Fluorescence PCR Quantification of Cyclin D1 Expression
2002; Elsevier BV; Volume: 4; Issue: 2 Linguagem: Inglês
10.1016/s1525-1578(10)60686-1
ISSN1943-7811
AutoresKojo S.J. Elenitoba‐Johnson, Sandra D. Bohling, Stephen D. Jenson, Zhaosheng Lin, Kimberly A. Monnin, Megan S. Lim,
Tópico(s)Cancer-related Molecular Pathways
ResumoWe have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A β-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL. We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A β-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL. Mantle cell lymphoma (MCL) is a distinct clinicopathologic entity that is characterized by the presence of the t(11;14)(q13;q32) chromosomal translocation.1Banks PM Chan J Cleary ML Delsol G De Wolf-Peeters C Gatter K Grogan TM Harris NL Isaacson PG Jaffe ES Mason D Pileri S Ralfkiaer E Stein H Warnke RA Mantle cell lymphoma: a proposal for unification of morphologic, immunologic, and molecular data.Am J Surg Pathol. 1992; 16: 637-640Crossref PubMed Scopus (435) Google Scholar, 2Campo E Raffeld M Jaffe ES Mantle-cell lymphoma.Semin Hematol. 1999; 36: 115-127PubMed Google Scholar, 3Harris N Jaffe E Stein H Banks P Chan J Cleary M Delsol G De Wolf-Peeters C Falini B Gatter K Grogan T Isaacson P Knowles D Mason D Muller-Hermelink H-K Pileri S Piris M Ralfkiaer E Warnke R A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar The t(11;14) results in juxtaposition of the bcl-1 locus in close proximity to the immunoglobulin heavy chain enhancer, resulting in deregulation and overexpression of cyclin D1, an important regulator of G1/S progression in the cell cycle.4Tsujimoto Y Yunis J Onorato-Showe L Erikson J Nowell PC Croce CM Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation.Science. 1984; 224: 1403-1406Crossref PubMed Scopus (523) Google Scholar, 5Tsujimoto Y Jaffe E Cossman J Gorham J Nowell PC Croce CM Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation.Nature. 1985; 315: 340-343Crossref PubMed Scopus (342) Google ScholarMCL shares some histological and immunophenotypic features with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and other low-grade B-cell lymphomas. Because MCL is a clinically aggressive neoplasm, its distinction from CLL/SLL and other low-grade B-cell lymphomas is important. In this regard, the detection of the t(11;14) has served as a good discriminator of MCL from other entities exhibiting similar histopathological features. The translocation can be detected in 90 to 95% of MCLs by fluorescence in situhybridization,6Vaandrager JW Schuuring E Zwikstra E de Boer CJ Kleiverda KK van Krieken JH Kluin-Nelemans HC van Ommen GJ Raap AK Kluin PM Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization.Blood. 1996; 88: 1177-1182PubMed Google Scholar 70 to 80% by conventional cytogenetics,7Cuneo A Bigoni R Rigolin GM Roberti MG Bardi A Piva N Milani R Bullrich F Veronese ML Croce C Birg F Dohner H Hagemeijer A Castoldi G Cytogenetic profile of lymphoma of follicle mantle lineage: correlation with clinicobiologic features.Blood. 1999; 93: 1372-1380PubMed Google Scholar 60 to 70% by Southern blot hybridization,8Williams ME Meeker TC Swerdlow SH Rearrangement of the chromosome 11 bcl-1 locus in centrocytic lymphoma: analysis with multiple breakpoint probes.Blood. 1991; 78: 493-498PubMed Google Scholar and 30 to 40% using polymerase chain reaction (bcl-1, major translocation cluster/immunoglobulin joining).9Luthra R Hai S Pugh WC Polymerase chain reaction detection of the t(11;14) translocation involving the bcl-1 major translocation cluster in mantle cell lymphoma.Diagn Mol Pathol. 