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Human Immunodeficiency Virus Infection, Bcl-2, p53 Protein, and Ki-67 Analysis in Ocular Surface Squamous Neoplasia
Amina Mahomed, MMed, FCS;
Runjan Chetty, FRCPath, PhD
Arch Ophthalmol. 2002;120:554-558.
ABSTRACT
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Objectives To determine the age and human immunodeficiency virus (HIV) status of
patients with ocular surface squamous neoplasia (OSSN), and to analyze tumor
proliferation, Bcl-2, and p53 oncoprotein expression in OSSN.
Methods Only patients with histologically proved neoplasia were included in
this study. The HIV status was obtained only with informed consent. Monoclonal
antibodies to p53 and Bcl-2 protein were used after microwave antigen retrieval
to enhance immunohistochemical staining of the sections. Proliferation was
assessed by means of Ki-67 antigen expression. Positive staining in each specimen
was expressed as a percentage and graded accordingly.
Results Forty-one eyes in 40 black patients with a mean age of 37 years were
found to have OSSN. Of the 41 lesions, 35 represented in situ or invasive
carcinoma. The remaining 6 had mild or moderately dysplastic lesions. Seventeen
patients agreed to an HIV test and, of these, 12 (70.6%) were HIV positive.
All 12 were younger than 50 years, and 11 had either carcinoma in situ or
invasive lesions. Twenty-two of 40 lesions expressed significant (greater
than 50% of neoplastic cells) p53 positivity, while Bcl-2 expression was detected
in 10. Ki-67 expression was low, even in the HIV-positive lesions.
Conclusions At our institution, OSSN occurs in young patients, many of whom are
HIV positive. Expression of p53 is a common finding, whereas Bcl-2 immunoexpression
occurs in the minority of cases. Ki-67 analysis showed that OSSN is a slow-growing
tumor, even in the presence of HIV infection.
INTRODUCTION
OCULAR SURFACE squamous neoplasia (OSSN) is a rare condition, but interest
in this disease has been revived in the literature through association with
the human immunodeficiency virus (HIV) epidemic.1-5
The diagnosis of OSSN includes a spectrum of conditions ranging from mild
squamous dysplasia of the conjunctiva through moderate dysplasia, carcinoma
in situ (CIS), and, in the advanced stage, invasive squamous carcinoma.6 To the best of our knowledge, the relationship between
OSSN and HIV infection has not yet been explored in Kwa-Zulu Natal, South
Africa. Furthermore, whereas Dushku et al7
reported p53 expression to be confined to the limbal basal stem cells of pterygia
and limbal tumors, other genetic and protein abnormalities in OSSN have not
yet been reported.
Genes can be roughly categorized into 2 types: oncogenes and tumor suppressor
genes. The latter exist as proto-oncogenes that then become converted into
tumor-promoting genes as a result of a variety of genotoxic events that lead
to translocations, mutations, deletions, etc. The tumor suppressor genes have
a protective function in the normal state, and any genetic abnormality in
these genes results in loss of this protective role. All types of genes produce
proteins that are amenable to detection by immunohistochemistry. This is a
simple, relatively inexpensive, and reproducible technique that uses specific
monoclonal and polyclonal antibodies directed against the protein of interest.
A chromagen is used for visualization purposes.
Tumor cell progression results from the accumulation of multiple mutations
in both tumor suppressor genes and oncogenes. Tumor suppressor genes and their
proteins play a major role in preventing tumorigenesis by negatively regulating
cell growth. The p53 is a prototype tumor suppressor
gene. It consists of 11 exons and is located on chromosome
17p13.8-9 Mutations and deletions of p53 are said to be the most common genetic abnormality in human cancers.10 The missense base substitutions that occur in the
highly conserved region of the p53 gene produce mutant
p53 protein that accumulates within tumor cells.11
Immunohistochemistry is a good indicator of p53 gene
mutation because mutant protein is more stable and has a longer half-life
than the wild-type protein.
Induction of mutant p53 leads to decreased
expression of Bcl-2 protein, which is a 25-kd integral membrane protein within
the cell. It is necessary for cell longevity and it facilitates a healthy
immune system. The effect of mutant p53 and diminished
Bcl-2 is an increase in cell turnover within the tumor. Overexpression of
Bcl-2 protein is related to the t(14;18) chromosomal translocation. This protein
impairs apoptosis by an antioxidant pathway through suppression of lipid peroxidation.
