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Clinical Cancer Research Vol. 11, 8304-8311, December 1, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Human Leukocyte Antigen and Antigen Processing Machinery Component Defects in Astrocytic Tumors

Angelica Facoetti1, Rosanna Nano1, Paola Zelini1, Patrizia Morbini2, Eugenio Benericetti4, Mauro Ceroni3,5, Michael Campoli6 and Soldano Ferrone6

Authors' Affiliations: 1 Department of Animal Biology, University of Pavia and Center of Study for Histochemistry, Consiglio Nazionale delle Ricerche; 2 Department of Pathology, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo; 3 Department of Neurological Sciences, Istituto di Ricovero e Cura a Carattere Scientifico C. Mondino Institute, University of Pavia, Pavia, Italy; 4 Neurosurgery Division, Hospital of Parma, Parma, Italy; 5 Policlinico of Monza, Monza, Italy; and 6 Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Soldano Ferrone, Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-8534; Fax: 716-845-7613; E-mail: soldano.ferrone{at}roswellpark.org.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To determine the frequency of abnormalities in human leukocyte antigen (HLA) and antigen processing machinery (APM) component expression in malignant brain tumors. This information may contribute to our understanding of the immune escape mechanisms used by malignant brain tumors because HLA antigens mediate interactions of tumor cells with the host's immune system.

Experimental Design: Eighty-eight surgically removed malignant astrocytic tumors, classified according to the WHO criteria, were stained in immunoperoxidase reactions with monoclonal antibody recognizing monomorphic, locus-specific, and allospecific determinants of HLA class I antigens, ß2-microglobulin, APM components (LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin), and HLA class II antigens.

Results: HLA class I antigens were lost in ~50% of the 47 glioblastoma multiforme (GBM) lesions and in ~20% of the 18 grade 2 astrocytoma lesions stained. Selective HLA-A2 antigen loss was observed in ~80% of the 24 GBM lesions and in ~50% of the 12 grade 2 astrocytoma lesions stained. HLA class I antigen loss was significantly (P < 0.025) correlated with tumor grade. Among the APM components investigated, tapasin expression was down-regulated in ~20% of the GBM lesions analyzed; it was associated, although not significantly, with HLA class I antigen down-regulation and tumor grade. HLA class II antigen expression was detected in ~30% of the 44 lesions analyzed.

Conclusion: The presence of HLA antigen defects in malignant brain tumors may provide an explanation for the relatively poor clinical response rates observed in the majority of the T cell–based immunotherapy clinical trials conducted to date in patients with malignant brain tumors.

Astrocytomas are heterogeneous intracranial neoplasms that develop from astrocytes. These tumors can range from the highly aggressive glioblastoma multiforme (GBM, grade 4) to the indolent, low-grade pilocytic astrocytoma (grade 1; refs. 1, 2). Typical survival rates are >5 years for diffuse astrocytoma (grade 2), 2 to 5 years for anaplastic astrocytoma (grade 3), and <1 year for the majority of patients with GBM (grade 4; refs. 1, 2). The lack of effective treatment for advanced-stage astrocytomas by conventional therapies (3, 4) has highlighted the need to develop alternative therapeutic strategies. Among them, immunotherapy has attracted much attention because of the promise of offering a tumor-specific treatment for patients with brain tumors. T cell–based immunotherapy has been emphasized due in part to the convincing evidence in animal model systems (5, 6) and more recently in patients with malignant disease (7–9) that MHC class I antigen-restricted, tumor antigen–specific CTLs play a major role in the control of tumor growth. Taking advantage of the tumor antigens identified in astrocytoma cells (7–9) and of the multiple strategies to enhance an immune response (10), a number of clinical trials, aimed at inducing CTL-mediated immune responses in patients with astrocytoma have been implemented. Contrary to the expectations, irrespective of the tumor antigen or immunization strategy used, clinical responses have only occasionally been observed and disease frequently recurs in spite of induction and/or persistence of tumor antigen-specific CTL-mediated immune responses (7–9). In view of the genetic instability of tumor cells (11), the discrepancy between induction of tumor antigen–specific immune response and lack of clinical response is likely to reflect the ability of malignant cells to escape from immune recognition and destruction.

