Taqman PCR In The Detection And Quantification Of Chromosomal Translocations In Follicular Lymphoma, Mantle Cell Lymphoma And Chronic Myeloid Leukemia: Taqman Real-time PCR Assay
F Vega, J Medeiros, R Luthra
chronic myeloid leukemia, follicular lymphoma, mantle cell lymphoma, medicine, pathology, taqman real-time
F Vega, J Medeiros, R Luthra. Taqman PCR In The Detection And Quantification Of Chromosomal Translocations In Follicular Lymphoma, Mantle Cell Lymphoma And Chronic Myeloid Leukemia: Taqman Real-time PCR Assay. The Internet Journal of Genomics and Proteomics. 2003 Volume 1 Number 1.
Real-time polymerase chain reaction (PCR) methods are relatively recent technological advances that allow routine quantitative analysis of nucleic acid sequences (DNA and RNA). In this review, we provide a brief overview of 5\'-3\' exonuclease-based real-time PCR assays (also known as TaqMan real-time PCR) used in own and in many other laboratories. We discuss the theory, protocols, advantages, drawbacks and utility of these methods in the assessment and quantification of some of the common chromosomal translocations associated with hematopoietic tumors.
I. TaqMan Real-Time PCR assay
The real-time polymerase chain reaction (PCR) assay is a relatively recent technological advance that is quickly becoming widely accepted for routine quantitative analysis of nucleic acid sequences (DNA and RNA). TaqMan Real-time PCR, sometimes referred to as kinetic PCR, or 5' exonuclease-based PCR assay, exploits the 5'-3' exonuclease activity of Taq polymerase first decribed by Holland and collaborators (1) in 1991. This assay integrates fluorogenic PCR with a laser-based instrumentation system, the PRISM 7700 Sequence Detector (PE Applied Biosystems, Foster City, CA), to detect and quantitate specific PCR amplicons as the reactions proceed.
The real-time PCR reaction includes all the components of a conventional PCR reaction (the four nucleotides in an appropriate buffer containing an optimum concentration of magnesium, primers and DNA polymerase), however, a TaqMan probe is also required. The TaqMan probe is the key feature of the real-time PCR assay and consists of a non-extendable oligonucleotide complementary to the target sequence that is labeled with a 5'-reporter dye and a 3' quencher dye. The fluorescent dye 6-carboxy-fluorescein (FAM), generally used as reporter dye is covalently linked to the 5' end of the oligonucleotide. Other dyes such as JOE (2,7 dimethoxy-4,5-dichloro-6-carboxyfluorescein) and VIC may also be used as reporter dyes. The reporter dye is quenched by TAMRA (6-carboxy-tetramethylrodamine), located at the 3' end. When the probe is intact, the fluorescence emission of the reporter dye is quenched owing to the physical proximity of the reporter and quencher dyes (Foster-type energy transfer) (2,3). During PCR, forward and reverse primers hybridize to a specific sequence of the target of DNA and the TaqMan probe hybridizes to the target sequence internal to the primer sequences. During the extension phase of PCR, the 5' exonuclease activity of Taq polymerase hydrolyzes the TaqMan probe. The reporter dye and quencher dyes are separated upon cleavage resulting in increased fluorescence of the reporter (Figure 1).
This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence is measured, and is proportional to the amount of target amplification during PCR. Both primer and probe must hybridize to the target for amplification and cleavage to occur. Because of this requirement, non-specific amplification is not detected. Fluorescence increases in proportion to the concentration of the DNA templates, which rises geometrically in the presence of DNA amplification, but only linearly in its absence (Figure 2).
As the amount of amplicon produced in any given cycle within the exponential phase of PCR is proportional to the initial number of template copies, a standard curve can be generated using the fluorescence data from the serial dilution study (Figure 3). The threshold cycle (y-axis) is the cycle at which the fluorescence signal of the reporter dye rises above the baseline signal of the dye. Such standard curves can be utilized to determine the relative number of cells in a test sample carrying tumor associated fusion sequences, such as t(14;18), t(11;14) or t(9;22) chromosomal translocations.
II. Advantages and drawbacks of the real-time PCR assay
Real-time PCR methods have several advantages over conventional PCR techniques that require gel electrophoresis-based amplicon detection and quantitation techniques. The majors advantages are:
No post-PCR manipulation is required. Thus, the results are available as soon as PCR is completed (i.e., within two hours) decreasing the turnaround time and additionally reducing the risk of PCR contamination.
This methodology allows co-amplification and detection of a normalizer gene (housekeeping gene and/or positive internal control) in the same tube to obtain accurate quantitative measurements and to confirm the presence of amplifiable DNA in a test sample.
