Proc. Nat. Acad. Sci. USAVol. 71, No. 3, pp. 685-689, March 1974

Human Leukemia-Associated Anti-Nuclear Reactivity(immunology/neoplasms/anti-complement fluorescence/myeloblasts/lymphomas)

GEORGE KLEIN*t, MELITA STEINER*, FRANCIS WIENER*, AND EVA KLEIN*t

*Department of Tumor Biology, Karolinska Institutet, S 104 01 Stockholm 60, Sweden; and tDepartment of Immunology,Hadassah Medical School, Jerusalem, Israel

Contributed by George Klein, October 27, 1973

ABSTRACT A brilliant, coarsely granular nuclearantigen was detected by anti-complement immuno-fluorescence in the nuclei of acute myeloid leukemiamyeloblasts. Designated as LANA (leukemia-associatednuclear antigen), the reactivity differs from that of theEpstein-Barr-virus-determined nuclear antigen (EBNA)in immunological specificity and morphological appear-ance, although it is visualized by the same method. Serumfrom acute myeloid leukemia patients gave positivereactions in 73% of the cases. In acute lymphatic leukemia,chronic myeloid leukemia, chronic lymphatic leukemia,and Burkitt's lymphoma the sera were positive in 35, 14, 19,and 24%, respectively. Two of five polycythemia and twoof eleven myeloma sera were also positive. Among 61healthy controls, 58 were negative, whereas three showed adiffuse nuclear staining with a different pattern. Among24 carcinoma patients, 18 were negative, whereas six gavea nuclear staining with a different, diffuse pattern. Serafrom 20 patients who had recovered from infectiousmononucleosis were all negative. In addition to theblasts of acute myeloid leukemia, a similar reactivity wasseen with two Epstein-Barr virus DNA and EBNA-negativeAfrican lymphoma biopsies and in a short-lived tissueculture line derived from one of them. LANA could be afetal or tissue-specific antigen, a virally determined anti-gen, or a specific form of anti-nuclear reactivity.

We have recently described (1) a nuclear antigen, EBNA,determined by Epstein-Barr virus (EBV). It is present in allhuman lymphoblastoid cell lines that carry the EBV genomeand in Burkitt's lymphoma (BL) biopsies. Between 80 and95% of all nuclei stain for EBNA. Its presence does notdepend on virus production; both producer and nonproducerlines carry it regularly. EBNA is absent from lines that lackthe viral genome. Negative lines include a human lympho-blastoid line of probable T (thymus-derived)-lymphocyteorigin (2) and simian lymphoblastoid lines that carry herpes-virus saimiri. Superficially at least, EBNA resembles theT-antigens of polyoma-, SV40-, and adenovirus-transformedcells. It differs from the T-antigens in one important respect,however: T-antigens are in the nuclear sap and enter thecytoplasm during mitosis, whereas EBNA remains associatedwith the chromosomes (1).

Abbreviations: EBV, Epstein-Barr virus; EBNA, Epstein-Barrvirus-determined nuclear antigen; BL, Burkitt's lymphoma; T,thymus-derived; ACIF, anti-complement immunofluorescence;ALL, acute lymphatic leukemia; CLL, chronic lymphatic leu-kemia; AML, acute myelogenous leukemia; CML, chronic myelo-genous leukemia; AMML, acute monocytic myelogenous leukemia;LANA, leukemia-associated nuclear antigen; BSS, balanced saltsolution; FITC, fluorescein isothiocyanate; WBC, white blood cell.

In spite of its constant presence in EBV-genome-carryingcells, EBNA cannot be demonstrated by direct or indirectfluorescence, presumably because its quantity is too small inrelation to the sensitivity threshold of the eye. Anti-comple-ment immunofluorescence (ACIF) gives a brilliant staining,however (1). This is in line with the higher sensitivity of thistest (3-5).

