Laboratory for Clinical and Molecular Virology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK1
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK2
Leukaemia Research Fund Virus Centre, University of Glasgow, Bearsden, Glasgow G61 1QH, UK3
Author for correspondence: Dorothy Crawford. Fax +44 131 650 3711. e-mail d.crawford{at}ed.ac.uk
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Abstract |
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Main text |
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PTLD biopsy material is scant and does not generally grow in vitro. The few cell lines available do not reflect initial tumour phenotype (Cen et al., 1993 ; Itoh et al., 1993
). Additionally, biopsies generally contain heavy infiltrates of non-malignant T cells (Perera et al., 1998
) which can limit their use in molecular studies. The aim of this study was to expand original PTLD biopsies in scid mice, which readily accept human xenografts due to lack of functional B and T cells (Bosma et al., 1983
; Johannessen & Crawford, 1999
).
PTLD biopsies were obtained from five solid organ graft patients (designated patient 15; for patient details, see Table 1). For each biopsy, 25x10650x106 cells (denoted biopsy) were injected intraperitoneally (i.p.) into a scid mouse within 12 h of biopsy (sample from patient 2 was inoculated into two animals). Tumours (denoted scid tumour) formed in all mice. It was possible to passage material from patient 3. Patients 3 and 5 experienced primary EBV infection following transplantation whereas patients 1, 2 and 4 were persistently infected. All five biopsies gave rise to i.p. tumours in scid mice. EBER in situ hybridization was performed on all sample material using standard methods (Howe & Steitz, 1986
). Similar to biopsies, all scid tumours were EBER+ve [for an assessment of the relative proportion of EBV+ve cells in the sample material, see (2) Cell phenotype below].
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(1) Clonality. In order to ascertain whether scid tumours represented the corresponding biopsy, samples were assessed for Ig and EBV clonality. Ig clonality was analysed by PCR using primers specific for a conserved region of the variable (V) segment of framework (Fr) 3 and junction (J) segments of the Ig heavy chain (IgH) gene locus (McCarthy et al., 1990 ; Stetler-Stevenson et al., 1990
). Fr3 PCR products were analysed on an ABI PRISM 310 Genetic Analyser. Primers derived from Fr1 were used to analyse samples from patient 2. Based on sequence analysis of the amplification products, a TaqMan PCR assay specific for the rearrangement in this case was designed and performed using standard methodology (Kuppers et al., 1995
). PCR analysis showed that biopsy and scid tumours from patients 1, 2 and 5 consisted of monoclonal material (for patient 2, see Fig. 1A
; data not shown for patients 1 and 5). Matching clones were found in each sample set demonstrating that each tumour pair was identical. Sequence analysis of material from patient 2 confirmed the identical clonal nature of biopsy and scid lesions. TaqMan PCR analysis confirmed these results and further detected the malignant clone in a peripheral blood leukocyte sample taken from patient 2 at time of PTLD. Biopsy and scid tumour from patient 3 were oligoclonal with an identical dominant rearrangement (Fig. 1B
). Passaged scid tumours were clonal, one of which was identical to the dominant biopsy and scid rearrangement. Biopsy from patient 4 contained a dominant clone in a polyclonal background (Fig. 1C
). The scid tumour was clonal, but did not reflect the biopsy. EBV clonality was assessed using the standard Gardella gel technique (Gardella et al., 1984
; Raab-Traub & Flynn, 1986
). The number of reiterated 500 bp EBV genome terminal direct repeats involved in forming the virus episome following infection of a target B cell is characteristic of any infection event. Progeny EBV+ve cells retain the same episome thus giving rise to an EBV clonal population which can be studied using the Gardella gel technique (Hurley & Thorley-Lawson, 1988
). Material from cases 2, 4 and 5 was analysed with this assay. All lesions contained a single band indicating virus clonality (data not shown). Each set of samples from patients 2 and 5 contained bands of identical size, indicating a common infection event in each case, whereas material from patient 4 contained different EBV clones, suggesting that the scid tumour arose from a latently infected, non-malignant B cell.
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(4) Cytokine expression. Extensive T cell infiltrates (26% of all cells; see Table 2
) were apparent in all biopsies as previously described (Perera et al., 1998
). Conversely, only occasional T cells were identified in corresponding scid tumours. To address the possible role of T cell-derived factors in tumour growth, human cytokine gene expression of all material from patients 15 was analysed by RTPCR (Fig. 2
). Analysis of human cytokine mRNAs [interleukin (IL)-2, -4, -6, -10 and interferon (IFN)-
] was carried out using published conditions (Yamamura et al., 1991
, 1992
). Primers for IL-10 did not cross-amplify EBV-encoded viral IL-10. Biopsy and scid lesions demonstrated a similar cytokine profile with expression of the B cell growth factors IL-2, -4, -6, -10 and IFN-
suggesting a key role in tumour growth. In situ hybridization studies in our laboratory have demonstrated that these growth factors are expressed by PTLD cells themselves (unpublished observations). The results suggest that T cells may contribute growth factors in the original malignancy whilst lack of T cells in scid tumours indicates that tumour cells supply themselves with necessary factors in an autocrine fashion supporting autonomous growth. This is in line with our previously proposed model of PTLD pathogenesis (Johannessen et al., 2000
).
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To our knowledge, this is the first report demonstrating outgrowth of a panel of PTLD biopsies in scid mice and contrasts with an earlier study by Randhawa et al. (1997) suggesting that biopsies did not grow in vivo. Analysis of passaged scid tumours from patient 3 showed progression from oligoclonal to monoclonal tumour (Fig. 1B
), thus providing possible insight into PTLD clonal progression.
Our study provides an adequate quantity of homogeneous PTLD material from four graft recipients which is uncontaminated with infiltrating non-malignant cells, thus providing opportunity for further molecular studies on PTLD pathogenesis.
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Acknowledgments |
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The authors also wish to thank Dr P. L. Amlot (Royal Free Hospital School of Medicine, London, UK) and Dr M. Burke (Harefield Hospital, Harefield, UK) for providing tumour material as well as Dr G. J. Bancroft, Mr J. P. Kelly, and Mr A. R. Turner (London School of Hygiene & Tropical Medicine) for supplying and maintaining the scid mice.
I.J. was supported by the British Council, a British ORS Award, a NATO Science Award, and awards from the Icelandic Jonsdottir & Kristjansson and Magnusdottir & Bjarnason Funds. This work was supported by the Medical Research Council, the United Kingdom Children Cancer Study Group and the Leukaemia Research Fund.
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Received 22 August 2001;
accepted 1 October 2001.