression was not in the range observed in MCLs. In MALT lymphomas originating in the lungs and stomach, the cyclin D1 expression overlapped to some extent with the expression observed in MCLs (12/19 MALT lymphomas from the lungs and stomach showed cyclin D1 overexpression). Thus, the cyclin D1 expression level was not helpful to separate the lung and gastric MALT lymphomas from MCLs with certainty despite the fact that there was a significant difference (P<0.0001) between MCLs and MALT lymphomas in general and even between MCLs and MALT lymphomas from the lungs and stomach (P<0.0001).JOURNAL/dimp/04.03/00019606-200803000-00007/figure2-7/v/2021-02-17T195944Z/r/image-jpeg Distribution of ΔCP values in MALT lymphomas (MALT L), MCLs, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 3.750 for MALT Ls from the lungs or stomach (MALT L l/s), 5.080 for MALT Ls from tissues other than the lungs or stomach (MALT L other), and 1.040 for MCLs. The result of the Kruskal-Wallis test was P<0.0001. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances.PCR Targeting MTC, FISH, and qRT-PCRGenomic PCR targeting MTC detected t(11;14) only in 10/40 specimens from MCL patients tested, and the detection rate (25%) was consistent with the results published in the literature. FISH detected t(11;14) in 52/55 specimens from MCL patients. In 3 patients, the specimens were not evaluable owing to insufficient samples. However, qRT-PCR yielded satisfactory results in all specimens. Thereby, qRT-PCR is more sensitive and applicable for the molecular diagnosis of MCL. None of the patients with B-NHLs other than MCL (including all MALT lymphomas with the high cyclin D1 mRNA level) showed positive PCR and/or FISH result for t(11;14). As expected from the qRT-PCR results, a good correlation was observed between qRT-PCR of cyclin D1 mRNA and the presence of t(11;14) detected by interphase FISH and/or PCR. The 2 MCL patients (no. 9 and no. 43) with low expression of cyclin D1 mRNA and positive FISH for t(11;14) are of interest. As PCR targets only MTC and no other breakpoints, a possible positional effect of the IgH enhancer on the cyclin D1 transcription may be inferred by comparing cyclin D1 mRNA levels in PCR-positive and PCR-negative cases. This comparison showed no effect of the proximity of the breakpoint site on cyclin D1 mRNA expression levels (Fig. 3), which is in accordance with data presented by Thomazy et al.30 Similarly, there was no apparent correlation between the nuclear staining intensity, as revealed by IHC, and the precise location or presence of a detectable translocation.17JOURNAL/dimp/04.03/00019606-200803000-00007/figure3-7/v/2021-02-17T195944Z/r/image-jpeg Comparison of ΔCp values in PCR-positive (MTC+) and PCR-negative (MTC−) cases, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 0.295 for MTC+ and 1.260 for MTC−. The result of the Mann-Whitney test was P>0.2449. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances.IHC Staining and qRT-PCRCases with available histologic material were subjected to IHC staining for the cyclin D1 protein. The cyclin D1 protein expression was examined by the immunohistochemical analysis in 95/116 B-NHLs. Forty-one cases of MCL showed strong nuclear cyclin D1 staining, 12 weak nuclear cyclin D1 staining, and in 2 patients we were unable to interpret cyclin D1 staining results. In 1 case (no. 20), which was previously shown by FISH to carry t(11;14), the biopsy failed to reveal the cyclin D1 staining. In the remaining specimen (no. 38), the tissue was poorly preserved and neither cyclin D1 IHC nor FISH for t(11;14) yielded satisfactory results. However, qRT-PCR was able to detect the cyclin D1 overexpression in these 2 cases. Elevated cyclin D1 mRNA levels examined by qRT-PCR were always accompanied by the cyclin D1 overexpression detected by IHC. None of the B-NHLs other than MCL showed a positive result of IHC for cyclin D1, as expected from this group of lymphomas, which is in complete concordance with the qRT-PCR. We analyzed immunohistochemically the cyclin D1 protein expression in all MALT lymphomas. None of the MALT lymphomas revealed the cyclin D1 positive staining in tumor cells. Actually, IHC staining identified a significant proportion of the cyclin D1 positive