In view of the fact that Burkitt's lymphoma biopsy cells,known to carry the EBV genome (6, 7), regularly containEBNA, we looked for the antigen in buffy coat smears pre-pared from various human leukemias and lymphomas. Wehave set up a three-point screening test against each cellspecimen, with an EBNA-negative control serum, an EBNA-positive reference reagent and the autochthonous serum ofeach cell donor. We have failed to demonstrate EBNA inperipheral leukemic cells derived from acute or chronic,lymphatic or myelogenous leukemias (ALL, CLL, AML, andCML). In the course of this screening, another nuclear reac-tion was found, however, in an autochthonous leukemia cell-serum combination. The responsible antigen is tentativelydesignated LANA (leukemia-associated nuclear antigen).It can be demonstrated by the anti-complement fluorescencetechnique, like EBNA, but it is immunologically differentand has another morphological appearance.

MATERIALS AND METHODS

Target Cells. Buffy coat leukocytes, obtained by the sedi-mentation of cells of heparinized blood, lymphoblastoid celllines maintained in serially passaged stationary suspensioncultures, and Burkitt's lymphoma (BL) biopsy specimenswere used. Lymphoblastoid cell lines were propagated andBL biopsy cell suspensions prepared as described elsewhere(1,8).

Anti-Complement Immunofluorescence (ACIF) Test. Smearswere prepared by spreading a concentrated suspension ofwashed buffy coat or tissue culture cells on clear slides (ap-proximately 3 X 105 cells per slide), air drying and fixationin chilled 1:1 acetone-methanol mixture. After fixation for5 min, the smears were dried and stored at -200.For staining, fixed smears were washed with 0.075 M KC1.

Subsequently, they were treated with a mixture of 0.1 mlof 1: 4-diluted test serum inactivated at 560 for 30 min and 10,ul of complement (C') at 370 for 30 min in a humid chamber.Human EBV-negative (viral capsid antibody titer <10) serumthat gave no EBNA reaction in the standard test with Rajicells (1) was used as the source of complement. All dilutions

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FIG. 1. Negative reaction of AD cells (AML blasts), withEBNA-positive reference serum from a patient with Burkitt'slymphoma, complement, and FITC-anti #IC/#,A. Evans bluecounter stain. X 1000.

FIG. 2. Positive LANA reaction with AD blast cells and AMLpatient serum, complement, and FITO-anti #,C/#%A. Evans bluecounter stain. X 1000.

FIG. 3. Positive LANA reaction of AD blasts with serum froman AML patient, complement, and FITC-anti ,6,C/#83A. Evansblue counter stain. X540.

were made in balanced salt solution (BSS: 0.8% NaCl, 0.014%CaCl2, 0.04% KCl, 0.02% MgSO4 * 7H20, 0.06% KH2PO4,0.06% Na2HPO4. 2H20, pH 6.9). Subsequently, the smearswere washed with stirring for 30 min, stained with 1: 30 dilutedfluorescein isothiocyanate (FITC)-conjugated anti-human

FIG. 4. Positive EBNA reaction, between EBV-genome-carrying human lymphoblastoid cells (Raji line) and the sameBurkitt patient reference serum as used for the test in Fig. 1,complement, and FITC-anti #1C/iA. Evans blue counter stain.X540.FIG. 5. Positive LANA reaction between AML blasts of

patient BE and LANA-positive AML serum, complement, andFITO-anti jSIC/#,A. Evans blue counter stain. X540.

FIG. 6. Positive LANA reaction between AML blasts ofpatient HK and LANA-positive AML serum, complement, andFITC-anti ,BIC/#,1A. Evans blue counter stain. X 540.

#IC/#,A globulin (Hyland Laboratories, Los Angeles, Calif.)at 40 for 2 hr, washed, and mounted in BSS: glycerol 1: 1. Theslides were examined in a Leitz Ortholux microscope equippedwith a vertical Ploem-type illuminator at oil immersion(X54or X100).

