Apoptosis-associated proteins in oral lymphomas from HIV-positive patients

Apoptosis-associated proteins in oral lymphomas from HIV- positive patients Joseph A. Regezi, DDS, MS, a Alex McMillan, PhD, b Nusi Dekker, MA, a Troy E. Daniels, DDS, MS, a Sol Silverman, Jr., DDS, a Michael Schoelch, DMD, a,c and Barry L. Ziober, PhD, a San Francisco, Calif. UNIVERSITY OF CALIFORNIA AT SAN FRANCISCO

Extranodal oral lymphomas, seen with increasing frequency in HIV infection, may have dysfunctional apoptotic mech- anisms that favor tumor progression. The purpose of this study was to evaluate extranodal lymphomas from HlV-positive patients for expression of apoptosis-associated proteins. Correlations were made with 10 histologically comparable extranodal lymphomas from HlV-negative patients and 6 hyperplastic lymph nodes from otherwise healthy young adults. Formalin-fixed tissue sections were immunohistochemically stained for apoptosis-associated proteins (Bcl-2, Bcl-x, Bax, Bak, p53, MDM2, BHRF). In situ hybridization was also done on deparaffinized sections for Epstein-Barr virus EBER mRNA. Eighteen consecutive oral lymphomas were studied in HIV/AIDS-positive patients. Four of 5 intermediate-grade lymphomas expressed Bcl-2 to a greater degree than did high-grade lymphomas (4 of 13). Most lymphomas were positive for Bcl-x and Bax, and few expressed Bak. The staining patterns for these proteins were similar to those seen in HIV-negative patients. Staining patterns were relatively consistent in the hyperplastic lymph nodes, whereas such patterns were irregular in lymphomas. Positive p53 staining was seen in 11 of 18 HIV-positive cases; 9 of these were also MDM2-positive. Double stains suggested that both p53 and MDM2 proteins were expressed in the same cells in these nine cases. Epstein-Barr virus-EBER mRNA was detected in 14 of 18 cases and in 3 of 10 cases from HIV-negative patients. BHRF staining was evident in onlya few cells of three HIV-positive lymphomas. The irreg- ular expression of Bcl-2, Bcl-x, Bax, and Bak in oral lymphomas indicates dysfunctional apoptotic mechanisms in these tumors. Bc[-2 staining differs with tumor grade. Positive staining for p53 and MDM2 proteins is a notable feature of lymphomas in HIV- positive patients and may relate to binding of MDM2 to wild-type p53. Epstein-Barr virus is more commonly associated with oral lymphomas in HlV-positive patients, although the Epstein-Barr virus-produced protein BHRF, which has Bcl-2-1ike activity, is minimally expressed. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;86:196-202)

Non-Hodgkin 's lymphomas are relatively common neoplasms (53,600 new cases estimated for 1997) 1 and often occur in extranodal head and neck sites, espe- cially in cases of HIV infection (AIDS). 2-6 Until recently, extranodal oral lymphomas, appearing as either primary or secondary oral disease, were uncommon.7, 8 With the emergence of HIV infection, oral HIV-associated lymphomas are now seen with increasing frequency and are accompanied by a distinct shift to predominately high-grade tumors associated with Epstein-Barr virus (EBV) infection. 9-14

Apoptosis is a genetically controlled cell death mechanism that can be activated by a number of stimuli and detected by morphologic alterations15,16 and biochemical changes (endonuclease-associated nuclear fragmentation).17 Apoptosis-associated gene products have either proapoptotic or antiapoptotic functions. 18-20 Loss of apoptosis control is a potentially important aspect of tumor biology.

