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
). 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
). 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
). 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
). 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
), 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
), 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
). 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
). 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
). 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
). 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
). 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.