American Journal of Pathology, Vol. 128, No. 3, September 1987Copyright © American Association of Pathologists

Immunohistochemical Evidence ofActive ThymocyteProliferation in Thymoma

Its Possible Role in the Pathogenesis ofAutoimmune Diseases

MARCO CHILOSI, ANTONIO IANNUCCI,FABIO MENESTRINA, MAURIZIO LESTANI,

ALDO SCARPA, FRANCO BONETTI,LUCIANO FIORE-DONATI,

BRUNO DIPASQUALE, GIOVANNI PIZZOLO,GIORGIO PALESTRO, GIUSEPPE TRIDENTE,

and GEORGE JANOSSY

Eight cases ofhuman thymoma have been analyzed oncryostat sections with the monoclonal antibody Ki67,which reacts with cells in the proliferative phases ofthecell cycle. The aim was to assess the proportion ofpro-liferating thymocytes among lymphoid cells in thethymoma samples. In all cases a large number of cells(mean, 58.75%; range, 35-80%), recognized as thymo-cytes by morphology and lack of cytokeratin expres-sion in a combined immunohistochemical assay, ex-

HUMAN thymomas are neoplasms of epithelial ori-gin that are frequently associated with an exuberantlymphoid component.1 Recent studies have estab-lished that the epithelial and lymphoid componentsin thymoma closely mimick the cell organization ob-served in the normal thymus.2-10 In most cases ofthymoma with high lymphocyte/epithelial ratiosneoplastic epithelial cells exhibit differentiation anti-gens ofthymic cortical epithelium311 ' and appear to beintermingled with lymphocytes exhibiting a "corti-cal" phenotype as observed in the normal cortex(TdT+, T6+, T4+, and T8+, etc.).2'3'6'9-12 Residualareas of "medullary differentiation" are also presentwith mature T cells (TdT-, T6-, T3+), and medullaryepithelial cells as defined by their peculiar pheno-type.3'7 Cases showing predominantly medullary epi-thelial type have been also occasionally seen.4'7 Thus,in lymphoid-predominant thymomas the followingcharacteristic features are seen: a) an abundance ofimmature thymocytes and b) an abnormal neoplasticepithelial microenvironment which frequently ex-

From Istituto di Anatomia Patologica, di Scienze Immunologicheed Ematologia, Universita' di Verona, and Istituto di AnatomiaPatologica, Universita' di Torino, Italy, and Department oJImmunology Royal Free Hospital School of Medicine. London,England

hibited nuclear Ki67 staining. These findings differfrom the reactivity pattern observed in age-matchednonneoplastic thymuses where lower growth activityof cortical thymocytes was observed (15-20% Ki67+cells). Intensive thymocyte proliferation in thymomasmay represent one ofthe factors which lead to autoim-munity in myasthenia gravis and thymomas. (Am JPathol 1987, 128:464-470)

hibits hyperkeratinization and changes of HLA-DRexpression.3'11 Autoimmune phenomena are also fre-quently associated with thymoma. These include,most frequently, myasthenia gravis' 7 and, occasion-ally, cytopenias, systemic lupus erythematosus, rheu-matoid arthritis, thyroiditis, and other diseases.13-15 Itis not known whether the large numbers of thymo-cytes observed in these cases derive from an increasedproliferation of cortical thymocytes or from an exag-gerated accumulation of end-stage nondividing thy-mocytes. This is an important question because theincreased proliferation is likely to be associated withan increased output of cells from the thymus into the

Supported in part by the Associazione Italiana per la Ri-cerca sul Cancro, Milano, and Progetto Finalizzato CNR"Oncologia", Italy.Accepted for publication April 27, 1987.Address reprint requests to M. Chilosi, Istituto di Anato-

mia Patologica, Universita' di Verona, Policlinico BorgoRoma, 37100 Verona, Italy.

464

PROLIFERATING THYMOCYTES IN HUMAN THYMOMA 465

periphery. For this reason, in our study we evaluatedthe replicating sets of thymocytes in 8 cases of thy-moma using a quantitative immunostaining by themonoclonal antibody Ki67. This reagent binds to nu-clear antigens expressed by cells in the proliferativephasesG 1, G2, M, and S.16 These data were comparedwith the quantitative observations obtained withKi67 staining on samples of normal thymuses.

