Journal of General Virology (1998), 79, 573–579. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Persistence of parvovirus B19 DNA in testis of patients with testicular germ cell tumours Anne Gray,1 Louis Guillou,1 Jade Zufferey,2 François Rey,3 Anne-Marie Kurt,5 Patrice Jichlinski,4 Hans-Jurg Leisinger4 and Jean Benhattar1 1,2,3,4

Institute of Pathology1, Institute of Microbiology2, Department of Endocrinology3 and Department of Urology4, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland 5 University Institute of Pathology, Centre Me! dical Universitaire, CH-1205 Geneva, Switzerland

Germ cell tumours (GCT) of the testis are the most common malignant tumours occurring in young adults. In view of the young age of patients, the increasing incidence of GCT and the overexpression of wild-type p53 observed in a majority of tumours, the possibility of the involvement of a virus in the development of this cancer was considered. Testicular GCT were analysed for the presence of cytomegalovirus and Epstein–Barr virus (EBV), which are known to cause overexpression of wildtype p53 protein, and parvovirus B19. The testicular tissue of 39 patients with testicular GCT and 12 patients with healthy testicular tissues was tested for presence of viral DNA by PCR. Neither cytomegalovirus nor EBV DNAs were detected in the 39 tumours analysed, but parvovirus B19 DNA

Introduction Testicular cancer is a treatable, often curable cancer that usually develops in young to middle-aged adults. A recent increase in the incidence of testicular cancer has been noted (Forman & Moller, 1994). In contrast to the pattern for incidence rates, cancer mortality rates are now declining and the 5 year relative survival rate is now over 85 %. Very little is known about the aetiology of germ cell tumours (GCT), though several risk factors have been implicated, including cryptorchidism, hereditary factors and testicular dysgenesis. Other factors, including viruses, have been suggested to play a role (Newell et al., 1984). Knowing that testicular GCT occur in young patients and that testicular tumour cells express high levels of wild-type Author for correspondence : Jean Benhattar. Fax ­41 21 3147115. e-mail Jean.Benhattar!chuv.hospvd.ch

sequences were demonstrated in the testicular tissue of 85 % (33/39 cases) of patients with GCT. The sera of 16 of the 39 patients with GCT were tested for the presence of parvovirus B19 IgM and IgG. B19-specific IgG was detected in the sera of 11 patients (69 %). Only one case was positive for parvovirus B19 IgM, which was also shown to have B19 genome sequences in the serum by PCR, indicating that in a majority of cases an acute B19 infection can be excluded as being the source of the B19 DNA sequences in the testis. B19 DNA could not be detected in normal testicular tissue and thus parvovirus B19 could play a role, direct or indirect, in the development of testicular GCT or have tropism for the tumour cells.

p53 (Guillou et al., 1996), a role for a virus or viruses in GCT pathogenesis might be considered. Virus-associated human tumours constitute a substantial fraction of total tumours worldwide (zur Hausen, 1986). There is now evidence that Epstein–Barr virus (EBV), hepatitis B virus (HBV), several types of human papillomaviruses (HPV) and human T-cell leukaemia}lymphoma virus (HTLV) type I, and possibly type II, are linked to particular forms of human tumours (Masucci, 1993). Testicular GCT have been investigated for the presence of EBV and cytomegalovirus (CMV) as these viruses are known to cause overexpression of wild-type p53 protein (Allday et al., 1995 ; Jault et al., 1995 ; Muganda et al., 1994). EBV, a human herpesvirus, is found as an asymptomatic infection in more than 90 % of the population worldwide (Young & Rowe, 1992). Several studies have searched for evidence of EBV in testicular tumours. In one study, an elevated titre of antibodies against EBV in 80 % of GCT

