Major histocompatibility antigens and antigen-processing molecules in retinoblastoma

Major Histocompatibility Antigens and Antigen-Processing Molecules in Retinoblastoma

Subramanian Krishnakumar, M.D.1

Amirthalakshmi Sundaram, M.S., M.L.T.1

Dhiraj Abhyankar, M.D.2

Vanitha Krishnamurthy, B.Sc., D.M.L.T.1

Mahesh Palanivelu Shanmugam,D.O., M.D., Ph.D.

3

Lingam Gopal, M.S., M.D.3

Tarun Sharma, M.S., M.D.3

Jyotirmay Biswas, M.D.1

1 Department of Ocular Pathology, Medical andVision Research Foundations, Chennai, India.

2 Evanston Northwestern Healthcare, Evanston, Il-linois.

3 Department of Ocular Oncology, Medical andVision Research Foundations, Chennai, India.

Supported by the Vision Research Foundation(Chennai, India).

Address for reprints: Subramanian Krishnakumar,M.D., Vision Research Foundation, Sankara Neth-ralaya, 18 College Road, Chennai 600 006, TamilNadu, India; Fax: (011) 91 4428254180; E-mail:[email protected]

Received August 26, 2003; accepted December10, 2003.

BACKGROUND. Malignant transformation of cells is frequently associated with

abnormalities in human leukocyte antigen (HLA) expression. These abnormalities

may play a role in the clinical course of the disease, because HLAs mediate

interactions of tumor cells with cytotoxic T lymphocytes (CTLs) and natural killer

(NK) cells. Retinoblastoma is the most common intraocular malignant tumor in

childhood and is characterized by direct spread to the optic nerve and orbit as well

as hematogeneous and lymphatic spread. Little is known about the role of HLA

expression in the progression of this malignant disease.

METHODS. HLA Class I antigen, �2-microglobulin (�2-m), HLA Class II antigens,

and the antigen-processing molecules (APMs) of the HLA Class I pathway, includ-

ing proteasomal subunits (low–molecular mass polypeptide 2 [LMP-2] and LMP-

10), the transporter-associated protein (TAP-1) subunit, the binding protein tapa-

sin, and the chaperone molecule calnexin, were studied in 30 archival

retinoblastoma specimens by immunohistochemistry. Immunoanalysis was per-

formed based on the International Histocompatibility Working Group Project

Description.

RESULTS. HLA Class I antigen, �2-m, HLA Class II antigen, and APMs were positive

in 12 tumors with no invasion and were decreased in 13 tumors with choroidal and

optic nerve invasion. The difference in HLA and APM expression between the 2

groups was statistically significant (P � 0.001).

CONCLUSIONS. Decreased expression of HLA was observed in aggressive tumors

and in poorly differentiated tumors. The current findings support a role for both

CTLs and NK cell-mediated control of tumor growth in the clinical course of

retinoblastoma. Cancer 2004;100:1059 – 69. © 2004 American Cancer Society.

KEYWORDS: antigen-processing molecules, calnexin, cytotoxic T lymphocytes, hu-man leukocyte antigen, immunohistochemistry, retinoblastoma, tapasin.

Retinoblastoma is the most common primary intraocular tumor inchildren. In the United States, this disease presents most fre-

quently as unilateral sporadic tumors and less frequently as bilateralhereditary tumors. When it is left untreated, retinoblastoma is almostalways fatal. Prognosis is affected by many risk factors, the mostimportant of which is the extent of invasion of the retinoblastomainto ocular coats and the optic nerve.1 Although many tumors are notdetected until they are large enough to be visible to the parents, verysmall tumors sometimes may be treated effectively using laser ther-apy or cryotherapy. More commonly detected larger tumors oftenrequire removal by enucleation of the affected eye. When retinoblas-toma is treated in the early stages by enucleation, the cure ratesapproach 95%.2– 4 In addition to the loss of vision, this therapeuticapproach may leave the child with a facial deformity that worsensthroughout life.5 More advanced disease may require radiotherapy or

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© 2004 American Cancer SocietyDOI 10.1002/cncr.20062

chemotherapy in addition to enucleation. Both ofthese additional therapeutic regimes increase theprobability that the surviving child will develop addi-tional malignancies later in life. Currently, there is nosuccessful therapy for the treatment of patients whodevelop metastatic disease. In recent years, howeverphysicians have explored alternative therapies in anattempt to salvage the eyes and vision of the patient.

