Lack of association of polyomavirus and herpesvirus types 6 and 7 in human lymphomas

Authors


Abstract

BACKGROUND

The association of viruses with several human tumors has been studied for almost 100 years, and it remains a very controversial issue. Due to the fact that the presence of polyomaviruses and herpes viruses reportedly are associated with lymphomas, albeit with striking results and differences between the many studies, the authors undertook a study into the presence of viral sequences of polyomavirus (BK virus, JC virus, and especially simian virus 40 [SV40]) in human lymphomas in an attempt to explain this contradictory association. To complete the study, the presence of different virus types from the herpesviriridae family were analyzed, such as herpesvirus type 6 (HHV6), HHV7, HHV8, and Epstein–Barr virus, in human lymphomas.

METHODS

DNA was isolated from 83 frozen human lymphoma samples, and different polymerase chain reaction techniques were used to find polyomavirus and herpesvirus sequences in these samples. To assess the incidence of the presence of sequences in lymphomas, a parallel analysis was made of 53 samples from normal donor spleen lymphocytes. Positive samples were analyzed for polyomavirus sequences by immunohistochemistry.

RESULTS

Polyomavirus sequences were detected in 9 of 83 lymphomas (11%), and SV40 sequences could be confirmed in only 1 lymphoma. Immunohistochemistry for large-T antigen was negative in all samples. Herpesviruses were detected in 53 of 83 lymphomas (63.9%), were detected more frequently in Hodgkin lymphomas (80%) than in non-Hodgkin lymphomas (58.7%), and were detected in > 60% of normal spleen lymphocytes.

CONCLUSIONS

The current results did not support a clear association of polyomavirus and HHV6 or HHV7 with lymphomas; HHV6 and HHV7 sequences were detected in a similar percentage of normal samples and lymphomas. The lack of significant differences between normal and malignant lymphocytes and the absence of viral protein expression in the tumor cells did not allow the establishment of a clinical correlation between polyomaviruses or HHVs (HHV6, HHV7, HHV8) and lymphomas. Nevertheless, because viral products can be lost during tumor progression, and because host factors can modulate the oncogenic role of some viruses, the hypothetical role of these viruses cannot be discarded completely. Cancer 2005. © 2004 American Cancer Society.

Viruses have been studied largely as carcinogenetic agents, and some of them have demonstrated the capacity of transformation. Several viruses previously were associated with the development of human lymphomas, including herpesviruses, and especially Epstein–Barr virus (EBV) and human herpesvirus type 8 (HHV8), and only 1 retrovirus, the T-cell lymphotropic virus type I.1–3 In these lymphomas, the viral products can be detected by different polymerase chain reaction (PCR) methods and immunohistochemistry, and the causation or cocarcinogenetic effect appears to be unquestionable. In fact, lymphomas associated with these viruses include approximately 50% of Hodgkin lymphomas, almost all Burkitt lymphomas and posttransplantation lymphoproliferative disorders, acquired immunodeficiency syndrome-related non-Hodgkin lymphomas, T-cell/natural killer cell lymphomas, primary serous effusion lymphomas, and adult T-cell leukemia lymphomas.1, 4

Whereas the EBV virus-lymphoma association is clear, over the last few years, other viruses, such as adenoviruses and polyomaviruses, reportedly have been associated with human lymphomas.5–10 Both virus families are ubiquitous in humans, and it is believed that lymphoid cells are reservoirs that do not elicit viral replication. Although adenoviruses have > 40 different serotypes, polyomaviruses include 3 different types that can affect humans: simian virus 40 (SV40), BK virus (BKV), and JC virus (JCV). Moreover, SV40 is a monkey virus that has been proven oncogenic in laboratory animals and, in humans, has been associated with brain tumors, bone tumors, mesotheliomas, and lymphomas. How the SV40 polyomavirus became introduced in humans is questionable and seems to be related with poliomyelitis vaccines that were contaminated between 1955 and 1963. It is noteworthy that SV40-neutralizing antibodies have been detected in approximately 10–15% of individuals.11 Two recently published reports stated that > 40% of human lymphomas harbor SV40 sequences5, 6, 11; however, other investigators did not find this association.12–14 The percentage differences found in previous reports are remarkable, with the incidences ranging from 0% to 43% of lymphomas. Other polyomaviruses, JCV and BKV, have been associated with some human tumors, principally central nervous system (CNS) tumors and colorectal carcinomas, and a recent report found JCV sequences in 87% of CNS lymphomas.7, 15, 16

