J Virol . 2008 Oct; 82(19): 9329–9336. doi: 10.1128/JVI.00646-08 PMCID: PMC2546968 PMID: 18632860 Human Endogenous Retrovirus K (HML-2) Elements in the Plasma of People with Lymphoma and Breast Cancer ▿ † ,1,‡ ,1,‡ ,4 ,4 ,5 ,5 ,6 ,7 ,2,3 ,2,8 and 1,* Rafael Contreras-Galindo Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Rafael Contreras-Galindo Mark H. Kaplan Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Mark H. Kaplan Philippe Leissner Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Philippe Leissner Thibault Verjat Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Thibault Verjat Ilaria Ferlenghi Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Ilaria Ferlenghi Fabio Bagnoli Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Fabio Bagnoli Fabiola Giusti Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Fabiola Giusti Michael H. Dosik Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Michael H. Dosik Daniel F. Hayes Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Daniel F. Hayes Scott D. Gitlin Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by Scott D. Gitlin David M. Markovitz Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 Find articles by David M. Markovitz Author information Article notes Copyright and License information Disclaimer Divisions of Infectious Diseases,1 Hematology/Oncology, Department of Internal Medicine,2 Breast Oncology Program, Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109,3 Departments of Oncology and Human Genetics, BioMérieux SA, Grenoble 38024, France,4 Department of Cellular Microbiology and Informatics, Novartis Vaccines & Diagnostics, Siena 53100, Italy,5 Department of Evolutionary Biology, University of Siena, Siena 53100, Italy,6 North Shore Hematology/Oncology Associates, Setauket, New York 11733,7 Division of Hematology/Oncology, Department of Internal Medicine, Veterans Affairs Health System, Ann Arbor, Michigan 481048 *Corresponding author. Mailing address: University of Michigan Medical Center, 5220 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5640. Phone: (734) 647-1786. Fax: (734) 764-0101. E-mail: Corresponding author. Mailing address: University of Michigan Medical Center, 5220 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5640. Phone: (734) 647-1786. Fax: (734) 764-0101. E-mail: ude.hcimu@vokramd ‡These authors contributed equally. Copyright © 2008, American Society for Microbiology

Abstract Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material. They subsequently acquired multiple mutations, leading to the assumption that these viruses no longer replicate. However, certain human tumor cell lines have been shown to release endogenous retroviral particles. Here we show that RNA from human endogenous retrovirus K (HERV-K) (HML-2), a relatively recent entrant into the human genome, can be found in very high titers in the plasma of patients with lymphomas and breast cancer as measured by either reverse transcriptase PCR or nucleic acid sequence-based amplification. Further, these titers drop dramatically with cancer treatment. We also demonstrate the presence of reverse transcriptase and viral RNA in plasma fractions that contain both immature and correctly processed HERV-K (HML-2) Gag and envelope proteins. Finally, using immunoelectron microscopy, we show the presence of HERV-K (HML-2) virus-like particles in the plasma of lymphoma patients. Taken together, these findings demonstrate that elements of the endogenous retrovirus HERV-K (HML-2) can be found in the blood of modern-day humans with certain cancers.