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A. Gray and others

patients has been observed versus 20 % in the control group (Algood et al., 1988), though in another study no significant difference was found between patients with testicular cancer and controls (Heinzer et al., 1993). An in situ hybridization analysis was also performed, but no EBV DNA was detected (Heinzer et al., 1993). One study employing PCR techniques was also unable to detect EBV in these tumours (Rajpert-De Meyts et al., 1994). However, one group has identified EBV by in situ hybridization in some seminomas (Fend et al., 1995). CMV is a beta herpesvirus that infects almost all humans at some time during life. Several types of human malignancy have been linked to the presence of CMV by seroepidemiological evidence or molecular hybridization (Boldogh et al., 1983 ; Koffa et al., 1995). There has been one serological study which linked CMV to testicular cancer (Mueller et al., 1988), whereas another group found no evidence of CMV by either serology or in situ hybridization (Heinzer et al., 1993). Parvovirus B19 was first discovered in 1975 (Cossart et al., 1975). In 1983 it was identified as the causative agent of erythema infectiosum (also known as the ‘ fifth disease ’), a mild, acute exanthematous disease (Anderson et al., 1983). It is the aetiological agent of many diseases in children as well as in young adults. A growing list of pathologies are now attributed to B19. During pregnancy, parvovirus B19 infection can be a risk factor for hydrops foetalis and foetal death (Anand et al., 1987 ; Finch et al., 1995 ; Wright et al., 1996). Parvovirus infection may cause chronic anaemia, due to persistent bone marrow insufficiency in immunocompromised patients (Weiland et al., 1989 ; Brown & Young, 1996). Patients suffering from lymphoproliferative diseases and undergoing immunosuppressive treatment have an increased risk of acquiring a chronic infection (Kurtzman et al., 1988). The anaemia in these patients improves after cessation of immunosuppression. Arthritic symptoms following acute B19 infection are common among adults, especially in women (Taylor et al., 1992). Three cases of encephalopathy attributed to B19 (Umene & Nunoue, 1995) and a report of two patients with liver dysfunction (Tsuda, 1993) following B19 acute infection have been published. To determine whether viral DNA is present in testicular GCT, we investigated a group of 39 GCT for EBV, CMV and parvovirus B19 sequences. To eliminate an infection unrelated to testicular GCT, 12 apparently normal testes were also tested for presence of viral DNA. In addition, 16 patients were investigated for the presence of anti-parvovirus B19 IgG and IgM, and serological and PCR data were compared.

Methods + Patients. Two different groups of patients were studied. The first group was composed of 39 patients from Lausanne and Geneva (Switzerland) who presented with seminomatous and non-seminomatous GCT of the testis between 1990 and 1994. Tumours were classified according to the World Health Organization International Histological Classification. No treatment had been given prior to tumour resection.

