Download Use of the Polymerase Chain Reaction to Detect

CLINICAL SCIENCES
Use of the Polymerase Chain Reaction
to Detect B- and T-Cell Gene Rearrangements
in Vitreous Specimens From Patients
With Intraocular Lymphoma
Valerie A. White, MD, FRCPC; Randy D. Gascoyne, MD, FRCPC; Katherine E. Paton, MD, FRCSC
Objective: To determine whether the polymerase chain
reaction for B- and T-cell gene rearrangements could be
applied to vitreous specimens to aid in the diagnosis of
intraocular lymphoma.
Methods: Vitreous washing specimens from 4
patients were received in balanced saline solution and
centrifuged, and a portion of the pellet was used to
make routine cytospins. The remainder was used to
make a crude extract of DNA that was amplified for
immunoglobulin heavy chain and T-cell receptor g
gene rearrangements and the 14;18 translocation by
polymerase chain reaction.
minor cluster region breakpoint of the bcl-2 oncogene,
indicating the presence of a 14;18 translocation. One patient showed an immunoglobulin heavy chain gene rearrangement indicating a B-cell lymphoma. Two patients showed rearrangements of the T-cell receptor g
gene, indicating the presence of a T-cell lymphoma.
Conclusions and Clinical Relevance: Vitreous washing specimens can be used successfully to detect B- and
T-cell gene rearrangements by polymerase chain reaction. This may be useful to confirm the diagnosis of intraocular large cell lymphoma in cases suggestive of the
diagnosis. Prompt handling of the specimens is necessary to prevent degradation of the DNA.
Results: One patient had 2 specimens 2 years apart. In
each, there was an identical band corresponding to the
Arch Ophthalmol. 1999;117:761-765
T
From the Departments of
Pathology (Drs White and
Gascoyne) and Ophthalmology
(Drs White and Paton),
Vancouver Hospital and Health
Sciences Centre; Department of
Pathology, British Columbia
Cancer Agency (Dr Gascoyne);
and Departments of Pathology
(Drs White and Gascoyne) and
Ophthalmology (Dr Payton),
University of British Columbia,
Vancouver.
HE DIAGNOSIS of large-cell
lymphoma involving the
vitreous in the absence of
central nervous system
(CNS) disease can be difficult. Typically, the clinical picture is characterized by initial misdiagnosis and treatment for inflammatory uveitis, delaying
definitive diagnosis. Diagnosis requires an
invasive procedure, and often sufficient
material is received to only perform routine cytological examination. Procedures
such as immunohistochemical analysis,
which might aid in diagnosis, cannot often be used. The major diagnostic difficulties with vitreous washings are in the
distinction of inflammatory lymphoid infiltrates from intraocular lymphoma or,
when only a few atypical cells are present, being confident of the diagnosis of
lymphoma. The polymerase chain reaction (PCR) method for the detection of
immunoglobulin heavy chain (IgH) gene
rearrangements, T-cell receptor gene rearrangements, and the bcl-2 translocation has been used for several years to confirm the diagnosis of lymphoma at other
sites.1 Recently, PCR has been found to be
useful in the diagnosis of lymphomatous
meningitis in cerebrospinal fluid specimens.2 We sought to determine whether
PCR could be applied to vitreous samples
in a similar manner for diagnosis.
REPORT OF CASES
CASE 1
A 30-year-old woman was examined in
September 1992 because of a red, irritated right eye, blurred vision, and 2 seizures that had occurred over the past 12
to 18 months. A computed tomographic
(CT) scan showed a left parietal abnormality, and a magnetic resonance image
showed multiple periventricular enhancing lesions that were initially diagnosed as
multiple sclerosis. Visual acuity was 20/200
OD and 20/20 OS. After an 18-month
course of corticosteroid treatment for uveitis, vitrectomy was performed in the right
eye in December 1992, and intraocular
large-cell lymphoma was diagnosed. The
patient underwent whole-brain and bilateral orbital irradiation of 3500 cGy in 20
fractions and also received systemic che-
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MATERIALS AND METHODS
no further investigation or treatment was carried out, and
CNS involvement did not recur. After cataract extraction, the patient’s visual acuity was 20/20 bilaterally.
CYTOLOGICAL METHODS
CASE 2
Vitreous specimens were received in balanced saline
solution (Balanced Salt Solution Plus; Alcon Canada
Inc, Mississauga, Ontario) in the vitrectomy cassette
within 2 hours of surgery. They were centrifuged in a
benchtop centrifuge (Silencer model 103NAD; Silencer, Japan) at 1500 rpm for 10 minutes. The supernatant was removed and the cell pellet resuspended in
1 to 2 mL of Hanks buffered saline solution. Aliquots
(0.5 mL) of the cell pellet were spun onto glass slides
in a cytospin 2 (Shandon Southern Instruments Inc,
Sewickley, Pa) at 500 rpm for 5 minutes. The slides were
postfixed for at least 2 minutes in Clark fixative (12%
glacial acetic acid in 70% alcohol) and stained with a
rapid hematoxylin-eosin stain, or air dried and stained
with Giemsa stain. If lymphoma was identified on the
initial slides, the remainder of the specimen was stored
overnight at 4°C and DNA was extracted the next day.
