Malignant lymphomas are neoplasms that arise from B

Transcription

1 Overview of the Role of Molecular Methods in the Diagnosis of Malignant Lymphomas L. Jeffrey Medeiros, MD; Jeanne Carr, PhD Objective. To review the role of molecular genetics in the diagnosis of malignant lymphomas. Data Sources and Study Selection. Primary research studies and reviews published in the English literature that focus on molecular genetics and malignant lymphoma, in particular, clonality, chromosomal translocations, tumor suppressor genes, and Hodgkin disease. Data Extraction and Synthesis. Molecular genetics has an important role in the assessment of malignant lymphomas. Clonality, detected by Southern blot analysis or the polymerase chain reaction, is helpful for establishing the diagnosis of lymphoma in lesions with ambiguous morphologic and immunophenotypic findings. Southern blot analysis is the gold standard for clonality assessment, but the process is labor-intensive and time-consuming. Polymerase chain reaction analysis is more convenient, but a potentially significant false-negative rate exists in the analysis of some antigen receptor genes as a result of using consensus primers and the process of somatic hypermutation. Chromosomal translocations, which result in oncogene activation, occur in many types of B- and T-cell lymphomas, and their detection is helpful in classification as well as in establishing a diagnosis of malignancy. Gene rearrangements and chromosomal translocations also can be used to monitor minimal residual disease. Tumor suppressor genes, although their analysis is relatively less useful for diagnosis, are involved in both pathogenesis and tumor progression and will be more important diagnostically as this field continues to expand. Molecular genetic analysis has played a major role in improving our understanding of Hodgkin disease. Conclusions. Molecular genetic tests are currently important ancillary tools for the diagnosis and classification of malignant lymphomas, and their role is likely to increase in the future. (Arch Pathol Lab Med. 1999;123: ) Malignant lymphomas are neoplasms that arise from B or T cells at various stages in normal lymphocyte development. The large number of malignant lymphoma types and their wide range of morphologic features is a reflection of the complexity of the immune system. Although malignant lymphomas are assessed initially using histologic or cytologic features, it is now abundantly clear that morphology alone is inadequate to classify many malignant lymphomas. With the advent of immunophenotypic and molecular genetic methods in the 1980s, we learned that histologically similar lymphoid neoplasms are immunophenotypically and molecularly heterogeneous. Immunophenotyping to determine lineage and clonality, performed by utilizing either flow cytometry or immunohistochemical techniques, is usually the first ancillary test. As immunophenotypic techniques continue to evolve, immunophenotyping alone is usually adequate for establishing the correct diagnosis. With 5- or 6-parameter Accepted for publication June 17, From the Division of Pathology and Laboratory Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, Tex (Dr Medeiros); and William Beaumont Hospital, Department of Clinical Pathology, Royal Oak, Mich (Dr Carr). Presented at the Eighth Annual William Beaumont Hospital DNA Technology Symposium, DNA Technology in the Clinical Laboratory, March 25 27, Reprints: L. Jeffrey Medeiros, MD, Division of Pathology and Laboratory Medicine, Hematopathology Section, Box 85, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX analysis, flow cytometry immunophenotypic methods are very sensitive for the detection of B-cell clonality and are capable of detecting 1 monoclonal cell among 10 3 or 10 4 polyclonal cells, which is essentially equivalent to the detection capabilities of molecular techniques. Nevertheless, limitations of immunophenotypic analysis remain. T cells do not express clonal markers analogous to the immunoglobulin (Ig) light chains. Furthermore, immunophenotypic methods assess proteins and rely on gene expression. Molecular genetic techniques provide the advantage of assessing the genes directly. In general, molecular genetic methods provide a second level of testing, which is particularly useful for the analysis of neoplasms in which the histologic and immunophenotypic data are not conclusive. In this review article, we provide an overview of molecular genetic methods and their utility in the diagnosis of malignant lymphomas. We focus on 2 commonly used applications of molecular genetics for the diagnosis of malignant lymphomas: assessment of clonality and chromosomal translocations. We also briefly discuss tumor suppressor genes and the role molecular genetics has played in increasing our understanding of Hodgkin disease. CLONALITY Molecular Genetic Basis of Antigen Receptor Gene Rearrangements Malignant lymphomas are neoplasms that arise from lymphoid cells of either B-cell or T-cell lineage. B cells express surface immunoglobulins and T cells express T-cell receptors (TCRs). Immunoglobulins and TCRs are antigen receptors. 1 Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1189

2 Figure 1. Germline configuration of immunoglobulin heavy (IgH), Ig and Ig light-chain genes. Each gene is composed of variable (V), joining (J), and constant (C) regions. The IgH gene also has diversity (D) regions. The immunoglobulin molecule is a multidimeric protein composed of 2 heavy and 2 light (either or ) polypeptide chains. Similarly, the TCR is a heterodimeric protein that is analogous in structure to immunoglobulin, of which there are 2 types, / and /. Approximately 95% of mature T cells express the / TCR, which is composed of and polypeptide chains. Approximately 5% of mature T cells express the / TCR, composed of and polypeptide chains. Each antigen receptor gene, in its germline (or embryonic) configuration, is composed of discontinuous segments of DNA (Figures 1 and 2). 1 4 These discontinuous segments are known as variable (V), diversity (D), joining (J), and constant (C) regions. Variable, J, and C regions are present in all antigen receptor genes. Diversity regions are components of only the immunoglobulin heavy (IgH) and the TCR - and TCR -chain genes. Early in normal lymphocyte differentiation within the bone marrow, the antigen receptor genes rearrange, forming a continuous segment of V(D)J DNA and resulting in the synthesis of immunoglobulins in B cells and TCRs in T cells. For those genes without D regions, such as the Ig lightchain gene, one of many V segments is directly joined with a J segment. This step alone generates many different DNA sequences that code for slightly different Ig light-chain proteins. For other genes with D regions, such as the IgH gene, rearrangement begins with D-J joining, followed by V-DJ joining (Figure 3). Because there are numerous V, D, and J segments in the IgH gene, many more combinations are possible than for the Ig light-chain gene. Each combination of segments results in slightly different DNA sequences being used to form an antigen receptor gene and corresponding protein with unique specificity. No rearrangement occurs between the fused VJ or VDJ segments and the C regions. The message encoded by the C region is incorporated into the immunoglobulin or TCR molecules at the time of transcription into VDJC messenger RNA (mrna) and is then translated into protein. Using the gene rearrangement process, a relatively limited amount of genetic information inherited in the germline is translated into a large number of possible gene sequences that encode the antigen-binding sites of either immunoglobulin or TCR molecules. The actual shuffling and joining of these discontinuous segments explains, at a statistical level, the diversity of antigen receptors that can be generated by the human immune system. In addition, 3 other factors add to the diversity. First, there is flexibility at the joining sites between the V-J, D-J, and V-D regions, which can alter the open reading frame of the sequence. Second, nontemplated nucleotides, so-called N insertions, are added between the joined V, D, and J segments by the enzyme terminal deoxynucleotidyl transferase. 