FHE

The age of the patients ranged from 2 to 62 years (median age 33). The second group included 12 patients (7 with prostatic cancer and 5 autopsy cases) with apparently normal testes. + DNA extraction. DNA was extracted from freshly frozen testicular tissues using a standard procedure (Sambrook et al., 1989) with modifications. Thick frozen sections were cut from each frozen tissue sample. Following an overnight incubation with proteinase K, further proteinase K was added and the temperature increased to 55 °C for 2 h to make sure that viral capsid proteins were digested. DNA was subsequently extracted with phenol followed by phenol–chloroform and precipitated with ethanol. To avoid contamination the following precautions were taken : (1) new scalpel blades and gloves were used for each new case and only four cases were extracted at a time, (2) pipetting was done with barrier pipette tips and (3) pre-made commercial solutions were used. DNA was also extracted from the serum of 16 cases (the same cases were used for serology) using the same procedure, except the precipitation in ethanol was with glycogen and was left overnight at ®20 °C. + PCR detection. The extracted DNA (200–500 ng) was added to a PCR mixture containing 10 mM Tris–HCl, pH 8±8, 50 mM KCl, 1±8 mM MgCl and 250 µM dNTPs in a total volume of 30 µl. Samples were # heated to 98 °C for 8 min. Then 4 µl of a mixture containing the oligonucleotide primer pair (0±5 µM final concentration for each primer) and 1 unit Taq DNA polymerase were added. Amplifications were carried out for 40 cycles of 94 °C for 30 s, 55 °C (EBV, NSA–NSB and AL–BS) or 58 °C (CMV, PA–PB and VPA–VPB) for 45 s and 72 °C for 45 s. The amplified products were examined by 1±5 % agarose gel electrophoresis and ethidium bromide staining. The specificity of the PCR-positive products was confirmed by Southern hybridization with a specific internal DIG 5« end-labelled oligonucleotide probe (Table 1). The primers were selected according to the sequence published by Shade et al. (1986) of an isolate from a patient (Au) who had an aplastic crisis caused by B19. The primer sequences were compared to the corresponding regions of another strain (Wi) (Blundell et al., 1987) and they were found to be identical. To improve our ability to detect parvovirus B19 DNA by PCR we used four sets of primers (Table 1 and Fig. 1), one localized in the non-structural (NS) region and the other three in the capsid (VP) region. A negative control consisting of all reagents necessary for the PCR reaction with water instead of DNA template was always included. For B19 detection plasmid pYT103 (kindly provided by Peter Tattersall, Yale School of Medicine, CT, USA) was used as positive control at a concentration of 2000 copies per reaction. + Single strand conformation polymorphism (SSCP) analysis. To determine the diversity of parvovirus B19, SSCP analysis was performed using PCR-positive cases from the PA–PB region. Two µl of PCR product was denatured in 10 µl 50 mM NaOH and 1 mM EDTA at 50 °C for 10 min. After the addition of 1±5 µl formamide dye, the samples were immediately analysed in a 40 % Mutation Detection Enhancement acrylamide gel (AT Biochem) in 0±5¬ TBE, with a constant voltage of 20 V}cm for 4–5 h at 20 °C. After electrophoresis, the gel was stained by a silver staining technique (Chaubert et al., 1993). + Serological assay for parvovirus B19. The sera available from 16 patients with testicular GCT were used for this analysis. All sera were collected at the time (³2 days) of testicular surgery. The sera from 47 healthy men were also used as control for B19 seroprevalence. Parvovirus B19-specific IgG and IgM were assayed by an enzyme immunoassay (IDEIA, Dako) in sera of patients with GCT. Testing was performed according to standard procedures. These tests contained recombinant

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Parvovirus B19 in testicular tumours

Table 1. Oligonucleotide primers used for detection of EBV, CMV and parvovirus B19 DNA by PCR For EBV detection the oligonucleotide primers were from the long internal direct repeated region. For CMV, the primers were selected within the late antigen gp64 (LA) gene. The location of parvovirus B19 PCR primers is indicated according to the sequence described by Shade et al. (1986). The DIG 5« end-labelled oligonucleotides were used as internal probes for Southern hybridization of B19 PCR products.

PCR EBV

Oligonucleotide primers (5«–3«) EBV-A EBV-B CMV-LA CMV-LB NSA NSB

GTT CGC GTT GCT AGG CCA CC AGG ACC ACT TTA TAC CAG GG TGG CTA CGG TTC AGG GTC AG GCT GCC ATA CGC CTT CCA AT GGT GGT CTG GGA TGA AGG TAT T GCT CTT TTA AGG CTT TAG CAT GTA

PA–PB (B19)

DIG PA PB

CGG GAA CAC TAC AAC AAC TG AAA TGG TGG GAA AGT GAT GAT AA ACG AGC AAC TAA GTC AAA CAG AGA

AL–BS (B19)

DIG AL BS

ATC CCC TAG AAA ACC CAT CC ACC CAA GCA TGA CTT CAG TT GCG GGA GAA AAC ACC TTA T

VPA–VPB (B19)

DIG VPA VPB

CAA AAG CAT GTG GAG TGA GG TTA GGG CAA GGT CAG GAT AC GGG CAC TGG AGG AAA CTT AT

DIG

CAG CTT TTA GGT ACA GGA GG

CMV NSA–NSB (B19)

Location and PCR product size (bp)

Protein location

PCR : 108 PCR : 99 1539–1558 1724–1743 PCR : 207 1704–1723 2462–2484 2615–2638 PCR : 177 2595–2614 3117–3136 3285–3304 PCR : 187 3187–3206 3611–3630 3819–3838 PCR : 228 3782–3801