A number of immune escape mechanisms have been identified and characterized in astrocytoma cells. These include tumor cell secretion of transforming growth factor-ß (12) and/or interleukin-10 (13), tumor cell expression of Fas (CD95; ref. 14), and/or human leukocyte antigen (HLA)-G (15) as well as CD70-mediated apoptosis of immune effector cells (16). An additional potential escape mechanism is represented by defects in HLA antigen as well as antigen processing machinery (APM) component expression and/or function because these molecules play a crucial role in the interactions between malignant cells and the host's immune system (17). In this regard, HLA class I antigen and APM component defects have been convincingly shown to be associated with malignant transformation of cells, although with different frequency in various types of malignancies, and to have a negative effect on the clinical course of the disease in some malignancies as well as the outcome of T cell–based immunotherapy (18, 19). In spite of the critical role played by HLA antigens and APM components in the interactions between malignant cells and immune cells (17), very little information is available regarding HLA antigen as well as APM component expression in malignant brain tumors. Therefore, in the present study, we have used a panel of monoclonal antibody (mAb) recognizing monomorphic, locus-specific and allospecific determinants of HLA class I antigens, ß2-microglobulin, APM components (LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin) and HLA class II antigens to analyze HLA antigen and APM component expression in 88 surgically removed malignant astrocytic tumors by immunohistochemical staining. To investigate HLA antigen and APM component expression, we have selected immunohistochemical staining instead of Western blot and reverse transcription-PCR because (a) at variance with Western blot, the direct visualization of stained cells allows the analysis of the expression of markers in both normal and malignant cells and (b) at variance with reverse transcription-PCR, immunohistochemical staining allows one to assess marker expression at the protein level. Furthermore, to assess the potential clinical significance of HLA antigen and APM component defects, we have correlated HLA antigen and APM component expression with the histopathologic characteristics of the lesions and the clinical features of the disease.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissues. Eighty-eight primary astrocytic tumors were obtained from patients who underwent surgery in the Neurosurgery Division, Ospedale Maggiore, Parma, Italy. Normal brain samples were collected from patients undergoing neurosurgery for frontal and lateral lobe aneurysms. The astrocytic tumors were diagnosed histopathologically by two skilled neuropathologists according to the classification system established by WHO for tumors affecting the central nervous system. They included 5 pilocytic astrocytoma lesions (grade 1), 18 diffuse astrocytoma lesions (grade 2), 18 anaplastic astrocytoma lesions (grade 3), and 47 GBM lesions (grade 4). Tissues were processed within 15 minutes following their surgical removal. One part of the tissue sample was fixed in 10% neutral buffered formaldehyde and routinely processed. Another part of the specimen was frozen in liquid nitrogen and stored at –80°C until use. Four-micrometer-thick cryostat sections were dried and fixed in absolute acetone for 1 minute. Under these fixation conditions, cryostat sections could be stored for at least 3 months at –20°C without loss of reactivity with antibodies.

Monoclonal antibody and polyclonal antibodies. The mouse ß2-microglobulin-specific mAb VF19-LL26;7 mAb HC10, which recognizes a determinant expressed on ß2-microglobulin-free HLA-A3, HLA-A10, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, and HLA-B (excluding HLA-B5702, HLA-B5804, and HLA-B73) heavy chains (20, 21); mAb TP25.99, which recognizes a determinant expressed on ß2-microglobulin-associated HLA-A, HLA-B, and HLA-C heavy chains and on ß2-microglobulin-free HLA-A1, HLA-A3, HLA-A9, HLA-A11, HLA-A30, HLA-B, and HLA-C heavy chains (22); mAb LGIII-147.4.1, which recognizes a determinant restricted to ß2-microglobulin-associated HLA-A heavy chains, excluding HLA-A23, HLA-A24, HLA-A25, HLA-A32 heavy chains (23); HLA-A2, HLA-A28-specific mAb KS1 (24); mAb LGII-612.14, which recognizes a linear determinant expressed on HLA-DR, HLA-DQ, and HLA-DP ß chains (25); LMP2-specific mAb SY-1 (26); LMP7-specific mAb HB-2 (26); calnexin-specific mAb TO-5 (27); calreticulin-specific mAb TO-11 (27); and tapasin-specific mAb TO-3 (27) were developed and characterized as described. TAP1-specific mAb TO-1 and TAP2-specific mAb NOB-2 were developed and characterized using the strategy described elsewhere (27). Briefly, the TAP1-specific mAb TO-1 and the TAP2-specific mAb NOB-2 are secreted by hybridomas derived from the fusion of murine myeloma cells P3-X63-Ag8.653 with spleenocytes from BALB/c mice immunized with partial-length TAP1 recombinant protein (435-748) and keyhole limpet hemocyanin–conjugated TAP1 peptides (684-695) and (777-795) and with partial length TAP2 recombinant protein (316-703), respectively. The specificity of the selected mAb was determined by their reactivity with the corresponding antigens when tested with lymphoid cell lysates with the appropriate phenotype in Western blotting. The specificity of TAP1-specific mAb TO-1 and TAP2-specific mAb NOB-2 was corroborated by their lack of reactivity with a lysate of the T2 cell line, which does not express these molecules (28). CD34-specific mAb QBEnd, CD45-specific mAbs 2B11 and PD7/26, and CD68-specific mAb PG-M1 were purchased from DAKOCytomation (Carpinteria, CA).