686 Inununology: Klein et al.

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Leukemia-Associated Nuclear Antigen (LANA) 687

TABLE 1. ACIF tests with serum from patient AD againsta spectrum of target cells

Target cell Sources Diagnosis Reaction Notes

AD (autoch- BI CML, 94% Receivedthonous) acute Positive, from Dr.

blast CGPb Dickcrisis Killander

MLC TC HVS- Neg. Ref. 9simian"

1670 TC HVS- Neg. Ref. 10simian"

Raji TC BL, EBV- +++ Ref. 12genome- FGPdcarrying

AK BI CLL Neg. Receivedfrom Dr.Bo Jo-hansson

MI BI CLL Neg.AG BI CLL Neg.B-JA TC EBV- 16% Received

genome Positive, fromand CGPb Drs.EBNA- Jos6negative Menezes"Bur- andkitt's Wolf-lym- gangphoma" e Leibold

* TC, established tissue culture line; BI, in vivo material(heparinized blood or biopsy).

b Coarsely granular nuclear fluorescence pattern (compareFig. 2).

¢ Simian lymphoblastoid line transformed by herpesvirussaimiri.

d Finely granular pattern of EBNA type (1).eLindahl, T., Klein, G., Jondahl, M., Leibold, W., Menezes, J.,

Sundstrom, C., and Nilsson, K., submitted for publication.

RESULTS

The Initial Finding. The reaction was found accidentally,in the course of an unsuccessful search for EBNA in leukemiacells. CLL cells from 12 different donors were tested by ACIF,against an EBNA-positive reference serum (00, from a BLpatient), an EBNA-negative healthy control serum (JE),and the patiert's own serum. The results were uniformlynegative. Subsequently, a similar test was performed on aCML patient (AD) who entered an acute and eventuallylethal blast crisis. At the time of the test, the white blood cell(WBC) count was 40,000 with 91% blasts. The EBV-negative(JE) and -positive (00) reference sera gave again no reaction,whereas the patient's own serum (AD) showed a brilliant,coarsely granular nuclear staining of virtually all blast cells(Figs. 1-3). The pattern was different from the character-istic diffuse, finely granular EBNA staining of EBV-carryingLCL (Fig. 4).

Specificity Controls. Conceivably, the reaction of the ADserum against the AD cells could be due to conventional anti-nuclear antibodies. However, when a spectrum of target cellswas tested against theAD serum (Table 1), the autochthonousAD cells gave again a strong reaction, whereas the EBV-

TABLE 2. Disease-associated serum reactivity againstLANA (Each serum represents one patient)

No. of No. PercentDiagnosis sera tested positive positive

AML 78 57 73ALL 31 11 35CML 36 5 14AMML& 1 0PCVb 5 2CLL 37 7 19BL (African) 50 12 24Myeloma 11 2Carcinoma 24 6" 25"Post-IMd 20 0 0Healthy control

(adult) 61 30 50

Total 354 105 30

aAcute monocytic myelogenous leukemia.b Polycythemia vera.a Atypical staining.d Infectious mononucleosis.

and fresh buffy coat cells from three cases of chronic lympho-cytic leukemia were negative. This reduces the probabilitythat a conventional anti-nuclear factor could be responsible forthe reaction. When tested against the EBV-genome-positiveRaji cell, theAD serum gave a typical EBNA staining pattern.This could be attributed to the positive (1: 20) anti-EBV (viralcapsid antibody) titer of this serum. However, since thebrilliant and diffuse EBNA pattern may cover other patterns,it is not possible to decide whether the Raji cell line also car-

ries the nuclear antigen demonstrated in the AD cell, inaddition to EBNA. For the same reason, other EBNA-positivecell lines and the usual EBNA-positive BL biopsy cell (1)could not give any information regarding the presence of thenew antigen.An exceptional, EBV-genome-negative and EBNA-negative

lymphoma biopsy was obtained from Nairobi (Dr. S. Singh),taken from a child (JA) with the clinical and histopathologicaldiagnosis of BL. A cell line (B-JA) was established from thistumor by Drs. W. Leibold and J. Menezes that grew as a

stationary suspension culture (Lindahl, T., Klein, G., Jondahl,M., Leibold, W., Menezes, J., Sundstr6m, C., and Nilsson, K.,submitted for publication). When tested against the ADserum, 16% of the cells gave a pattern of nuclear stainingsimilar to that of the AD blast cells. The serum of the same

patient was subsequently tested against the AD cell; it stained63% of the cells, with the same coarsely granular pattern as

the autochthonous serum, suggesting the presence of anti-bodies with similar specificities.The AD antigen could not be demonstrated by indirect

immunofluorescence. In the ACIF test, staining was de-pendent on active complement and disappeared when heat-inactivated complement was used.