Recent advances in tumor immunology have pro-vided the necessary information to develop immuno-therapies to achieve this goal. There are several rea-sons why these therapies may be highly successful intreating patients with ocular tumors. Ocular tumorsusually are detected early, when the tumor is relativelysmall. This allows initiation of treatment early in thedevelopment of the disease, which is important be-cause it is believed that immunotherapies provide thegreatest benefit when the tumor burden is low. Tu-mors also grow within a contained space, and surgicaltechniques are available for the delivery of intratumorinjection of cytokine and/or lymphocytes.

Major histocompatibility complex (MHC) and an-tigen-processing molecules (APMs) have generated in-terest in immunotherapy in many tumors.6 MHC ClassI antigens are responsible for presenting antigen tocytotoxic T lymphocytes (CTLs). In fact, reduced oreliminated Class I antigen expression on tumor cells iscorrelated inversely with their rejection. The pathwayof antigen-processing machinery involved in Class Iantigen recently was characterized. This pathway in-volves proteasomal complexes, consisting of low–mo-lecular mass polypeptide 2 (LMP-2), LMP-7, and therecently identified LMP-10 or multicatalytic endopep-tidase complex-like-1, which is necessary for LMP-2expression; all generate antigenic peptide fragments.The transporter-associated proteins (TAP), TAP-1 andTAP-2, and the different endoplasmic reticulum (ER)resident chaperones, such as calnexin, calreticulin, thebinding protein BiP, ER60, and tapasin, stabilize MHCClass I molecules during their folding and assembly inthe ER or assist in their binding to peptides. Deficien-cies in LMP, TAP, and the chaperone proteins reducethe supply and repertoire of peptides available forbinding to MHC Class I molecules. Decreased humanleukocyte antigen (HLA) Class I antigen levels result-ing from a decrease in APMs have been observed inmany tumors.7

There have been several studies on HLAs in reti-noblastoma. Previous studies focused on cell lines andused monomorphic antibodies W6/32 against HLA-A,HLA-B, and HLA-C heavy chains and BBM.1 againstthe associated �2-microglobulin (�2-m) light chainand on transgenic animal models of retinoblastoma.Studies showed that both Y-79 and WERI-Rb1 retino-

blastoma cell lines exhibited significant levels of HLA-A, HLA-B, and HLA-C antigens; however, over time,subcultured Y-79 tumor cell lines lost Class I surfaceHLA expression, and there was a paucity of HLA ex-pression in the Rb 355-7 cell line.8 In separate studies,it was found that MHC Class I and Class II antigenexpression was negative in the Y-79 cell line but wasinduced by interferon (IFN) in Y-79 cells.9 –11 Similarly,MHC Class I antigens were absent in RbSF81 cells(originally derived from Y-79 cells) but were inducedlater by retinoic acid.12 It is possible that the morpho-logic and genetic characteristics of cultures grown forextended periods may have contributed to the varia-tion in the expression of HLAs in those cell lines.Studies in a transgenic mouse model of retinoblas-toma showed that tumor cells expressed high levels ofMHC Class I antigen and tumor-associated antigen(Tag), which induced a high T-cell response in whichCD8 positive (CD8�) T cells recognized peptide frag-ments of Tag presented by the MHC Class I molecules.Further studies showed that CTLs failed to lyse trans-genic retinoblastoma tumor target cells that haddown-regulated HLA Class I molecules. However, italso was observed that retinoblastoma cells treatedwith IFN-� up-regulated Class I expression and thenwere lysed by T cells from the transgenic mice.13,14

Thus, these earlier studies showed that HLAs are ex-pressed in retinoblastoma and that they are immuno-genic tumors.

Nonetheless, to our knowledge, APMs and HLAsin retinoblastoma and their correlation with invasive-ness have not been investigated. Therefore, in thecurrent study, we investigated the immunoreactivityof HLA Class I antigen, �2-m, HLA Class II antigen, andAPM (including proteasomal subunits LMP-2, LMP-10, the transporter protein TAP-1 subunit, and thechaperone molecules tapasin and calnexin) in archivalspecimens of retinoblastomas and correlated the re-sults with regard to differentiation and invasion.

MATERIALS AND METHODSPatientsPreviously, we published a large series involving 232Asian Indian children with retinoblastoma.15 In thatstudy, a higher incidence of choroidal and optic nerveinfiltration was noted among Asian Indian childrenthan among children from the Western world. Weconcluded that this finding may have been due todelayed diagnosis or to a difference in the biologicbehavior of tumors occurring in the Asian Indian pop-ulation. Thirty retinoblastoma lesions were obtainedfrom 12 male children and 18 female children with anage range of 1– 8 years at the time of enucleation. Allpatients were evaluated in the Ocular Oncology Clinic

1060 CANCER March 1, 2004 / Volume 100 / Number 5

at our hospital between 2000 and 2002. The tumorswere divided into two groups: Group A tumors, withno invasion of the choroid or the optic nerve and nometastasis; and Group B tumors, with invasion of thechoroid, optic nerve, and orbit and with metastasis.

