LymphoTrack^® Dx IGHV Leader Somatic Hypermutation Assay Panel - MiSeq^TM

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 1,250 kilobases.

  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 protein are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating cell-specific 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 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 they 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 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.

  This LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay – MiSeq represents a significant improvement over existing clonality assays using fragment analysis, as it efficiently detects the majority of IGH gene rearrangements using a single multiplex master mix, and identifies the DNA sequence specific for each clonal gene rearrangement. Therefore, this product has three important and complementary uses: it aids in the detection of initial clonal populations, identifies sequence information required to track those clones in subsequent samples, and provides detailed sequence information required to calculate the degree of SHM.

  Our single multiplex master mix targets the Leader (VHL) and the joining (J) gene regions of IGH. Primers included in the master mixes are designed with Illumina adapters and up to 24 different indices. This method allows 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 and a somatic hypermutation positive control are included in the kit.

  Note: For a more thorough explanation of the locus and the targeted deep sequencing strategy, please refer to Principle of Immunoglobulin and T Cell Receptor Gene Rearrangement.⁴

• 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 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. Different populations of the B and T lymphocytes arise as a result of these genetic rearrangements.

  The antigen receptor genes that undergo rearrangements are the immunoglobulin heavy chain (IGH) and light chain loci (IGK and IGL) in B-cells, and the T-cell receptor gene loci (TRA, TRB, TRG and TRD) in T-cells. Each B- and T-cell has one or two productive V – J rearrangements that are 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 that are 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 IGH 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 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.

  Multiplexing Amplicons
  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 acts 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 – MiSeq kits 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. When 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 need to 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 (MiSeq v3) can generate 20-25 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.

  IGHV Somatic Hypermutation (SHM) Evaluation
  When analyzing the somatic hypermutation status of samples, the bioinformatics software will provide the mutation rate based upon the percent mismatch of the clonal amplicons as compared to germline reference genes, a prediction of whether the protein translation would be in or out of frame, a prediction of whether mutations or gene rearrangements result in a pre-mature stop codon, and the percentage of V_H gene coverage for the region targeted by the assay.

• Specimen Requirements
  • The minimum input quantity is 50 ng of high-quality DNA.
  • This assay tests extracted and purified genomic DNA. DNA must be quantified with a method specific for double-stranded DNA (dsDNA) and be free of PCR inhibitors.
  • 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.