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 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). Digestion products were electrophoresed overnight in an 0.8% agarose gel, as described (10). After nicking of the DNA by light to decrease the aver- age chain length, DNA fragments were transferred out of the WI'a FIG. 2. Autoradiograms from agarose gels onto filters as described by _ lymphoma and control DNA hy- Southern (11). The filters were then hybridized with radiola- bridized with immunoglobulin gene beled plasmid DNA carrying immunoglobulin DNA probes. (A) Case 1. (B) Case 2. pBR322 for heavy-chain and K fragments. Hybridization reactions were carried out under Analyses 50% formamide light-chain gene rearrangements conditions described elsewhere (12), using were carried out on DNA digested at 420C. After extensive washing and drying of filters, auto- * with the BamHI restriction en- radiography was carried out at -70'C against a single inten- zyme; the X light-chain locus was sifying screen for 12-72 hr. analyzed with DNA digested with Human genomic DNA fragments specific for the K (13, 14) .J A the EcoRI . constant regions (C) and the heavy-chain joining (J) i8 r Lanes 1, control DNA; lanes 2, and (15) DNA. Dashes have and ,u regions (16) were isolated from recombinant bacte- lymphoma kindly provided by P. Leder (Harvard Medical been placed alongside germ-line riophage bands, arrows are beside rear- were subcloned into the Esche- Jo School). These fragments ranged bands. Based on marker richia coli plasmid pBR322 using standard procedures (17). DNA fragments coelectropho- The positions of the DNA probe fragments with respect to resed with DNA digests in these the immunoglobulin genes are shown in Fig. 1B. Plasmids blots but not shown in the figure, containing immunoglobulin DNA were isolated from E. coli the germ-line bands are of expect- and nick-translated in vitro with [a-32P]dNTPs as described ed size: about 19.3 kb (heavy and to a activity of 3-5 x 108 cpm/pgg. chain); 12 kb (K light chain); elsewhere (19), specific and 8 kb light chain). Radiolabeled dNTPs were obtained from Amersham. 16, 14, (X Downloaded by guest on September 27, 2021 Medical Sciences: Cleary et aL Proc. Natl. Acad. Sci. USA 81 (1984) 595

Table 1. Correlation of immunoglobulin gene rearrangements (immunogenotype) with immunoglobulin antigens (immunophenotype) in various lymphoproliferative conditions Immunogenotype Immunophenotype Case Histologic diagnosis K A / K X 1 Diffuse large cell ML R R G + + 2 Diffuse large cell ML R R G + + 3 Reactive follicular hyperplasia G G G ND ND ND 4 Large cell immunoblastic ML R R G - - - 5 Diffuse small cleaved cell ML R R G + + 6 Small noncleaved cell ML, non-Burkitt R R G + + 7 Small lymphocytic ML R R G + + 8 Unclassified ML R R G + + 9 Lymphocytic ML, intermediate differentiation R R R + - + 10 Diffuse large cell ML R R G 11 Lymphoblastic MLt G G G - - - ML, malignant lymphoma; R, rearranged immunoglobulin gene; G, germ-line; ND, not determined. *Rearrangements of the heavy-chain locus detected with heavy-chain J-specific probe were confirmed with the C, probe. In each case, the C,J probe hybridized to at least one rearranged band detected with the J probe. tT-cell differentiation determined by analysis of surface markers (Leu-1+, Leu-2a', Leu-3a+, Leu-4', la-).

