PCR Testing in Hematopathology

The Hematopathology laboratory at the University of Washington is pleased to announce two new molecular diagnostic tests for the diagnosis and monitoring of lymphoma. These tests take advantage of the polymerase chain reaction (PCR), a technique that is being used with increasing frequency in clinical laboratories for everything from genetic testing to detection of microorganisms and viruses. We have used this technology to develop two tests: one to detect clonal immunoglobulin gene rearrangements for the diagnosis of lymphoma, and the other to detect the common t(14;18) translocation of follicular lymphoma. Detailed information about these tests is provided on the following pages.

Specimen Information
Testing can be performed on blood, bone marrow,body fluids, fresh or frozen tissue specimens, or paraffin-embedded, fixed tissues

Arrangements for testing should be scheduled with the Hematopathology Laboratory at (206) 598-6215.

Scientific and Clinical Information
Daniel E. Sabath, MD, PhD, Hematopathology Laboratory Director, is available for discussion. He can be reached at (206) 598-6131.

B Cell Clonality Analysis

During lymphocyte development, both B and T cells rearrange the DNA encoding their respective antigen receptors. In the case of B cells, the immunoglobulin gene DNA is rearranged. This rearrangement mechanism enables the immune system to generate the antibody diversity necessary to detect millions of different antigens. When the immunoglobulin heavy chain rearranges, different gene segments that are widely separated in unrearranged DNA are brought into proximity. These segments are referred to as the V (variable), D (diversity), and J (joining) segments (see Figure 1). In a random

Figure 1. VDJ Joining. During B cell development, one of several V, D, and J segments are brought together during gene rearrangement. In addition, random nucleotides are inserted at each of the joining sites. PCR is performed using the V and J primers. In a random population of B cells, a variety of PCR products are produced. In a B cell lymphoma, a single PCR product is produced.

population of B cells, all the cells use different combinations of V, D, and J segments, and consequently they all have different immunoglobulin gene structures. In the case of B cell lymphoma, all the cells are descended from a single clone, and therefore the cells all have the same immunoglobulin gene structure. We take advantage of this property of B cells when we perform genomic DNA blot (Southern blot) analysis of B cell clonality, the current technology used for gene rearrangement studies.

At the same time that V-D-J joining occurs, random numbers of DNA basepairs are inserted between at the V-D and D-J junctions. Consequently, all non-neoplastic B cells have slightly different distances between the V and J segments. The polymerase chain reaction allows one to produce large amounts of DNA, the ends of which are determined by the primers one uses. For B cell clonality analysis, we use two PCR primers: one that corresponds to a consensus sequence common to most V segments, the other a consensus sequence common to most J segments. When the DNA is amplified, the length of the PCR product is determined by the number of random nucleotides added at the time of VDJ joining. In a non-neoplastic population of B cells, each PCR product is slightly different in size. When these products are separated by electrophoresis, a smear is seen on the gel. In the case of lymphoma, all the B cells have the same VDJ rearrangement. Therefore, the neoplastic cells all produce a PCR product of the same size, resulting in the appearance of a discrete band on the gel.

The advantages of the PCR method over the Southern blot technology currently in use include the ability to use less DNA, shorter turnaround time, and the ability to analyze paraffin-embedded specimens. For Southern blot analysis, 30 mg of DNA are used, and it takes up to two weeks from the time a specimen is received to when a result is obtained. For PCR clonality analysis, only 0.5 mg of DNA is needed. This means that B cell clonality analysis can be performed on small specimens such as endoscopic biopsies that would have yielded insufficient DNA for blot analysis. Currently, PCR analysis will be performed twice a week, meaning that a result can be obtained within 5 working days from receipt of a specimen, compared with up to two weeks for Southern blot. Finally, paraffin-embedded specimens can be used as a source of DNA for clonality analysis. This was not possible for Southern blot analysis, since high molecular weight DNA is needed for that technique and fixation degrades DNA. Partially degraded DNA can be used for PCR analysis, since only a small segment of the DNA needs to be intact for amplification. The ability to perform gene rearrangement studies on fixed tissue can result in substantial cost savings for patients who in the past would have had repeat biopsies to obtain fresh or frozen tissue for gene rearrangement studies when frozen tissue was not saved initially.

Compared to Southern blot analysis, the PCR method is somewhat less sensitive. Clonality analysis by DNA blot can detect a clonal population if it represents 5% of the total cells in the specimen. The sensitivity of PCR analysis depends on the type of the specimen. Unlike Southern blotting, where all cells, including non-lymphoid cells, contribute to the background signal, only B cells contribute to background signal in PCR analysis. Thus, in tissues where there are large numbers of normal B cells, such as lymph nodes, the PCR technique is less sensitive than in tissues where there are small numbers of B cells, including peripheral blood, bone marrow, and non-hematopoietic tissue. In a preliminary study where 55 cases of known lymphoma were analyzed by PCR, 38 cases were positive, for a sensitivity of 69%, compared with 90% for DNA blot analysis. The largest group of PCR negative cases is follicular lymphoma where amplification may not occur due to inability of the consensus primers to bind to the DNA. In most cases where primer binding occurs, the PCR technique probably has equal or better sensitivity to the Southern blot.

