Expression of a T-Cell Antigen (Leu-1) by

B-Cell Lymphomas

BRUCE F. BURNS, MD, FRCP(C),ROGER A. WARNKE, MD,

REUBEN S. DOGGETT, MD, andROBERT V. ROUSE, MD

The Leu-1 antigen has been defined by monoclonalantibodies (L17F12, T101, and OKT-1) as a pan-T-cellantigen present on all human peripheral blood T cellsand thymocytes. Although originally thought to beconfined to T-cell lineage, some cases of B-cell chroniclymphocytic leukemia have been found to react withthese antibodies. Using a frozen section immunoper-oxidase staining technique, 125 lymphomas with B-celldifferentiation were examined for the presence ofLeu-1antigen. Leu-1 antigen was detected in 4 of 11 cases ofdiffuse small lymphocytic lymphoma (Rappaport'sDWDL) and 3 of 4 cases of diffuse intermediate lym-phocytic lymphoma. Follicular lymphomas less oftenexpressed this antigen-2 of 29 cases of the smallcleaved cell type (Rappaport's NPDL), none of 13 casesofmixed small cleaved and large cell type (Rappaport'sNM), and 1 of 6 cases of large cell type (Rappaport'sNH). Diffuse lymphomas of presumed follicular center

SINCE the advent of the hybridoma monoclonal an-tibody technique a wide variety of antigenic determi-nants have been found that appear to be specific forT lymphocytes. One of these is Leu-1, a 67,000-dal-ton complex found on the T-cell membrane.' Thisantigen is found on thymocytes, normal T-lympho-cytes, and most neoplastic T cells but is not detect-able on normal B cells.2 Recently, however, this anti-gen4'5 or a similar antigen6 has also been found on thecell membrane in some cases of B-type chronic lym-phocytic leukemia (B-CLL). These B-CLL cells boreimmunoglobulin (Ig+) and lacked the E-rosette re-ceptor for sheep red blood cells. There have been iso-lated reports of Leu-1-positive, Ig-positive B-cell ma-lignant lymphomas7'9 but only limited data on the in-cidence of this phenomenon in the various histologicsubtypes of these tumors is available.10'"1

In this report we studied the immunologic pheno-types of 125 non-Hodgkin's lymphomas of B-cell dif-

From the Department of Pathology, Stanford UniversityMedical Center, Stanford, California

cell origin expressed this antigen infrequently aswell-1 of 3 cases of the small cleaved cell type (Rap-paport's DPDL), neither of 2 cases of mixed smallcleaved and large cell type (Rappaport's DM), and 3 of43 of large cell type (cleaved/noncleaved) (Rappaport'sDH). Diffuse large cell, immunoblastic lymphoma ofB-cell type expressed Leu-1 in 1 of 6 cases. None of the3 cases of Burkitt's lymphoma or of the three smallnoncleaved non-Burkitt's lymphoma (Rappaport'sundifferentiated) expressed detectable Leu-1. B-lym-phoblastic lymphoma (1 case) and B-cell unclassifiedlymphoma (1 case) both failed to express detectableLeu-1. It appears that this pan-T-cell antigen is mainlyfound on those B-cell lymphomas composed predomi-nantly of small lymphocytes. This finding may be ofuse in distinguishing extranodal neoplastic collectionsof small lymphocytes from lymphocytic hyperplasias.(Am J Pathol 1983, 113:165-171)

ferentiation, looking specifically for the expression ofLeu-I antigen on neoplastic cells.

Materials and Methods

Staining Technique

All lymphoma biopsies used in this study were re-ceived fresh and unfixed. They were cut into 3-mmcubes and placed into embedding medium (OCT,Miles Laboratories, Naperville, Ill) in air-tight plastic

Dr. Burns was a Hematopathology Fellow, supported inpart by a McEachern Fellowship from the Canadian Can-cer Society and by CA-05838 from the National Institutesof Health.Accepted for publication June 13, 1983.Address reprint requests to Dr. Roger Warnke, Depart-

ment of Pathology, Stanford University Medical Center,Stanford, CA 94305.