Materials and MethodsPatients

Fresh frozen tissue samples were obtained from 8patients at surgery for the diagnosis and therapy ofthymoma (Table 1). The patients received no im-munosuppressive therapy before thymectomy. Foursamples ofnormal thymus were also analyzed as con-trols. The antibodies applied for the study ofepithelialand lymphoid antigens of the thymomatous samplesare listed in Table 2.3

Methods

The samples were covered with OCT medium(Ames), snap-frozen in liquid nitrogen, and processedfor immunohistochemistry as previously described.17Briefly, 5-,u sections were cut in a cryostat, dried ontoglass slides covered with polylysine adhesive (Sigma),and fixed in cold chloroform/acetone mixed in a 1: 1ratio. A modification of the original technique fordemonstrating Ki67 antigen'6 was used for improve-ment ofthe fixation ofthe nuclear antigens. This pro-cedure included a second fixation step with 10% cal-cium-formalin for 10 minutes. After fixation the

Table 2-Conventional and Monoclonal Antibodies Used forImmunohistochemical StudyImmunologic

reagentOKT4 (CD4)OKT8 (CD8)OKT3 (CD3)OKT6 (CD1)TdTKeratinKi67HLA-DRMR3RFD4

MMMMRRMMMM

SpecificityHelper T cellsSuppressor T cellsT cellsCortical thymocytesCortical thymocytesEpithelial cellsReplicating cellsB cells, etc.Thymic epithelium (cortex)Thymic epithelium (medulla)

Source

OrthoOrthoOrthoOrtho(Ref 11)DakoDakoBecton Dickinson(Ref 19)(Ref 11)

R, rabbit; M, monoclonal.

slides were washed in buffered saline (PBS) and la-beled with Ki67 by means ofthe alkaline phosphataseanti-alkaline phosphatase method (APAAP).18 A gen-tle hematoxylin counterstaining was performed forassessing the morphology and percentage of Ki67+cells. Ki67 antibody (Code M722) and the APAAPreagent (Code K670) were purchased from Dako-patts, Denmark.

Additional sections were also stained using a com-bined immunocytochemical staining for Ki67 andcytokeratin to confirm the nature ofthe Ki67+ cells inthe given samples. In addition to the staining for Ki67(APAAP with red chromogen), the samples were si-multaneously incubated with a rabbit anti-keratinantibody (Dakopatts, Code A575) followed by anti-rabbit serum and peroxidase anti-peroxidase (PAP)complex (Dakopatts, Code K548) with diaminoben-zidine as a brown chromogen.The samples were first viewed with the two-color

immunochemical method and also with labeling for

Table 1-Evaluation of Replicative Fractions of Thymocytes in Thymomatous and Control SamplesAssociateddisease

MyastheniaMyastheniaMyasthenia*MyastheniaMyastheniaNoneMyastheniaMyasthenia*

Thyroid adenomaHyperparathyroidism

CardiopathyHodgkin's disease

*Ki67+ percentage confirmed on smear preparations (Case 3, 41%; Case 8, 68%).

Cases

12345678

Age-matchedcontrol

thymuses910

Controlthymuses

1112

Sex

MFMMMMMF

MF

MM

%Ki67+Age4061694142193154

3736

215

HistologyThymomaThymomaThymomaThymomaThymomaThymomaThymomaThymoma

Normal thymusNormal thymus

Normal thymusNormal thymus

40%70%35%80%60%70%45%70%

20%15%

60%48%

Vol. 128 · No. 3

466 CHILOSI ET AL

RFD4 antigen present on medullary epithelium. Theoverall distribution of cortical and medullary areashas been determined. Fields with predominant epi-thelial components and with signs of medullary dif-ferentiation (such as Hassal corpuscles) were not in-cluded.

In all cases fields with lymphoid predominancewere viewed, and more than 2000 cells were evalu-ated for Ki67 nuclear staining with a 40X objective.Five different areas were selected in all samples, andthe percentages of positive cells within the lymphoidcell population were counted.

In 2 cases fresh cell suspensions were also available,and cytospin preparations were obtained. Immuno-cytochemical staining on these preparations allowedthe precise evaluation of the proportions of Ki67+thymocytes within the TdT+, T6+ cortical thymocytepopulations and comparison of these findings withthe observations on cryostat sections.

ResultsThe cases examined were encapsulated multinodu-

lar neoplasms with the typical histologic pattern ofthecortical type ofthymoma and were characterized bynodules ofdifferent size, mainly composed by lymph-oid cells and minor proportions of epithelial cells.Epithelial cells had round-ovoid nuclei and loosechromatin structure. Lymphoid mitoses were com-mon. In three cases areas ofmedullary differentiationwith Hassall's corpuscles were present. In another 2cases areas of medullary differentiation were only de-tected by immunohistochemistry, revealing mature

lymphocytes (TdT-, T6-, T3+) among remnants ofanepithelial network exhibiting the medullary pheno-type (RFD4+) when studied on adjacent sections.