5 These N insertions also can alter the open reading frame of the sequence. Third, point mutations occur in the V and J segments, particularly within the immunoglobulin complementarity-determining regions of the V segments, the regions that appear to be most responsible for encoding antigen receptor specificity. This phenomenon, known as somatic hypermutation, occurs commonly within the immunoglobulin genes, particularly in B cells after they have been exposed to antigen selection, and does not occur (or is rare) in the TCR genes. The IgH, Ig, and TCR genes have more than one C region, which further increases the number of immunoglobulin molecules with different specificity. The IgH gene C regions also may rearrange, subsequent to VDJ joining, through a mechanism that allows an antigen receptor en Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr

3 Figure 2. Germline configuration of T-cell receptor (TCR) / -, -, and -chain genes. Each gene is composed of variable (V), joining (J), and constant (C) regions. The TCR and TCR genes also have diversity (D) regions. Figure 3. Schematic illustration of immunoglobulin heavy-chain gene rearrangement. Diversity-joining (D-J) joining occurs initially, followed by V-DJ joining. During the process of transcription, VDJ RNA is joined with constant (C) region RNA. Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1191

4 Table 1. Summary of Immunoglobulin (Ig) and T-Cell Receptor (TCR) Gene Rearrangements in Lymphoid Neoplasms Detected Using Southern Blot Hybridization* Neoplasm IgH Ig TCR TCR TCR B-cell lymphoblastic lymphoma/ acute lymphoblastic leukemia Mature B-cell lymphoma Rare T-cell lymphoblastic lymphoma/ acute lymphoblastic leukemia Mature T-cell lymphomas Deleted Natural killer cell lymphomas Hodgkin disease Unknown * All values are presented as percentages. This result does not include the results of single cell polymerase chain reaction studies. coded by a single VDJ rearrangement to become contiguous with any of the C-region genes that encode IgM, IgG, IgA, IgD, or IgE. This phenomenon is known as IgH switching, and in lymph nodes it occurs within the follicle germinal centers. It is estimated that the mechanisms of gene rearrangement can generate more than 10 8 different immunoglobulins and TCRs, more than sufficient to counteract the large number of foreign antigens that humans may encounter. Developmental Hierarchy of Antigen Receptor Gene Rearrangements In B cells the immunoglobulin genes rearrange sequentially. 6 At the earliest stage of B-cell differentiation, the IgH-chain gene rearranges, followed by Ig light-chain gene rearrangement. Either Ig light chain may rearrange initially, although Ig usually rearranges first. Productive rearrangement of either Ig allele inhibits rearrangement of Ig (or vice versa). This molecular mechanism allows an individual mature B cell to express only one immunoglobulin light chain, known as the principle of allelic exclusion. In T cells, the TCR gene appears to rearrange first, followed by the TCR, TCR, and TCR genes. 7 Lineage Infidelity The occurrence of TCR gene rearrangements in B-cell lymphomas and immunoglobulin gene rearrangements in T-cell lymphomas is known as lineage infidelity (or lineage promiscuity). 3,8 Lineage infidelity is common in neoplasms arising from precursor B cells and T cells and is much less common in neoplasms arising from mature lymphoid cells. Since the mechanisms of immunoglobulin and TCR gene rearrangement are similar, and both use the same recombinase enzymes, it is perhaps not a surprise that lineage-inappropriate gene rearrangements occur. Why lineage infidelity is more common in neoplasms derived from precursor cells than from mature cells is unknown, as are the exact mechanisms that generally restrict immunoglobulin gene rearrangement to B cells and TCR gene rearrangement to T cells. Knowledge of lineage infidelity must be kept in mind when assessing lineage using clonality results, and more than 1 gene must be evaluated in the form of a panel. Also, certain genes, such as Ig, are rarely rearranged in T-cell neoplasms and therefore are more reliable for use in assessing lineage (Table 1). Clonality and the Diagnosis of Malignancy The gene rearrangement process is not involved in mechanisms that lead lymphoid cells to become malignant. Instead, the gene rearrangement process is normal and usually occurs prior to neoplastic transformation. 1 Thus, all of the neoplastic cells carry an identical immunoglobulin or TCR gene rearrangement, in contrast to benign lesions within which each B or T cell carries its own unique gene rearrangement. However, since the gene rearrangement process is not involved in neoplastic transformation, absence of gene rearrangements cannot always be equated with a benign process, nor can the presence of gene rearrangements always be equated with malignancy. For example, lymphomas that arise from lymphocytes at an early stage of maturation, prior to when the immunoglobulin or TCR genes rearrange, will not have gene rearrangements. This situation is unusual, but may provide an explanation for the absence of gene rearrangements in some subsets of malignant lymphoma (eg, some anaplastic large cell lymphomas). On the other hand, monoclonal gene rearrangements may be detected in patients with compromised immune systems (eg, post organ transplantation and in acquired immunodeficiency syndrome). 9,10 In general, the following statement holds true: In immunocompetent patients who develop lymphoid proliferations, detection of a gene rearrangement that represents 1% to 5% of all cells in the biopsy specimen (the lower limit of the sensitivity of Southern blot hybridization) highly correlates with malignant lymphoma. Application of molecular methods that are more sensitive than Southern blot analysis may result in the detection of very small monoclonal B-cell or T-cell populations in histologically benign lymphoid lesions from patients with intact immune systems. The clinical significance of these small monoclonal populations in this setting is uncertain. Detection of Antigen Receptor Gene Rearrangements Southern Blot Hybridization (Restriction Fragment Length) Analysis. Method. To perform Southern blot analysis, DNA must first be extracted from fresh or frozen cells or tissues using standard methods. One of the common protocols uses chemical methods, phenol and chloroform, to remove cell lipids and protein contaminants, followed by precipitation of high-molecular-weight genomic DNA using cold ethanol and salt. Proper disposal of organic reagents is inconvenient, and therefore use of nonorganic reagents is popular. Occasionally, DNA extracted by nonorganic methods is resistant to digestion by restric Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr

5 tion enzymes. This resistance may be overcome if organic methods are employed to purify the DNA further. If a specimen is to be anticoagulated, EDTA is a preferred anticoagulant. Heparin is less desirable owing to problems with restriction enzyme digestion. Concentration and purity of DNA is determined using ultraviolet spectrophotometry. The second step is to cut DNA into relatively small fragments, allowing the DNA to be analyzed by electrophoresis. DNA can be cut by restriction enzymes, bacterial enzymes that cut specific or restricted sequences of bases. For the analysis of antigen receptor genes, restriction enzymes that cut 6 base sequences of DNA are used, most commonly EcoRI, HindIII, and BamHI. The restricted DNA is then electrophoresed in a gel. Agarose gels are popular because they allow adequate separation of the DNA restriction fragments and are relatively inexpensive. An agarose concentration of 0.7% is commonly used. Following separation, a solid matrix such as a nylon membrane (or blot) is laid over the gel, and the DNA fragments are transferred onto the membrane. One of the simplest methods of accomplishing transfer is to place the gel and nylon membrane between filter paper below and blotting paper above. The filter paper has long wicks immersed in buffer, and the blotting paper is stacked in the form of a tower. Capillary pressure causes buffer to migrate up the filter paper wicks, through the gel, and into the tower carrying the DNA fragments onto the nylon membrane. Equipment that uses vacuum or positive pressure to aid in DNA transfer is commercially available as well. After transfer, the blot contains an exact imprint of the fragments separated on the gel. The location of the restriction fragment carrying the gene of interest is then detected by hybridizing the blot in a liquid solution containing a labeled probe. Both the DNA fixed to the blot and the probe DNA need to be denatured (ie, converted to single strands) to allow hybridization. The probe will seek out complementary single strands of DNA on the blot and hybridize with them. Unbound probe is then washed away under precise conditions of temperature and salt concentration so that only specifically hybridized probe remains on the blot. If the probe is radioactive, the blot is exposed to x-ray film, and the resulting autoradiograph is developed to reveal the position of the restriction fragment containing the gene sequence recognized by the probe. Multiple probes are available commercially, and systems that use nonradioactively labeled probes and colorimetric or chemiluminescent detection methods are also available. Sensitivity. The distinction between a monoclonal and a polyclonal process, as determined by Southern blot hybridization, relies on the sensitivity of the assay. In a polyclonal lesion, each B or T cell has its own rearranged immunoglobulin or TCR genes. However, these rearrangements are not sufficient in number to be detected as a single band by this technique. For an immunoglobulin or TCR gene rearrangement to be detected, cells containing the identical gene rearrangements must represent at least 1% to 5% of the total cell population, making this technique 1% to 5% sensitive Southern blot methods are more sensitive than standard 2-color flow cytometric immunophenotypic analysis for detecting clonality. However, with 3- or 4-color analysis, the sensitivity of flow cytometry approximates (or is better than) Southern blot analysis. Utility. The gene rearrangement process is well suited for detection by Southern blot hybridization because the process of DNA rearrangement deletes intervening DNA sequences, including restriction enzyme sites, resulting in a change in size from the germline restriction fragment size (Figures 4 and 5) Thus, to interpret results one must know the germline configuration of the gene being analyzed and, in particular, the location of relevant restriction enzyme sites. For diagnostic purposes, 2 genes (IgH and TCR ) are usually analyzed for clonality. If the results are uncertain, the Ig gene is also studied. 13 Other antigen receptor genes are not routinely assessed for a variety of reasons. For example, the Ig locus is highly polymorphic, resulting in a number of germline bands that complicate interpretation of patient specimens. Interpretation of the TCR locus also can be problematic. In this instance, the limited V region repertoire results in a number of germline bands in polyclonal T-cell lesions. Distinguishing the bands of polyclonal T cells from a population of monoclonal T cells can be difficult, particularly when the polyclonal T cells are numerous ( 30% of all cells). 14 The TCR gene, situated within the TCR gene between the V and J regions, is routinely deleted in normal / T cells and in most T-cell lymphomas. 15 The TCR gene is large, with a J region of up to 80 kilobases (kb), and therefore cannot be analyzed conveniently using routine Southern blot methods. The clonality and lineage of most lymphoid neoplasms can be determined using probes specific for the IgH, Ig, and TCR genes in conjunction with Southern blot analysis (Table 1). In general, J-region probes are superior to C-region probes. For example, both the C and C regions of the immunoglobulin genes are often deleted as a result of IgH switching for the former and site-specific deletions for the latter. 1,3 UseofJHandJ probes avoids these potential pitfalls. Although the C region of the TCR -chain gene is not deleted, the configuration of the gene prevents the assessment of rearrangements in EcoRI and HindIII DNA digests simultaneously. In the germline configuration, an EcoRI or HindIII restriction enzyme site is situated between the J 2 andc 2 orthej 1 andc 1 regions, respectively (Figure 6). Use of J probes avoids this problem. Polymerase Chain Reaction. Method. The polymerase chain reaction (PCR) technique allows the synthesis and amplification of double-stranded DNA in vitro. 16 This technique accommodates the unique characteristics of DNA polymerase, which proceeds only in the 5 to 3 direction. To begin, all of the components of DNA must be added to a test tube. Thus, the 4 nucleotides in an appropriate buffer containing an optimum concentration of magnesium (generally mmol/l) are added. Primers are also needed. Typically, primers are synthesized oligonucleotides, 20 to 25 bases long, that are complementary to the template DNA. The primers serve 2 functions. First, they provide a 3 end of DNA that can be extended by DNA polymerase. Second, the primers are designed to anneal with the 5 end of each template strand of DNA, flanking the target DNA sequence to be amplified, and therefore they provide the specificity of the reaction. The last ingredient to be added is DNA polymerase. Taq polymerase, isolated from the bacterium Thermus aquaticus, is heat stable, allowing PCR to be performed as a closed system in a fully automated thermal cycler. Although Taq Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1193

