Immunoglobulin gene rearrangements in adult non-hodgkin's lymphoma

lmmunoglobulin Gene Rearrangements in Adult Non-Hodgkin’s Lymphoma

ALAN

C.

AISENBERG,

BARBARA

M.

JOSEPH

0.

NANCY

L.

M.D.,

WILKES,

JACOBSON, HARRIS,

Southern blotting was employed to analyze the immunoglobulin heavy and light chain genes and the gene for the T cell receptor beta chain in genomic DNA derived from the tumor specimens of 120 adults with pathologically classified and immunotyped non-Hodgkin’s lymphoma and B cell chronic lymphocytic leukemia. In a consecutive series of 100 patients, one or two rearranged heavy chain genes could be detected in each of the 80 samples expressing clonal surface immunoglobulin. The kappa gene was rearranged in 70 percent of kappabearing tumors and in 23 percent of lambda-bearing specimens. Furthermore, a rearranged immunoglobulin gene was also observed in 21 of 29 lymphomas (nine from the consecutive series and 20 selected for surface immunoglobulin-negative status) in which B cell lineage was in doubt because of absent clonal surface immunoglobulin. These findings indicate that most cases of lymphoma and lymphocytic leukemia in adults are of B cell lineage, even when phenotypic evidence is inconclusive. The exceptional cases (only 3 percent in the consecutive series) were of either follicular lymphoma or diffuse large cell (histiocytic) lymphoma subtype; the lineage in cases of diffuse lymphocytic lymphoma or chronic lymphocytic leukemia was never in doubt. Although the convenience of surface marker analysis assures its continuing clinical application, gene study resolves indeterminate cases and extends the understanding of the pathogenesis of lymphoproliferative disease.

Ph.D.

B.S. M.D. M.D.

Boston, Massachusetts

From the Hematology/Oncology Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts. This work was supported by Research Grant CA-30020-05 from the National Institutes of Health. Requests for reprints should be addressed to Dr. Alan C. Aisenberg, Massachusetts General Hospital, Boston, Massachusetts 02114. Manuscript submitted October 24, 1986, and accepted December 10, 1986.

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The assembly of complete genes for the heavy and light chains of immunoglobulin from physically separated gene segments is a central event in B cell maturation [l-3]. The variable portion of the immunoglobulin heavy chain gene is assembled from one of each of the multiple nonidentical variable, diversity, and joining segments; the kappa and lambda light chains require variable and joining segments only. The light and heavy chain variable region genes are joined to their respective constant region genes prior to transcription and processing as RNA, translation of the RNA into protein, and assembly of the completed chains into immunoglobulin. The rearranged variable region genes of the light and heavy chains provide the physical basis of antibody diversity. With the modern technique of Southern blotting and specific DNA probes, these gene rearrangements are readily detected. In a monoclonal B cell lymphoma, a new band replaces the germline immunoglobulin band present in cells that are not of 6 cell lineage; in a benign B cell population, the polyclonal immunoglobulin gene rearrangements are not visualized on Southern analysis. Thus, immunoglobulin gene rearrangement provides a sensitive index of a clonal B cell population. Since the alpha, beta, and gamma

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TABLE I

IN NON-HODGKIN’S

LYMPHOMA-AISENBERG

Rearrangement of the lmmunoglobulin Heavy and Kappa Light Chain Genes in Lymphomas Lambda Surface Phenotype and in Those Lacking Surface lmmunoglobulin

S&kappa

JH Chronic lymphocytic leukemia Diffuse poorly differentiated lymphocytic lymphoma Diffuse large cell lymphoma Follicular lymphoma (lymphocytic (or mixed) Miscellaneous lymphomas All of above R = rearrangement;

13113

JH

ii/15

Ckappa

JH

AND

Ckappa

JH

ET AL

of Kappa and Double Rearrangements (RR/R + RR)