Horseradish peroxidase–conjugated goat anti-mouse Fc antibodies were purchased from DAKOCytomation.

Immunohistochemical staining of tissues. Indirect immunoperoxidase staining of formalin-fixed, paraffin-embedded tissue sections for HLA class I and class II antigen expression with mAb HC10 and LGIII 612.14, respectively, was done using horseradish peroxidase–conjugated goat anti-mouse Fc antibodies and DAKOCytomation EnVision staining system with 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and 4-chloro-2-methylbenzenediazonium salt Azoic Diazo No. 11 (Fast Red TR Salt; Sigma) as chromogen. Indirect immunoperoxidase staining of frozen tissue sections for HLA-A antigen, HLA-A2 antigen, and APM components LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin expression with the corresponding mAb was done using horseradish peroxidase–conjugated goat anti-mouse Fc antibodies and DAKOCytomation EnVision staining system with 3,3'-diaminobenzidine tetrahydrochloride and Fast Red TR Salt as chromogens. The immunostaining procedure has been described in detail elsewhere (27). At least 10 fields at x40 magnification were randomly selected in each stained tumor section for analysis of immunostaining results. Necrotic tumor sections were avoided in all cases because immunohistochemical staining of necrotic tissue yields unreliable results. The percentage of stained malignant cells in each lesion was evaluated independently by two investigators. Variations in the percentage of stained cells enumerated by the two investigators were within a 10% range. Results were classified according to the criteria established by the HLA and Cancer component of the 12th International Histocompatibility Workshop (29). According to these criteria, lesions are scored as positive, heterogeneous, and negative, when the percentage of stained tumor cells in the entire lesion is >75% (positive), between 75% and 25% inclusive (heterogeneous), and <25% (negative), respectively.

Statistical methods. The differences in HLA class I antigen expression were analyzed using the {chi}2 test. P < 0.01 (for 1 degree of freedom) and 0.025 (for 2 degrees of freedom) were considered statistically significant. The association of HLA antigen expression with disease-free interval and survival was analyzed using the log-rank test.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Human leukocyte antigen class I expression in astrocytic tumors. Preliminary studies investigated the expression of HLA class I antigens and ß2-microglobulin in normal brain samples collected from patients undergoing neurosurgery for frontal and lateral lobe aneurysms; mAb recognizing monomorphic, locus-specific and allospecific determinants of HLA class I antigens and ß2-microglobulin-specific mAb were used as probes. Both astrocytes and infiltrating lymphocytes stained positive with all of the mAb tested.

The results of immunoperoxidase staining of 44 frozen and 44 formalin-fixed, paraffin-embedded lesions with mAb recognizing monomorphic, locus-specific and allospecific determinants of HLA class I antigens and ß2-microglobulin-specific mAb are summarized in Tables 1Go–3 and in Fig. 1. Representative staining patterns with mAb HC10, which recognizes a determinant expressed on ß2-microglobulin-free HLA-A3, HLA-A10, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, and HLA-B (excluding HLA-B5702, HLA-B5804, and HLA-B73) heavy chains (19, 20) are shown in Fig. 2. The expression of the antigenic determinant recognized by each mAb used in the patient from whom the malignant astrocytic lesion being tested had been removed was monitored by analyzing the staining of surrounding normal cells (i.e., vascular endothelial cells, lymphocytes, and microglial cells present in the same tissue section). Vascular endothelial cells and tumor infiltrating lymphocytes stained positive with all of the mAb tested. HLA class I antigens were not detected in 22 of the 47 GBM lesions and 3 of the 18 grade 2 astrocytoma lesions. HLA-A antigens were not detected in 11 of the 24 GBM lesions but were expressed in all 12 grade 2 astrocytoma lesions. Similar results were obtained by staining frozen tissue sections with ß2-microglobulin-specific mAb. GBM and grade 2 astrocytoma lesions were surgically removed from 24 and 12 HLA-A2+ patients, respectively. HLA-A2 antigen was not detected in 19 of the 24 GBM lesions and in 6 of the 12 grade 2 astrocytoma lesions.