6 Figure 4. Hypothetical schematic illustration of immunoglobulin light-chain gene rearrangement. In this example, the gene rearrangement deletes an EcoRI restriction enzyme site, and therefore changes the size of the DNA fragment recognized by a J probe in EcoRI-digested DNA from 9.4 to 5.8 kb. polymerase is the most well known, other genetically modified DNA polymerases also are used. Polymerase chain reaction amplification is based on repetitive cycles, with each cycle composed of 3 reactions differing only in temperature and time of incubation. In the first reaction (denaturing), high temperature (eg, C) is used to denature double-stranded template DNA into single strands. The second reaction (annealing) is conducted at a relatively low temperature (eg, C) to maximize the opportunity for the primers to anneal with complementary sequences flanking the target region. In the last reaction (extending), an intermediate temperature (eg, 72 C) is used to optimize DNA polymerase activity, with DNA synthesis occurring at the 3 ends of the annealed primers. Following 1 such cycle, the target region of DNA template is exactly duplicated. An important feature of PCR is that all synthesized DNA sequences can then serve as templates for additional DNA synthesis in subsequent cycles. Theoretically, after the third cycle DNA amplification becomes geometric (Figure 7). In other words, the target DNA sequence is exponentially amplified 2 n, with n representing the number of cycles. Commonly, 30 to 40 cycles are performed to amplify the target. Once amplification is complete, the amplified products are analyzed by gel electrophoresis, which is followed by staining the gel with ethidium bromide. Alternatively, these products can be transferred to a nylon membrane by Southern blotting and the membrane can be hybridized with a labeled probe that is specific for the target sequence amplified. A successful PCR will yield a discrete band that corresponds to the expected size of the product, as predicted from the sites of the primers. Agarose and polyacrylamide gels may be used, depending on the PCR application. Compared with polyacrylamide gels, agarose gels are less expensive and easier to prepare, and amplified products are more easily transferred to a solid membrane that can be hybridized with a specific internal probe. However, the separation of PCR products with very small differences in size is suboptimal in agarose gels and far better in polyacrylamide gels. Denaturing chemicals, such as urea or formamide, can be added to polyacrylamide gels to create denaturing gradient gels. The concentration of urea or formamide required for denaturation of double-stranded DNA into single strands depends on the nucleotide sequence and can be used to separate DNA molecules. Single-stranded DNA migrates through the gel more slowly than double-stranded DNA. Using denaturing gradient gels, DNA molecules that differ by only a single base change can be separated. Utility. Although Southern blot analysis remains the gold standard for detecting antigen receptor gene rearrangements, PCR-based methods are faster, less expensive, and can be performed using small amounts of relatively low-quality DNA, such as that obtained from fixed, paraffin-embedded tissue. 17 To date, most efforts using PCR to assess clonality have been directed at the analysis of the IgH- and the TCR -chain genes. 18,19 The logic behind the use of PCR-based assays to detect antigen receptor gene rearrangements is as follows: in the germline configuration, the V and J regions are widely separated; thus, PCR amplification of intervening DNA 1194 Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr

7 Figure 5. Southern blot analysis of the T-cell receptor -chain gene. Three specimens are shown, each digested with EcoRI, BamHI, and HindIII restriction enzymes, run in agarose gels, transferred to a nylon membrane, and hybridized with a radioactively labeled J probe. Two specimens, one (negative control) in lanes 2, 5, and 8, and the second in lanes 3, 6, and 9, reveal only germline bands and are polyclonal. The third specimen, in lanes 4, 7, and 10, has evidence of gene rearrangement in all 3 lanes and is monoclonal. Lane 1 shows the markers. Lane 11 is a 5% sensitivity control. using standard methods does not usually occur. 18 In contrast, VJ or VDJ gene rearrangement brings the flanking V and J primers into close proximity, allowing successful amplification of intervening DNA. Monoclonal cells contain 1 or 2 rearranged alleles; therefore, PCR will amplify 1 band or 2 predominant bands. In contrast, polyclonal cells each carry a distinctive gene rearrangement of slightly different sizes, which PCR will amplify in a smear pattern. Any of the V and J segments of an antigen receptor gene may be used in the process of gene rearrangement. Thus, primers must be designed to anneal with as many of the V and J region primers as possible, while keeping the number of PCR reactions to a minimum for practical considerations. For example, the IgH gene has 88 V segments, of which approximately 50 are functional, and 6 J segments. Similarly, the TCR gene has 50 to 75 V and 2 sets of J regions, J 1 andj 2, composed of 6 and 7 J segments, respectively. Using a different set of primers for each possible combination is impractical. Thus, investigators have taken 2 approaches to overcome this problem. In one approach, investigators have deliberately chosen a simple gene with limited V and J regions. For example, the TCR gene has 4 small families of V segments and 5 J segments (2 highly homologous) that can be assayed using only a small number of V and J primers (Figure 7). 19 However, most of the antigen receptor genes are not simple. As an alternative, investigators have searched for consensus sequences, shared by all V segments or all J segments, which can be used to detect all possible combinations. 18 Since the number of J segments is relatively small and there is conservation of sequences among the J segments, good consensus J primers are available. However, an ideal primer that will anneal with all V segments for a particular antigen receptor gene is much more difficult to identify, owing to the relatively large number and the lack of conservation of sequences among the V segments. The best conservation of DNA sequences is found in the framework of the complementarity-determining regions of the V segments. However, even in the complementarity-determining regions there is no sequence shared by all V segments, and thus an ideal primer that can anneal to all possible V segments involved in the gene rearrangement process cannot be designed. False-Negative Results. It therefore follows that the major disadvantage of using PCR-based assays to detect antigen receptor gene rearrangements is the occurrence of false-negative results. For a simple gene like TCR, in which the primers are theoretically designed to anneal with all V and J segments, false-negative results occur rarely. 