Inclusion and Exclusion CriteriaThe inclusion criterion was that all patients weretreated by enucleation. Exclusion criteria included pa-tients who had received preoperative adjunctive treat-ments, such as chemotherapy, which could influencethe interpretation of immunohistochemistry.

Tumor SamplesNeoplastic tissues were obtained in enucleation ma-terial from the patients. Each sample was processedfor conventional histopathologic diagnosis. Histologicsections were prepared from tissues fixed in 10% buff-ered neutral formalin for 48 hours and embedded inparaffin. Hematoxylin and eosin–stained, 6 �m sec-tions were prepared from the central region of thetumor and were reviewed for differentiation and inva-sion. Blocks were obtained from consecutive patients.

DifferentiationRetinoblastomas were graded microscopically intothree groups according to the predominant pattern ofdifferentiation: 1) poorly differentiated cells, with highnuclear-to-cytoplasmic ratios and high mitotic indices(pseudorosettes and Homer–Wright rosettes werefound in such areas); 2) moderately differentiated cells,with moderate nuclear-to-cytoplasmic ratios, moder-ate mitotic indices, and possible pseudorosettes andFlexner–Winter Steiner rosettes; and 3) well differen-tiated cells, with low nuclear-to-cytoplasmic ratios,low mitotic indices, and the presence of Flexner–Win-ter Steiner rosettes and florets. There were 6 well dif-ferentiated tumors, 6 moderately differentiated tu-mors, and 18 poorly differentiated tumors.

InvasionAll tumor slides were reviewed, and choroidal invasionwas staged according to the new grading system de-vised by Schilling et al.16

Staging of Choroidal InvasionChoroidal invasion was staged as follows: Stage 0, nochanges in retinal pigment epithelium and choroids;Stage 1, changes in retinal pigment epithelium with-out involvement of Bruch membrane; Stage 1a, simpledefects of the retinal pigment epithelium cell layer;Stage 1b, tumor growth under the elevated retinalpigment epithelium cell layer with or without reactivecell proliferation; Stage 2, complete defect of the ret-

inal pigment epithelium, including the basementmembrane, without tumor invasion into the chorio-capillaries; Stage 3, proof of tumor cells in the chorio-capillaries and superficial infiltration around the largechoroidal vessels; and Stage 4, complete infiltration ofthe choroid with lateral extension.

Optic Nerve InvasionFor optic nerve invasion analyses, prelaminar inva-sion, postlaminar invasion, and invasion of the surgi-cal end of the optic nerve were considered. There were12 tumors with no invasion and 18 tumors with inva-sion. Three tumors had invasion of the choroid only,six tumors had invasion of the choroid and the opticnerve, and nine tumors had invasion of the opticnerve. There was one tumor that had metastasized tothe submandibular lymph node. The various stages ofchoroidal invasion and optic nerve invasion are shownin Table 1, and the immunochemistry results areshown in Table 2.

Monoclonal AntibodiesThe affinity purified mouse antihuman, locus-specificmonoclonal antibody (mAb) HC-10, which recognizesa determinant expressed on HLA-A10, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, and HLA-A33heavy chains and on virtually all HLA-B heavy chains;the anti-�2-m mAb L368; the anti-HLA Class II mAbLGII-612.1417–19; and the APMs in the HLA Class Ipathway, including the anti-LMP-2 mAb SY-1, the an-ti-LMP-10 mAb TO-6, the anti-TAP-1 mAb TO-1, theanti-tapasin mAb TO-4, and anticalnexin mAb TO-5,which are target-specific and exhibit no cross reactiv-ity, were used.20 The antibodies were gifts from Dr.Soldano Ferrone (Department of Immunology, Ro-swell Park Cancer Institute, Buffalo, NY). A labeledstreptavidin kit was purchased from DAKO Laborato-ries (Copenhagen, Denmark).

ImmunohistochemistryImmunostaining of tissue sections was performed usinglabeled streptavidin with an indirect immunoperoxidasetechnique. Briefly, 4-�m, formalin fixed paraffin sectionswere used for the study. Tissue sections were thendeparaffinized and rehydrated. Endogenous peroxidasewas blocked with hydrogen peroxide for 10 minutes atroom temperature. Antigen retrieval was performed be-fore antibody incubation using a microwave oven. Tis-sue sections were then rinsed in Tris-buffered saline(TBS), pH 7.6, and incubated with respective primaryantibodies over night at 4 °C. This was followed by asequential, 40-minute incubation with biotinylated sec-ondary antibody and streptavidin-labeled horseradishperoxidase (DAKO Laboratories). Sections were washed

HLAs in Retinoblastoma/Krishnakumar et al. 1061

TABLE 2Immunohistochemistry Results from Group B: Retinoblastoma with Invasion

Patient no.