If there was no suggestion of lymphoma when it was
strongly suspected, the remainder of the specimen was
used to prepare more slides.
MOLECULAR METHODS
The remainder of each cell pellet was used to make
a crude extract of DNA by digesting overnight at 55°C
with proteinase K (60 µg/mL). After digestion, the
samples were heated to 95°C for 10 minutes to denature the proteinase K, centrifuged in a microfuge
for 30 seconds to clear the supernatant, and diluted
1:4 before PCR. The DNA was amplified for IgH3 and
T-cell receptor gene rearrangements4 and the 14;18
translocation as previously described.5 With all amplifications, known positive controls and samples containing no patient DNA were run. All patients’ specimens were amplified for the b-globin gene (510 base
pairs) as a test for DNA integrity. As negative controls, 14 vitreous specimens from 14 patients whose
cytological studies showed mixed inflammation, consisting of lymphocytes, histiocytes, plasma cells,
and/or occasional neutrophils, were also examined
by PCR for evidence of gene rearrangement.
motherapy consisting of methotrexate, dexamethasone,
doxorubicin hydrochloride, vincristine sulfate, and procarbazine hydrochloride.
She did well until about 22 months later, when she
began to see black spots in front of the previously unaffected left eye. On examination, there were +3 cells in
the anterior vitreous, and the fundus showed a white deposit on the surface of the retina adjacent to the superonasal vascular arcade. A second vitrectomy specimen in
January 1995 also showed large-cell lymphoma. She was
ineligible for further irradiation because of full bilateral
treatment initially, and was therefore treated with 6 subTenon injections of methotrexate, cytarabine, and dexamethasone. Her vision improved and vitreous cells cleared
during the next 9 months. During 24 months of followup, some cells were present in the anterior vitreous, but
An 83-year-old woman was seen in August 1995, 3 months
after phacoemulsification surgery because of inflammation that developed in her right eye. This had been treated
with prednisone, 40 mg daily, but had failed to clear. Before vitrectomy her visual acuity had been counting fingers OD and 20/50 OS. The family reported that the patient had seemed tired and weak for approximately 21
months, but that this had worsened along with a decreased
appetite, weight loss of 9 kg, and some confusion in the
last 2 months. A CT scan of the brain showed hypodense
lesions in the central white matter, thought to be lacunar
infarcts. A cerebrospinal fluid specimen did not show lymphoma cells, but the protein level was increased. A CT scan
of the orbits showed no optic nerve thickening. A vitrectomy performed in September 1995 disclosed large-cell
lymphoma. After vitrectomy, the patient’s visual acuity was
20/200 OD and 20/60 OS. Fundus examination showed
an atrophic choroid with no evidence of lymphomatous
deposits. She subsequently received 2000 cGy of radiation
in 5 fractions over 5 days to the right anterior orbit and
eye. The patient was alive 21⁄2 years after diagnosis, but
her general status was unchanged.
CASE 3
A 73-year-old woman was seen in July 1996 because of
a 1-month history of increasing floaters in both eyes and
a feeling of looking through fog. Chest radiograph was
unremarkable, but the result of skin testing for purified
protein derivative, Candida, and Trichophyton was anergic. There was no notable previous illness, and review
of systems was negative, with no weight loss. Visual acuity was 20/25 OD and 20/30 OS. There were mild lenticular changes in both eyes, and intraocular pressure was
18 mm Hg in both eyes. Fundus examination showed
moderate vitreous cells in both eyes that appeared crenated and of varying shapes. There were small macular
drusen and pigmentary changes.
A pars plana vitrectomy was performed in October
1996 and intraocular large-cell lymphoma was diagnosed. There was no evidence of systemic lymphoma; CT
scans of the brain, orbits, abdomen, and pelvis were normal, and bone marrow and cerebrospinal fluid were negative for lymphoma. She received 3500 cGy of radiation
in 20 fractions over 4 weeks to the brain, orbits, base of
the skull, and brain stem to C2, and intravenous chemotherapy consisting of methotrexate, folinic acid, dexamethasone, doxorubicin, vincristine, and procarbazine.
During the ensuing months, the patient developed a
cataract in the left eye, and cataract extraction and intraocular lens placement were performed in September 1997.