Total

m

%la”bda

C kappa

Ckappa

JH

Ckappa

lO/lO

l/IO

6/6

417

29/29

16132

15119

O/16

0

14114

9114

5/a

l/9

1 l/l6

lO/lO

7110

414

214

0

14114

13115

515

215

213

l/3

21122

16123

6/11

717

l/7

315

315

21/13

10123

7116

2110

616

316

0

l/l

l/l

717

4/a

417

o/4

54/ 54 (100%)

40157 (70%)

12/15 (80%)

9/16 (56%)

92195 (97%)

551100 (55%)

42166 (64%)

5155 (9%)

ll/ll

Slg = surface

26126 (100%)

immunoglobulin;

0 6126 (23%) JH = immunoglobulin

heavy

chain

gene;

Ckappa = kappa

light chain

2116

gene.

were obtained from Ortho Pharmaceutical (Raritan, New Jersey), with the exception of the CALLA and Bl (Coulter Immunology; Hialeah, Florida) and HLA-DR (Becton-Dickinson; Sunnyvale, California) antiserum preparations. The lymphocytes were washed after incubation with the monoclonal antibody and then reincubated with a fluoresceinconjugated F(ab’)* fraction of goat anti-mouse gamma globulin antibody (Tago; Burlinghame, California). After washing again, the cells were suspended in buffered glycerine and examined with a Zeiss ultraviolet microscope equipped with an Osram HBO 200 mercury arc lamp and a fluorescein isothiocyanate 485 nm excitation primary filter. At least 200 cells were examined after staining with each antibody. Surface immunoglobulin was assessed with fluorescein-conjugated heteroantiserum specific for the human IgM and IgG heavy chains, and the kappa and lambda light chains (Meloy Laboratories; Springfield, Virginia). The methods have been described in detail [6]. In an additional group of 20 lymphoma biopsy specimens (Table II), surface marker analysis was performed on stored frozen tissue employing the immunoperoxidase technique [7]; these specimens were selected for their absence of staining with anti-immunoglobulin antibodies. Southern Blot Analysis of Genomic DNA. High-molecularweight DNA was prepared from each of the immunotyped specimens, and 6 to 10 pg was digested with restriction enzymes (obtained from New England Biolabs; Beverly, Massachusetts) and subjected to electrophoresis on 0.8 percent agarose gel slabs. The restriction enzymes EcoRl and Sacl were employed to study the immunoglobulin heavy chain gene, BarnHI to study the immunoglobulin kappa light chain gene, and BamHI, EcoRI, and HindIll were employed to study the T cell receptor beta chain gene. After denatura-

chain genes of the T cell receptor undergo similar rearrangement [4], with appropriate probes, T cell proliferations can be analyzed in identical fashion. In the study reported herein, immunoglobulin and T cell receptor gene rearrangements were analyzed in an extensive series of pathologically classified and immunotyped non-Hodgkin’s lymphoma. We found that non-Hodgkin’s lymphoma in adults is of B cell lineage, almost without exception. PATIENTS

GENES

METHODS

Patients and Surface Marker Analysis. Surface markers were analyzed in cell suspensions prepared from 100 consecutive adult patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukemia (Table I). Fresh lymphoma tissue was obtained directly from the operating room, sieved through a stainless steel mesh employing medium 199, and studied without storing. All biopsy specimens were subclassified according to a modified Rappaport system for non-Hodgkin’s lymphoma [5]. The diagnosis of chronic lymphocytic leukemia was based on conventional morphologic criteria; viable mononuclear cells were obtained from the heparinized peripheral blood of consecutive untreated patients (excluding those with a T cell phenotype) by centrifugation through a Ficoll-Hypaque gradient. An aliquot of lymphocytesf106 cells in 0.05 ml of medium 199) was incubated with 0.05 ml of each of the following monoclonal antibodies: CALLA (common acute leukemia antigen); OKTl (peripheral and thymic T cells); OKT4 (inducerhelper T cells); OKT8 (cytotoxic-suppressor T cells): OKTl 1 (sheep cell receptor); HLA-DR (HLA-DR antigen); and Bi (B cells). Nonfluoresceinated monoclonal mouse antibodies