The extent of cellular proliferation is an indication of tumor aggression,
and this may be measured immunohistochemically by the MIB-1 index. MIB-1 is
an antibody to Ki-67 antigen in the nuclei of neoplastic cells. Ki-67 antigen
is expressed in cycling cells during G1, S, G2, and
M phases and is therefore an indicator of cellular proliferation.
The interest in p53 and Bcl-2 oncoprotein lies in its implication for
the development of cancer therapies and in its prognostic merit. Tumors negative
for p53 do not respond well to DNA-damaging radiotherapy and chemotherapy.
Consequently, conventional therapy will actually select for genomic instability
in these tumors.12 Immunoexpression of Bcl-2
generally confers a better prognosis, while p53 is associated with high grade
and stage in several tumors.11, 13
In prostate cancer, however, p53 and Bcl-2 are predictors of recurrence after
surgery.14
The purpose of this study was to determine the pattern of presentation
of OSSN (with respect to age and HIV infection), tumor proliferation as assessed
by the MIB-1 index, and p53 and Bcl-2 immunoexpression in OSSN.
PATIENTS AND METHODS
Patients with localized conjunctival lesions who were first seen at
the King Edward VIII Hospital eye clinic, Natal, between January 1, 1995,
and December 31, 1997, were operated on under local anesthesia by means of
a "no-touch" modified Mohs micrographic technique, which aims for total excision
by means of histologic surveillance of tumor margins.15-16
Large infiltrating lesions were first examined by biopsy, and exenteration
was offered thereafter. Specimens were submitted to the Pathology Department
at the Nelson R. Mandela School of Medicine, University of Natal, Natal, for
histopathologic analysis. Only histologically proved neoplastic lesions were
included in this study. Forty patients were found to have OSSN. After informed
consent was obtained, an enzyme-linked immunosorbent assay test for HIV antibodies
was performed.
One of the 41 histologic specimens could not be retrieved and, therefore,
immunostaining was not performed on this lesion, which was an invasive squamous
carcinoma in a 66-year-old woman whose HIV status was not known.
Sections 3 µm thick, embedded in paraffin wax, were picked up
onto poly-L-lysine–coated slides and incubated for 10 minutes at 70°C.
Sections were cleared through xylene and rehydrated through descending grades
of alcohol. Microwave antigen retrieval was accomplished by placing the specimens
in 0.01M buffered sodium citrate at pH 6.0 at 85°C for 10 minutes in a
microwave oven processor (H2500; Energy Beam Sciences, Inc, Agawam, Mass).
After cooling in water, the specimens were placed in a jar of phosphate-buffered
saline (PBS) at pH 7.4. The slides were then incubated in 3% hydrogen peroxide
in a microwave oven (Sharp carousel R7280, 650 W; Sharp Electronics Corp,
Mahwah, NJ) at low heat for 2 minutes 30 seconds. Each section was rinsed
with PBS; the specific antibody was placed over the tissue and thereafter
heated in a microwave oven for 4 minutes 30 seconds. The antibodies used were
MIB-1 (Biomed Corp, Foster City, Calif; dilution, 1:40); monoclonal mouse
anti–human Bcl-2 oncoprotein (clone 124; DAKO, Glostrup, Denmark; dilution,
1:40), and the monoclonal mouse anti–human p53 protein (DO7; DAKO; dilution,
1:100). The latter reacts with wild-type and mutant p53 protein.
Appropriate positive controls were run simultaneously: high-grade lymphoma
for p53, a reactive lymph node for Bcl-2, and a reactive tonsil for MIB-1.
Each specimen was rinsed thoroughly with PBS and then incubated with biotinylated
link antibody in the microwave oven for 3 minutes 30 seconds. After washing
in PBS, sections were incubated with peroxidase-labeled streptavidin in the
microwave oven for 3 minutes 30 seconds. The slides were again washed in PBS
and a substrate chromogen (DAB; DAKO, Carpinteria, Calif) was placed over
each specimen. After incubating with the substrate chromogen for 5 minutes
at room temperature, the sections were rinsed in distilled water, then counterstained
with Mayer hematoxylin for 1 minute and rinsed in running tap water. Finally,
sections were rinsed in ammoniated water and tap water, then dehydrated in
alcohol and xylene and coverslipped with dibutylphthalate-xylene mountant.
