18 Next-Generation Sequencing (NGS) | CE-IVD Assays Invivoscribe 2019 | 19 NGS CE-IVD Assays LymphoTrackDx IGH FR1/2/3 Assays Assay Description LymphoTrack Dx IGH FR1 Assays The LymphoTrack Dx IGH FR1 Assays for the Illumina® MiSeq® , or Thermo Fisher Scientific® Ion PGM™ and Ion S5™ are in vitro diagnostic products intended for next-generation sequencing (NGS) based determination of the frequency distribution of Vh-JH gene rearrangements as well as the degree of somatic hypermutation (SHM) within Framework Region 1 (FR1) of the IGH locus, of rearranged genes in patients suspected of having lymphoproliferative disease. These assays help identify lymphoproliferative disorders, and they offer an aid in the identification of disease prognosis. LymphoTrack Dx IGH FR2 Assays The LymphoTrack Dx IGH FR2 Assays are in vitro diagnostic products intended for NGS analysis for the Illumina® MiSeq® , or Thermo Fisher Scientific Ion PGM™ and Ion S5™ instruments. The assays will determine the frequency distribution of VH-JH gene rearrangements within Framework Region 2 (FR2) of the IGH locus in patients suspected of having lymphoproliferative disease. These assays aid in the identification of lymphoproliferative disorders. LymphoTrack Dx IGH FR3 Assays The LymphoTrack Dx IGH FR3 Assays are in vitro diagnostic products intended for NGS analysis for the Illumina® MiSeq® , or Thermo Fisher Scientific Ion PGM™ and Ion S5™ instruments. The assays will determine the frequency distribution of VH-JH gene rearrangements within Framework Region 3 (FR3) of the IGH locus in patients suspected of having lymphoproliferative disease. These assays aid in the identification of lymphoproliferative disorders. Summary and Explanation of the Test The LymphoTrack Dx IGH Assays represent a significant improvement over conventional clonality assessment methods utilizing fragment analysis by providing three important and complementary uses in a single workflow: 1. Detection of clonal populations. 2.  Identification of sequence information required to track clonal rearrangements in subsequent samples. 3.  The LymphoTrack Dx IGH framework 1 FR1 master mixes provide the degree of SHM in the immunoglobulin variable heavy chain (IGHV) gene locus. The ability to track sequences in subsequent samples with the Invivoscribe LymphoTrack MRD* Software. For more information, please refer to page 48 and 49. These assays utilize a single multiplex master mix to target each conserved IGH Framework Region (FR1, FR2, and FR3) within the VH and the JH regions described in lymphoid malignancies. The included primers are designed with Illumina® or Thermo Fisher Scientific adapters and indices (8-24 and 12, respectively). This allows up to 24 samples on MiSeq® and 12 samples on Ion PGM™ and Ion S5™ to be sequenced at the same time with any of the individual FRs. In addition, amplicons generated with different FR master mixes or Invivoscribe LymphoTrack Dx kits (such as IGK or TRG) can be pooled together in the same sequencing library to reduce testing costs. The associated LymphoTrack Dx Software provides interpretation of the data via a simple and streamlined method of analysis and visualization. By following the guidelines provided in the instructions for use, samples can be interpreted for evidence of clonality and SHM rates. Positive clonal (SHM negative) and negative polyclonal DNA controls are included in kits. A clonal SHM positive control can be purchased separately (cat#: 4-088-0008). Background The human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 non- functional variable (VH), 27 functional diversity (DH), and 6 functional joining (JH) gene segments. The VH gene segments can be further broken down into three conserved frameworks (FR) and three variable