Over the course of millions of years, actively replicating retroviruses entered into the human genome and ultimately became a stable part of the inherited genetic material (3, 35, 43, 45). These viruses are termed human endogenous retroviruses (HERVs), and endogenous retroviral elements make up approximately 8 percent of the human genome (3, 34, 45, 48). HERVs exist in the genome in a proviral form and consist of three genes (gag, pol, and env) flanked by two long terminal repeats (3, 44). After entering the genome, HERVs subsequently acquired multiple mutations and deletions, leading to the assumption that none of them are competent to replicate. In fact, almost all of the proviruses thus far characterized in the human genome appear to be nonfunctional and fixed. However, some have recently been found to contain polymorphisms in different humans, suggesting that they have integrated into the genome relatively recently in human history and may still be evolving (4, 7, 8, 38, 40, 41, 55). The endogenous retrovirus HERV-K (HML-2) is the most recent entrant into the human genome, having entered 200,000 to 5 million years ago (4, 55), as well as being the most transcriptionally active (29, 47, 49, 51, 54, 57, 59). In the past 2 years, one group has created an infectious clone of HERV-K (HML-2) (23), and another has shown that virus can be generated using several transcomplementary plasmids (32). Thus, at least in its reanimated form with mutations corrected to reintroduce open reading frames, HERV-K (HML-2) can be shown to replicate. Debate exists as to whether HERV-K (HML-2) is still capable of replication in modern humans. HERV-K (HML-2) has been linked to oncogenesis. Indeed, this virus first came to the notice of biologists due to its similarity to mouse mammary tumor virus (MMTV), a virus that causes mammary tumors in mice, replicates in lymphocytes, and has an exogenous phase and an endogenous phase (9, 37). Consistent with the similarity of HERV-K (HML-2) to MMTV, HERV-K (HML-2) env has been found to be overexpressed in breast cancer tissue, where it also exhibits novel, alternatively spliced forms (24, 56, 57). The overexpression of HERV-K gag has additionally been seen in the peripheral blood cells of leukemia patients (21). It has also been shown that HERV-K (HML-2) is capable of forming viral particles in megakaryocytes from patients with essential thrombocythemia and in cell lines from melanoma, breast cancer, and teratocarcinoma, although it has not been demonstrated that these particles are infectious (10, 16, 36, 39, 42, 49). The mechanisms by which HERV-K (HML-2) might prove to be oncogenic, if it is at all, have only recently begun to be elucidated. In this regard, HERV-K (HML-2), which has two forms, type 1 and type 2, can encode at least two putative oncoproteins (1, 2, 15, 20). Type 1 encodes the newly identified Np9 oncoprotein, whereas type 2 encodes an accessory protein, Rec, which is necessary to export unspliced RNA from the nucleus to the cytoplasm like its counterparts human immunodeficiency virus type 1 (HIV-1) Rev and human T-cell leukemia virus type 1 Rex (38, 58). Both Np9 and Rec have been shown to be capable of cellular transformation under certain circumstances, and Rec can induce carcinoma in situ in mice (1, 2, 6, 11, 15, 20, 25, 38). We recently made the observation that HERV-K (HML-2) RNA can be found in the plasma of HIV-1-infected patients (18, 19). In view of this and the factors cited above, we investigated whether HERV-K (HML-2) RNA might also be present in the blood of patients with either lymphoma or breast cancer. We report that these patients indeed have extremely high titers of viral RNA in their blood, and these titers fall precipitously when patients are treated for lymphoma. We further demonstrate that individuals with lymphoma who have high titers of HERV-K (HML-2) RNA also have reverse transcriptase activity and viral Gag and Env proteins in the same plasma fractions in which viral RNA is found. Finally, using electron microscopy (EM) and immunogold staining we visualized, for the first time, the presence of HERV-K (HML-2)-like particles in the plasma of people (lymphoma patients). These findings demonstrate that HERV-K (HML-2) viral elements can be found circulating in the blood of humans with lymphoma and breast cancer.

