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depleted in their 3 patients. T-cell chimerism before DLI, therefore, was
mixed and not already predominantly of donor type, as in our cases.
Gardiner et al state that their technique using lineage-specific STR-PCR
provides reliable results on the chimeric profile post-BMT and may be more
useful than the FICTION assay in situations with low cell numbers.
Although the important aspect of morphology and individual cell by cell
analysis is lost with the PCR approach, we agree that STR-PCR is a
most valuable tool in this context and therefore complementary to our
FICTION analysis. However, we doubt that subtle changes in the degree
of mixed chimerism can be seen with a semiquantitative technique according
to which mixed chimerism is defined as a percentage of recipient cells
between less than 90% and greater than 10%. Indeed, in the work of
Gardiner et al, significant changes in every case were detected only
concomitantly to the onset of clinical graft-versus-host disease (GVHD),
a result similar to that obtained in our cases when using the AmpliType
Polymarker PCR kit to document non–lineage-specific DNA chimerism. In contrast, when using our quantitative techniques, ie, FICTION
and competitive differential bcr-abl RT-PCR as an additional diseasespecific marker, the beginning of the critical switch period could be
detected several weeks before the onset of clinical GVHD. Although we
therefore very much support a larger scale study to better understand the
kinetics of the GVL response after DLI in T-cell–depleted and non—T-cell–
depleted BMT recipients, we strongly suggest the use of true quantitative
measures of lineage-specific chimerism. The FICTION method is
especially helpful in small centers, because it can be performed without
larger equipment or in situations in which morphological control of the
selected population is of additional value. Another attractive tool is the
combination of FACS sorting and quantitative multiplex STR-PCR with
fluorescent primers.3 This technique has been successfully applied by
members of our group to study subtle changes of subset chimerism in
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patients undergoing nonmyeloablative stem cell transplantation and was
shown to be predictive of response, GVHD, and disease recurrence.4
Herrad Baurmann
BMT Center
Deutsche Klinik fu¨r Diagnostik
Wiesbaden, Germany
Christian Thiede
Stefan Nagel
Thomas Binder
Andreas Neubauer
Wolfgang Siegert
Dieter Huhn
REFERENCES
1. Gardiner N, Lawler M, O’Riordan JM, DeArce M, McCann SR:
Monitoring of lineage specific chimaerism allows early prediction of
response following donor lymphocyte infusions for relapsed chronic
myeloid leukemia. Bone Marrow Transplant 21:711, 1998
2. Baurmann H, Nagel S, Binder T, Neubauer A, Siegert W, Huhn D:
Kinetics of the graft versus leukemia response after donor leukocyte
infusions for relapsed chronic myeloid leukemia after allogeneic bone
marrow transplantation. Blood 92:3582, 1998
3. Thiede C, Florek M, Bornha¨user M, Ritter M, Mohr B, Brendel C,
Ehninger G, Neubauer A: Rapid quantification of mixed chimerism
using multiplex amplification of short tandem repeat markers and
fluorescence detection. Bone Marrow Transplant (in press)
4. Thiede C, Brendel C, Mohr B, Florek M, Oelschlagel U, Ritter M,
Naumann R, Geissler G, Ehninger G, Neubauer A, Bornha¨user M: Comparative analysis of chimerism in the early post-transplantation period in cellular
subsets of patients undergoing myeloablative and non-myeloablative allogeneic blood stem cell transplantation. Blood 92:132a, 1998 (abstr, suppl 1)
Detection of Human Herpesvirus-8 and Epstein-Barr Virus DNA in Primary Intraocular Lymphomas
To the Editor:
Two herpesviruses, human herpesvirus-8 (HHV-8) and Epstein-Barr
virus (EBV), are able to contribute to lymphomagenesis in humans.1
HHV-8 is documented not only in a strong association with Kaposi’s
sarcoma, but also in a high percentage of primary effusion lymphoma,
Castleman’s disease, and multiple myeloma. Most recently, HHV-8
genome has been associated with primary central nervous system
lymphoma (PCNSL) of patients with and without acquired immunodeficiency syndrome (AIDS).2 However, the role of HHV-8 in the
pathogenesis of PCNSL remains controversial.3 In vitro, EBV efficiently transforms human B lymphocytes, causing them to proliferate
continuously.1 EBV and the latent membrane protein can be detected in
tumor cells of almost all AIDS-related PCNSL.
Primary intraocular lymphoma, a component of PCNSL, is a large B-cell,
non-Hodgkin’s lymphoma.4 The disease is aggressive, with a 5-year
survival rate of less than 33%. In the past 15 years, the incidence of the
tumor has increased dramatically, coincident with the AIDS epidemic.