A positive reaction was indicated by brown staining of the nucleus (p53 and
MIB-1) or perinuclear region and cytoplasm (Bcl-2).
Immunostaining for MIB-1, p53, and Bcl-2 was assessed on each slide
by counting the number of positive cells and the total number of cells in
at least 10 fields at x40 magnification. Positivity was expressed as
a percentage in each field, and the average percentage for each slide was
scored as follows: zero, less than or equal to 5%; 1+, 6% to 25%; 2+, 26%
to 50%; 3+, 51% to 75%; and 4+, more than 75%.
RESULTS
Forty-one eyes in 40 patients were found to have OSSN during the period
of this study. There were 20 men and 20 women. The ages of the patients ranged
from 24 to 76 years, with a mean age at presentation of 37 years. Of the 40
patients, 27 (67.5%) were younger than 50 years. Clinical illustrations are
seen in Figure 1. Of the 41 lesions,
35 were either CIS or invasive squamous carcinomas. Table 1 indicates the ages of patients and the histologic grade
of their tumors.
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Figure 1. A, Invasive squamous carcinoma
infiltrating the orbit and destroying the globe. B, Localized invasive conjunctival
squamous carcinoma.
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Table 1. Patient Age, HIV Status, and Histologic Findings*
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Results of the HIV test confirmed that 12 of the 17 patients were infected
with the virus. All 12 were younger than 50 years. Three of the 5 HIV-negative
patients were younger than 50 years. A 52-year-old woman with bilateral OSSN
was HIV negative. The histologic findings of 18 lesions excised from these
17 patients are shown in Table 1.
Eleven of the 12 HIV-positive patients had CIS or invasive squamous cancer
at the initial examination.
Details of p53, Bcl-2, and MIB-1 staining in the spectrum of OSSN are
given in Table 2. MIB-1–positive
staining limited to the basal cells of the conjunctival epithelium was not
considered part of the lesional pattern of staining, as these are normally
dividing cells and are expected to be MIB-1 positive. Thirteen specimens displayed
1+ MIB-1 positivity and 5 specimens were 2+. MIB-1 positivity of 3+ was seen
in 1 case of CIS. Twenty-one cases of OSSN showed less than 5% staining.
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Table 2. p53, Bcl-2, and MIB-1 Staining in the Spectrum of Ocular Surface
Squamous Neoplasia*
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Positive staining for p53 was present within the nuclei of neoplastic
cells (not confined to the basal cells) in 28 lesions, representing 70% of
cases. Positivity for p53 of 3+ and 4+ was present in 10 cases each of CIS
and invasive squamous carcinoma (Figure 2).
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Figure 2. Full-thickness dysplasia (carcinoma
in situ) showing more than 75% of the dysplastic cells exhibiting nuclear
immunolabeling for p53 protein (anti-p53, original magnification x50).
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Expression of Bcl-2 was found to be minimal or absent in 30 specimens.
Eight cases showed 1+ staining. One specimen representing CIS expressed focal
2+ staining, and another case of CIS showed areas of 3+ positivity.
Results of the specimens belonging to the subgroup of HIV-positive patients
are summarized in Table 2. Seven
cases showed either 3+ or 4+ p53 positivity. Three HIV-negative cases showed
a similar staining intensity as well. Expression of Bcl-2 was minimal, and
MIB-1 activity was similar in HIV-positive and HIV-negative groups.
COMMENT
Ocular surface squamous neoplasia occurs more commonly in younger patients
(younger than 50 years) at King Edward VIII Hospital. This may or may not
be related to the high HIV prevalence (31%) in the 20- to 40-year age group
in Kwa-Zulu Natal. Twelve (70.6%) of 17 patients tested were HIV positive,
and they were all younger than 50 years. Of all OSSN lesions encountered in
this study, 35 (85.4%) were either CIS or frank invasive squamous carcinomas.