MATERIALS AND METHODS Study subjects. Following informed consent, plasma samples were obtained from human subjects following protocols approved by the Institutional Review Boards of the University of Michigan and the North Shore University Hospital. Subjects included 18 healthy individuals and patients with rheumatoid arthritis, breast cancer, HIV infection with diffuse large B-cell lymphoma, non-HIV-associated diffuse large B-cell lymphoma, and HIV infection with Hodgkin lymphoma. Viral RNA extraction. For reverse transcriptase PCR (RT-PCR), plasma was treated with 200 U RNase-free DNase (Roche Laboratories) for 2 h. RNA was extracted from 140 μl of plasma using the QIAamp viral RNA minikit following the manufacturer's instructions (Qiagen, Valencia, CA). Absence of DNA contamination was confirmed by PCR. For nucleic acid sequence-based amplification (NASBA), RNA was extracted using the NucliSENS easyMAG extraction method (Biomerieux, France) (17). Synthesis of RNA transcripts in vitro and real-time RT-PCR. Synthesis of RNA transcripts for calibration and real-time RT-PCR were performed as described previously (18). Primers were designed to amplify and quantify the HERV-K gag (KgagRTF, 5′-AGC AGG TCA GGT GCC TGT AAC ATT-3′; KgagRTR, 5′-TGG TGC CGT AGG ATT AAG TCT CCT-3′) and the HERV-K env tm (KenvTMF, 5′-GCT GTA GCA GGA GTT GCA TTG-3′; KenvTMR, 5′-TAA TCG ATG TAC TTC CAA TGG TC-3′). NASBA. NASBA amplification was performed as described previously (17), using primers specific to the HERV-K (HML-2) env (Ktype1F, 5′-AGA AAA GGG CCT CCA CGG AGA TG-3′; Ktype1R, 5′-AAT TCT AAT ACG ACT CAC TAT AGG GAG AAG GCT CTC CCT AGG CAA ATA GGA-3′; Ktype2F, 5′-AGA CAC CGC AAT CGA GCA CCG TTG A-3′; and Ktype2R, 5′-AAT TCT AAT ACG ACT CAC TAT AGG GAG AAG GAT CAA GGC TGC AAG CAG CAT ACT C-3′) with molecular beacons specific for type 1 and type 2 viruses labeled with the fluorophore FAM (6-carboxyfluorescein) or ROX (6-carboxy-X-rhodamine) at the 5′ end and a quencher (Dabsyl) at the 3′ end. Probe sequences were Ktype1P (5′-FAM-CGA TCG ACG GAG ATG GTA ACA CCA GTC ACA TGG ACG ATC G-3′) and Ktype2P (5′-ROX-CGA TCG AAG TTG CCA TCC ACC AAG AAG GCA GAC GAT CG-ROX-3′). Iodixanol gradients and viral particle purification. One milliliter of each plasma sample was diluted in 10 ml phosphate-buffered saline (PBS) and centrifuged at 3,000 rpm for 10 min. Supernatants were overlaid on 20% iodixanol cushions (Optiprep density gradient medium; Sigma, St. Louis, MO) and centrifuged at 45,000 × g for 2 h. Pellets were resuspended in 500 μl PBS and overlaid in 50% iodixanol solution in 0.85% NaCl. A self-gradient was achieved through ultracentrifugation in a vertical fixed-angle rotor at 350,000 × g for 6 h. Fractions of 350 μl were collected, and their density was calculated by measuring the absorbance at 340 nm and also reading the refraction index. RT assays. The RT activity was measured in 5 μl of each fraction using the EnzCheck RT assay kit (Invitrogen) as described by the manufacturer. Serial dilutions of murine leukemia virus RT (Stratagene) were used as calibrators. Western blotting. Proteins were precipitated from the fractions with chloroform-methanol, separated on 10% sodium dodecyl sulfate-polyacrylamide gels, and blotted onto nitrocellulose membranes. The membranes were blocked in 10% milk for 1 h and incubated with primary anti-HERV-K Env (HERM-1811-5) or anti-HERV-K Gag (HERM-1841-5) monoclonal antibody (Austral Biologicals) in blocking solution. As a control for nonspecific cross-reactivity, blots were incubated with mouse serum. Membranes were washed five times in PBS containing 1% Tween. The bound primary antibody was then detected with horseradish peroxidase-conjugated goat anti-mouse secondary antibody using the Super Signal West Pico system (Pierce Chemical Co., Rockford, IL). EM. Plasma fractions obtained by iodixanol gradients were diluted in PBS, and particles were pelleted at 250,000 × g. Negative staining, conventional EM, and immunogold labeling of particles were performed as described previously (30). Briefly, particles were absorbed to 300-mesh nickel grids and blocked in Tris-buffered saline-1% bovine serum albumin. Particles were incubated with primary mouse monoclonal anti-HERV-K Env (HERM-1811-5, diluted 1:10 in blocking solution) (Austral Biologicals) for 1 h. In addition, another set of grids was incubated with purified mouse immunoglobulin G (IgG) for 1 h to control for cross-reactivity of mouse serum with plasma proteins. Grids were washed five times and incubated with gold-labeled anti-mouse secondary antibody for 1 h. In the experiments done with 10-nm gold particles, the secondary antibodies came from Sigma (St. Louis, MO), and in experiments done with 5-nm gold particles, the secondary antibodies came from BB International (Madison, WI). Particles were fixed in 2% glutaraldehyde, negative stained in 2% uranyl acetate, and visualized with a Philips CM-100 (Ann Arbor, MI) or a CM-10 (Siena) transmission electron microscope operating at 80 kV.

