Test Name IGH FR1 MRD clonality assay IGH FR2 MRD clonality assay IGH FR3 MRD clonality assay IGHV Leader MRD Somatic Hypermutation Clonality Assay Assay Type Next-Generation Sequencing (NGS) This test is performed by using the LymphoTrack® Assay from Invivoscribe. Data is analyzed using the LymphoTrack MRD Data Analysis Tool (RUO). Method Description To track and identify previously detected IGH clonal sequences in post treatment follow up samples, a multiplex master mix targeting the conserved framework region 1, framework region 2, or framework region 3, and the joining region is used for PCR amplification. Next-generation sequencing of the PCR products is used to identify DNA sequences specific to previously identified clonal rearrangements detected at diagnosis. Bioinformatics tools facilitate the detection of these specific sequences present at MRD levels up to 10-6 . The assay requires a sample taken at diagnosis as well as the post treatment follow up samples. If the patient has previously been tested by LabPMM for IGH clonality, no diagnosis sample is needed. Indications for Testing •  Identify tumor-specific markers for post-treatment monitoring • Monitor and evaluate disease recurrence Interpretation Turn-around Time Specimen Requirements Shipping Conditions Storage Conditions An interpretive report will be issued indicating whether IGH MRD was detected 5 to 14 business days •  3 mL of peripheral blood in EDTA •  1 mL of bone marrow in EDTA •  700-3000 ng of previously isolated DNA depending on level of sensitivity required  Ambient or Cool;  Do not freeze • Room Temp up to 72 hours •  2-8 °C up to 7 days MRD Tests LabPMM Services Catalog 2019 | 47 Clinical Information Combinations of chemotherapy, radiation therapy and bone marrow transplantation are potentially curative for several hematologic malignancies. However, in some patients, occult tumor cells exist and are thought to increase the patient’s risk of relapse1 . These subclinical levels of residual leukemia are termed minimal residual disease (MRD) and can be evaluated using more sensitive assays. The tracking of antigen-receptor gene rearrangements for clonality analyses and MRD monitoring can be applied to virtually all patients. During early B-cell development, the germline variable (VH), diverse (DH), and joining (JH) fragments of the human immunoglobulin heavy chain (IGH) locus become rearranged through the random deletion or insertion of nucleotides within the junctional region, generating specific and unique sequences within each lymphocyte. As a result, cancer cells that arise from alterations in single lymphoid precursors acquire clonal IGH junctional regions, which can be used as tumor-specific markers2-3 . MRD detection by Next-Generation Sequencing has demonstrated utility in predicting clinical outcomes and in generating clinically actionable results – allowing early intervention, confirmation of disease status prior to transplant, and increased confidence in remission status. IGH MRD Clonality Assays References 1.  Rezuke WN et al. (1997) Molecular diagnosis of B- and T-cell lymphomas: fundamental principles and clinical applications. Clin Chem 43:1814-23. 2.  Gazzola A et al. (2014) The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies. Ther Adv Hematol. 5:35-47. 3.  González D et al. (2007) Immunoglobulin gene rearrangements and the pathogenesis of multiple myeloma. Blood 110:3112-21. 46