CASE 4
A 50-year-old man was examined in October 1997 with
left vitreitis. Although lymphoma was considered, it was
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Figure 1. Case 1. High-power photomicrograph showing a cluster of large
malignant lymphoma cells in the second vitrectomy specimen
(hematoxylin-eosin, original magnification 3650).
excluded because of lack of other signs and his young
age. In January 1998, he had a CT scan that disclosed a
mass in the corpus callosum suggestive of a lymphoma.
At this time he was slightly confused. Visual acuity was
20/100 OS and 20/20 OD. A diagnostic vitrectomy was
carried out and a diagnosis of lymphoma made. He began undergoing treatment.
Figure 2. Case 2. High-power photomicrograph of a single large malignant
lymphoma cell and occasional karyorrhectic cells (hematoxylin-eosin,
original magnification 3650).
mcr
M
N
+
95
92
RESULTS
CYTOLOGICAL FINDINGS
The slides from the first vitrectomy on the right eye in patient 1 showed necrotic cells and karyorrhectic debris. Numerous large lymphocytes with high nuclear to cytoplasmic ratio, irregular nuclear membranes, and prominent
nucleoli were present singly and in groups within the necrotic debris. Occasional histiocytes and lymphocytes were
also present. The second vitrectomy, performed 2 years
later, showed the left eye to be less cellular, but with otherwise similar findings (Figure 1).
The specimen from patient 2 was moderately cellular, with large numbers of necrotic cells. Only occasional
large malignant lymphocytes were present, scattered
throughout the necrotic debris (Figure 2).
The specimen from patient 3 showed 8 to 10 large
malignant lymphocytes on 2 slides. These were admixed with other inflammatory cells, including histiocytes, small lymphocytes, and plasma cells.
The specimen from patient 4 was paucicellular and
showed only occasional large atypical, degenerated cells
in a background of macrophages, lymphocytes, plasma
cells, and neutrophils.
Figure 3. Case 1. Polyacrylamide gel showing identical rearrangements of
the bcl-2 oncogene at the minor cluster region (mcr) in both specimens (95,
92) with approximate molecular size of 350 base pairs (arrowhead). M
indicates molecular size marker; N, no DNA; and plus sign, positive control.
MOLECULAR FINDINGS
Both specimens from patient 1 showed identical rearrangements of the bcl-2 oncogene at the minor cluster
region, indicating the presence of a follicle center cell–
derived B-cell lymphoma (Figure 3). No IgH gene rearrangement was present by PCR.
In cases 2 and 3, the specimens showed rearrangements of the T-cell receptor g gene, indicating the pres-
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Tγ
2
N
+
M
Figure 4. Case 2. Polyacrylamide gel showing rearrangement of the T-cell
receptor g gene (Tg), with approximate molecular size of 300 base pairs
(lane 2, left arrowhead). N indicates no DNA; plus sign, positive control; and
M, molecular size marker (right arrowhead at 123 base pairs). The gel from
case 3 gave a similar result.
ence of a T-cell lymphoma (Figure 4). No IgH gene rearrangements were present.
The specimen from patient 4 showed a strong rearrangement of the IgH gene. There was no rearrangement of the bcl-2 gene or the T-cell receptor gene. Controls gave the appropriate results in all cases.
Amplification failed in 2 of the 14 cases with mixed
inflammation, indicating degraded DNA. In 12 cases, 1,
2, or 3 of the above sites could be amplified, and these
all gave nonclonal results.
COMMENT
These 4 cases demonstrate that the PCR can be used successfully with vitreous specimens to confirm the diagnosis of large-cell lymphoma. In the first 2 cases, the cytological diagnosis was not in doubt, as large malignant
lymphocytes were present. However, in the third and
fourth cases, only a few atypical cells were present, admixed with inflammatory cells, making the diagnosis more
difficult. To our knowledge, this is the first report in which
the diagnosis of intraocular lymphoma together with lineage assignment has been confirmed by molecular techniques on vitreous fluid alone, rather than on biopsy material from another site, in patients with ocular disease
(MEDLINE, 1966-1998).
We did not do a cell count on the vitreous as received for a number of reasons: it was admixed with a
variable amount of vitrectomy fluid; the count would have
included inflammatory as well as malignant cells; and we
did not feel that we could spare any specimen for this
extra test. For this last reason, it was impossible to do
rigorous testing of different vitrectomy solutions to determine how these would affect viability of the cells, and
we could not test how long a specimen would survive
before degradation of the DNA; this would also depend
on how viable the cells were when removed from the patient. We did not think that testing these variables would
add to the information obtained, as we were most interested in making a definitive diagnosis by cytological examination and PCR, and we handled the specimens as
soon as was reasonably possible. Sensitivities were not
established directly on the vitrectomy specimens themselves for similar reasons. We have established sensitivities for our methods, as previously reported.4,5
It should be emphasized that, while a positive result for clonality testing in the presence of malignant or
atypical lymphocytes confirms the presence of lymphoma, a negative result does not necessarily rule it out.