gle germ-line band obtained from peripheral granulocyte (data not shown). When present, the surface or cytoplasmic DNA. The second band, which migrated in a position below light chain identified in frozen sections corresponded to the that of the germ-line band, represents clonal rearrangement light-chain class that showed gene rearrangement (Table 1). of one heavy-chain immunoglobulin allele. Separate blots Both K and X gene rearrangements were found in a single prepared from the same DNA and hybridized with a probe specimen containing only X surface immunoglobulin (case 9), for the K light-chain gene also revealed a single clonally rear- but no X gene rearrangement was found in lymphomas with K ranged band that migrated slightly ahead of the position of surface immunoglobulin. This observation conforms to the the germ-line band as well as a weaker band that comigrated proposed hierarchy of immunoglobulin gene rearrangement, with the germ-line band from granulocyte control DNA. such that rearrangements of the X gene occur only after de- Autoradiograms obtained from blots hybridized with the X fective or nonproductive rearrangements of both K gene al- light-chain probe showed identical patterns for the control leles (4, 21). and lymphoma . The results obtained in this case are Immunoglobulin Gene Rearrangements Are Detectable in consistent with a clonal rearrangement of at least one allele Lymphomas Lacking Cellular Immunoglobulin. Two of the of the heavy-chain and K-chain loci and correlate with immu- cases examined did not show staining for surface or cyto- nologic phenotyping offrozen sections of this patient's lym- plasmic immunoglobulin but contained immunoglobulin gene phoma, which revealed u and K surface immunoglobulin (Ta- rearrangements. Both cases 10 and 4 (Table 1) are diffuse ble 1). large cell lymphomas that lack distinctive markers except for A similar analysis was carried out (Fig. 2B) for a diffuse B1 (22) and Ia antigens. Despite the absence of immunoglob- large cell lymphoma that developed in a patient with Wis- ulin production in both cases, clonal heavy-chain immuno- kott-Aldrich syndrome. Nonlymphoma control DNA was globulin gene rearrangements (Fig. 3) support the B-cell lin- extracted from autopsy liver, which was grossly and micro- eage of these tumors. Moreover, light-chain K gene rear- scopically free of tumor. When the heavy-chain J probe was rangements were found in each instance, while the X genes used in the hybridization, three bands were seen, two of were in a germ-line configuration. which migrated faster than the unrearranged germ-line band present in the control DNA. This finding can be explained by rearrangement of both heavy-chain alleles in the lymphoma 3 4 5 6 7 8 9 10 11 cells. The K light-chain probe showed a single rearranged band representing a fragment larger than the germ-line frag- ment in addition to a band that comigrated with the control DNA band. Hybridization of DNA from control and lympho- ma tissue with the X light-chain probe produced identical pat- terns. As in Fig. 2A, these data are consistent with a clone of malignant cells that expressed A-K immunoglobulin, in agree- ment with surface marker analyses, as shown in Table 1. Different amounts of germ-line band were present in anal- yses of both lymphoma specimens. These bands may have resulted from either an unrearranged immunoglobulin allele within tumor cells or from contamination of the tumor tissue with normal polyclonal lymphocytes, blood cells, fibrous tis- FIG. 3. Autoradiograms obtained with DNA prepared from sue, and blood vessels. lymph nodes with various proliferative disorders and hybridized Immunoglobulin Gene Rearrangements Are Detectable in with a heavy-chain J-specific probe. Analyses for heavy-chain gene Various Histologic Subtypes of B-Cell Lymphoma. Fig. 3 rearrangements were carried out using BamHI-digested DNA ex- shows results obtained when DNA isolated from lymph node tracted from lymph node biopsies. The numbers coincide with the biopsies representing a variety of conditions was analyzed case numbers in Table 1. Dashes indicate the germ-line 19.3-kb with heavy-chain J-region DNA probes. Rearrangements band; arrows show rearranged heavy chain J-specific bands. In most were seen in all cases involving of the autoradiograms, there is a faint 12-kb band of unknown origin histologic subtypes of B-cell that hybridizes weakly with the J-specific probe under the condi- lymphoma, as summarized in Table 1. Light-chain rear- tions used. This band is also detected in blots prepared from non- rangements were also seen in all cases of B-cell lymphoma lymphoid DNA. Downloaded by guest on September 27, 2021 596 Medical Sciences: Cleary et al. Proc. Natl. Acad Sci. USA 81 (1984)