epithelial cells in the MALT lymphoma specimens with high cyclin D1 mRNA. The proportion of the cyclin D1 expressing epithelial cells was considerably higher in this group of MALT lymphomas than the number of epithelial cells present in other specimens with low cyclin D1 mRNA or in MCLs. Thus, qRT-PCR compares favorably with the results of IHC. However, the level of cyclin D1 mRNA did not seem to correlate with the protein staining intensity by IHC. IHC allows only a semiquantitative assessment, and it is less sensitive and reproducible than qRT-PCR. Moreover, although IHC staining for cyclin D1 is reproducible and reliable in epithelial cells and is applicable in most cases, it is still technically difficult to obtain standard results in all cases of lymphoid tissues. qRT-PCR permits a precise, simple, rapid, and reproducible quantitative assessment of the cyclin D1 expression in both, well-preserved and poorly preserved specimens. The results of quantitative PCR, therefore, have a great advantage over IHC and will be especially of value in identifying a smaller number of tumor cells (in bone marrow or after therapy).Cyclin D1 Expression Level in MCLs in Relation to Sex and AgeThe median of relative cyclin D1 levels was higher in men (1.250 for male, 0.725 for female), although the expression patterns were not statistically different when we compared male and female patients (Fig. 4). This observation is in accordance with Thomazy et al30 who did not obtain any sex-related differences in the cyclin D1 levels. We did not observe any correlation between age of patients and ΔCp (Spearman rank correlation coefficient: −0.0044, P>0.9727).JOURNAL/dimp/04.03/00019606-200803000-00007/figure4-7/v/2021-02-17T195944Z/r/image-jpeg Comparison of ΔCp values between males and females, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 0.725 for females and 1.250 for males. The result of the Mann-Whitney test was P>0.4970. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances.qRT-PCR Assay—Reproducibility, Reliability, and UtilityNo major difference in relative cyclin D1:β2-microglobulin expression levels were observed in cases if multiple lymph node specimens from the same patient were examined.We tested the reproducibility of the assay by running 58 samples repeatedly. In 55/58 cases we obtained reproducible results. None of them failed to amplify. The median variability for the Cp values between the runs was 0.090 for cyclin D1 and 0.095 for β2-microglobulin, as calculated by running repetitious assays. Thus, there were no significant shifts in Cp values when the analysis was repeated or the reverse transcription product was changed.Duplicate amplifications produced nearly identical overlapping amplification curves from which the Cp values were calculated.We tested the utility of the assay on routinely formalin-fixed, paraffin-embedded tissues. A quantifiable result for β2-microglobulin was obtained from every specimen analyzed, even in the case in which qualitative PCR failed to amplify the control gene. All MCLs displayed ΔCp values between −8.8 and 3.71, irrespective of the type of tissue used (frozen or formalin-fixed) (Fig. 5). The median value for formalin-fixed, paraffin-embedded tissue was 0.960 and for frozen specimens it was 1.128. Although the corresponding Cp values for the fresh-frozen specimens for both cyclin D1 and β2-microglobulin were lower than the Cp values, resulting from the formalin-fixed cases, the consequent ΔCp values were not statistically different between frozen and formalin-fixed, paraffin-embedded tissues. To compare expression level measurements by real time qRT-PCR in formalin-fixed, paraffin-embedded tissues with those obtained in fresh-frozen tissues, we analyzed cyclin D1 mRNA in 3 matching MCLs (data not shown) and did not find any significant difference. Thus, we were able to demonstrate the applicability of this assay to formalin-fixed, paraffin-embedded tissues.JOURNAL/dimp/04.03/00019606-200803000-00007/figure5-7/v/2021-02-17T195944Z/r/image-jpeg Comparison of ΔCp values in formalin-fixed, paraffin-embedded (FFPE) and frozen specimens, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 0.960 for FFPE and 1.128 for frozen specimens. The result of the Mann-Whitney test was P>0.6789. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances.DISCUSSIONEstablishing the diagnosis of MCL may be difficult, but to distinguish MCL from other low-grade B-cell lymphoproliferative disorders is important because of the different biology of MCL and therapeutical reasons. Current WHO guidelines for the diagnosis of MCL rely on a morphologic assessment supplemented with an analysis of the cyclin D1 translocation or overexpression.The translocation t(11;14)(q13;q32) is reported to be detectable in 40% to 50% of MCL by PCR, 70% to 80% by Southern blot, 60% to 80% cytogenetically, and in almost all cases by FISH.3,31–33 Of these methods, FISH is the most reliable method, and PCR has the lowest reported sensitivity. An alternative approach to obtain support for the diagnosis is to determine the expression levels of the CCND1 gene. As the cyclin D1 expression is very low to undetectable in normal lymphocytes, staining the cells for increased levels of cyclin D1 provides an excellent marker for making a specific diagnosis. The cyclin D1 expression is reported in a majority of MCLs and rarely in hairy cell leukemia and multiple myeloma.25 Hairy cell leukemia and multiple myeloma are morphologically easily distinguishable from MCL. In MCL, the cyclin D1 overexpression has been studied by different methods, including Northern blot analysis,34 quantitative and qualitative RT-PCR,21 in situ hybridization,35 Western blot analysis,24 and immunohistochemical analysis.24 Although the blotting techniques and in situ hybridization either require relatively large amounts of tumor tissue or are technically demanding, immunohistochemical analysis is commonly, and in a majority of cases, used successfully in the diagnosis of MCL. However, this method is technically challenging and the result also depends on the quality of tissue fixation. Therefore, false negative results are observed, which decreases the sensitivity of the method. Moreover, IHC is susceptible to subjective interpretation and more importantly, it does not allow quantification of the protein expression, which is important if monitoring of the disease dynamics is needed. In the case of cyclin D1, IHC is technically difficult and it is not applicable to frozen tissues. In the case of lymphocytes, it is still complicated to obtain results with sufficient sensitivity and reproducibility of the staining in all cases, limiting the cyclin D1 IHC in the diagnosis of MCL. RT-PCR is not convenient either, because it demonstrates the cyclin D1 expression in the majority of lymphomas other than MCLs22 owing to low expression levels in the neoplastic cells and in the non-neoplastic cells present in the sample. We obtained positive results by RT-PCR even from reactive lymphoid tissues. Therefore, these techniques are not commonly used for diagnosis of MCL.In this study, we examined the level of the cyclin D1 expression using qRT-PCR in a group of various B-NHLs and have established a reliable cutoff value, which is applicable to the differential diagnosis of MCL.We tested fresh-frozen and formalin-fixed, paraffin-embedded tissue samples for the cyclin D1 mRNA expression and normalized these concentrations to the concentrations of β2-microglobulin mRNA. We found that the assay has the potential to distinguish MCL from other B lymphoproliferative malignancies. Previous studies have shown the detection limit of qRT-PCR to be 1 tumor cell in 104 to 105 nucleated cells in the specimen.28,36We detected some cyclin D1 expression in all B-NHLs and reactive lymph nodes. Despite the fact that some groups have reported that reactive lymphoid tissues and B-NHLs other than MCL do not express cyclin D1,35,37 other groups (including our data) have detected low levels of cyclin D1 mRNA or the protein in non-neoplastic B-lymphocytes and B-NHLs other than MCL by Northern or Western blot or RT-PCR.23,30,34 We were unable to discriminate between MCLs and other B-NHLs until we examined the level of cyclin D1 mRNA.Our results have shown that measurement of cyclin D1 mRNA can distinguish MCL from other B-NHLs. By the criteria set in this study, the cyclin D1 mRNA overexpression was detected in 60/63 MCL patients and was not detected in any of the reactive lymph nodes and “non-MCL” B-NHLs, except