Molecular Analysis of Genetic Markers for Non‐Hodgkin Lymphomas

Lynette M. Sholl1, Janina Longtine2, Frank C. Kuo1

1 Brigham and Women's Hospital, Boston, Massachusetts, 2 Yale New Haven Hospital, New Haven, Connecticut
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 10.14
DOI:  10.1002/cphg.37
Online Posting Date:  April, 2017
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Abstract

Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen‐receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to therapy. The polymerase chain reaction (PCR) is typically used to detect antigen‐receptor gene rearrangements as well as specific translocations that can be supplemented by fluorescence in situ hybridization (FISH) and karyotype analysis. © 2017 by John Wiley & Sons, Inc.

Keywords: antigen receptor gene rearrangements; lymphoma; translocations

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Detection of Clonal Immunoglobulin Heavy Chain Gene Rearrangements by PCR (BIOMED‐2 Protocol)
  • Support Protocol 1: DNA Isolation from Paraffin‐Embedded Samples, Fixed Cell Pellets, Small Frozen Biopsies, and Bodily Fluids
  • Alternate Protocol 1: Detection of Clonal T Cell Receptor‐γ Gene Rearrangements by PCR (BIOMED‐2 Protocol)
  • Basic Protocol 2: Detection of Chromosomal Translocations in DNA Samples by PCR—t(14;18) for Follicular Lymphoma
  • Alternate Protocol 2: Detection of Chromosomal Translocations in DNA Samples by PCR—t(11;14) for Mantle Cell Lymphoma
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Clonal Immunoglobulin Heavy Chain Gene Rearrangements by PCR (BIOMED‐2 Protocol)

  Materials
  • BIOMED IGH Gene Clonality Assay for gel detection (InVivoScribe, cat. no. 1‐101‐0020) or BIOMED IGH Gene Clonality Assay for ABI fluorescence detection (InVivoScribe, cat. no. 1‐101‐0021)
  • AmpliTaq Gold (Applied Biosystems)
  • 100 ng sample DNA (see protocol 2Support Protocol)
  • DEPC‐treated water
  • 6% non‐denaturing polyacrylamide gel prepared in TBE [unit 2.7 (Jarcho, ) & (Warren et al., )]
  • TBE running buffer ( appendix 2D)
  • Bromophenol blue, ice cold
  • Ethidium bromide or HI DI formamide (Applied Biosystems)
  • Genescan 400HD ROX size standard (Applied Biosystems)
  • 0.2‐ml PCR tubes
  • 1.5‐ml microcentrifuge tubes
  • Thermal cycler
  • 94°C heating block
  • Polyacrylamide gel electrophoresis equipment or ABI Genetic Analyzer
  • UV light box
  • 96‐well plates
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7; Jarcho, ), nondenaturing polyacrylamide gel electrophoresis [ appendix 3F (Albright & Slatko, ); unit 7.4 (Warren et al., )], and capillary electrophoresis (unit 7.1; Larsen, Christiansen, Vuust, & Anderson, )

Support Protocol 1: DNA Isolation from Paraffin‐Embedded Samples, Fixed Cell Pellets, Small Frozen Biopsies, and Bodily Fluids

  Materials
  • Tissue samples or bodily fluids
  • Xylene
  • 100% ethanol
  • QIAamp DNA mini kit (QIAGEN, cat. no. 51304) containing:
    • QIAamp mini spin columns
    • 2‐ml collection tubes
    • Buffer AL
    • Buffer ATL
    • Buffer AW1
    • Buffer AW2
    • Buffer AE
    • Protease and solvent
    • Proteinase K
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 15‐ml tubes
  • Disposable weigh boats
  • Scalpel
  • Transfer pipets
  • Vortexer
  • 62°C water bath
  • 70°C dry bath incubator
  • Spectrophotometer
  • Additional reagents and equipment for isolation of genomic DNA ( appendix 3B; Gilbert & Vance, )

Alternate Protocol 1: Detection of Clonal T Cell Receptor‐γ Gene Rearrangements by PCR (BIOMED‐2 Protocol)

  Additional Materials (also see protocol 1)
  • BIOMED TCRG Gene Clonality assay for gel detection (IVS, cat. no. 1‐207‐0020) or BIOMED TCRG Gene Clonality assay for ABI fluorescence detection (IVS, cat. no. 1‐207‐0021)
  • AmpliTaq Gold (Applied Biosystems)
  • DNA sample (see protocol 2Support Protocol)
  • DEPC‐treated water ( appendix 2D)
  • 0.2‐ml PCR tubes
  • Thermal cycler (e.g., ABI 2700)
NOTE: Steps 1 through 3 should be performed in a PCR hood to reduce the risk of contamination.

Basic Protocol 2: Detection of Chromosomal Translocations in DNA Samples by PCR—t(14;18) for Follicular Lymphoma

  Materials
  • 10 × PCR amplification buffer (see recipe)
  • 25 mM MgCl 2
  • 10 mM deoxynucleotide triphosphate solution (dNTP)
  • DEPC‐treated water
  • 10 μM primers (primer sequences detailed in Fig. ):
    • primer 1‐MBRα (forward)
    • primer 2‐MBR3′ (reverse)
    • primer 3‐JHα (reverse)
    • primer 4‐mcrα (forward)
    • primer 5‐MBRβ (reverse)
    • primer 6‐JHβ (reverse)
    • primer 7‐mcrβ (forward)
  • 5 U/μl Taq polymerase
  • DNA sample (see protocol 2Support Protocol)
  • MBR positive control DNA (InVivoScribe) diluted into normal placenta DNA at 1:100 (0.1 μg/μl)
  • Mcr positive control DNA (InVivoScribe) diluted into normal placenta DNA at 1:100 (0.1 μg/μl)
  • 100‐bp molecular weight marker
  • 1.5‐ml microcentrifuge tubes
  • 0.5‐ml PCR tubes
  • Thermal cycler
  • Vortexer
  • Microcentrifuge
  • Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7; Jarcho, )

Alternate Protocol 2: Detection of Chromosomal Translocations in DNA Samples by PCR—t(11;14) for Mantle Cell Lymphoma

  Additional Materials (also see protocol 4)
  • Primers:
  • BCL1/MTC primer: 5′‐GGA TAA AGG CGA GGA GCA TAA‐3′
  • JH consensus primer: 5′‐CTT ACC TGA GGA GAC GGT GAC C‐3′
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Figures

Videos

Literature Cited

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Key References
  Bagg, (2008). See above.
  Comprehensive review of the biology of antigen receptor gene rearrangement, clinical indications, and common pitfalls in clonality assessment.
  van Dongen et al., (2003). See above.
  Overview of the rationale, design, and validation of the BIOMED‐2 PCR assays for antigen receptor gene rearrangement and t(14;18) and t(11;14) detection.
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