VD 0 <0) 0 (') -' diagnosis of lymphoma was made by morphologic criteria se Cv' 0 0) alone. Surface and cytoplasmic immunoglobulin was ana- lyzed in these cases as well. When present, immunoglobulin markers correlated with the pattern of immunoglobulin gene rearrangements, so that any tumor expressing either K or X light chains always showed a rearrangement in at least one allele for the corresponding gene. Normal and reactive lymph nodes, a variety of non-B-cell lymphomas, and several nonlymphoid cancers have been FIG. 4. Sensitivity of the Southern blot hybridization technique tested for immunoglobulin gene rearrangements. These have for detecting immunoglobulin gene rearrangements. Mixtures con- been consistently negative for both heavy- and light-chain taining a total of 10 jug of DNA were prepared with lymphoma DNA gene rearrangements. As shown in Fig. 3 (case 3), normal or node DNA. The DNA and various amounts of nonmalignant lymph reactive lymph nodes show only germ-line bands. There are with BamHI restriction enzyme. After electrophoresis was digested innumerable rearranged immunoglobulin genes and transfer, the samples were hybridized with a radiolabeled CK- presumably specific probe, washed, and autoradiographed for 48 hr. The per- in such tissues; however, apparently no single rearranged centage of lymphoma DNA within each mixture is indicated above DNA fragment is sufficiently abundant to be detectable by the lanes of the autoradiogram. Dash indicates the position of the the Southern blot technique. On the basis of these findings, germ-line band; arrows indicate the position of two rearranged K we conclude that detection of a rearranged immunoglobulin bands in the lymphoma DNA. band in a Southern blot analysis of lymphoid DNA is a reli- able test for clonal proliferation and, within the limits dis- Immunoglobulin Gene Rearrangements Are Not Seen in cussed above, for B-cell lymphoma. Tissue Specimens Other Than B-Cell Lymphomas. We have Our failure to detect immunoglobulin gene rearrangements examined four T-cell lymphomas-for example, a lympho- in human T-cell lymphomas contrasts with studies on small blastic lymphoma (Fig. 3, case 11). Afl showed only germ- numbers of mouse T-cell tissue culture lines, in which occa- line bands and no detectable immunoglobulin gene rear- sional heavy-chain gene rearrangements were found in the rangements. We have also examined DNA from lymph absence of light-chain rearrangements (23, 24). In 11 of 12 nodes containing a wide variety of benign and reactive con- cases of human T-cell acute lymphoblastic leukemia (ALL), ditions, including reactive follicular hyperplasia (case 3), an- Korsmeyer et al. (3) found germ-line configurations of gioimmunoblastic lymphadenopathy, rheumatoid arthritis, immunoglobulin genes. A single T ALL-derived cell line in and sarcoidosis. Other non-B-cell neoplastic processes were this series contained a nonproductive heavy-chain gene rear- examined; these included acute myelocytic leukemia, acute rangement but retained germ-line configurations of the K and monocytic leukemia, malignant histiocytosis, Hodgkin dis- X genes. The significance of rearrangements in cell lines is ease, metastatic carcinomas and sarcomas, thymoma, and unclear, however, because rearrangements may have oc- Warthin's tumor of the salivary gland. DNAs from none of curred in these cells at some point after they were put into these specimens showed bands other than those comigrating culture. At the present time, available information, summa- with unrearranged germ-line bands. rized above, suggests that if immunoglobulin gene rearrange- High Sensitivity of the Southern Blotting Technique for De- ments occur in human T-cell tumors they are rare and do not tecting Clonal Immunoglobulin Gene Rearrangements. To involve light-chain loci. Therefore, analysis of immunoglob- test the sensitivity of the Southern blotting technique for de- ulin gene rearrangements does not seem applicable to diag- tecting minor subpopulations of malignant lymphocytes nosis of T-cell lymphoma. within biopsies, blots were prepared from known lymphoma As a diagnostic method for B-cell lymphoma, analysis of DNA mixed with various amounts of nonmalignant lymph immunoglobulin gene rearrangements suffers from the time node DNA. After hybridizing with a K-specific probe, rear- necessary to complete the test (5-10 days) and the use of ranged alleles could be confidently detected down to a level radioactive probes required by the present form of the of 1-2.5% lymphoma relative to nonlymphoma DNA (Fig. Southern blot technique. On the other hand, analysis of 4). immunoglobulin gene rearrangements possesses several ad- vantages over conventional diagnostic methods. Above all it DISCUSSION offers a method that is not dependent on subjective criteria Clinically apparent malignant lymphoma results from the and the experience of the observer. In addition, the tech- clonal proliferation of neoplastic lymphocytes (1). Clonality nique requires small amounts of tissue (as little as 1 mg per is not, however, the equivalent of malignancy. Benign