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  		Table 1. HLA class I and class II antigen expression in formalin-fixed, paraffin-embedded astrocytic tumor sections

 

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  		Table 2. HLA class I and class II antigen expression in frozen astrocytic tumor sections

 

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  		Table 3. HLA class I antigen expression in malignant brain tumors

 


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  		Fig. 1. Frequency of HLA class I antigen defects in surgically removed astrocytoma lesions, as determined by immunohistochemical staining with mAb that recognize monomorphic antigenic determinants. Grades 1, 2, 3, and 4 astrocytoma lesions were divided into groups according to their reactivity in the immunoperoxidase reaction with mAb, which recognize monomorphic determinants of HLA-A, HLA-B, and HLA-C antigens. Lesions were scored as positive (P), heterogeneous (H), and negative (N), when the percentage of stained tumor cells in the entire lesion was >75%, between 75% and 25% inclusive, and <25%, respectively.

 


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  		Fig. 2. HLA class I antigen down-regulation in a surgically removed GBM lesion. Immunoperoxidase staining with mAb HC10 resulted in the staining of only gemistocytes (black arrows) (A, x100) and vascular endothelial cells and infiltrating lymphocytes (black arrows) (B, x100) in a surgically removed GBM lesion.

 
To investigate the potential clinical significance of HLA class I antigen abnormalities in astrocytoma lesions, their expression was correlated with the histopathologic characteristics of the lesions and with clinical variables of the disease. Association of HLA class I allospecificity loss with tumor grade could not be tested because only 17 lesions were analyzed. Total HLA class I antigen loss was significantly [P < 0.025; low-grade astrocytoma (grades 1 and 2) versus high-grade astrocytoma (grades 3 and 4), 2 degrees of freedom] correlated to tumor grade. No association was found between the frequency of total HLA class I antigen loss in surgically removed GBM and grade 2 astrocytoma lesions and disease-free interval and survival. The latter finding is most likely due to the small number of lesions analyzed.

Antigen processing machinery component expression in astrocytic tumors. The results of immunoperoxidase staining of 2 normal brain tissue sections, 12 diffuse astrocytoma grade 2, 8 anaplastic astrocytoma grade 3, and 24 GBM frozen tissue sections with APM component-specific mAb are summarized in Fig. 3. Representative staining patterns are shown in Fig. 4. All normal brain tissue sections displayed a diffuse cytoplasmic staining pattern with all the APM component-specific mAb tested. LMP2, LMP7, TAP1, and TAP2 were expressed in 42, 40, 37, and 40 of the 44 lesions tested, respectively. Both TAP1 and TAP2 were down-regulated in two GBM lesions. All tissue sections were positive for calnexin and calreticulin expression. In contrast, tapasin was down-regulated (i.e., lesions were scored as heterogeneous and/or negative, in 2 of 12 of the astrocytoma grade 2 and in 19 of the 24 GBM lesions tested). It is noteworthy that tapasin down-regulation was associated with HLA class I antigen down-regulation as well as tumor grade. However, this association did not reach the level of statistical significance (P < 0.01 for 1 degree of freedom; P < 0.025 for 2 degrees of freedom) most likely due to the small number of lesions analyzed.



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  		Fig. 3. Differential APM component expression in surgically removed astrocytoma lesions of different histologic grades. Grade 1, 2, and 3 astrocytoma and GBM lesions were divided into groups according to their reactivity in the immunoperoxidase reaction with calnexin-specific mAb TO-5 ({04-2588fx01g}), calreticulin-specific mAb TO-11 ({square}), and tapasin-specific mAb TO-3 ({blacksquare}). Lesions were scored as positive, heterogeneous, and negative, when the percentage of stained tumor cells in the entire lesion was >75%, between 75% and 25% inclusive, and <25%, respectively.