We analyzed HHV-8 and EBV DNA sequences in 13 primary intraocular lymphomas with and without AIDS. Of the 13 specimens, 5 were whole
eyes, 1 was a vitreoretinal biopsy, and 7 were vitrectomy samples. Slides
with cells from 2 noninfectious uveitis were also used as control.
The 13 patients (6 Americans and 7 Europeans) include 2 AIDS patients (1
American and 1 French patient): the American patient developed an early
lymphoma and the French patient had an advanced stage, aggressive
lymphoma.5 At the time at which the 13 specimens were obtained, only
3 (2 eyes and 1 vitrectomy specimen) had a proven diagnosis of PCNSL
involving the eyes. Of the 5 eyes, 2 had received radiation therapy. Nine
patients were being investigated for possible primary intraocular
lymphoma and underwent diagnostic procedures (7 vitrectomies, 1
vitreoretinal biopsy, and 1 enucleation). The eye of the American AIDS
patient was diagnosed with primary intraocular lymphoma at necropsy.
On examination of the histology and cytology slides of the 13 cases, only 6
specimens (3 of 5 eyes, 3 of 7 vitrectomies, and 0 of 1 vitreoretinal biopsy)
showed typical large B-cell lymphoma (Fig 1). Of the 7 cases without
obvious typical lymphoma cells, 5 (4 vitreous and 1 vitreoretinal biopsy)
were also available for cytokine analysis and had high vitreous interleukin-10
(IL-10) levels with high ratios of IL-10 to IL-6, suggesting primary
intraocular lymphoma.6 Finally, 2 cases (1 eye with AIDS and 1 eye that had
received radiation) showed only a few atypical lymphocytes in the subretinal
space. The microdissected morphologically suspicious abnormal cells
in all 13 cases had presented a rearrangement in FR3A of the IgH gene
that confirmed the diagnosis of primary intraocular lymphoma (Fig 2).5
Lymphoma cells of these 13 specimens, normal lymphocytes of 2
vitrectomy specimens, and the 2 uveitic cases were microdissected from
either deparaffinized sections or cytological slides.5 DNA of these cells
were extracted for polymerase chain reaction (PCR) amplification and
Southern hybridization for HHV-8 or EBV genome. Multiple primers
and positive and negative controls were also used to confirm the results.
Four cases were positive for HHV-8 (Fig 2). Two were the AIDSrelated lymphoma eyes and 2 were non-AIDS vitreous samples. The 2
non-AIDS patients were diagnosed with suspicious primary intraocular
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2750
CORRESPONDENCE
Fig 1. Microphotograph showing typical intraocular lymphoma (arrow) in the subretinal space (A; case no. 8; insert, higher magnification of
the lymphoma cells; R, retina; C, choroid) and a vitrectomy specimen (B; case no. 7; arrowheads, normal lymphocytes). (Original magnifications:
A, hematoxylin & eosin, ؋200, insert, ؋400; B, Diff-Quick, ؋400.)
lymphoma both clinically and cytopathologically. These 4 positive
patients included 2 Americans and 2 French. Three samples (1 American
AIDS eye and 2 vitreous specimens) were also microdissected to obtain
nonmalignant cells (retinal and optic nerve cells from the eye and
infiltrating lymphocytes from the other 2 vitreous specimens) for
identification of the viral genome. HHV-8 DNA was undetectable in the
inflammatory or ocular cells without tumor infiltration in the 3 early
cases with primary intraocular lymphoma. EBV DNA was detected in only
1 of the 13 samples, ie, the French AIDS eye with a diffuse lymphoma.
This investigation has shown HHV-8 DNA sequences in 2 of 2
Fig 2. PCR amplification showing rearrangement of third framework region in the VH region of
heavy chain Ig gene in the lymphoma cells of all 13 cases, HHV-8
genome in 4 cases (lanes 3 and 4,
AIDS patients; lanes 7 and 9, nonAIDS patients), and EBV genome
in 1 case (lane 4, AIDS with lymphoma involving the orbit and
eye). Lanes 1 through 13, cases
no. 1 through 13, respectively;
lane 14, negative control; lane 15,
positive control.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
CORRESPONDENCE
2751
AIDS-related intraocular lymphoma and in 2 of 11 non-AIDS intraocular lymphoma. The HHV-8 genome encodes homologs of cyclin D1, a
cell-cycle control element; certain cytokines, regulators, and receptors;
and Bcl-2, an antiapoptotic protein.7 The HHV-8 sequence also contains
homologs of vial IRFs that may be involved in modulating both HHV-8
replication and HHV-8–associated tumorigenicity.8 The viral genome
could therefore contribute to cellular growth and transformation through
activation of the cell cycle. Although the virus could simply be an
innocent passenger in this lymphoma, the so-far ubiquitous association
of HHV-8 with AIDS-related PCNSL and relatively low incidence in
non-AIDS patients, plus most other lymphoproliferative disorders in
both AIDS and non-AIDS patients, strongly favor a causal role. In this
series, HHV-8 was detected in 100% (2 cases: 1 American and 1 French)
of AIDS-related intraocular lymphoma and in 1 of the 6 European and 1
of the 5 American, non-AIDS cases. Furthermore, HHV-8 DNA was
found only in the neoplastic cells. We speculate that the nonneoplastic B
lymphocytes in those AIDS patients who eventually develop primary
intraocular lymphoma may be infected with HHV-8, HIV, or other viruses
into the eye, in which ultimate transformation into malignant cells occurs.