165

166 BURNS ET AL

Table 1 -Antibodies Employed

Antibody Specificity

L 17F12* Leu-1 (pan-T-cell)SK1 * Leu-2a (cytotoxic/suppressor T-celIl)SK3* Leu-3a (helper/inducer T-cell)SK7* Leu-4 (pan-T-cell)ATM 1.1 Leu-5 (pan-T-cell, sheep E-rosette receptor)Anti-K, anti-A* x and A lg light chainsAnti-lAt A lg heavy chain63D3 or 61 D3t Monocyte/histiocyte seriesB1§ Pan-B-cellT015l1 Pan-B-cell

* Becton-Dickinson, Mountain View, California.1314t Courtesy of Dr. R. Miller, Stanford, California.t Courtesy of Dr. J. D. Capra, Dallas, Texas.15§ Coulter-Clone, Hialeah, Florida.16Courtesy of Drs. D. Y. Mason and H. Stein, Oxford, England."

capsules. These were snap-frozen in isopentane anddry ice and stored at - 60 C prior to sectioning. Thebiopsies were sectioned at 4-A thickness in a cryostat,immediately fixed in acetone for 3 seconds and al-lowed to air-dry. They were then stored at - 20 C un-til staining (up to 2 weeks). Immediately prior tostaining they were fixed again for 10 minutes in coldacetone, then hydrated in PBS. The details of thisstaining procedure have been published previously.12Briefly, it consisted of a first-stage incubation withone of the monoclonal antibodies mentioned below.After washing, this was followed by biotin-conju-gated goat anti-mouse Ig F(ab')2 antibody fragments(Tago, Burlingame, Calif) prior to a third stage ofavidin-conjugated horseradish peroxidase (VectorLabs Inc., Burlingame, Calif). The sections werethen incubated with diaminobenzidine (DAB) fol-lowed by copper sulfate and counterstained withmethylene blue.

Monoclonal Antibodies Employed

In the study of these lymphomas the followingmonoclonal antibodies were used in varying com-binations (Table 1).

Case Selection and Interpretation

All cases of malignant lymphoma studied immuno-enzymatically in our laboratory from May 1980 toOctober 1982 were reviewed with the use of a conven-tional light microscope. Included for review were alllymphomas stained as diagnostic cases as well asthose included in series of follicular lymphomas andlarge cell lymphomas stained for research purposes.Those cases that demonstrated staining of neoplasticcells for two or more T-cell antigens (Leu-1, -2, -3,

-4, or -5) or monocyte/histiocyte antigens (with anti-bodies 63D3 or 61D3), or lacked any lineage specificmarkers at all were excluded. The remaining casescomprising this study demonstrated features of B-celldifferentiation (expressing x or A light chains and/or ,uheavy chain and/or expression of pan-B-cell markersdefined by monoclonal antibodies BI or T015). All ofthese cases of B-cell lymphoma were also examinedfor Leu-l antigen expression. Most cases in this se-ries, and any cases expressing Leu-l antigen, werealso stained with another pan-T-cell monoclonal an-tibody, anti-Leu-4. This was used to assess the num-ber of reactive T cells present in the tumor and servedas a negative control for the tumor cells. The patternand intensity of Leu-I expression and other charac-teristics of the immunologic phenotype were re-corded.

In every case additional biopsy material was for-malin-fixed and paraffin-embedded, and sectionswere stained with hematoxylin and eosin for histo-pathologic assessment. The NIH Working Formula-tion of non-Hodgkin's lymphomas,17 with its Rappa-port classification equivalent, were used in subtypingthe lymphomas. Those cases of diffuse small lympho-cytic lymphoma were differentiated from CLL by vir-tue of having absolute lymphocyte counts of less than

