Objectives: To determine whether VH family usage in B-cell lymphoproliferative disorders can be deduced from polymerase chain reaction (PCR) product-length information obtained through the BIOMED-2 (Invivoscribe, San Diego, CA) clonality assay.
Methods: We develop an algorithm that uses the sizing information of the BIOMED-2 immunoglobulin heavy chain (IGH) clonality assay to deduce VH family usage. PCR with family-specific primers on 51 clinical samples containing 54 rearranged alleles were used to validate the algorithm.
Results: The clonal PCR products in different framework reactions contain the same NDN segment (because they are from the same allele). Subtracting the size of the framework III product from the size of the framework I and II products yields the relative position of the framework primer binding sites for the VH segment used. The VH family can be assigned with these relative positions because they are VH family specific in the BIOMED-2 assay. The VH family assigned by the algorithm was concordant with family-specific PCR results for 49 (96%) of the 51 specimens.
Conclusions: We have developed an algorithm that can correctly assign VH family usage when all three BIOMED-2 framework reactions produced clonal products. Given the wide adoption of BIOMED-2 assay, the algorithm can facilitate collection of IGH VH usage data without additional cost to the laboratories.
Materials and Methods
The immunoglobulin heavy chain (IGH) component of human antibodies contains a variable region encoded by the variable (VH), diversity (DH), and joining (JH) gene segments. During B-cell maturation, these gene segments rearrange and join together via VDJ recombination, thus enabling B cells with different antigen-binding specificity to be produced. Based on sequence similarities, the 229 different human VH gene segments can be grouped into seven families: VH1 to VH7.
Several types of B-cell malignancies exhibit preferential VH family usage. Mortuza et al1 first showed that VH6 is preferentially used in adult acute lymphoblastic leukemia. Donisi et al,2 Rosenquist et al,3 and Pritsch et al4 reported selective use of VH1 and VH5 in patients with chronic lymphocytic leukemia (CLL). Crowther-Swanepoel et al5 and Ghiotto et al6 confirmed these findings and provided a more detailed analysis of the VH repertoires in CLL. Sato et al7 reported relatively infrequent use of VH4 in thyroid large cell lymphomas but not, however, in thyroid mucosa-associated lymphoid tissue lymphomas. Coupland et al8 found that VH3 is most commonly used in ocular lymphomas. Camacho et al9 reported VH family usage for mantle cell lymphomas that are similar to those seen in peripheral blood B cells. Rettig et al10 reported the VH family usages for multiple myeloma and noted the absence of VH4-34. Together, these findings suggest that the tumor-initiating events for some mature B-cell neoplasms may be linked to the immunoglobulin proteins expressed by the particular B cells and that antigen stimulation may be a contributing factor in the lymphomagenesis of these neoplasms.
Despite this evidence for preferential VH family usages, few prognostic or therapeutic differences have (to date) been shown to correlate with specific VH family usage. This is in part due to the fact that VH family usage is usually not determined when IGH clonality assays are performed in diagnostic laboratories. Consequently, large-scale, comprehensive studies to determine the clinical significance (if any) of VH family usage have not been performed. Similarly, limited information is available for the significance of light chain variable region usage in different lymphomas.
Unlike the clonality assays for T-cell β and γ receptors, the interpretation of an IGH clonality assay is generally straightforward. Polyclonal samples produce a Gaussian distribution of multiple peaks, while clonal samples produce tall peaks above the background in some or all of the reactions. Occasionally, it is necessary to measure the peak heights and employ a cutoff or ratio to determine the significance of a peak. Several reports have validated the sensitivity, specificity, and usefulness of the BIOMED-2 IGH assay.11‐15 Because the assay is PCR based, a small quantity of DNA is usually sufficient to yield valuable diagnostic information.14 The DNA can be isolated from fresh or formalin-embedded, paraffin-embedded tissues or from specimens such as cerebrospinal fluid, vitreous fluids, or fine-needle aspirates. Although the assay uses VH family-specific primers, the identity of the specific VH segment involved in the rearranged allele remains unknown in clonal cases. Identifying the specific VH segment involved would require steps that are not routinely performed in diagnostics laboratories (ie, purification of the clonal PCR products and sequencing them by Sanger or next-generation methods).16
In this report, we show that when clonal products are present in all three framework reactions, VH family usage can be determined from the PCR product lengths of the standard BIOMED-2 IGH PCR clonality assay with little added cost or laboratory work. To validate our method, we performed family-specific PCR reactions on 51 clinical specimens (54 alleles, because three cases had biallelic IGH rearrangements) that had at least one clonal peak in each of the three framework reactions as determined by the BIOMED-2 clonality assay. For 52 of the 54 alleles, there was a concordant result between our method and the family-specific PCR.