1995; 4: 4-7Crossref PubMed Scopus (62) Google Scholar, 10Pinyol M Campo E Nadal A Terol MJ Jares P Nayach I Fernandez PL Piris MA Montserrat E Cardesa A Detection of the bcl-1 rearrangement at the major translocation cluster in frozen and paraffin-embedded tissues of mantle cell lymphomas by polymerase chain reaction.Am J Clin Pathol. 1996; 105: 532-537PubMed Google Scholar On the other hand, cyclin D1 protein expression is demonstrable in approximately 70% of cases of MCL by immunohistochemical methods.11Zukerberg LR Yang WI Arnold A Harris NL Cyclin D1 expression in non-Hodgkin's lymphomas: detection by immunohistochemistry.Am J Clin Pathol. 1995; 103: 756-760Crossref PubMed Scopus (141) Google Scholar, 12Vasef MA Medeiros LJ Koo C McCourty A Brynes RK Cyclin D1 immunohistochemical staining is useful in distinguishing mantle cell lymphoma from other low-grade B-cell neoplasms in bone marrow.Am J Clin Pathol. 1997; 108: 302-307PubMed Google Scholar, 13Aguilera NS Bijwaard KE Duncan B Krafft AE Chu WS Abbondanzo SL Lichy JH Taubenberger JK Differential expression of cyclin D1 in mantle cell lymphoma and other non-Hodgkin's lymphomas.Am J Pathol. 1998; 153: 1969-1976Abstract Full Text Full Text PDF PubMed Scopus (70) Google ScholarWhile highly characteristic of MCL, elevated cyclin D1 protein has been demonstrated in other lymphoproliferative processes such as hairy cell leukemia,11Zukerberg LR Yang WI Arnold A Harris NL Cyclin D1 expression in non-Hodgkin's lymphomas: detection by immunohistochemistry.Am J Clin Pathol. 1995; 103: 756-760Crossref PubMed Scopus (141) Google Scholar, 14Bosch F Campo E Jares P Pittaluga S Munoz J Nayach I Piris MA Dewolf-Peeters C Jaffe ES Rozman C Montserrat E Cardesa A Increased expression of the PRAD-1/CCND1 gene in hairy cell leukaemia.Br J Haematol. 1995; 91: 1025-1030Crossref PubMed Scopus (98) Google Scholar, 15Miranda RN Briggs RC Kinney MC Veno PA Hammer RD Cousar JB Immunohistochemical detection of cyclin D1 using optimized conditions is highly specific for mantle cell lymphoma and hairy cell leukemia.Mod Pathol. 2000; 13: 1308-1314Crossref PubMed Scopus (93) Google Scholar plasma cell dyscrasias,11Zukerberg LR Yang WI Arnold A Harris NL Cyclin D1 expression in non-Hodgkin's lymphomas: detection by immunohistochemistry.Am J Clin Pathol. 1995; 103: 756-760Crossref PubMed Scopus (141) Google Scholar, 12Vasef MA Medeiros LJ Koo C McCourty A Brynes RK Cyclin D1 immunohistochemical staining is useful in distinguishing mantle cell lymphoma from other low-grade B-cell neoplasms in bone marrow.Am J Clin Pathol. 1997; 108: 302-307PubMed Google Scholar rare cases of B-cell CLL/SLL,11Zukerberg LR Yang WI Arnold A Harris NL Cyclin D1 expression in non-Hodgkin's lymphomas: detection by immunohistochemistry.Am J Clin Pathol. 1995; 103: 756-760Crossref PubMed Scopus (141) Google Scholar, 12Vasef MA Medeiros LJ Koo C McCourty A Brynes RK Cyclin D1 immunohistochemical staining is useful in distinguishing mantle cell lymphoma from other low-grade B-cell neoplasms in bone marrow.Am J Clin Pathol. 1997; 108: 302-307PubMed Google Scholar and epithelial malignancies.16Donnellan R Chetty R Cyclin D1 and human neoplasia.Mol Pathol. 1998; 51: 1-7Crossref PubMed Scopus (303) Google Scholar Elevated cyclin D1 mRNA expression has been demonstrated by Northern blot17Bosch F Jares P Campo E Lopez-Guillermo A Piris MA Villamor N Tassies D Jaffe ES Montserrat E Rozman C Cardesa A PRAD-1/cyclin D1 gene overexpression in chronic lymphoproliferative disorders: a highly specific marker of mantle cell lymphoma.Blood. 