complementarity-determining regions (CDRs). During development of lymphoid cells, antigen receptor genes undergo somatic gene rearrangements. Specifically during B-cell development, IGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements generating VH-DH-JH combinations 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. In addition, the IGHV hypermutation status obtained with the LymphoTrack Dx IGH FR1 master mixes, provides important prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). The SHM rate has been shown to have clinical relevance for B-CLL per 280389, as there is a clear distinction in the median survival of patients with and without SHM. Specimen Requirement 50 ng of high-quality genomic DNA. References 1. S Tonegawa. Nature 302: 575-581 (1983). 2.  JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 30.2.7.13 and 30.2.7.18. 3. KJ Trainor et al., Blood 75: 2220-2222 (1990). 4. P Ghia. Leukemia 21: 1-3(2007). 5. Ofran et al., J Immunol 2008; 181:6230-6235 The LymphoTrack Dx IGH FR1/2/3 Assays contain components from respective individual FR kit A's or panels. LymphoTrack Dx IGH FR1 Components LymphoTrack Dx IGH FR2 Components LymphoTrack Dx IGH FR3 Components Master Mix Name Index # Master Mix Name Index # Master Mix Name Index # IGH FR1 MiSeq 01 A001 IGH FR2 MiSeq 01 A001 IGH FR3 MiSeq 01 A001 IGH FR1 MiSeq 02 A002 IGH FR2 MiSeq 02 A002 IGH FR3 MiSeq 02 A002 IGH FR1 MiSeq 03 A003 IGH FR2 MiSeq 03 A003 IGH FR3 MiSeq 03 A003 IGH FR1 MiSeq 04 A004 IGH FR2 MiSeq 04 A004 IGH FR3 MiSeq 04 A004 IGH FR1 MiSeq 05 A005 IGH FR2 MiSeq 05 A005 IGH FR3 MiSeq 05 A005 IGH FR1 MiSeq 06 A006 IGH FR2 MiSeq 06 A006 IGH FR3 MiSeq 06 A006 IGH FR1 MiSeq 07 A007 IGH FR2 MiSeq 07 A007 IGH FR3 MiSeq 07 A007 IGH FR1 MiSeq 08 A008 IGH FR2 MiSeq 08 A008 IGH FR3 MiSeq 08 A008 IGH FR1 MiSeq 09 A009 IGH FR2 MiSeq 09 A009 IGH FR3 MiSeq 09 A009 IGH FR1 MiSeq 10 A010 IGH FR2 MiSeq 10 A010 IGH FR3 MiSeq 10 A010 IGH FR1 MiSeq 11 A011 IGH FR2 MiSeq 11 A011 IGH FR3 MiSeq 11 A011 IGH FR1 MiSeq 12 A012 IGH FR2 MiSeq 12 A012 IGH FR3 MiSeq 12 A012 IGH FR1 MiSeq 13 A013 IGH FR2 MiSeq 13 A013 IGH FR3 MiSeq 13 A013 IGH FR1 MiSeq 14 A014 IGH FR2 MiSeq 14 A014 IGH FR3 MiSeq 14 A014 IGH FR1 MiSeq 15 A015 IGH FR2 MiSeq 15 A015 IGH FR3 MiSeq 15 A015 IGH FR1 MiSeq 16 A016 IGH FR2 MiSeq 16 A016 IGH FR3 MiSeq 16 A016 IGH FR1 MiSeq 18 A018 IGH FR2 MiSeq 18 A018 IGH FR3 MiSeq 18 A018 IGH FR1 MiSeq 19 A019 IGH FR2 MiSeq 19 A019 IGH FR3 MiSeq 19 A019 IGH FR1 MiSeq 20 A020 IGH FR2 MiSeq 20 A020 IGH FR3 MiSeq 20 A020 IGH FR1 MiSeq 21 A021 IGH FR2 MiSeq 21 A021 IGH FR3 MiSeq 21 A021 IGH FR1 MiSeq 22 A022 IGH FR2 MiSeq 22 A022 IGH FR3 MiSeq 22 A022 IGH FR1 MiSeq 23 A023 IGH FR2 MiSeq 23 A023 IGH FR3 MiSeq 23 A023 IGH FR1 MiSeq 25 A025 IGH FR2 MiSeq 25 A025 IGH FR3 MiSeq 25 A025 IGH FR1 MiSeq 27 A027 IGH FR2 MiSeq 27 A027 IGH FR3 MiSeq 27 A027 KIT A Kit A's contain indices IGH FRX A001 to A008. Panels contain all master mixes listed above. Controls in Individual FR (1, 2, or 3) Kit A's Controls in Individual FR (1, 2, or 3) Panels Controls in Combo FR 1/2/3 Kit A Controls in Combo FR 1/2/3 Panel IGH POS (+) Qty. 1 IGH POS (+) Qty 3 IGH POS (+) Qty. 2 IGH POS (+) Qty. 6 NGS NEG (-) Qty. 1 NGS NEG (-) Qty 3 NGS NEG (-) Qty. 2 NGS NEG (-) Qty. 6 VH family primers VL VH-FR1 VH-FR2 VH-FR3 JH primers JH DH Simplified Representation of the IGH Gene Simplified depiction of variable (VH) and downstream consensus joining (JH) region genes involved in gene rearrangements. KIT A KIT A Reagents - MiSeq® Detection 1. *MRD Software can be used to track sequences generated by either LymphoTrack Assays - MiSeq® or Ion S5/PGM™ MRD applications are for Research Use Only. To obtain a copy, please contact your local distributor or send an e-mail to MRD@invivoscribe.com