NS NS VP1 VP1 VP1 VP1}VP2 VP1}VP2 VP1}VP2

Detection of parvovirus B19 DNA in testicular tissues by PCR

Fig. 1. Schematic representation of the parvovirus B19 genome. The NS, VP1 and VP2 coding regions are indicated above the bar and the localization of each of the four amplified PCR products is shown below the bar.

viral capsid antigen VP2. Briefly, IgGs were detected by a two-step assay based on streptavidin-coated microwells and IgM by a µ-capture assay.

Results Search for EBV and CMV DNA sequences by PCR

Neither EBV nor CMV were detected by PCR in the tumour tissues of the 39 GCT analysed. DNAs obtained from known EBV-infected lymphomas and from placental tissue infected with CMV were used as positive controls.

Using PCR 85 % (33}39) of cases were found to be positive for B19 DNA with at least one set of primers, 43 % (17}39) of cases were positive with at least two sets of primers and three cases (T8, T9 and T38) were positive with all four sets of primers. These results were confirmed by Southern blotting of the amplification products and hybridization with an internal probe. An example using the PA–PB set of primers is shown in Fig. 2. The sensitivity of PCR assays was assessed by serial tenfold dilutions of the B19 plasmid. The assays were sensitive enough to detect between 1 and 10 copies of plasmid DNA per PCR reaction. The majority of the positive cases were found with primers from the VP coding region and only nine GCT were positive for the NS protein area. Of the 39 testicular GCT, 20 (51 %) were positive for AL–BS primers, 16 (41 %) were positive for VPA–VPB primers, 15 (38 %) were positive for PA–PB primers and 9 (23 %) were positive for NSA–NSB primers. There was no significant difference between seminoma and non-seminoma GCT with regard to the presence of B19 (P ¯ 0±42). All 12 samples from apparently normal testes were negative after amplification with the four sets of primers and Southern hybridization.

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Table 2. Testicular cancers and parvovirus B19 detection : correlation of DNA detection and serological markers

(a)

Serology

(b)

Fig. 2. Demonstration of PCR amplification of the VP1 region of parvovirus B19 performed on DNA extracted from testicular GCT (T1–T13) using the PA–PB set of primers. (a) Agarose gel. (b) Southern hybridization of gel shown in (a) with a non-radioactive DIG-labelled B19 probe. Lanes : 1, marker (100 bp ladder) ; 2, negative control ; 3, positive control containing 2000 copies of plasmid pYT103 ; 4–16, individual samples corresponding to cases T1–T13, respectively.

PCR*

Case

IgM

IgG

Serum

Testis

T15 T16 T17 T18 T23 T24 T26 T29 T30 T33 T34 T35 T36 T37 T38 T39

– – ­ – – – – (­)† – – – – – – – –

­ ­ ­ – ­ – – – ­ ­ ­ – ­ ­ ­ ­

– – ­­ – – – – – – – – – – – – –

­ ­­ ­­ ­­ – ­ – – ­ ­ ­­ – ­ ­­ ­­ ­­

* Parvovirus B19 DNA detected by PCR in serum and in testicular tissues. ­, positive PCR result with only one set of primers ; ­­, positive PCR result with at least two sets of primers. † IgM false positive results due to positivity for EBV.

one case (lane 5) seems identical to the B19 control (lane 7). Altogether, 11 strains of parvovirus B19 were demonstrated out of the 15 cases tested using the SSCP assay. This indicates that the variability of the sequence of the VP region is high. It also shows that in the majority of cases the positive results cannot be due to contamination by a laboratory virus strain.

Parvovirus B19 serology and PCR detection in serum

Fig. 3. SSCP analysis of PCR products from the VP1 region of parvovirus B19. The figure shows a mobility shift of denatured single-stranded DNA from the B19-positive cases observed in Fig. 2. Lanes : 1–6, individual samples corresponding to testicular GCT T2, T5, T8, T9, T10 and T13, respectively ; 7, plasmid pYT103.