1994; 84: 2726-2732PubMed Google Scholar, 18de Boer CJ van Krieken JH Kluin-Nelemans HC Kluin PM Schuuring E Cyclin D1 messenger RNA overexpression as a marker for mantle cell lymphoma.Oncogene. 1995; 10: 1833-1840PubMed Google Scholar and in situ hybridization analyses in MCLs.19Williams ME Nichols GE Swerdlow SH Stoler MH In situ hybridization detection of cyclin D1 mRNA in centrocytic/mantle cell lymphoma.Ann Oncol. 1995; 6: 297-299PubMed Scopus (31) Google Scholar Similarly, RNA expression studies have also shown elevated levels of cyclin D1 transcripts in the majority of MCLs and in a minority of other lymphoproliferative disorders by conventional end-point reverse trancription-polymerase chain reaction (RT-PCR)-based analyses.13Aguilera NS Bijwaard KE Duncan B Krafft AE Chu WS Abbondanzo SL Lichy JH Taubenberger JK Differential expression of cyclin D1 in mantle cell lymphoma and other non-Hodgkin's lymphomas.Am J Pathol. 1998; 153: 1969-1976Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Uchimaru K Taniguchi T Yoshikawa M Asano S Arnold A Fujita T Motokura T Detection of cyclin D1 (bcl-1, PRAD1) overexpression by a simple competitive reverse transcription-polymerase chain reaction assay in t(11;14)(q13;q32)-bearing B-cell malignancies and/or mantle cell lymphoma.Blood. 1997; 89: 965-974PubMed Google Scholar Such end-point PCR-based methods suffer the drawback of including the non-quantitative plateau phase of the amplification in the final determination of relative cyclin D1 expression levels. Furthermore, these methods are labor intensive, and may yield variable results. The purpose of this study, therefore, was to apply continuous fluorescence PCR monitoring, which reliably determines the onset of the exponential phase of PCR, for quantification of cyclin D1 mRNA levels. Using this methodology, we also sought to determine the sensitivity and specificity of cyclin D1 mRNA overexpression for the diagnosis of MCL.Materials and MethodsSample SelectionArchived snap-frozen tissue samples of a total of 57 cases of lymphoproliferative disorders including mantle cell lymphoma (n = 10), CLL/SLL (n = 11), follicular lymphoma (n = 6), peripheral T-cell lymphoma (n = 3), anaplastic large cell lymphoma (n = 3), acute lymphoblastic leukemia/lymphoma (n = 15), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) were selected for study. Reactive follicular hyperplasia (n = 5) and healthy peripheral blood lymphocytes (n = 6) were also examined. All clinical samples were classified according to the Revised European American Lymphoma (REAL) classification,3Harris N Jaffe E Stein H Banks P Chan J Cleary M Delsol G De Wolf-Peeters C Falini B Gatter K Grogan T Isaacson P Knowles D Mason D Muller-Hermelink H-K Pileri S Piris M Ralfkiaer E Warnke R A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar and demonstrated the characteristic histological and immunophenotypic profiles of the diagnosis rendered. Fourteen cell lines (10 hematopoietic, 4 non-hematopoietic) were also evaluated for cyclin D1 mRNA expression (Table 1).Table 1Cyclin D1 mRNA Expression Normalized to β-Globin and Relative to Follicular Hyperplasia: Cell LinesSampleDiagnosisCyclin D1 mRNA overexpressionCyclin D1 normalized quantity* CyclinD1 normalized quantity=E^[CT(Test cyclin D1)-CT(Follicular hyperplasia cyclin D1)]E^-[CT(Test β-globin)-CT(Follicular hyperplasia β-globin)] CT = crossing threshold. +, positive. −, negative. E = efficiency of reaction.GrantaMantle cell lymphoma+15.35NCEBMantle cell lymphoma−0.82SUPB-15B-cell ALL−0.30K-562Chronic myelogenous leukemia−0.52Molt-4Precursor T-cell ALL−0.31REHPrecursor B-cell ALL−0.25697Precursor B-cell ALL−0.20NB-4Acute promyelocytic leukemia+1.56Kasumi-1Acute myeloid leukemia−0.20Karpas 299t(2;5) positive T-cell lymphoma−0.22SW1271Small cell carcinoma+59.30HT1080Fibrosarcoma+290.02PA-1Ovary teratocarcinoma+390.72SK-N-SHNeuroblastoma+18.77Samples with a normalized cyclin D1 expression level greater than 1.00 were defined as overexpressing cyclin D1 MRNA.