The IgH gene at chromosome 14q32 includes clusters of multiple V, J, D, and C gene segments. Somatic recombination is an orderly process in which D is first rearranged to J (with deletion of the intervening DNA) and then V is rearranged to DJ (with deletion of the intervening DNA) (Fig. 8.2). Approximately 150 V, 30 D, and six functional J genes are known. The V genes are grouped into major families. The VDJ product is joined to the C region at the messenger RNA level as transcribed noncoding intron sequences are spliced out. The VDJ is initially joined to Cμ (and adjacent Cδ) but heavy chain switching, which is a separate gene rearrangement event that occurs later in B-cell development, can result in the VDJ being joined to other C gene segments (γ, α, and ε).

When performing Southern blot analysis of the IgH gene, both joining region (JH) and constant region (Cμ) probes are available. Cμ probes were first available historically, but JH probes are more reliable for detecting IgH gene rearrangements (Fig. 8.3) because the Cμ region is deleted when Ig heavy chain class switching occurs.

The Igκ gene at chromosome 2p11 is organized like the IgH gene except that there are no D segments and only a single
C region. However, nearly 100 V and five J segments can recombine to produce diversity. When the Igκ gene rearranges nonproductively, a κ-deleting element, located 3′ to the Cκ region, is responsible for deleting Cκ and sometimes also Jκ (11). This occurs in some B cells expressing Igκ and in all B cells expressing Igλ. For this reason, Jκ probes are most informative when this gene is analyzed by Southern blot analysis.

The Igλ gene at chromosome 22q11 is organized differently from that of the other Ig genes: in addition to multiple V gene segments, J and C gene segments occur in tandem. Because of polymorphisms in the human population, the number of J and C gene segments can vary between six and nine (15). Cλ probes are available for Southern blot analysis. However, the number of Cλ regions and the polymorphic nature of this region yields a variable number of germline bands, making it difficult to appreciate a true gene rearrangement. For these reasons, the Igλ gene is not usually evaluated by Southern blot analysis.

The TCRβ chain gene at chromosome 7q34 is composed of multiple V gene segments, and two sets of D, J, and C gene segments arranged in tandem. Each set includes one D, six J, and one C gene segment. Following DJ rearrangement, VDJ gene rearrangement can involve either the Jβ1 or Jβ2 gene segments. Southern blot analysis of the TCRβ gene is the most reliable means of assessing T-cell clonality, detecting TCRβ rearrangements in 90% to 95% of T-cell lymphomas and leukemias.

Cβ probes, which were available initially, detect gene rearrangements involving either the Cβ1 or Cβ2 constant regions. The location of an EcoRI restriction enzyme site between Jβ2 and Cβ2 renders rearrangements involving Jβ2 undetectable in EcoRI-digested DNA. Similarly, a Hind III site between Jβ1 and Cβ2 renders rearrangements involving Jβ1 undetectable in Hind III-digested DNA. Jβ probes do not suffer from this limitation; typically, a mixture of Jβ1 and Jβ2 probes is used.

One advantage of using a Cβ probe is that it allows one to estimate the percentage of polyclonal T cells. In EcoRI-digested DNA, the intensity of the 12–kb Cβ1 germline band diminishes proportionally to the percentage of polyclonal T cells in the sample (Fig. 8.4). This is because each polyclonal T cell that has a gene rearrangement involving Cβ1 migrates differently according to a log-linear relationship of molecular mass versus gel distance. In contrast, Cβ2 is not diminished in intensity, in comparison with the germline band, when either Cβ1 or Cβ2 is rearranged because the EcoRI site is 5′ to the Cβ2 allele (1).

The TCRγ gene at chromosome 7q15 is far simpler than TCRβ, consisting of 11 functional V gene segments that can be subdivided into four families. In addition, J and C segments occur in tandem, with five total J segments (three Jγ 1 and two Jγ2) and two total C segments (Cγ1 and Cγ2). TCRγ gene rearrangements are retained in nearly all normal and neoplastic T cells expressing TCRαβ protein, including most peripheral and cutaneous T-cell lymphomas.