Apart from EBV and HHV8, which largely have been proven to be associated with the development of lymphomas, other viruses of the herpesviridae family, such as cytomegalovirus (CMV), HHV6, and HHV7, have been detected in different human diseases.17, 18 HHV6, which is present in some carcinomas and lymphomas, and especially in angioimmunoblastic lymphomas, has demonstrated transforming potential in vitro.19 HHV7 is highly homologous to HHV6 and has selective tropism for CD4-positive T lymphocytes. It has been detected in Hodgkin disease,18 but it has not been proven oncogenic. Both viruses seem to be in low concentrations in tumor tissues; therefore, these viruses are undetectable by Southern blot analysis, and the PCR technique is required.

The objective of this article was to report our results from a study of the association of these polyomaviruses and herpesviruses with human lymphomas. The lack of significant differences between normal and malignant lymphocytes and the absence of viral protein expression in the tumor cells did not allow us to establish a clinical correlation between polyomaviruses or HHV6/HHV7 and lymphomas.

MATERIALS AND METHODS

An analysis was made of 83 lymphomas, 6 reactive lymphadenopathies, and 46 normal donor spleen lymphocytes from Clínica Puerta de Hierro (Madrid, Spain). The series included 20 Hodgkin lymphomas and 63 non-Hodgkin lymphomas, 17 of which were of T-cell origin. Patient age ranged from 30 years to 82 years. None of the patients were human immunodeficiency virus (HIV) positive or had any other immunosuppression.

We used 200–500 ng of DNA obtained by phenol-chloroform from frozen tissues. All samples were tested for suitability of amplification with primers specific for a 200-base pair (bp) fragment of the human glyceraldehyde-3-phosphate dehydrogenase gene.10

Several approaches, including PCR with the primers PYV forward /PYV reverse employed in most previous reports,6, 20 were used to study polyomavirus sequences. In addition, the Diagnostic Microbiology Service of the Instituto de Salud Carlos III (Madrid, Spain) developed a new, sensitive method for detecting SV40, JCV, and BKV in the same reaction.21 This method consists of a multiplex, nested PCR with a sensitivity similar to Taqman PCR, which is < 10 virus copies per reaction. All PCR analyses were repeated at least twice in two different laboratories, and positive samples were sequenced.

The same procedure that was used in the previous study was followed to study the herpes virus family, detecting DNA sequences of EBV, HHV6 (types A and B), HHV7, HHV8, and CMV from the different aliquots. The method has been described previously by the same group at the Instituto de Salud Carlos III in Madrid, Spain22 (see Fig. 1).

Figure 1.

The presence of human herpesvirus 6 and 7 (HHV6 and HHV7) was detected in human lymphomas. The same multiplex polymerase chain reaction analysis detected several types of herpes viruses in human lymphomas. WM: weight marker; SC: sensitivity control; IC: internal control; CMV: cytomegalovirus; EBV: Epstein–Barr virus.

Immunohistochemistry for SV40 T-antigen was performed with the polyclonal antibody 416 (Oncogene Science), and EBV immunohistochemistry was performed with the antibody LMP CS.1-4 (DakoCytomation) according to the manufacturer's suggested protocols (see Fig. 2). Data were analyzed with the Statistical Package for Social Science (version 11; SPSS, Chicago, IL). To study correlations between variables, a chi-square analysis was performed.

Figure 2.

(A,B) The presence of Epstein–Barr virus (EBV) in lymphomas is shown in hematoxylin and eosin-stained samples. The presence of EBV sequences in human lymphoma was detected by polymerase chain reaction analysis and confirmed by immunohistochemistry. Original magnification, × 10 (A); × 20 (B).