Table 2- Leu-1 Expression in Various Subtypes ofNon-Hodgkin's Lymphoma

Number of Leu-1-positive cases/Histologic subtype Total number examined

Small lymphocytic 4/11Lymphocytic intermediate 3/4

differentiationFollicular small cleaved 2/29

cellFollicular mixed small 0/13

cleaved and large cellFollicular large cell 1/6Diffuse small cleaved 1/3

cellDiffuse mixed small 0/2

cleaved and large cellDiffuse large cell, cleaved 3/43and noncleaved

Diffuse large cell 1/6immunoblastic (B-celltype)

Burkitt's 0/3Undifferentiated, Non- 0/3

Burkitt'sB-lymphoblastic* 0/1B-unclassified 0/1

Totals 15/125

* An unusual gastric lymphoma with morphologic features of theconvoluted lymphoblastic type. Immunostaining demonstrated X lightchains, JA heavy chains, la antigens, and expression of the commonacute lymphocytic leukemia (ALL) antigen (J5).

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T-CELL ANTIGEN IN B-CELL LYMPHOMAS 167

4500/cumm. Four cases, not readily classifiable inthe working formulation, were felt to have featuresof the intermediate lymphocytic type.18

Results

The number of lymphomas showing Leu-1, Leu-4- tumor cells in each histologic subtype is shown inTable 2. In no case did the neoplastic cell populationstain for the Leu-2, -3, or -4 antigens.Lymphomas of the small lymphocytic type and

those of intermediate lymphocytic type had the high-est incidence of reactivity, 4 of 11 cases in the formergroup and 3 of 4 in the latter. In contrast, the follicu-lar forms of lymphoma had a much lower incidenceof Leu-1 expression on tumor cells; three out of a to-tal of 48 follicular lymphomas of all cell types. Thediffuse forms of lymphoma of presumptive follicularcenter cell origin in this series (based on cytologic fea-tures and the presence of Ig or other B-cell markers)expressed Leu- 1 in 4 of 48 cases. Burkitt's lym-phoma, also currently thought to be of follicular cen-

ter cell origin, failed to express Leu-l in the threecases examined. None of the undifferentiated non-

Burkitt's lymphomas expressed Leu-1, but one Blarge cell immunoblastic lymphoma did.The immunologic phenotypes in the 15 lymphomas

expressing Leu-I are presented in Table 3. All butthree cases demonstrated unequivocal evidence of re-

stricted light chain expression, and all but one were

found to have ,u heavy chain.Leu-l expression was generally weaker on the neo-

plastic B cells than on the T cells invariably found as-

sociated with the lymphomas (Figures 1 and 2). TheseT cells, also showing strong Leu-4 positivity, were

randomly distributed in the diffuse lymphomas. Inthe follicular lymphomas they were concentrated inthe interfollicular regions; there were only scatteredcells in the neoplastic follicles (Figure 2B).

There did not appear to be any definite correlationbetween the strength of Leu-l expression on the neo-

plastic lymphocytes and the histologic subtype oflymphoma. While the small lymphocytic formstended to show rather discrete membrane staining forLeu-1, the large cell lymphomas usually had a

coarser, less well defined pattern of staining.An additional case, not included in this group of

lymphomas expressing Leu-1, was originally diag-nosed as chronic lymphocytic leukemia with lympha-denopathy evolving into a diffuse large cell lym-phoma (Richter's syndrome). A lymph node from thesupraclavicular area showed a diffuse infiltration ofsmall lymphocytes, which were demonstrated to ex-

press x light chains, y heavy chains, and Leu-l anti-gen. A second lymph node biopsy from the same re-

gion less than 1 year later showed only small areas ofresidual small lymphocytic infiltrate with large celllymphoma effacing the lymph node in a diffuse pat-tern. In this specimen the large cells also showed xlight chain and , heavy chain but lacked demonstra-ble Leu-1.