The simplicity of the TCRγ gene makes it better suited for analysis by PCR rather than Southern blot hybridization. Southern blot analysis has a sensitivity of 1% to 5%. The limited number of possible Vγ–Jγ gene rearrangements means that Southern blot analysis will detect discrete bands when numerous polyclonal T cells are present. This results in confusing band patterns when diagnostic biopsy specimens composed of monoclonal and polyclonal T-cell populations are analyzed (16). For these reasons, Southern blot analysis of the TCRγ gene can be problematic for molecular diagnosis.

The TCRα gene at chromosome 14q11 is very large and not suitable for Southern blot analysis. The Jα region is approximately 80 kb, three to four times the size of the largest DNA fragment (20 to 25 kb) routinely studied by Southern blot analysis, and it has approximately 50 Jα gene segments that require multiple probes for analysis (1,2). However, this region can be evaluated in a research environment with the use of pulsed-field gel electrophoresis, which is capable of resolving extremely large DNA fragments.

The TCRδ gene is a relatively small gene, located within the TCRα gene at chromosome 14q11, flanked by Vα and Jα gene segments. The TCRδ gene contains approximately five Vδ, two or three Dδ, and three Jδ segments. During normal T-cell development, TCRδ gene rearrangement plays a role in determining whether a T cell expresses the TCRαβ or TCRγ/δ receptor. If the TCRδ gene rearranges successfully, the cell goes on to become a γ/δ T cell. Otherwise, the TCRδ gene is deleted via Vα to Jα gene rearrangement, which deletes all intervening DNA sequences, including the TCRδ gene. TCRδ gene rearrangements can be detected in precursor lymphoblastic lymphoma/leukemia corresponding to the earliest stages of lymphoid differentiation and also in T-cell leukemias and lymphomas that express the TCRγδ receptor (17). Jδ probes are most helpful for Southern blot analysis.

PCR is a versatile molecular technique for assessing clonality, chromosomal translocations, minimal residual disease, gene mutations, and the presence of infectious agents. The lymph
node sample can be fresh, frozen, stored in buffer, or formalin-fixed and routinely processed. The target molecule can be either genomic DNA (standard PCR) or messenger RNA, which, by using reverse transcriptase (RT), is converted to complementary DNA before standard PCR is performed. This is known as reverse transcriptase PCR (RT-PCR). Repetitively altering the reaction temperature during heat denaturation, primer annealing, and extension geometrically amplifies the template DNA sequence 2^N times, where N equals the number of cycles (2,3). Multiplex PCR assays also can be set up. A multiplex PCR assay simply combines two or more PCR reactions within the same test tube. Setting up a successful multiplex PCR assay is more complex, because the primers and PCR conditions must be optimized for all the PCR reactions in the tube.

In clinical PCR applications, the DNA target usually spans 2,000 bp or less. In fixed, paraffin-embedded tissues, targets of less than 200 bp are ideal. Prior knowledge of the sequences upstream (5′) and downstream (3′) of the target is required to synthesize short, complementary oligonucleotides (20 to 30 bp), also called primers or amplimers. The primers specifically anneal to the template DNA and provide the initial sequences for Taq DNA polymerase extension. Selecting optimal sequences for both 5′ and 3′ primers often involves compromise; the desire for equivalent guanine and cytosine (G+C) content, which affects melting temperature, must be balanced against the need to avoid complementarity between the 3′ ends, which promotes wasteful primer–dimer formation instead of amplifying the template (18,19,20).

Most DNA-based PCR strategies to detect Ig or TCR gene rearrangements exploit the change in distance between V and J segments that occurs after gene rearrangement (2,3,4). In the germline configuration, V, D, and J gene segments are too widely dispersed for PCR to generate VDJ or VJ products (also known as amplicons) (Fig. 8.1). By contrast, in VDJ or VJ gene rearrangements, the V and J segments are in close proximity and can be amplified by PCR (Fig. 8.2). Upstream and downstream consensus primers are designed to recognize homologous DNA sequences of V genes, such as the framework regions or leader sequence, and the 5′ end of J genes, respectively.