SSCP analysis

SSCP analysis was performed to evaluate the diversity of the B19 DNA sequences in the different samples. The 15 positive cases detected with the PA–PB primers were used. Six examples are shown in Fig. 3. Five different migration patterns can be observed, indicating variations in the sequence. Only

FHG

Results of anti-parvovirus B19 IgG and IgM assays were obtained for 16 patients with testicular GCT. Eleven (69 %) of them were IgG antibody-positive (Tables 2 and 3). Only one case was positive for both IgG and IgM. Among these 11 patients seropositive for parvovirus B19, 10 were positive for B19 DNA as shown by PCR. In contrast, PCR results were positive only in two cases (T18 and T24) with undetectable anti-parvovirus B19 antibodies (Tables 2 and 3). The seroprevalence of B19 in patients with GCT (69 %) was similar to that observed in healthy men (70 %). In both groups the seroprevalence increased slowly with age. B19 DNA was detected by PCR only in the serum of the patient who was IgM-positive (Table 2, case T17). All other cases showed no evidence of B19 DNA in the serum using the four sets of B19 primers.

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Parvovirus B19 in testicular tumours

Table 3. Correlation between the presence of parvovirus B19 DNA in testicular tissues and anti-parvovirus B19 IgG and IgM antibodies (ELISA) in serum of patients with testicular GCT

B19 DNA in testicular tissues ­ ­ ­ ­ – –

IgM (serum)

IgG (serum)

No. of cases (%)

­ ­ – – – –

­ – ­ – ­ –

1 (6) 0 9 (56) 2 (13) 1 (6) 3 (19)

Discussion Newell et al. (1984) hypothesized that earlier exposure to viral agents could be involved in the aetiology of testicular GCT. Viruses might play a role for three reasons : (1) the young age of patients with GCT, (2) increasing incidence of GCT and (3) overexpression of wild-type p53 in most cases. But to date the only virus detected has been EBV and only in a minority of cases (Fend et al., 1995). In this study, we did not detect EBV or CMV DNA. Parvovirus B19 DNA sequences, however, were detected in 85 % (33}39) of the testicular GCT tested. A chance infection by B19 is unlikely since all apparently normal testes showed no evidence of B19. Although we have not shown the exact location of B19 in the testis, the results of serological and PCR assays (of the 16 patients where the serum at the time of surgery was available) prove the presence of B19 it is not due to an acute infection. The virus was probably deposited in the testis during prior B19 viraemia. It is known that the detection of B19 can be extremely difficult (Durigon et al., 1993). Even using PCR many positive cases go undetected. To circumvent this problem we and others have used PCR primers corresponding to multiple regions of B19. Another approach is to use nested PCR reactions to detect small quantities of B19 DNA in tissue or blood samples (Cassinotti et al., 1993 ; McOmish et al., 1993 ; Carrie' re et al., 1993). We avoided nested PCR because it is prone to contamination problems. The virus load in testicular GCT could be very low. When we performed a Southern analysis on genomic DNA, none of the cases analysed gave a positive result, indicating that the concentration of viral DNA in testicular tissues is at least lower than one parvovirus B19 genome per 10 cells. Using SSCP analysis we also found great diversity in the B19 genome in GCT. This diversity may explain why, in some cases, B19 DNA was detected with one set of primers and not another. A mutation within a primer binding site would reduce or prevent amplification. The marked diversity observed in part of the VP region of the B19 genome