Disease-Associated Serum Reactivity. Table 2 shows thedisease-associated serum reactivity against the nuclear anti-gen in the AD cells. All reactive sera showed the typical,coarsely granular nuclear fluorescence pattern, except nine(as indicated by a footnote to Table 2). Among 78 AML

genome-negative herpesvirus-saimiri-transformed simian lines sera tested, 57, i.e., 73%, gave a reaction, in contrast to 35%

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TABLE 3. LANA reactivity of peripheral white cells fromleukemia patients

Patient

A&AAD

BEBGCLGSHKJHJKKSSM

HENOXLIOJICNIVMILFNAASBKBLBSLE

VKBLEDTMG

MLNKPSIBE

IWRIKGITMICDCAISGPTACFO

HA

Diagnosis

AMLCML in acute

blast crisisdAMLAMLAMLAMLAMLAMLAMLAMLAMLAMLAMLAMLAMLAMLAMMLALLALLCMLCMLCMLCMLCML

CMLCMLCMLCML

CMLCMLCMLAML -CMLe

CMLCMLCMLCMLCMLCLLCLLCLLCLLCLL

WBCcount

(cells/mm')

4,400

40,0004,6007,32014,30014,80066,000

1,8001,75019,0003,5003,40063,0008,90055,00020,9002,0002,900

179,00016,00048,0003,60062,000

9,20048,0009,700

240,000

15,00040,8009,70098,000

2,80087,00019,0005,800

37,600275,00048,0006,00056,0003,600

Serumreactivityagainst

LANA LANA-pos.Differential reactions target

count of cells cellab

e Neg. 4-

91% Myeloblsts +++ +++

63% Myeloblasts +++ ++

1% Myeloblasts Neg. +

76% Promyelocytes Neg.87% Myeloblasts ++76% Myeloblasts +++ ++

57% Blasts ++a Neg. +

0.5% Myeloblasts Neg. +

60% Myeloblasts + ++

13.5% Blasts Neg.a Neg.

17.5% Blasts Neg.32% Blasts ++ Neg.67% Blasts +++ +++

48% Monocytes Neg. ++

Neg.e Neg.

3% Myeloblasts Neg.a Neg.

31% Myelocytes Neg.a Neg.

2% Myeloblasts,20% promyelocytes Neg.

a Neg.31% Myelocytes Neg.

a Neg.8.5% Blasts,36% myelocytes Neg.

15% Myelocytes Neg.Neg.

12% Promyelocytes Neg.60% Eosinophils,35% neutrophils Neg. +++

Neg.- Neg. 4e Neg.e Neg.

Neg.Neg. +++

Neg.Neg.

5% Lymphoblasts Neg.e Neg.

Coarsely granular nuclear fluorescence with AD and JA sera.None of the cells listed in Table 3 reacted with the healthycontrol (IE) or the EBNA-positive BL reference serum (00).

b Tested against AD, HK, or GS cells.No significant deviation from normal.

d Classified as AML.e Originally AML, changing into CML. Classified as CML.

of the ALL, 14% of the CML, 19% of the CLL, and 24% ofthe BL sera. Two of five polycythemia and two of elevenmyeloma-patient sera were also positive. Among 61 healthycontrol sera, 58 were negative, whereas three showed a diffuse,atypical nuclear staining. Similar atypical staining was ob-tained with six among 24 carcinoma patient sera, whereas therest were negative. All 20 post-infectious mononucleosis sera

tested were negative.

Search for LANA-Like Reactivity in Different Target Cells.Two sera that gave regularly positive reactions against ADcells, the AD serum itself, and the serum of the EBNA-negative LANA-positive BL biopsy donor JA (vide supra),were selected in combination with different target cells andused in the tests described below.