Once amplification is complete, the PCR products must be detected. Traditionally, amplification products were subjected to electrophoresis, during which they separate according to their size (21,22). Monoclonal populations, yielding one or two overrepresented homogeneous PCR species, appear as sharp bands (Fig. 8.5); polyclonal populations yielding many heterogeneous PCR species, each differing in length by a few base pairs, appear as a smear (2,3,4). Alternative gel systems, either single-stranded conformation polymorphism gel electrophoresis or denaturing gradient gel electrophoresis, also can be used (23). In these systems, the PCR product nucleotide sequence determines gel mobility by affecting secondary or tertiary DNA structure.

More recently, fluorescent dyes are being incorporated into primers so that the PCR amplicons are fluorescent (23,24,25,26). Products are then separated by capillary electrophoresis and analyzed by genetic analyzers armed with sophisticated software (e.g. GeneScan) (Fig. 8.6). This new approach has many advantages. Turnaround time is faster; the number of setup steps is reduced, thereby reducing technician error; the detection of fluorescent products is more sensitive than naked-eye examination of a gel; and amplicons can be sized reliably, thereby allowing the comparison of product size over time in minimal residual disease applications (23,24,25,26).

Because of the high sensitivity of PCR assays, very small monoclonal B- or T-cell populations can be identified in histologically and immunophenotypically benign specimens. These may be viewed as “false-positive” results, although the assay is truly detecting a small monoclonal cell population. However, the clinical and biologic significance of this small clone in the clinicopathologic setting is unknown. In general, when evaluating a histologically equivocal biopsy specimen, a positive PCR result is less persuasive than a positive Southern blot analysis result—because the latter is less sensitive, it detects a larger monoclonal population of cells. One must also avoid the overinterpretation of faint bands or a peak in a background of polyclonal cells seen as an underlying smear or bell curve of peaks. These results are often truly false-positive. Clinicopathologic correlation is always required in the analysis of PCR data.

PCR assays can also yield false-negative results. In the assessment of the IgH gene by PCR, two major causes of false-negative results are possible: (a) consensus V region primers may not anneal with all possible V regions involved in gene rearrangement, and (b) somatic mutations of the immunoglobulin V regions can result in poor annealing of the consensus V primer with the DNA target (2,3). Certain types of B-cell lymphoma (e.g., follicular lymphoma) are much more prone to somatic mutation than other types, meaning that the false-negative rate is also related to lymphoma type (2,19). By using multiple primer sets and analyzing both the IgH and Igκ genes, the false-negative rate can be substantially reduced but usually not eliminated in modern PCR protocols (27,28).

PCR-based methods have replaced Southern blot analysis for the routine assessment of Ig and TCR antigen receptor gene rearrangements (Fig. 8.4). The sensitivity of PCR-based methods for the detection of monoclonal antigen receptor gene rearrangements is approximately 1%. The major advantages of PCR-based methods over Southern blot analysis are that they allow faster turnaround time, are less laborious, can be performed on very small biopsy specimens, and allow assessment of formalin-fixed, paraffin-embedded tissue specimens. PCR-based assays, unlike Southern blot analysis, are also more specific in the sense that they only analyze lymphoid cells; Southern blot analysis analyzes all cells in a specimen (e.g., stromal cells,
histiocytes, etc.). This results in PCR-based assays being more sensitive than Southern blot analysis in the evaluation of specimens with a mixture of cell types. Most laboratories now use PCR-based methods initially or exclusively to assess for antigen receptor gene rearrangements. As discussed earlier, the major disadvantage of PCR-based assays is that that can yield false negative results. However, by assessing multiple antigen receptor genes for clonality (e.g., IgH and Igκ or TCRγ and TCRβ), false-negative results are infrequent. Although the most recent protocols using multiplex assays and the analysis of multiple antigen receptor genes have decreased the percentage of false-negative results, they still occur in a subset of cases (27,28). Thus, Southern blot analysis of the antigen receptor genes is still considered by some molecular pathologists to be the most reliable test to assess clonality (Table 8.1).