 


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  		Fig. 4. Tapasin down-regulation in a surgically removed GBM lesion. Immunoperoxidase staining with tapasin-specific mAb TO-3 resulted in a heterogeneous staining pattern of astrocytoma cells in a surgically removed GBM lesion (x100). (black arrow, positive; clear arrow, negative).

 
Human leukocyte antigen class II expression in astrocytic tumors. The results of immunoperoxidase staining of 44 formalin-fixed, paraffin-embedded lesions with mAb LGII-614.12, which recognizes a linear determinant expressed on HLA-DR, DQ, and DP ß chains are summarized in Table 1. Representative staining patterns are shown in Fig. 5. Only 13 of the 44 formalin-fixed, paraffin-embedded lesions analyzed were stained by HLA class II antigen-specific mAb. Both tumor-infiltrating microglia and microglia surrounding tumor margins, which had the typical morphology of preactivated microglia, were stained by HLA class II antigen-specific mAb. It is noteworthy that tumor-infiltrating microglia showed a stronger intensity of staining than microglia surrounding the tumor mass.



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  		Fig. 5. HLA class II antigen expression in a surgically removed GBM lesion. Immunoperoxidase staining of a surgically removed GBM lesion with mAb LGII-612.14 resulted in the staining of microglia bordering (A, x100) and infiltrating (B, x200) the tumor cells.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To the best of our knowledge, the present study is the first one to have analyzed HLA antigen and APM component expression in surgically removed malignant astrocytic tumors by immunohistochemical staining. A total of 88 malignant astrocytic tumors, classified according to the WHO criteria, were investigated. HLA class I antigens were lost in ~50% of the 47 GBM lesions and in ~20% of the 18 grade 2 astrocytoma lesions analyzed. The frequency of HLA class I antigen loss we have found in grade 2 astrocytoma lesions is similar to that found in cervical carcinoma and melanoma lesions, whereas that of GBM is similar to that found in head and neck squamous cell, breast, lung, renal cell, colon, and prostate carcinoma lesions (18). In view of the role of immunoselection in the generation of malignant cell populations with HLA class I antigen defects (30–32), the differences in the frequency of HLA class I antigen abnormalities may reflect the complex interplay between the level of T cell–selective pressure, the extent of genetic instability in malignant cells, the time length between onset of tumor and diagnosis, and/or the characteristics of the patient population investigated.

The present study has shown for the first time a high frequency (i.e., 70%) of selective HLA-A down-regulation in both surgically removed grade 2 astrocytoma and GBM lesions. Although caution should be exerted when interpreting these data, due to the relative small sample size analyzed, this frequency of down-regulation of the gene products of HLA-A locus is higher than that described for melanoma, head and neck squamous cell, breast, lung, renal cell, colon, and prostate carcinoma lesions (18).

A number of investigations have shown that staining of malignant cells with mAb recognizing monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss (18). Therefore, to determine whether these findings apply also to brain cancer, in the present study we have analyzed the expression of HLA-A2 antigen. This allospecificity was chosen because (a) it is expressed in ~50% of the patient population (33); (b) it is selectively lost in some malignant lesions with a frequency of at least 20% (18); (c) it presents immunodominant epitopes derived from a number of tumor antigen, including HER-2/neu, tyrosinase, tyrosinase-related proteins 1 and 2, gp100, MAGE-1, and MAGE-3, which have been shown to be expressed in astrocytoma lesions (7–9, 34, 35); (d) it is used as a restrictive element by tumor antigen–specific CTL that lyse glioblastoma cells (7–9, 34, 35); and (e) HLA-A2-specific mAb capable of detecting HLA-A2 antigen determinants in surgically removed frozen tissue sections by immunohistochemical staining are readily available to us. HLA-A2 antigen was not detected in ~80% of the surgically removed GBM lesions and ~50% of the surgically removed grade 2 astrocytoma lesions. It should be noted that lesion CR46 was scored as negative for HLA-A antigen expression but heterogeneous for HLA-A2 antigen expression. The latter finding can be attributed to (a) the low percentage of tumor cells stained by HLA class I antigen locus-specific and HLA-A2 antigen-specific mAb in this lesion [i.e., HLA-A (21%) and HLA-A2 (28%)] and/or (b) selective loss of the determinant recognized by the HLA-A antigen-specific mAb. It is noteworthy that selective loss of antigenic determinants expressed on HLA class I molecules has recently been documented in melanoma lesions (36). Undetected selective loss of a HLA class I allele may account for the unexpected poor prognosis of the disease in patients with high expression of HLA class I antigens in primary and/or metastatic lesions, as measured by staining with mAb to monomorphic determinants of HLA class I antigens. Furthermore, selective loss of a HLA class I allospecificity is likely to have a negative effect on the efficacy of T cell–based immunotherapy, which uses the lost HLA class I allele as a restricting element.