% stained cells (staining intensity)

HLA ProteasomeTransporterprotein Chaperone protein

HLA Class I �2-m HLA Class II LMP-2 LMP-10 TAP-1 Tapasin Cainexin

1 � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 ()2 40 (�/�) 40 (�/�) 30 (�/�) 40 (�/�) 30 (�/�) 40 (�/�) 50 (�/�) 40 (�/�)3 0 (�) 0 (�) 0 (�) 0 (�) 0 (�) � 25 (�/�) � 25 (�/�) 40 (�/�)4 � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) 30 (�/�) 30 (�/�)5 50 (�/�) 50 (�/�) 40 (�/�) 50 (�/�) 30 (�/�) 50 (�/�) 50 (�/�) 50 (�/�)6 40 (�/�) 40 (�/�) 30 (�/�) 40 (�/�) 60 (�/�) 30 (�/�) 60 (�/�) 40 (�/�)7 � 25 (�/�) � 25 (�/�) � 25 (�/�) 0 (�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�)8 � 25 (�/�) � 25 (�/�) 0 (�) � 25 (�/�) 0 (�) � 25 (�/�) � 25 (�/�) � 25 (�/�)9 � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�)10 � 25 (�/�) � 25 (�/�) � 25 (�/�) 0 (�) � 25 (�/�) 0 (�) � 25 (�/�) � 25 (�/�)11 � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) 0 (�) � 25 (�/�)12 50 (�/�) 50 (�/�) 40 (�/�) 50 (�/�) 30 (�/�) 40 (�/�) 50 (�/�) 50 (�/�)13 0 (�) 0 (�) 10 (�/�) 0 (�) 0 (�/�) 0 (�) � 25 (�/�) � 25 (�/�)14 � 25 (�/�) � 25 (�/�) � 25 (�/�) � 25 (�/�) 0 (�) � 25 (�/�) � 25 (�/�) � 25 (�/�)15 50 (�/�) 50 (�/�) 60 (�/�) 30 (�/�) 50 (�/�) 40 (�/�) 50 (�/�) 50 (�/�)16 50 (�/�) 50 (�/�) 50 (�/�) 50 (�/�) 40 (�/�) 30 (�/�) 50 (�/�) 50 (�/�)17 40 (�/�) 40 (�/�) 40 (�/�) 30 (�/�) 40 (�/�) 30 (�/�) 40 (�/�) 40 (�/�)18 0 (�) 0 (�) 0 (�) 0 (�) 0 (�) 0 (�) 0 (�) 0 (�)

HLA: human leukocyte antigen; LM: low–molecular mass polypeptide; �2-m: beta 2 microglobulin; TAP: transporter-associated protein; (�): absent staining intensity; (�/�): dull staining intensity.

TABLE 1General Characteristics of the Cohort with Retinoblastoma Exhibiting Invasion

Patientno. Age (yrs) Gender Clinicopathologic parameters

Choroidinvasion

Optic nerveinvasion

1 7.0 Female OS, mod diff — Surgical end2 1.0 Male OD, poorly diff — Prelaminar3 2.0 Female OD, poorly diff Stage 4 Surgical end4 4.0 Male OU, OS, poorly diff Stage 4 Surgical end5 4.0 Female OD, mod diff — Lamina cribrosa6 2.0 Male OD, poorly diff — Lamina cribrosa7 1.1 mos Female OD, poorly diff Stage 4 Postlaminar8 3.0 Male OD, poorly diff — Postlaminar9 4.0 Female OD, mod diff Stage 3 —10 2.5 Female OD, poorly diff Stage 4 —11 2.0 Female OD, poorly diff — Postlaminar12 3.0 Male OD, poorly diff — Postlaminar13 5.0 Male OD, poorly diff Stage 4 Surgical end14 5.0 Female OU, (OD) well diff Stage 3 Prelaminar15 2.0 Female OU, (OS) poorly diff Stage 4 —16 2.0 Male OD, poorly diff — Postlaminar17 1.0 Female OU, (OD) well diff — Lamina cribrosa18 2.0 Female Poorly diff metastasis to submandibular lymph node Stage 4 Postlaminar

Mod: moderately; diff: differentiated; OS: left eye; OD: right eye; OU: both eyes; OD: right eye.


with TBS between incubations. The peroxidase reactionwas developed for 5 minutes using commercially avail-able 3,3�-diaminobenzidine and was counterstainedwith Harris hematoxylin.