IGH PCR and Electrophoresis
DNA from clinical specimens was isolated from fresh or formalin-fixed, paraffin-embedded specimens with DNA isolation kits from Qiagen (Valencia, CA) according to the manufacturer’s instructions and stored at –20°C. The concentration of DNA was measured by the NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). DNA quality was assessed using a multiplexed control PCR ladder with amplicons of 100, 200, 300, 400, and 600 base pairs (bp) included in the BIOMED-2 kit. The pathologic diagnoses for the samples used in the validation are listed in Table 1.
BIOMED-2 IGH clonality assay kits were purchased from Invivoscribe, and the manufacturer’s instructions were followed when performing the assay. JH primers and family-specific primers for FR1, FR2, and FR3 were purchased from Invitrogen (Carlsbad, CA). Individual family-specific PCR reactions were performed with the standard BIOMED-2 protocol (50 µL final volume with 1× ABI buffer II, 100-400 ng DNA, 10 pmol each primer, 200 µmol/L dNTP, 1.5 mmol/L MgCl2, and 1 U Taq enzyme, with all the reagents being purchased from Applied Biosystems, Foster City, CA) and standard BIOMED-2 cycling conditions (7 minutes at 95°C followed by 35 cycles of 30 seconds at 95°C, 45 seconds at 60°C, 90 seconds at 72°C, and then 10 minutes at 72°C). For the BIOMED-2 assay, the samples were analyzed with an ABI genetic analyzer 3130xl and Gene Scan software (Applied Biosystems) following the manufacturer’s instruction. For the family-specific PCR reactions, the products were run on a 2% agarose gel and visualized following ethidium bromide staining.
Implementation of the Algorithm
We next reviewed 559 specimens that had been submitted for IGH clonality testing to the Center for Advanced Molecular Diagnostics at our institution during 2009. Of the 559 specimens that we reviewed, 104 samples were clonal by the BIOMED-2 assay, and they all had a corresponding histopathologic diagnosis of a B-cell clonal process (Table 1). Ninety-five specimens were monoallelic and nine specimens were biallelic, giving us a total of 113 clonally rearranged alleles. Thirteen (11.5%) alleles had a clonal PCR product in only one of the three reactions (five with FR1 only, six with FR2 only, and two with FR3 only), and 40 (35.6%) alleles were clonal in two of the three reactions. Most alleles in this latter group lacked a clonal FR3 peak (26 of 40). These results are similar to those reported by others, and the underlying cause has been attributed to frequent somatic mutations that alter the sequences for FR3 binding.14,18‐21 A lack of the FR1 peak is often due to partially fragmented DNA that has been isolated from older paraffin-embedded specimens.
Sixty alleles had clonal PCR products in each of the three reactions and were thus suitable for the VHFS algorithm. For these 60 alleles, the length of each of the PCR products was recorded and then used to calculate the values for FR1-FR2 and FR2-FR3. The results of these calculations were used to assign each allele to a VH family according to the VHFS algorithm. Six alleles had FR1, FR2, and FR3 sizes outside the acceptable ranges, which suggested that either a large insertion or deletion had occurred.22 For the 54 remaining alleles, we then performed family-specific IGH PCR reactions with the common JH primer and the seven family-specific FR1 primers. Analysis of the PCR products on agarose gel was used to determine the actual VH family usage and compared with those assigned by VHFS. The VHFS VH family assignment was concordant with the family-specific PCR results in 52 (96%) of the 54 alleles. In the two discordant alleles, the VHFS assigned them to VH4, but both had VH1 by PCR and gel analysis Table 3.
Molecular pathologists have long appreciated the importance of PCR product lengths in IGH clonality assays and have used them to assess clonal relationships between primary disease and relapses and to compare lesions at multiple sites or with disparate morphologies (as illustrated in our earlier example). In this report, we show that pairwise subtractions of the length of PCR products from one another (FR1-FR2, FR2-FR3, and FR1-FR3) in the BIOMED-2 IGH clonality assay produce a set of three values that are VH family specific. When CE is used to size the PCR products, the values are sufficiently accurate to assign VH family usage in a high percentage of the cases (52 of 54 in our test) when all three reactions have a clonal peak. This adds another utility to the routine recording and reporting of the PCR product lengths of IGH clonality analysis. Any laboratory already using the BIOMED-2 IGH assay with CE can obtain VH family usage information without additional cost or laboratory work. The information can then facilitate the study of VH family usage and its significance in B-cell lymphomas.