mono- immunoglobulin chain analysis) that needs no special han- clonal gammopathy and expansion of an isolated B-cell clone dling or preparation. In fact, we have obtained high quality in response to an antigenic stimulus are examples of clonal autoradiograms with DNA extracted from autopsy tissue processes that are not malignant. Benign monoclonal gam- several days after death. This contrasts with the difficulty mopathy seldom if ever involves lymph nodes, and neither experienced in microscopic examination of lymph node bi- monoclonal nor oligoclonal immune response has ever been opsies, both morphologically and for marker studies, if tis- documented as a cause of clinical lymphadenopathy. Never- sue is either not properly fixed or not frozen immediately on theless, the existence or potential existence of such process- removal from the patient. Also, DNA immunoglobulin es indicates that clonal B-cell proliferation in a biopsy should probes are stable and easy reagents to use. They can be pre- be regarded as strongly correlated with, but not an absolute pared in large quantities and stored indefinitely in the freez- criterion of, malignancy. The results described in this report er. There is very little variation in quality from preparation show that analysis of immunoglobulin gene rearrangement is to preparation, a problem sometimes encountered with anti- an accurate and practical method of detecting B-lymphocyte sera used in immunologic marker studies. clones. Used in the proper clinical context and in conjunc- A possible difficulty associated with analysis of immuno- tion with available morphologic information, this technique globulin gene rearrangements in lymphoma is the detection provides valuable evidence for B-cell lymphoma in biopsy of spurious rearrangements that are actually due to inherited specimens. polymorphisms in immunoglobulin gene DNA. An absolute In this report we present data from cases of unequivocal control against such occurrences is parallel analysis of non- lymphoma to show the applicability and validity of this tech- lymphocytic DNA from the same patient, as shown for two nique. In each case, rearrangements were found in which a cases in this report. In practice, such polymorphic mutations Downloaded by guest on September 27, 2021 Medical Sciences: Cleary et aL Proc. NatL. Acad. Sci. USA 81 (1984) 597

appear to be rare for the heavy-chain and K loci, none having diagnostic methods. This situation creates no special prob- been encountered by us for the restriction enzymes used in lem for immunoglobulin gene analysis. Sensitivity may be this report in specimens of normal or non-B-cell tissues from even more critical in staging of lymphoma or monitoring pa- over 40 patients. Others have reported similar experiences tients after treatment. For instance, small numbers of malig- (3, 4). However, the X light-chain locus does show occasion- nant cells may be detected in lymph nodes or blood in relaps- al fragment polymorphisms that involve the smallest of the ing patients or in those with persistent disease. We have suc- three EcoRI restriction fragments detected in the germ-line cessfully isolated DNA from bone marrow needle biopsies DNA of most patients (Figs. 1 and 2). These polymorphisms and analysis of such samples could be of value in following consist of acquisition or loss of multiples of a 5-kb DNA se- treated lymphoma patients as well as those who have under- quence resulting in a small set of predictable variant bands gone bone marrow transplantation. (15). This complicates but usually does not prevent interpre- tation without parallel germ-line controls. This work was supported by Grants NP-376 from the American Clonal immunoglobulin gene rearrangements were found Cancer Society and CA 34233 from the National Institutes of in every histologic subtype of B-cell lymphoma tested. Anal- Health. M.L.C. is a Postdoctoral Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. J.S. is a Fellow of the John ysis of immunoglobulin gene rearrangements fails to distin- A. Hartford Foundation. guish between these subtypes and, therefore, provides no information about expected biologic behavior (e.g., clinical 1. Levy, R., Warnke, R., Dorfman, R. F. & Haimovich, J. (1977) aggressiveness), as can be obtained from the morphologic J. Exp. Med. 145, 1014-1028. characteristics of the tumor. Nevertheless, it may be possi- 2. Aisenberg, A. C., Wilkes, B. M., Long, J. C. & Harris, N. L. ble to predict features of the biological behavior of tumors (1980) Am. J. Med. 68, 206-213. based on the extent of immunoglobulin gene rearrange- 3. Korsmeyer, S. J., Arnold, A., Bakshi, A., Ravetch, J. V., Sie- ments. For example, a tumor showing rearrangements of benlist, U., Hieter, P. A., Sharrow, S. O., LeBien, T. W., only heavy-chain genes and no rearrangements of light-chain Kersey, J. H., Poplack, D. G., Leder, P. & Waldmann, T. A. genes may have a biologic behavior and response to therapy (1983) J. Clin. Invest. 