Peripheral Blood of Leukemia Patients. Table 3 summarizesthe tests with peripheral blood specimens from leukemia

patients. Cells from eight of 16 AML patients gave a positivereaction with AD and JA serum, showing the same coarselygranular pattern as the AD cells (Figs. 5 and 6). They wereall negative with the EBNA-positive reference serum (00)and the healthy control serum (IE). The frequency of myelo-blasts was 91, 63, 87, 76, 57, 60, 32, and 67%, respectively,in the eight positive specimens. In the eight negative AMLsamples, three showed no significant deviation from a normaldifferential WBC count, four had 0.5, 1.0, 13.5, and 17.5%blasts, respectively, and one had 76% relatively well-differ-entiated myelocytes. Although the sample size is small, thefigures suggest that the reactivity was dependent on a pre-ponderance of blast cells. It is interesting that two patients(BE and IOJ) with relatively low WBC counts (4,600 and8,900) but a considerable frequency of blasts (63 and 32%)gave clearly positive reactions (Fig. 5) whereas patients CLand AL with a much higher WBC count but a low frequencyof blasts, were negative.Two ALL, one AMML, 18 CML, and five CLL cases were

all negative in spite of high WBC counts (up to 240,000 inCML patient MG and 275,000 in CLL patient ISG).The contrasting clinical histories of patients AD and IBE

are of special interest. AD went from CML to acute blastcrisis; his cell population contained 91% blast cells and washighly positive for LANA. On the other hand, IBE went fromAML to CML and while he still had 98,000 WBC and astrongly positive serum at the time of cell sampling, his cellpopulation had virtually no blasts and was completely nega-tive.Each negative cell sample was also tested against the

donor's own serum. There were no detectable positive reac-tions.To compare the reactivity of the different positive target

cells, selected collections of AML and control sera were testedin parallel. Since identical patterns were obtained in this andother, similar tests, it was concluded that the AML blasts ofdifferent patients carry the same antigen.

Morphologically, the appearance of the antigen showedslight differences between different donors (Figs. 5 and 6).The cells of some patients contained a more finely granularantigen, whereas in others the material was collected in largerblocks. The similarities were more impressive than the differ-ences, however.

Tumor Biopsies. An attempt to test BL biopsy cells waslargely unsuccessful, because most BL biopsies contain EBNAand most of our sera were EBNA-positive as well. The ex-ceptional, EBNA- and EBV-DNA-negative BL biopsy of thepatient JA mentioned above, and the reactivity of the EBNA-negative B-JA line (compare Table- 1) encouraged us, how-ever, to test other EBNA-negative lymphoma biopsies.Another such case (KM) was encountered in a lymphomabiopsy, kindly. provided by Dr. S. Singh of the KenyattaNational Hospital. It came from a 12-year-old Mkambaboy, admitted with a large tumor in the right parotid regionand temporal fossa. Histologically, it was not regarded ashaving the features characteristic for a Burkitt's lymphoma.ACIF tests were carried out with the same serum spectrum

against the LANA-positive AD blast cell and the KM biopsycells in parallel. One EBNA-LANA- and 14EBNA+LANA-sera were all negative against KM. Among 13 EBNA+LANA+sera, nine were positive against KM, with a coarsely granularnuclear fluorescence pattern, similar to that of the AD blast

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Leukemia-Associated Nuclear Antigen (LANA) 689

cells. The reactive sera included AD and JA, the referencesera used to demonstrate LANA in the cell spectrum listedin Table 3. Two EBNA+LANA+ sera gave doubtful reactions(±) against KM and two were negative. These results suggestthat KM cells contain LANA.

Positive reference sera were also tested against biopsy cellsfrom three cases of Hodgkin's disease and against peripheralblood from two cases of infectious mononucleosis, with neg-ative results.

Tests Against Established Tissue Culture Lines. Two basi-cally different kinds of tests have been carried out. In oneseries, six different EBV-genome-carrying lymphoblastoidlines were tested against LANA-reactive sera. These testscannot be interpreted, since the LANA-positive sera ADand JA were also EBNA-reactive. The question whether ornot EBNA-positive lines also contain LANA will have toawait the selection of appropriate LANA+EBNA- refer-ence sera.

