Regis A Vilchez, Charles R Madden, Claudia A Kozinetz, Steven J Halvorson, Zoe S White, Jeffrey L Jorgensen, Chris J Finch, Janet S Butel

Departments of Medicine (R A Vilchez MD), Molecular Virology and Microbiology (R A Vilchez, C R Madden PhD, S J Halvorson BS, Z S White BS, J S Butel PhD), Pediatrics (C A Kozinetz PhD), and Pathology (J L Jorgensen MD, C J Finch MD), and Baylor Center for AIDS Research (R A Vilchez, C A Kozinetz, J L Jorgensen, C J Finch, J S Butel), Baylor College of Medicine, Houston, TX, USA

Background Non-Hodgkin lymphoma has increased in frequency over the past 30 years, and is a common cancer in HIV-1-infected patients. Although no definite risk factors have emerged, a viral cause has been postulated. Polyomaviruses are known to infect human beings and to induce tumours in laboratory animals. We aimed to identify which one of the three polyomaviruses able to infect human beings (simian virus 40 [SV40], JC virus, and BK virus) was associated with non-Hodgkin lymphoma.

Methods We analysed systemic non-Hodgkin lymphoma from 76 HIV-1-infected and 78 HIV-1-uninfected patients, and non-malignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumours; 54 colon and breast carcinoma samples served as cancer controls. We used PCR followed by Southern blot hybridisation and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses.

Findings Polyomavirus T antigen sequences, all of which were SV40-specific, were detected in 64 (42%) of 154 non-Hodgkin lymphomas, none of 186 non-malignant lymphoid samples, and none of 54 control cancers. This difference was similar for HIV-1-infected patients and HIV-1-uninfected patients alike. Few tumours were positive for both SV40 and Epstein-Barr virus. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B-cell and follicular-type lymphomas.

Interpretation SV40 is significantly associated with some types of non-Hodgkin lymphoma. These results add lymphomas to the types of human cancers associated with SV40.

Lancet 2002; 359: 817-23

Non-Hodgkin lymphoma comprises a biologically diverse group of haematological malignancies with clinical courses ranging from indolent to highly aggressive. During the past 30 years, the reported incidence and death rate of the disease have increased strikingly, nearly doubling since 1970.¹ About 55 000 new cases of non-Hodgkin lymphoma are estimated to be diagnosed annually in the USA,¹ and deaths related to the disorder are ranked fourth and fifth among all cancer deaths in women and men, respectively. Although the reasons for the increase in incidence are not fully understood, a substantial number of cases of non-Hodgkin lymphoma are linked to the HIV-1 epidemic. Indeed, non-Hodgkin lymphoma is a common malignancy in HIV-1-infected patients and the incidence can be up to 300 times higher than in HIV-1-negative individuals.¹

No obvious risk factors have emerged for non-Hodgkin lymphoma in the general population, but a viral cause has been postulated.² Some cases of non-Hodgkin lymphoma in HIV-1-infected patients have been attributed to deficient immune surveillance of oncogenic herpesviruses, such as Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8), or perhaps to chronic antigenic stimulation and defective immune regulation.³ EBV is suspected of having a major role in primary central-nervous-system non-Hodgkin lymphoma in HIV-1-infected patients, since most of those tumours contain EBV DNA, but it is detected less frequently (<40%) in systemic non-Hodgkin lymphoma in HIV-1-infected patients.^1-3 EBV is found even less commonly in non-Hodgkin lymphoma from HIV-1-negative patients. HHV-8 is specifically associated with multicentric Castleman's disease and primary effusion lymphoma, which often occurs in a setting of profound immunosuppression.^2,4

Because EBV and HHV-8 are absent from many cases of non-Hodgkin lymphoma, other viral agents should be considered as possible causes. The small DNA-containing polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus) are known to infect human beings, to have oncogenic potential, and to be associated with some human cancers.^2,5,6 SV40 DNA sequences have been found repeatedly in some brain and bone cancers and mesotheliomas.⁵ Polyomaviruses typically establish subclinical and persisting infections in their natural host, with persistence or latency in several organs, including kidney, brain, and spleen.² Studies have identified SV40, JC virus, and BK virus DNA sequences in B lymphocytes from HIV-1-infected and HIV-1-uninfected patients, suggesting that polyomaviruses are lymphotropic in man.^7-9 Polyomaviruses are known to induce tumour formation in animals, including the production of B-cell lymphomas by SV40.¹⁰ The major types of tumours induced by SV40 in laboratory animals are the same as the human cancers found to contain SV40 DNA, with the exception of lymphomas.⁵ In animals, oncogenesis is mediated by the polyomavirus large tumour (T) antigen.^2,5,6 The large T antigen is a multifunctional protein that stimulates host cells to enter S phase and is required for initiation of viral DNA synthesis. Fundamental to the effects of T antigen on host cells is binding to cellular tumour-suppressor proteins p53 and members of the pRB family.^2,5,6