Discussion

Leu-I antigen was originally defined as an antigenon human peripheral T-lymphocytes, thymocytes,

Table 3-Immunologic Phenotypes of Lymphomas Expressing Leu-1

Pattern andintensity

Other of Leu-1Case Lymphoma Immunoglobulin B-cell expressionno. subtype light chain markers on tumor cells

1 Diffuse small lymphocytic x Uniform, weak2 Diffuse small lymphocytic ±t X; Uniform, weak3 Diffuse small lymphocytic A Uniform, moderate4 Diffuse small lymphocytic A Uniform, moderate5 Diffuse intermediate lymphocytic +x' A Variable, most weak6 Diffuse intermediate lymphocytic A IA Uniform, strong7 Diffuse intermediate lymphocytic x ; Uniform, weak8 Follicular small cleaved cell x M Uniform, weak in follicles9 Follicular small cleaved cell A ; Uniform, weak in follicles10 Follicular large cell x B,T015t Uniform, moderate11 Diffuse small cleaved cell A ; Uniform, moderate12 Diffuse large cleaved cell x pA, B, Uniform, weak13 Diffuse large cell pA Uniform, weak14 Diffuse large cell A jA, B, Uniform, weak15 Diffuse large cell immunoblastic None A, B, Uniform, weak

Probable weak positivity but high background rendered interpretation difficult.t Only Cases 10, 12, 14, and 15 were tested for B,, and only Case 10 for T015.

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168 BURNS ET AL

Figure 1 -Case 1. Cryostat sections oflymph node from a patient with diffuse,small lymphocytic lymphoma (DWDL). (Im-munoperoxidase with methylene bluecounterstain, all x 300) A-Anti-Leu-1. Virtually all cells show uniform weakstaining. Scattered, strongly staining lym-phocytes (arrowheads) are probably Tcells. B-Anti-Leu-4. Most cells do notstain. Scattered T cells stain strongly (ar-rowheads). C-Anti-n. Virtually allcells show uniform strong staining, con-firming the B-cell nature of the lymphoma.

AJP * November 1983

T-CELL ANTIGEN IN B-CELL LYMPHOMAS 169

Figure 2-Case 8. Cryostat sections oflymph node from a patient with follicularsmall cleaved cell lymphoma (NPDL). (Im-munoperoxidase with methylene bluecounterstain, all x 120) A-Anti-Leu-1. Most cells show weak staining in thisnodule. In the central region a cluster ofstrongly staining cells is seen (arrow-heads), corresponding to the T cells iden-tified in b. B - Anti-Leu-4. A cluster ofstrongly staining T cells is present in thecenter of this nodule with scattered cellsin the two adjacent follicles. Most of thecells in the neoplastic follicles do notstain. C-Anti-.. Here the neoplasticcells in the follicle stain strongly, whilethe central cluster of T cells (arrowhead)does not stain.

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170 BURNS ET AL

and T-lymphoblastic leukemia cells and can be de-fined by the OKT1, TIOI, 10.2, A-50, and L17F12antibodies.2-46 Since the introduction of these anti-bodies, however, a number of investigators have re-ported finding this antigen on chronic lymphocyticleukemia cells3'4-6'1 of B-cell origin.The present study demonstrates that this antigen

may also be expressed on a small number of lympho-mas of B-cell origin. This is, perhaps, not surprisingin the small lymphocytic subtype of malignant lym-phoma in view of its close relationship to B-CLL,both on histogenetic and pathologic grounds,19 al-though none of the 15 cases were leukemic. Indeed itis within this group and the closely related lympho-cytic, intermediate (differentiation) subtype that theincidence of Leu-I positivity was highest (7 of 15cases in all). Previous suspension studies of B-CLLcells have found Leu-1 expression in frequenciesranging from 44% to 100%/, with most greater than80%. The slightly lower incidence in our series ofsmall lymphocytic lymphomas may reflect a lesser de-gree of sensitivity of immunoperoxidase, comparedwith fluorescence activated cell sorter analysis or mayreflect actual differences in antigen expression. How-ever, the expression of this antigen was not restrictedto these lymphomas, because Leu-1 was also found in3 cases of follicular lymphoma, 1 case of diffusesmall cleaved cell type, 3 cases of diffuse large celltype (cleaved/noncleaved), and 1 case of large cellimmunoblastic type.Our findings, in 10 of the cases, of relatively