MALT lymphomas. From the pattern of the cyclin D1 expression in MCLs and B-NHLs other than MCL, we concluded that ΔCp value higher than 4 can be considered as a low expression, whereas a result less than 4 should be taken as a high expression. Thus, our data permit an estimation of a cutoff value of ΔCp for the diagnosis of MCL below which all cases of MCL fall. However, we strongly recommend that all laboratories establish their own reliable cutoff limit, discriminating between MCL and other B-NHLs during the test development and validation process. The cutoff value set at 4 in the reported system resulted in nearly 100% sensitivity and specificity for the diagnosis of MCL in lymphoid tissue specimens. None of the B-NHLs other than MCL or reactive lymphadenopathies demonstrated ΔCp value below 4 (100% specificity). Of the 63 MCLs, 2 specimens (nos. 9, 59) showed a low expression of the cyclin D1 transcript, because the sample available for the qRT-PCR analysis did not contain the tumor cell population as revealed by histopathology. Only 1 case of 63 MCLs (1.59%) (case no. 43) was in the non-MCL B-NHLs range. The lack of the cyclin D1 mRNA or protein expression has been reported in 5% to 10% of MCL patients.18,19,34 Recently, a new subtype of MCL which is cyclin D1 negative and shares the unique gene expression signature of MCL, was identified.38,39 However, the specimen no. 43 was positive for cyclin D1 by IHC and positive for t(11;14) by FISH and had morphologic findings consistent with a classic form of MCL. A posttranscriptional mechanism induced by an increased stability of the cyclin D1 transcript, resulting in an enhanced translation of cyclin D1,40 or a translational mechanism,23 or a polymorphism, or a mutation of the affected allele may prevent the assay from detecting the mRNA overexpression. These facts are possible explanations of the discrepancy between genomic, transcriptional, and protein observations. Similarly, Thomazy et al30 observed 1 case with a moderate level of cyclin D1 mRNA by qRT-PCR and a cyclin D1 overexpression by the immunohistochemical analysis. The cyclin D1 mRNA overexpression was detected in the absence of the cyclin D1 protein in rare cases of peripheral T-cell lymphoma by Elenitoba-Johnson et al.41 Discrepancies between cyclin D1 mRNA and protein levels suggest that regulatory controls at posttranscriptional and/or translational levels persist in malignant cells of MCL. Alternatively, we may speculate that a simultaneous decrease in the cyclin D1 expression and increase in the β2-microglobulin gene expression may occur. Also, it is unknown whether β2-microglobulin was changed in the case. However, it is reported that β2-microglobulin is the most suitable normalizing transcript tested because its variation between different sample origins and within distinct cell types is the lowest.29We have demonstrated a 98.41% specificity of qRT-PCR for the diagnosis in the MCL group and 100% specificity in the group of B-NHLs other than MCLs. Thus, for the ancillary diagnosis of MCL, qRT-PCR represents a diagnostic improvement as the detection rates for t(11;14) by PCR (40% to 50%) or the immunohistochemical detection of the cyclin D1 expression (85%) are lower.Contrary to our expectation, 12/19 MALT lymphomas from the lungs and stomach expressed comparable levels of cyclin D1 with levels observed in MCLs. We interpreted high levels of β2-microglobulin–normalized cyclin D1 values in these cases as caused by admixed non-neoplastic cells rather than a true cyclin D1 overexpression in the tumor. Contaminating epithelial cells, which show an immunohistochemically detectable expression of cyclin D1, may falsely decrease ΔCp values in these extranodal lymphomas. This fact was confirmed by microscopic analysis of the cyclin D1 IHC of extranodal lymphomas. The cyclin D1 positive epithelia were observed in all the extranodal specimens. The cyclin D1 mRNA level reflected the proportional composition of the extranodal specimen rather than the staining intensity of the epithelium. The cyclin D1 mRNA was higher in the specimens in which the cyclin D1 negative tumor population formed a minor component. Samples with predominating tumor cells showed lower cyclin D1 mRNA. Generally, the MALT