LymphoTrack^® Dx IGH FR3 Assay - S5/PGM™

Product Details

• Summary and Explanation of the Test

  The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 non-functional variable (V_H) gene segments, 27 functional diversity (D_H) gene segments, and 6 functional joining (J_H) gene segments spread over 1250 kilobases. The V_H gene segments contain three conserved framework (FR) and two variable complementarity-determining regions (CDRs).

  Lymphoid cells are different from other somatic cells in the body. During development, the antigen receptor genes in lymphoid cells undergo somatic gene rearrangements.¹ For example, during B-cell development, genes encoding the IGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating V_H – D_H – J_H combinations that are unique in both length and sequence. Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect IGH clonal rearrangements can be useful in the study of B- and T-cell malignancies.

  In addition, immunoglobulin heavy chain variable region (IGHV) gene hypermutation status provides important prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).  The presence of IGHV somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline V_H gene sequence, whereas less than 2% difference is considered evidence of no SHM. The status of SHM for clone(s) has clinical relevance for B-CLL, as there is a clear distinction in the median survival of patients with and without SHM. Hypermutation of the IGHV region is strongly predictive of a good prognosis while lack of mutation predicts a poor prognosis.²

  Initially, clonal rearrangements were identified using Restriction Fragment, Southern Blot Hybridization (RF-SBH) techniques. However, these tests proved cumbersome and labor-intensive, they required large amounts of DNA, and were not suitable for analysis of many of the less diverse antigen receptor loci.

  During the last several decades, the use of RF-SBH assays has been supplanted by PCR-based clonality tests developed by Alexander Morley,³ and are considered the current gold standard method. PCR-based assays identify clonality on the basis of overrepresentation of amplified V_H – D_H – J_H (or incomplete D_H – J_H products) gene rearrangements following their separation using gel or capillary electrophoresis.  Though sensitive and suitable for testing small amounts of DNA, these assays cannot readily differentiate between clonal populations and multiple rearrangements that might lie beneath a single-sized peak, and are not designed to identify the specific V_H – J_H DNA sequence that is required to track clonal populations in subsequent analyses.

  The LymphoTrack Dx IGH (FR1, FR2 and FR3) Assays for the Ion S5™ and Ion PGM™ (sold separately and as a set) represent a significant improvement over existing clonality assays using fragment analysis as they efficiently detect IGH gene rearrangements, and at the same time identify the DNA sequence specific for each clonal gene rearrangement.  Therefore, these products have two important and complementary uses: they provide critical evidence on the existence of clonality and identify sequence information required to track those clones in subsequent samples.  The LymphoTrack Dx IGH FR1 Assay additionally provides detailed sequence information on the degree of SHM.

  Each single multiplex master mix for IGH targets one of the conserved framework regions (FR1, FR2 or FR3) within the V_H and the J_H regions described in lymphoid malignancies.  Targeting all three framework regions significantly reduces the risk of not being able to detect the presence of clonality, as somatic hypermutations in the primer binding sites of the involved V_H gene segments can impede DNA amplification.⁴  Data from all three framework regions is needed to determine evidence of clonality for a sample.

  Primers included in the master mixes are designed with Thermo Fisher Scientific adapters and 12 different indices.  These assays allow for a one-step PCR reaction and pooling of amplicons from several different samples and targets (generated with other LymphoTrack Dx Assays for the Ion S5™ or Ion PGM™ instrument) onto one Ion S5 or Ion PGM chip, allowing for up to 12 samples per target to be analyzed in parallel in a single run.

  The associated LymphoTrack Dx Software – S5/PGM provides direct interpretation of the data generated from LymphoTrack Dx Assays via a simple and streamlined method of analysis and visualization.  By following the guidelines provided in the Instructions for Use (IFU) the sample results summarized in the software can be easily interpreted for the presence or absence of clonality and somatic hypermutation.  The results of molecular clonality tests should always be interpreted in the context of clinical, histological and immunophenotypic data.

  Positive and negative controls for clonality are included in the kit.

  Note:  For a more thorough explanation of the locus and the targeted sequencing strategy, please refer to Principle of Immunoglobulin and T Cell Receptor Gene Rearrangement.⁵

  Note:  Life Technologies is a subsidiary of Thermo Fisher Scientific.

• Principles of the Procedure

  Polymerase Chain Reaction (PCR)

  PCR assays are routinely used for the identification of clonal B- and T-cell populations. These assays amplify the DNA between primers that target the conserved framework (FR) of the V_H regions and the conserved J_H regions of antigen receptor genes. These conserved regions, where primers target, lie on either side of an area where programmed genetic rearrangements occur during the maturation of all B and T lymphocytes. It is a result of these genetic rearrangements that different populations of the B and T lymphocytes arise.

  The antigen receptor genes that undergo rearrangements are the immunoglobulin heavy chain (IGH) and light chains (IGK and IGL) in B-cells, and the T-cell receptor genes (TRA, TRB, TRG, TRD) in T-cells. Each B- and T-cell has a single productive V – J rearrangement that is unique in both length and sequence. Therefore, when DNA from a normal or polyclonal population is amplified using DNA primers that flank the V – J region, amplicons unique in both sequence and length, reflecting the heterogeneous population, are generated. In some cases, where lymphocyte DNA is not present, no amplicons will be generated. For samples containing clonal populations, the yield is one or two prominent amplified products of the same length and sequence that are detected with significant frequency of occurrence, within a diminished polyclonal background amplified at a lower frequency.