Abnormalities in HLA class I antigen expression seem to have clinical significance because, in the present study, the frequency of HLA class I antigen defects was significantly correlated with tumor grade, an important prognostic marker in astrocytoma (1, 2). Furthermore, the association between HLA class I antigen abnormalities and tumor grade argues in favor of a potential role of HLA class I antigen abnormalities in the clinical course of astrocytoma, although the frequency of total HLA class I antigen abnormalities was not associated with disease-free interval and survival in this study. The latter finding is most likely due to the small number of lesions analyzed. It is noteworthy that the association between HLA class I antigen abnormalities and tumor grade is not unique to astrocytoma lesions because a similar association has been found in several malignancies, including carcinomas of the larynx, lung, breast, colon, kidney, bladder, and cervix as well as melanoma. In the latter malignancies, the frequency of HLA class I antigen abnormalities is significantly associated with poor histologic differentiation, abnormal DNA content, and advanced clinical stage (tumor grading), each criteria suggestive of a more aggressive malignant phenotype (18).

In the last several years, the realization of the potential negative effect of APM component defects on the recognition of malignant cells by the immune system of host (17) has stimulated interest in the analysis of APM component expression in malignant lesions (19). Alterations in APM component expression (i.e., LMP2, LMP7, TAP1, TAP2, and tapasin) have been identified in many tumors of different histotype (19). These aberrations tend to correlate with defects in HLA class I antigen expression and, in some malignancies, are significantly associated with the clinical course of the disease (19). Brain tumors are no exception to this rule because tapasin was down-regulated in a high proportion of GBM lesions and its down-regulation was frequently associated with defects in HLA class I antigen expression. Tapasin down-regulation has also been documented in renal cell carcinoma (32) and head and neck squamous cell carcinoma (37, 38) lesions. In the latter malignancy, this down-regulation is significantly associated with poor prognosis (37). This association is likely to reflect abnormalities in tumor cell recognition by CTL because of defects in the generation and presentation of HLA class I antigen-peptide complexes. These results suggest that abnormalities in APM component expression may underlie defective HLA class I antigen phenotypes in GBM lesions.

In the present study, the frequency of HLA class II antigen expression in 44 formalin-fixed, paraffin-embedded astrocytoma lesions was ~30%. Caution should be exercised when interpreting these results because the number of lesions analyzed in the present study is too low to draw definitive conclusions regarding the frequency of HLA class II antigen expression. However, induction of HLA class II antigens has been documented in a number of malignancies, including melanoma as well as carcinomas of the colon, cervix, and ovaries (39–47). In the present study, no correlations could be drawn between HLA class II antigen expression and clinical course of the disease, which is at variance with melanoma (39, 42). Despite this finding, HLA class II antigen expression on astrocytoma cells is likely to play a role in their interactions with the immune system of host given their ability to activate CD4+ T lymphocytes that seem to be an important immune component in the eradication of tumor cells (48). Therefore, the results of the present study emphasize the need to perform more comprehensive studies to assess the functional role of HLA class II antigen expression in the clinical course of astrocytoma.

In conclusion, this study has evaluated for the first time the frequency and the histopathologic significance of HLA antigen and APM component expression in surgically removed astrocytoma lesions, providing information regarding the frequency of HLA class I antigen abnormalities in this type of tumor. These findings suggest that abnormalities in HLA class I antigen as well as in APM components may provide a mechanism for astrocytoma cells to escape immune recognition and killing by HLA class I antigen–restricted, tumor antigen–specific CTL. Moreover, these findings emphasize the need to monitor HLA class I antigen and APM component expression in astrocytoma lesions to select patients to be treated with T cell–based immunotherapy.


   Footnotes
 
Grant support: USPHS grants P30 CA16056, RO1 CA67108, and T32 CA85183 awarded by the National Cancer Institute, Department of Health and Human Services and Department of Defense predoctoral fellowship BC030039.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 S. Ferrone et al., unpublished data. Back

Received 12/21/04; revised 7/26/05; accepted 7/28/05.


   References
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 Materials and Methods
 Results
 Discussion
 References
 

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