Determination of VH Family Usage in B-Cell Malignancies via the BIOMED-2 IGH PCR Clonality Assay

The BIOMED-2 IGH clonality assay (Invivoscribe, San Diego, CA) is widely used in molecular laboratories throughout the world, as driven by the need and desire for standardization. More than 50% of the laboratories that participated in the 2014 and 2015 College of American Pathologists proficiency test for B-cell clonality used the BIOMED-2 IGH clonality assay. The BIOMED-2 assay consists of three semi-multiplexed polymerase chain reactions (PCRs) Figure 1. Each reaction targets one of the three framework regions (FRs) of the variable (V_H) segments. At the 5′ end, fluorescently labeled primers bind to the FR1, FR2, and FR3 regions of each of the seven V_H families (in the FR1 reaction, six primers are used instead of seven because the V_H1 and V_H7 families share the same consensus FR1 sequence). At the 3′ end, a common degenerate consensus primer binds to the joining (J_H) region of the IGH gene. The fluorescently labeled PCR products are analyzed via capillary electrophoresis (CE).

Figure 1

Open in new tabDownload slide

BIOMED-2 immunoglobulin heavy chain (IGH) clonality assay. A schematic diagram of a rearranged IGH allele (top) and the three polymerase chain reaction products (red bars) of the BIOMED-2 IGH clonality assay is shown. Blue arrows represent primers for the three framework regions and the common J_H region. The precise position of the V_H primer for each V_H family differs. The relative positions of primer binding sites for FR1, FR2, and FR3 do not change for a given rearranged allele, even though the NDN has variable lengths. As a result, the relative size differences (namely, FR1-FR2, FR2-FR3, and FR1-FR3) do not vary for different rearranged alleles within a particular V_H family.

Figure 1

Open in new tabDownload slide

BIOMED-2 immunoglobulin heavy chain (IGH) clonality assay. A schematic diagram of a rearranged IGH allele (top) and the three polymerase chain reaction products (red bars) of the BIOMED-2 IGH clonality assay is shown. Blue arrows represent primers for the three framework regions and the common J_H region. The precise position of the V_H primer for each V_H family differs. The relative positions of primer binding sites for FR1, FR2, and FR3 do not change for a given rearranged allele, even though the NDN has variable lengths. As a result, the relative size differences (namely, FR1-FR2, FR2-FR3, and FR1-FR3) do not vary for different rearranged alleles within a particular V_H family.

Materials and Methods

The immunoglobulin heavy chain (IGH) component of human antibodies contains a variable region encoded by the variable (V_H), diversity (D_H), and joining (J_H) 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 V_H gene segments can be grouped into seven families: V_H1 to V_H7.

Several types of B-cell malignancies exhibit preferential V_H family usage. Mortuza et al¹ first showed that V_H6 is preferentially used in adult acute lymphoblastic leukemia. Donisi et al,² Rosenquist et al,³ and Pritsch et al⁴ reported selective use of V_H1 and V_H5 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 V_H repertoires in CLL. Sato et al⁷ reported relatively infrequent use of V_H4 in thyroid large cell lymphomas but not, however, in thyroid mucosa-associated lymphoid tissue lymphomas. Coupland et al⁸ found that V_H3 is most commonly used in ocular lymphomas. Camacho et al⁹ reported V_H family usage for mantle cell lymphomas that are similar to those seen in peripheral blood B cells. Rettig et al¹⁰ reported the V_H family usages for multiple myeloma and noted the absence of V_H4-34. 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 V_H family usages, few prognostic or therapeutic differences have (to date) been shown to correlate with specific V_H family usage. This is in part due to the fact that V_H 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 V_H 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 V_H family-specific primers, the identity of the specific V_H segment involved in the rearranged allele remains unknown in clonal cases. Identifying the specific V_H 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

Results

DNA Isolation

In this report, we show that when clonal products are present in all three framework reactions, V_H 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. J_H 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.

The 229 human V_H segment sequences were obtained from the National Center for Biotechnology Information IgBlast site¹⁷ and used to map the coordinates of the primer binding sites. The interprimer distances (FR1-FR2 and FR2-FR3) were determined and used to construct a decision tree to assign V_H family usage Figure 2. A computer program was implemented using Microsoft (Microsoft, Redmond, WA) VB.NET programming language and rendered as a webpage. The user enters the sizes of the three FR1, FR2, and FR3 clonal PCR products as measured by capillary gel electrophoresis and the webpage returns the V_H family assignment (http://bwhpathology.partners.org/igh_family.aspx).

