Proc. Natl. Acad. Sci. USA Vol. 81, pp. 593-597, January 1984 Medical Sciences Immunoglobulin gene rearrangement as a diagnostic criterion of B-cell lymphoma (Southern blot hybridization technique/cancer/DNA probe/clonal analysis/immunogenotyping) MICHAEL L. CLEARY, JAMES CHAO, ROGER WARNKE, AND JEFFREY SKLAR Department of Pathology, Stanford University, Stanford, CA 94305 Communicated by Paul Berg, September 26, 1983 ABSTRACT We describe the use of the Southern blot hy- large numbers of cells within a histologic section or cell bridization technique to diagnose B-cell lymphoma by detect- suspension bear the same antigenic markers if the prolifera- ing clonal immunoglobulin gene rearrangements in lymph tion is neoplastic. For instance, homogeneous neoplastic node and other biopsy tissues. DNA was isolated from a wide proliferation of B lymphocytes that synthesize detectable variety of neoplastic and non-neoplastic specimens and ana- immunoglobulin will show only a single immunoglobulin lyzed for the presence of rearranged immunoglobulin genes light chain, K or X, when analyzed for these two polypep- using radiolabeled DNA probes specific for the heavy- and tides. Although this immunophenotyping technique is rapid light-chain immunoglobulin constant region genes. Among the and has proved to be helpful in evaluating certain biopsy specimens examined, clonal immunoglobulin gene rearrange- specimens, it suffers from several disadvantages. One fre- ments were found only in biopsy samples of B-cell lymphoma quent problem is that malignant B-cell proliferation within and not in samples containing reactive lymphoid processes or lymph nodes is often intermixed with various amounts of non-B-cell cancers. In lymphomas, the presence of rearrange- normal B cells, in which case this technique may depend on ments for either the K or A light-chain gene correlated with detecting small deviations from the 2:1 ratio of K- to A-bear- expression of one or the other of these chains when cellular ing B cells found in normal human lymphoid tissue. Other immunoglobulins could be detected by frozen-section immuno- problems include artifacts associated with suboptimal han- phenotyping techniques. The analysis of immunoglobulin gene dling or fixation of tissues, the requirement for good anti- rearrangements offers several advantages over conventional body reagents directed against antigenic markers, and the diagnostic methods for lymphomas, including improved sensi- absence of markers in certain lymphoid tumors. tivity in detecting minor populations of neoplastic lymphocytes Recently we have explored an alternative approach to the composing as little as 1% of the total cell population. In addi- diagnosis of B-cell lymphoma. This approach relies on de- tion, clonal immunoglobulin gene rearrangements are demon- tecting uniform rearrangements of immunoglobulin genes strable in a subset of lymphomas that lack detectable surface within clonal populations of B lymphocytes, as detected pre- or cytoplasmic immunoglobulin, thus offering positive evi- viously in human B-cell leukemias (3-5). Our work is based dence for both malignancy and the B-cell origin of these tu- on the fact that B lymphocytes must undergo a series of mors. Our studies indicate that detection of immunoglobulin DNA rearrangements prior to immunoglobulin production gene rearrangements is a valuable method for diagnosis and (6, 7). In germ-line cells the variable and constant domains of classification of various lymphoproliferative disorders that are each type of immunoglobulin chain (one heavy chain and difficult to evaluate histologically or that lack distinctive anti- two light chains, K and X) are encoded in separate discontinu- genic markers. ous regions of specific chromosomes. During B-lymphocyte maturation, an initial event in immunoglobulin synthesis is Diagnosis of malignant lymphoma depends on histologic the somatic recombination of the separated variable and con- evaluation of tissue biopsies. However, distinguishing be- stant gene segments. This results in the removal of interven- tween malignant and benign disorders in lymph nodes and ing DNA and the close apposition of specific variable and other lymphoid tissues by light microscopy remains one of constant DNA sequences to form an active immunoglobulin the pathologist's most difficult tasks. Although the majority gene (see Fig. 1A). The high degree of variability with which of such biopsies are unambiguous, a significant minority immunoglobulin gene segments are rearranged and the fact pose serious problems for even the most expert histopathol- that each individual B cell is capable of expressing only a ogist. In large part, the biological cause for this difficulty is single antibody idiotype make the configuration of rear- that antigenically stimulated lymphocytes may morphologi- ranged immunoglobulin gene segments an entirely specific cally resemble neoplastic lymphocytes. Conversely, so- marker for a given B cell and for any clone that may