Epithelial ComponentIn all cases the neoplastic epithelial cells expressed

cytokeratin and the antigen detected by MR3 mono-clonal antibody. This reagent is one of the most spe-cific reagents for cortical epithelium.'9 HLA-DR wasabsent or heterogeneously expressed on epithelialcells, unlike in cortical epithelium ofthe normal con-trol samples. RFD4, an antibody specific for subcap-sular and medullary epithelium, only reacted withsmall medullary-like areas as described earlier.3

Lymphoid ComponentIn all cases the large majority oflymphocytes (more

than 60-80%) were of the T type and exhibited a"cortical" phenotype as defined by TdT and T6 posi-tivity together with the simultaneous presence of T4and T8 antigens. Similar proportions of T6+, TdT+lymphoid cells (62% and 70%) were observed in thetwo samples which were studied on cell suspension.The lymphocytes homing within the areas ofmed-

ullary differentiation were mostly TdT and T6 nega-tive and expressed either T4 or T8 antigen.

Ki67 ExpressionIn all the cases examined

revealed the presence of aKi67 immunostaininglarge number (range,

Figure 1-Cryostat section ofthymoma of cortical type (Case3). A proportion of thymocytes(evaluated as 35% on direct tis-sue examination) express theproliferative marker Ki67.

AJP * September 1987

PROLIFERATING THYMOCYTES IN HUMAN THYMOMA 467

35-80%; mean, 58.75%) ofpositive cells with nuclearstaining ofheterogeneous intensity. The positive cellsshowed a lymphoid morphology (Figure 1). In dou-ble-stained sections keratin-positive epithelial cells(PAP, brown staining) were almost completely devoidof nuclear Ki67 staining (APAAP, red staining) (notshown).

In the 2 cases with areas of medullary differentia-tion these were characterized by a lack of (or few)Ki67-positive thymocytes and clearly appeared asnegative islands within the tissue (Figure 2).

Figure 2-Cryostat sections ofthymoma of cortical type withmedullary differentiation (Case2). A large number of TdT-posi-tive "cortical" thymocytes areshown (a) around a small medul-lary area. In a serial section (b)most cortical cells express theproliferative marker Ki67. Themedullary area is nearly devoidof positive cells.

The quantitative evaluation of Ki67-positive thy-mocytes was performed on cryostat sections. Thymo-cytes were easily recognized by morphology and bylack of cytokeratin expression. The proportions ofKi67-positive thymocytes in thymomas ranged be-tween 35% and 80% and were higher than those ob-served in the normal age-matched thymuses in our(15-20%) and Steinmann's (10%) series.20

In the 2 cases that were studied on cytospin prepa-rations, the proportions ofTdT+, T6+, Ki67+ thymo-cytes could be carefully analyzed. In these 2 cases the

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Vol. 128 * No. 3

468 CHILOSI ET AL

results on cytospin and cryostat preparations werecomparable (Table 1 and Figure 3).

DiscussionHuman thymoma is a peculiar epithelial neoplasm

where "abnormal" epithelium supports the differen-tiation of polyclonal T lymphocytes.-1''21 We re-cently confirmed the polyclonality of the lymphoidcomponent ofthymoma at the gene level using probesfor the beta-chain ofthe T-cell receptor (Scarpa et al,manuscript in preparation).The immunophenotypic studies have shown that

most lymphocytes in thymoma are identical to nor-mal cortical thymocytes.2" In some cases minor pro-portions of mature T lymphocytes can be also foundin islands which show "medullary differentiation."These areas are most frequently small, but normallyorganized.3'6'7

It has already been demonstrated by conventionalhistology that thymomas contain lymphocytes with a"stimulated" or "activated" appearance, and somemitoses are also seen.' 2' Nevertheless, the replicativepotential of the lymphoid component in thymomaneeds further study with more quantitative methods.Unfortunately, autoradiographic analysis or the mi-togen-responsive capacity of thymoma cells mea-sured in vitro22 may not give relevant informationabout their proliferative features in vivo. Recent ob-servations with Ki67 antibody indicate that withinthe lymphoid lineage there is a close relationship be-