DISCUSSION Virus-like particles generated by mammalian tissues have often been found to have their genesis in endogenous retroviral proviruses. Mouse tissues generate such endogenous retrovirus-derived virus-like particles under both pathological and normal conditions, including in the developing embryo (31, 46, 60). In addition to mice, the presence of endogenous retroviruses and retroviral particles has been linked to cancer in several other animals, including humans. Recently, Tarlinton et al. isolated a new retrovirus from koala bears, termed KoRV. This virus was isolated from koalas that developed lymphomas. The authors showed that KoRV is a new endogenous koala retrovirus currently in transition between an exogenous and endogenous phase. Furthermore, KoRV is found in high titers in the blood of certain koalas as demonstrated by real-time RT-PCR (52, 53). Interestingly, in these koalas there is a strong association between plasma viral load, as assessed by RNA levels, and the development of leukemia/lymphoma (51). HERV-K (HML-2) virus-like particles have previously been found in cell lines from human teratocarcinoma, melanoma, and breast cancer (10, 42, 49). Here we demonstrate, for the first time, that HERV-K (HML-2) RNA can be found in the blood of human patients with lymphoma, as well as in patients with breast cancer. In addition, these patients have remarkably high plasma titers of HERV-K (HML-2) RNAs, and these titers drop dramatically with successful treatment of lymphoma. By analogy to MMTV, it is likely that mammary or lymphocytic tumor cells are the main source of production of HERV-K (HML-2) particles, the number of which appears to be drastically decreased in the plasma after the tumor burden is reduced by chemotherapy. However, ongoing experiments in our laboratory aim to clarify that this is indeed the case. It must be cautioned that what we are detecting as HERV-K (HML-2) plasma RNA could conceivably be viral DNA that is released in response to the increased cellular proliferation and turnover seen with malignancy (27, 28). Several factors suggest that this is not likely the case, however. First, we detected no amplification of HERV-K (HML-2) RNA when RT was not added into the reaction mixtures (reference 19 and data not shown). Second, the RT-PCRs are performed in the presence of DNase, and, perhaps more significantly, we obtained very similar results with both RT-PCR and NASBA, the latter being a method of amplifying RNA that does not require thermocycling and is not interfered with by the presence of free DNA. In addition, even intentional contamination of NASBA reactions did not lead to amplification of DNA (see Fig. S1 in the supplemental material). Thus, while it is impossible to completely rule out DNA contamination, these considerations, as well as the presence of viral particles and proteins in the plasma of these patients, suggest that it is much more likely to be HERV-K (HML-2) RNA than DNA that we are detecting in these studies. In addition to HERV-K (HML-2) RNA, we have also presented evidence suggesting the presence of other viral elements in the plasma of lymphoma patients. First, when plasma is separated over iodixanol gradients, the fractions corresponding to the appropriate density for a retrovirus contain RT activity, HERV-K (HML-2) gag and env RNA, and Gag and Env proteins, whereas no RT activity, viral RNA, or viral proteins are seen in the plasma of control individuals. Further, likely because the titers of virus-like particles are so high, we were able to visualize HERV-K (HML-2)-like particles in the plasma of the lymphoma patients. Immunogold staining demonstrated that these particles are indeed quite likely to be HERV-K (HML-2), as suggested by the Western blots, RT-PCR, and NASBA results. To our knowledge, this is the first demonstration that, similar to the case for the supernatants of human malignant cell lines, HERV-K (HML-2) particles can be found in the sera of actual cancer patients. Although the presence of these viral elements correlates with disease, whether HERV-K (HML-2) plays an actively pro-oncogenic role remains to be elucidated. However, should these viral elements in the plasma ultimately prove to be from truly infectious viral particles (see discussion below), targeting HERV-K (HML-2) with antiretroviral compounds might ultimately emerge as a therapeutic strategy in patients with lymphoma or breast cancer. Therefore, our findings have the potential to affect both the understanding of viral oncogenesis and therapies for important malignancies. Whether or not replicating HERV-K (HML-2) plays a role in the pathogenesis