Another possibility may be the promotion of a second oncogenic virus.
Nine EBV genes are known to express as proteins in EBVtransformed B-lymphoblastoid cell lines.9 One of the latent membrane
proteins of the EBV engages members of the tumor necrosis factor
receptor-associated molecules and then activates NF-␬B–driven expression of multiple viral and cellular genes. However, only certain subsets
of AIDS-non Hodgkin’s lymphoma appear to be EBV-related, although
a high frequency of EBV is reported in AIDS-PCNSL.10 Our data do not
show a strong association between EBV and primary intraocular
lymphoma, suggesting that EBV may not play a major role in the
early development of primary intraocular lymphoma with or without
AIDS.
Chi-Chao Chan
De Fen Shen
Scott M. Whitcup
Robert B. Nussenblatt
National Eye Institute
National Institutes of Health
Bethesda, MD
Phuc LeHoang
Francois G. Roberge
Nathalie Cassoux
Department of Ophthalmology
Pitie-Salpetriere Hospital
Paris, France
Carl Herbort
Department of Ophthalmology
Lausanne, Switzerland
Zhengping Zhuang
National Cancer Institute
National Institutes of Health
REFERENCES
1. Lyons SF, Liebowitz DN: The roles of human viruses in the
pathogenesis of lymphoma. Semin Oncol 25:461, 1998
2. Corboy JR, Garl PJ, Kleinschmidt-DeMasters BK: Human herpesvirus 8 DNA in CNS lymphomas from patients with and without AIDS.
Neurology 50:335, 1998
3. Epstein LG: Does human herpesvirus 8 have a pathogenetic role
in primary CNS lymphoma? Neurology 50:322, 1998
4. Whitcup SM, de Smet MD, Rubin BI, Palestine AG, Martin DF,
Burnier MJ, Chan CC, Nussenblatt RB: Intraocular lymphoma. Clinical
and histopathologic diagnosis. Ophthalmology 100:1399, 1993
5. Shen DF, Zhuang Z, LeHoang P, Boni R, Zheng S, Nussenblatt
RB, Chan CC: Utility of microdissection and polymerase chain
reaction for the detection of immunoglobulin gene rearrangement and
translocation in primary intraocular lymphoma. Ophthalmology 105:
1664, 1998
6. Whitcup SM, Stark-Vancs V, Wittes RE, Solomon D, Podgor MJ,
Nussenblatt RB, Chan CC: Association of interleukin-10 in the vitreous
and cerebral spinal fluid and primary central nervous system lymphoma.
Arch Ophthalmol 115:1157, 1997
7. Russo JJ, Bohenzky RA, Chien MC, Chen J, Yan M, Maddalena
D, Parry JP, Peruzzi D, Edelman IS, Chang Y, Moore PS: Nucleotide
sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc
Natl Acad Sci USA 93:14862, 1996
8. Moore PS, Boshoff C, Weiss RA, Chang Y: Molecular mimicry of
human cytokine and cytokine response pathway genes by KSHV.
Science 274:1739, 1996
9. Liebowitz D: Epstein-Barr virus and a cellular signaling pathway
in lymphomas from immunosuppressed patients. N Engl J Med
338:1413, 1998
10. MacMahon EM, Glass JD, Hayward SD, Mann RB, Becker PS,
Charache P, McArthur JC, Ambinder RF: Epstein-Barr virus in AIDSrelated primary central nervous system lymphoma. Lancet 338:969,
1991
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1999 93: 2749-2751
Detection of Human Herpesvirus-8 and Epstein-Barr Virus DNA in Primary
Intraocular Lymphomas
Chi-Chao Chan, De Fen Shen, Scott M. Whitcup, Robert B. Nussenblatt, Phuc LeHoang, Francois G.
Roberge, Nathalie Cassoux, Carl Herbort and Zhengping Zhuang
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