Figure 2

Open in new tabDownload slide

V_H family signature. An algorithm to assign V_H family usage is shown. Start by recording the polymerase chain reaction product sizes for a given specimen. Calculate FR1-FR2 by subtracting FR2 from FR1 and calculate FR2-FR3 by subtracting FR3 from FR2. Follow the algorithm from top to bottom to find the V_H family assignment. All numbers indicate number of base pairs. If X or Y falls out of the ranges in any of the box, then either the rearranged allele has suffered an insertion/deletion or the clonal peaks used in the calculation do not belong to the same allele (as can be seen in cases with more than one rearranged allele).

Figure 2

Open in new tabDownload slide

V_H family signature. An algorithm to assign V_H family usage is shown. Start by recording the polymerase chain reaction product sizes for a given specimen. Calculate FR1-FR2 by subtracting FR2 from FR1 and calculate FR2-FR3 by subtracting FR3 from FR2. Follow the algorithm from top to bottom to find the V_H family assignment. All numbers indicate number of base pairs. If X or Y falls out of the ranges in any of the box, then either the rearranged allele has suffered an insertion/deletion or the clonal peaks used in the calculation do not belong to the same allele (as can be seen in cases with more than one rearranged allele).

Rationale

Discussion

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 V_H 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 J_H primer and the seven family-specific FR1 primers. Analysis of the PCR products on agarose gel was used to determine the actual V_H family usage and compared with those assigned by VHFS. The VHFS V_H 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 V_H4, but both had V_H1 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 V_H family specific. When CE is used to size the PCR products, the values are sufficiently accurate to assign V_H 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 V_H family usage information without additional cost or laboratory work. The information can then facilitate the study of V_H family usage and its significance in B-cell lymphomas.

The method for V_H 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 V_H 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 V_H 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 V_H segments using the downloaded sequences and the standard formula of molecular weight: (A_n × 313.21) + (T_n × 304.2) + (C_n × 289.18) + (G_n × 329.21) – 61.96 + 79.0. The results showed that the calculated molecular weights of the different V_H segments within a particular V_H 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 V_H 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 V_H 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 V_H 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-J_H 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 V_H 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 V_H family based on the VHFS, then it suggests that they share the same NDN-J_H region and therefore are more likely derived from a single allele.

Mapping of V_H 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 V_H 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 V_H family usage and different types of lymphomas.

V_H Family Signature

We came across an interesting case during the course of our analysis that illustrates the value of VHFS. A patient had CLL, splenomegaly, and a mass in the colon. A segmental resection of the colon and a splenectomy were performed. In the spleen, the pathologic diagnosis was involvement by the patient’s CLL. The mass in the colon, however, showed a diffuse large B-cell lymphoma. The sections of the spleen and colon were both submitted for IGH clonality analysis to determine whether the diffuse large B-cell lymphoma was clonally related to the CLL. On the BIOMED-2 assay, both lesions displayed clonal peaks in all three PCR reactions Figure 4A. The sizes of the FR1, FR2, and FR3 products from the spleen were 355, 290, and 155 bp, respectively, while those from the colon were 322, 258, and 115 bp, respectively. Although the lengths of these PCR products were different, the question remained as to whether a deletion in the IGH allele could explain these differences. Using the VHFS, the splenic CLL was assigned to the V_H3 family, while the diffuse large B-cell lymphoma was assigned to V_H4. This additional information established the independent nature of these two neoplastic processes without further purification and sequencing steps. Subsequent V_H family-specific PCR reactions and gel analysis confirmed the V_H family assignment by VHFS Figure 4B.

Figure 4

Open in new tabDownload slide

A case that illustrates the added value of V_H family signature (VHFS). A case with two concurrent lymphomas illustrates the value of the VHFS. A, Electrophoretogram of the two lesions analyzed with the BIOMED-2 immunoglobulin heavy chain assay followed by capillary electrophoresis (CE). From top to bottom are the three reactions: FR1, FR2, and FR3. B, The results of seven V_H family-specific PCR reactions followed by agarose gel electrophoresis. The FR1-FR2 and FR2-FR3 were calculated from the peak sizes obtained from CE. The V_H family assignment is according to the VHFS algorithm described in Figure 2 and the text. CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma.

Figure 4

Open in new tabDownload slide

A case that illustrates the added value of V_H family signature (VHFS). A case with two concurrent lymphomas illustrates the value of the VHFS. A, Electrophoretogram of the two lesions analyzed with the BIOMED-2 immunoglobulin heavy chain assay followed by capillary electrophoresis (CE). From top to bottom are the three reactions: FR1, FR2, and FR3. B, The results of seven V_H family-specific PCR reactions followed by agarose gel electrophoresis. The FR1-FR2 and FR2-FR3 were calculated from the peak sizes obtained from CE. The V_H family assignment is according to the VHFS algorithm described in Figure 2 and the text. CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma.

References

Experimental Validation of the Algorithm