19 In contrast, for the more complex genes, such as the IgH- and TCR -chain genes, there is a significant false-negative rate. 18 There are a variety of explanations for false-negative results. First, the use of imperfect consensus primers predicts a priori that PCR will be falsely negative in a subset of cases. Second, PCR using consensus V primers may not detect antigen receptor gene rearrangements that result from chromosomal translocation or partial DJ rearrangements. In these circumstances, the V and J segments are not brought into proximity and intervening DNA cannot be amplified. Third, the V and J segments of the immunoglobulin genes (and rarely the TCR genes) commonly undergo somatic hypermutation. These mutations can prevent the primers from annealing to the DNA, further adding to the false-negative rate. Somatic mutations in the V and J segments normally occur in B cells after they are exposed to antigen selection, which in lymph nodes occurs in the follicle germinal centers. Malignant lymphomas that arise from B-cell precursors that are not exposed to antigen (eg, mantle cell lymphoma) have a very low rate of somatic mutation, and therefore false-negative results are not a problem In contrast, malignant lymphomas that arise from B-cell precursors that are exposed to antigen (eg, follicular lymphomas) arise from cells that commonly have somatic mutations in the V and J segments. The false-negative rate of PCR for analyzing the immunoglobulin genes in these neoplasms is therefore relatively high (Table 2) Polymerase chain reaction-based assays for detection of TCR and immunoglobulin gene rearrangements are best used as a screening method in daily practice. 17,18 A negative result should not be accepted as negative unless confirmed by Southern blot analysis. Sensitivity. Polymerase chain reaction methods that assess the antigen receptor genes for a monoclonal B- or T- cell population are less sensitive than PCR methods for detecting a chromosomal translocation. A chromosomal translocation is not normally present (or is very rare) in normal cells. Therefore, PCR assays designed to detect chromosomal translocations are very sensitive and can identify 1 positive cell among 10 5 negative cells. In contrast, in IgH gene analysis, one is using a PCR technique to identify a monoclonal B-cell population in a background of polyclonal B cells. The background noise explains the lower sensitivity of the technique for this purpose. Polymerase chain reaction methods can identify 1 monoclonal B or T cell in 10 2 or 10 3 B or T cells, respectively. If the PCR sensitivity is 10 2, how is it any more sensitive than Southern blot analysis, which also is sensitive at 10 2? Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1195

8 Figure 6. Germline configuration of the T-cell receptor (TCR) -chain gene showing joining (J), diversity (D), and constant (C) regions and known EcoRI (E), BamHI (B), and HindIII (H) restriction enzyme sites. A HindIII site between the J 1 and C 1 regions precludes detection of TCR gene rearrangement using a C probe and HindIII-digested DNA. Similarly, an EcoRI site between the J 2 and C 2 regions precludes detection of TCR gene rearrangement using a C probe and EcoRI-digested DNA. Figure 7. Schematic illustration of amplification by the polymerase chain reaction, using the example of T-cell receptor (TCR) -chain gene rearrangement. The sensitivity is greater because of the specificity of the PCR technique. For example, the sensitivity of Southern blot analysis for assessing the IgH gene is 1 monoclonal B cell in 100 cells of all types, including B and T cells as well as histiocytes, fibroblasts, etc. In contrast, the PCR method will detect 1 monoclonal B cell in 100 B cells. In a biopsy or peripheral blood specimen in which T cells outnumber B cells by 10 to 1 or 100 to 1, the PCR is more sensitive than Southern blot analysis by a 1 or 2 logs, respectively. Clone-Specific Probes. As mentioned, one of the roles of the enzyme terminal deoxynucleotidyl transferase is to insert nontemplated nucleotides (so-called N insertions) at the V-D and D-J junctions during the process of antigen receptor gene rearrangement. 5 Up to 30 nucleotides can be inserted by this mechanism. The insertion of these nu Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr

9 Table 2. False-Negative Rate* of Polymerase Chain Reaction Based Assays for Detecting Immunoglobulin Heavy-Chain Gene Rearrangements in B-Cell Non-Hodgkin Lymphomas False-Negative B-Cell Lymphoid Neoplasms Rate, % Lymphoblastic lymphoma/acute lymphoblastic 10 leukemia Small lymphocytic lymphoma/chronic lymphocytic 5 leukemia Mantle cell lymphoma 5 Lymphoplasmacytoid lymphoma Follicular lymphoma Marginal zone B-cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphoma Plasma cell myeloma * False-negative rates are based on analysis of these neoplasms with the most commonly used set of consensus polymerase chain reaction primers, VH-FRIII and JH. Use of additional primer sets can reduce these rates significantly. These numbers represent a summary of the results previously reported in the literature by others and our own experience. cleotides occurs in a combination that is essentially unique and is therefore a fingerprint of a particular gene rearrangement. 5 The process of N insertion allows one to design PCR clone specific primers. Typically, this application is used to monitor minimal residual disease. At the time of de novo diagnosis of malignant lymphoma (or acute leukemia), a monoclonal B- or T-cell population is amplified by traditional PCR, isolated, cloned, and sequenced. Once the sequence is known, primers specific for the particular clone can be designed, and this clone can be specifically searched for in all follow-up biopsy specimens. 25 Although this technique has proved to be useful for monitoring residual disease, it must be remembered that somatic mutations of the IgH variable and J regions can interfere with this approach in 2 ways. At time of initial diagnosis, the immunoglobulin gene rearrangement of the neoplasm may not be amplifiable by PCR. Also, in neoplasms with amplifiable immunoglobulin gene rearrangements at diagnosis, somatic mutations may occur subsequently, thereby leading to false-negative results at the time of recurrence. CHROMOSOME TRANSLOCATIONS Nonrandom chromosome translocations are another form of gene rearrangement, but unlike antigen receptor gene rearrangements, translocations do not occur or occur rarely in normal cells. During chromosome translocation, a portion of one chromosome becomes separated and attached to a different chromosome. 