American Journal of Pathology, Vol. 142, No. 6, June 1993 Copyright © American Society for Investigative Pathology Detection of Clonal Immunoglobulin Gene Rearrangements by Polymerase Chain Reaction Amplification and Single-Strand Conformational Polymorphism Analysis Thomas H. Davis, Courtland E. Yockey, and Steven P. Balk From the Department of Medicine, Division of Hematology-Oncology, Beth Israel Hospital, Boston, Massachusetts Analysis ofimmunoglobulin gene rearrangements by Southern blotting is a sensitive and specific method for detecting B ceUl malignancies but re- quires a relatively large amount of intact DNA. It cannot be utilized in many cases where only a smaU amount of tissue is available or where the tissue has been fixed. this report demonstrates that polymerase chain reaction (PCR) amplifica- tion in conjunction with single-strand conforma- tional polymorphism (SSCP) analysis can be uti- lized to detect clonal immunoglobulin heavy chain (IgH) gene rearrangements. IgHgene rearrange- mentsfrom a series offrozen orformalin-flxed B ceUl malignancies were PCR-amplifted using oli- gonucleotide primers, based upon consensus se- quences in the IgH variable and joining regions. Analysis of the single-stranded PCR products on nondenaturing polyacrylamide gels revealed dis- crete SSCPs corresponding to the malignant B ceUs. These SSCPs were detectable when the ma- lignant cells represented asfew as 0.2% of the to- tal mononuclear ceUs in peripheral blood. PCR amplification in conjunction with SSCP analysis thus provides a sensitive and specific method to detect clonal IgH rearrangements from minute amounts offresh, frozen, orfixed tissue. (Am J Pathol 1993, 142:1841-184 7) The rearrangement of immunoglobulin or T-cell anti- gen receptor genes during a lymphocyte's differen- tiation produces a region of DNA sequence unique to that cell and its progeny.1 This clone-specific se- quence (N region) is created by random incorpora- tion of nucleotides between imprecisely spliced vari- able (V), diversity (D), and joining (J) gene segments. In the majority of non-Hodgkin's lymphomas and lym- phoid leukemias, Southern blot hybridization analysis can detect nongermline bands representing the T cell antigen receptor, immunoglobulin heavy chain (IgH), or light chain gene rearrangements of the malignant clone.2- The presence of these nongermline bands demonstrates that the process is clonal, and the pat- tern can serve as a fingerprint for a particular lym- phocyte clone. Though powerful, this technique has several dis- advantages. First, a clonal population can be de- tected only if it represents at least 1% of the total cells.4 Second, the tissue must be fresh or frozen, because DNA extracted from paraffin-embedded tis- sue is generally too degraded for Southern blot anal- ysis. Third, a relatively large amount of DNA is re- quired, often precluding analysis of minute specimens for which morphological diagnosis can be difficult and confirmation of clonality would be most useful. We have circumvented these shortcomings by combining polymerase chain reaction (PCR) ampli- fication of IgH gene rearrangements with single- strand conformational polymorphism (SSCP) analy- SiS.7 PCR amplification of IgH rearrangements has been reported previously,813 but a sensitive method to separate amplified clonal IgH rearrangements from the polyclonal rearrangements of admixed normal B cells has not been readily available. SSCP analysis, which employs electrophoresis of single-stranded DNA on nondenaturing polyacrylamide gels, sepa- rates PCR products based upon differences in con- Supported by grants from the NIH Hematology Career Training Program (HL 07516), the Council for Tobacco Research, and an American Cancer Society Junior Faculty Research Award to SPB. Accepted for publication December 11, 1992. Address reprint requests to Dr. Steven Balk, Division of Hematology-Oncology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215. 1841