of breast cancer or lymphoma, it appears that, consistent with the koala data (52, 53), HERV-K (HML-2) viral loads may prove to be an important new biomarker in these diseases. First, whereas some biomarkers in clinical use show changes over a range of a single log unit, the titers of HERV-K (HML-2) RNA in patients versus controls are markedly different: normal individuals have titers of 102 copies/ml on average, whereas patients with lymphoma can have titers of up to 1010 copies/ml. In addition, while the specificity of these findings will require significantly greater investigation before conclusions are reached, it does appear that there are differences within cancer groupings that are not simply based on overall titer. For example, patients with Hodgkin disease have very high titers of HERV-K (HML-2) type 1 but have negligible titers of HERV-K (HML-2) type 2 (Fig. ). Finally, we find that when patients are successfully treated for lymphoma (Fig. ), the titers of HERV-K (HML-2) RNA in the blood return to low or even undetectable levels. Thus, HERV-K (HML-2) titers have the potential to be developed into a badly needed biomarker for this important cancer. However, it must be emphasized that further work must be done before the true clinical utility of HERV-K (HML-2) as a biomarker is established. Endogenous retroviral elements make up approximately 8 percent of the human genome (33, 45), and they are generally considered to have lost the ability to replicate due to having acquired multiple mutations. Further, there is a paucity of complete functional elements [those that encode functional copies of all HERV-K (HML-2) genes contiguously] to be found in the human genome. Two groups have recently shown that reanimated versions of HERV-K (HML-2), made from cloned constructs in which mutations have been corrected, are able to replicate (23, 34). These elegant experiments demonstrate that HERV-K (HML-2) could indeed replicate in the past, but they do not directly address whether HERV-K (HML-2) can still replicate in modern humans. Interestingly, at least one full-length HERV-K with intact open reading frames, HERV-K 113, has been found to be present in about 15 to 30 percent of individuals who have been tested, and genetic analysis reveals that it is a very recent addition to the human genome (14, 55). This led the authors to suggest that HERV-K 113 is a candidate for active replication in modern humans, but three groups have now produced evidence that HERV-K113 is by itself likely defective, at least in cell culture assays (5, 13, 22). Belshaw and colleagues have shown that HERV-K (HML-2) has been under continuous purifying selection, a finding that they interpret to suggest that proliferation of this family has been almost entirely due to germ line reinfection (7, 8). Conservation of the env gene was further thought to support this idea, as it would suggest a need for ongoing reinfection in the life cycle of these viral elements. Finally, at least eight elements from the HERV-K (HML-2) family appear to be polymorphic with respect to their presence in the human population, indicating that they have inserted into the human genome subsequent to the last common ancestor of humans and chimpanzees (7, 8, 38, 40, 41). Therefore, while the majority of investigators today believe that HERV-K (HML-2) is no longer capable of replication in modern humans, some evidence to the contrary does exist. Our finding of HERV-K (HML-2) RNA, RT activity, processed viral proteins, and what appear to be mature viral particles in the blood of cancer patients raises the issue of whether active HERV-K (HML-2) replication might take place in these patients under some circumstances. However, proof to that effect will require transmission of the modern virus in the laboratory.

Acknowledgments We thank Rino Rappuoli, John Donnelly, John Moran, Steve Goff, and Joseph Pagano for their thoughtful comments on the work; Donna Gschwend for manuscript preparation; Dotty Sorenson for help with the electron microscopy studies; and Judith Estes and Kathryn Jacobi for patient recruitment. This work was supported primarily by a generous grant to M.H.K. from the Concerned Parents for AIDS Research (05-5089), with additional funding coming from a gift from students at Roslyn High School, Roslyn, NY. D.F.H. was supported by the Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale. S.D.G. was supported by a grant from the Michigan Institute for Clinical and Health Research. D.M.M. was supported by grant R01 AI062248 from the National Institutes of Health and is the recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.