tween antibody reactivity and the cycling of the cell(in Gi, S, or M phase).16

In this study we have determined the replicativefraction of thymocytes in thymomas using the im-munohistochemical detection of Ki67 antigen.'6 Wefound a marked activity of thymocytes in all thymo-matous samples. An internal control for each samplehas been provided by the remnants ofmedullary areaswhere all cells were virtually devoid ofKi67 staining.The proportions of Ki67-positive thymocytes foundin thymomas (35-80%) are similar to those observedin normal control thymuses from younger patientsbut significantly higher than those observed in thecortical areas ofage-matched control thymuses in ourstudy (15-20%) and in a series of cases recently re-ported by Steinman et al.20 This observation suggeststhat in thymoma the accumulating thymocytes donot merely represent a population of long-lived end-stage cells which divide rarely. It seems that this popu-lation actively proliferates within the epithelial net-work, which has been shown to synthetize thymichormones23 and which, in normal thymus, may havea determinant role in the selection ofimmatureT cellsfor self MHC specificity.24 There is also evidence inboth experimental animals and man that thymocytesacquire their antigen specificity within the cortex,where the rearrangement of genes coding for T-cellreceptor takes place.25'26 It appears from our studythat the proliferating T-cell precursors in thymomaare part of a microenvironment constituted by anabnormal cortical epithelium, which is frequently

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Figure 3-Smear preparationsobtained from a cell suspensionof thymoma of cortical type(Case 8). The majority of cellsshow the immature "cortical"marker TdT (a) and the prolifera-tive marker Ki67 (b). A Ki67-negative epithelial cell is shown(arrow).

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Vol. 128 * No. 3 PROLIFERATING THYMOCYTES IN HUMAN THYMOMA 469

HLA-DR negative, as it has been demonstrated inanother disease, the "bare lymphocyte" syndrome.27Education in such a microenvironment in thymomamay lead to the export of functionally handicappedT-cell clones. The search of such immature T cells inthe peripheral blood of patients with thymoma is anongoing project in our laboratories, but it is relevanthere that lymphocytes of intermediate phenotype(T4+, T8+, T6-) can be demonstrated in some patientswith myasthenia gravis.28

In addition to the cortical overproduction of thy-mocytes, we have previously detected another impor-tant contributing defect in thymoma. Proliferatingcortical thymocytes may avoid medullary influenceexerted at the cortical-medullary junction prior toentry into the circulation. It has been recently demon-strated that medullary interdigitating dendritic cellsinduce self-tolerance.29'30 These factors, taken to-gether, may represent the key factors in the frequentdevelopment of autoimmunity in thymomas.

References

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18. Cordell JL, Falini B, Erber WN, Ghosh AK, AbdulazizZ, MacDonald S, Pulford KAF, Stein H, Mason DY:Immunoenzymatic labeling of monoclonal antibodiesusing immune complexes of alkaline phosphatase andmonoclonal anti-alkaline phosphatase (APAAP com-plexes). J Histochem Cytochem 1984, 32:219-229

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20. Steinman GG: Changes of the human thymus duringaging,7 pp 43-88

21. Levine GD, Polliack A: The T-cell nature of the lym-phocytes in two human epithelial thymomas: A com-parative immunologic, scanning and transmissionelectron microscopic study. Clin Immunol Immuno-pathol 1975, 4:199-208

22. Musiani P, Maggiano N, Aiello FB, Larocca LM, Pian-telli M: Phenotypical characteristics and proliferativecapabilities ofthymocyte subsets in human thymoma.Clin Immunol Immunopathol 1986, 40:385-392

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24. Kyewski BA: Thymic nurse cells: Possible sites ofT-cellselection. Immunol Today 1986, 7:374-379

25. Royer HD, Acuto 0, Fabbi M: Genes encoding the Tibeta subunit of the antigen/MHC receptor undergo re-arrangement during intrathymic ontogeny prior to sur-face T3-Ti expression. Cell 1984, 39:261-266

26. Snodgrass HR, Kisielow P, Kiefer M, Steinmetz M,von Boehmer H: Ontogeny ofthe T-cell antigen recep-tor within the thymus. Nature 1985, 313:592-595

27. Schuurman HJ, Wijngaert FP, Huber J, SchuurmanRKB, Zegers BJM, Roord JJ, Kater L: The thymus in"bare lymphocyte" syndrome: Significance of expres-sion of major histocompatibility antigens on thymicepithelial cells in intrathymic maturation. Hum Im-munol 1985, 13:69-82

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470 CHILOSI ET AL AJP * September 1987

and Leu2a antigens on circulating T lymphocytes inmyasthenia gravis. J Neuroimmunol 1986, 11:311-319

29. Kyewski BA: Thymic dendritic cells present blood-borne antigens to medullary thymocytes in vivo : Apossible role in the generation of the T-cell repertoire,Haematology and Blood Transfusion. Vol 29: ModemTrends in Human Leukemia VI. Edited by Neth,Gallo, Greaves, Janka. Berlin, Springer-Verlag, 1985, p486

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Acknowledgments

We thank Mrs. L. Montagna and Mrs. D. Toffali for theirskillful technical assistance.