Decreased variability, because the collection of the data is performed during the exponential phase of the PCR, thus the results are not influenced by limited reagents.
As the TaqMan probe is complementary to the target, the assay is target specific.
High sensitivity, 1 tumor cell in 100,000 normal cells can be detected.
The major disadvantages are:
Real-time PCR methods do not provide size determination of the PCR products. Due to this drawback, one cannot easily exclude contamination between samples and compare amplicon sizes when multiple samples from the same patient are analyzed.
The assay requires that the size of the target to be amplified to be limited to a less than 150 base pairs to obtain a maximum efficiency.
III. Taqman PCR in the Detection and Quantification of Chromosomal Translocations
Detection of t(14;18)(q32;q21) in Follicular Lymphoma
Follicular lymphoma (FL) is a neoplasm of follicle center B cells and represents 22% of non-Hodgkin's lymphomas (NHL) (4). Most cases of FL are cytogenetically characterized by the t(14;18)(q32;q21) translocation detected by conventional cytogenetics in approximately 90% of FL, as well as 20-30% of diffuse large B-cell lymphomas (DLBCL), the latter presumably of follicular center origin (5,6). In the t(14;18), the
Real-time PCR and Follicular Lymphoma
Although the majority of patients with indolent FL achieve clinical remission after induction therapy, they continue to have low-level disease that eventually leads to clinical relapse. Several studies have recently demonstrated that achievement of molecular complete remission is a desirable goal of new therapies because patients with molecular complete remission have longer disease free status (9,10,11). The clinical importance of achieving molecular response has been demonstrated in FL patients treated with several regimens, including rituximab (11,12) and stem cell therapy (13).
Since our original report on the application of real-time PCR for t(14:18), several investigators have demonstrated the utility and reliability of this technique in quantifying tumor burden in FL patient samples (8,13,14,15,16,17). For example, Hirt and Dolken (13) have demonstrated that quantitative detection of circulating t(14;18) by real-time PCR in follow up samples of FL patients after autologous bone marrow transplantation predicts clinical course of the disease. Similarly, Ladetto and collaborators (17) have shown that tumor burden in stem cell harvests detected by real-time PCR for t(14;18) can predict the effectiveness of therapeutic intervention in FL patients. These studies demonstrate that real-time PCR is a reliable tool that can be used for monitoring minimal residual disease and in the evaluation of treatment effectiveness in FL patients.
Detection of t(11;14)(q13;q32) in Mantle Cell Lymphoma
Mantle cell lymphomas (MCL) represent approximately 6% of NHLs (4). The t(11;14)(q13;q32) is present in virtually all cases of MCL and is also found in a small subset of cases of plasma cell myeloma (18A). The t(11;14) also has been rarely reported in other types of NHL and lymphoid leukemias. However, most of these cases, in retrospect, are examples of MCL that were misclassified because they were either high-grade or had marked leukemic involvement (18B).
As a result of the t(11;14),
Translocations involving the MTC are amenable to routine PCR analysis, and thus 30% to 40% of cases of MCL can be shown to carry the t(11;14) translocation by PCR methods.
Real-time PCR and Mantle Cell Lymphoma
Our study using real-time PCR for the detection of the t(11;14) offered promising results. This assay effectively identified
An alternative approach to obtain support for the diagnosis of MCL is to determine expression levels of the
Several investigators have demonstrated the utility and reliability of the real-time PCR assay detection of
Detection of t(9;22)(q34;q11) in Chronic Myeloid Leukemia
The Philadelphia (Ph) chromosome, which can be found in 90-95% of cases of chronic myeloid leukemia (CML), is the cytogenetic hallmark of this disease. The t(9;22)(q34;q11) in CML involves juxtaposition of sequences from c-
Depending on the breakpoint in
The major forms of
The (9;22) translocation is detected by conventional cytogenetics in approximately 90% of patients with a clinical presentation consistent with CML. Three to five percent of patients show a normal chromosome 22 but molecular evidence of the
Real-time RT PCR and Chronic Myeloid Leukemia
Real-time RT PCR technique is a reliable method for monitoring CML patients before and after therapy (33-37). As new therapies improve the rate of complete cytogenetic response, molecular monitoring is likely to become increasingly important in the management of patients with CML.
Rajyalakshmi Luthra, Ph.D University of Texas M.D. Anderson Cancer Center NA01.091, 8515 Fannin Houston, TX 77054 Tel: 713-794-5443 Fax: 713-794-4773 Email: email@example.com