Studies have reported the detection of SV40 DNA sequences in non-Hodgkin lymphoma from HIV-1-infected and HIV-1-uninfected patients,^9,11,12 and the amplification of JC virus DNA sequences from systemic non-Hodgkin lymphoma of HIV-1-infected children.¹³ These findings suggest a possible role for polyomaviruses in lymphoproliferative disorders, but the small size of the study populations, the lack of screens for other tumour viruses, and the limited confirmation of identity of the viral sequences detected made conclusion of whether polyomaviruses were definitely associated with non-Hodgkin lymphoma difficult. We aimed to determine the frequency of detection of polyomavirus T antigen DNA sequences in non-Hodgkin lymphoma among HIV-1-infected and HIV-1-uninfected patients, to identify which one of the three polyomaviruses able to infect humans (SV40, JC virus, and BK virus) was associated with non-Hodgkin lymphoma in adult patients, and to establish clinical correlations between the presence of viral sequences and non-Hodgkin lymphoma among HIV-1-infected and HIV-1-uninfected patients. The HIV-1-infected population was included in this study because of their high incidence of non-Hodgkin lymphoma and because immunocompromised individuals are known to be at risk of development of virus-mediated neoplasms.²
 

We studied 28 adult patients with HIV-1 infection and 35 HIV-1-uninfected patients who were diagnosed with systemic non-Hodgkin lymphoma between January, 1996, and August, 2001, at the Harris County Hospital District, the Veterans Administration Medical Center, and the Methodist Hospital, all of which are affiliated with Baylor College of Medicine, Houston, TX, USA. Additionally, the AIDS and Cancer Specimen Bank of the US National Cancer Institute, through collaboration with the Baylor Center for AIDS Research, provided non-Hodgkin lymphoma samples and clinical data from 48 HIV-1-positive and 43 HIV-1-negative adult patients diagnosed between November, 1987, and May, 2000, at different medical centres in the USA. The histological types of non-Hodgkin lymphoma among HIV-1-infected and HIV-1-uninfected patients were categorised according to WHO Classification for Neoplastic Diseases of the Lymphoid Tissues.¹⁴ No lymphomas of the central nervous system were included in this study.

Two types of control sample were analysed. Peripheral-blood leucocytes and hyperplastic lymph nodes from 79 HIV-1-positive and 107 HIV-1-negative patients without non-Hodgkin lymphoma or any type of cancer from the Harris County Hospital District, the Methodist Hospital, and the AIDS and Cancer Specimen Bank served as the non-malignant lymphoid control samples. 26 samples of colon carcinoma and 28 of breast carcinoma from patients diagnosed with these malignancies between January and August, 2001, at the Methodist Hospital served as the cancer control group. A preliminary analysis of some non-Hodgkin lymphoma specimens was included in an earlier report.³ Institutional Review Board approval was obtained for this study.

All sample processing was done in a laminar flow hood within a biosafety level 3 facility free from viruses and plasmids at the Department of Molecular Virology and Microbiology, Baylor College of Medicine. Total cellular DNA from non-Hodgkin lymphoma and control samples was extracted as previously described.¹⁵

All PCR assays were set up in the PCR Clean Rooms core facility of the Department of Molecular Virology and Microbiology at Baylor College of Medicine to avoid contamination of reaction mixtures. As a further precaution, positive-displacement pipetters and barrier-tip pipettes were used. Oligonucleotide primers used for PCR and DNA sequence analysis have been described previously.^15-19 All DNA samples were tested for suitability for amplification with primers specific for a fragment of the human ß-haemoglobin gene (primers PC03/KM38). Only specimens from which cellular ß-globin gene sequences could be amplified were then examined for viral sequences by PCR amplification with primer sets specific for a region of the large T antigen gene (PYVfor/PYVrev) conserved among all three polyomaviruses capable of infecting humans (SV40, JC virus, and BK virus), for the EBV latent membrane protein 2a (LMP-2a) gene (TP1Q5/TP1Q3), or for a region of the HHV-8 capsid gene (KS1/KS2).^18,19 Primers were obtained from Integrated DNA Technologies (Coralville, IA, USA).

