Proc. Natl. Acad. Sci. USAVol. 81, pp. 593-597, January 1984Medical Sciences

Immunoglobulin gene rearrangement as a diagnostic criterionof B-cell lymphoma

(Southern blot hybridization technique/cancer/DNA probe/clonal analysis/immunogenotyping)

MICHAEL L. CLEARY, JAMES CHAO, ROGER WARNKE, AND JEFFREY SKLARDepartment of Pathology, Stanford University, Stanford, CA 94305

Communicated by Paul Berg, September 26, 1983

ABSTRACT We describe the use of the Southern blot hy-bridization technique to diagnose B-cell lymphoma by detect-ing clonal immunoglobulin gene rearrangements in lymphnode and other biopsy tissues. DNA was isolated from a widevariety of neoplastic and non-neoplastic specimens and ana-lyzed for the presence of rearranged immunoglobulin genesusing radiolabeled DNA probes specific for the heavy- andlight-chain immunoglobulin constant region genes. Among thespecimens examined, clonal immunoglobulin gene rearrange-ments were found only in biopsy samples of B-cell lymphomaand not in samples containing reactive lymphoid processes ornon-B-cell cancers. In lymphomas, the presence of rearrange-ments for either the K or A light-chain gene correlated withexpression of one or the other of these chains when cellularimmunoglobulins could be detected by frozen-section immuno-phenotyping techniques. The analysis of immunoglobulin generearrangements offers several advantages over conventionaldiagnostic methods for lymphomas, including improved sensi-tivity in detecting minor populations of neoplastic lymphocytescomposing as little as 1% of the total cell population. In addi-tion, clonal immunoglobulin gene rearrangements are demon-strable in a subset of lymphomas that lack detectable surfaceor cytoplasmic immunoglobulin, thus offering positive evi-dence for both malignancy and the B-cell origin of these tu-mors. Our studies indicate that detection of immunoglobulingene rearrangements is a valuable method for diagnosis andclassification of various lymphoproliferative disorders that aredifficult to evaluate histologically or that lack distinctive anti-genic markers.

Diagnosis of malignant lymphoma depends on histologicevaluation of tissue biopsies. However, distinguishing be-tween malignant and benign disorders in lymph nodes andother lymphoid tissues by light microscopy remains one ofthe pathologist's most difficult tasks. Although the majorityof such biopsies are unambiguous, a significant minoritypose serious problems for even the most expert histopathol-ogist. In large part, the biological cause for this difficulty isthat antigenically stimulated lymphocytes may morphologi-cally resemble neoplastic lymphocytes. Conversely, so-called well-differentiated neoplastic lymphocytes may be cy-tologically indistinguishable from normal unstimulated lym-phocytes. There are also cases in which reactive conditionscoexist with and obscure malignancy, further complicatinghistologic interpretation. Occasionally, poorly differentiatedmetastatic carcinoma or melanoma may be mistaken for lym-phoma.An important method devised to deal with these problems

is the analysis of immunologic markers on the surface or inthe cytoplasm of lymphoid cells in tissue sections or in cellsuspensions of lymph node biopsies (1, 2). This methodtakes advantage of the clonal nature of malignancy, such that

large numbers of cells within a histologic section or cellsuspension bear the same antigenic markers if the prolifera-tion is neoplastic. For instance, homogeneous neoplasticproliferation of B lymphocytes that synthesize detectableimmunoglobulin will show only a single immunoglobulinlight chain, K or X, when analyzed for these two polypep-tides. Although this immunophenotyping technique is rapidand has proved to be helpful in evaluating certain biopsyspecimens, it suffers from several disadvantages. One fre-quent problem is that malignant B-cell proliferation withinlymph nodes is often intermixed with various amounts ofnormal B cells, in which case this technique may depend ondetecting small deviations from the 2:1 ratio of K- to A-bear-ing B cells found in normal human lymphoid tissue. Otherproblems include artifacts associated with suboptimal han-dling or fixation of tissues, the requirement for good anti-body reagents directed against antigenic markers, and theabsence of markers in certain lymphoid tumors.