Assessment of Immunohistochemical ResultsTissue sections were read independently by two ocu-lar pathologists (S.K. and J.B.), each without knowl-edge of the results obtained by the other investigator.Furthermore, each investigator read all slides twicewithout the knowledge of the results obtained in theprevious reading. The staining intensity was scored asabsent (�), dull (�/�), or bright (�). The tumors weregraded as follows: positive (� 75% of cells stained with� intensity), heterogeneous (25–75% of cells stained,mainly at �/� intensity; percentages of cells are ex-pressed to the nearest 10%), and negative (� stainingor � 25% of cells stained with �/� intensity).21

Variations in the percentage of stained cells enu-merated by the 2 investigators were smaller than 10%.The staining intensity of adjacent normal structures(i.e., lymphoid and endothelial cells) was used as aninternal control to evaluate the staining intensity ofmalignant cells. For negative controls, the primaryantibody was omitted, and nonimmune serum wasused in the immunostaining. The study was reviewedand approved by the local ethics committee of ourinstitute, and the committee deemed that it con-formed to the generally accepted principles of re-search, in accordance with the Helsinki Declaration

Statistical AnalysisFor statistical analyses, negative, heterogeneous, andpositive expression of HLAs and APM expression werecompared between tumors with no invasion and tu-mors with invasion using the Mann–Whitney U test.

RESULTSThe clinical and immunohistochemical data on pa-tients who had retinoblastoma with invasion areshown in Tables 1 and 2, and the data for patients whohad retinoblastoma without invasion are shown inTables 3 and 4.

Immunoreactivity of HLA Class I Antigens and �2-m inRetinoblastomaImmunoreactivity for HLA Class I antigens and �2-mwas concurrent. HLA Class I antigens and �2-mstained positively (� 75% of cells stained with � in-tensity) in the 12 tumors in Group A with no invasion.In Group B (n � 18), heterogeneous expression wasobserved in 8 tumors (30 –50% of cells stained with�/� intensity) and was not observed in 10 tumors (7

tumors with � 25% of cells stained with �/� intensityand 3 tumors with absent staining intensity).

Immunoreactivity of HLA Class II Antigens inRetinoblastomaHLA Class II antigen staining was positive (� 75% ofcells stained with � intensity) in the 12 tumors inGroup A with no invasion. In Group B (n � 18), het-erogeneous expression was observed in 7 tumors (30 –50% of cells stained with �/� intensity) and was neg-ative in 11 tumors (8 tumors with � 25% of cellsstained with �/� intensity and 3 tumors with absentstaining intensity).

Immunoreactivity of Proteasomal Subunits inRetinoblastomaExpression levels of LMP-2 and LMP-10 were concor-dant. LMP-2 and LMP-10 expression levels were pos-itive in 12 tumors in Group A (� 75% of cells stainedwith � intensity). In Group B (n � 18), heterogeneousexpression was observed in 7 tumors (30 –50% of cellsstained with �/� intensity). LMP-2 was negative in 11tumors (6 tumors with � 25% of cells stained with�/� intensity and 5 tumors with absent staining in-tensity). LMP-10 was negative in 11 tumors (5 tumorswith � 25% of cells stained with �/� intensity and 6tumors with absent staining intensity).

Immunoreactivity of the TAP-1 Subunit in RetinoblastomaTAP-1 expression was positive in 12 tumors in GroupA (� 75% cells stained with � intensity). In Group B (n� 18), heterogeneous expression was observed in 7tumors (30 –50% of cells stained with �/� intensity)and was negative in 11 tumors (8 tumors with � 25%

TABLE 3General Characteristics of the Cohort with Retinoblastoma ExhibitingNo Invasion

Patientno. Age (yrs) Gender Clinicopathologic parameters

1 2.0 Male OU, (OD) well differentiated2 5.0 Male OS, poorly differentiated3 1.0 Female OD, well differentiated4 2.0 Male OU, (OD) well differentiated5 8.0 Male OS, poorly differentiated6 4.0 Female OS, poorly differentiated7 1.0 Female OS, poorly differentiated8 5.0 Female OD, poorly differentiated9 1.0 Female OU, (OD) moderately differentiated10 2.0 Female OU, OD, moderately differentiated11 2.0 Female OU, OS, moderately differentiated12 1.5 Male OD, well differentiated

OU: both eyes; OD: right eye; OS: left eye.


of cells stained with �/� intensity and 3 tumors withabsent staining intensity).