arise called well-differentiated neoplastic lymphocytes may be cy- from that B cell. tologically indistinguishable from normal unstimulated lym- In this report, we show that detection of immunoglobulin phocytes. There are also cases in which reactive conditions gene rearrangements in biopsy tissue by the Southern blot coexist with and obscure malignancy, further complicating hybridization procedure affords an accurate and highly sen- histologic interpretation. Occasionally, poorly differentiated sitive means of identifying clonal lymphoid proliferations in metastatic carcinoma or melanoma may be mistaken for lym- a wide variety of B-cell malignancies. In addition, benign re- phoma. active processes and non-B-cell malignancies are distin- An important method devised to deal with these problems guished by the absence of detectable immunoglobulin gene is the analysis of immunologic markers on the surface or in rearrangements. This technique, therefore, provides a valu- the cytoplasm of lymphoid cells in tissue sections or in cell able adjunct to currently available methods for diagnosing B- suspensions of lymph node biopsies (1, 2). This method cell lymphoma. Furthermore, this technique avoids many of takes advantage of the clonal nature of malignancy, such that the problems associated with immunologic marker studies and conventional morphologic diagnosis. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: kb, kilobase(s); C and J, constant andjoining regions in accordance with 18 U.S.C. §1734 solely to indicate this fact. of immunoglobulin chains. 593 Downloaded by guest on September 27, 2021 594 Medical Sciences: Cleary et al. Proc. NatL Acad Sci. USA 81 (1984) B A Probe CK JH Cli Germline 123456 1234 Germline Mu c Gene Gene Allele $ lkb h~ f 4 4 12345 t CDi Probe JK CK t VK1 VK 2VK3 VIK4VK5VK6 JK CK t 12345 Germline Kappa Gene I I DNA REARRANGEMENT 1 kb C K Probe 3 CAK-eOF Rearranged f f f Germline CAMcg CXKOe0 Lymphoma 9 VA l-. k1U' F.i Lambda Allelev v I Gene K 1 K2 VK1C3VK4JKPo43 CK b 7 2k 2 kb C X Probe i BanH1 Cal Probe # EcoRI FIG. 1. (A) Hypothetical gene rearrangement for the light-chain K locus. During B-cell maturation prior to immunoglobulin production, one of the multiple K variable-region genes (V44) undergoes somatic recombination with one of several separate J segments (1K3) that lie directly upstream from a single K constant-region gene (CK). DNA rearrangement leaves the downstream BamHI restriction site (downward arrow to the right of CK) unaltered while the BamHI site upstream and nearest to the C gene is changed in the rearranged lymphoma allele. This allows distinction between germ-line and rearranged K genes, because the C,, probe will detect different-sized BamHI DNA fragments (bracketed lines at top and bottom) by the Southern blot hybridization procedure. [Figure based on Cossman et al. (18).] (B) Chromosomal map of the germ-line J-region configuration for the heavy-chain, K, and X C genes. The probes used in this work are indicated by hatched boxes. The heavy-chain probe consisted of a 6.5-kilobase (kb) BamHI/HindIII DNA fragment. A probe specific for the C,. gene consisted of a 1.4-kb EcoRI fragment, which includes the first, second, and part of the third exons of the human C,. gene. The J probe is preferable to the C,. probe for showing heavy- chain rearrangements because the C,. gene segment may be deleted during heavy-chain class switching. The light-chain CK probe contained a 2.5-kb EcoRI fragment spanning the entire human C,. gene. The CA locus consists of a family of at least six closely linked related genes-e.g., Mcg, Ke7Oz-, and Ke-Oz+ (15). A combined C, probe was used consisting of a 3.5-kb EcoRI/HindllI fragment containing the Ke-Oz-C>, gene and a 2.5-kb EcoRI/HindIII fragment containing the Mcg CA gene. METHODS All lymphoma specimens were categorized histopathologi- as (20). Analysis of immunologic surface and Biopsy tissues were routinely collected from the operating cally described markers was carried out in frozen sections as room, frozen in airtight plastic capsules by immersion in a cytoplasmic dry ice/isopentane bath, and stored at -70'C for up to five described (8). years (8). Normal control tissues for two cases studied con- RESULTS sisted of peripheral blood granulocytes and autopsy liver tis- Immunoglobulin Gene Rearrangements Are Present in sue. but Not in Nonlymphoid Control Tissue from the and other Lymphomas DNA was extracted from lymph node biopsies Same Patient. Fig. 2A shows the data obtained from a lymph tissues and purified according to standard procedures (9). node biopsy diagnosed histologically as a diffuse large cell The starting material represented 10-25 mg (wet weight) of lymphoma. The heavy-chain J probe detected two bands in tissue. the lymphoma DNA, one of which comigrated with the sin- After purification, high molecular weight DNA was digest- ed with appropriate restriction enzymes according to condi- the supplier (Bethesda Research tions recommended by J K A Laboratories).