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TABLE II

GENES

IN NON-HODGKIN’S

LYMPHOMA-AISENBERG

ET AL

Rearrangement of the lmmunoglobultn Heavy and Kappa Light Chain Genes in Lymphomas Lacking Surface Immunoglobulin by lmmunoperoxidase Study

Diffuse large cell lymphoma Follicular lymphoma (lymphocytic or mixed) Diffuse mixed lymphoma All of above

lmmunoglobulin Heavy Chain Genes

Kappa Lighl Chain Genes

14*/19 419

IO/19 319

l/l 19/29

l/l 14129

chain gene were demonstrated in each of the 80 specimens of either kappa or lambda surface phenotype. Rearrangement of the kappa light chain gene was detected in 70 percent (40 of 57 specimens) of the kappa-bearing specimens, but in only 23 percent (six of 26 samples) of the lambda-bearing samples. Double rearrangements (RR) of the heavy chain genes were much more likely to be observed (42 of 66 rearrangements, or 64 percent, were double) than of the kappa’light chain (only five of 55 rearrangements, or 9 percent, were double). In 15 surface immunoglobulin-negative samples (Table I), heavy chain gene rearrangement was detected in 12; one of three diffuse large cell (histiocytic) lymphomas and two of five follicular lymphomas showed a germline pattern of the immunoglobulin heavy chain gene. In the entire consecutive series (both surface immunoglobulin-positive and surface immunoglobulin-negative specimens), heavy chain rearrangement was detected in 97 percent, and kappa light chain rearrangement was found in 55 percent. To examine immunoglobulin genes in surface immunoglobulin-negative lymphomas more closely, we prepared genomic DNA from an additional group of 20 stored frozen specimens whose surface immunoglobulin-negative status had been established by the immunoperoxidase technique. (It should be noted that the immunoperoxidase technique, although excellent for antigen localization, may not detect lambda light chains and may provide false results even when frozen tissue is employed.) Table II is a summation of the findings in the immunoperoxidasestudied specimens and the surface immunoglobulin-negative lymphoma specimens presented in Table I; the combined results support the earlier conclusion based on specimens whose surface markers were studied in suspension. In 19 of 29 samples, at least one rearranged heavy chain gene was detected, and in 14, a rearranged kappa light chain gene was found. Since two of the rearranged kappa genes were observed among the 10 with germline heavy chain genes, 2 1 of 29 surface immunoglobulin-negative tumors reveal genetic evidence of B cell lineage. The entire series was assessed for rearrangement of the beta chain of the T cell receptor employing &M-k, a restriction enzyme that reliably reveals T cell receptor beta chain gene rearrangements in tumors of known T cell lineage [I 1,13,14]. A single non-germline T cell receptor beta chain gene band was observed in seven of the initial 100 consecutive specimens whose surface markers were studied in suspension: three of B cell chronic lymphocytic leukemia, two of follicular lymphoma, one of diffuse large cell lymphoma, and one of diffuse mixed lymphoma. Rearranged bands could not be readily identified with other restriction enzymes, raising the possibility of polymorphism rather than rearrangement. In all seven cases, a rearranged immunoglobulin heavy chain gene was found, in four a kappa light chain gene was

* One sample with germline immunoglobulin heavy and kappa light chain genes showed an arranged gene for the beta chain of the T cell receptor, and one each with germline and with deleted immunoglobulin heavy chain genes revealed rearranged kappa light chain genes.