PCR analysis has proven to be exquisitely specific. In a preliminary analysis of 36 cases that were negative for lymphoma, no positive PCR results were obtained, for a specificity of 100%. Thus, for clinical use, we recommend PCR clonality analysis as a first step in determining whether a patient has a clonal population of B cells in a specimen. If the result is positive (or if a clear polyclonal population is seen), no further workup is necessary. If the result is negative or equivocal, Southern blot confirmation is recommended if there is sufficient DNA.

B cell clonality analysis can be performed on peripheral blood, bone marrow, fresh or frozen tissue, body fluids, or paraffin-embedded specimens. It can be ordered in two forms, PCR alone , or PCR with Southern blot confirmation . If ordered alone, when a clear result (clonal or polyclonal) is obtained, that result will be reported by telephone within 5 working days, with a printed report to follow. If the analysis is technically inadequate or equivocal, a Southern blot will be performed. If Southern blot confirmation is ordered, both tests will be performed. The cost of both tests is less than if they were ordered separately. The cost of PCR alone is less than that of Southern blot alone. For paraffin-embedded tissue or very small specimens, Southern blot confirmation will not be available.

T Cell Clonality Analysis

As of May 1, 1996, a PCR method will be available for detecting clonal T cell populations in clinical specimens. The principle of this test is identical to that for the B cell clonality test, and the advantages of PCR testing such as smaller specimen requirements and the ability to use fixed tissue are available for the T cell method. Three sets of consensus primers for the T cell receptor gamma chain are used in the analysis. A small amount of radioactivity is included in the PCR reaction, and the PCR products are analyzed on a DNA sequencing gel. A polyclonal T cell population results in a smear of PCR products on the gel, and a monoclonal population results in one or two distinct bands. The sensitivity and specificity for the T cell PCR method are similar to those for the B cell PCR method.

As with B cell clonality testing, T cell clonality testing can be performed on peripheral blood, bone marrow, fresh or frozen tissue or fixed paraffin-embedded tissue (note that certain fixatives, particularly B-5, can result in more DNA fragmentation than formalin, so these specimens often cannot be used for PCR. A similar situation exists for decalcified specimens). As with B cell testing, the T cell PCR testing can be ordered with or without Southern blot confirmation.

Bcl-2 Translocation Analysis

In most cases of follicular lymphoma, there is a translocation involving chromosomes 14 and 18. The translocation results in insertion of the Bcl-2 gene from chromosome 18 into the immunoglobulin heavy chain locus on chromosome 14 (Figure 2). The result is overexpression of the Bcl-2 protein, which

Figure 2. The t(14;18) associated with follicular lymphoma. Detection of this translocation is accomplished by the polymerase chain reaction using Bcl-2 and JH primers.

is involved in prevention of cell death by apoptosis. The resulting increased life span of the affected cell is thought to make the cell susceptible to other mutational events leading to neoplasia. This rearrangement of DNA can be detected by the polymerase chain reaction using primers that span the translocation breakpoint. The clinical test to detect t(14;18) uses two primers, one recognizing sequences from the Bcl2 gene, the other recognizing immunoglobulin sequences. There are two major breakpoint cluster regions, so we actually use a number of Bcl-2 primers to increase sensitivity. If a positive result is obtained, cells containing the translocation are present. We have been able to detect as few as one in 105 translocation-containing cells using the primers for the major breakpoint region in our preliminary studies. If a negative result is obtained, either less than one in 105 cells contain the translocation, or the DNA was not able to be amplified. To rule out this second possibility, we perform PCR using primers for a different gene (the cystic fibrosis gene) on negative specimens.

This test has two main uses. First, it can be used to differentiate follicular lymphoma from reactive lymphoid hyperplasia. A positive result can confirm a diagnosis of follicular lymphoma if the morphology is correct. The other use for the test is to detect small numbers of lymphoma cells in patients with known follicular lymphoma. We have been able to detect small numbers of lymphoma cells in the peripheral blood and bone marrow of lymphoma patients. In addition, this test has been used to determine whether lymphoma cells are present in peripheral stem cell or bone marrow products being used for autologous transplantation. Finally, the test can be used to monitor therapy of patients in whom a clinical remission has been achieved.

As with the B cell clonality analysis, Bcl-2 translocation analysis can be performed on peripheral blood, bone marrow, fresh or frozen tissue, body fluids, or paraffin-embedded tissue. This test is offered in two forms. The first is the Bcl-2 translocation screen , which is designed for patients who have not had Bcl-2 PCR analysis before. PCR is performed using all primer sets in order to determine which breakpoint, if any, is detectable. The second test is the Bcl-2 translocation analysis for minimal residual disease . This test is designed for patients who have previously had a positive PCR result. For these patients, only the diagnostic primer set is used, resulting in a lower cost. The test is performed weekly; a positive result can usually be obtained within 5 working days. If there is a negative result, the amplification with CF primers will be performed to rule out technical problems; these results will require an additional few working days.

Last updated: 6/22/00