Human B-cell antigen receptor genes include the immunoglobulin (Ig) heavy-chain locus (IGH) at 14q32, the kappa light-chain locus (IGK) at 2p11, and the lambda light-chain locus (IGL) at 22q11. In the germline or nonrearranged state, antigen receptor loci are composed of separate coding segments distributed in a region of DNA estimated to be several hundred kilobases (kb) in length. The IGH locus consists of 38 to 46 variable (V) genes (depending on the patient’s haplotype), 27 diversity (D) genes, 6 joining (J) genes, and 5 isotype-specific constant (C) genes.¹

A similar architecture is present at the light-chain kappa (IGK) and lambda (IGL) loci. These loci include V, J, and C, but not D, genes. The combinatorial diversity available from rearranging these loci is also constrained by differences in the structure of the J and C cassettes. The IGK locus has a single C region. At the IGL locus, the J regions are interspersed between the C regions, so a given J is always paired with the corresponding C.

In the pre–B-cell stage of development, B cells undergo V(D)J recombination, in which they rearrange these loci to create complete Ig heavy-chain open reading frames. This process provides the combinatorial diversity needed to assemble a complete immune repertoire. The cell first attempts to rearrange one IGH allele. An IGH D gene is fused to a J gene, and this DJ segment is fused with a V gene to form a functional VDJ exon that encodes the variable antigen recognition site of the IGH protein. If the result is nonfunctional, the cell will try to rearrange the other allele. Cells may therefore harbor two IGH rearrangements. If both alleles fail to recombine, the cell undergoes apoptotic death.

If an open reading frame has been successfully assembled at one IGH locus, the cell will, upon reaching the immature B stage, attempt to rearrange light-chain loci, starting with IGK (kappa light chain). If the result at the first IGK allele is nonfunctional, the allele is deleted and the other allele is rearranged. If both alleles fail to rearrange productively, IGL (lambda light chain) rearrangement occurs at one or both loci. If a functional light chain still has not been created, the cell undergoes apoptotic death. IGL rearrangement is attempted only in the event that IGK rearrangement fails, which explains the observation that kappa B cells are more common than lambda B cells.

The main source of diversity in Ig sequences is the many available combinations of V, J, and D (for heavy chains) segments in the IGH, IGK, and IGL genes. A secondary source of diversity is nucleotide loss and addition at the D–J and V–D junctions by the enzyme terminal deoxynucleotidyl transferase (TdT), which is active during rearrangement. Subsequent events introduce further diversity as part of affinity maturation. V segment substitution and somatic hypermutation occur in germinal centers. Isotype switching also occurs, under the influence of T helper cells.

Developing thymocytes have T-cell receptor (TCR) α, β, γ, and δ loci, which are rearranged to form functional TCR open reading frames. Similarly to the Ig loci, the TCR loci consist of multiple cassettes. TCRA and TCRG have V, J, and C genes, whereas TCRB and TCRD have V, D, J, and C.

TCRB, TCRG, and TCRD undergo simultaneous rearrangement early in development. Successful TCRB rearrangement prompts TCRA rearrangement and commitment to the αβ lineage. The TCRD locus is entirely embedded in an intron of TCRA, so that TCRA rearrangement results in deletion of TCRD. The TCRG locus, however, is not deleted in the event of successful TCRA/TCRB rearrangement, and remains present and rearranged in T cells of either αβ or γδ lineage.

IGH and TCR rearrangement studies are a useful ancillary diagnostic tool in patients suspected of harboring clonal lymphoid processes. The presence of a clonal T/B-cell population is suggestive of T/B-cell malignancies, particularly when clinical or histologic suspicion is high. However, clonal populations can also be seen in nonneoplastic processes. Clinical correlation is therefore required in all cases and should be explicitly recommended in reports of clonality testing studies.

By performing IGH and TCR rearrangement studies on the same sample, it is possible to infer whether a clonal process originates from B or T lymphocytes, respectively. However, clonal lymphoid populations may show lineage infidelity. B-cell neoplasms are particularly likely to harbor TCR rearrangements; these have been described in half of precursor B-cell neoplasms and as many as 5% to 10% of mature B-cell neoplasms.¹

In PCR-based clonality testing, the amplicon arising from the clonally rearranged IGH or TCR gene has a characteristic size, reported in nucleotides. Because the size resolution of capillary electrophoresis is <1 nt, an amplicon of the same size can be sought in subsequent samples. Identifying an amplicon of the same size in different samples from the same time point (e.g., two different skin sites) can suggest a neoplastic process rather than a reactive one. Recovering an amplicon of the same size at a subsequent time can clarify that a biopsy shows recurrent/residual disease rather than a new neoplasm. Identification of additional amplicons can suggest clonal evolution. Any reasoning based on amplicon size requires that the assays be performed with the same methodology and primer sets, which is often the case.

PCR-based IGH clonality testing has a role in prognostication for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Patients with a pre–germinal center phenotype have a significantly worse prognosis than those with a post–germinal center phenotype.^2,3 The phenotype can be ascertained by detecting features of post–germinal center B cells, including loss of ZAP-70 expression and somatic hypermutation of the rearranged IGHV gene.^4,5,6 Although ZAP-70 immunohistochemistry was previously in clinical use, this stain was technically difficult to interpret and is now deprecated.