tion and neutralization, the DNA was transferred to nitrocellulose paper by the technique of Southern [a]. Hybridization was carried out at 68% in a solution of 5 X SET (1 X SET = 15 mM sodium chloride, 1 mM EDTA, 30 mM Tris hydrochloride; pH 8.0), 1 X Denhardt’s solution, 0.5 percent sodium dodecyl sulfate, IO percent dextran sulfate, 20 mM sodium phosphate (pH 7.0), 40 pg/ml salmon sperm DNA, and 0.025 to 0.10 pg of nick-translated 32P-labeled (IO7 dpm) DNA probe. Human genomic DNA probes specific for the immunoglobulin heavy chain joining region [9], the kappa immunoglobulin light chain constant region [lo], and the constant region of T cell receptor beta chain gene (a BgllllEcoRV fragment of YT35 [ 1 I]) were employed. Filters were prehybridized for two hours at 68’C in prehybridization buffer, and hybridized for 15 to 20 hours at the same temperature. Following hybridization, filters were washed successively for one-hour periods at 68’C with 2 X SET containing 0.5 percent sodium dodecyl sulfate, 1 X SET with 0.25 percent sodium dodecyl sulfate, and twice with 0.5 X SET containing 0.125 percent sodium dodecyl sulfate. Autoradiography of the washed filters was carried out for one to seven days at -7O’C with two intensifying screens. Details of the standard methods are available

[121* RESULTS 1 illustrates rearrangements of the immunoglobulin heavy chain gene (genomic DNA digested with EcoRI) and immunoglobulin light chain gene (&rI-lI digestion) in 12 representative patients with non-Hodgkin’s lymphoma. It should be noted that germline bands may be derived from the nonmalignant cells of lymphoma tissue. Further, not all rearranged genes are detected with a single restriction enzyme, although rearrangement of the heavy chain gene is rarely overlooked if both EcoRl and Sacl are employed. Table I summarizes the results of immunoglobulin gene studies in specimens from 100 consecutive adult patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. One or more rearrangements of the heavy Figure

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GENES

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LYMPHOMA-AISENBERG

ET AL

rearranged as well, and in three clonal surface immunoglobulin was present; in five of the seven specimens, clonal surface immunoglobulin and/or a rearranged kappa band was detected in addition to the rearranged heavy chain gene. One additional non-germline band was detected in the immunoperoxidase series of surface immUnoglobulin-negative specimens; in this sample of a diffuse large cell lymphoma developing on a background of lymphomatoid granulomatosis, the genes for both the light and heavy immunoglobulin chains were in germline configuration. In summary, a non-germline T cell receptor beta chain gene band was observed in four of the entire group of 29 surface immunoglobulin-negative tumors, but the available evidence favored a T cell lineage in only one. COMMENTS

The rapid progress of the past two decades in understanding malignant lymphoma is a direct result of the dramatic advances in immunobiology and immunogenetics. Although histologic subtype continues to guide patient management, the novel techniques derived from basic science define lymphoma phenotype and genotype, provide fundamental insights into pathogenesis, and supply the foundation of future progress. Close upon recognition that lymphocytes could be divided into bone marrow-derived B cells and thymus-processed T cells, surface marker techniques (first heteroantiserum and later monoclonal antibodies) were applied to human lymphoma [ 15,161. In addition to allowing identification of B cell populations through the presence of surface immunoglobulin, surface marker analysis can establish the monoclonal character of B cell proliferations through the exclusion of one immunoglobulin light chain; in the absence of clonal surface immunoglobulin, other phenotypic markers can provide convincing, albeit not conclusive, evidence of B lineage. Surface markers can also identify a T cell subset, but do not establish clonality, since no characteristic similar to immunoglobulin light chain restriction has been recognized in T cells. In the past five years, the powerful techniques of molecular genetics have also been applied to malignant lymphoid populations. Korsmeyer et al [ 17,181 first demonstrated that the clonal rearrangement of immunoglobulin genes that takes place during B cell ontogeny can identify clonal proliferations of leukemic B cells; not only do B lineage chronic lymphocytic leukemia cells exhibit such immunoglobulin gene rearrangement, but cells of the common (non-T, non-B) variant of acute lymphocytic leukemia could be assigned to the B cell lineage on the basis of similar genetic alterations. When probes specific for the T cell receptor became available, we [ 131 and others [ 11,14] observed that these techniques could, for the first time, identify clonal proliferations of T lymphocytes. The present study was prompted by the need for a