26,27 Commonly, chromosome translocations in lymphoid neoplasms are reciprocal; in other words, segments from 2 different chromosomes exchange places. Reciprocal translocations may be further characterized as balanced or unbalanced. In balanced translocations there is no net loss of DNA. The mechanisms involved in chromosomal translocation are not well understood. In some instances, the processes active in normal antigen receptor gene rearrangement are involved, and this appears to be true for the t(8;14) translocation. 28 In other instances, specific DNA sequences, such as alternating purine-pyrimidine tracts or Alu repeat regions, may be able to promote translocation. 26 Table 3. Oncogene c-myc CCND-1/PRAD1/bcl-1 bcl-2 bcl-3 bcl-6 bcl-8 bcl-9 bcl-10 pax-5 alk tpm3 mmset c-maf src2 Ski c-rel N-ras K-ras Tal1/Scl/tcl-5 LMO1 LMO2 Ttf lyt-10 Bob1/obf1 api2/c-iap2 mlt Oncogenes Involved in Malignant Lymphomas Chromosome Location 8q24 11q13 18q21 19q13 3q27 15q q21 1q22 9p13 2p23 1q25 4p16 16q23 1p36 1q11 2p p p p32 11p15 11p13 4p13 10q24 11q23 11q21 18q21 Chromosomal translocations are specific for various types of lymphoid neoplasms and are thought to be integrally involved in their pathogenesis Typically, chromosomal translocations activate oncogenes, therefore we provide a brief discussion of oncogenes here. Oncogenes Oncogenes can be defined as normal genes that are usually actively transcribed in embryogenesis or early development and that are tightly controlled and expressed at relatively lower levels in mature cells. 26,30 Oncogenes typically encode for proteins that are involved in the control of cell proliferation or differentiation. The majority of known oncoproteins are growth factors, growth factor receptors, intracellular signal transducers (eg, c-abl), and nuclear transcription factors (eg, c-myc). There are also oncogenes that do not fit these categories, such as CCND-1, which encodes for a cyclin that acts to drive the cell cycle, and bcl-2, which encodes a protein that inhibits apoptosis (programmed cell death). 30 Oncogenes may be activated by a variety of mechanisms, including chromosomal translocation, point mutation, DNA amplification, and viral insertion into oncogene DNA. 26,30 Chromosome translocations are the most common mechanism of oncogene activation in malignant lymphomas. Translocated oncogenes also can undergo point mutation. A brief list of oncogenes involved in the pathogenesis of malignant lymphomas is provided in Table 3. Chromosome Translocations There are 2 major groups of chromosomal translocations. 26,27 In the first group, which is the case for most non- Hodgkin lymphomas, the translocation juxtaposes an oncogene with an antigen receptor gene, resulting in a quantitative increase of qualitatively normal oncoprotein. For example, t(14;18)(q32;q21) is found in 90% of follicular Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1197

10 Table 4. Summary of Chromosomal Translocations, Associated Lymphoid Neoplasms, and the Affected Genes Translocation Non-Hodgkin Lymphoma Genes Involved t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11) t(9;14)(p13;q32) t(11;14)(q13;q32) t(14;18)(q32;q21) t(14;19)(q32;q13) t(3;var)(q27;var)* t(14;15)(q32;q11-13) t(1;14)(q21;q32) t(11;18)(q21;q21) t(1;14)(p22;q32) t(2;5)(p23;q35) t(11;14)(p15;q11) t(4;14)(p16;q32) t(14;16)(q32;q23) t(16;22)(q23;q11) Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma Lymphoplasmacytoid lymphoma Mantle cell lymphoma Follicular lymphoma B-cell chronic lymphocytic leukemia Diffuse large B-cell lymphoma Diffuse large B-cell lymphoma Pre B acute lymphoblastic leukemia, mantle cell lymphoma Low-grade B-cell MALT lymphoma Low-grade B-cell MALT lymphoma T/null-cell anaplastic large cell lymphoma Pre T acute lymphoblastic leukemia, lymphoblastic lymphoma Plasma cell myeloma Plasma cell myeloma Plasma cell myeloma c-myc and IgH Ig and c-myc c-myc and Ig pax-5 and IgH CCND-1 (bcl-1) and IgH IgH and bcl-2 IgH and bcl-3 bcl-6 and IgH* IgH and bcl-8 bcl-9 and IgH api2 and mlt bcl-10 and IgH alk and npm LMO1 and TCR mmset and IgH IgH and c-maf c-maf and IgH * 14q32 (IgH) is most commonly involved in translocations involving 3q27 (bcl-6), but in less than 50% of cases; other sites that can be involved in bcl-6 translocations include 2p12, 22p11, 8q24, 11q13, and 5q31. Very few cases of mantle cell lymphoma have been assessed for bcl-9 rearrangements. MALT indicates mucosa-associated lymphoid tissue. lymphomas. 31 In this translocation, the bcl-2 gene at 18q21 is juxtaposed with the IgH gene, and bcl-2 is overexpressed. 32 In the second group, the translocation splits 2 different gene loci, and recombination results in the formation of a novel gene. This mechanism, although common in leukemias, is uncommon in non-hodgkin lymphomas. The t(2;5)(p23;q35) translocation that occurs in a subset of T- and null-cell anaplastic large cell lymphomas is a conspicuous exception. In this translocation, 2 oncogenes, npm and alk, are disrupted and spliced together to form a fusion gene, npm-alk, from which novel mrna is transcribed. 33 A list of the common and more recently described chromosomal translocations that occur in malignant lymphomas is provided in Table 4. Detection of Chromosomal Translocations Southern Blot Hybridization. The method of Southern blot hybridization, although time-consuming, is an excellent means of detecting gene rearrangements that are the result of chromosomal translocations. 3 Analogous to the assessment of antigen receptor gene rearrangement, the germline size of the affected oncogene locus is known, and the translocation deletes restriction enzyme sites, resulting in a different-sized fragment. Use of an oncogene probe and Southern blot techniques, however, provides only an indirect assessment of a chromosomal translocation. For example, if one is assessing the bcl-2 gene locus with a specific probe, the detection of bcl- 2 gene rearrangement only proves the presence of disruption of the bcl-2 locus. Although in most instances disruption results from the t(14;18) translocation, the Southern blot result does not prove that chromosome 14 is involved, and theoretically other partner chromosomes could be involved. One can go a step further to prove the presence of t(14; 18) by assessing genomic DNA with probes specific for both bcl-2 and IgH. The detection of a non germline restriction fragment that hybridizes with both probes is proof that the restriction fragment is composed of portions of chromosomes 14 and 18, in other words the t(14;18) translocation. Polymerase Chain Reaction. Polymerase chain reaction is a far more convenient method of detecting chromosomal translocations if the breakpoints are tightly clustered and the sequences flanking the breakpoints are known. 16 For example, in the t(14;18) translocation, there are a variety of breakpoints within the bcl-2 gene on chromosome The majority of cases with t(14;18) involve two breakpoint cluster regions. Breakpoints on chromosome 14 are also clustered within the JH5 and JH6 segments of the IgH gene J region. The sequences flanking the breakpoints on chromosomes 14 and 18 are known, allowing the design of primers that flank these sites of t(14;18) fusion. 