lymphomas with high cyclin D1 mRNA showed a significantly higher number of admixed cyclin D1 positive epithelial cells than the extranodal MALT lymphomas with low cyclin D1 mRNA or any other lymphoma specimen. Similarly, Bijwaard et al28 found cyclin D1 mRNA near the cutoff value in the transbronchial biopsy and adenoids because of the cyclin D1 expression in the epithelium overlying the lymphoid tissue. Elenitoba-Johnson et al41 noted that higher levels of cyclin D1 transcript were detected in 2 reactive tonsils in which tonsillar epithelium was present. False positive results were obtained in a high number of extranodal tissues containing epithelia42 if the cyclin D1 expression was normalized to the housekeeping gene. Therefore, in MALT lymphomas, which represent a distinct clinical entity from MCL, concordance is needed between morphologic, immunohistochemical, flow cytometric, cytogenetic, and/or molecular examination and the cyclin D1 status. In contrast to nearly 100% specificity for the diagnosis of MCL in the group of B-NHLs other than MALT lymphomas, 63% of MALT lymphomas from the lungs and stomach showed results of qRT-PCR similar to those observed in MCLs. All other MALT lymphomas expressed cyclin D1 mRNA comparable with the non-MCL B-NHLs' range.The analytical sensitivity of qRT-PCR may be increased if an expressed gene specific for B cells was used instead of β2-microglobulin as the normalizing transcript. A gene expressed only in B cells and expressed in MCL cells and in almost all B-NHLs would decrease the background variability, and this can be especially useful if large numbers of nonlymphoid cells are included in the specimens, in the way they are in MALT lymphomas. The contribution of a B-cell fraction to the total cyclin D1 was estimated by using CD20 as a normalizer by Thomazy et al.30 However, the latter authors and Ginaldi et al43 reported CD20 mRNA at slightly different levels in various B-NHLs. Thus, the use of another B-cell specific marker is recommended. As CD19 is generally present at similar concentrations in MCLs and other B-NHLs,43 it is an accurate B-cell specific normalizer increasing the specificity and sensitivity of qRT-PCR analysis of cyclin D1 mRNA.36 The detection limit of this assay was 15-fold to 20-fold higher compared with the assay normalized, to β2-microglobulin. Cyclin D3 mRNA has not been reported in epithelia either. Therefore, if cyclin D3 was used as a normalizer, all the non-MCL B-NHLs were separated from the MCL samples, including extra-nodal specimens containing epithelial cells.42 Using cyclin D1/cyclin D3 ratio would not lead to false positive diagnoses of MCL in extra-nodal presentations.The differences in the level of the cyclin D1 mRNA overexpression in MCL cases, resulting in a non-normal distribution of the mRNA copy numbers, suggest heterogeneity within the MCL group. This observation is in accordance with the results of Thomazy et al,30 who reported that the heterogeneity was in correlation with immunohistochemical findings. They hypothesized that the observed variability possibly reflected the lack of regulatory constraint on the translocated cyclin D1 gene. The variation in cyclin D1 levels may reflect a biologic heterogeneity of MCLs.38Bijwaard et al,28 Hui et al,44 and this study confirm that the qRT-PCR assay could be readily applied to analyze RNA extracted from a routine archival material, even in cases in which the end point control PCR fails. This allows the use of vast tissue resources stored as archived paraffin blocks. The stored samples used in the present study were tissue blocks archived up to 6 years, and amplifiable RNA was obtained in all cases. The dependence on optimal fixation and processing, in case of qRT-PCR, is obviated by normalizing the level of cyclin D1 mRNA to a reference message. As the messages of both the tested and the reference gene are similarly affected, a valid relative level can be obtained even if the RNA yield is low owing to a suboptimal recovery. Fixation does not influence qRT-PCR results as long as small amplicons are used. It is in accordance with observations of Specht et al,45 who demonstrated that the expression level determinations from formalin-fixed, paraffin-embedded tissue were comparable with