* CyclinD1 normalized quantity=E^[CT(Test cyclin D1)-CT(Follicular hyperplasia cyclin D1)]E^-[CT(Test β-globin)-CT(Follicular hyperplasia β-globin)]CT = crossing threshold.+, positive.−, negative.E = efficiency of reaction. Open table in a new tab Immunohistochemical StudiesImmunohistochemistry for cyclin D1 was performed on formalin-fixed, paraffin-embedded tissue sections of all tissue samples, but not on cell lines. Antigen retrieval was performed using microwave heat pretreatment.21Bhan A Immunoperoxidase.in: Colvin R Bhan A McCluskey R Diagnostic Immunopathology. ed 2. Raven Press, New York1995: 711-723Google Scholar An avidin-biotin peroxidase method was performed using an automated immunostainer (Ventana Medical System, Tuscon, AZ). We used a commercially available antibody against cyclin D1 (Neomarkers, Union City, CA).RNA Extraction and Reverse TranscriptionTotal RNA was extracted from archived fluid and tissue samples taken from patients from the University of Utah Health Sciences Center, Salt Lake City, Utah, and from the Sunnybrook and Women's College Health Sciences Center, Toronto, Ontario, Canada. RNA was extracted using Trizol (Gibco BRL, Life Technologies, Rockville, MD) or the RNeasy Mini RNA extraction kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Reverse transcription was performed using Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco BRL, Life Technologies, Rockville, MD) and random hexamers (Promega, Madison, WI) in the presence of RNase inhibitor (Amersham-Pharmacia, Piscataway, NJ). cDNA quantity was assessed using absorbance at 260 nm.Primer and Probe DesignThe primers and probes used for the cyclin D1 (GenBank Accession no. Z23022) and β-globin (GenBank Accession no. AF181989) RT-PCR are summarized in Table 2, and were designed using Primer Designer software version 4.0 (Sci-Ed Software, Durham, NC).Table 2Oligonucleotide Primer and Probe Sequences for Fluorescent Cyclin D1 and β-Globin PCRPrimers and probesSequenceGenBank accession no.Basesβ-GlobinβG-555′-GAGAAGTCTGCCGTTACTGC-3′AF18198955–74βG-268c5′-GGTGAGCCAGGCCATACTA-3′AF181989249–268βG-151-F5′-CAGAGGTTCTTTGAGTCCTTTGGGGATCTG-fluorescein-3′AF181989151–180βG-181-7055′-LCRed705-TCCACTCCTGATGCTGTTAT-phosphate-3′AF181989181–200Cyclin D1D1-11095′-CCTCCTCTCCGGAGCATTT-3′Z230221109–1127D1-1315c5′-CTGTAGCACAACCCTCCTCC-3′Z230221296–1315D1-1140-F5′-GGAAAGCTTCATTCTCCTTGTTGTTGGTTG-fluorescein-3′Z230221140–1169D1-1171-6405′-LCRed640-TTTTTCCTTTGCTCTTTCCC-phosphate-3′Z230221171–1190 Open table in a new tab Fluorescence RT-PCRQuantitative PCR was performed using sequence-specific hybridization probes for cyclin D1 and β-globin mRNA transcripts. Rapid cycle amplification was performed using a thermal cycler integrated with a fluorimeter (Lightcycler, Roche Molecular Biochemicals, Indianapolis, IN). Fifty ng of template cDNA were amplified in a 10 μl reaction containing 1X PCR buffer (50 mmol/L Tris [pH 8.3], 3.0 mmol/L MgCl2, and 500 μg/ml bovine serum albumin), deoxynucleotide triphosphates at 200 mmol/L each, and 0.4 units Promega Taq polymerase (Madison, WI) with 11 ng/μl of TaqStart antibody (ClonTech, Palo Alto, CA). The primers specific for cyclin D1 were used at a concentration of 0.5 μmol/L per reaction, while the β-globin primers were used at 0.2 μmol/L per reaction (Table 2). Reactions also included a