This study was supported in part by NIH grant PO1-DE-07946. aDepartmeut of Stomatology, UCSE bUCSF Cancer Center. CVisiting Investigator from the US Navy. Received for publication Jan. 15, 1998; returned for revision Mar. 23, 1998; accepted for publication Apr. 7, 1998. Copyright © 1998 by Mosby, Inc. 1079-2104/98/$5.00 + 0 7/14/90964

Important apoptosis-associated genes are members of the Bcl-2 family (Bcl-2, Bcl-x, Bax, Bak, and others). Bcl-2 protein is antiapoptotic. 21,22 Bcl-x represents two proteins (Bcl-x L and Bcl-xs) that have opposing func- tions, 23-25 whereas Bax 26,27 and Bak 28,29 are proapop- totic. The many competing proteins in the Bcl-2 family regulate apoptosis by their relative quantities and formation of competing dimers.26, 30 Overexpression of Bcl-2 has been described in follicular lymphomas31, 32 and other lymphomas, including HIV-associated and oral lymphomas. 33-35 Other Bcl-2-associated proteins are also involved in the pathogenesis of lymphomas (non-HIV and HIV-associated), inasmuch as both Bcl-x and Bax have been detected in these tumors.

The protein designated p53, which is a tumor suppressor gene product, can arrest the cell cycle in the G 1 phase to mediate DNA repair, or it can induce apop- tosis through regulation of apoptosis-associated genes (Bcl-2 suppression and increased Bax expression). 36,~7 p53 mutations resulting in defective protein contribute to cell proliferation. Although wild-type and mutated p53 overexpression is frequently seen in lymphomas, many cases are not associated with gene mutation. 38 p53-associated cell proliferation can also occur when wild-type protein is bound and inactivated by other cellular proteins (MDM2 protein) and viral proteins (human papillomavirus E6 protein, EBV EBNA5, and

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Table I. Antibodies used to identify apoptosis-associated proteins

Antigen Type Clone~type Source Dilution

Bcl-2 Antiapoptosis 124 (IgGlk) Dako, Carpinteria, Calif. 1:100 Bcl-Xs/l* Apoptosis (Xs) and Polyclonal Santa Cruz Biotechnology, 1:1000

antiapoptosis (X1) Santa Cruz, Calif. Apoptosis Coulter/Immunotech, Westbrook, Me. Apoptosis Oncogene Science, Cambridge, Mass. Proliferation Oncogene Science Apoptosis and Novocastra0 Burlingame, Calif. antiproliferation Antiapoptosis

Bax 4F11 (IgG2b) 1:200 Bak TC-100 (IgG2k) 1:25 MDM2 1F2 (IgG2bk) 1:25 p537 DO-7 (IgG2b) 1:200

BHRF1 5B 17 (IgG1) Advanced Biotech 1:600

*Antibody to Bcl-x does not discriminate between long and short isoforms of Bcl-x. ?Antibody to p53 identifies both wild-type and mutant proteins.

Table II. Summary of results of immunohistochemical and in situ hybridization reactions in HIV-positive oral lymphomas, HIV-negative oral lymphomas, and lymphoid hyperplasias

Bcl-2 Bcl-x Bax Bak MDM2 p53 EBER BHRF

HIV-positive 8/18* 16/18 17/18 7/18 9/18 11/18 14/18 3/18~ HIV-negative 5/10 9/10 9/10 9/10 1/10 4/10 3/10 0/10 Hyperplastic nodes 6/6:~ 5/6§ 6/6 0/6 0/6 0/6 0/6 0/6

*Positive/total specimens. ?Few cells positive in these 3 cases. $Staining seen predominantly in small lymphocytes. §Staining seen predominantly in large lymphoid cells.