Recently we have explored an alternative approach to thediagnosis of B-cell lymphoma. This approach relies on de-tecting uniform rearrangements of immunoglobulin geneswithin clonal populations of B lymphocytes, as detected pre-viously in human B-cell leukemias (3-5). Our work is basedon the fact that B lymphocytes must undergo a series ofDNA rearrangements prior to immunoglobulin production(6, 7). In germ-line cells the variable and constant domains ofeach type of immunoglobulin chain (one heavy chain andtwo light chains, K and X) are encoded in separate discontinu-ous regions of specific chromosomes. During B-lymphocytematuration, an initial event in immunoglobulin synthesis isthe somatic recombination of the separated variable and con-stant gene segments. This results in the removal of interven-ing DNA and the close apposition of specific variable andconstant DNA sequences to form an active immunoglobulingene (see Fig. 1A). The high degree of variability with whichimmunoglobulin gene segments are rearranged and the factthat each individual B cell is capable of expressing only asingle antibody idiotype make the configuration of rear-ranged immunoglobulin gene segments an entirely specificmarker for a given B cell and for any clone that may arisefrom that B cell.

In this report, we show that detection of immunoglobulingene rearrangements in biopsy tissue by the Southern blothybridization procedure affords an accurate and highly sen-sitive means of identifying clonal lymphoid proliferations ina wide variety of B-cell malignancies. In addition, benign re-active processes and non-B-cell malignancies are distin-guished by the absence of detectable immunoglobulin generearrangements. This technique, therefore, provides a valu-able adjunct to currently available methods for diagnosing B-cell lymphoma. Furthermore, this technique avoids many ofthe problems associated with immunologic marker studiesand conventional morphologic diagnosis.

Abbreviations: kb, kilobase(s); C and J, constant and joining regionsof immunoglobulin chains.

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594 Medical Sciences: Cleary et al.

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VK1 VK 2VK3 VIK4VK5VK6 JK

DNA REARRANGEMENT

Rearranged f f 3

CK Probe

tCK

Lymphoma 9 VA U'l-. k1 F.iAllelev v VK VK4JK3 CK IAlleleK 1 K2 1C3Po4b

Cal Probe

GermlineMu cGene

BJH

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Germline Kappa Gene

Cli1234

lkb h~CDi Probe

JK CKt 12345

1 kb

GermlineLambdaGene 7

I I

CK Probe

CAK-eOFCAMcg CXKOe0

C X Probe

i BanH1

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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

configuration for the heavy-chain, K, and X C genes. The probes used in this work are indicated by hatched boxes. The heavy-chain J-region

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.

METHODSBiopsy tissues were routinely collected from the operatingroom, frozen in airtight plastic capsules by immersion in a

dry ice/isopentane bath, and stored at -70'C for up to fiveyears (8). Normal control tissues for two cases studied con-

sisted of peripheral blood granulocytes and autopsy liver tis-sue.DNA was extracted from lymph node biopsies and other

tissues and purified according to standard procedures (9).The starting material represented 10-25 mg (wet weight) oftissue.

After purification, high molecular weight DNA was digest-ed with appropriate restriction enzymes according to condi-tions recommended by the supplier (Bethesda ResearchLaboratories). Digestion products were electrophoresedovernight in an 0.8% agarose gel, as described (10). Afternicking of the DNA by ultraviolet light to decrease the aver-

age chain length, DNA fragments were transferred out of theagarose gels onto nitrocellulose filters as described bySouthern (11). The filters were then hybridized with radiola-beled pBR322 plasmid DNA carrying immunoglobulin DNAfragments. Hybridization reactions were carried out underconditions described elsewhere (12), using 50% formamideat 420C. After extensive washing and drying of filters, auto-radiography was carried out at -70'C against a single inten-sifying screen for 12-72 hr.Human genomic DNA fragments specific for the K (13, 14)

and (15) constant regions (C) and the heavy-chain joining (J)and ,u regions (16) were isolated from recombinant bacte-

riophage kindly provided by P. Leder (Harvard MedicalSchool). These fragments were subcloned into the Esche-richia coli plasmid pBR322 using standard procedures (17).The positions of the DNA probe fragments with respect to

the immunoglobulin genes are shown in Fig. 1B. Plasmids

containing immunoglobulin DNA were isolated from E. coliand nick-translated in vitro with [a-32P]dNTPs as described

elsewhere (19), to a specific activity of 3-5 x 108 cpm/pgg.Radiolabeled dNTPs were obtained from Amersham.