Immunoreactivity of Chaperone Molecules inRetinoblastomaTapasin and calnexin expression levels were positivein 12 tumors in Group A (� 75% of cells stained with� intensity). In Group B (n � 18), tapasin exhibitedheterogeneous expression in 8 tumors (30 –50% ofcells stained with �/� intensity) and no expression in10 tumors (8 tumors with � 25% of cells stained with�/� intensity and 3 tumors with absent staining).Calnexin exhibited heterogeneous expression in 9 tu-mors (30 –50% of cells stained with �/� intensity) andno expression in 9 tumors (8 tumors with � 25% ofcells stained with �/� intensity and 1 tumor withabsent staining intensity).

Correlation of Immunoreactivity of HLA Class I Antigensand LMP-2, LMP-10, TAP-1, Tapasin, and Calnexin inRetinoblastomasThe expression levels of HLA Class I antigens and ofLMP-2, LMP-10, TAP-1, tapasin, and calnexin wereconcordant in most of tumors from Group A andGroup B. Staining for HLA Class I antigens and LMP-2,LMP-10, TAP-1, tapasin, and calnexin was positive intumors without invasion and was decreased in tumorswith invasion of the choroid and optic nerve. Thedifference in HLA and APM expression between the 2groups was statistically significant (P � 0.001). Figure1A–D shows positive immunoreactivity findings forHLA Class I, HLA Class II, LMP-2, and TAP-1; Figure1E–H shows heterogeneous immunoreactivity find-

ings for HLA Class I, LMP-2, and TAP-1; and Figure 1I,Jshows negative immunoreactivity findings for HLAClass I antigens.

DISCUSSIONThe current study is the first to examine HLAs andAPMs on archival paraffin sections in retinoblastoma,with results analyzed for correlations in terms of in-vasion of the choroid and the optic nerve. Our resultsare in accordance with the results from previous stud-ies on HLAs in retinoblastoma.8 –14 The current studydemonstrates that HLA Class I, �2-m, and HLA Class IIantigens are expressed in retinoblastomas with noinvasion and exhibit decreased expression when inva-sion of the choroid and the optic nerve are present.The difference in immunoreactivity was significant (P� 0.001). There was no correlation with tumor differ-entiation. The components of the antigen-processingpathway exhibited concurrent expression with HLAClass I antigen. Similar decreased expression of HLAClass I antigen with decreased expression of the com-ponents of the APMs has been reported in numerousother tumors7 and also in uveal melanoma, which isanother type of intraocular tumor found in adults.22

Thus, APMs are necessary for efficient peptidedelivery for MHC Class I antigen expression. Theselinked patterns of expression have led to the use of theterm HLA Class I coordinome to indicate a set of col-laborating gene products. The coordinome is onion-like and is organized hierarchically in multiple layers,suggesting the description of global changes in theantigen-processing machinery of neoplastic cells.23

The majority of invading tumors in our cohort

TABLE 4Immunohistochemistry Results for Group A: Retinoblastoma with No Invasion

Patient no.

HLA antigens ProteasomeTransporterprotein Chaperone protein

HLA Class I �2-m HLA Class II LMP-2 LMP-10 TAP-1 Tapasin Cainexin

1 Pos Pos Pos Pos Pos Pos Pos Pos2 Pos Pos Pos Pos Pos Pos Pos Pos3 Pos Pos Pos Pos Pos Pos Pos Pos4 Pos Pos Pos Pos Pos Pos Pos Pos5 Pos Pos Pos Pos Pos Pos Pos Pos6 Pos Pos Pos Pos Pos Pos Pos Pos7 Pos Pos Pos Pos Pos Pos Pos Pos8 Pos Pos Pos Pos Pos Pos Pos Pos9 Pos Pos Pos Pos Pos Pos Pos Pos10 Pos Pos Pos Pos Pos Pos Pos Pos11 Pos Pos Pos Pos Pos Pos Pos Pos12 Pos Pos Pos Pos Pos Pos Pos Pos

HLA: human leukocyte antigen; LM: low–molecular mass polypeptide; �2-m: beta 2 microglobulin; TAP: transporter-associated protein; Pos: positive.


expressed HLA Class I antigen and members of theantigen-processing machinery at low levels. This givesthe tumor an advantage, because retinoblastoma hasmultiple modes of invasion and metastasis, unlikeuveal melanoma, which metastasizes hematogenouslyto the liver.24 Human retinoblastomas exhibit fourpatterns of invasion and metastasis: 1) direct, invasivespread along the optic nerve to the brain, which alsocan seed the orbital tissue and adjacent bone, thenasopharynx via the sinuses, or the cranium via theforamina; 2) tumor cells that have invaded the opticnerve and leptomeninges and then disperse into thecirculating subarachnoid fluid are characteristic of thesecond pattern of metastasis; 3) hematogenous dis-semination that results in widespread metastasis tothe lungs, bones, brain, and other viscera (metastasisafter orbital invasion and, to a lesser degree, choroidalinvasion often is through this route); and 4) lymphaticspread, which occurs when the tumor is located ante-riorly or when massive extraocular invasion has oc-curred. Only tumors with these characteristics canspread through the lymphatic system, because thereare no lymphatic vessels in the eye or orbit. Whendisease reaches the regional lymph nodes, hematoge-nous spread also can occur.