BZLF proteins). 39,4° MDM2 protein is a negative regu- lator of p53 protein, and when overexpressed it has a tumor-promoting effect similar to that of mutated p53. This mechanism is active in some sarcomas and lymphomas.38,41-45

EBV association with lymphomas is well known. In addition to producing p53-binding proteins, EBV may also contribute to tumorigenesis through other proteins, such as LMP1, which blocks p53-triggered apoptosis through up-regulation of Bcl-2 gene46, 47 and BHRF1 that functions similarly to Bcl-2 protein. BHRF1 protects the cell from apoptosis even in the presence of some anticancer chemotherapeutic agents. 48-50

The purpose of this investigation was to evaluate oral lymphomas from HIV-positive patients for expression of apoptosis-regulating proteins (Bcl-2, Bcl-x, Bax, Bak, p53, MDM2). Tumors were also evaluated for EBV-associated BHRF protein and EBER mRNA tran- scripts by in situ hybridization.

MATERIAL AND METHODS Study subjects

Tissue sections of formalin-fixed oral lymphomas were reviewed independently by two pathologists for classification purposes. Eighteen consecutive acces- sions from patients known to be HIV-positive or to have been diagnosed with AIDS were identified. Classified according to the National Institutes of Health Working Formulation, there were 5 interme-

diate-grade lymphomas (diffuse large cell) and 13 high-grade lymphomas (3 large cell immunoblastic, 7 large cell anaplastic, and 3 small noncleaved). Classified according to the Revised European- American Lymphoma (REAL) scheme, there were 15 diffuse large B-cell lymphomas and 3 Burkitt's or Burkitt-like lymphomas. For comparison, 10 compa- rably diagnosed oral lymphomas were included from patients presumed to be HIV-negative (biopsy before AIDS epidemic or no HIV risk factors). These included eight diffuse large cell lymphomas, one large cell immunoblastic lymphoma, and one small noncleaved lymphoma; according to the REAL classification system, these were 9 diffuse large B-cell lymphomas and one Burkitt's or Burkitt-like lymphoma. Formalin- fixed hyperplastic lymph nodes, removed for diag- nostic purposes from the buccal mucosa of six patients (mean age, 16 years; age range, 9-27 years; gender distribution, 2 male and 4 female) between 1970 and 1980 served as lymphoid "controls."

The clinical profile for the 18 HIV-positive patients with lymphoma was as follows: mean age, 46 years; age range, 35 to 65 years; gender distribution, 18 male and 0 female; location, 5 palate, 4 buccal mucosa and vestibule, 4 gingiva, 4 jaw, and 1 lip; presenting sign, 10 ulcerated mass and 8 mass. For all patients in this group, oral lesions represented the first sign of their lymphomas. The profile for the 10 patients in the HIV- negative group was as follows: mean age, 61 years; age

198 Regezi et aL ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August 1998

Fig. 1. Immunohistochemical patterns for Bcl-2 family of proteins in oral lymphomas: Bcl-2 (top left; HIV-negative patient); Bcl-x (top right; HIV-positive patient); Bax (bottom left; HIV-positive patient); Bak (bottom right; HI-V-negative patient; original magnification xl000).

range 41 to 80 years; gender distribution, 5 male and 5 female; location, 5 gingiva, 2 buccal mucosa, 1 palate, 1 jaw, and 1 lip; presenting sign, 4 ulcer and 6 mass. Oral lesions were the first sign of lymphoma in eight patients in this group, and two had known systemic disease.

Immunohistochemistry Paraffin-embedded, formalin-fixed tissue sections

were used in a standard immunohistochemistry protocol. Sections were microwaved in 0.1 mol/L citrate buffer for 7 minutes. They were incubated for 1 hour with antibodies listed in Table I and then with a biotinylated supersensitive multilink peroxidase agent (BioGenex, San Ramon, Calif.). Antigen-antibody

reactions were visualized with the chromogen aminoethylcarbazole. Normal mouse (or rabbit) serum containing mixed immunoglobulins at a concentration approximating that in primary antibodies was used as a negative control. Immunohistochemically stained sections were evaluated through use of a semiquantita- tive grading scale: <25% cells positive, 1+; 25% to <50% cells positive, 2+; 50% to <75% cells positive, 3+; >75% cells positive, 4+.