RESULTS

Polyomavirus sequences were detected only in 9 of 83 lymphomas and were detected in none of the 6 reactive lymphadenopathies or the 46 normal spleen mononuclear cells. The percentages of positivity were ≈ 11% of total lymphomas (10% of non-Hodgkin lymphomas and 15% of Hodgkin lymphomas). However, most notably, after employing specific primer sets for the various members of the polyomavirus family, SV40 could be confirmed in only 1 lymphoma. In all other samples, there were JCV sequence in 4 of 83 lymphomas and BKV sequences in 5 of 83 lymphomas (see Table 1). Similar results were obtained with the PYV forward and PYV reverse primers under high-stringency conditions (data not shown). Immunohistochemistry for SV40 was negative in all samples. No significant association between polyomaviruses and controls and lymphomas (BKV, P = 0.18; JCV, P = 0.10; and SV40, P = 0.42).

Table 1. The Presence of Polyomavirus in Human Lymphomas (Polymerase chain reaction analysis)
DiagnosisNo. of patientsNo. of patients (%)Total virus
JCVBKVSV40
  1. JCV and BKV: the JC and BK polyomavirues, respectively; SV40: simian virus 40; MALT: mucosa-associated lymphoid tissue; CLL: chronic lymphocytic leukemia.

Follicular lymphoma151 (7)2 (13) 3 (20)
Mantle cell lymphoma2 1 (50) 1 (50)
MALT2   0 (0)
Large B-cell lymphoma221 (4)  1 (4)
Anaplastic T-cell lymphoma21 (50)  1 (50)
Lymphoblastic lymphoma3   0 (0)
Peripheral T-cell lymphoma151 (7)  1 (7)
Hodgkin lymphoma201 (5)2 (10)1 (5)3 (15)
Cutaneous B-cell lymphoma1   0 (0)
CLL1   0 (0)
Donor spleen lymphocytes46   0 (0)
Reactive lymphadenitis6   0 (0)
Non-Hodgkin lymphoma633 (5)3 (5)0 (0)6 (10)
Control samples520 (0)0 (0)0 (0)0 (0)
Lymphomas834 (5)5 (6)1 (1)9 (11)
Total1354 (3)5 (4)1 (1)9 (7)

Herpes viruses were detected in 53 of 83 lymphomas (63.9%) and were detected more frequently in Hodgkin lymphomas (80.0%) than in non-Hodgkin lymphomas (58.7%). They also were detected in nontumor samples, including 28 samples of normal spleen lymphocytes (60.9%) and in 5 samples of lymphadenopathies (83.3%) (see Table 2). Almost 50% of lymphomas showed sequences of EBV, including ≈ 70% of the Hodgkin lymphoma samples and 48% of the non-Hodgkin lymphoma samples. Statistical analysis of herpes viruses and lymphomas showed significant correlations between EBV and Hodgkin lymphomas (P = 0.002). The results for the remaining herpes viruses studied were as follows: HHV6 was positive in 39 of 135 samples (28.9%), HHV7 was positive in 33 of 135 samples (24.4%), and CMV was positive in 3 of 135 samples (3.6%) (see Table 2). No statistical correlation with lymphomas was observed for remaining viruses studied. Using immunohistochemistry, EBV products were detected in 32% of lymphomas, including 10 Hodgkin lymphomas, 2 anaplastic T-cell lymphomas, 3 large B-cell lymphomas, and 2 T-cell lymphomas.

Table 2. The Presence of Herpesvirus in Human Lymphomas (Polymerase chain reaction analysis)
DiagnosisNo. of patientsNo. of patients (%)Total herpes
EBVHHV6HHV7CMV
  1. EBV: Epstein–Barr virus; HHV6: human herpesvirus 6; HHV7: human herpesvirus 7; CMV: cytomegalovirus; MALT: mucosa-associated lymphoid tissue; CLL: chronic lymphocytic leukemia.