All lymphoma specimens were categorized histopathologi-cally as described (20). Analysis of immunologic surface andcytoplasmic markers was carried out in frozen sections as

described (8).

RESULTSImmunoglobulin Gene Rearrangements Are Present in

Lymphomas but Not in Nonlymphoid Control Tissue from theSame Patient. Fig. 2A shows the data obtained from a lymphnode biopsy diagnosed histologically as a diffuse large celllymphoma. The heavy-chain J probe detected two bands inthe lymphoma DNA, one of which comigrated with the sin-

J K A

WI'a FIG. 2. Autoradiograms from_ lymphoma and control DNA hy-

bridized with immunoglobulin geneprobes. (A) Case 1. (B) Case 2.Analyses for heavy-chain and K

light-chain gene rearrangementswere carried out on DNA digested

* with the BamHI restriction en-zyme; the X light-chain locus was

analyzed with DNA digested with.J A the EcoRI restriction enzyme.

i8 r Lanes 1, control DNA; lanes 2,lymphoma DNA. Dashes havebeen placed alongside germ-line

Jo bands, arrows are beside rear-

ranged bands. Based on markerDNA fragments coelectropho-resed with DNA digests in theseblots but not shown in the figure,the germ-line bands are of expect-ed size: about 19.3 kb (heavychain); 12 kb (K light chain); and16, 14, and 8 kb (X light chain).

GermlineGeneAllele f

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Proc. NatL Acad Sci. USA 81 (1984)

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Proc. Natl. Acad. Sci. USA 81 (1984) 595

Table 1. Correlation of immunoglobulin gene rearrangements (immunogenotype) with immunoglobulin antigens (immunophenotype) invarious lymphoproliferative conditions

Immunogenotype ImmunophenotypeCase 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 ND4 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 G11 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,Jprobe 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 granulocyteDNA. The second band, which migrated in a position belowthat of the germ-line band, represents clonal rearrangementof one heavy-chain immunoglobulin allele. Separate blotsprepared from the same DNA and hybridized with a probefor the K light-chain gene also revealed a single clonally rear-ranged band that migrated slightly ahead of the position ofthe germ-line band as well as a weaker band that comigratedwith the germ-line band from granulocyte control DNA.Autoradiograms obtained from blots hybridized with the Xlight-chain probe showed identical patterns for the controland lymphoma DNAs. The results obtained in this case areconsistent with a clonal rearrangement of at least one alleleof the heavy-chain and K-chain loci and correlate with immu-nologic phenotyping of frozen sections of this patient's lym-phoma, which revealed u and K surface immunoglobulin (Ta-ble 1).A similar analysis was carried out (Fig. 2B) for a diffuse

large cell lymphoma that developed in a patient with Wis-kott-Aldrich syndrome. Nonlymphoma control DNA wasextracted from autopsy liver, which was grossly and micro-scopically free of tumor. When the heavy-chain J probe wasused in the hybridization, three bands were seen, two ofwhich migrated faster than the unrearranged germ-line bandpresent in the control DNA. This finding can be explained byrearrangement of both heavy-chain alleles in the lymphomacells. The K light-chain probe showed a single rearrangedband representing a fragment larger than the germ-line frag-ment in addition to a band that comigrated with the controlDNA band. Hybridization ofDNA 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 ofmalignant 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 haveresulted from either an unrearranged immunoglobulin allelewithin tumor cells or from contamination of the tumor tissuewith normal polyclonal lymphocytes, blood cells, fibrous tis-sue, and blood vessels.Immunoglobulin Gene Rearrangements Are Detectable in

Various Histologic Subtypes of B-Cell Lymphoma. Fig. 3shows results obtained when DNA isolated from lymph nodebiopsies representing a variety of conditions was analyzedwith heavy-chain J-region DNA probes. Rearrangementswere seen in all cases involving histologic subtypes of B-celllymphoma, as summarized in Table 1. Light-chain rear-rangements were also seen in all cases of B-cell lymphoma