71, 301-313. 4. different from a tumor showing rearrangements of both Korsmeyer, S. J., Hieter, P. A., Ravetch, J. V., Poplack, D. G., Waldmann, T. A. & Leder, P. (1981) Proc. Natl. Acad. heavy- and light-chain genes. Sci. USA 78, 7096-7100. Although analysis of immunoglobulin gene rearrange- 5. Hieter, P. A., Korsmeyer, S. J., Waldmann, T. A. & Leder, ments does not permit histologic subtyping of B-cell tumors, P. (1981) Nature (London) 290, 368-372. this technique is helpful in distinguishing B-cell neoplasms 6. Hozumi, N. & Tonegawa, S. (1976) Proc. Natl. Acad. Sci. among lymphoid tumors that fail to show the definitive im- USA 73, 3628-3632. munologic markers for either T- or B-cell differentiation (non 7. Leder, P., Max, E. E. & Seidman, J. G. (1980) in Immunology T/non B or "null cell" lymphomas). The work of Warnke et 80, eds. Fougereau, M. & Dausset, J. (Academic, London), al. (25) indicates that these tumors may represent up to 25% pp. 34-50. of large cell lymphomas or 10-15% of all non-Hodgkin lym- 8. Wood, G. S. & Warnke, R. (1981) J. Histochem. Cytochem. 29, 1196-1204. phomas. In extensive studies of human B-cell leukemias, 9. Pettersson, V. & Sambrook, J. (1973) J. Mol. Biol. 73, 125- Korsmeyer and colleagues (3, 4) have shown that heavy- 130. chain immunoglobulin gene rearrangements occur early in 10. Robbins, J., Rosteck, P., Jr., Haynes, J. R., Freyer, G., human B-cell differentiation before the acquisition of all Cleary, M. L., Kalter, H. D., Smith, K. & Lingrel, J. B. known B-cell markers except Ta. However, their studies did (1979) J. Biol. Chem. 254, 6187-6195. not reveal Ia antigen on any B-cell precursors lacking heavy- 11. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. chain rearrangements. Furthermore, this antigen is not en- 12. Alwine, J. C., Kemp, D. J. & Stark, G. R. (1977) Proc. Natl. tirely specific for B cells, being present on some activated T Acad. Sci. USA 12, 5350-5354. cells and on cells of certain other tissues. The early appear- 13. Hieter, P. A., Max, E. E., Seidman, J. G., Maizel, J. V. & P. Cell 197-207. ance of gene rearrangements Leder, (1980) 22, immunoglobulin in B-cell de- 14. Hieter, P. A., Maizel, J. V. & Leder, P. (1982) J. Biol. Chem. velopment means that this genetic marker may be present in 257, 1516-1522. cells lacking definitive immunologic B-cell markers. This sit- 15. Hieter, P. A., Hollis, G. F., Korsmeyer, S. J., Waldmann, uation is shown in cases 4 and 10 of this report. The presence T. A. & Leder, P. (1981) Nature (London) 294, 536-540. of heavy- and light-chain immunoglobulin gene rearrange- 16. Ravetch, J. V., Siebenlist, V., Korsmeyer, S., Waldmann, T. ments in these cases reveals the B-cell origin of both tumors. & Leder, P. (1981) Cell 27, 583-591. These cases also show that analysis of immunoglobulin gene 17. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular rearrangements provides for the first time a means of docu- Cloning: A Laboratory Manual (Cold Spring Harbor Labora- menting clonality for at least some null-cell lymphomas. tory, Cold Spring Harbor, NY), pp. 390-401. 18. Cossman, J., Neckers, L. M., Arnold, A. Analysis of immunoglobulin gene rearrangements is an ex- & Korsmeyer, S. J. (1982) N. Engl. J. Med. 307, 1251-1254. tremely sensitive test for the presence of a minor clonal pop- 19. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) ulation of lymphocytes. This feature may be important in the J. Mol. Biol. 113, 237-251. diagnosis of a biopsy in which there is a small cluster of ma- 20. The Non-Hodgkin's Lymphoma Pathologic Classification Proj- lignant cells that may escape careful examination under the ect (1982) Cancer 49, 2112-2135. microscope or even miss being sectioned. Because DNA 21. Hieter, P. A., Korsmeyer, S. J., Waldmann, T. A. & Leder, analysis of biopsy tissue screens a three-dimensional frag- P. (1981) Nature (London) 290, 368-372. ment of tissue and can easily be scaled up to several grams of 22. Bhan, A. K., Nadler, L. M., Stashenko, P., McCluskey, R. T. starting material, the likelihood of failing to detect a clonal & Schlossman, S. F. (1981) J. Exp. Med. 154, 737-749. 23. rearrangement in a small portion of a biopsy is low as long as Cory, S., Adams, J. M. & Kemp, D. J. (1980) Proc. Natl. the clonal fraction of cells within the biopsy exceeds Acad. Sci. USA 77, 4943-4947. the 24. Kurosawa, Y., von Boehmer, H., Haas, W., Sakano, H., threshold of sensitivity of the method. Alternatively, diffuse Trauneker, A. & Tonegawa, S. (1981) Nature (London) 290, admixtures of malignant clones with nonclonal benign lym- 565-570. phocytes within a tissue section, a rare but occasionally en- 25. Warnke, R., Miller, R., Grogan, T., Pederson, M., Dilley, J. & countered situation, are difficult to evaluate by conventional Levy, R. (1980) N. Engl. J. Med. 303, 293-300. Downloaded by guest on September 27, 2021