CAT #: 91210119
LymphoTrack® Dx IGH FR3 Assay Panel - MiSeqTM
Intended Use (LymphoTrack Dx IGH FR1/2/3 Assays – MiSeq)
The LymphoTrack Dx IGH FR1 Assay for the Illumina MiSeq is an in vitro diagnostic product intended for next-generation sequencing (NGS) based determination of the frequency distribution of IGH gene rearrangements as well as the degree of somatic hypermutation of rearranged genes in patients suspected with having lymphoproliferative disease. This assay aids in the identification of lymphoproliferative disorders as well as providing an aid in determining disease prognosis.
This LymphoTrack Dx IGH FR2 Assay is an in vitro diagnostic product intended for next-generation sequencing (NGS) for the Illumina MiSeq instrument. The assay will determine the frequency distribution of IGH VH – JH gene rearrangements in patients suspected with having lymphoproliferative disease. This assay aids in the identification of lymphoproliferative disorders.
The LymphoTrack Dx IGH FR3 Assay is an in vitro diagnostic product intended for next-generation sequencing (NGS) for the Illumina MiSeq instrument. The assay will determine the frequency distribution of IGH VH – JH gene rearrangements in patients suspected with having lymphoproliferative disease. This assay aids in the identification of lymphoproliferative disorders.
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 (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) gene segments spread over 1250 kilobases. The VH gene segments contain three conserved framework (FR) and two variable complementarity-determining regions (CDRs).
Lymphoid cells are different from the 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 VH – DH – JH 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 VH gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation. The status of somatic hypermutation for clone(s) has clinical relevance for B-CLL, as there is a clear distinction in the median survival of patients with and without somatic hypermutation. 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 over-representation of amplified VH – DH – JH (or incomplete DH – JH 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 VH – JH DNA sequence that is required to track clonal populations in subsequent analyses.
The LymphoTrack Dx IGH (FR1, FR2 and FR3) Assays for MiSeq® (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 information 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 targets one of the conserved IGH framework regions (FR1, FR2 or FR3) within the VH and the JH 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 VH 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 Illumina adapters and up to 24 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 Illumina MiSeq instrument) onto one MiSeq flow cell, allowing for up to 24 samples per target to be analyzed in parallel in a single run.
The associated LymphoTrack Dx Software – MiSeq provides direct interpretation of the data generated from LymphoTrack Dx Assays via a simple and streamlined method of analysis and visualization of data. 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
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 VH regions and the conserved JH 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.
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.
Purified amplicons are quantified using the KAPA Library Quantification Kits for Illumina platforms. Purified and diluted PCR amplicons and a set of six pre-diluted DNA standards are amplified by quantitative (qPCR) methods, using the KAPA SYBR® FAST qPCR Master Mix and primers. The primers in the KAPA kit target Illumina P5 and P7 flow cell adapter oligo sequences.
The average Ct score for the pre-diluted DNA Standards are plotted against log10 to generate a standard curve, which can then be used to calculate the concentration (pM) of the PCR amplicons derived from sample DNA. Calculating the concentration of PCR amplicons allows equal amplicon representation in the final pooled library that is loaded onto the MiSeq 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 the LymphoTrack Dx master mixes are quantified, pooled, and loaded onto a flow cell for sequencing with an Illumina MiSeq sequencing platform. Specifically, the amplified products in the library are hybridized to oligonucleotides on a flow cell and are amplified to form local clonal colonies (bridge amplification). Four types of reversible terminator bases (RT-bases) are added and the sequencing strand of DNA is extended one nucleotide at a time. To record the incorporation of nucleotides, a CCD camera takes an image of the light emitted when fluorescently labeled nucleotides are added to the sequencing strand. A terminal 3’ blocker is added after each cycle of the sequencing process and any unincorporated nucleotides are removed prior to the addition of four new RT-bases.
This product was 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 24. Each of these 24 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 flow cell. An example would be to sequence a combination of products from several Invivoscribe LymphoTrack Dx Assay kits for the MiSeq such as IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK and TRG together. 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. For example, when multiplexing a combination of IGH FR1, IGH FR2, IGH FR3, IGK and TRG amplicons together, the MiSeq v2 (500 cycle) or v3 (600 cycle) sequencing kit should be used. When multiplexing any of these amplicons together with IGHV Leader, the MiSeq v3 (600 cycle) sequencing kit should be used. If multiplexing only IGH FR3 and TRG amplicons together, which both have shorter amplicon sizes, MiSeq v2 (300 or 500 cycle) sequencing kits can be used, but the cycle settings must be adjusted in the sample sheet.
The number of samples that can be multiplexed onto a single flow cell is also dependent on the flow cell that is utilized. Illumina’s standard flow cells can generate 12-15 million reads. To determine the number of reads per sample, the total number of reads for the flow cell should be divided by the number of samples that will be multiplexed and the number of reads for each sample should be sufficient for valid interpretation.
- 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.
1. Tonegawa, S. (1983). Nature 302, 575-581.
2. Ghia, P. et al., (2007). Leukemia 21, 1-3.
3. Trainor, KJ. et al., (1990). Blood 75, 2220-2222.
4. Evans, P. A. et al., (2007). Leukemia 21, 207-14.
5. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition., sections 188.8.131.52 and 184.108.40.206).
These are in vitro diagnostic products and available in regions that accept CE-IVD products.
This product is covered by one or more patents and patent applications owned by or exclusively licensed to Invivoscribe, Inc., including United States Patent Number 7785783, United States Patent Number 8859748, United States Patent Number 10280462, European Patent Number EP 1549764B1 (validated in 16 countries, and augmented by related European Patents Numbered EP2418287A3 and EP 2460889A3), Japanese Patent Number JP04708029B2, Japanese Patent Application Number 2006-529437, Brazil Patent Application Number PI0410283.5, Canadian Patent Number CA2525122, Indian Patent Number IN243620, Mexican Patent Number MX286493, Chinese Patent Number CN1806051, and Korean Patent Number 101215194.
Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use reagents, amplification enzymes or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.
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