Figure 1. Rearrangement of the immunoglobulin heavy chain (JH, upper panel) and light chain (C,,,,,, lower panel) genes in 72 cases of malignant lymphoma: lanes 1 to 3, follicular lymphoma; lanes 4 and 6, diffuse poorly differen% ated lymphocytic lymphoma; lanes 5, 10, and 11, diffuse large cell (histiocytic) lymphoma; lanes 7 to 9, diffuse welldifferentiated lymphocytic lymphoma; lane 12, intermedb ate cell lymphoma. The surface immunoglobulin of the specimens in lanes 4, 6, and 11 is of lambda light chain type, and the surface immunoglobulin of the remainder of the specimens is of kappa type. The far right lane contains a Hind/l/-digested lambda DNA-size marker. The solid bars on the left indicate the position of the germline bands, and arrows point to rearrangements. The samples in the upper panel were digested with EcoRI, and the those in the lower panel with BamHI. Digestion of fhe specimen in lane 3 with Sacl revealed a single rearranged band after probing with JH.

comprehensive investigation of the various subtypes of non-Hodgkin’s lymphoma with the new genetic techniques. Thus, we examined immunoglobulin and T cell receptor genes in 120 adult lymphomas and chronic lymphocytic leukemias subclassified by a modified Rappapot-i classification and characterized phenotypically with a panel of monoclonal antibodies. Our findings indi-

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LYMPHOMA-AISENBERG

ET AL

lymphoma arising in a patient with lymphomatoid granulomatosis; in this surface immunoglobulin-negative sample, the immunoglobulin genes were in germline configuration and a non-germline T cell receptor beta chain gene band was identified. Of the subtypes of adult lymphoma and lymphocytic leukemia we st@ed, diffuse lymphocytic lymphomas and chronic lymphocytic leukemia never presented a problem in lineage assignment on the basis of surface marker and molecular genetic criteria. All our cases showed immunoglobuljn gene rearrangements; the cases of typical chronic lymphocytic leukemia lacking detectible surface immunoglobulin [23,24] may reflect an inability to detect the very low concentrations of surface immunoglobulin that characterize some of these proliferations [25]. Immunoglobulin gene rearrangements confirm the B cell lineage of such surface immunoglobulin-negative cases. The two subclasses of lymphoma that present some difficulty in lineage assignment are diffuse large cell (histiocytic) lymphoma and follicular lymphoma. Most investigators are unable to establish a B cell lineage on the basis of surface marker study in one fourth to one third of the cases of diffuse large cell lymphoma [6,26] and in a smaller fraction of follicular lymphomas [6]. We now repot-t that immunoglobulin gene rearrangements indicate that at least two thirds of the surface immunoglobulinnegative cases of diffuse large cell lymphoma are of B cell lineage, and that one half of surface immunoglobulinnegative follicular lymphomas can be similarly assigned. These findjngs confirm and extend earlier immunoglobulin investigations from other laboratories [27-301. A small fraction of non-Hodgkin’s lymphomas remain that cannot be assigned a B cell lineage by either surface markers or molecular genetic analysis-in the present series, about 10 percent of the cases of follicular lymphoma and diffuse large cell lymphoma. In on!y one of eight such cases in our series, a large cell lymphoma with lymphotiatoid granulomatosis morphologic features, did a rearranged T cell receptor gene raise the possibility of T cell lineage. The inability to assign a lineage in this small group of cases may have several explanations. Most plausibly, the difficulty arises from the insensitivity of Southern blotting in the mixed populations of cells that characterize some lymphomas. In our experience, a IO percent admixture of tumor cell DNA is required to reliably detect a clonal gene rearrangement, rather than the 1 percent figure others have reported [31]. To be sure, the latter figure can sometimes be realized with judicious selection of probe, tissue, and restriction enzyme; however, under the usual conditions of the assay, 10 percent sensitivity is a more representative figure (Figure 2). Since many follicular lymphomas may be admixed with a large preponderance of nonmalignant T cells [32], the inability of both surface markers and molecular genetics