those obtained from matching fresh-frozen tissue.In this well-defined group of patients with MCL, we have shown an assay allowing assessment of the cyclin D1 expression level in all specimens. When the results of the qRT-PCR analysis are compared with those obtained from immunophenotyping and/or PCR, it may be concluded that qRT-PCR is the most useful, reliable, rapid, reproducible, sensitive, and specific method broadening our diagnostic tools in hematopathology. In comparison with interphase FISH in paraffin sections, qRT-PCR is less technically and time demanding and, furthermore, is more sensitive in detecting small changes in mRNA level. 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Link]1124547600019606-200803000-0000700006374_1991_6_449_rosenberg_overexpression_|00019606-200803000-00007#xpointer(id(citation_FROM_JRF_ID_d2166e948_citationRF_FLOATING))|11065405||ovftdb|SL000063741991644911065405citation_FROM_JRF_ID_d2166e948_citationRF_FLOATING[Medline Link]201140000019606-200803000-0000700006702_1991_88_9638_rosenberg_centrocytic_|00019606-200803000-00007#xpointer(id(citation_FROM_JRF_ID_d2166e982_citationRF_FLOATING))|11065213||ovftdb|SL00006702199188963811065213citation_FROM_JRF_ID_d2166e982_citationRF_FLOATING[CrossRef]10.1073%2Fpnas.88.21.963800019606-200803000-0000700006702_1991_88_9638_rosenberg_centrocytic_|00019606-200803000-00007#xpointer(id(citation_FROM_JRF_ID_d2166e982_citationRF_FLOATING))|11065405||ovftdb|SL00006702199188963811065405citation_FROM_JRF_ID_d2166e982_citationRF_FLOATING[Medline Link]1682919 Clinical Data of Patients With MCL, Biopsy Site, Result of PCR and/or FISH Analysis of t(11;14), IHC Detection of the Cyclin D1 Protein, and Relative Cyclin D1 mRNA Clinical Data of “non-MCL” B-NHL Patients, Biopsy Site, Result of PCR and/or FISH Analysis of t(11;14), IHC Detection of the Cyclin D1 Protein, and Relative Cyclin D1 mRNA Clinical Data of Patients With MALT Lymphomas, Biopsy Site, Result of PCR and/or FISH Analysis of t(11;18), Results of FISH Analysis of t(11;14), IHC Detection of the Cyclin D1 Protein and Relative Cyclin D1 mRNA Distribution of ΔCP values in “non-MCL” B-NHLs (B-NHL), MCLs, reactive lymph nodes (RLN), and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 6.695 for non-MCL B-NHLs, 1.040 for MCLs, and 6.215 for RLNs. The result of the Kruskal-Wallis test was P<0.0001. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances. Distribution of ΔCP values in MALT lymphomas (MALT L), MCLs, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 3.750 for MALT Ls from the lungs or stomach (MALT L l/s), 5.080 for MALT Ls from tissues other than the lungs or stomach (MALT L other), and 1.040 for MCLs. The result of the Kruskal-Wallis test was P<0.0001. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances. Comparison of ΔCp values in PCR-positive (MTC+) and PCR-negative (MTC−) cases, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 0.295 for MTC+ and 1.260 for MTC−. The result of the Mann-Whitney test was P>0.2449. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances. Comparison of ΔCp values between males and females, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 0.725 for females and 1.250 for males. The result of the Mann-Whitney test was P>0.4970. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances. Comparison of ΔCp values in formalin-fixed, paraffin-embedded (FFPE) and frozen specimens, and a normal quantile plot. The greater the Cp value, the lower the expression of cyclin D1 in the specimen. The median values were 0.960 for FFPE and 1.128 for frozen specimens. The result of the Mann-Whitney test was P>0.6789. The normal quantile plot shows both the difference in the values (vertical position) and the variances (slopes) for each group. The normality is judged by how well the points follow a straight line. The standard deviations are the slopes of the straight lines. Lines with steep slopes represent the distribution with greater variances.Quantitative Measurement of Cyclin D1 mRNA, a Potent Diagnostic Tool to Separate Mantle Cell Lymphoma From Other B-cell Lymphoproliferative DisordersBrizova Helena MSc; Kalinova, Marketa MSc; Krskova, Lenka PhD; Mrhalova, Marcela PhD; Kodet, Roman MD, PhDOriginal ArticlesOriginal Articles117p 39-50