Thus, both CTLs and natural killer (NK) cells mustplay a role in the spread of retinoblastoma. MHC ClassI antigen down-regulation in tumor cells is an impor-tant factor in the ability of these cells to escape rec-ognition and destruction by HLA Class I antigen-re-stricted, tumor-associated, antigen-specific CTLs.25 Incontrast, MHC Class I antigen loss may result in in-creased sensitivity of malignant cells to NK cells.26

However, the majority of invading tumor cells hadsome amount of HLA expression in the current study.A global loss of MHC Class I alleles, which occursinfrequently in tumors, may be due to the expressionof killer-inhibitory receptors on NK cells.27 Killer-in-hibitory receptor molecules recognize MHC Class I,and when they engage their ligand molecules, NKcell–mediated lysis is inhibited.28,29 Therefore, a tumorcell that is completely devoid of MHC Class I expres-sion may be able to evade specific CTL recognition butwould remain a good NK cell target. Some tumorsseem to lose expression of single HLA-A or B alleleswhile retaining the expression of other HLA al-leles.30 –32 Such a partial HLA loss may be advanta-geous to the tumor, because it potentially allows forthe escape of tumor cells from CTLs directed againstimmunodominant epitopes, presented by the lostHLA allele, while maintaining resistance to NK celllysis.

Thus, tumors only gain time for the accumulationof critical mutations and/or derangements in the ex-

pression of malignancy-causing genes, which eventu-ally will provide these tumors with uncontrollable in-vasive potential. Early-stage tumors manage to survivethe immune attack by maneuvering HLA expressionlevels within a rather narrow optimal window and bymodifying the expression of the individual HLA alleles,LMP, TAP, and tapasin without deviating excessivelyfrom the balance inherited from normal cells.

In the current study, HLA Class II antigens weredetected in retinoblastoma localized to the eye withno invasion. HLA Class II molecules play an importantrole in tumor progression, due to their ability topresent antigenic tumor peptides to T lymphocytes. Inthis regard, experimental findings have shown thatselected Tags are presented effectively by HLA Class IIantigens to CD4� T cells. Furthermore, it has beendemonstrated that solid tumor cells themselves canact as nonprofessional antigen-presenting cells (APCs)and are able to stimulate directly the proliferation ofautologous CD4� T cells. However, it also has beensuggested that tumor cells drive T cells in an anergystate that results in immunosuppression of tumor-reactive T-cell clones.33

HLA Class II antigens have been found in a num-ber of malignancies, albeit with variable frequency.Solid malignancies of different histology can expressHLA Class II antigens. However, limited data are avail-able on their functional role. HLA Class II antigenexpression is associated with poor prognosis and dis-ease progression in patients with cutaneous melano-ma34 and osteogenic sarcoma.35 However, an associ-ation with improved prognosis has been described inpatients with squamous cell carcinoma, breast carci-noma, colorectal carcinoma, cervical carcinoma, la-ryngeal carcinoma, and conjunctival carcinomas.36 – 42

In the current study, we observed low HLA Class IIexpression levels in all invasive tumors. What are thepossible implications of these low expression levels inretinoblastoma cells? HLA Class I expression on tumorcells is essential for killing by CD8� cytotoxic T cells.Nonetheless, recent studies also suggest an importantrole for CD4� helper-T cells and, consequently, forHLA Class II expression on tumor cells.43– 45 In addi-tion, cytotoxic CD8� cells are effective only after acti-vation by CD4� helper-T cells (i.e., when both CD8�

and CD4� cells recognize tumor-specific antigenic de-terminants on the same APCs).46 Thus, loss of bothHLA Class I and Class II expression may result inimpaired activation of cytotoxic CD8� T cells and mayfacilitate the growth of tumor cells. This finding sug-gests that not only host factors but also tumor cellcharacteristics, such as loss of HLA Class I and IIexpression, contribute to the phenomenon of immuneprivilege.