(data not shown). When present, the surface or cytoplasmiclight chain identified in frozen sections corresponded to thelight-chain class that showed gene rearrangement (Table 1).Both K and X gene rearrangements were found in a singlespecimen containing only X surface immunoglobulin (case 9),but no X gene rearrangement was found in lymphomas with Ksurface immunoglobulin. This observation conforms to theproposed hierarchy of immunoglobulin gene rearrangement,such that rearrangements of the X gene occur only after de-fective or nonproductive rearrangements of both K gene al-leles (4, 21).Immunoglobulin Gene Rearrangements Are Detectable in

Lymphomas Lacking Cellular Immunoglobulin. Two of thecases examined did not show staining for surface or cyto-plasmic immunoglobulin but contained immunoglobulin generearrangements. Both cases 10 and 4 (Table 1) are diffuselarge cell lymphomas that lack distinctive markers except forB1 (22) and Ia antigens. Despite the absence of immunoglob-ulin production in both cases, clonal heavy-chain immuno-globulin gene rearrangements (Fig. 3) support the B-cell lin-eage of these tumors. Moreover, light-chain K gene rear-rangements were found in each instance, while the X geneswere in a germ-line configuration.

3 4 5 6 7 8 9 10 11

FIG. 3. Autoradiograms obtained with DNA prepared fromlymph nodes with various proliferative disorders and hybridizedwith a heavy-chain J-specific probe. Analyses for heavy-chain generearrangements were carried out using BamHI-digested DNA ex-tracted from lymph node biopsies. The numbers coincide with thecase numbers in Table 1. Dashes indicate the germ-line 19.3-kbband; arrows show rearranged heavy chain J-specific bands. In mostof the autoradiograms, there is a faint 12-kb band of unknown originthat hybridizes weakly with the J-specific probe under the condi-tions used. This band is also detected in blots prepared from non-lymphoid DNA.

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FIG. 4. Sensitivity of the Southern blot hybridization techniquefor detecting immunoglobulin gene rearrangements. Mixtures con-taining a total of 10 jug ofDNA were prepared with lymphoma DNAand various amounts of nonmalignant lymph node DNA. The DNAwas digested with BamHI restriction enzyme. After electrophoresisand transfer, the samples were hybridized with a radiolabeled CK-specific probe, washed, and autoradiographed for 48 hr. The per-centage of lymphoma DNA within each mixture is indicated abovethe lanes of the autoradiogram. Dash indicates the position of thegerm-line band; arrows indicate the position of two rearranged Kbands in the lymphoma DNA.

Immunoglobulin Gene Rearrangements Are Not Seen inTissue Specimens Other Than B-Cell Lymphomas. We haveexamined four T-cell lymphomas-for example, a lympho-blastic lymphoma (Fig. 3, case 11). Afl showed only germ-line bands and no detectable immunoglobulin gene rear-rangements. We have also examined DNA from lymphnodes containing a wide variety of benign and reactive con-ditions, including reactive follicular hyperplasia (case 3), an-gioimmunoblastic lymphadenopathy, rheumatoid arthritis,and sarcoidosis. Other non-B-cell neoplastic processes wereexamined; these included acute myelocytic leukemia, acutemonocytic leukemia, malignant histiocytosis, Hodgkin dis-ease, metastatic carcinomas and sarcomas, thymoma, andWarthin's tumor of the salivary gland. DNAs from none ofthese specimens showed bands other than those comigratingwith unrearranged germ-line bands.High Sensitivity of the Southern Blotting Technique for De-

tecting Clonal Immunoglobulin Gene Rearrangements. Totest the sensitivity of the Southern blotting technique for de-tecting minor subpopulations of malignant lymphocyteswithin biopsies, blots were prepared from known lymphomaDNA mixed with various amounts of nonmalignant lymphnode DNA. After hybridizing with a K-specific probe, rear-ranged alleles could be confidently detected down to a levelof 1-2.5% lymphoma relative to nonlymphoma DNA (Fig.4).