Follicular lymphoma154 (27)2 (13)1 (7)0 (0)5 (33)
Mantle cell lymphoma11 (50)0 (0)0 (0)0 (0)1 (50)
MALT20 (0)1 (50)1 (50)0 (0)1 (50)
Large cell lymphoma2211 (50)3 (14)1 (4)2 (9)13 (59)
Anaplastic T cell lymphoma22 (100)2 (100)0 (0)0 (0)2 (100)
Lymphoblastic lymphoma31 (33)0 (0)1 (33)0 (0)2 (67)
Peripheral T-cell lymphoma1511 (73)5 (33)4 (27)1 (7)13 (87)
Hodgkin lymphoma2014 (70)8 (40)4 (20)0 (0)16 (80)
Cutaneous B-cell lymphoma10 (0)0 (0)0 (0)0 (0)0 (0)
CLL10 (0)0 (0)0 (0)0 (0)0 (0)
Donor spleen lymphocytes4610 (22)14 (30)18 (39)0 (0)28 (61)
Reactive lymphadenitis64 (67)3 (50)3 (50)1 (17)5 (83)
Non-Hodgkin lymphoma6330 (48)14 (22)8 (13)3 (5)37 (59)
Control samples5214 (26)17 (33)21 (40)1 (2)33 (64)
Lymphoma8344 (53)22 (27)12 (15)3 (4)53 (64)
Total13558 (43)39 (29)33 (24)4 (3)86 (64)

DISCUSSION

The results concerning polyomaviruses clearly contradict previous reports, which indicated that the percentage of polyomavirus sequences was similar in normal mononuclear cells and lymphomas,8 that SV40 was present in a high proportion of lymphomas,5, 6 and that JCV sequences were present in CNS lymphomas.7

Several points have to be discussed and highlighted to explain these results. First, the population or geographic origin of the tumors is a relevant point that we are unable to clarify with absolute certainty. Although SV40-contaminated vaccines were inoculated in Spain during the late 1950s,11 there are insufficient epidemiological studies about the incidence of antibodies detected in the Spanish population. In this sense, a recently published study indicated that 9.5% of control patients in Spain showed antibodies against SV40, but only 5.9% of patients with lymphoma showed such antibodies.11 In the current study, we were unable to demonstrate SV40 sequences in normal controls. In addition, the presence of SV40 in mesotheliomas also is controversial, in that it has been described in some articles,23 whereas other authors did not detect any positive SV40 mesotheliomas,24, 25 and some authors have explained that these differences are related to the location in the countries where the patients live. Similar epidemiological differences with SV40 and lymphomas also may apply in Spain, and this possibility cannot be excluded. Nevertheless, we do not believe that specific differences can be found between countries like Italy and Spain. It is noteworthy that one of the world's reference virus laboratories, the H zur Hausen group in Germany, was unable to detect SV40 sequences in human tumors.26 Second, the analytic method used probably is the most crucial point for discussion and consideration in intrepreting the possible association of polyomaviruses and lymphomas. In the current study, we employed very specific PCR methods, which are as sensitive as those employed previously. Therefore, we can discard any lack of sensitivity in our procedure. During the last 5 years, the specific nested PCR described by the Spanish Virus Research Center Virological Research Group has been tested extensively in numerous patients and in the general population. Moreover, we did not detect protein expression of the large-T antigen in the positive lymphomas, as predicted by the PCR results, in which > 45 cycles or nested PCRs are required to visualize some agarose bands. Previous reports on SV40 sequences detected by PCR lacked immunohistochemistry studies of positive samples, which may have resulted in a stronger association of SV40 in those series of lymphomas.5, 6 More recently, an exhaustive work into mesotheliomas did not find any SV40 sequences.25

A third important point to consider is the presence of false-positive results due to PCR contamination. These false-positive PCR studies may be a result of the amplification of viral vectors or viral controls used in many laboratories or the detection of presumably DNA human sequences with a high homology to SV40 DNA. This recent observation pointed out by the Martini group26, 27 is important in the verification of SV40-positive lymphomas. Because SV40 Tag sequences between 4476–4140 bp in N′ and 2274–2630 bp in C′ are 97% homologous to the human telomeres region of chromosome 10 and 11, and as at least 1 PYD primer and the SV40 probe (4458–4479 bp) are within that region, other complementary studies should be undertaken. In some series, analyses of different regions have been made with similar results; whereas, in other series, the regulatory region mostly has been negative.28 In addition, an important factor that only some groups have considered25 is that SV40 sequences are found in diverse vectors employed in many research laboratories, and this may be the origin of the false PCR results obtained by some groups.