fluorescein isothiocyanate (FITC)-labeled probe and a LightCycler Red (LCRed) 640-labeled probe specific for cyclin D1, and a FITC-labeled probe and a LCRed705 labeled-probe specific for β-globin. FITC-labeled probes were used at a 0.1 μmol/L concentration, while the LCRed- labeled probes were used at 0.2 μmol/L (Table 2). The reaction mixture was subjected to rapid PCR amplification consisting of denaturation at 95°C for 0 seconds, annealing at 55°C for 10 seconds, and extension at 72°C for 10 seconds. The fluorescence readings were plotted against the cycle number over 45 cycles. The log-linear portion of this graph was used todetermine a fractional cycle number for threshold fluorescence (Figure 1).Quantitative Fluorescence PCRThe second derivative maximum function included in the LightCycler software was used to determine the fractional cycle numbers used for quantification.22Rasmussen R Quantification on the LightCycler.in: Meuer S Wittwer CT Nakagawara K Rapid Cycle Real-Time PCR. Springer, Mannheim, Germany2001: 21-34Crossref Google ScholarDetermination of Relative Cyclin D1 Transcript QuantityThe exponential phase of a PCR amplification is described by the equation Tn = T0En, where Tn is the amount of target sequence at a particular cycle number (n),T0 is the initial target quantity,E represents the efficiency of the amplification reaction and n is cycle number. The log-linear equivalent of the above equation: log Tn=log T0+ n*log E, permits determination of E from the standard curves. Efficiency was calculated from LightCycler software plots using log E = −1/slope.22Rasmussen R Quantification on the LightCycler.in: Meuer S Wittwer CT Nakagawara K Rapid Cycle Real-Time PCR. Springer, Mannheim, Germany2001: 21-34Crossref Google Scholar Our relative quantification assay using β-globin as an external standard was configured such that the PCR amplifications for both cyclin D1 (E = 2 ± 0.1) and β-globin (E = 2 ± 0.1) yielded very similar amplification efficiencies (data not shown). Quantification of mRNA was accomplished by analysis of fluorescence curves and determination of crossing threshold (the cycle at which the fluorescent signal rises above background) for each sample. Samples with a higher pre-amplification target concentration show an earlier cycle threshold (Figure 2). The difference in cycle threshold obtained for samples with high levels of cyclin D1 mRNA and those samples with normal levels of cyclin D1 mRNA is used to calculate a relative quantity of cyclin D1 mRNA. This value is normalized to a β-globin transcript to adjust for differences in the amount of mRNA present in the sample. The cyclin D1 quantity relative to β-globin andnormalized to the cyclin D1 quantity of follicular hyperplasia was calculated using the following equation: Normalized cyclin D1 quantity=E∧-[CT(Test cyclin D1)-CT(Follicular hyperplasis cyclin D1)]E∧-[CT(Test β-globin)-CT(Follicular hyperplasis β-globin)](CT=crossing threshold)E=efficiencyThe cut-off value for overexpression of cyclin D1 was defined as greater than the highest normalized transcript level for cyclin D1 obtained for reactive follicular hyperplasia.Figure 2Determination of cyclin D1 mRNA expression level normalized to β-globin and relative to follicular hyperplasia. Schematic representation of the cycle shifts using cyclin D1 and β-globin primer/probes. Quantification of mRNA expression is accomplished by analysis of the fluorescence curves from each sample. Samples with a higher pre-amplification target concentration show an earlier cycle threshold. The difference in cycle threshold obtained for samples with high levels of cyclin D1 mRNA and those samples with normal levels of cyclin D1 mRNA is used to calculate a relative quantity of cyclin D1 mRNA transcripts. β-globin is used to normalize for the amount of mRNA present in the sample. The differences in cycle threshold were used to calculate the normalized cyclin D1 mRNA expression level as indicated in the text. ΔC = cycle threshold difference.