DISCUSSIONClinically apparent malignant lymphoma results from theclonal proliferation of neoplastic lymphocytes (1). Clonalityis not, however, the equivalent of malignancy. Benign mono-clonal gammopathy and expansion of an isolated B-cell clonein response to an antigenic stimulus are examples of clonalprocesses that are not malignant. Benign monoclonal gam-mopathy seldom if ever involves lymph nodes, and neithermonoclonal nor oligoclonal immune response has ever beendocumented as a cause of clinical lymphadenopathy. Never-theless, the existence or potential existence of such process-es indicates that clonal B-cell proliferation in a biopsy shouldbe regarded as strongly correlated with, but not an absolutecriterion of, malignancy. The results described in this reportshow that analysis of immunoglobulin gene rearrangement isan accurate and practical method of detecting B-lymphocyteclones. Used in the proper clinical context and in conjunc-tion with available morphologic information, this techniqueprovides valuable evidence for B-cell lymphoma in biopsyspecimens.

In this report we present data from cases of unequivocallymphoma to show the applicability and validity of this tech-nique. In each case, rearrangements were found in which a

diagnosis of lymphoma was made by morphologic criteria

alone. Surface and cytoplasmic immunoglobulin was ana-

lyzed in these cases as well. When present, immunoglobulinmarkers 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-celllymphomas, and several nonlymphoid cancers have been

tested for immunoglobulin gene rearrangements. These havebeen consistently negative for both heavy- and light-chaingene rearrangements. As shown in Fig. 3 (case 3), normal or

reactive lymph nodes show only germ-line bands. There are

presumably innumerable rearranged immunoglobulin genes

in such tissues; however, apparently no single rearrangedDNA fragment is sufficiently abundant to be detectable by

the Southern blot technique. On the basis of these findings,we conclude that detection of a rearranged immunoglobulinband in a Southern blot analysis of lymphoid DNA is a reli-

able test for clonal proliferation and, within the limits dis-

cussed above, for B-cell lymphoma.Our failure to detect immunoglobulin gene rearrangements

in human T-cell lymphomas contrasts with studies on smallnumbers of mouse T-cell tissue culture lines, in which occa-

sional heavy-chain gene rearrangements were found in theabsence of light-chain rearrangements (23, 24). In 11 of 12

cases of human T-cell acute lymphoblastic leukemia (ALL),Korsmeyer et al. (3) found germ-line configurations ofimmunoglobulin genes. A single T ALL-derived cell line inthis series contained a nonproductive heavy-chain gene rear-

rangement but retained germ-line configurations of the K andX genes. The significance of rearrangements in cell lines isunclear, however, because rearrangements may have oc-

curred in these cells at some point after they were put intoculture. At the present time, available information, summa-

rized above, suggests that if immunoglobulin gene rearrange-

ments occur in human T-cell tumors they are rare and do not

involve light-chain loci. Therefore, analysis of immunoglob-ulin gene rearrangements does not seem applicable to diag-nosis of T-cell lymphoma.As a diagnostic method for B-cell lymphoma, analysis of

immunoglobulin gene rearrangements suffers from the timenecessary to complete the test (5-10 days) and the use ofradioactive probes required by the present form of theSouthern blot technique. On the other hand, analysis ofimmunoglobulin gene rearrangements possesses several ad-vantages over conventional diagnostic methods. Above all itoffers a method that is not dependent on subjective criteriaand the experience of the observer. In addition, the tech-nique requires small amounts of tissue (as little as 1 mg per

immunoglobulin chain analysis) that needs no special han-dling or preparation. In fact, we have obtained high qualityautoradiograms with DNA extracted from autopsy tissueseveral days after death. This contrasts with the difficultyexperienced in microscopic examination of lymph node bi-opsies, both morphologically and for marker studies, if tis-sue is either not properly fixed or not frozen immediately on

removal from the patient. Also, DNA immunoglobulinprobes are stable and easy reagents to use. They can be pre-

pared in large quantities and stored indefinitely in the freez-er. There is very little variation in quality from preparationto preparation, a problem sometimes encountered with anti-sera used in immunologic marker studies.A possible difficulty associated with analysis of immuno-

globulin gene rearrangements in lymphoma is the detection

of spurious rearrangements that are actually due to inherited

polymorphisms in immunoglobulin gene DNA. An absolute

control against such occurrences is parallel analysis of non-

lymphocytic DNA from the same patient, as shown for two

cases in this report. In practice, such polymorphic mutations

seVD 0

Proc. Natl. Acad Sci. USA 81 (1984)

Proc. NatL. Acad. Sci. USA 81 (1984) 597

appear to be rare for the heavy-chain and K loci, none havingbeen encountered by us for the restriction enzymes used inthis report in specimens of normal or non-B-cell tissues fromover 40 patients. Others have reported similar experiences(3, 4). However, the X light-chain locus does show occasion-al fragment polymorphisms that involve the smallest of thethree EcoRI restriction fragments detected in the germ-lineDNA of most patients (Figs. 1 and 2). These polymorphismsconsist of acquisition or loss of multiples of a 5-kb DNA se-quence resulting in a small set of predictable variant bands(15). This complicates but usually does not prevent interpre-tation without parallel germ-line controls.