In a second series of tests, EBNA-negative cell lines wereused. The herpesvirus-saimiri-transformed simian lympho-blastoid lines 1670 and MLC gave no nuclear reactions. Theline derived from the exceptional EBV-DNA- and EBNA-negative Burkitt's lymphoma patient B-JA gave a clearLANA pattern. It never acquired the EBNA antigen. Freshlyexplanted LANA-positive cells of the AML patient BE (com-pare Table 2) gave a clear LANA pattern during the first 2weeks of their in vitro maintenance. Subsequently, an EBV-carrying EBNA-positive lymphoblastoid line emerged. Thisis obviously not representative of the originally explantedblast cell population.

DISCUSSIONIt is thus clear that the LANA reactivity is different fromthat of EBNA with regard to immunological specificity andmorphological appearance. It is mainly associated with AMLblast cells. It disappears when AML changes into a morechronic, highly differentiated form, with myelocytes andgranulocytes. Contrariwise, LANA appears when CMLenters into an acute blast crisis. It has not been found in CLLblast cells or in the small sample of ALL blasts so far tested.

Conceivably, the antigen could be a tissue-specific antigen,expressed at a certain early stage during the differentiationof the myeloid series. On the other hand, a similar reactivityhas been also seen in two EBNA-negative lymphomas. Onewas histologically and clinically indistinguishable fromEBNA- and EBV-DNA-positive Burkitt's lymphomas,whereas the other was not a typical Burkitt-type lymphoma.We could only receive serum from one of the patients; it gavea strongly positive reaction.An association of the reactivity with leukemias other than

AML and with lymphomas was also confirmed by the disease-related serological study. Whereas 73% of the AML serawere positive, ALL, CML, CLL, and BL sera were reactive in35, 14, 19, and 24% of the cases, respectively. Two of five

polycythemia and two or eleven myeloma patient sera werealso positive. In contrast, among 61 normal healthy controls,none gave similar reactions. Fifty-eight were negative, whereasthree showed a certain diffuse nuclear staining.An an alternative to the possibility that we are dealing with

a tissue-specific or fetal antigen preferentially activated inlympho- and myeloproliferative conditions, the association ofLANA with leukemic and lymphoma cells and sera may reflectthe presence of a leukemia-associated virus, particularlyprevalent in AML, but also present in the same or cross-reactive form in other leukemias and lymphomas. Yet anotherpossibility cannot be excluded: that the positive sera reactwith the DNA or nucleoprotein of the blast cells, whichreaction is detected by the highly sensitive ACIF method.

Note added in proof. Recent experiments demonstrated thepresence of LANA in normal lymphocytes transformed by phyto-hemagglutinin and in two EBV-genome-free human lympho-blastoid lines, MOLT4 and 698. This speaks against the viralhypothesis and favors the two other alternatives mentioned in theDiscussion.

We wish to express our sincere appreciation for cell and serumspecimens kindly provided by Dr. S. Singh, Kenyatta NationalHospital, Nairobi; Drs. Dick Killander, Peter Reizenstein, G6staGahrton, and Farkas Vanky, Karolinska Sjukhuset, Stockholm;Prof. Lars Engstedt, Sddersjukhuset, Stockholm; Prof. GabrielIzak, Hadassah Medical School, Jerusalem. Our thanks are dueto Mrs. Britt-Marie Xkerman, Miss Mina Feigis, Miss IngridEriksson, Miss Ingrid Bostr6m, and Miss Lotta Vrang for valu-able technical assistance. We thank Dr. Jose Menezes and Wolf-gang Leibold for tissue culture materials. This work was sup-ported by Contract no. NO1 CP 3316 within the Virus CancerProgram of the U.S. National Cancer Institute, the SwedishCancer Society, King Gustaf V:s Jubilee Fund, Ake Wiberg'sFoundation and Harald and Greta Jeansson's Foundation.

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