A specimen may be falsely negative on PCR testing because of degradation of or insufficient DNA, or lack of
amplification by the primers used. If a patient is suspected of having lymphoma, but this cannot be proved
by either cytological examination or PCR, it may be necessary to repeat the procedure.
Recently, Katai et al6 reported on 2 patients with previous B-cell lymphomas of the orbit and chest wall who
developed vitreitis after initial treatment. Although vitrectomies did not disclose malignant cells, IgH gene rearrangement was detected in each case, and the authors
diagnosed ocular involvement by malignant lymphoma.
Rhodes et al2 recently described their experience with
the use of PCR to aid in the diagnosis of lymphomatous
meningitis. Of 20 specimens that were negative for or suggestive of lymphoma, PCR for IgH gene rearrangement
was clonal in 10. Two of 4 specimens that were positive
on cytological examination were also clonal. The specimens were not tested for rearrangements of the T-cell receptor genes or the bcl-2 oncogene. Rhodes et al postulated that useful DNA may be present in karyorrhectic
nuclei as well as free in the cerebrospinal fluid, making
it important to ensure that specimens are handled
promptly to prevent further degradation of DNA.
Intraocular lymphoma may be present with primary CNS lymphoma, and multiple procedures are often required before a definitive diagnosis can be established. Whitcup et al7 studied 12 patients with intraocular
lymphoma and found that 3 had normal findings on vitrectomies initially: 1 case was diagnosed on the second
vitrectomy, 1 on the third, and 1 on enucleation of a blind
eye. Five of 11 patients eventually had findings positive
for lymphoma on lumbar puncture, but 3 only on the second attempt. Peterson et al8 studied 24 patients with intraocular lymphoma, 23 of whom had CNS lymphoma
at some point in their course. Vitrectomy was positive
for lymphoma in 10 of 15 patients, but only on the second attempt in 2. Their 7 nondiagnostic vitrectomies were
all in patients who had received previous corticosteroid
therapy, a problem also mentioned by Whitcup et al. Freeman et al9 published an earlier series of 32 cases of intraocular lymphoma, of which only 18 were diagnosed
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on vitrectomy. Four were diagnosed by enucleation and
10 at autopsy. The use of molecular techniques should
be useful in eliminating false-negative vitrectomy specimens, such as those described above, thus allowing
prompt intervention.
Primary T-cell lymphoma of the vitreous is rare.
Brown et al10 presented 2 cases, reviewed the literature,
and found that 12 (21%) of 57 cases of intraocular lymphoma in which cell marker studies had been performed were of T-cell lineage. Two well-documented cases
have been described. Novak and Katzin11 presented a case
of primary T-cell lymphoma of the CNS in which the patient initially manifested bilateral vitreitis. Although a
vitrectomy was performed, insufficient cells were obtained for immunophenotypic analysis. The diagnosis of
T-cell lymphoma was made on material obtained from a
brain biopsy specimen that showed that 100% of cells were
CD8 positive by flow cytometry and that multiple rearranged bands were detected by the T-cell receptor b-chain
gene probe with the use of Southern analysis. Goldey et
al12 reported a case of a patient with unilateral anterior
uveitis in which all of the aspirated cells were large granular lymphocytes that were CD3 positive. This patient was
subsequently found to have a high-grade T-cell lymphoma involving the small bowel and retroperitoneal
lymph nodes, which was confirmed by Southern analysis. Intraocular lymphoma is often associated with primary CNS lymphoma. In a review, Ferracini et al13 stated
that 27 cases of primary T-cell lymphoma of the CNS were
reported in the literature from 1983 to 1992.
Jellinger and Paulus14 studied 5 cases of primary CNS
lymphoma by immunohistochemistry and Southern analysis and found no bcl-1 or bcl-2 rearrangements; therefore, our case 1 may be unique in this regard. Bcl-2 gene
rearrangements can be found at diagnosis in systemic diffuse large-cell lymphoma with frequencies ranging from
14% to 40%.15-17
As the incidence of primary CNS lymphoma and
therefore of intraocular lymphoma appears to be increasing, PCR gene rearrangement studies on vitreous specimens for the determination of both lineage and clonality offer a useful adjunct for diagnosis in difficult cases.18
Accepted for publication January 8, 1999.
Cases 1 and 2 were presented at the International Society of Ophthalmic Pathology biennial meeting, Chicago,
Ill, October 25, 1996.
Corresponding author: Valerie A. White, MD,
FRCPC, Department of Pathology, Vancouver General
Hospital, 910 W 10th Ave, Vancouver, British Columbia,
Canada V5Z 1L8 (e-mail: [email protected]).
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