FIGURE 1. Immunohistochemistry of retinoblastoma. (A) Microphotograph showing the positive immunoreactivity of human leukocyte antigen (HLA) Class I

antigens, with bright intensity in tumor cells with no invasion (3,3�-diaminobenzidine [DAB] chromogen with hematoxylin counterstain). (B) Microphotograph showing

the positive immunoreactivity of HLA Class II antigens, with bright intensity in tumor cells with no invasion (DAB chromogen with hematoxylin counterstain). (C)

Microphotograph showing the positive immunoreactivity of low–molecular mass polypeptide 2 (LMP-2) in tumor cells arranged around the blood vessels (DAB

chromogen with hematoxylin counterstain). (D) Microphotograph showing positive immunoreactivity of the transporter-associated protein 1 (TAP-1) subunit in tumor

cells of well differentiated retinoblastoma with no invasion (DAB chromogen with hematoxylin counterstain). (E) Microphotograph showing the heterogeneous

immunoreactivity (50% of cells stained with dull intensity) of HLA Class I antigens in tumor cells arranged around the blood vessels in retinoblastoma with choroidal

invasion (DAB chromogen with hematoxylin counterstain). (F) Microphotograph showing the heterogeneous immunoreactivity (40% of cells stained with dull intensity)

of LMP-2 in retinoblastoma with choroidal invasion (DAB chromogen with hematoxylin counterstain). (G) Microphotograph showing the heterogeneous immuno-

reactivity (50% of cells stained with dull intensity) of the TAP-1 subunit in retinoblastoma with choroidal invasion (DAB chromogen with hematoxylin counterstain).

(H) Microphotograph showing the heterogeneous immunoreactivity (40% of cells stained with dull intensity) of the TAP-1 subunit in tumor cells invading the optic

nerve (DAB chromogen with hematoxylin counterstain). (I) Microphotograph showing the negative immunoreactivity of HLA Class I antigens in tumor cells invading

the postlaminar portion of the optic nerve (DAB chromogen with hematoxylin counterstain). (J) Microphotograph showing the negative immunoreactivity of HLA Class

I antigens in tumor cells at the metastatic site (submandibular region; DAB chromogen with hematoxylin counterstain). Original magnification �20 (C); �40 (D,F,G);

�100 (A,B,H–J); �200 (E).


To date, the molecular basis of low HLA Class Iantigen expression in retinoblastoma has not beenelucidated. In this regard, the concordant expressionof HLA Class I antigen and APMs in our study suggestsdefects in the regulatory mechanisms that controltheir expression. However locus-specific down-regu-lations may be mediated transcriptionally or may becaused by genetic defects. Immunohistochemical de-fects cannot easily discriminate between these twopossibilities. Analysis using cell lines have shown thatMHC Class I and Class II antigen expression was up-regulated after retinoic acid and IFN, suggesting thattranscriptional mechanisms are implicated in themodulation of HLA expression in retinoblastoma.

Although our understanding of the genetic eventsassociated with retinoblastoma metastasis is begin-ning to emerge, information on the mechanisms oftumor invasion and metastasis, which may allow thedevelopment of new diagnostic and therapeutic strat-egies to counteract the development of metastatic dis-ease, remains limited. Several factors, such as earlygenetic events (including increased copy numbers ofchromosomes 6p and 1q) and late events (such as highlevels of telomerase activity, loss of chromosome 1p,and p53 inactivation), contribute to the aggressivenessof retinoblastoma. In addition, increased angiogene-sis, deregulation of cell-to-cell adhesion molecules,and changes in integrins also contribute to tumoraggressiveness.47 In our earlier work on retinoblas-

toma, we observed that levels of Fas (APO-1 or CD95,a type I membrane protein of 43 kD and a member ofthe tumor necrosis factor receptor family),48 nm23protein (a metastasis suppressor protein),49 and tet-raspanin protein KAI1/CD82 were decreased in reti-noblastoma exhibiting invasion of the choroid and theoptic nerve.50

Because the eye is an immune privileged site,tumors must use multiple mechanisms to escape fromimmune-mediated rejection. These include igno-rance, impaired antigen presentation, expression ofimmunosuppressive factors and molecules, tolerance,apoptosis resistance, and counterattack. Our prelimi-nary data suggest that by expressing decreased levelsof HLAs on their surfaces, aggressive tumor cells es-cape recognition and destruction by HLA Class I an-tigen-restricted, tumor-associated, antigen-specificCTLs, and they also escape NK cells in travelingthrough the blood stream.

Clinically, HLAs are of limited interest comparedwith known histologic factors; such as invasion of thechoroid, optic nerve, and orbit; which currently areused to generate prognoses for patients with retino-blastoma. Nonetheless, biologically, our findings sug-gest a potential role for HLAs in tumor progression.Thus, further studies are required to elucidate themechanism of expression of HLAs in retinoblastoma.Resolution of this issue is of great importance to thedevelopment of potential therapeutic regimens.

FIGURE 1. (continued)


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Major histocompatibility antigens and antigen-processing molecules in retinoblastoma

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