The method for VH family assignment described in this report depends on accurate measurement of PCR product size. Gel-based methods (agarose and polyacrylamide) do not have the sufficient resolution for this purpose. We found that CE was sufficiently accurate to support our algorithm. In most commercial CE instruments, PCR product sizes are determined by running a set of size standards followed by a regression fitting (“local Southern method”). The reported margin of error with this approach is typically ±2 nucleotides.23 This margin of error is acceptable for our method given the known primer binding positions. The other consideration is the actual sequence of the VH segments. In CE, the sample DNA is denatured and then run as single-stranded molecules and separated by molecular weight. Because the molecular weights of the four nucleotides (A, G, T, and C) are different, two DNA molecules of the same length may migrate differently if one of the molecules has significantly more Gs than the other. If different VH segments within the same family have very different GC content, then conceivably they may be sized differently even when they are of the same length. To examine this issue, we calculated the molecular weights of the 229 VH segments using the downloaded sequences and the standard formula of molecular weight: (An × 313.21) + (Tn × 304.2) + (Cn × 289.18) + (Gn × 329.21) – 61.96 + 79.0. The results showed that the calculated molecular weights of the different VH segments within a particular VH family were within 300 Daltons of each other (ie, less than the molecular weight of one nucleotide). This is consistent with our finding that 52 of 54 alleles had sizes expected for their respective VH family.
Somatic hypermutation of the VH segments can introduce point mutations, small insertions, and/or deletions to the VH segment. These alterations can affect primer binding and thus reduce the sensitivity of IGH clonality testing. They can also cause changes in the length of the PCR products as measured in CE. These and other changes in PCR fragment length can be significant enough to cause the VHFS algorithm to make incorrect assignments. This could potentially explain the two alleles in our validation set that had sizes different from those expected from their VH family assignment. As the algorithm is more widely tested and used, the extent of the impact of somatic hypermutations and other small insertions/deletions on PCR product length can be assessed more accurately.
Six alleles in our validation set had FR1-FR2 or FR2-FR3 values that differed significantly from those predicted in any VH family and suggest that a large insertion or deletion occurred between the primer binding sites, similar to findings described by Vargas et al.22 In reviewing our clinical samples since 2005, we found that, for 2005 to 2009, about 10% of the rearranged alleles had sizes that similarly fell outside the predicted ranges. Oversized or undersized PCR products can be the result of insertions/deletions or unusually large or small NDN-JH regions. With VHFS, one can potentially differentiate between these two possibilities.
When a specimen has two or more peaks in each of the three reactions, it suggests that there may be more than one clonally rearranged allele. Without the VHFS, it is natural to assume that the longer PCR products from each reaction are derived from one allele and the shorter products are from the other alleles. The VHFS algorithm offers a rationale for associating the appropriate peaks to a rearranged allele by examining if the FR1-FR2 and FR2-FR3 intervals of the selected peaks fit into a VH family. In addition, the VHFS can potentially be helpful in evaluation of paucicellular specimens, in which only a few peaks are present, raising the question of whether these peaks are from a single rearranged allele or are part of a “pseudoclonal” pattern. If the FR1-FR2 and FR2-FR3 values for the peaks can be assigned to one specific VH family based on the VHFS, then it suggests that they share the same NDN-JH region and therefore are more likely derived from a single allele.
Mapping of VH Primers to All Known VH Segments
In summary, we report a new insight about the BIOMED-2 IGH clonality test that is widely used in molecular diagnostic laboratories. We believe that the method described in this report can make VH family assignment possible for most of the rearranged alleles when all three reactions produce a clonal peak. It may also shed light on the relationship between VH family usage and different types of lymphomas.
VH Family Signature
|VH Family||Assignment by VHFS Algorithm, No.||Assignment by Family-Specific PCR, No.|
|VH3 (or VH6)b||18||15|
|VH Family||Assignment by VHFS Algorithm, No.||Assignment by Family-Specific PCR, No.|
|VH3 (or VH6)b||18||15|
PCR, polymerase chain reaction; VHFS, VH family signature.
VH family assignment was concordant with PCR results in 52 (96%) of the 54 alleles. The table shows the number of alleles that were assigned to each VH family by the VHFS and the family-specific PCR. Two specimens assigned to VH4 by VHFS had VH1 by family-specific PCR.
VHFS cannot differentiate VH3 from VH6 (see text).
Open in new tab