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analytical SensitivitycDNA extracted from the Granta-519 cell line was serially diluted into cDNA obtained from peripheral blood lymphocytes. The samples were analyzed using our fluorescent RT-PCR method and the normalized quantities of cyclin D1 mRNA were calculated (Figure 3).Figure 3Dilutional analysis for cyclin D1 mRNA quantification using sequence-specific hybridization probes. A: cDNA from the mantle cell lymphoma cell line, Granta-519, was diluted into cDNA obtained from peripheral B-lymphocytes in log dilutions. When Granta-519 cDNA was diluted in peripheral blood lymphocyte-derived cDNA in a 1:10 ratio, it was still possible to demonstrate an earlier fractional cycle for the 10% Granta-519 sample when compared to 100% peripheral blood lymphocyte cDNA. H2O, no template control. B: Standard curve generated using dilutions of Granta-519. The error bars represent the crossing threshold standard deviation (SD) of three replicates for each dilution (100% Granta: SD = 0.22; 10% Granta: SD = 0.12; 1% Granta: SD = 0.54; and 0.1% Granta: SD = 0.78).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Statistical AnalysesThe statistical tool included in the Microsoft Excel program (Microsoft, Redwood, WA) was used for the determination of p-values (paired two-tailed Students' t-test) for the normalized quantities of cyclin D1 in the MCLs in comparison to the CLL/SLLs, and the mean values of normalized quantities of cyclin D1 transcripts.ResultsImmunohistochemical StudiesImmunohistochemical reactivity for cyclin D1 was demonstrated in 8 of 10 MCLs. These immunohistochemically positive samples corresponded to those identified by RT-PCR analysis for cyclin D1 mRNA. No reactivity for cyclin D1 was demonstrable in all other (n = 57) biopsy samples (data not shown).Quantitative Fluorescence RT-PCRThe hybridization probe assay results are summarized in Table 1, Table 3. The cyclin D1 mRNA expression level normalized to β-globin and relative to the highest cyclin D1 quantity obtained for a follicular hyperplasia sample was determined as described in the methods section. Samples with a normalized cyclin D1 quantity greater than 1.00 were defined as overexpressing cyclin D1 mRNA. By this criterion, cyclin D1 mRNA overexpression was detected in 8 of 10 MCLs, 1 of 6 T-cell lymphomas, 1 acute promyelocytic leukemia derived cell line (NB-4), and all 4 of 4 non-hematopoietic cell lines. All CLL/SLL cases (n = 11) showed relatively low levels of cyclin D1 transcript expression (generally two to three orders of magnitude less than MCL). Using our method, none of the cases of hairy-cell leukemia (0 of 3) showed elevated cyclin D1 transcript levels (Table 3).Table 3Cyclin D1 mRNA Expression Normalized to β-globin and Relative to Follicular Hyperplasia: Biopsy SamplesDiagnosisNumber of samples% of samples showing cyclin D1 mRNA overexpressionRange of relative normalized cyclin D1 quantityMantle cell lymphoma1080%0.611–62.25Chronic lymphocytic leukemia/small lymphocytic lymphoma110%0.00064–0.395Hairy cell leukemia30%0.0063–0.0105Follicular lymphoma (grades I & II)60%0.066–0.702Anaplastic large cell lymphoma30%0.014–0.0415Peripheral T-cell lymphoma333%0.110–6.63Other non-Hodgkin's