allows us to exclude false positive PCR results. A major source of error during the detection of virus by PCR is the possible contamination of extracted genomic DNA with other sources of DNA (e.g. plasmid or genomic DNA) containing all or part of the same virus. The immune response of volunteers who developed viraemia after B19 inoculation is characteristic of primary systemic virus infection (Anderson et al., 1985). Specific antibody is first detected a few days after infection in the form of IgM–B19 immune complexes. After clearing of the viraemia, IgM antibody to B19 is detectable for a period of about 7 days before IgG develops in the third week after inoculation. IgM persists for up to 4 weeks, after which it decreases to zero, usually within 2–3 months. It seems that IgG remains at a high level for years and in most cases throughout life (Pattison, 1990). In this study, IgM serology was negative in the majority of GCT cases, thus acute infection at the time of surgery can be excluded. The prevalence of anti-parvovirus B19 IgG antibodies was 69 % in the patients with GCT and 70 % in controls with normal testes. It is interesting to note that 13 % (2}16) were B19-positive using PCR but serologically negative. This could be due to defective antibody production, epitope differences between parvovirus B19 strains not detectable by ELISA or to the fact that in persistent infection in immunocompromised individuals, serology is known to give misleading negative results (Kurtzman et al., 1987). By detecting EBV antibodies (90 % of the general population has antibodies against this virus) it was established that the immune status of the patients in this study was adequate, thus eliminating the latter possibility. Saal et al. (1992) found similar results in patients with rheumatoid arthritis. They observed that 40 % of their patients with parvovirus B19 DNA present in the synovial membrane were IgG-negative in both serum and synovial fluid. The variability of B19 from patients with erythema infectiosum seems to be minimal. A study comparing nine isolates collected over 20 years analysed the regions encoding the VP1-specific and VP1–VP2 junction portions of the capsid and showed that there has been minimal change in the genome (Gallinella et al., 1995). A study of the NS region by Kerr et al. (1995) also found very little variability. Originally it was thought there was only one B19 sub-group but now the situation is unclear. In one study of restriction site polymorphism of 48 B19 strains isolated in seven countries, six groups of B19 strains were identified (Mori et al., 1987). No correlation between genome type and a particular clinical manifestation was found. There is a tendency for increased variability, both at DNA and protein levels, in parvovirus B19 isolated from patients suffering from arthritis or persistent B19 infection (Hemauer et al., 1996). Our SSCP results indicate that the PA–PB region (in the VP1 gene) is variable in testicular tumours. Variants of B19 may have a function in testicular GCT pathogenesis. It is known from other parvoviruses that

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variants can cause different pathologies. For example, the cell type specificity and pathogenicity of the related autonomous parvovirus, minute virus of mice (MVM), can depend on small sequence differences in the capsid protein gene (Antonietti et al., 1988 ; Ball-Goodrich & Tattersall, 1992). The non-structural protein of MVM is known to be cytotoxic and to interfere with the cell cycle in proliferating cells by inducing accumulation of cells in the G2 phase (Op De Beck et al., 1995). In contrast, Telerman et al. (1993) found that certain cells were resistant to the effects of the autonomous murine H-1 parvovirus. These resistant cells were found to express wild-type p53 and the authors concluded that p53 contributes to resistance against the cytopathic effect of H-1 parvovirus. We have observed previously that wild-type p53 is overexpressed in all the testicular GCT tested (Guillou et al., 1996). Thus, in the testis it may be that p53 is overexpressed as a cellular defence against toxic effects of the non-structural protein of parvovirus B19. The questions of the localization and form of parvovirus B19 in the testis have not been specifically addressed in this study. The form, for example, could either be episomal or integrated into the human genome. It is clear that further experiments are needed to answer these questions. Determining the relationship between a virus infection and a cancer is no easy task (Henderson, 1989) as there is a long latent period between infection by a tumour virus and a low prevalence of cancer in those infected. In conclusion, we believe that parvovirus B19 might play some role, direct or indirect, in the aetiology or development of testicular GCT based on our findings, i.e. the presence of parvovirus B19 DNA sequences in the majority of GCT investigated, the absence of B19 in non-tumour testis and, with the exception of one patient, the absence of acute B19 infection at the time of the surgery. Alternatively, B19 may infect the tumour tissue. We thank Professor Bernhard Hirt and Dr Peter Beard for critical reading of the manuscript and for continual support and interest. We acknowledge Sarah Chappuis and Genevie' ve Metthez for technical assistance and Jacques Maillardet for photography. This work was supported in part by a grant from the Fondation pour la Lutte Contre le Cancer (Zurich, Switzerland).

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