Twenty-eight (70.0%) of 40 specimens showed some p53 positivity. Furthermore,
in 22 of these lesions, more than 50% of the tumor cells expressed p53 protein.
Significant p53 expression occurred more commonly in higher-grade OSSN lesions
(20 of 22). Expression of p53 was not affected by the HIV status of the patient.
The p53 gene causes arrest of cell division
in G1 and late G2 phases by affecting transcription
of key cell cycle control genes. Overexpression of p53 protein can occur from
stress or DNA damage in the absence of p53 gene mutations.
Tumorigenesis occurs within an environment characterized by high levels of
proliferation and rapid apoptosis.
The absence of Bcl-2 expression as seen in 30 of 40 OSSN specimens analyzed
in this study may be indicative of cells undergoing more rapid death, since
Bcl-2 expression confers longevity to cells. The resultant synergy between
the effect of increased mutant p53 and absence of Bcl-2 expression may promote
rapid clonal expansion, leading to progressively increased genomic instability
and ultimately to malignancy.12 Minimal Bcl-2
positivity was present in 5 cases of CIS and 4 invasive squamous carcinomas.
Only 1 CIS lesion contained more than 50% of cells exhibiting Bcl-2 positivity.
These findings differ slightly from Bcl-2 expression in oral squamous neoplasia
reported by Singh et al,17 who concluded that
expression of Bcl-2 oncoprotein was directly proportional to the degree of
epithelial dysplasia and that Bcl-2 was down-regulated in differentiating
carcinomas.
Overexpression of Bcl-2 was also not seen in HIV-infected cases. The
Tat protein of HIV modulates the expression of various cellular genes.18 Human immunodeficiency virus causes aberrant cytokine
activation, and HIV protease promotes apoptosis by depleting the cell of Bcl-2
and increasing Bax protein, which has apoptosis-inducing properties.18-19 Infection with HIV and Bcl-2 deficiency
promotes neoplasia by impairing immune surveillance, thereby allowing the
unchecked survival of mutation-bearing cells. However, HIV infection cannot
solely be implicated as the cause of the low Bcl-2 immunoexpression encountered
in this study because both HIV-positive and HIV-negative cases displayed a
similar Bcl-2 immunoprofile.
MIB-1 analysis showed a low proliferation index for OSSN, which may
explain the lack of extraorbital metastases and the low mortality associated
with this tumor. One case of CIS expressed MIB-1–positive staining in
more than 50% of the dysplastic cells. This tumor was excised from a 76-year-old
woman whose HIV status was unknown. The tumor also expressed p53 positivity
in 75% of the dysplastic cells and lacked Bcl-2 expression. In the group of
5 patients requiring exenteration, 3 tumors expressed less than 50% MIB-1
positivity. Even in the HIV-positive subgroup of 12 patients, MIB-1 index
was low. These findings support the theory that OSSN is a lesion with a low
proliferation index, but they do not explain recent reports of aggressive
OSSN in young, HIV-positive patients.4-5,20
Perhaps a different mechanism accounts for the aggression in some cases associated
with HIV infection. The notion that HIV infection confers greater tumor aggression
as measured by cell proliferation is certainly challenged by the immunohistochemical
findings of this study.
The clinical role of p53 and Bcl-2 in OSSN needs further exploration.
Their use as possible markers for tumor recurrence and metastatic potential
needs investigation and may influence primary management protocols. While
wide margin excision remains the gold standard, the benefit of radiotherapy
and chemotherapy to preserve vision or limit morbidity in large, infiltrating,
and poorly localized lesions may ultimately depend on p53 status of the tumor.
AUTHOR INFORMATION
Submitted for publication June 28, 2001; final revision received January
14, 2002; accepted January 24, 2002.
Corresponding author: Amina Mahomed, MMed, FCS, Department of Ophthalmology,
University of Natal Medical School, Private Bag X10, Dalbridge 4014, South
Africa (e-mail: bellamina4u{at}yahoo.com).
From the Department of Ophthalmology, King Edward VIII Hospital (Dr
Mahomed) and the Departments of Ophthalmology (Dr Mahomed) and Pathology (Dr
Chetty), Nelson R. Mandela School of Medicine, University of Natal, Natal,
South Africa.
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