lymphomas60%0.0091–0.165Precursor acute lymphoblastic leukemia/lymphoma150%9.02E-07–0.078Reactive follicular hyperplasia50%0.0204–1.00Reactive peripheral blood lymphocytes60%9.64E-05–0.00012Samples with a normalized cyclin D1 expression level greater than 1.00 were defined as over-expressing cyclin D1 mRNA.Other Non-Hodgkin's lymphomas included Burkitt (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1). Open table in a new tab Analytical SensitivityDilutional analysis using Granta-519 cell line cDNA diluted into cDNA obtained from peripheral blood lymphocytes (PBL) revealed that cyclin D1 mRNA expression was higher in a 10% dilution of the Granta-519 cell line cDNA, when compared to the cyclin D1 expression in 100% PBL cDNA (Figure 3). This suggests that cyclin D1 mRNA overexpression may only be distinguished when the sample analyzed contains at least 10% cyclin D1 overexpressing tumor cells. The within run standard deviation (three replicates each) for the fractional threshold cycles for the cyclin D1 quantitative PCR using the Granta-519 cell line at 100%, 50%, and 10% dilutions into cDNA derived from unstimulated peripheral blood lymphocyte-derived mRNA were 0.22, 0.15, and 0.12, respectively.Statistical AnalysesCyclin D1 mRNA overexpression clearly distinguished MCL from CLL/SLL (p = 0.07) (paired two-tailed Student's t-tests), but was less discriminatory in the other lymphoproliferative disorders (Figure 4).Figure 4Comparison of normalized cyclin D1 mRNA expression levels in mantle cell lymphoma and other non-Hodgkin's lymphomas, leukemias, and cell lines derived from hematopoietic and non-hematopoietic neoplasms. The difference in cycle threshold between the cyclin D1 and β-globin reactions was calculated and then normalized against the cycle threshold difference for reactive hyperplasia. Samples with a normalized cyclin D1 quantity greater than 1.00 (dashed line) were defined as overexpressing cyclin D1 mRNA. The horizontal black bars indicate the median expression level for each sample type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionSeveral methods have been described for the quantitative analysis of nucleic acid sequences. In general, these methods have been based on end-point or competitive PCR analyses measuring band intensities in ethidium bromide stained gels or hybridization-based approaches using radioisotopic labeling and densitometry.23Wang AM Doyle MV Mark DF Quantitation of mRNA by the polymerase chain reaction.Proc Natl Acad Sci USA. 1989; 86: 9717-9721Crossref PubMed Scopus (1611) Google Scholar, 24Raeymaekers L A commentary on the practical applications of competitive PCR.Genome Res. 1995; 5: 91-94Crossref PubMed Scopus (107) Google Scholar, 25Ferre F Quantitative or semi-quantitative PCR: reality versus myth.PCR Methods Appl. 1992; 2: 1-9Crossref PubMed Scopus (43) Google Scholar, 26Clementi M Menzo S Bagnarelli P Manzin A Valenza A Varaldo PE Quantitative PCR and RT-PCR in virology.PCR Methods Appl. 1993; 2: 191-196Crossref PubMed Scopus (127) Google ScholarHiguchi et al27Higuchi R Dollinger G Walsh PS Griffith R Simultaneous amplification and detection of specific DNA sequences.Biotechnology (N Y). 1992; 10: 413-417Crossref PubMed Scopus (744) Google Scholar, 28Higuchi R Fockler C Dollinger G Watson R Kinetic PCR analysis: real-time monitoring of DNA amplification reactions.Biotechnology (N Y). 1993; 11: 1026-1030Crossref PubMed Scopus (1621) Google Scholar introduced fluorescence monitoring at each cycle for quantitative PCR analysis using ethidium bromide to monitor DNA synthesis. Since then, real-time PCR has gained increasi
Referência(s)