To determine whether p53 (wild-type and mutant proteins) and MDM2 were coexpressed in tumor cells, double immunohistochemical stains were done on 10 cases (9 HIV-positive, 1 HIV-negative) in which single stains for both of these antigens were positive. A modi- fied Vector Laboratories (Burlingame, Calif.) protocol

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Regezi et al. 199 Volume 86, Number 2

Fig. 2. Immunohistochemical stains show positive nuclear reaction for p53 protein (left) and MDM2 protein (right) in oral lymphoma (HIV-positive patient; original magnification xl000).

was used to identify two nuclear antigens with two different mouse monoclonal antibodies. After sections were deparaffinized, antigen enhancement was achieved by microwaving in citrate buffer. After hydrogen peroxide block and BSA/PBS bath, sections were incubated with the first primary antibody for 1 hour and then with BioGenex Multilink avidin/biotin peroxidase and substrate Vector SG (blue). Sections were washed, treated again with BSA/PBS, and incu- bated with the second primary antibody for 1 hour. BioGenex Multilink avidin/biotin peroxidase and substrate aminoethylcarbazole (red) were applied. No counter stain was used. In additional sections, the sequence of primary antibodies was reversed but the order of the detection systems was the same.

In situ hybridization Paraffin sections were stained for EBV-EBER mRNA

transcripts through use of an in situ hybridization kit (BioGenex).5 ~-53 Detection of EBER mRNA in formalin- fixed, paraffin-embedded tissue is possible when sensi- tive in situ hybridization methods are used. A 0 to 4+ scoring system was used, as described previously.

RESULTS Generally, expression of apoptosis-associated

proteins in oral lymphomas from HIV-positive patients was unpredictable. Cytoplasmic expression of Bcl-2 was seen in 8 of 18 cases (Table II), ranging from 1+ to 4+. In intermediate-grade lymphomas (Working Formulation) 4 of 5 cases were positive, and in high- grade lymphomas 4 of 13 cases were positive. Bcl-x and Bax proteins were detected in the cytoplasm of most tumor cells of most lymphomas. Staining was generally intense (3+ to 4+) for these proteins. Positive

Bak protein staining was seen in 7 of 18 HIV-positive lymphomas, usually in less than 25% of cells (Fig. 1). Positive p53 staining (1+ to 4+) was seen in 11 of 18 lymphomas, and in 9 of these MDM2 staining was also positive (1+; Fig. 2). Double stains for p53 and MDM2 showed that most positively stained cells exhibited a smudgy purple-brown color suggesting expression of both proteins by the same cell (Fig. 3). Occasionally, some cells showed only a red-orange or blue color, indicating detection of only one of the two target proteins. EBER mRNA was detected in 14 of 18 HIV- positive lymphomas (Fig. 4), and BHRF protein staining was seen in only a few cells of three cases.

The expression of Bcl-2, Bcl-x, Bax, and Bak in lymphomas from HIV-negative patients was compa- rable to expression in HIV-positive patients (Table II). Positive p53 staining was evident in four cases, and one of these also stained positive for MDM2. A signif- icant difference in EBER mRNA staining was detected: 3 of 10 HIV-negative cases vs 14 of 18 HtV- positive cases.

In hyperplastic lymph nodes, relatively consistent staining patterns were seen. Bcl-2 was expressed by small lymphocytes in mantle and interfollicular zones in all of these specimens (Table II). There was a statis- tically significant difference (by Fisher's Exact Test) with respect to Bcl-2 expression between hyperplastic lymph nodes and all of the lymphomas evaluated. Although the number of lymphomas expressing Bcl-x was not significantly greater than the number of lymph nodes expressing this protein, quantitative differences were noted; 1+ to 2+ in lymph nodes vs 1+ to 4+ in lymphomas. Differences among HIV-posi- tive, HIV-negative, and hyperplastic nodes were not significant for Bax expression. The remainder of the

200 Regezi et al. ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August 1998

Fig. 3. Double immunohistochemical stains for p53 protein (blue) and MDM2 protein (red). Note that double-stained cells (arrows) appear as dark purple nuclei (HIV-positive patient; original magnification x 1000).

