114 Gel & Capillary | RUO Assays Invivoscribe 2019 | 115 Gel & Capillary RUO Assays BCL2/JH Translocation Assay Assay Use The BCL2/JH Translocation Assay is useful for studies involving: •  Monitoring and evaluation of follicular lymphomas and other B-cell lymphomas • Distinguishing lymphoma from benign lymphoid hyperplasia •  Distinguishing follicular lymphoma from other B-cell lymphomas that may have a similar appearance • Monitoring and evaluation of disease recurrence • Detection and assessment of residual disease • Evaluation of new research and methods in malignancy studies Summary and Explanation of the Test Four master mixes are included in this assay. Three are used to identify translocations in the major breakpoint region (MBR) and minor cluster region (mcr) of BCL2. The Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result. This assay includes negative control DNA and positive control DNAs for both the MBR and mcr. PCR products can be analyzed using standard gel electrophoresis with ethidium bromide staining. A BCL2 translocation is indicated if any one of the master mixes generates product(s) within the valid size range. Background The BCL2 t(14;18)(q32;q21) translocation is found in 80-90% of follicular lymphomas and 30% of diffuse large cell lymphomas. The translocation is rarely present in other lymphoproliferative diseases. The t(14;18) brings about juxtaposition of BCL2 with the Ig heavy chain joining segment. This leads to a marked increase in expression of BCL2 driven by the Ig heavy chain gene enhancer. The BCL2 protein inhibits programmed cell death (apoptosis) leading to cell accumulation. The majority of breakpoints on 18q21-22 occur within the major breakpoint region (MBR) in the 3’ untranslated region of exon 3 (60- 70% of the cases), and the minor cluster (mcr) region located 3’ to BCL2 exon 3 (20-25% of the cases). Some breakpoints occur at distant loci and will not be identified by this particular test. Therefore, a negative result does not completely exclude the presence of a BCL2/IGH gene rearrangement in the sample1 . In comparison, Southern blot analysis requires 1-2 weeks, is significantly less sensitive, and has more restrictive Specimen requirements. The performance characteristics of this assay have been independently determined by the EuroClonality/BIOMED-2 Group. This consortium of 47 molecular diagnostic laboratories tested hundreds of clinical samples, taking into account their lymphoproliferative status as defined by the WHO classifications2 . Specimen Requirements 1.  5 mL of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; or, 2. Minimum 5 mm cube of tissue; or, 3. 2 μg of genomic DNA; or, 4. Formalin-fixed, paraffin-embedded (FFPE) tissue or slides. Reference 1. PAS Evans et al., Leukemia 17: 2298-2301 (2003). 2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-309-0020 BCL2/JH Translocation Assay - Gel Detection 33 reactions 1-309-0040 BCL2/JH Translocation Assay MegaKit - Gel Detection 330 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were run on a 2% agarose/TBE gel. Lane 1 is data generated testing an alternative 100% clonal control DNA; lane 2 is data generated testing the recommended 100% clonal control DNA; lane 3 is data generated testing a 1% dilution of the recommended clonal control DNA; and, lane 4 is data generated testing IVS-0000 Polyclonal Control DNA (Cat# 4-092-0010). The IVS-0000 control is used as a negative control for many of our tests. A standard 100 base pair DNA size ladder was run in the lanes flanking the test samples. Figure Legend: Schematic diagram of the IGH-BCL2 t(14;18) translocation showing the BCL2 gene on the left and the Ig heavy chain (IGH) gene on the right. Shown are the relative positions and orientations for the major breakpoint region (MBR) primers, the minor cluster region (mcr) primers, and the JH primer, which are included in the 3 BCL2/JH master mix tubes. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0030 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-P002 Clonal Control DNA 1600 pg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0031 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0000 Polyclonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in Assay MegaKit BCL2/JH Tube A BCL2 MBR + IGH JH 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH Tube B BCL2 3’ MBR + IGH JH 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH Tube C BCL2 mcr + IGH JH 1 x 1500 μL tube 10 x 1500 μL tubes Specimen Control Size Ladder Multiple Genes 1 x 1500 μL tube 10 x 1500 μL tubes This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.