weaker Leu-l staining on the neoplastic cells whencompared with the admixed reactive T cells is not sur-prising. Wormsley et al,20 using a monoclonal anti-body, TIOI, which reacts with a T-cell antigen withcharacteristics similar if not identical to those ofLeu-1, found that antigen density was lower onB-CLL cells than on normal peripheral blood T cells.However, 5 of our cases showed little detectable dif-ference in the intensity of staining between the neo-plastic B cells and reactive T cells as assessed by tissuesections stains. In these situations comparison withthe number of T cells staining with Leu-4 provided anapproximation of the relative numbers and distribu-tion of reactive cells in the tumor. We do not feel thatthis represents expression of B-cell antigens by T-celllymphomas, for in none of the cases did we detectany other T-cell antigens on the neoplastic cells, andin all cases we found evidence of expression of atleast two B-cell markers.The murine pan-T-cell antigen Lyt-I exhibits ho-

mologies to Leu-l in structure and in expression bydifferent T-cell populations. Lanier et al2l have de-

scribed expression and production of Lyt-I by other-wise typical mouse B-cell lymphomas and Hayakawaet a122 have recently described a small subpopulationof nonneoplastic murine B lymphocytes that bear theLyt-1 antigen. According to current theories regard-ing the histogenesis of non-Hodgkin's lymphomas,23each of these different histologic subtypes representsan example of a clone of neoplastic lymphocytes ar-rested at a particular point in the differentiation ortransformation sequence. However, Leu-I expres-sion has not been noted on transforming follicularcenter cells in a number of reactive follicular hyper-plasias also studied at our institution,2 although oc-casional cells in follicles expressing Leu-l are inter-preted as T cells because Leu-4 stains a similar num-ber of cells.24 Whether there is a "hidden" populationof Leu-1- and Ig-bearing normal B cells is not cer-tain, but a subpopulation of normal B cells that formmouse RBC rosettes and also express Leu-1 has beennoted by others.23 In the mouse, a small populationof peripheral lymphocytes is Lyt-l-positive but lacksthe T-cell antigen Thy-i; some of these cells havebeen identified in B cell zones of lymph nodes andspleen.26 Other explanations, as pointed out byLanier et al,21 for the expression of a T-cell antigenby B-cell neoplasms include gene derepression, or se-lective induction during lymphomagenesis of an anti-gen which is either not expressed or expressed at un-detectable levels by normal B cells.

Expression of Leu-I antigen on neoplastic B cellsmay lead to erroneous impressions in lymphomasstudied by cell suspension or tissue section techniquesif only anti-Leu-1 is used to detect T cells and if Igstaining is equivocal (not uncommon in small lym-phocytic neoplasms, which may express low levels ofIg). In fact, one of the cases in our series was origi-nally thought to be a T-cell lymphoma because of thenearly 1000/o Leu-1 expression. However, further ex-amination of frozen tissue showed that this samepopulation of cells did not express Leu-4 and showedlight-chain restriction, confirming its B-cell origin.Although we have not yet observed a lymphoma ex-pressing solely Leu-1 with no other antigens (B or T),our results suggest that such a finding may not be un-equivocal evidence of T-cell differentiation.

Sometimes distinguishing lymphocytic hyperplasiafrom diffuse, small lymphocytic lymphoma can bedifficult in extranodal locations such as the skin, or-bit, gastrointestinal tract, and bone marrow becauseof high serum Ig background and/or low level Ig pro-duction, as mentioned above. Here the finding ofLeu-1 expression, without Leu-4 expression, in thesecollections of small lymphocytes may be useful in

AJP * November 1983

Vol. 113 * No. 2 T-CELL ANTIGEN IN B-CELL LYMPHOMAS 171

confirming their neoplastic nature. The incidence ofthis finding may approach 50Gb if results from ourstudy and others10 are representative.

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Acknowledgments

We wish to acknowledge the photographic assistance ofM. Phil Horne and the secretarial assistance of Mrs. Mar-garet Beers in the preparation of the manuscript.