Fig. 4. In situ hybridization for Epstein-Barr virus EBER mRNA transcripts in oral lymphoma. Positive nuclear signal (red) is seen in most tumor cells (HIV-positive patient; original magnifi- cation x400).

apoptosis-associated proteins were not detected in these hyperplastic tissues.

DISCUSSION Tumor progression, which is directly associated

with increased cell proliferation, is influenced by the rate of cell death (apoptosis). An apoptosis-associated growth advantage may be controlled, at least in part, by abnormal expression of the Bcl-2 family of genes. Our results show that the Bcl-2 group of proteins, which either positively or negatively regulate apop- tosis, are variably altered in oral lymphomas regard- less of HIV status.

Comparison of Bcl-2 protein expression in interme- diate-grade lymphomas and high-grade lesions showed reduced staining in the high-grade lesions--an obser- vation previously noted in systemic lymphomas. 34 This loss of Bcl-2 expression may have importance in tumor pathogenesis. Reduced expression of an apparently important antiapoptotic protein could be compensated for by increases in other antiapoptotic proteins, such as Bcl-x L. Although antibody to Bcl-x does not discrimi- nate between the two isoforms with opposing effects, Bcl-x L is believed to be the dominant isoform. 24,54

Bax has an important role in limiting the life span of normal lymphoid cells. Lymphomas showed Bax

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Regezi et al. 201 Volume 86, Number 2

staining that was similar to that of hyperplastic nodes.

Almost all tumors that expressed Bax also expressed

Bcl-2 or Bcl-x. The apoptotic effects of Bax, and in a few cases Bak, could be counteracted by Bcl-x or Bcl-

2 and contribute to tumor cell longevity. The ratio of

apoptosis- inducer proteins to apoptosis- inhibitor

proteins could not be determined.

It has been shown recently that Bax may act as a

tumor suppressor gene, being transcriptionally acti-

vated by p53 protein. 55 This being the case, Bax levels

would be expected to be reduced in cases in which

mutant p53 protein is produced. However, there were

few tumors that were Bax-negative and p53-positive.

This suggests the possibility that in the cases that were

both p53-positive and Bax-positive, p53 may not be mutated. 38 MDM2 protein binding to wild-type p53

protein may account for positive staining in many of these cases. 44,45 The double staining (p53 and MDM2)

of tumor cells adds support to this notion.

As expected, there was evidence for the presence of

EBV in oral lymphomas from HIV-positive patients.

This indicat ion of latent EBV infection (an in situ

hybridization signal for EBER mRNA) is consistent

with reports of other HIV-associated lymphomas and

supports the belief that EBV may play a role in the pathogenesis of HIV-associated lymphomas.51, 52 The

EBV-produced protein BHRF, which has Bcl-2-1ike

antiapoptotic activity, was absent in all but one case,

which suggests a minor role for this protein in the pathogenesis of oral lymphomas.

We conclude that Bcl-2, Bcl-x, and Bax are altered

and prominently expressed in oral lymphomas regard-

less of HIV status, indicating dysfunctional apoptotic

mechanisms in these tumors. Bak expression, less

prominent in lymphomas and not apparent in hyper-

plastic nodes, may also be involved in few cases.

Positive staining for p53 and MDM2 proteins appears

to be a feature of lymphomas from HIV-positive

patients. EBV also is more commonly associated with

oral lymphomas in HIV-positive patients, although the

EBV-produced protein BHRF, which has Bcl-2-1ike

activity, is minimally expressed.

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Reprint requests: Joseph A. Regezi, DDS, MS 513 Parnassus, S-512 University of California San Francisco, CA 94143-0424