Clonal immunoglobulin gene rearrangements were foundin every histologic subtype of B-cell lymphoma tested. Anal-ysis of immunoglobulin gene rearrangements fails to distin-guish between these subtypes and, therefore, provides noinformation about expected biologic behavior (e.g., clinicalaggressiveness), as can be obtained from the morphologiccharacteristics of the tumor. Nevertheless, it may be possi-ble to predict features of the biological behavior of tumorsbased on the extent of immunoglobulin gene rearrange-ments. For example, a tumor showing rearrangements ofonly heavy-chain genes and no rearrangements of light-chaingenes may have a biologic behavior and response to therapydifferent from a tumor showing rearrangements of bothheavy- and light-chain genes.Although analysis of immunoglobulin gene rearrange-

ments does not permit histologic subtyping of B-cell tumors,this technique is helpful in distinguishing B-cell neoplasmsamong lymphoid tumors that fail to show the definitive im-munologic markers for either T- or B-cell differentiation (nonT/non B or "null cell" lymphomas). The work of Warnke etal. (25) indicates that these tumors may represent up to 25%of large cell lymphomas or 10-15% of all non-Hodgkin lym-phomas. In extensive studies of human B-cell leukemias,Korsmeyer and colleagues (3, 4) have shown that heavy-chain immunoglobulin gene rearrangements occur early inhuman B-cell differentiation before the acquisition of allknown B-cell markers except Ta. However, their studies didnot reveal Ia antigen on any B-cell precursors lacking heavy-chain rearrangements. Furthermore, this antigen is not en-tirely specific for B cells, being present on some activated Tcells and on cells of certain other tissues. The early appear-ance of immunoglobulin gene rearrangements in B-cell de-velopment means that this genetic marker may be present incells lacking definitive immunologic B-cell markers. This sit-uation is shown in cases 4 and 10 of this report. The presenceof heavy- and light-chain immunoglobulin gene rearrange-ments in these cases reveals the B-cell origin of both tumors.These cases also show that analysis of immunoglobulin gene

rearrangements provides for the first time a means of docu-menting clonality for at least some null-cell lymphomas.

Analysis of immunoglobulin gene rearrangements is an ex-

tremely sensitive test for the presence of a minor clonal pop-

ulation of lymphocytes. This feature may be important in thediagnosis of a biopsy in which there is a small cluster of ma-lignant cells that may escape careful examination under themicroscope or even miss being sectioned. Because DNAanalysis of biopsy tissue screens a three-dimensional frag-ment of tissue and can easily be scaled up to several grams ofstarting material, the likelihood of failing to detect a clonalrearrangement in a small portion of a biopsy is low as long as

the clonal fraction of cells within the biopsy exceeds thethreshold of sensitivity of the method. Alternatively, diffuseadmixtures of malignant clones with nonclonal benign lym-phocytes within a tissue section, a rare but occasionally en-

countered situation, are difficult to evaluate by conventional

diagnostic methods. This situation creates no special prob-lem for immunoglobulin gene analysis. Sensitivity may beeven more critical in staging of lymphoma or monitoring pa-tients after treatment. For instance, small numbers of malig-nant cells may be detected in lymph nodes or blood in relaps-ing patients or in those with persistent disease. We have suc-cessfully isolated DNA from bone marrow needle biopsiesand analysis of such samples could be of value in followingtreated lymphoma patients as well as those who have under-gone bone marrow transplantation.

This work was supported by Grants NP-376 from the AmericanCancer Society and CA 34233 from the National Institutes ofHealth. M.L.C. is a Postdoctoral Fellow of the Jane Coffin ChildsMemorial Fund for Medical Research. J.S. is a Fellow of the JohnA. Hartford Foundation.

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