The extent of mutation in the variable region of the IGH gene can also be used to differentiate pre–germinal center B-cell clones from those having a post–germinal center phenotype. Hypermutation is strongly predictive of a better prognosis, whereas lack of hypermutation predicts a poorer prognosis. Hypermutation is defined as ≥2% difference between the clone’s IGHV sequence and that of the germline, while <2% difference is considered lack of hypermutation.

Hypermutation is detected by amplifying, then sequencing, the clonally rearranged IGHV segment, and comparing the patient’s sequence to the reference (nonhypermutated) IGHV sequence. This assay has clinical significance only in the setting of a new diagnosis of CLL/SLL, and is not indicated as part of disease monitoring or at any other point in the course of disease.

PCR-based clonality testing uses a forward primer located in an IGH or TCR V gene, and a reverse primer located in a J gene. The method takes advantage of the fact that there is an upper limit on the size of the PCR products that can be made by Taq polymerase. This limit is on the order of 500 to 1000 nt, depending on the specific polymerase and other conditions. In the germline (nonrearranged) state, the primers used for clonality testing are too far from one another (hundreds of kilobases) to allow for productive PCR. In rearranged loci, however, the primer sites are brought closer together, and a PCR amplicon can be made.

The test further takes advantage of the fact that all members of a clonal population can be expected to contain the same IGH and/or TCR gene rearrangements, whereas polyclonal populations of lymphocytes will contain many different gene rearrangements. In clonal populations, clonality testing will therefore reveal a restricted repertoire of gene rearrangements. Polyclonal populations will yield many different PCR products, with a Gaussian distribution of sizes, testifying to the diversity of gene rearrangements present therein.

The test is performed on DNA extracted from fluid or tissue containing a suspected clonal lymphoid population. The most common sample type is formalin-fixed, paraffin-embedded (FFPE) tissue. Paraffin sections can be cut onto slides and then macrodissected with a hollow-core needle or other tool to enrich the sample in the cell population of interest, or scrolls can be collected in a microcentrifuge tube. DNA is extracted from such samples by use of an organic solvent, typically xylene or Citrasolv, to remove paraffin, followed by a standard manual or automated DNA extraction. A major advantage of FFPE specimens is that they will typically have also been examined by histopathologic methods, so that the clonality testing results can be correlated with a pathologic impression. Fresh (unfixed) tissue is suitable from a laboratory perspective, but is clinically less desirable, as there will typically be no closely associated histologic correlate (at best, there may be an FFPE specimen obtained from a nearby site). Clonality testing can be performed on fine needle aspirates, peripheral blood, or bone marrow aspirate specimens. Appropriate laboratory data such as aspirate or peripheral blood cell counts must be integrated into the eventual clinical interpretation for such specimens.

The European BIOMED-2 network (EuroClonality consortium) developed between 2002 and 2012 a series of standardized primers and guidelines to facilitate PCR-based clonality testing.⁷ This project was intended to improve interlaboratory reproducibility and make testing more widely available, including in smaller or less experienced laboratories lacking the resources to design a lab-developed test. Standardization also allows results of tests conducted in different laboratories to be compared. Because amplicons of the same size will be detected from the same disease with standard reagents, recurrent/residual disease can be distinguished from new disease regardless of where the testing is performed.

BIOMED-2 primer sets for rearrangement of IGH, IGK, IGL, TCRB, TCRD, and TCRG are available as analyte-specific reagents from Invivoscribe Technologies (San Diego, CA). These kits are labeled for research use only, and analytical and clinical validation is required before they can be used for clinical laboratory testing. Like any ancillary method, IGH and TCR clonality studies cannot independently establish any diagnosis of malignancy, and can at best be used to support a diagnosis suspected on the basis of routine morphologic examination.

The BIOMED-2 investigators suggest an algorithm to be used for sequential testing of suspected B-cell proliferations, T-cell proliferations, and lymphoid proliferations of unknown origin.⁷ By using multiple primer sets for testing (e.g., IGH V_H-J_H, IGK V_K-J_K, IGK Kde [kappa-deleting element], IGH D_H-J_H, and IGL), one increases the sensitivity, but this approach also increases the cost, complexity, and time of testing, with the trade-off depending in part upon whether the tests are performed simultaneously or sequentially.

Much of the benefit of testing can be obtained from using a smaller number of primer sets. In T-cell lymphomas, TCRG V_γ−J_γ primer sets were found to have a sensitivity (89%) comparable to TCRB V_β−J_β + D_β−J_β primer sets (91%) for detecting clonal rearrangements, but the combination was found to be even more sensitive (94%).⁸ Similarly, in B-cell lymphomas, using IGH V_H–J_H primer sets alone gave a sensitivity of 88% for detecting clonal rearrangements, but adding the results of D_H−J_H, V_K–J_K, and Kde primer sets allowed clonal peaks to be detected in 99%.⁸

In practice, a desirable cost–benefit trade-off is obtained by using a single test for TCR clonality (typically TCRG) and a single test for Ig clonality (typically IGH). The present chapter focuses on this approach, with the caveat that some laboratories may choose to implement additional assays to capture the subset of clones that will not be ascertained using a single assay.