32 Chromosomal translocations that have widely dispersed breakpoint sites or involve an oncogene that is translocated with multiple partner chromosomes are less amenable to detection by PCR-based assays. t(11;14)(q13;q32) is an example of a translocation in which the breakpoints are widely dispersed. More than 95% of mantle cell lymphomas carry the t(11;14) translocation, involving the bcl-1 locus at 11q13 and the IgH gene at 14q32. However, the breakpoints on chromosome 11 are widely dispersed over more than 200 kb. 34 Currently, PCR assays can detect one tightly clustered region, the bcl-1 major translocation cluster, which accounts for 30% to 40% of all cases. 35 Other breakpoints on chromosome 11 are widely scattered and cannot be assessed by routine PCR assays. Chromosomal translocations involving the bcl-6 gene at chromosome locus 3q27 exemplify the problem of multiple-partner chromosomes. The bcl-6 gene can be translocated with a number of different chromosome loci. 36 Obviously, primers that anneal to the bcl-6 locus can be designed, but multiple primers specific for the partner chromosomes also are needed. Thus, routine PCR analysis for bcl-6 translocations is currently not practical. For translocations that involve one gene locus with multiple-partner chromosomes, Southern blot hybridization 1198 Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr

11 remains valuable. Using the example of bcl-6, a specific bcl- 6 probe will demonstrate disruption of this locus when it is involved in translocation, no matter which partner chromosome is involved. 36 Reverse-Transcriptase PCR (RT-PCR). In many chromosomal translocations, for example, t(2;5) in anaplastic large cell lymphoma and t(9;22) in chronic myeloid leukemia, the breakpoints are widely scattered within large introns. As a result, a standard PCR assay to amplify intronic DNA involved in the translocation is not practical, as many primer sets would be needed. However, primers can be designed to anneal with the involved exons, which are constant, although the presence of the intervening introns precludes amplification by standard PCR, which has an upper limit of approximately 1 kb. An alternative approach is to synthesize complementary DNA (cdna) from mrna by using reverse transcriptase. In the process of normal transcription, introns are spliced out of mrna and exons are brought into close proximity (Figure 8). Polymerase chain reaction primers specific for these exons are therefore close together in cdna, allowing standard PCR methods to be used. Long-Range PCR. Long-range PCR is an alternative approach to the problem of chromosomal translocations that have widely scattered breakpoints within introns. 37 In this technique, primers and conditions are designed to optimize the efficiency of PCR, allowing amplification of relatively long segments of DNA. In our laboratory, Sarris and colleagues 38 have used a long-range PCR method to amplify npm/alk genomic DNA in cases of anaplastic large cell lymphoma. Fluorescence In Situ Hybridization. A detailed description of fluorescence in situ hybridization (FISH) methods is beyond the expertise of the authors, and the reader is referred to reviews by others. 39 Fluorescence in situ hybridization can be used to study either interphase or metaphase preparations. For chromosomal translocations with widely dispersed breakpoints, FISH assays can be very helpful because FISH probes are typically large and span long ( 100 kb) regions of DNA, allowing detection of chromosomal breakpoints that are widely dispersed. For example, others have shown that FISH probes can detect almost all of the 8q24 breakpoints of the t(8;14) translocation in Burkitt lymphomas, which are dispersed over 400 to 500 kb. 39 Similar to Southern blot analysis, FISH assays using a single probe can detect breakpoints within a chromosome and provide presumptive evidence of translocation. Single probe assays are generally easier and cheaper to perform and can be very helpful diagnostically. However, disruption of the gene does not prove the presence of a partner chromosome. For this purpose, 2 FISH probes labeled with different fluorescent colors are needed. Usually, green and red probes are employed. Using the example of the t(8;14) translocation, intact chromosomes 8 and 14 are green and red, respectively. In contrast, both green and red probes hybridize with and are juxtaposed on the t(8;14) site, resulting in a yellow color. Specific Chromosomal Translocations Involved in Malignant Lymphoma Translocations Assessable by Southern Blot and PCR Analysis. t(11;14)(q13;q32). The t(11;14) translocation has been identified in up to 95% of cases of mantle cell lymphoma. 34 This translocation also has been identified in approximately 20% of B-cell prolymphocytic leukemias, 3% to 5% of plasma cell myelomas, and in rare cases ( 1%) of B-cell chronic lymphocytic leukemia. 40 In t(11;14), the bcl-1 locus at 11q13 is juxtaposed with the IgH gene locus. The bcl-1 locus does not encode for a gene, but t(11;14) is responsible for deregulating the CCND-1 (cyclin D1) gene that is located approximately 120 kb upstream (5 ) tothebcl-1 locus. The cyclin D1 protein product normally functions at the G1 to S checkpoint of the cell cycle and is involved in transition from G1 to S phase. Cyclin D1 overexpression drives cell proliferation. In cases of mantle cell lymphoma, the chromosome 14 breakpoints are generally clustered in the J region of the IgH gene, but the chromosome 11 breakpoints are widely scattered (Figure 9). 41,42 Approximately 30% to 40% occur in a tight cluster in the bcl-1 locus (80 to 100 bp) known as the major translocation cluster region. 41 The remaining breakpoints are widely scattered and have been found upstream, within the CCND-1 gene locus (120 kb 5 to bcl- 1), and up to 330 kb further upstream in cases of plasma cell myeloma. 34,39 The rate of detection of t(11;14) is method-dependent (Table 5). A number of genomic chromosome 11 probes have been cloned, and use of these probes and Southern blot analysis has identified bcl-1 rearrangements in up to 75% of cases of mantle cell lymphoma. 40,42 The major translocation cluster region is amenable to routine PCR analysis, and thus 30% to 40% of cases of mantle cell lymphoma can be shown to carry the t(11;14) translocation by PCR (Figure 10). 35 Obviously, neither of these methods is ideal for routine molecular diagnosis. Fluorescence in situ hybridization assays also may be used, and the large size of FISH probes allows detection of most chromosome 11 breakpoints. 39,43 An alternative approach has been detection of cyclin D1 RNA or protein, using either RT-PCR or immunohistochemical analysis, respectively. Since all possible variations of t(11;14) theoretically lead to increased cyclin D1 expression, detection of overexpression is a surrogate indicator of the presence of the translocation. 44 t(14;18)(q32;q21). The t(14;18) translocation has been identified in up to 90% of follicular lymphomas and 20% to 30% of diffuse large B-cell lymphomas, the latter presumably of follicle center cell origin. 31,45 In this translocation, the bcl-2 gene locus at 18q21 is juxtaposed with the IgH gene locus at 14q32 and is brought under the influence of the immunoglobulin enhancer elements, resulting in bcl-2 deregulation. 32 The bcl-2 gene has 3 exons, and it encodes an oncogene product, Bcl-2 protein, which normally functions to inhibit apoptosis (ie, programmed cell death). Thus, cells that overexpress Bcl-2 have a prolonged life span and are at increased risk for secondary genetic events that lead to neoplastic transformation. Although the bcl-2 gene was initially cloned from a case of follicular lymphoma, it has since become clear that bcl-2 plays a major role in all cells, and Bcl-2 is overexpressed in a wide variety of malignant hematologic and nonhematologic neoplasms. 46 The bcl-2 gene is also the prototype of a large class of genes that either inhibit apoptosis, such as bcl-2, or promote apoptosis, such as bax. 46 The chromosome 14 breakpoints are clustered in the J region of IgH. The breakpoints on chromosome 18 are widely scattered (Figure 11), but there are 2 tightly clustered regions. 32 The major breakpoint cluster region (MBR), located within the 3 untranslated portion of the Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1199