Figure 2. Sensitivity of the Southern blot hybridization technique for detecting rearrangement of the immunoglobulin heavy chain (J”) gene. Mixtures confaining a total of 6 pg of DNA from a lymphocytic lymphoma and from a nonmalignant lymph node were digested with either EcoRl (lanes 1 to 5) or Sac/ (lanes 7 to 1 I). The percentage of lymphoma DNA is indicated above the autoradiogram. The solid bars indicate the germline position of the gene, and the open arrows indicate the rearranged band(s). Lane 6 is the Hind/l/-digested DNA-size marker.

cate that almost all non-Hodgkin’s lymphoma in adults is of B cell lineage; we identified immunoglobulin gene rearrangements in 97 percent of 100 consecutive cases studied, and in 2 1 of 29 selected because of the absence of detectable immunoglobulin. Seven cases assigned a B cell lineage on the basis of rearranged immunoglobulin genes with or without surface immunoglobulin, displayed a non-germline band after digestion with the restriction enzyme BarnHI and hybridization with the probe for the T cell receptor beta chain gene; five of the seven could be securely assigned a B cell lineage on the basis of rearranged heavy chain genes and either clonal surface immunoglobulin or a rearranged kappa chain gene. The B lineage assignment of the other two was less secure since it was based only on rearranged heavy chain genes [ 171. T cell receptor beta chain gene rearrangements in non-T cell neoplasms have been described by ourselves [ 191 and others [20,2 I], but the significance of the finding remains unclear. In the present work, the available evidence raises the possibility that some of the non-germline T cell receptor beta chain gene bands represent polymorphism [22] or another mechanism rather than rearrangement. The balance of evidence favored T cell lineage in only one specimen in our entire series, a diffuse large cell

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to detect a clonal B cell population in some of these tumors may simply reflect the sensitivity limits of the two techniques. A similar argument can be applied to diffuse large cell lymphomas, but a proviso should be added: some of these neoplasms may not be of lymphoid origin. Thus, the accumulating evidence indicates that lymphomas of T cell lineage are rare in the adult population seen at institutions such as ours. It should be emphasized that cases of lymphoma and leukemia in children and adolescents were excluded from consideration (hence the absence of cases of T cell lymphoblastic lymphoma and T cell acute lymphoblastic leukemia). The chronic lymphocytic leukemias of T cell lineage in adults were also excluded from this study. These rare T cell chronic lymphocytic leukemias include cutaneous T cell lymphoma

GENES

IN NON-HODGKIN’S

LYMPHOMA-AISENBERG

ET AL

(Sezary syndrome), adult T cell lymphoma/leukemia (HTLV-I associated disease of the Japanese/Caribbean type), chronic T-8 lymphocytosis (with and without neutropenia and/or splenomegaly) and miscellaneous still unclassified chronic T cell leukemias. However, with the exclusion of the childhood lymphomas and these uncommon chronic leukemias of T cells, non-Hodgkin’s lymphoma in adults is, with rare exception, a B cell neoplasm. ACKNOWLEDGMENT We are grateful to Dr. Philip Leder for the immunoglobulin probes that were supplied as recombinant bacteriophage, and to Dr. Tak W. Mak for the probe for the T cell receptor beta chain.

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Siminovitch KA, Jensen JP, Epstein AL, Korsmeyer SJ: lmmunoglobulin gen rearrangements and expression in diffuse histiocytic lymphomas reveal cellular lineage, molecular defects and sites of chromosomal translocations. Blood 1986; 67: 391-397. Cleary ML, Chao J, Warnke R, Sklar J: lmmunoglobulin gene rearrangement as a diagnostic criterion of B-cell lymphoma. Proc Natl Acad Sci USA 1984; 81: 593-597. Aisenberg AC, Wilkes BM, Long JC, Harris NL: Surface phenotype in lymphoproliferative disease. Am J Med 1980; 68: 206-213.