Interpretation Turn- around Time Specimen Requirements Shipping Conditions Storage Conditions  An interpretive report will be issued indicating the level of IGH SHM along with the rearrangement class for the dominant clones and the specific sequence for the dominant clone. 5 to 10 business days •  3 mL Peripheral Blood in EDTA, ACD or Heparin •  1 mL bone marrow in EDTA, ACD or Heparin •  500 ng of previously isolated DNA • FFPE tissue samples Ambient or Cool; Do not freeze • Room Temp up to 72 hours • 2-8 °C up to 7 days Test Name IGH somatic hypermutation assay IGHV leader assay Assay Type Next-Generation Sequencing (NGS) This test is performed by using the LymphoTrack® Assay from Invivoscribe. Method Description For detection of the vast majority of clonal IGH VH-JH rearrangements, including the associated VH-JH region DNA sequences, a multiplex master mix targeting the conserved framework region 1 (FR1) or leader and the joining region is used for PCR amplification. Next-generation sequencing of the PCR products is used to identify the frequency distribution of VH region and JH region segment utilization, as well as for the definition of the extent of somatic hypermutation present in the IGH gene. Bioinformatics tools facilitate the characterization of sequences present at greater than 2.5% or 5% of the population and the level of somatic hypermutation present in the dominant clone. Bioinformatics also identify clonal rearrangements that involve the V3-21 gene, which has been associated with a poor prognosis in CLL, independent of SHM status. Indications for Testing •  Identify clonality in atypical lymphoproliferative disorders •  Support a differential diagnosis between reactive lesions and hematologic malignancies •  Assign presumptive lineage in mature monoclonal lymphoproliferative disorders • Monitor and evaluate disease recurrence Clonaltiy Tests LabPMM Services Catalog 2019 | 33 Clinical Information Lymphoid cells are different from the other somatic cells in the body as during development, the antigen receptor genes in these cells undergo somatic gene rearrangement1 . During B-cell development, genes encoding the human immunoglobulin heavy chain (IGH) proteins 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 for each cell2-3 . An additional level of diversity is generated by point mutations in the variable regions, also known as somatic hypermutations (SHM). Leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, which means that 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-cell malignancies. Immunoglobulin variable heavy chain gene hypermutation status provides important prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). The presence of IGH 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 SHM. The status of SHM for clone(s) has clinical relevance, as there is a clear distinction in the median survival of patients with and without SHM. Hypermutation of the IGH variable region is strongly predictive of a good prognosis, while lack of mutation predicts a poor prognosis4 . In addition, this assay identifies clonal rearrangements involving the V3-21 gene, which has been associated with a poor prognosis in CLL independent of SHM status. This assay has been shown to further stratify CLL patients5 . IGH Somatic Hypermutation References: 1. Tonegawa S (1983). Somatic Generation of Antibody Diversity. Nature 302:575-581. 2.  Trainor KJ et al. (1990). Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 75:2220-222 2. 3. JE Miller et al., Molecular Genetic Pathology (2013, 2nd ed.) Springer Science & Business Media 302.2.7.13 and 30.2.7.18. 4.  P. Ghia, et al. (2007). ERIC recommendations on IGHV gene mutational status in chronic lymphocytic leukemia. Leukemia 21:1-3. 5.  Stamatopoulos, B et al. (2017). Targeted deep sequencing reveals clinically relevant subclonal IgHV rearrangements in chronic lymphocytic leukemia. Leukemia 31(4):837-845. 32