  Amplicon Purification

  PCR amplicons are purified to remove excess primers, nucleotides, salts, and enzymes using the Agencourt^® AMPure^® XP system. This method utilizes solid-phase reversible immobilization (SPRI) paramagnetic bead technology for high-throughput purification of PCR amplicons. Using an optimized buffer, PCR amplicons that are 100 bp or larger are selectively bound to paramagnetic beads while contaminants such as excess primers, primer dimers, salts, and unincorporated dNTPs are washed away. Amplicons can then be eluted and separated from the paramagnetic beads resulting in a more purified PCR product for downstream analysis and amplicon quantification.

  Amplicon Quantification

  Purified amplicons are quantified utilizing the Agilent Technologies 2100 Bioanalyzer.  These are electrophoretic methods that utilize the principles of traditional gel electrophoresis to separate and quantify DNA on a chip based platform. Quantification is achieved by running a marker of known concentration alongside PCR amplicons and then extrapolating the concentration of the amplicons. Calculating the concentration of PCR amplicons allows equal amplicon representation in the final pooled library that is loaded onto the Ion S5 or Ion PGM for sequencing.

  Next-Generation Sequencing (NGS)

  Sanger sequencing methods represent the most popular in a range of ‘first-generation’ nucleic acid sequencing technologies. Newer methods, which leverage massively parallel sequencing approaches, are often referred to as next-generation sequencing (NGS). NGS technologies can use various combination strategies of template preparation, sequencing, imaging, and bioinformatics for genome alignment and assembly.

  NGS technologies used in this product rely on the amplification of genetic sequences using a series of consensus forward and reverse primers that include adapter and index tags. Amplicons generated with LymphoTrack Dx master ixes are quantified, pooled, and loaded onto a chip for sequencing with a Thermo Fisher Scientific Ion S5 or Ion PGM instument. The Ion S5 and Ion PGM require that the pooled library of DNA fragments is bound to individual beads prior to sequencing, (one unique sequence per bead) through a process known as emulsion PCR. Once bound to the beads the DNA fragments are amplified until they cover the surface of the bead. Beads are then loaded onto a semi-conductor chip, where they find their own well to occupy and where sequencing occurs. Sequencing is conducted by flooding the chip with individual unincorporated nucleotides one base at a time (dATP, dCTP, dGTP, dTTP). The Ion S5 and Ion PGM instruments detect the addition of nucleotides when hydrogen ions are released during DNA polymerization and causes a change in pH of the wells, which can be measured as a change in voltage. The voltage changes proportionally to the number of nucleotides added. After nucleotides are incorporated, unincorporated nucleotides are washed away and the process begins again with a new dNTP.

  Multiplexing Amplicons

  These products were designed to allow for two different levels of multiplexing in order to reduce costs and time for laboratories. The first level of multiplexing originates from the multiple indices that are provided with the assays, up to 12. Each of these 12 indices can be considered to act as a unique barcode that allows amplicons from individual samples to be pooled together after PCR amplification to generate the sequencing library. Later, the resulting sequences can be sorted by the bioinformatics software to identify those that originated from an individual sample.

  The second level of multiplexing originates from the ability of the accompanying software to sort sequencing data by both index and target. This allows amplicons generated with targeted primers (even those tagged with the same index) to be pooled together to generate the library to be sequenced on a single sequencing chip. An example would be to sequence products from several Invivoscribe LymphoTrack Dx Assay kits for the Ion S5 and Ion PGM together in the same run. Thermo Fisher Scientific Ion 316™ Chip v2 can generate 2-3 million reads so it is recommended to multiplex no more than three different gene targets together. Up to five different gene targets can be multiplexed together on the Ion PGM Ion 318™ Chip v2 BC (4-5.5 million reads), Ion S5 Ion 520™Chip (3-6 million reads) and Ion S5 Ion 530™ Chip (15-20 million reads).  To determine the number of reads per sample, the total number of reads for the sequencing chip should be divided by the number of samples that will be multiplexed.

  When multiplexing amplicons of different gene targets it is important to use the appropriate sequencing chemistry. The number of sequencing cycles must be sufficient to sequence the largest amplicon in the multiplex.  Two or more sequencing libraries generated from the same LymphoTrack Dx gene target master mixes (e.g., two IGH FR1 sequencing libraries, either from the same or different kit lots) can also be multiplexed together into a single sequencing library as long as each index for that master mix is only included once per sequencing run.

• Specimen Requirements
  • This assay tests extracted and purified genomic DNA. DNA must be quantified with a method specific for double-stranded DNA (dsDNA) and free of inhibitors of PCR amplification.
  • Resuspend DNA in an appropriate solution such as 0.1X TE (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0, prepared with molecular biology grade water) or molecular biology grade water alone.
  • The minimum input quantity is 50 ng of high quality DNA.