Positive control plasmids were added to the control PCR reactions outside the core facility after tubes containing negative controls and test DNA were closed. The positive controls for polyomavirus PCR reactions were plasmid DNAs containing cloned SV40 (pSVSph21-N), JC virus (pBRJC-MAD-1), or BK virus (pBRBKV-Dunlop) genomes. The SV40 control genome contains an engineered restriction site that distinguishes it from natural isolates.¹⁵ The positive control for EBV reactions was DNA extracted from the EBV-positive Burkitt's lymphoma cell line Namalwa; that for HHV-8 was a plasmid DNA containing cloned viral sequences of the capsid antigen gene. Negative controls for PCR assays were reactions without added DNA template. PCR amplifications (45 cycles) were done with a GeneAmp PCR system 2400 thermocycler (Perkin-Elmer, Norwalk, CT, USA). High-stringency annealing temperatures specific for each primer set have been described elsewhere.^8,15,17-19 PCR amplification products were analysed by agarose gel electrophoresis.¹⁵

Probes specific for each virus were used to discriminate among amplified N-terminus T antigen polyomavirus sequences.^11,16 These specific oligoprobes are 39-labelled with a tail of dUTP-fluorescein by terminal transferase. Electrophoresed polyomavirus PCR products were transferred to a nylon membrane and the DNA was cross-linked to filters by ultraviolet irradiation for 2 min. The fluorescent hybrid was detected with an anti-fluorescein horseradish-peroxidase-conjugated antibody. Autoradiography was done at room temperature for 15 min. Additionally, representative polyomavirus PCR products were cloned into a TA cloning vector (Invitrogen, Carlsbad, CA, USA); multiple clones were screened by PCR and then sequenced with the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (USB, Cleveland, OH, USA) to confirm the identity of polyomavirus-specific DNA from the tumours.

The necessary sample size for the study was calculated a priori from previously published reports. The presence of polyomavirus SV40 neutralising antibodies has been reported in 16% of HIV-1-infected and 11% of HIV-1-uninfected patients.²⁰ Published estimates of detection of SV40 DNA sequences in non-Hodgkin lymphoma range from 10% to 20%.^9,11,12 Therefore, we assumed a conservative rate of 15% for the detection of polyomavirus large T antigen sequences in non-Hodgkin lymphoma in HIV-1-uninfected patients. The rate of polyomavirus T antigen DNA in non-Hodgkin lymphoma was expected to be at least 30% among HIV-1-infected patients. On those assumptions, 120 individuals in each group (HIV-1-infected and HIV-1-uninfected patients) would be necessary to observe a difference of that magnitude assuming a power of 80%, a two-sided test, and a test significance level of 0·05. Post-hoc estimates, however, indicated that, with 75 participants, our study had more than 99% power to identify SV40 detection in non-Hodgkin lymphoma compared with control.

Statistical methods were used to address the third objective of this research. ² analysis was used to compare the distribution of viral sequences in non-Hodgkin lymphoma between HIV-1-infected and HIV-1-uninfected patients. The t test was used to compare the mean age of patients with SV40-positive non-Hodgkin lymphoma between the two groups, and non-parametric analysis of variance was used to compare the difference in CD4 cell count among HIV-1-infected patients with systemic non-Hodgkin lymphoma. Statistical analysis was done with the SAS/PC statistical software package.

The funding sources had no role in study design; in the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.
 

Table 1 shows the demographic characteristics of the 154 HIV-1-infected and HIV-1-uninfected patients with systemic non-Hodgkin lymphoma. The distribution of histological types of non-Hodgkin lymphoma analysed among HIV-1-infected and HIV-1-uninfected individuals was indicative of the frequency of non-Hodgkin lymphoma in these two populations of patients in general.¹⁴ Diffuse large B-cell lymphoma was the most common histological type of non-Hodgkin lymphoma in HIV-1-infected and HIV-1-uninfected patients. The mean CD4 cell count of HIV-1-infected patients at the time of diagnosis of systemic non-Hodgkin lymphoma was 165/µL (SD 185, range 2-901).

Polyomavirus large T antigen PCR products were generated from 64 of 154 (42%) samples of non-Hodgkin lymphoma, including from 25 (33%) HIV-1-infected patients and 39 (50%) HIV-1-negative patients (table 2, figure 1). Polyomavirus sequences were not detected in the non-cancer controls (peripheral-blood leucocytes and lymph-node samples from HIV-1-infected and HIV-1-uninfected patients without non-Hodgkin lymphoma) or cancer controls (colon and breast carcinomas). EBV DNA was detected in 30 (39%) of 76 non-Hodgkin lymphoma samples from HIV-1-infected patients and in 12 (15%) of 78 samples in the HIV-1-negative group. HHV-8 sequences were not detected in any of the non-Hodgkin lymphoma samples from either group of patients. Only 11 (7%) of 154 non-Hodgkin lymphoma samples (seven HIV-1-infected and four HIV-1-uninfected patients) were positive for both EBV and polyomavirus sequences.

Figure 1: Agarose gel electrophoresis and staining with ethidium bromide of PCR-amplified polyomavirus sequences (upper panel), and Southern blotting (lower panels) with probes for individual polyomaviruses M=molecular-weight markers. SV40+ Home Mesothelioma Cancer Mesothelioma Treatment Compensation for Mesothelioma Cancer Contact Us Disclaimer & Terms of Use