12 Figure 8. Schematic illustration of t(2;5)(p23;q35). The translocation occurs within introns, and the breakpoints are widely scattered (not shown here), precluding amplification of genomic DNA using one set of primers and standard polymerase chain reaction (PCR) methods. The exons are constant, allowing use of one set of exon-specific primers, but the distance between the exons is too great to perform standard PCR. However, if messenger RNA is transcribed into complementary DNA by reverse transcriptase, exon-specific primers are brought into close proximity allowing PCR amplification using standard methods. Alternatively, genomic t(2;5) DNA can be amplified using long-range PCR methods. Figure 9. Germline configuration of chromosome 11q13 region involved in t(11;14)(q13;q32). The CCND-1 (cyclin D1) gene is indicated as an open box. The major translocation cluster region (open arrow) accounts for 30% to 40% of all translocations. Minor breakpoint clusters are indicated as mtc1 and mtc2. The asterisk indicates breakpoints that occur far 5, which have been reported in cases of plasma cell myeloma. third exon, accounts for 50% to 60% of all cases of t(14; 18). The minor breakpoint cluster region (MCR), located 20 kb downstream (3 ), accounts for 10% to 20% of cases. Other small breakpoint cluster regions have been reported and are widely scattered in follicular lymphomas. In addition, another breakpoint cluster region, 5 to the first exon, occurs in 5% of B-cell chronic lymphocytic leukemias. Routine analysis of the t(14;18) translocation, using either Southern blot or PCR analysis, is practical in the clinical laboratory 47,48 ; however, the rate of detection is dependent on the method used (Table 6). For Southern blot analysis, 2 genomic probes specific for the MBR and MCR will detect bcl-2 gene rearrangements in up to 80% of follicular lymphomas, about 10% less than can be detected by conventional cytogenetics. Polymerase chain reaction, using 2 sets of primers specific for the MBR and MCR, will detect t(14;18) in approximately 60% to 70% of cases. The detection rate by PCR is lower than that of Southern blot hybridization because PCR assays are highly focused and 1200 Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr

13 Table 5. Detection Rate of Different Methods for t(11;14)(q13;q32) in Mantle Cell Lymphoma Method* DNA fiber FISH Conventional FISH with chromosome 11 probes Conventional cytogenetics Southern blot hybridization Polymerase chain reaction, bcl-1 MTC/JH Detection Rate, % * FISH indicates fluorescence in situ hybridization; MTC, major translocation cluster. Summary of literature. assess only on the MBR and MCR breakpoints. In contrast, genomic probes used for Southern blot analysis detect additional breakpoints close to the MBR and MCR. Fluorescence in situ hybridization methods have been used to detect t(14;18), and the detection rate with this approach may be superior to other methods, including conventional cytogenetics. In one study, Poetch and colleagues 49 identified the translocation in all 28 follicular lymphomas analyzed. t(14;18) in Benign Tissues. Although many chromosomal translocations are known to be tumor-specific, the t(14; 18) translocation has been shown to be present at very low frequency in approximately 50% of benign lymphoid tissues. 50 The translocation was identified in benign tissues by investigators who used modified PCR methods with greatly enhanced sensitivity. Usually, the low frequency of t(14;18) in benign tissues is below the level of detection using standard PCR methods. Thus, PCR rarely detects the t(14;18) translocation in biopsy specimens of follicular hyperplasia, although it may detect a faint signal in a subset of cases. In contrast, PCR analysis of follicular lymphomas yields a very strong signal. Nevertheless, in the setting of minimal residual disease, the interpretation of t(14;18) results may be problematic, and sequencing of the amplified products with comparison to the original neoplastic clone may be required. t(2;5)(p23;q35). The t(2;5) translocation has been identified in 20% to 60% of anaplastic large cell lymphomas of either T-cell or null-cell type, most commonly in neoplasms that occur in younger patients. 38 Recent data suggest anaplastic large cell lymphomas with the t(2;5) translocation respond better to therapy and have longer survival times than anaplastic large cell lymphomas without this translocation; thus, detection of t(2;5) has important prognostic implications. 51 In the t(2;5) translocation, the alk (anaplastic lymphoma Figure 10. Polymerase chain reaction study of the t(11;14) major translocation cluster region in a case of mantle cell lymphoma. After amplification, the products were transferred to a membrane by Southern blot transfer and hybridized with a radioactively-labeled internal probe. From the left to right: lane 1, markers; lane 2, positive control; lane 3, blank; lane 4; mantle cell lymphoma with amplification product (case tested); lane 5, blank; lane 6, water control without DNA template (H 2 0). Table 6. Detection Rate of Different Methods for t(14;18)(q32;q21) in Follicular Lymphoma FISH Conventional cytogenetics Southern blot hybridization Polymerase chain reaction bcl-2 MBR/JH bcl-2 MCR/JH Method* Detection Rate, % * FISH indicates fluorescence in situ hybridization; MBR, major breakpoint cluster region; and MCR, minor breakpoint cluster region. Summary of literature. kinase) gene at 2p23 is juxtaposed with the npm (nucleophosmin) gene at 5q35, forming a novel npm/alk gene located on the derivative 5 chromosome. 33 The 5 npm portion of the fusion gene includes a strong promoter that drives alk overexpression, which is thought to be the cause Figure 11. Germline configuration of chromosome 18q21 and bcl-2 gene involved in t(14;18)(q32;q21). The exons of bcl-2 are indicated as open boxes. Most of the breakpoints occur in the major (mbr) and minor (mcr) breakpoint cluster regions. The variant translocation cluster region (vcr) is involved in t(2;18) and t(18;22) reported in 5% of cases of B-cell chronic lymphocytic leukemia. Arch Pathol Lab Med Vol 123, December 1999 Molecular Methods in Diagnosis of Malignant Lymphomas Medeiros & Carr 1201