In the association of herpesviruses with lymphomas, our results showed that EBVs were detected in approximately two-thirds of patients with Hodgkin disease (70%); however, it is interesting to note that the proportion of non-Hodgkin lymphomas was greater than expected (48%) and was high in the control samples (26%). It is clear that the highly sensitive PCR method used was capable of detecting herpesviruses more than other studies based on immunohistochemistry or in situ hybridization. The most frequent EBV-positive non-Hodgkin lymphomas were T-cell anaplastic lymphomas, as described by other authors.2

HHV6 is a ubiquitous virus in the adult human population and has been detected in lymphomas in proportions varying from 12%19 to 73%3 of lymphomas. The virus remains latent in human cells and can reactivate in immunocompromised patients. In fact, it has been detected in reactive lymph nodes of normal population, and 1 study even demonstrated that all benign lymphadenopathies were positive for HHV6 in a PCR analysis.29 The same study demonstrated immunohistochemically that HHV6 positivity is in histiocytes and plasma cells, whereas neoplastic cells consistently are negative. In our series, HHV6 sequences were present in similar proportions in both normal samples and lymphomas. No significant differences were found between the diverse lymphomas types. It is interesting to note the association between HHV6 and EBV. It has been shown that HHV6 can activate EBV replication in vitro.30 In our series, we found sequences of both viruses in 7 of 20 Hodgkin lymphomas and in 6 of 13 T-cell lymphomas that were EBV-positive. Most of the samples that were HHV6-positive had EBV sequences. All of these findings suggest that HHV6 does not play a major role in lymphomagenesis but may play a part in EBV infections.

HHV7 is a CD4 T-lymphotropic virus that has been detected in Hodgkin and non-Hodgkin lymphomas as well as in reactive lymphadenopathies, but it has never been proven oncogenic. The current results showed a greater proportion of HHV7 sequences in the normal population with respect to lymphomas, with HHV7 found more frequently in Hodgkin lymphomas. Other series showed a greater frequency of HHV7 in lymphomas, with proportions of 77% in non-Hodgkin lymphomas, 68% in Hodgkin lymphomas, and 32% of reactive lymphadenopathies.18 In these lymphomas, in situ hybridization showed positivity in small T-cell lymphocytes.

In summary, polyomavirus sequences, and especially SV40, have been demonstrated in > 40%,5, 6 19%,13 14%,8 10%,14 14%,28 and 0%12 of lymphomas. No significant differences have been found between HIV-positive and HIV-negative patients. There also are striking differences in normal blood cells, ranging from 0%5, 6 to 23%.28 In the current series, we found ≈ 10% positive samples but only 1 SV40-positive sample. Striking percentages of BKV and JCV have been described in normal blood cells in numbers that range from 0% to 90%.9 A recent study found that JCV was present in 1–3% of healthy individuals, and BKV was present in ≈ 7% of healthy individuals.9 Therefore, the current results and the results reported by other investigators do not support a clear association between polyomaviruses and lymphomas. HHV6 and HHV7 have been detected in similar proportions between the general population and patients with lymphoma, as reported previously.

The association between polyomaviruses and human tumors, including lymphomas, remains a very controversial issue. The fact that viral sequences of polyomavirus and herpes virus can be detected in normal tissue and the well known situation that some virus can induce molecular alterations and then can be eliminated from the cells makes it more difficult to study and interpret the results. This hit-and-run hypothesis has been demonstrated previously in animal models, and it is an attractive idea that will require studies for further clarification. Nevertheless, with the data that we have to date and with the technology that we are employing, we cannot conclude that polyomavirus SV40 is involved in the development of lymphomas. We can only suggest that the follow-up of those children and adults with anti-SV40 and polyomaviruses antibodies probably will demonstrate whether or not there is a greater incidence of lymphomas with respect to the population without antibodies at any age.

Acknowledgements

The authors thank Martin Hadley-Adams for his assistance with the English language and reparation of the article.

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